Formulation of krill oil and silymarin

Abstract

The invention relates to a novel formulation or composition of krill oil and silymarin and use thereof, in particular for the treatment and prophylaxis of liver diseases.

Description

[0001] Novel formulation of krill oil and silyniarin

[0002] Description

[0003] The invention relates to a novel formulation or composition of krill oil and silymarin and use thereof, in particular for the treatment and prophylaxis of liver or bile diseases.

[0004] The milk thistle (Silybum marianum or Carduus marianus) is a cultivated plant that is particularly widespread in southwestern and central Europe and has naturalised in Eurasia, North America and South America.

[0005] The drug is known to be effective in the prevention and treatment of various forms of liver and gallbladder dysfunction, as well as cancer and tumour diseases. The drug consists of the ripe fruits, freed from the pappus, with a minimum silymarin content of 1.5%. Tinctures (usually alcoholic-aqueous extracts of the drug) from milk thistle have been known since ancient times. In particular, isolated silymarin is particularly important and suitable (e.g. DE 1 923 082 (Madaus) ) . Milk thistle extracts are common medicines and are also an ingredient of combination preparations, such as the phytopharmaceutical Iberogast®. A well-known milk thistle fruit dry extract product is Legalon forte®, for example .

[0006] Silymarin is a f lavonolignan complex or polyhydroxyphenylchromanone and was first isolated from the plant in the 1960s (dissertation by Janiak Bernhard, June 1960, FU-Berlin (DE 2020407) , Belter A., Hansel R., Tetrahedron Letters, 25, (1968) ) .

[0007] Silymarin consists of an isobaric mixture of f lavonolignans with the main components silybin A and B (silibinin A and B) , isosilybin A and B ( isosilibinin A and B) , silydianin (silidianin) and silychristin ( silichristin) (cf. Kim, N.-C.; Graf, T. N.; Sparacino, C. M.; Wani, M. C.; Wall, M. E., Complete isolation and characterization of silybins and isosilybins from milk thistle (Silybum marianum) . Organic & Biomolecular Chemistry 2003, 1, 1684-1689.; Smith, W. A.; Lauren, D. R.; Burgess, E. J.; Nigel, B. P.; Martin, R. J., A Silychristin Isomer and Variation of Falavonolignan Levels in Milk Thistle (Silybum marianum) Fruits Planta Medica 2005, 71, 877-880) .

[0008] Other known secondary constituents are 2 , 3-dehydrosilybin, desoxysilydianin (silymonin) , silandrin, silybinom, silyhermin and neosilyhermin .

[0009] The declared requirements for a dry milk thistle extract are a content of preferably 65-85% by weight of silymarin (other ranges are possible) , with the silymarin content being composed as follows:

[0010] 40-65% by weight (relative proportion of total silymarin) : silybin A and B (synonymously silibinin) (diastereomeric mixture, C25H22OH10 MW 482.4 (CAS 22888-70-6) ) and 10-20 wt . % (relative proportion of total silymarin) : isosilybin A and B (diastereomeric mixture, C25H22OH10 MW 482.4 (CAS 72581-71-6) ) and

[0011] 20-45% by weight (relative proportion of total silymarin) : silydanin and silychristin (C25H22OH10 MW 482.4) . These f lavonolignans are insoluble or di f ficult to dissolve in water ( the solubility of pure silymarin at pH 6 . 9 is approx . 0 . 08 mg / ml ) and have low bioavailability .

[0012] In the context of the present invention, " silymarin" is a mixture of substances which contains ( at least ) the four substances silybin, silydianin, silychristin and isosilybin in di f ferent concentrations . It is immaterial in which proportions these substances are present in relation to each other and whether other substances are present in the mixture . This variation may depend on the origin and the genotype and phenotype of the milk thistle used (plant drug) . However, it is preferable that these substances ful fil the requirements of the Ph . Eur . or DAB in the respective applicable version . According to the invention, this is the case .

[0013] Krill oil is a dietary supplement derived from tiny shrimplike crustaceans called krill , which are found in the oceans , particularly in the Antarctic . Krill oil is made through a series of processes that involve harvesting, extraction, and puri fication . The krill are then subj ected to extraction processes to obtain the oil . Common methods include solvent extraction and cold-pressing . Solvent extraction involves using food-grade solvents to separate the oil from the krill , while cold-pressing physically extracts the oil without the use of heat or chemicals .

