CHEMICAL DETERMINATION OF PACs

The PACs content of Oximacro® is determined by the BL-DMAC method, but this method DOES NOT DISTINGUISH between PACs-A and PACs-B.

When we measured the standards of PAC-A and PAC-B using the BL-DMAC method, we found that the same amount of PAC reacted differently with various absorbance spectra. In particular, a higher absorbance (at 640 nm) was found for PAC-B than PAC-A even if a calibration curve was prepared with a PAC-A2 standard. Thus, in a cranberry sample, the presence of PAC-B overestimates the total PAC-A content.

The plot below shows the absorbance spectrum of PAC-A and PAC-B standards. PAC-B shows a higher absorbance with respect to PAC-A, when the two PACs are used at the same concentration.

The PACs content of Oximacro® is determined by the BL-DMAC method
HPLC-ESI-MS/MS ANALYSIS OF OXIMACRO® IS THE ONLY METHOD ABLE TO ASSESS THE PRECISE CONTENT OF PACs-A, WHICH ARE THE ONLY BIOACTIVE COMPOUNDS FOR UTI AND ANTIVIRAL ACTIVITIES.

The figure below shows the HPLC-ESI-MS/MS chromatogram performed in MRM mode showing the presence of PAC-A dimers typical of Oximacro®.

MS/MS spectra in negative mode of a typical PAC-B dimer

The plot below shows MS/MS spectra in negative mode of a typical PAC-B dimer ([M- H]-1 577 m/z) and of a typical PAC-A dimer ([M-H]-1 575 m/z).

PAC-B A PAC-A

 

Based on BL-DMAC and HPLC-MS analyses, Oximacro® contains:
360 mg/g Total PACs of wich 86% PACs-A

A VALID SUPPORT FOR URINARY TRACT HEALTH

To assess the effect of Oximacro®, we recruited participants from a population of volunteers (10 male and 60 female). The inclusion criteria included any woman or man at least 18 years of age to over 51 years of age with at least 2 culture-documented symptomatic UTIs in the calendar year prior to recruitment. The experimental group (5 males and 30 females) received 1 capsule containing 36% PAC-A twice per day (morning and evening = 72 mg PACs-A per day) for 7 days, and the placebo group (5 males and 30 females) was given the same number of capsules with no PACs. A score (from 0, representing no effect, to 10, representing a maximum effect of Oximacro® in preventing UTI) was recorded for all volunteers and the data were log-transformed.

After 7 days of Oximacro® administration (72 mg PACs-A), a significant difference was found between the placebo and Oximacro® groups for both females (Mann-Whitney U-test = 875; P < 0.001; N = 60) and males (Mann- Whitney U-test = 24; P = 0.016; N = 10) (Below panel).

a significant difference was found between the placebo and Oximacro® groups

When the female and male age ranges were analysed separately, the female age range 31-35 showed only slightly significant differences between the placebo and Oximacro® groups (Mann-Whitney U-test =20.5; P = 0.095; N = 10), whereas all other female age ranges showed highly significant differences between the placebo and Oximacro® groups (Mann-Whitney U-test = 25; P = 0.008; N = 10). All males showed significant responses to Oximacro® administration (right panel).

highly significant differences between the placebo and Oximacro® groups

The results of this work are in agreement with previous randomized, double-blind versus placebo multicentre studies examining the effects of 72 mg of PAC-standardized cranberry. Furthermore, our results show that 72 mg PAC-A is highly effective, and we suggest the use of dosages based on PACs-A instead of the total PACs in UTI prevention. In fact, due to the impossibility of BL-DMAC in discriminating between PAC-A and PAC-B, the sole total PACs quantification may not be sufficient in providing the required amount of PAC-A needed to significantly inhibit UTIs.

For a complete description of methods and results please refer to the paper:

 

Occhipinti, A., Germano, A., Maffei, M.E. (2016)
Prevention of urinary tract infection with Oximacro®, a cranberry extract with a high content of A-type Proanthocyanidins (PAC-A). A pre-clinical double-blind controlled study.
Urology Journal, 13: 2640-2649 (download pdf).

ANTIVIRAL ACTIVITY AGAINST HERPES

In the absence of efficient preventive vaccines, topical microbicides offer an attractive alternative to nucleoside analogues in the prevention of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Because of its recognized anti-adhesive activity against bacterial pathogens, Oximacro® may represent a natural source of new antiviral microbicides.

