IT202200025929A1 - COMPOSITIONS COMPRISING BERGAMOT FRUIT FRACTIONS - Google Patents

Description

Description of the patent for industrial invention entitled:

?COMPOSITIONS COMPRISING BERGAMOT FRUIT FRACTIONS?

The invention relates to bergamot fruit fractions obtained by atomization of the juice thickened with gum arabic. The invention also relates to solid oral compositions comprising said fractions and their use in the treatment of dyslipidemia.

State of the art

Non-alcoholic fatty liver disease (NAFLD) is one of the most common causes of liver damage, often associated with cirrhotic degeneration and hepatocellular carcinoma. The origin of NAFLD has not been fully clarified. Many studies have shown that some inflammatory markers such as TNF-? and other cytokines play an important role in the pathogenesis. Additional factors are obesity and metabolic disorders related to insulin resistance. At present there are no available drugs and lifestyle modification, attention to nutrition and weight loss are the only measures currently recommended for subjects affected by NAFLD.

Bergamot (Citrus bergamia Risso & Poiteau), a citrus fruit grown in restricted areas of Calabria, has been used above all for the properties of its essential oil, particularly sought after in perfumery.

Bergamot juice, rich in polyphenols and flavonoids (brutieridin and melitidin), has hypocholesterolemic and hypoglycemic properties (Mollace et al., Fitoterapia, vol.

82, n? 3, 2011, pp.309-316).

US 8,741,362 describes a phytoextract obtained from the albedo and juice of the fresh fruit of Citrus aurantium var. bergamia that is able to normalize lipid and glycemic parameters in patients affected by diet-dependent or familial genetic dysmetabolism. In pathological hyperlipidemias, a reduction in total cholesterol and LDL cholesterol at concentrations between 20 and 32% has been demonstrated, with a 30% increase in HDL cholesterol. These important variations are associated with a clear reduction in VLDL cholesterol and an increase in the size of LDL with consequent reduction in their oxidation. Bergamot extract also improved endothelial function with vascular protection attributable to flavonoids. In a model of hepatic steatosis in transgenic mice developing a pathology overlapping with that of humans, bergamot extract was shown to be effective in normalizing the lipid and glycemic parameters in patients with diet-dependent or familial genetic dysmetabolism. was able to significantly counteract perisinuidal fibrosis which remains the most difficult condition to modify in NAFLD.

Description of the invention

A fraction of bergamot fruit has now been found with significant antioxidant, anti-inflammatory and anti-dyslipidemic activity. The fraction of the invention, containing polyphenols, organic acids, betaines, soluble and insoluble fibres, is obtained by pressing the peeled fruits, removing the residual solid part by filtration or centrifugation, concentrating to 20-40 degrees Brix, adding gum arabic in a ratio of 20 to 30% by weight, adjusting the pH in the range from 4 to 5, and drying in an atomizer or by freeze-drying.

The fraction of the invention has a polyphenol content (including naringenin, hesperidin, neohesperidindisosmetin, erocitrin, neoeriocitrin, luteolin, apigenin, brutierudin and their glucosides) of approximately 10 - 15% by weight and is particularly rich in betonicin, stachydrin, luteolin 6-C-glucoside, apigenin-6-C-glucoside, N-(1-deoxy-1-fructosyl)leucine, isosakuranine. Other components of the fraction include quinic acid, citric acid, feruloyl glucosides, pectins.

A distinctive feature of the fraction of the invention, in addition to the presence of substantial quantities of pectins (up to 18-20 g/100 g), is represented by the presence of nanovesicles, as demonstrated by electron microscopy analyses.

The fraction of the invention can be formulated in forms suitable for oral administration, for example tablets, capsules, granules, suspensions or solutions, using conventional techniques and excipients.

The compositions thus obtained may contain other active ingredients with complementary, synergistic or in any case useful activity for the uses considered. Examples of other active ingredients include extracts of Cynara scolimus, Cynara cardunculus, Olea europaea, Berberis aristata, Olea oleracea, Vitis vinifera, Cyclanthera pedata, Gymnema silvestris, Eugenia jambolana, vitamins, policosanols.

The unit dose of the atomized bergamot fruit fraction in the compositions, which constitute a further object of the invention, is preferably between 50 and 500 mg.

The compositions of the invention, despite a reduced content of polyphenols and flavonoids compared to that of other known bergamot extracts, have been found to be surprisingly more active in normalizing the lipid profile both in experimental models and in clinical trials.

The presence of pectins, partly derived from gum arabic as well as from bergamot juice itself, provides a significant contribution to the therapeutic activity by virtue of the interactions between soluble fibers and bile acids, as reported for example in Nutrients, 2019, 11, 1424 and in Int. J. Mol. Sci., 2020, 21,6495.

The process for the preparation of the bergamot juice fraction of the invention comprises in detail the following stages, starting from the fruits:

- Washing, removal of dust and leaves in steel tanks with drinking water;

- Peeling the flavedo and centrifuging;

- Pressing of the fruits without the flavedo and separation of the juice from the solid residue;

- Depulping by decanter and centrifuge;

- Concentration at 20-40 Brix, preferably 40 Brix;

- Mixing with Senegal Acacia extract (acacia gum);

- Atomization in spray drier or freeze-drying.

The solid residue can be subjected to pectin extraction by countercurrent washing with a water:solid residue (mash) ratio of 1.5:1, with subsequent pressing with double screw presses and repetition of this operation until the sugars and other components that hinder drying have been eliminated.

The metabolic and vascular effects of the compositions of the invention were studied in a three-arm, double-blind, placebo-controlled clinical trial. 90 patients were treated, divided into three groups, treated with placebo and with two different doses for up to 12 weeks.

At the end of treatment, statistically significant changes in the lipid profile were observed compared to placebo. In particular, statistically significant decreases were observed in triglycerides, HOMA index, LDL cholesterol and total cholesterol.

The invention will be illustrated in more detail in the following examples.

Example 1 - Preparation and characterization of the fraction

10,000 kg of fresh bergamot fruits are subjected to flavedo scraping and centrifugation.

The peeled fruits (9950 kg) are squeezed with a double screw press. 2985 kg of juice and 6965 kg of solid residue (mash) are obtained which, after pulp washing with a water/mash ratio of 1.5:1, is dried in rotating ovens and sent for extraction of pectins for industrial use.

The pulp (about 12%) is removed from the juice with a decanter and centrifuge and the clear extract (9194 kg) is concentrated from 3 to 40 Brix in an evaporator to give 689.71 kg of concentrated juice at 40 Brix.

After adding 20% by weight of gum arabic and adjusting the pH to 4.5 with KOH, it is dried in a spray drier to obtain 275.88 kg of a powder, indicated below with the name of Endo@berg, having a density between 0.4 and 0.6.

The presence of nanovesicles was observed by transmission electron microscopy.

Qualitative profiling was performed using high-resolution mass spectrometry (HR-MS).

MS analysis was performed in both ion modes (positive and negative) and Xcalibur 4.0 software was used to analyze the spectra. Data were evaluated using a targeted approach based on an in-house compiled database and an untargeted approach.

Presumptive identification of compounds is based on accurate mass, isotope and fragmentation pattern evaluation. A total of 113 compounds were identified: 105 in negative polarity, 8 in positive polarity and 29 confirmed in both polarities. Figure 1 shows the Endo@berg? TIC in negative ion mode, while Figure 2 shows the Endo@berg? TIC in positive ion mode. The relative polyphenol content is 12,950 × 0.562 wt%.

The presence of nanovesicles was determined on the fraction subjected to ultracentrifugation.

The sample was diluted in saline solution and analyzed by Zeta View (Particle Metrix, Germany), to measure particle concentration and mean diameter.

The sample was fixed on 200 mesh nickel Formvar carbon-coated grids (Electron Microscopy Science, USA) for 20 minutes and negatively stained with NanoVan (Nanoprobes, USA) and observed using a JEOL JEM-1400 electron microscope (Jeol, Tokyo, Japan).

Nanoparticle Tracking Analysis (NTA) showed a population of particles with characteristics comparable to that of nanovesicles (NVs).

The particle concentration was 6.9E+12 particles/mL and the mean particle diameter was 101.8 × 37.3 nm, mean (X50) 93.0 nm.

TEM analysis showed the presence of NVs with classical spherical morphology and shape. The outer membrane and electron-dense core typical of NVs are observed.

In conclusion, NTA analysis showed the presence of particles in the tested sample. The sample showed a comparable particle concentration, in the order of 10<12 >particles/ml, and a size of 101 nm. The concentration and size parameters observed for the studied sample are in the range of NV particles [Th?ry C et al. J Extracellular Vescicol. 2018-11-23;7(1):1535750].

TEM analysis confirmed that bergamot juice contains particles with shape, size and morphology comparable to nanovesicles.

Example 2 - Evaluation of antioxidant activity in vitro

Antioxidant activity was assessed by the DPPH assay. A 50:50 (% v/v) solution of H2O:EtOH of Endo@berg? and a commercial bergamot extract (BPF) was prepared and diluted to obtain final extract concentrations in the range of 50 - 1000 μg/mL and 10 - 500 μg/mL, respectively. The reaction mixture was prepared by mixing aliquots of 500 μL of extract, 1 mL of acetate buffer (100 mM, pH 5.5), 1 mL of ethanol and 500 μL of an ethanolic solution of DPPH (500 μM). After 90 min incubation in the dark, the absorbance was 1.5 μL. was read at 517 nm with a Shimadzu UV 1900 spectrophotometer (Shimadzu, Milan, Italy). The percentage of inhibition was calculated using equation 2 and the results are expressed as mean ? SD (Table 1); the IC50 values obtained were compared with those of trolox and ascorbic acid chosen as reference compounds for their antioxidant activity.

