Isolation of punicalagin from Punica granatum rind extract using mass-directed semi-preparative ESI-AP single quadrupole LC-MS

We are living in a era of alarming increases in microbial resistance to currently available antibiotics, and there is a growing need for new pharmaceutical products to treat infectious diseases. The pomegranate is an edible fruit that has virucidal and antimicrobial activities which is primarily attributable to the high concentration of hydrolysable tannins. Punicalagin, a high molecular weight tannin (1084.7), accounts for approximately 70% of the total and is concentrated in the fruit exocarp (rind). It is the focus of much research, although it is prohibitively expensive to purchase which presents an obstacle to further exploitation and development. Here we describe a method for the isolation of punicalagin from pomegranate rind extract and total pomegranate tannins using an Agilent preparative mass-directed LC–MS single quadrupole ESI-API system, where the ionization was set as negative and the mass signal acquired in Single Ion Monitoring. Thanks to the automatic fraction collector, the method can be used in automatic mode and is capable of producing punicalagin with >95% purity

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 6 h, and maintained for up to 24 h. 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

Co-administration of fish oil with signal transduction inhibitors has anti-migration effects in breast cancer cell lines, in vitro

Background: There is an urgent need for new therapies to treat cancer metastasis. Fish oil, with high omega 3 fatty acid content, has shown anticancer activity and signal transduction inhibitors have shown anti-metastatic properties. Objective: To provide preliminary in vitro data on the anti-migration potential of signal transduction inhibitors and co-administered fish oil. Methods: MCF-7, TamR and FasR breast cancer cell lines were used to determine the effects of combinations of PD98059, LY294002 and fish oil in growth assays. Modulations of p-Src and COX-2, both mediators of motility and invasion, were then determined by Western blotting and IHC to ascertain effects on migration potential. Results: Migration rates for the three cell lines examined were ranked: FasR>TamR>MCF-7 (p <0.05). Addition of fish oil reduced the number of TamR cells migrating after 48h (p <0.05), while the addition of PD98059 and LY294002 also decreased migratory potential of TamR cells (p <0.05). Addition of PD98059 and LY294002 to TamR cells did not result in a significant decrease in p-Src levels; as was the case when PD98059, LY294002 and 4-hydroxytamoxifen were added to MCF-7 cells. However, the co-administration of fish oil markedly reduced p-Src and COX-2 expression in both cell lines. Conclusion: Co-administration of a commercial fish oil with signal transduction inhibitors results in decreased cell migration via an unknown co-operative mechanism and could constitute a novel approach for the treatment of breast cancer metastasis

Topical delivery of a Rho-kinase inhibitor to the cornea via mucoadhesive film

The application of inhibitors of the Rho kinase pathway (ROCK inhibitors) to the surface of the eye in the form of eyedrops has beneficial effects which aid the recovery of diseased or injured endothelial cells that line the inner surface of the cornea. The aim of this study was to test the plausibility of delivering a selective ROCK inhibitor, Y-27,632, to the cornea using a thin polymeric film. Mucoadhesive polymeric thin films were prepared incorporating Y-27,632 and diffusional release into PBS was determined. Topical ocular delivery from the applied film was investigated using freshly excised porcine eyes and eyedrops of equivalent concentration acted as comparators; after 24 h the formulations were removed and the corneas extracted. Drug-loaded thin polymeric films, with high clarity and pliability were produced. ROCK inhibitor Y-27,632 was weakly retained within the film, with release attaining equilibrium after 1 h. This in turn facilitated its rapid ocular delivery, and an approximately three-fold greater penetration of Y-27,632 into cryoprobe-treated corneas was observed from the thin film (p < 0.01) compared to eyedrop. These findings support the further development of ROCK inhibitor delivery to the cornea via release from thin mucoadhesive films to treat vision loss cause by corneal endothelial dysfunction

Antimicrobial Potential of Pomegranate Extracts

The search for plant extracts with efficacious antimicrobial activity remains important, partly due to fears of the side effects associated with conventional antibiotics and to counter the emergence of resistant microorganisms. Pomegranate extracts have been used for millennia for their anti-infective properties, with activity more recently being attributed to its rich composition of ellagitannins and other secondary polyphenolic compounds. This chapter highlights the growing number of publications that have probed the activity of pomegranate extracts against microbes. Research generally supports folklore claims and has shown that pomegranate extracts possess unusual and potent broad-spectrum activities against Gram-positive and Gram-negative bacteria (planktonic and biofilm), fungi, viruses and parasites. Possible pathways/mechanisms of antimicrobial activity of pomegranate extracts are discussed and enhancement/potentiation of such activity using metal ions considered

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

Evaluation of the in vitro oral wound healing effects of pomegranate (Punica granatum) rind extract and punicalagin, in combination with Zn (II)

Pomegranate (Punica granatum) is a well-established folklore medicine, demonstrating benefits in treating numerous conditions partly due to its antimicrobial and anti-inflammatory properties. Such desirable medicinal capabilities are attributed to a high hydrolysable tannin content, especially punicalagin. However, few studies have evaluated the abilities of pomegranate to promote oral healing, during situations such as periodontal disease or trauma. Therefore, this study evaluated the antioxidant and in vitro gingival wound healing effects of pomegranate rind extract (PRE) and punicalagin, alone and in combination with Zn (II). In vitro antioxidant activities were studied using DPPH and ABTS assays, with total PRE phenolic content measured by Folin–Ciocalteu assay. PRE, punicalagin and Zn (II) combination effects on human gingival fibroblast viability/proliferation and migration were investigated by MTT assay and scratch wounds, respectively. Punicalagin demonstrated superior antioxidant capacities to PRE, although Zn (II) exerted no additional influences. PRE, punicalagin and Zn (II) reduced gingival fibroblast viability and migration at high concentrations, but retained viability at lower concentrations without Zn (II). Fibroblast speed and distance travelled during migration were also enhanced by punicalagin with Zn (II) at low concentrations. Therefore, punicalagin in combination with Zn (II) may promote certain anti-inflammatory and fibroblast responses to aid oral healin

Enhanced delivery of naproxen to the viable epidermis from an activated poly N-isopropylacrylamide (PNIPAM) Nanogel: Skin penetration, modulation of COX-2 expression and rat paw Oedema

Stimulus-responsive drug-loaded poly (N-isopropylacrylamide) nanogels were examined as a means of enhancing the delivery of naproxen into skin ex vivo. Following massaging into skin, the epidermis was probed (with and without base activation) for depth penetration and transdermal delivery of drug, and anti-inflammatory activity in the relative levels of COX-2 expression. Rat paw oedema testing was used to determine anti-inflammatory effects in vivo. When activated by sodium carbonate, particle size reduced by 19%. Tape stripping revealed significantly greater delivery of naproxen into the epidermis for the activated nanogel and the steady state flux was enhanced 2.8-fold. With base-activation COX-2 was 50% lower than non-activated, and this trend was confirmed by immunostaining, and by the reduction of rat paw swelling which provided ex vivo – in vivo corroboration. A mechanism of action is proposed. In conclusion, stimulus-responsive nanogels have potential for enhancing dermal drug delivery and therapeutic outcomes in inflammatory skin diseases

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 ± 0.048 nM cm− 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 6 h. Overall, a hydrogel containing 1.25 mg mL− 1 PRE and 0.25 M 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

Bioactive molecules from plants: discovery and pharmaceutical applications

The plant kingdom is one of the richest sources of bioactive compounds with pharmaceutical potential [...