Antibacterial Activity of Passion Fruit Purple Variant (Passiflora edulis Sims var. edulis) Seeds Extract Against Propionibacterium acnes

Introduction

Plants have been used over thousands of years as a source of therapeutic medicine. Tropical fruits are largely known to have not only antioxidant and anti-inflammatory activities but also antibacterial properties, and have a long history of use as traditional herbal medicines.¹ Passiflora edulis Sims is a variety of passion fruit that is mostly used commercially for consumption, in refreshment drinks and in syrup production. This plant exhibits various pharmacological properties and possesses a complex phytochemistry. Most of the pharmacological investigations into P. edulis have addressed its central nervous system (CNS) activities, such as anxiolytic, anticonvulsant, and sedative actions.² In recent years, many studies have been conducted on the purple variant, Passiflora edulis Sims var. edulis, and revealed a high concentration of piceatannol. This polyphenol is largely found in the seeds and shows high antioxidant and potential antibacterial activity.^3,4

Propionibacterium acnes is a Gram-positive anaerobic bacillus normally found on human skin and plays an important role in the pathogenesis of acne vulgaris. People with acne lesions have higher numbers of P. acnes compared with controls. Increased colonies are found as a result of sebum accumulation and infundibulum hyperkeratosis. Furthermore, P. acnes was found to be capable of inducing the release of chemoattractants, eliciting the inflammatory response. Therefore, the clinical manifestations appear to be the result of bacterially induced inflammation of plugged sebaceous glands.^4,5 Docherty et al reported that piceatannol has antibacterial activity against P. acnes and this is comparable with erythromycin and benzoyl peroxide, which are commonly used as first line topical antimicrobial therapy in acne vulgaris.⁴ For this reason, an in vitro study is necessary to evaluate the antibacterial property of passion fruit extract, which contains a high concentration of piceatannol. Therefore, the aim of this study is to evaluate the inhibitory effect of P. edulis Sims var. edulis seed extract against P. acnes.

Materials and Methods

Seeds of P. edulis Sims var. edulis were collected from a farm in Brastagi, Karo, North Sumatera, Indonesia, from January to February 2019. The extract was prepared by maceration in the Department of Biological Pharmacy, Faculty of Pharmacy, Universitas Sumatera Utara, Medan, Indonesia. First, the seeds were separated from the pulp, dried at 40°C, and mashed with a blender into powder. Then, the powder was soaked in 96% ethanol in a 2:3 ratio for 5 days, and evaporated at 40–50°C to produce the seed extract (Figure 1). The extract was dissolved in DMSO into multiple concentrations, of 1.25%, 2.5%, 5%, 10%, 20%, and 40% (Figure 2A).

Figure 1 Passiflora edulis Sims var. edulis: (A) wet seeds, (B) dry seeds, (C) simplicia, and (D) seed extract.

Figure 2 (A) Seed extract of Passiflora edulis Sims var. edulis; (B) isolated P. acnes swabbed over BBS 5% agar plate for 24 hours; (C) disc diffusion method of various extract concentrations compared with clindamycin (center); (D) measuring the inhibition zone using calipers.

Propionibacterium acnes used in this study was obtained from ATCC (strain 11827). In the first step, the antimicrobial activity of the plant extracts was evaluated using the agar disc diffusion method. The inoculum size of each clinical isolate was standardized matching a turbidity equivalent to a 3 McFarland standard. A sterile cotton swab was dipped into the suspension, and rotated several times on the inside wall of the tube above the fluid level to remove excess inoculum from the swab. The swab was drawn over the entire surface of already prepared plates of Brucella blood sheep (BBS) 5% agar to achieve uniform distribution of bacteria (Figure 2B). Four discs impregnated with the extracts at various concentrations and one disc in the center with clindamycin as control were placed on the BBS 5% agar plates (Figure 2C). Three separate plates were prepared in a similar manner for the positive controls of the bacterial strains. We also compared the 1% extract with erythromycin in conjunction with clindamycin as the most common topical antibiotic preparation used in acne vulgaris. Cultures were incubated in an anaerobic gas jar with an anaerobic gas pack, creating anaerobic conditions at 37°C, and the inhibition zone diameter was measured at 24 hours (Figure 2D). The entire procedure was repeated for three sets of plates. Data were analyzed to find the mean diameter of the zone of inhibition and the Mann–Whitney test was used to compare the inhibition zones of the extract, clindamycin and erythromycin.