[0014] The krill belong to the order Euphausiacea, and one of the most well-known species is the Antarctic krill (Euphausia superba ) . These tiny creatures play a crucial role in the marine ecosystem as they are a maj or food source for many larger animals . Krill are typically about 1 to 2 centimeters long and have a semi-transparent body . They feed primarily on phytoplankton, microscopic plants that dri ft near the ocean surface . Through their feeding activities , krill play a key role in the ocean ' s carbon cycle , helping to transport carbon from the surface waters to the deep sea .

[0015] Krill oil is rich in omega-3 fatty acids , primarily eicosapentaenoic acid (EPA) and docosahexaenoic acid ( DHA) , which are also found in fish oil . These omega-3 fatty acids are known for their potential health benefits , including supporting heart health, reducing inflammation, and improving brain function .

[0016] One of the key di f ferences between krill oil and fish oil is that the omega-3 fatty acids in krill oil are attached to phospholipids , which may make them more easily absorbed by the body compared to the triglyceride form found in fish oil . Additionally, krill oil contains astaxanthin, a powerful antioxidant that can help protect the oil from oxidation and add extra health benefits .

[0017] Because krill are lower on the food chain, they accumulate fewer toxins than larger fish, which can be an advantage over some fish oils .

[0018] Now, the inventors have found that krill oil improves or enhances the bioavailability of silymarin respectively silybin, namely about 255 % ( see Example 2 , Figure 1A) . It is stated by the inventors that , surprisingly, choline - the active ingredient in krill oil - plays an important role in the accumulation of silybin in the blood stream . Moreover, the inventive concept outlined in the Examples leads to improved cell viability, reduced oxidative stress and lipid accumulation, and supported colony formation . These results emphasise the superior protective ef fects of the combined treatments in enhancing cellular resilience under lipotoxic conditions .

[0019] A preferred krill oil in accordance with the invention can be characteri zed by the following features :

[0020] Total phospholipids , incl . phosphatidylcholine >= 56 g / 100 g

[0021] Total omega 3 fatty acids , incl . EPA and DHA >= 27 g / 100 g

[0022] Choline >= 7 g / 100 g

[0023] In a further preferred embodiment phosphatidylcholine has >= 48 g / 100 g, and EPA has >= 15 g / 100 g, and DHA >= 7 g / 100 g .

[0024] In a further preferred embodiment krill oil is solely derived from Euphausia superba .

[0025] In a further preferred embodiment according to the invention, an additional amount of , in particular, 100 mg to 2 , 000 mg of ( synthetic ) choline is added to 100 g of the formulation or composition or completed to the total amount of 100 g of the formulation or composition, preferably in the form of a salt , for example choline bitartrate . The choline salt is preferably completely dissolved .

[0026] The problem addressed by the present invention is therefore to provide a novel formulation or composition for improving or enhancing the bioavailability of silybin in a subj ect .

[0027] The obj ect or problem is solved by the technical teaching as provided in at least one of the claims . For this purpose, the applicant has performed comprehensive tests with several weight ratio of silymarin to krill oil (see examples and drawings) .

[0028] Surprisingly, for a composition of silymarin and krill oil the bioavailability of silymarin can be improved or enhanced in a subject as outlined (supra) .

[0029] Hence, the present invention provides a composition comprising :

[0030] (a) milk thistle extract containing silymarin, and

[0031] (b) krill oil, wherein a weight ratio (w / w) of silymarin to the krill oil is

[0032] 1 : 2 to 1 : 40, in particular 1 : 5 to 1 : 30.

[0033] In a further preferred embodiment of the invention the composition comprises:

[0034] (a) milk thistle extract containing silymarin, and

[0035] (b) krill oil, wherein a weight ratio (w / w) of silymarin to the krill oil is

[0036] 1 : 8 to 1 : 20, in particular 1 : 10 to 1 : 15.

[0037] Other preferred weight ratios (w / w) of silymarin to the krill oil is: 1 : 8 to 1 : 10, 1 : 10 to 1 : 20, 1 : 13 to 1 : 17, 1 : 20 to 1 : 35 or 1 : 25 to 1 : 30.

[0038] In a further preferred embodiment of the invention the composition comprises:

[0039] (a) milk thistle extract containing silymarin, and

[0040] (b) krill oil, wherein a weight ratio (w / w) of silymarin to the krill oil is 35 mg - 160 mg : 450 mg - 1200 mg, in particular 35 mg - 160 mg : 450 mg - 700 mg, in particular 51 mg : 570 mg, in particular 40 mg - 80 mg : 1000 mg - 1350 mg, or 80 mg - 150 mg : 1000 mg - 1350 mg, in particular 40 mg - 80 mg : 400 mg - 800 mg or 80 mg - 150 mg : 400 mg - 800 mg .