Antiviral activity of Oximacro® was tested in vitro on HSV-1 and HSV-2 replication.
Pretreatment of Vero cells with Oximacro® 1 h before infection produced a significant concentration-dependent inhibition of both clinical isolates of HSV-1 and HSV-2 (see figure below).

Antiviral activity of Oximacro® on HSV-1 and HSV-2 replication

Oximacro® inhibits HSV adsorption

Analysis of the mode of action revealed that Oximacro® prevents adsorption of HSV-1 and HSV-2 to target cells. Prechilled Vero cells were treated with various concentrations of Oximacro®, or heparin at 4°C for 30 min and then infection was carried out with precooled HSV-1 or HSV-2 at a MOI of 0.002 for 3 h at 4°C in the presence of compounds as indicated in the figure below. Oximacro® impai- rs the attachment of HSV in a concentration-dependent manner and to a similar degree as observed in the virus yield.

Oximacro® inhibits HSV adsorption

Therefore, Oximacro®, a cranberry extract highly enriched in A-type PACs, exerts a potent dose-dependent antiviral activity against clinical isolates of HSV-1 and HSV-2, the mechanism for which involves the inhibition of the initial virus attachment to the surface of target cells.

OXIMACRO® PACs-A ARE THE ONLY TO EXHERT ANTIVIRAL ACTIVITY

The presence of A-type PACs in cranberry extract is central to its bioactivity. Gel filtration chromatography allowed us to fractionate Oximacro® into five major fractions, which were chemically characterized by HPLC-ESI-MS/MS and showed to contain anthocyanins, flavonoids, and PACs-A dimers and trimers (see figure below).

In the above figure: (A) Eluogram from Sephadex-LH20 column fractionation of Oximacro®, five fractions were separated. The blue line correspond to absorbance readings at 360 nm, dotted orange line correspond to BL-DMAC reading at 640 nm. Only fractions 3 and 4 react to the BL-DMAC reagent. (B) HPLC-MS analysis of Fraction 1 was mainly composed of delphinidin and cyanidin glycosides and rutin. (C) HPLC-MS analysis of Fraction 2 shows the presence of quercetin and isorhamnetin. HPLC-MS analysis of Fractions 3 and 4 were dominated by several isomers of PAC-A dimers (D) and trimers (E), whereas fraction 5 did not contain any detectable compound.

In order to evaluate whether the whole cranberry or the PAC-A only exerted a biological activity on Herpes virus we analysed the anti-HSV activity of the five Oximacro®-derived purified fractions. We found that only identified fractions 3 and 4, corresponding to dimers and trimers of PAC-A are responsible for the inhibitory activity of the whole extract (See table below).

Fraction Citotoxicity activity
(CC50 µg/ml)
Antiviral activity*
(IC50 µg/ml)
HSV-1 HSV-2
1 >200 >75 >75
2 >200 >75 >75
3 188.2 ± 2.3 19.8 ± 3.1 6.8 ± 3.2
4 106.9 ± 1.1 19.2 ± 3.7 7.6 ± 2.2
5 >200 >75 >75
* Oximacro® concentration that inhibits 50% HSV replication as determined by a plaque reduction assay. The values are means ± SD of three independent experiments performed in duplicate.

THEREFORE, PACs-A ARE THE ONLY RESPONSIBLE FOR THE ANTIVIRAL ACTIVITY OF THE CRANBERRY EXTRACT OXIMACRO®. OTHER FRACTIONS PRESENT IN THE EXTRACT DO NOT CONTRIBUTE TO THE BIOLOGICAL ACTIVITY AGAINST HSV-1 AND HSV-2.

HOW DOES OXIMACRO® WORKS AS AN ANTIVIRAL AGENT?

The schematic representation shown in the figure below shows the interaction between the A-type PACs present in Oximacro® and the HSV envelope glycoproteins. Oximacro® binds the ectodomain of HSV glycoproteins (gD and gB) in a concentration- and time-dependent manner, thus inhibiting their functions in virus attachment and entry. However, at present, it remains still unclear whether the interactions between A-type PACs of Oximacro® and HSV envelope glycoproteins result in alterations of specific protein domains, or whether the A-type PACs simply “coat” the whole glycoproteins (as shown in the figure below), thus preventing access to their normal binding partners on target cells.