The antibacterial assay was continued with a serial dilution method for several concentrations of the extract, namely 5%, 10%, 15%, 20%, and 25%. Brain–heart infusion (BHI) broth medium was prepared in dilution tubes. The cotton swab containing P. acnes was dipped into the dilution tubes and rotated several times. Then, each concentration of the extract was added to the dilution tubesand incubated anaerobically at 37°C for 24 hours (Figure 3). Thereafter, a sterile cotton swab was dipped into each dilution tube and swabbed over the entire surface of BBS 5% agar plates. The agar plates then were incubated anaerobically at 37°C for another 24 hours and P. acnes culture results were compared for each extract concentration to determine the minimum inhibition concentration (MIC) and minimum bactericidal concentration (MBC). The entire procedure was repeated for three sets of tubes.

Figure 3 All the tested dilution tubes were put into an anaerobic jar with a gas pack.

Results

In the agar disc diffusion method, all concentrations of P. edulis Sims var. edulis showed inhibitory activity on P. acnes (Figure 4). The minimum concentration of the extract to have an antibacterial effect was 5%, with a mean inhibition zone (MIZ) of 8.5 mm, followed by 10% with MIZ 10.08 mm, 20% 14 mm, and 40% 16 mm (Table 1). These results are comparable with or even superior to clindamycin, which had an MIZ of 8 mm. There was no statistically significant difference in inhibition zones between P. edulis Sims var. edulisseed 5% extract and clindamycin (p=0.121). However, the antimicrobial activity of P. edulis seedextract was inferior to erythromycin, which had an MIZ of 22.67 mm (p=0.046) (Table 2).

Figure 4 Agar disc diffusion method showing the inhibition zone of Passiflora edulis Sims var. edulis seed extract with concentrations of (A) 1.25%, 2.5%, 5%, and 10%, (B) 20% and 40%; (C) compared with clindamycin (M) and erythromycin (E).

Table 1 Inhibition Zone of Passiflora edulis Sims var. edulis Seed Extract at Several Concentrations Compared with Clindamycin and Erythromycin

Table 2 Comparison of Propionibacterium acnes Inhibition Zones in Passiflora edulis Sims var. edulis Seed Extract 5% with Clindamycin and Erythromycin

In serial dilution assays, P. acnes growth was still found at concentrations of 5%, 10%, 15%, and 20%. The passion fruit purple variant seed extract showed more than 50% colony inhibition at a concentration of 20%. Furthermore, no bacterial growth was found at 25% concentration of the extract (Figure 5). Therefore, the MIC and MBC of P. edulis Sims var. edulis seed extract against P. acnes were 20% and 25%, respectively.

Figure 5 Propionibacterium acnes growth on BBS 5% agar plates with different extract concentrations: (A) 5%, (B) 10%, (C) 15%, (D) 20%, (E) 25%.

