BIOENHANCER MINT

Abstract

The invention relates to a composition for oral administration, containing an extract of mint.

Description

[0001] The invention relates to a composition for oral administration, a method for improving the absorption of a plant ingredient, and the use of a composition according to the invention, an extract of mint and / or carvone.

[0002] Various plant compounds are known to exhibit biological activities after consumption that contribute to the health of humans and animals. These include a variety of plant metabolites such as polyphenols, alkaloids, terpenes, carotenoids, and flavonoids, which play an important role in nutrition and health.

[0003] Despite their great potential, many phytochemicals are considered to have low bioavailability. Due to their low water solubility, intensive metabolism, degradation, or reabsorption into the intestinal lumen, for example, only a limited amount of ingested polyphenols reach the bloodstream. Since the gut is not only involved in the digestion and absorption of nutrients but also prevents the penetration of foreign and potentially harmful substances, often only a small fraction of the active plant compounds are effectively absorbed and transported to their potential target sites in the body. An example of a health-promoting nutrient with low bioavailability is curcumin. The main source of the yellow pigment curcumin is the rhizome of Curcuma longa, better known as turmeric. Curcumin and its two main derivatives, bisdemethoxycurcumin and demethoxycurcumin, are called curcuminoids.

[0004] Bioenhancers are natural substances that increase the absorption and / or efficacy of concomitantly ingested molecules. Several mechanisms have been described by which bioenhancers improve the oral bioavailability of substances, including the inhibition of efflux transporters, altered membrane fluidity, or the modulation of tight junctions. Numerous natural compounds have been investigated for their potential as bioenhancers, including piperine, the main active ingredient in black pepper, which is among the most effective and best-studied bioenhancers.

[0005] However, there is a need for additional bioenhancers to improve the bioavailability of substances, especially plant-based compounds such as xanthophylls, isoflavones, and curcuminoids. Furthermore, existing bioenhancers offer potential for improvement, for example, regarding their spectrum of activity, potency, and cost.

[0006] Against this background, one object of the present invention is to provide a further bioenhancer. Preferably, the further bioenhancer can overcome disadvantages or limitations of known bioenhancers. Summary of the invention

[0007] One aspect of the efforts to solve this problem is a composition for oral administration containing an extract of mint.

[0008] From the efforts to solve this problem, a further aspect results in a method for improving the absorption of a substance, preferably a plant ingredient, wherein a composition according to the invention is taken orally.

[0009] A further aspect of the efforts to solve this problem is the use of i) a composition according to the invention, ii) an extract of mint and / or iii) carvone to improve the bioavailability of a substance, preferably a plant ingredient selected from polyphenols, alkaloids, terpenes, carotenoids, flavonoids, isoflavones and curcuminoids.

[0010] As a further aspect of the efforts to solve this problem, the use of i) a composition according to the invention, ii) an extract of mint and / or iii) carvone to inhibit the efflux transporter BCRP results.

[0011] The underlying idea of ​​the invention is that an extract from mint can act as a bioenhancer. Detailed description of the invention

[0012] The mint extract preferably comprises an extract selected from spearmint (Mentha spicata), peppermint (Mentha × piperita), or mixtures thereof. Particularly preferably, the mint extract is spearmint (Mentha spicata). In another preferred embodiment, the mint extract is peppermint (Mentha × piperita).

[0013] It has been found that mint extract can act as a bioenhancer. The presence of a bioenhancer allows the composition according to the invention to realize a nutrient transport concept (NTC). The mint extract is, for example, an extract from the aerial parts, preferably the flowering aerial parts. The extract can also be obtained by steam extraction or CO2 extraction.

[0014] The extract from mint, especially spearmint, can be described by one or more of the following characteristics: - at least 3% by weight carvone; - 35 wt.% to 85 wt.% carvone, in particular 40 wt.% to 80 wt.% carvone; - 40 wt.% to 80 wt.% (R)-(-)-Carvone and 0.1 wt.% to 3 wt.% (S)-(+)-Carvone; - at least 5 wt.% limonene, for example 5 wt.% to 20 wt.% limonene, in particular 5 wt.% to 15 wt.% limonene.

[0015] An extract with at least one of these characteristics has proven to be particularly effective.

[0016] An example composition of a suitable extract is also given in Table 1.

[0017] It was found, particularly for the substances described in the examples, that mint extract can act as a bioenhancer. Accordingly, compositions containing mint extract and the substances described in the examples are especially preferred.

[0018] In a preferred embodiment, the composition according to the invention therefore contains - a xanthophyll, preferably a xanthophyll selected from lutein and zeaxanthin, and / or - an isoflavone, preferably an isoflavone selected from daidzein, glycitein and genistein, and / or - an extract of turmeric (Curcuma longa), in particular a turmeric extract complexed with cyclodextrin.

[0019] In a preferred embodiment, the composition is a dietary supplement and / or a foodstuff.

[0020] The composition according to the invention can, in particular, be in a formulation selected from a powder, a liquid, an oil, a solution, and a dispersion. The composition according to the invention can have a particular formulation, which is described in the following embodiments.

[0021] In a preferred embodiment, the composition according to the invention contains glycerin.