For a complete description of methods and results, please refer to the paper:

TERLIZZI, M.E., OCCHIPINTI, A., LUGANINI, A., MAFFEI, M.E., GRIBAUDO, G. (2016)
Inhibition of Herpes Simplex Type 1 and Type 2 infections by Oximacro®, a cranberry extract with a high content of A-type Proanthocyanidins (PACs-A).
Antiviral Research 132: 154-164. (download pdf)

ANTIVIRAL ACTIVITY AGAINST INFLUENZA VIRUS

The influenza viruses type A and type B (IAV, IBV) are widespread major pathogens among human populations and are responsible for seasonal epidemics and pandemics. Annual influenza epidemics cause worldwide 3–5 million cases of serious disease and up to half a million deaths among high-risk groups, with an even greater impact in developing countries. The defense against influenza virus (IV) infections still poses a series of challenges. The current antiviral arsenal against influenza viruses is in fact limited; therefore, the development of new anti-influenza strategies effective against antigenically different viruses is an urgent priority. Oximacro® inhibits influenza A and B viruses (IAV, IBV) replication in vitro because of its high content of A-type proanthocyanidins (PAC-A) dimers and trimers.

Oximacro® and Its PACs-A Inhibit IAV and IBV Replication

Oximacro® inhibits both IAV and IBV replication in MDCK cells in a concentration-dependent manner. The calculated IC50 values were 4.5 ± 0.2 μg/ml for IAV, and 4.5 ± 0.5 μg/ml for IBV, respectively. The 50% cytotoxic concentration (CC50) was 141 ± 0.8 μg/ml, thus indicating that the antiviral activity of Oximacro® did not stem from a non-specific cytotoxicity. The selectivity index for IAV and IBV (CC50/IC50) was therefore 31.1 and 31.3, respectively.

Also against the influenza virus ONLY the PAC-A of Oximacro® have antiviral action

To pinpoint the active phytochemical of Oximacro® that was responsible for its inhibitory activity, antiviral assays were performed with purified fractions obtained through fractionation of Oximacro® (see above). Analysis of the fractions’ anti-IV activity identified fractions 3 and 4 as having inhibitory activity against IAV and IBV, thus indicating that the components responsible for the antiviral activity of Oximacro® were only PAC-A.

Oximacro® Targets Early Phases in the IV Replication Cycle

To understand the Oximacro® mechanism of action, prechilled MDCK cells were infected with either IAV or IBV in the presence of different concentrations of Oximacro® for 2 h at 4°C, so that to enable the attachment of IV particles, but not their entry into target cells. Oximacro® inhibited the attachment of both IAV and IBV in a concentration-dependent manner (Figure A). Next, to investigate the inhibitory activity of Oximacro® on IV entry, prechilled MDCK monolayers were infected with IV at 4°C for 2 h to enable viral attachment but not entry. Different concentrations of Oximacro® were then added and infected cultures were incubated at 37°C for 2 h to allow entry of adsorbed IV particles. Oximacro® prevented the entry of both IAV and IBV in a concentration-dependent manner (Figure B). Together, these results revealed that Oximacro® is able to interfere with both viral attachment and entry, therefore suggesting its suitability as an early-acting inhibitor of IV replication.

Oximacro® Interferes With IV Hemagglutinin

Influenza virus hemagglutinin (HA) is responsible for both the IV attachment and entry by mediating both the initial interaction with cell receptors containing sialic acid, and the fusion between endosome membranes and the viral envelope. Oximacro® is able to interact with HA in a concentration- and time-dependent fashion and suggest that interactions between Oximacro® and IV HA may hamper its functions in virus attachment and entry, and underlie the overall antiviral activity of Oximacro®. To support the occurrence of interactions between HA and Oximacro® PAC-A further, in silico docking simulations were performed. Using the crystal structure of HA (A/Puerto Rico/8/1934, PDB ID: 1RU7) of our tested IAV strain, docking analysis revealed that PAC-A dimers binds to the internal grooves of the HA structure first (Figure A, left panel), and subsequently to the surface of the HA structure (Figure A, right panel). The best binding pose is shown in Figure B, where HA residues (PHE 299, TRP 234, and ASN 210) forming hydrogen bonds to PAC-A2 are highlighted.