Discussion

Preliminary analysis of the crude extract from the leaf, stem, and fruit of Passiflora edulis revealed the presence of several biologically active compounds, such as carbohydrates, tannin, glycoside, flavonoid, saponin, alkaloids, volatile oil, phenol, resins, and balsam, which may have therapeutic value.⁶ Furthermore, Johnson et al reported the antimicrobial properties of the leaf and callus of P. edulis, which varied in effectiveness depending on the solvent type owing to the variety of contained compounds, including saponins, steroids, tannins, phenolics, triterpenoids, alkaloids, and flavonoids.⁷ Ripa et al observed that the chloroform leaf extracts were much more active than the petroleum ether extracts, having average zones of inhibition of 7–10 mm by the disc diffusion method.⁸ These studies showed that the stem extract has the highest activity against the growth of Vibrio mimicus, with a zone of inhibition of 17 mm. Besides this, the extract showed good activity against the growth of Vibrio parahaemolyticus (16 mm), Shigella dysenteriae (15 mm), and Shigella boydii (14 mm).^7,8 According to Akanbi et al, the fruit extract has significant antimicrobial activities in hexane and water solvents. The hexane extract showed MIZs of 16 mm for Klebsiella pneumoniae, and 14 mm for Staphylococcus aureus and Pseudomonas aeruginosa, and the water extract an MIZ of 16 mm for Salmonella paratyphi. Furthermore, the hexane leaf extract showed MIZs of 16 mm for S. paratyphi, 15 mm for S. aureus, 14 mm for P. aeruginosa, and 13 mm for K. pneumoniae. The water extract showed degree of growth inhibition of 15 mm for S. paratyphi and 13 mm for S. aureus. The stem extracts showed greater and significant activities for the hexane and methanol extracts, with MIZs of 16 and 14 mm, respectively, for K. pneumoniae. No activity was observed against any of the tested bacteria for the ethyl acetate and water stem extracts. They concluded that the antimicrobial activities were dependent on the part of the plant used as well as the type of extract used.⁶ However, there are no data available on the antibacterial effects of P. edulis seed extract on P. acnes.

In this study, the antimicrobial activity of P. edulis Sims var. edulis seed extract with ethanol solvent on P. acnes was evaluated using two different methods. The agar disc diffusion assay revealed that the seed extract with the minimum concentration of 5% already has antibacterial activity against P. acnes, which is comparable to clindamycin. A moderate inhibitory effect on P. acnes growth was observed with 10% concentration and this increased at higher concentrations. Hence, the 20% and 40% extracts of P. edulis Sims var. edulis seeds showed higher inhibitory activity against this Gram-negative anaerobic bacillus, which plays an important role in the pathogenesis of acne vulgaris. According to a 2019 publication that questioned the replicability of the agar disc diffusion method in evaluating the antimicrobial activity of plant extracts, serial dilution assays could be combined to determine the MIC and MBC of the plant extract, as in this study.⁹ In serial dilution assays, inhibition of P. acnes colonies was observed at a concentration of 20%. Bactericidal activity was noted with the 25% extract. The in vitro test results confirmed that P. edulis Sims var. edulis seeds showed potential antibacterial activity against P. acnes, with an MIC of 20% and MBC of 25%.

The high amount of piceatannol in the seeds of P. edulis Sims var. edulis was thought to play a significant role in the inhibitory effect on P. acnes growth. Matsui et al reported that 88% of total polyphenols of the fruit is found in the seeds, which comprised 33% of the freeze-dried seed compounds, and the majority of these is piceatannol (4.8 mg/g).³ Our results are in accordance with a study on the antimicrobial effect of piceatannol on P. acnes, which also showed significant inhibitory activity in comparison with resveratrol, benzoyl peroxide, and erythromycin. The average IC₅₀ and IC₁₀₀ of piceatannol were 123 and 234 mg/L, respectively, for three strains of P. acnes (ATCC 25746, 29399, and 33179). Benzoyl peroxide treatment had an average IC₅₀ of 164 mg/L and an average IC₁₀₀ of 295 mg/L. On the other hand, the average IC₅₀ for resveratrol was much lower, at 73 mg/L, and the average IC₁₀₀ was 187 mg/L. Furthermore, with erythromycin, the average IC₅₀ for all strains of P. acnes was only 1.5 mg/L and the IC₁₀₀ was 11 mg/L.⁴ Therefore, the seed extract of P. edulis Sims var. edulis in ethanol solvent has significant antimicrobial activity against P. acnes, and may be a promising alternative to clindamycin and erythromycin topical preparations, which are widely used as the first line antibacterial treatment for acne vulgaris.

Conclusion

The extract of P. edulis Sims var. edulis seeds showed good antimicrobial effects on P. acnes and the inhibitory activity increased with concentration of the extract. Comparable inhibitory effects with clindamycin and erythromycin may support the application of this extract in the management of acne vulgaris. Therefore, further clinical trials on the use of P. edulis Sims var. edulis seeds extract are necessary to show its efficacy in acne vulgaris patients.