[0022] In a preferred embodiment, the composition according to the invention contains lecithin and water.

[0023] In a preferred embodiment, the composition according to the invention contains lecithin, glycerin and water.

[0024] In a preferred embodiment, the composition according to the invention and / or the mint extract is present in micellized form. Such a composition can be achieved by mixing and homogenizing a mixture of the mint extract, water, and lecithin, as well as optionally other lipophilic substances and optionally glycerin.

[0025] In a preferred embodiment, the composition according to the invention comprises a polar phase, a non-polar phase, and a lecithin phase consisting of the lecithin in the composition. The polar phase preferably contains, based on the total weight of the polar phase, 40 wt.% to 96 wt.%, more preferably 60 wt.% to 90 wt.%, polyol, preferably glycerol, and 3 wt.% to less than 20 wt.%, more preferably 5 wt.% to 17 wt.% water. The composition preferably contains 0.5 g to 10 g of lecithin per 100 g of the composition, more preferably 1 g to 3.5 g of lecithin.

[0026] In these embodiments, the composition according to the invention enables the simultaneous application of hydrophilic and hydrophobic ingredients in a stable dosage form, and facilitates their particularly efficient absorption by the human body. The presence of a polar and a nonpolar phase allows the composition according to the invention, especially in micellized form, to implement a nutrient transport concept (NTC). This is because hydrophilic and lipophilic ingredients can dissolve in the polar and nonpolar phases, respectively, and are thus absorbed particularly quickly and to a high degree after application. The technology for combining lipophilic and hydrophilic substances in a carrier system can also be referred to as a microsolve. + -technology. In the case of Microsolve +This technology is a further development of microsolve technology, in which one or more lipophilic substances are finely dispersed and essentially pre-dissolved in the aqueous phase using natural, food-type solubilizers (e.g., soy lecithin) (e.g., via micellization). This facilitates the dispersion of the lipophilic nutrient in the gastrointestinal tract and thus supports optimal nutrient absorption. In the corresponding embodiments of the composition according to the invention, the lipophilic (synonym: nonpolar) phase remains finely and homogeneously dispersed in the aqueous (synonym: polar) phase for an extended period. Therefore, the composition according to the invention is physically stable in these embodiments. Only lecithins can be used as emulsifiers, and these can be used in small quantities relative to the substance to be emulsified. A further advantage is that lecithins are of natural origin.Furthermore, synthetic surfactants and monohydric alcohols such as ethanol or propanol are not required for the stability of the composition. The polyol contributes to preservation, and no additional preservatives are needed besides the aforementioned ingredients. This eliminates the need for synthetic or allergenic preservatives.

[0027] In a preferred embodiment, the composition according to the invention further comprises a hydrophilic (synonym: polar) ingredient, in particular selected from vitamin C, vitamin B1, vitamin B2, vitamin B3, vitamin B6, vitamin B7 (biotin), vitamin B 12 , minerals, trace elements and oligomeric proanthocyanidins. The hydrophilic (synonym: polar) component is primarily found in the polar phase.

[0028] In a preferred embodiment, the composition according to the invention further comprises a further lipophilic (synonym: nonpolar) ingredient, in particular selected from coenzyme Q10, omega-3 fatty acids, vitamin D2, vitamin D3, derivatives of vitamin D, vitamin A and derivatives thereof, vitamin E and derivatives thereof, vitamin K1, vitamin K2, isoprenoids, berry extract, hawthorn extract and artichoke extract. The further lipophilic ingredient is present in particular in the nonpolar (synonym: lipophilic) phase.

[0029] In a preferred embodiment, the composition according to the invention contains a lipophilic extract of ginger (Zingiber officinale), preferably from ginger rhizomes, wherein the ginger extract is preferably a CO2 extraction extract.

[0030] In a preferred embodiment, the composition according to the invention contains an extract from the bay laurel (Laurus nobilis). The extract can be obtained from the leaves and / or the fruit.

[0031] The ginger extract and the bay laurel extract can further increase the bioavailability of the ingredients.

[0032] The invention also relates to a method for improving the absorption of a substance, preferably a plant ingredient, and uses for improving the bioavailability of a substance, preferably a plant ingredient. The plant ingredient is preferably selected from lutein, zeaxanthin, daidzein, glycitein, genistein, bisdemethoxycurcumin, demethoxycurcumin, and curcumin. When using carvone, the carvone can be selected from S-(+)-carvone and R-(-)-carvone, as well as mixtures thereof, with the use of R-(-)-carvone being preferred.

[0033] The method and uses according to the invention are preferably non-medical methods and uses.

[0034] The invention can also relate to a method for improving the absorption of a pharmaceutical active ingredient and uses for improving the bioavailability of a pharmaceutical active ingredient. In other words, the substance can also be a pharmaceutical active ingredient. The background is that inhibition of the efflux transporter BCRP (BCRP = breast cancer resistance protein) can also improve the bioavailability of pharmaceutical active ingredients.

[0035] The embodiments and aspects within this document can be combined arbitrarily, unless the subject matter and the description of the embodiments clearly indicate otherwise.

[0036] The verbs “contain” and “encompass” and their conjugations also include the verb “consist of” with its conjugations.

[0037] The terms “ein” and “eine” do not represent a numerical limit, but are to be understood as “at least one” and “at least one”, unless the context clearly indicates otherwise.

[0038] The invention is illustrated below by means of examples which are not intended to be limiting. Examples Example 1: Increased curcuminoid uptake through mint extract in differentiated Caco-2 cells

[0039] The influence of an extract from spearmint (Mentha spicata) on the cellular uptake of the three curcuminoids bisdemethoxycurcumin, demethoxycurcumin and curcumin was investigated in differentiated Caco-2 cells.

[0040] Cyclodextrin-complexed curcumin extract (CU) was applied to cells for four hours at a final concentration of 2.95 µg / ml total curcuminoids, with or without the addition of spearmint extract. Cellular uptake was quantified by HPLC-FLD, normalized to cell protein content, and presented for each of the three curcuminoids individually as well as for the total curcuminoid concentration. Error bars represent the mean ± SD (n=11-12).

[0041] As in Fig.As shown in Figure 1, significantly higher intracellular concentrations of two of the three measured curcuminoids, and consequently also of the total curcuminoids, were observed during co-incubation with spearmint extract. The cellular uptake of the least abundant curcuminoid, bisdemethoxycurcumin, was increased by approximately 25%, although this increase was not statistically significant. In contrast, the spearmint extract significantly increased the uptake of both demethoxycurcumin and curcumin by approximately 65%. The total concentration of all three measured curcuminoids was increased by approximately 60%. Example 2: The enantiomer R-(-)-carvone increases cellular curcuminoid uptake to a significantly greater extent than the enantiomer S-(+)-carvone.

[0042] The influence of S-(+)-carvone and R-(-)-carvone on the cellular uptake of the three curcuminoids bisdemethoxycurcumin, demethoxycurcumin and curcumin was investigated in differentiated Caco-2 cells.

[0043] Cyclodextrin-complexed curcumin extract (CU) was applied to cells for four hours at a final concentration of 2.95 µg / ml total curcuminoids, with or without the addition of the pure substances S-(+)-carvone and R-(-)-carvone at a final concentration of 71.4 µg / ml. Cellular uptake was quantified by HPLC-FLD and normalized to protein content. Fig. Figure 2 shows the individual values ​​of the three curcuminoids (AC) as well as the sum of the curcuminoids (D). The error bars represent the mean ± SD (n=6).

[0044] S-(+)-Carvone slightly but significantly increased the absorption of demethoxycurcumin, curcumin, and the sum of the three measured curcuminoids by approximately 15%. Fig. 2B-D), while the enantiomer R-(-)-carvone led to a significantly increased absorption of all three curcuminoids by approximately 50% ( Fig.2A-D). In a direct comparison of the effects of the two enantiomers, R-(-)-carvone increased cellular curcuminoid uptake to a significantly greater extent than S-(+)-carvone ( Fig. 2B-D). Example 3: Inhibition of the efflux transporter BCRP.

[0045] To determine the mechanism that leads to increased curcumin uptake through spearmint extract, the involvement of the intestinal efflux transporters P-gp and BCRP was investigated.

[0046] These apically localized ABC transporters (ATP-binding cassette transporters) limit the bioavailability of already absorbed nutrients by transporting them back into the intestinal lumen. Therefore, the effect of spearmint (Mentha spicata) extract on the activity of the P-gp and BCRP transporters was investigated by efflux assays using the fluorescent substrates Rhodamin123 and Hoechst33342, respectively.

[0047] Fig.Figure 3 shows the effect of spearmint extract on the efflux of Rhodamin123 and Hoechst33342 in differentiated Caco-2 cells. After accumulation with the fluorescent P-gp and BCRP substrates (A) Rhodamin123 and (B, C) Hoechst33342, the respective inhibitors verapamil (50 µM) and Ko143 (10 µM), spearmint extract, (S)-(+)-carvone, and R-(-)-carvone were applied for one hour, followed by determination of the intracellular fluorescence intensity of the substrates. The error bars represent the mean ± SD (n=6-9).

[0048] When assessing BCRP activity, a significant reduction of 16% in Hoechst33342 efflux was observed for the inhibitor Ko143 ( Fig. 3B). The extract from spearmint also reduced Hoechst33342 efflux in a dose-dependent manner, with the two highest concentrations having significant effects, suggesting reduced BCRP-mediated efflux activity ( Fig.3B). A dose dependency was also observed for the two carvone enantiomers, leading to significantly reduced Hoechst33342 efflux rates at the two highest concentrations of (S)-(+)-carvone and at the highest concentration of R-(-)-carvone ( Fig. 3C).

[0049] Example 4: Spearmint extract increases the cellular uptake of lutein and zeaxanthin and the transport of isoflavones through a differentiated Caco-2 cell layer.

[0050] Intestinal efflux transporters such as BCRP cover a broad spectrum of ligands, and it is unlikely that the effect of spearmint extract is limited to curcuminoids. To test this hypothesis, the effect of the spearmint extract on the cellular uptake of (all-E)-lutein and meso-zeaxanthin, as well as on the apical to basolateral transport of soy isoflavones, was determined using a Caco-2 monolayer transwell assay. In the gut, isoflavone glycosides are normally enzymatically hydrolyzed by microbial glucosidases and other enzymes, yielding the three main aglycones genistein, daidzein, and glycitein. To mimic this biotransformation process by the gut microbiota, the isoflavone product was digested prior to cell treatment with β-glucosidase.