Overall, the described mechanism of action of Oximacro® against IV advocates its potential application as a valid support for influenza infections.

For a complete description of methods and results, please refer to the paper:

LUGANINI, A., TERLIZZI, M.E., CATUCCI, G., GILARDI, G., MAFFEI, M.E., GRIBAUDO, G. (2018)
The cranberry extract Oximacro® exerts in vitro virucidal activity against influenza virus by interfering with hemagglutinin.
Frontiers in Microbiology, 9: 1826 – (download pdf).

IN VITRO BIOFILM INHIBITION ASSAY

Oximacro® effect on Escherichia coli 7402 (pilus rod PAP + type 1 pili): in vitro inhibition bioassay

Oximacro® was tested for its capacity to inhibit in vitro Escherichia coli 7402, by evaluating the effect on the pilus rod (Pap) and type 1 pili. In uropathogenic infections caused by Escherichia coli, the fim operon encodes type 1 pili (expressing an hemagglutination which is mannose-sensitive), whereas the pap operon encodes P- or Pap-pili (which are able to interact with the digalactoside unit in the P-blood group antigen) (for more information see the publication by Terlizzi et al., 2017).

The bioassay performed on E. coli indicates that Oximacro® exerts its maximal inhibition activity at concentrations between 0.5 and 1.5 μg/ml, as shown in the below figure (average of 2 experiments, each with 5 replications).

For more information of UroPathogenic Escherichia coli, please refer to the paper:

Maria E. Terlizzi, Giorgio Gribaudo and Massimo E. Maffei (2017)
UroPathogenic Escherichia coli (UPEC) Infections: Virulence Factors, Bladder Responses, Antibiotic, and Non-antibiotic Antimicrobial Strategies.
Frontiers in Microbiology, 8: e1566 – (read online).

 

OXIMACRO® SHOWS AN ANTIOXIDANT ACTIVITY COMPARABLE TO RESVERATROL ON HUMAN HEPG2 CELLS

When tested on human HepG2 cells, Oximacro® shows a strong and direct antioxidant power. When used at 0.78 mg/ml, Oximacro® shows an Antioxidant Index (AOP) of 924 (theoretical maximum = 1000), and when used at 0.39 mg/ml the AOP is even higher (933). The EC50 of Oximacro® is 0.109 mg/ml and its strong antioxidant effect becomes significant from 0.062 mg/ml (EC10). 90% effect (EC90) is reached at 0.194 mg/ml. A direct comparison with one of the most potent antioxidants, resveratrol, shows that Oximacro® exerts a comparable antioxidant activity. For instance, AOP of resveratrol is 970 when used at 0.028 mg/l, whereas resveratrol EC50 = 0.016 mg/ml.

The Figure below shows the Kinetic data of Oximacro® obtained by the LUCS method, where the intracellular antioxidant effect is revealed by a time delay in the fluorescence increase triggered by the LED-dependent photo-activation of TO (see Oxi-P® facts for a better explanation of the LUCS test). The figure shows the fluorescence profiles of human HepG2 cells treated 4 h at different Oximacro® concentrations.

Intracellular antioxidant effect of Oximacro® on human HepG2 cells. Control (non-treated, Control Medium) curve is illustrated in black. Differences between control and Oximacro® Area Under Curves (AUC) allow to establishing AntiOxidant Power index that qualifies the intracellular antioxidant effect of Oximacro®.

Calculation of LUCS index at different Oximacro® concentrations allows to establish dose-effect profiles that fits well with sigmoid regression, allowing the evaluation of the EC10 (Efficacy Concentration 10%, threshold of cellular effect), the standard EC50 (Efficacy Concentration 50%) and the EC90 (Efficacy Concentration 90%, weakest dose at which the compound acts with a maximum effect).  Dose-Effect data are shown in the Figure below.

Dose-effect curve obtained after 4 hours treatment with Oximacro® on human HepG2 cells.

These quantitative results show that Oximacro® is a potent antioxidant agent able to act effectively at the cellular level as demonstrated with the bioassay on human HepG2 cells. These data may be used as human cell-based references to improve preparation processes of products based on Oximacro®.

For more information on LUCS technology please refer to the following links: