A Time-Kill Assay Study on the Synergistic Bactericidal Activity of Pomegranate Rind Extract and Zn (II) against Methicillin-Resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa.

There is a need for new antimicrobial systems due to increased global resistance to current antimicrobials. Pomegranate rind extract (PRE) and Zn (II) ions both possess a level of antimicrobial activity and work has previously shown that PRE/Zn (II) in combination possesses synergistic activity against Herpes simplex virus and Micrococcus luteus. Here, we determined whether such synergistic activity extended to other, more pathogenic, bacteria. Reference strains of methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, and Pseudomonas aeruginosa were cultured and subjected to challenge by PRE, Zn (II), or PRE + Zn (II), in time-kill assays. Data were obtained independently by two researchers using different PRE preparations. Statistically significant synergistic activity for PRE + Zn (II) was shown for all four bacterial strains tested compared to untreated controls, although the extent of efficacy and timescales varied. Zn (II) exerted activity and at 1 h, it was not possible to distinguish with PRE + Zn (II) combination treatment in all cases. PRE alone showed low activity against all four bacteria. Reproducible synergistic bactericidal activity involving PRE and Zn (II) has been confirmed. Potential mechanisms are discussed. The development of a therapeutic system that possesses demonstrable antimicrobial activity is supported which lends itself particularly to topical delivery applications, for example MRSA infections.

Correction: Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV.

[This corrects the article DOI: 10.1371/journal.pone.0179291.].

Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV.

BACKGROUND: There is a clinical need for new therapeutic products against Herpes simplex virus (HSV). The pomegranate, fruit of the tree Punica granatum L, has since ancient times been linked to activity against infection. This work probed the activity of pomegranate rind extract (PRE) and co-administered zinc (II) ions. MATERIALS AND METHODS: PRE was used in conjunction with zinc (II) salts to challenge HSV-1 and aciclovir-resistant HSV in terms of virucidal plaque assay reduction and antiviral activities in epithelial Vero host cells. Cytotoxicity was determined by the MTS assay using a commercial kit. RESULTS: Zinc sulphate, zinc citrate, zinc stearate and zinc gluconate demonstrated similar potentiated virucidal activity with PRE against HSV-1 by up to 4-fold. A generally parabolic relationship was observed when HSV-1 was challenged with PRE and varying concentrations of ZnSO4, with a maximum potentiation factor of 5.5. Punicalagin had 8-fold greater virucidal activity than an equivalent mass of PRE. However, antiviral data showed that punicalagin had significantly lower antiviral activity compared to the activity of PRE (EC50 = 0.56 µg mL-1) a value comparable to aciclovir (EC50 = 0.18 µg mL-1); however, PRE also demonstrated potency against aciclovir-resistant HSV (EC50 = 0.02 µg mL-1), whereas aciclovir showed no activity. Antiviral action of PRE was not influenced by ZnSO4. No cytotoxicity was detected with any test solution. CONCLUSIONS: The potentiated virucidal activity of PRE by coadministered zinc (II) has potential as a multi-action novel topical therapeutic agent against HSV infections, such as coldsores.

In vitro permeation and biological activity of punicalagin and zinc (II) across skin and mucous membranes prone to Herpes simplex virus infection.

Coadministration of pomegranate rind extract (PRE) and zinc (II) ions has recently been reported as a potential new topical treatment for Herpes simplex virus (HSV) infections. In the current work we examined the in vitro topical delivery of punicalagin (major phytochemical of PRE) and zinc from hydrogels across epithelial membranes that can become infected with HSV. Porcine epidermal, buccal and vaginal mucous membranes were excised and mounted in Franz diffusion cells and dosed with a simple hydrogel containing PRE and zinc sulphate (ZnSO4). The permeation of punicalagin and zinc were determined by HPLC and ICPMS respectively; punicalagin was also determined in the basal layers by reverse tape stripping. Receptor phases from the epidermal membrane experiment were also used to challenge HSV-1 in Vero host cells, and ex vivo porcine skin was used to probe COX-2 modulation. Punicalagin and zinc permeated each of the three test membranes, with significantly greater amounts of both delivered across the epidermal membrane. The amounts of punicalagin permeating the buccal and vaginal membranes were similar, although the amount of zinc permeating the vaginal membrane was comparatively very large - it is known that zinc interacts with vaginal mucosa. The punicalagin recovered by reverse tape stripping of the epidermal, buccal and vaginal membranes gave 0.47±0.016, 0.45±0.052 and 0.51±0.048nMcm-2 respectively, and were statistically the same (p<0.05). A 2.5 log reduction was achieved against HSV-1 using diffusion cell receptor phase, and COX-2 expression was reduced by 64% in ex vivo skin after 6h. Overall, a hydrogel containing 1.25mgmL-1 PRE and 0.25M ZnSO4 was able to topically deliver both the major bioactive compound within PRE and Zn (II) across all membranes and into the site specific region of Herpes simplex vesicular clusters, while maintaining potentiated virucidal and anti-inflammatory properties. This novel therapeutic system therefore has potential for the topical treatment of HSV infections.

Anti-inflammatory activity of Punica granatum L. (Pomegranate) rind extracts applied topically to ex vivo skin.

Coadministered pomegranate rind extract (PRE) and zinc (II) produces a potent virucidal activity against Herpes simplex virus (HSV); however, HSV infections are also associated with localised inflammation and pain. Here, the objective was to determine the anti-inflammatory activity and relative depth penetration of PRE, total pomegranate tannins (TPT) and zinc (II) in skin, ex vivo. PRE, TPT and ZnSO4 were dosed onto freshly excised ex vivo porcine skin mounted in Franz diffusion cells and analysed for COX-2, as a marker for modulation of the arachidonic acid inflammation pathway, by Western blotting and immunohistochemistry. Tape stripping was carried out to construct relative depth profiles. Topical application of PRE to ex vivo skin downregulated expression of COX-2, which was significant after just 6h, and maintained for up to 24h. This was achieved with intact stratum corneum, proving that punicalagin penetrated skin, further supported by the depth profiling data. When PRE and ZnSO4 were applied together, statistically equal downregulation of COX-2 was observed when compared to the application of PRE alone; no effect followed the application of ZnSO4 alone. TPT downregulated COX-2 less than PRE, indicating that tannins alone may not be entirely responsible for the anti-inflammatory activity of PRE. Punicalagin was found throughout the skin, in particular the lower regions, indicating appendageal delivery as a significant route to the viable epidermis. Topical application of TPT and PRE had significant anti-inflammatory effects in ex vivo skin, confirming that PRE penetrates the skin and modulates COX-2 regulation in the viable epidermis. Pomegranates have potential as a novel approach in ameliorating the inflammation and pain associated with a range of skin conditions, including cold sores and herpetic stromal keratitis.