[0051] The effects of spearmint extract on cellular uptake of lutein and transport of isoflavones in differentiated Caco-2 cells are described in Fig.Figure 4 illustrates the analysis of cellular uptake of lutein (all-E-lutein) and meso-zeaxanthin (A) and the apical to basolateral transport of the isoflavones daidzein, glycitein, and genistein (B). Lutein + zeaxanthin (LUT) at a final concentration of 11 µg / ml and isoflavones (IF) at a final concentration of 3.92 µg / ml were applied to cells for four hours, with or without the addition of mint extract. Cellular uptake was quantified by HPLC-DAD and normalized to cell protein content. The results are shown for each xanthophyll individually and for the sum of both. The concentration of isoflavone aglycones and their metabolites was quantified by HPLC-DAD to calculate the apparent permeability coefficients (Papp), which are shown for each aglycone individually and for the sum of the isoflavones. The error bars represent the mean ± SD (n=8-9) and (n=9).

[0052] Treatment with spearmint extract significantly increased the cellular uptake of (all-E)-lutein by 76% and of meso-zeaxanthin by 64%, resulting in a significant overall increase in the uptake of both xanthophylls by 74%, from 0.42 µg / mg in the untreated group to 0.73 µg / mg of cell protein in the spearmint-treated group ( Fig. 4A). Furthermore, the transport rate of three individual isoflavone aglycones, as well as the sum of the aglycones, was significantly increased upon simultaneous incubation with spearmint extract. The spearmint extract increased the transport rates of daidzein, glycitein, and genistein by approximately 10–15% compared to the untreated control ( Fig. 4B). The aglycone concentrations in the apical and basolateral compartments were used to calculate the permeability coefficient (P). app). When monitoring TEER levels, no significant differences in cell layer integrity were observed between the untreated and spearmint-treated groups.

[0053] In summary, the increased uptake and transport mediated by spearmint are not limited to curcuminoids, but have also been observed for lutein and isoflavones. Example 5:

[0054] By conducting further transport studies in differentiated Caco-2 cells on Transwell inserts, the effect of an extract of spearmint and peppermint on the apical to basolateral transport of soy isoflavones was investigated.

[0055] Isoflavones (IF) at a final concentration of 3.92 µg / ml after β-glucosidase digestion were applied to cells for four hours with or without the addition of spearmint (S) or peppermint (P) extract in the form of micelles (water, lecithin, lipophilic components). In the control group, an equal amount of the same micelle composition (water, lecithin, lipophilic components) was applied without mint extract. The concentrations of the isoflavone aglycones daidzein, glycitein, and genistein, and their metabolites, were quantified by HPLC-DAD. The effects of spearmint (S) or peppermint (P) extract were determined by calculating the apical-to-basolateral transport rate relative to the control for each aglycone individually (AC) and for the total isoflavone concentration (D). The error bars represent the mean ± SD (n=12).Statistically significant differences are marked with *(p ≤ 0.05) and **(≤ 0.01).

[0056] The relative transport of the individual isoflavone aglycones ( Fig. 5A-C) and the sum of the aglycones ( Fig. 5D) was significantly increased upon simultaneous incubation with extracts of spearmint and peppermint. The spearmint extract increased the relative transport rates of daidzein, glycitein, and genistein by approximately 8–15%, while the peppermint extract increased the transport of daidzein and genistein by approximately 8–20% compared to the untreated control. Example 6:

[0057] The effects of an extract of spearmint and peppermint on the cellular uptake of lutein and zeaxanthin were investigated in differentiated Caco-2 cells.

[0058] Lutein + zeaxanthin (LUT) at a final concentration of 11 µg / ml was applied to cells for four hours, with or without the addition of an extract of spearmint (S) or peppermint (P) in a micelle formulation (water, lecithin, lipophilic components). In the control group, the same amount of the micelle formulation (water, lecithin, lipophilic components) was applied without the mint extract. The cellular uptake of (all-E)-lutein and meso-zeaxanthin was quantified by HPLC-DAD and normalized to cell protein levels.

[0059] The effects of individual extracts are measured for the sum of both xanthophylls as cellular uptake in relation to the control group in Fig. Figure 6 is shown. The error bars represent the mean ± SD (n=6-9). Statistically significant differences are marked with **(p≤ 0.01) and ****(≤ 0.0001).

[0060] Treatment with spearmint and peppermint extract significantly increased the cellular uptake of lutein and zeaxanthin by 106% and 72%, respectively, compared to the untreated control. Fig. 6). Example 7:

[0061] Individual extracts from bay laurel, spearmint and peppermint, as well as various extract combinations, were investigated for their potential to improve the cellular uptake of curcuminoids by measuring the intracellular concentrations of the three main curcuminoids bisdemethoxycurcumin, demethoxycurcumin and curcumin.

[0062] Cyclodextrin-complexed turmeric extract (CU) was applied to cells for four hours at a final concentration of 2.95 µg / ml total curcuminoids, with or without the addition of bay leaf (L), spearmint (S), or peppermint (P) extracts and their combinations, in a micelle composition (water, lecithin, lipophilic components). In the control group, the same amount of micelle composition (water, lecithin, lipophilic components) but without mint extract was applied. Cellular uptake was quantified by HPLC-FLD and normalized to cell protein content. The cellular uptake of the total curcuminoids was compared to the control group for the individual extracts (A) as well as for individual extracts and their combinations (B) in the following ways: Fig.Figure 7 is shown. The error bars represent the mean ± SD (n=9-12). Statistically significant differences are marked with *(p ≤ 0.05), **(≤ 0.01), ***(≤ 0.001) and ****(≤ 0.0001), in gray for comparison with the control group and in black for comparison between the treatment groups.

[0063] When considering the effects of the individual extracts, significantly higher intracellular curcuminoid concentrations were observed when simultaneously incubated with an extract of bay laurel, spearmint, or peppermint ( Fig. 7A). The bay laurel extract led to 40%, the spearmint (Mentha spicata L.) extract to 67%, and the peppermint extract to 126% higher intracellular curcuminoid concentrations compared to the untreated control group ( Fig.7A). Furthermore, treatment with combinations of two extracts (L + S, S + P) as well as the combination of all three extracts (L + S + P) significantly improved cellular curcuminoid uptake by ~144-195% ( Fig. 7B). The combinations improved curcuminoid absorption, at least in part, to a significantly greater extent than the individual extracts ( Fig. 7B). Example 8:

[0064] 2.5 g of soy lecithin with a phosphatidylcholine content of 45% are completely dispersed in 92 g of an 85% (wt%) aqueous glycerol solution while stirring at 50°C.

[0065] To this dispersion, 0.3 g of an extract from spearmint (Mentha spicata L.) and 0.2 g of an extract from turmeric (Curcuma longa) are added and stirred homogeneously at 50 °C. Finally, homogenization is carried out using a high-pressure homogenizer.

[0066] The result is a stable dispersion whose components can be absorbed particularly well by the human body. Example 9:

[0067] 0.3 g of mint extract is mixed with plant-based ingredients and fillers to a total of 100 g and homogenized. The product is in powder form and can be mixed with other substances. The result is a composition whose components are particularly well absorbed by the human body. Example 10:

[0068] 0.3 g of mint extract is mixed with medium-chain triglycerides to a total of 100 g and homogenized. The product is liquid and can be mixed with other substances. The result is a composition whose components are particularly well absorbed by the human body. Table 1: Average Rt [min] Calculated RI Metabolite Peak area [counts × min] Before SPE After SPE 7,337 1106 β-Pinen 1.33 × 10 6 - 7,759 1120 β-phellandres 4.60 × 10 5 - 9,011 1166 β-Myrcen 5.46 × 10 5 - 9,83 1198 +- Limonen 4.22 × 10 7 1.36 × 10 7 9,98 1204 Eucalyptol 3.45 × 10 6 1.21 × 10 6 11,602 1272 o-Cymen 9.49 × 10 5 4.17 × 10 5 12,878 1330 3-Methylcyclohexanone 2.28 × 10 5 - 14,225 1394 3-Octanol 1.95 × 10 6 1.63 × 10 6 15,697 1468 cis-menthon 2.62 × 10 6 2.36 × 10 6 16,238 1497 trans-menthone 9.76 × 10 5 9.09 × 10 5 16,738 1524 β-Bourbons 4.03 × 10 5 6.18 × 10 5 17,5 1566 Isomenthyl acetate 6.39 × 10 5 7.31 × 10 5 18,394 1617 trans-Dihydrocarvon 1.81 × 10 6 1.83 × 10 6 18,834 1643 menthol 4.44 × 10 6 5.16 × 10 6 19,381 1675 Dihydrocarbyl acetate 1.63 × 10 6 1.85 × 10 6 19,802 1701 Ia-Terpineol 7.79 × 10 5 8.49 × 10 5 20,282 1730 6-Methoxy-4-([(5-vinyl-1-azabicyclo[2.2.2]oct-2-yl)oxy]methyl)quinoline 9.16 × 10 5 1.08 × 10 6 20,527 1746 Carvon 1.70 × 10 8 1.86 × 10 8 20,68 1755 Neodihydrocarveol 1.96 × 10 6 2.37 × 10 6 22,006 1840 trans-Carveol 5.58 × 10 5 6.49 × 10 5 22,479 1872 cis-Carveol 3.07 × 10 5 3.72 × 10 5 24,319 1998 Carophyllene oxide 3.55 × 10 5 8.06 × 10 5

[0069] Quantification of the enantiomers in the extract of spearmint showed that approximately 99% of the carvone was the (R)-(-)-carvone enantiomer. Details on the experimental procedure for solid-phase extraction (SPE)

[0070] Prior to all experiments, optional SPE fractionation of the mint extract was performed with slight modifications as described (Antonelli, A., & Fabbri, C. (1999). Essential oils: Spe fractionation. Chromatographia, 49(3-4), 125-130). Silica SPE cartridges (200 mg bed weight, Thermo Fisher Scientific) were conditioned with 1.5 ml of n-hexane and 10 ml of mint extract was applied to the bed. The first fraction was eluted with 1.2 ml of n-hexane, followed by 2.4 ml of a mixture of n-hexane and diethyl ether (1 / 1, v / v) for fraction 2, and finally 1.2 ml of diethyl ether for fraction 3. The fractions were combined in a glass reaction tube, and the solvents were carefully evaporated under a stream of nitrogen and, if necessary, diluted with n-hexane for subsequent GC-MS measurements. Cell culture

[0071] Human Caco-2 cells (DSMZ, Braunschweig, Germany) were cultured in Earl's Minimal Essential Medium (MEM) containing non-essential amino acids, supplemented with 10% fetal bovine serum (FBS) and 100 U / ml penicillin / 100 µg / ml streptomycin (all from PAN-Biotech, Aidenbach, Germany) under standard conditions at 37 °C, 5% CO2, and ≥ 95% relative humidity. For differentiation, the cells were seeded and incubated overnight. The following day, the growth medium was removed, and differentiation was induced within 48 hours as previously described (König, A. et al. (2023). Combined acid hydrolysis and fermentation improves bioactivity of citrus flavonoids in vitro and in vivo. Communications Biology, 6(1).). In short, the differentiation medium consisted of Dulbecco's Modified Eagle's Medium (DMEM, PAN-Biotech) supplemented with 100 U / ml penicillin / 100 µg / ml streptomycin, 0.1% MITO+ serum extender (Corning, NY, USA) and 5 mM butyric acid (Sigma-Aldrich).On the third day after sowing, cell differentiation was complete, which was confirmed by microscopic observation of the domes. The medium for the Fasted State Simulated Intestinal Fluid (FaSSIF) was prepared accordingly and contained Hanks' Balanced Salt Solution (HBSS, PAN-Biotech), supplemented with 0.75 mM lo-lecithin and 3 mM taurocholic acid (both from Sigma-Aldrich), adjusted to a pH of 6.5. Cell viability test

[0072] To assess the effects of the tested extracts on cell viability, a resazurin assay was performed. 15 × 10 4Cells per well were seeded into black 96-well plates (Greiner-Bio One, Kremsmünster, Austria), incubated overnight, and then differentiated as described above. The test substances were dissolved in FaSSIF medium and applied for four hours. The cells were then washed and incubated with 80 µM resozurin sodium salt (Sigma-Aldrich) in growth media for 90 minutes. Subsequently, the fluorescence of resorufin was determined using a microplate reader (POLARStar Omega, BMG LABTECH, Ortenberg, Germany) at excitation wavelengths of 544 nm and emission wavelengths of 590 nm. Cell viability results were normalized to the untreated control, and values ​​above 90% were considered non-toxic. The micellized extract of spearmint with 1% essential oil was diluted 1:100 in FaSSIF, resulting in a final concentration of 0.01% essential oil without significant effects on cell viability.The conditions in the experiments with the fractions and the pure substance carvone were adjusted to the same concentration of essential oil. Studies on the absorption of curcumin and lutein

[0073] The extracts used in the bioavailability studies were dissolved in a simulated fasting intestinal fluid (FaSSIF) prior to treatment.

[0074] For studies on curcumin intake, 5 × 10 6 Cells were seeded in 6 cm cell culture dishes (Greiner-Bio One) and 2 × 10⁻⁶ cells were used for studies on lutein uptake. 6Cells were seeded in 6-well plates (Greiner Bio One) followed by differentiation as described above. Treatment solutions were applied to the cells in FaSSIF at a final concentration of 2.95 µg / ml total curcuminoids or 11 µg / ml lutein + zeaxanthin for four hours, with or without the addition of 1% mint extract. The cells were washed twice with cold FaSSIF medium, followed by two extractions with n-hexane and isopropanol (3:2, v / v) for 10 minutes with continuous stirring. Subsequently, the extracts were evaporated at 45 °C until completely dry in a vacuum concentrator (Labconco, MO, USA) equipped with a vacuum pump (Vacuubrand, Wertheim, Germany). For HPLC measurements (see below), the dried samples were resuspended in 150 µl of 50% or 80% acetonitrile.For protein determination, the dry cell layers were lysed with 0.1 M NaOH and a micro-Bradford assay (BioRad, Hercules, CA, USA) was performed according to the manufacturer's instructions. The curcuminoid, lutein, and zeaxanthin concentrations were normalized to the amount of cell protein. Isoflavone transport studies

[0075] The extracts used in the bioavailability studies were dissolved in a simulated fasting intestinal fluid (FaSSIF) prior to treatment.

[0076] For transport studies, 16.5 × 10 4 Cells / Transwell insert (ThinCert, 0.336 cm²) 2, 0.4 µm pore diameter; Greiner-Bio One) were seeded into 24-well plates (Greiner-Bio One) and incubated overnight. Differentiation was induced as described above. To assess cell layer integrity before and after the experiment, the transepithelial electrical resistance (TEER) was measured using a Millicell ERS-2 volt-ohmmeter (Merck, Darmstadt, Germany) as previously described (Ollinger, N. et al. (2022). Anti-Hyperglycemic Effects of Oils and Extracts Derived from Sea Buckthorn - A Comprehensive Analysis Utilizing In Vitro and In Vivo Models. Molecular Nutrition & Food Research, 66(12), Article 2101133). For hydrolysis, the isoflavones were incubated with β-glucosidase (from almonds, 4 U / ml, Sigma-Aldrich), dissolved in 50 mM MES monohydrate buffer (pH 5.5, Carl Roth), for 2 hours at 37 °C and 600 rpm.After enzyme inactivation (95 °C, 5 min) and centrifugation, the isoflavones in the supernatant were adjusted to a final concentration of 3.92 µg / ml and applied to the cells with or without the addition of the mint extract. Aliquots of the treatment solutions were used for subsequent analyses to measure the initial isoflavone concentration. The differentiation medium was aspirated from the cells, and the inserts were transferred to a 24-well plate filled with pre-warmed HBSS, representing the basolateral compartment. The isoflavone treatment solutions were added to the apical side of the cell layer, and the cells were incubated for 4 hours. Subsequently, samples were taken from the apical and basolateral compartments.After centrifugation, the samples were diluted with 100% acetonitrile (HPLC grade, Sigma-Aldrich) to a final acetonitrile concentration of 15%, and the amount of each aglycone in the respective compartments was determined by HPLC measurements (see below). The apparent permeability coefficients (Pi, P ... app ) were calculated using the following equation (1), where V r The volume in the basolateral compartment, dC / dt the slope of the cumulative concentration of isoflavones in the basolateral compartment over time, A the area of ​​the Transwell insert, and C0 the initial concentration in the apical compartment (van Breemen, RB et al. (2005). Caco-2 cell permeability assays to measure drug absorption. Expert Opinion on Drug Metabolism & Toxicology, 1(2), 175-185). Papp=Vr×(dC) / (dt)×1 / AC0 Rhodamin 123 and Hoechst 33342 efflux assays

[0077] P-gp activity was assessed based on Rhodamine-123 efflux, and BCRP activity was assessed based on Hoechst33342 efflux (Mao, Q., & Unadkat, JD (2015). Role of the breast cancer resistance protein (BCRP / ABCG2) in drug transport—an update. The AAPS Journal, 17(1), 65–82). To assess efflux transporter activity in Caco-2 cells, 15 × 10 4Cells / wells were seeded into black 96-well plates, incubated overnight, and then differentiated for 48 hours as described above. After removal of the differentiation medium, the cells were washed with HBSS and incubated for 30 minutes with either 10 µM Rhodamin123 (Sigma-Aldrich) or 20 µM Hoechst33342 (MedChemExpress, Monmouth Junction, NJ, USA) in HBSS. The cells were then placed on ice and washed twice with ice-cold HBSS. Various concentrations of the mint extract and the P-gp inhibitor verapamil (50 µM, Sigma-Aldrich) or the BCRP inhibitor Ko143 (10 µM, MedChemExpress) in FaSSIF were applied to the cells for 60 minutes at 37 °C. The cells were again placed on ice and washed twice with ice-cold DPBS (PAN Biotech). Cell lysis was induced by adding 100 µL of RIPA lysis buffer (150 mM NaCl, 0.5% sodium dodecyl sulfate, 50 mM Tris-HCl at pH 8.0 and 1% Triton X-100) or by shaking for 30 min at room temperature.The fluorescence intensity of the intracellular substrates was measured using a microplate reader (POLARStar Omega, BMG LABTECH) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm for Rhodamin 123 and at an excitation wavelength of 355 nm and an emission wavelength of 460 nm for Hoechst33342. HPLC analysis of curcuminoids, lutein and isoflavones

[0078] HPLC analysis was performed as previously described (Blank-Landeshammer, B. et al. (2022). Improved Bioavailability and Bioaccessibility of Lutein and Isoflavones in Cultured Cells In Vitro through Interaction with Ginger, Curcuma and Black Pepper Extracts. Antioxidants (Basel, Switzerland), 11(10)). The target substances were analyzed by HPLC-FLD and HPLC-DAD using an external standard calibration. Separation was performed on an Ultimate 3000 HPLC system equipped with an Accucore C18 column (150 × 3 mm, 2.6 µm particle size, both Thermo Fisher Scientific) and 0.1% formic acid (FA, Carl Roth) in water as solvent A and 0.1% FA in acetonitrile as solvent B. For the detection of curcuminoids, the gradient was initiated at 12% B and maintained for 5 minutes, then increased to 38% B in 8 minutes, to 70% B in 5 minutes and to 80% B in 1 minute, which was held constant for 1.5 minutes.The solvent concentration was then reduced to 12% B and balanced for 10 minutes. The column temperature was maintained at 40 °C, and detection was performed using a fluorescence detector with excitation and emission wavelengths set to 432 nm and 535 nm, respectively. The same gradient and column parameters were used for the isoflavone separation, but the UV signals were recorded at wavelengths of 249 nm and 260 nm. The separation of lutein and zeaxanthin was achieved using a binary gradient, with the solvent concentration B starting at 80%, increasing to 94% over 4 minutes, held at 94% B for 10 minutes, then returning to 80% for 1 minute, and finally balancing for 10 minutes. The column temperature was maintained at 25 °C, and the signals were obtained at a wavelength of 445 nm. GC-MS measurement of the extract from spearmint

[0079] The volatile components of spearmint extract were analyzed by GC-MS. A Trace 1300 gas chromatograph was used in conjunction with an ISQ QD single quadrupole mass spectrometer equipped with a PTV injector and a TriPlusRSH autosampler (all Thermo Fisher Scientific). The injector temperature was maintained at a constant 240 °C, and 0.5 µl of the samples were injected onto a Stabilwax DA column (30 m × 0.25 mm, 0.25 µm film thickness; Restek, Centre County, PA, USA) at a split ratio of 1:20. Helium was used as the carrier gas at a constant flow rate of 1.0 mL / min. The oven temperature was held constant at 45 °C for 5 minutes, then increased to 240 °C at a rate of 7 °C / min, followed by a constant period of 10 minutes at 240 °C, before returning to the starting conditions. The injector and ion source temperatures were set to 250 °C and 240 °C, respectively.Complete scans of m / z 40–550 were recorded at a rate of 5 scans / s. Instrument operation and data analysis were performed using the Chromeleon 7.2 software package (Thermo Fisher Scientific), and the analytes were preliminarily identified using the NIST 11 spectral library in conjunction with the NIST 11 GC RI database. For external quantification of carvone, authentic standards of both (R)-(-)-carvone and (S)-(+)-carvone (both Thermo Fisher Scientific Acros, 99% and 98% purity, respectively) were diluted with n-hexane and subjected to GC-MS analysis. A 5-point calibration curve was generated, and linear regression analysis was performed using the software (R. 2 = 0.999). Quantification of carvone antiomers

[0080] To quantify the enantiomers (R)-(-)-carvone and (S)-(+)-carvone, the GC-MS system was equipped with a special chiral column (Rt-bDEXsa, 30 m × 0.25 mm, 0.25 µm film thickness, Centre County, PA, USA). The helium flow rate was set to 1.2 ml / min, and the temperature gradient was varied as follows: constant at 40 °C for 1 minute, then increased to 230 °C at 2 °C / min, held at 230 °C for 3 minutes, and then returned to the initial conditions. All other parameters were maintained as described above. For external quantification of the enantiomers, authentic standards of both (R)-(-)-carvone and (S)-(+)-carvone (both Thermo Scientific Acros, purity 99% and 98%, respectively) were used. A 5-point calibration curve was created and a linear regression analysis was performed (R). 2 = 0.995). Statistical analysis

[0081] Statistical analysis and data visualization were performed using GraphPad Prism version 9.5.1 (GraphPad Software, San Diego, CA, USA). Pooled experimental data are presented as mean ± SD (standard deviation). In this study, outliers were identified using the ROUT method, and all statistical tests were performed with a 95% confidence interval. The datasets were tested for normality using the Shapiro-Wilk test and for homogeneity of variance using the F-test and the Brown-Forsythe test. If the majority of the tested datasets were normally distributed and a maximum of three test groups did not follow a normal distribution, the data were considered normally distributed.For the comparison of two groups, a multiple unpaired t-test was performed, followed by the Holm-Sidäk method to correct for multiple comparisons. If the standard deviations (SD) differed significantly, a Welch correction was applied. When more than two groups were compared, an ordinary one-to-one analysis of variance (ANOVA) and a Tukey or Dunnett multiple comparison test were performed. If the data were not from a population with the same standard deviation, a Brown-Forsythe and Welch ANOVA test was performed. Statistically significant differences are indicated by * (p ≤ 0.05), ** (≤ 0.01), *** (< 0.001), and **** (≤ 0.0001). QUOTES INCLUDED IN THE DESCRIPTION

[0000] This list of documents cited by the applicant was automatically generated and is included solely for the reader's convenience. The list is not part of the German patent or utility model application. The DPMA accepts no liability for any errors or omissions. Cited non-patent literature

[0000] Antonelli, A., & Fabbri, C. (1999). Essential oils: Spe fractionation. Chromatographia, 49(3-4), 125-130

[0070] König, A. et al. (2023). Combined acid hydrolysis and fermentation improves bioactivity of citrus flavonoids in vitro and in vivo. Communications Biology, 6(1

[0071] Ollinger, N. et al. (2022). Anti-Hyperglycemic Effects of Oils and Extracts Derived from Sea Buckthorn - A Comprehensive Analysis Utilizing In Vitro and In Vivo Models. Molecular Nutrition & Food Research, 66(12), Article 2101133

[0076] van Breemen, R. B. et al. (2005). Caco-2 cell permeability assays to measure drug absorption. Expert Opinion on Drug Metabolism & Toxicology, 1(2), 175-185

[0076] Mao, Q., & Unadkat, J. D. (2015). Role of the breast cancer resistance protein (BCRP / ABCG2) in drug transport-an update. The AAPS Journal, 17(1), 65-82

[0077] Blank-Landeshammer, B. et al. (2022). Improved Bioavailability and Bioaccessibility of Lutein and Isoflavones in Cultured Cells In Vitro through Interaction with Ginger, Curcuma and Black Pepper Extracts. Antioxidants (Basel, Switzerland), 11(10

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