Table 1 - Antioxidant activity expressed as IC50.

Example 3 - Evaluation of anti-inflammatory activity

The anti-inflammatory activity of Endo@berg? and stachydrine was tested using the previous in vitro cell model described by Baron et al. Antioxidants 2021, 10, 141. Before cell incubation, the Endo@berg? sample was treated to remove the fibrous and insoluble fraction, in order to free the polyphenolic fraction incorporated into the matrix and make it more available to the cells.

The treatment consisted of sonicating Endo@berg? in a hydroalcoholic mixture (CH3 CH2 OH/H2O, 70/30 %v/v) in a ratio of 1:10 m/v for 30 minutes and centrifuging the mixture for 5 minutes at 10000 rpm. The supernatant was then separated from the fibrous pellet and dried with a centrifugal evaporator at 30 ?C for 18 hours. The percentage extraction yield was 77.922 ? 1.181 and the qualitative composition of the polyphenols was maintained.

For the anti-inflammatory cell assay, the R3/1 NF-kb cell line was seeded in a white 96-well plate (BRANDplates?, cell grade) at a density of 4000 cells/well. The cells were pretreated with different concentrations of treated and untreated Endo@berg? (1 ?g/mL - 250 ?g/mL) and standard stachydrine (1 - 200 ?M) for 18 h in complete medium (DMEM, 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin). Subsequently, an inflammatory state was induced for 6 h using 10 ng/mL TNF?. To evaluate the activity of luciferase, cells were washed once with 100 μL of 1x PBS and finally 50 μL of DMEM was added to each well. A 50 μL aliquot of ONE-Glo? Luciferase Assay Substrate (purchased from Promega Corporation, Madison, WI, USA) was added directly to the cells and then luciferase was measured with a luminometer (Wallac Victor2 1420, Perkin-Elmer? Life Science, Monza, Italy). Figure 3 shows the anti-inflammatory dose-response of treated (a) and untreated (b) Endo@berg? extract and stachydrine (c). Endo@berg? was effective only after treatment, indicating that polyphenols are incorporated in an inactive form into the matrix. A statistical analysis of the obtained results was performed using a one-way ANOVA with Bonferroni's multiple comparison test (p < 0.05 was considered significant). Cell viability was tested at all concentrations used in the anti-inflammatory assay by the MTT assay performed on the same cell line and under the same conditions. The treated and untreated extract and stachydrine had no negative effects on the viability of the cell line used.

Example 4 - Evaluation of antioxidant activity in cell cultures The HEK293 NRF2/ARE Responsive Luciferase cell line was used to evaluate antioxidant activity. Cells were seeded in white 96-well plates (BRANDplates?, cell grade) at a density of 10,000 cells/well and subsequently treated with Endo@berg? extract at different concentrations (1-250 μg/mL) for 6 and 18 hours in complete medium (DMEM, 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin) (Figure 4) and with different concentrations of stachydrine (1-250 μM). To avoid reading interference, the medium was removed and 50 μL of PBS was added to each well. 50 μL of ONE-Glo? Luciferase Assay Substrate (purchased from Promega Corporation, Madison, WI, USA) was added directly to the cells and then luciferase was measured with a luminometer (Wallac Victor2 1420, Perkin-Elmer?). Figure 4 shows the antioxidant dose-response of Endo@berg? extract after 6 and 18 h of incubation, while Figure 5 shows the dose-response of stachydrine after 18 h of incubation. Statistical analysis was performed on the results obtained using a one-way ANOVA with Bonferroni's multiple comparison test (p < 0.05 was considered significant). Cell viability was tested at all concentrations used in the antioxidant assay by the MTT assay performed on the same cell line after 18 h of incubation, and both Endo@berg? and stachydrine did not significantly affect cell viability.

Claims (7)

1. A fraction of bergamot fruit obtained by squeezing the peeled fruit, removing the residual solid part by filtration or centrifugation, concentrating to 20-40 degrees Brix, adding gum arabic in a ratio of 20 to 30% by weight, adjusting the pH in the range of 4 to 5, and drying in a spray dryer or by freeze-drying. 2. A fraction according to claim 1 comprising pectins, polyphenols, organic acids and nanovesicles. 3. Solid oral compositions comprising as active ingredient the fraction of claim 1 or 2. 4. Compositions according to claim 3 in the form of tablets comprising suitable excipients. 5. Compositions according to claim 3 or 4 comprising other active ingredients selected from extracts of Cynara scolimus, Cynara cardunculus, Olea europaea, Berberis aristata, Olea oleracea, Vitis vinifera, Cyclanthera pedata, Gymnema silvestris, Eugenia jambolana, vitamins, policosanols. 6. Compositions according to one or more of claims 2 to 5 wherein the unit dose of the atomized bergamot fruit fraction is between 50 and 500 mg. 7. Compositions of claims 2-6 for use in the treatment of dyslipidemias.