[0041] Other preferred weight ratios (w / w) of silymarin to the krill oil are : 40 mg : 1200 mg ( 1 : 30 ) , 80 mg : 1200 mg ( 1 : 15 ) ,

[0042] 150 mg : 1200 mg ( 1 : 8 ) .

[0043] In a further preferred embodiment of the invention the milk thistle extract contains 65- 85% by weight of silymarin .

[0044] In the context of this invention, liver diseases and disorders are preferably selected from the group consisting of hepatitis , Steatotic Liver Disease ( SLD) , Metabolic Dys function-associated Steatotic Liver Disease (MASLD) , Metabolic Dys function-associated Steatohepatitis (MASH) ( formerly : Non-Alcoholic Fatty Liver Disease (NAFLD) , Non- Alcoholic Fatty Liver (NAFL ) , Non-Alcoholic Steatohepatitis (NASH) ) , Alcoholic Liver Disease (ALD) or cirrhosis .

[0045] In the context of this invention, bile diseases and disorders refer to any inflammation of the gall , gallbladder or bile duct .

[0046] All of said diseases are described in Pschyrembel , 268th edition 2023 , De Gruyter (Berlin) and further information is available by the European Association for the Study of the Liver (EASL ) .

[0047] The ef fective agent containing the novel formulation or composition according to the invention can now advantageously be used for the treatment and prophylaxis of an ill patient or individual , subj ect or preferably human, and speci fically for the treatment and prophylaxis of liver diseases and disorders , preferably selected from the group consisting of hepatitis , Steatotic Liver Disease ( SLD) , Metabolic Dys function- associated Steatotic Liver Disease (MASLD) , Metabolic Dys function-associated Steatohepatitis (MASH) ( formerly : Non- Alcoholic Fatty Liver Disease (NAFLD) , Non-Alcoholic Fatty Liver (NAFL ) , Non-Alcoholic Steatohepatitis (NASH) ) , Alcoholic Liver Disease (ALD) or cirrhosis .

[0048] The agents can be administered in any desired quantities and dosages .

[0049] The galenic formulation of the agent according to the invention can be selected from the group consisting of in the form of drops , capsules , soft capsules or gel capsules or infusions , preferably for oral applications . The formulation may of course contain pharmaceutically standard excipients .

[0050] In another embodiment , the invention relates to a drug containing an agent according to the invention for use or application for the treatment and prophylaxis of liver diseases and disorders , preferably selected from the group consisting of hepatitis , Steatotic Liver Disease ( SLD) , Metabolic Dys function-associated Steatotic Liver Disease (MASLD) , Metabolic Dys function-associated Steatohepatitis (MASH) ( formerly : Non-Alcoholic Fatty Liver Disease (NAFLD) ) , Non-Alcoholic Fatty Liver (NAFL ) , Non-Alcoholic Steatohepatitis (NASH) ) , Alcoholic Liver Disease (ALD) or cirrhosis . Another preferred embodiment relates to a dietary supplement containing the agent according to the invention, in particular in the form of a dietary composition or balanced diet for the treatment and prophylaxis of liver diseases and disorders , preferably selected from the group consisting of hepatitis , Steatotic Liver Disease ( SLD) , Metabolic Dys function- associated Steatotic Liver Disease (MASLD) , Metabolic Dys function-associated Steatohepatitis (MASH) ( formerly : Non- Alcoholic Fatty Liver Disease (NAFLD) , Non-Alcoholic Fatty Liver (NAFL ) , Non-Alcoholic Steatohepatitis (NASH) ) , Alcoholic Liver Disease (ALD) or cirrhosis . A suitable physiologically compatible carrier can be added to the dietary supplement according to the invention .

[0051] The pharmaceutical preparations according to the invention may be prepared in the form of dosage units or formulation units . This means that the preparations are in the form of individual parts , preferably capsules and vials , the active-substance content of which corresponds to a multiple of an individual dose . The dosage units may e . g . contain 1 , 2 , 3 or 4 individual doses or 1 / 2 , 1 / 3 or 1 / 4 of an individual dose . An individual dose preferably contains the quantity of the agent according to the invention that is administered in an application and usually corresponds to a whole daily dose , hal f of a daily dose , a third of a daily dose or a quarter of a daily dose . Preferably, a dose is administered three times per day, preferably in the form of a capsule or drops , in particular in the morning, at lunchtime and in the evening, optionally at mealtimes . Therefore , the invention likewise relates to a pharmaceutical preparation containing an agent according to the invention together with excipients and additives .

[0052] It should be noted that features described in connection with an exemplary embodiment or an exemplary article may be used with any other exemplary embodiment or with any other exemplary article can be combined .

[0053] I f a term is designated with an indefinite or definite article , such as " a" in the singular, this also includes the term in the plural and vice versa, unless the context clearly states otherwise .

[0054] The term " comprise" as used herein not only includes the meaning of " contain" but can also mean " consist of" and " consisting essentially of" .

[0055] Examples and drawings :

[0056] The following examples and drawings ( figures ) are used solely to explain the invention, without limiting the invention to these examples .

[0057] Example 1 :

[0058] The in vitro assessment of the intestinal transepithelial transport of silymarin was performed using the well- established Caco-2 model . The Caco-2 cell line is derived from human epithelial colorectal adenocarcinoma cells and is commonly used as model for intestinal uptake . The cells are seeded on filter inserts and differentiated over 14-21 days for full performance.

[0059] The following test products were used:

[0060] Table 1 : Test substances and batch numbers

[0061] In vitro digest simulation

[0062] The in vitro model "artificial digest" simulated the enzymatic and pH conditions during the gastro-intestinal-passage. Metabolic changes of the active ingredients in the test product due to these conditions will be considered in the subsequent examples.

[0063] Therefore, the respective test product was added to an aqueous mucin / pepsin solution at a pH of 2 for 2 h at 37 °C. After adding pancreatin, trypsin, and bile extracts the pH of the mixture was adjusted to pH 7,5. The respective solutions were incubated for further 4 h at 37 °C. Digest solutions of compositions 1-3 and Silybum marianum extract can be used in a concentration of 2%.

[0064] Example 2 :

[0065] In a first step, a pre-test was made to check the sensitivity of the analytical method to make sure that the silymarin concentration tolerated by the CaCo-2 model as well as in the basolateral compartment is still above the detection limit.

[0066] Main Assay

[0067] The transepithelial transport of silybin from composition 1, 2 and 3 as well as from Silybum marianum extract was determined for the respective 2% digestion solution.

[0068] After 24 h incubation the silybin content in the basal compartment was determined by LC-MS / MS.

[0069] Silybin was not detectable in the digestion control samples. As can be seen in Figure 1, the concentration of silybin in the basal compartment is dependent on the concentration applied on top.

[0070] If composition 3 is compared with the corresponding initial concentration Silybum marianum extract (40 mg) the basal concentration is increased by approximately 2.5-fold (255%) .

[0071] Based on the data obtained, the following apparent permeability coefficient (Papp) values for the permeation of silybin from the apical to basolateral compartment were calculated (Artursson P., Karlsson J., Biochem Biophys Res Commun., 175(3) , 880-885 (1991) ) .

[0072] Table 2: Pappvalues of the formulations.

[0073]

[0074] There is a dose-dependent increase in transport rate with increasing silymarin concentration.

[0075] Comparing Pappfor composition 3 with that for the corresponding initial concentration of Silybum marianum extract concentration, there is an increase by approximately 28% in the transport rate of the formulation in krill oil.

[0076] For the other Silymarin components, silicristin, and isosilibin, no quantitative determination was made. However, different transport rates were observed for the other components, leading to a shift in their ratio to each other.

[0077] The ratio of silicristin : silibinin (silybin) : isosilibin in the supplemented solution was 19.2 - 26.8 : 46.8 - 55.3 : 21.6 - 26.4, in the basal compartment: 3.7 - 5.0 : 75.7 81.3 : 14.8

[0078] 19.2 From these observations it can be concluded that silicristin is rarely transported across the epithelial barrier .

[0079] Example 3 :

[0080] A comparison of composition 1 ( 150 mg in krill oil ) with corresponding initial concentration of Silybum marianum extract ( 150 mg) was made . As can be seen in Figure IB, the silybin concentration in the basal compartment is higher in the group with the krill oil formulation than in Silybum marianum extract group .

[0081] I f composition 1 is compared with the corresponding initial concentration of Silybum marianum extract ( 150 mg) the basal concentration is increased by approximately 1 . 5- fold .

[0082] Based on the data obtained, the following apparent permeability coef ficient ( Papp) values for the permeation of silybin from the apical to basolateral compartment were calculated .

[0083] Table 3 : Pappvalues of the di f ferent formulations .

[0084] Comparing Pappfor composition 1 with that for the corresponding initial concentration of Silybum marianum extract concentration, there is an increase by approximately 11% in the transport rate of the formulation in krill oil.

[0085] Example 4

[0086] A study focuses on investigating the role of a combined silymarin and krill oil treatment in human hepatocytes in the context of metabolic liver disease. To this end, a high-fat dietary model of hypernutrition is employed within newly established primary human and mouse in vitro systems comprising cell lines and organoids. This approach enables the functional effects of silymarin and krill oil treatment to be examined in this setting. i.) silymarin extract alone: Silymarin (80%) (5g) , ii.) krill oil alone: krill oil (5g) , iii.) silymarin + krill oil: Silymarin (80%) 0,4g + krill oil 4, 6g (5g) , i.e. Silymarin (80%) 8g + krill oil 92g (100g) , ratio: silymarin / krill oil: 11,5. iv.) silymarin + krill oil + slight amount of synthetic choline: Silymarin (80%) 0,4g + krill oil 4,57g + choline 0,03g (5g) , i.e. silymarin (80%) 8g + krill oil 91,4g, choline 0, 6g (100g) , ratio: silymarin / krill oil: 11,42.

[0087] (Total amount 5g or 100g) .

[0088] Example 5

[0089] Silymarin and krill oil dose-response curves To determine the dose-response curves of silymarin and krill oil , THLE5B cells were treated with various concentrations of each compound for 48 hours . The concentrations of silymarin ranged from 10 to 100 pg / mL and the concentrations of krill oil ranged from 100 to 3200 pg / mL (based on the active concentrations of these compounds found in the literature ) . Both compounds were dissolved in dimethyl sul foxide ( DMSO) , ensuring that the final treatment concentrations were lower than 0 . 5% . Cell viability was assessed using the water-soluble tetrazolium salt (WST- 1 ) assay ( see Figure 2 ) .

[0090] The dose-response curves were then used to determine the respective IC50 values for silymarin and krill oil . IC50 , representing the concentration at which 50% of cell viability is inhibited, was calculated as 34 . 47 pg / mL for silymarin, indicating relatively higher potency . In contrast , krill oil exhibited an IC50 of 441 . 2 pg / mL, suggesting that signi ficantly higher concentrations are required to achieve comparable cytotoxic ef fects . These values demonstrate that silymarin has a much stronger inhibitory ef fect on cell viability than krill oil under the tested conditions .

[0091] Based on the IC50 values obtained from the dose-response curves , the IC25 concentrations for both silymarin and krill oil were calculated to inform the design of subsequent experiments . These sub-cytotoxic concentrations were selected to avoid inducing cell death, while still allowing potential biological ef fects to be observed . The IC25 was determined to be 13 . 79 pg / mL for silymarin and 201 . 69 pg / mL for krill oil . To ensure consistency across treatment conditions , the concentration of silymarin was fixed at its IC25 value in all experimental groups. The corresponding Krill oil concentration in vials 3 and 4 (Silymarin + Krill oil and Silymarin + Krill oil + artificial choline, respectively) was then calculated (see Table 4) . This approach ensured that both compounds were present at equivalent concentrations across all tested combinations. Notably, the krill oil concentration used in these mixtures (198.24 pg / mL) closely approximates its calculated IC25 (201.69 pg / mL) , which supports the relevance of the chosen doses.

[0092] Table 4 :

[0093] *ln order to have the concentration of active silymarin equal to its IC25 (13,79 pg / mL)

[0094] Example 6

[0095] Viability assay

[0096] A WST-1 viability assay was performed on THLE5B cells that had been treated for 48 hours, in order to evaluate the effects of silymarin, krill oil and their combinations, in the context of metabolic dysfunction-associated steatotic liver disease (MASLD) . To model lipotoxic stress, a high-fat medium (HEM) containing 250 pM palmitic acid was used alongside a normal culture medium (DMEM) . The treatments included silymarin, krill oil, a mixture of silymarin and krill oil, and silymarin alone and in combination with HEM, and silymarin + krill oil + artificial choline, both alone and in combination with HEM. Figure 3 shows that HEM alone significantly reduced cell viability compared to the DMEM control , confirming its lipotoxic ef fect . Silymarin further decreased viability when combined with HEM, suggesting a limited protective ef fect under lipotoxic conditions at this concentration . In contrast , krill oil treatments and both mixture treatments ( silymarin + krill oil and silymarin + krill oil + choline ) showed similar viability levels with or without HEM, suggesting that these treatments may mitigate the impact of stress induced by a high- fat diet . Overall , these results imply that , while silymarin alone does not directly af fect viability, its combination with krill oil helps to maintain it , suggesting a potential stabilising or modulatory ef fect in MASLD-like conditions .

[0097] Example 7 :

[0098] Analysis of oxidative stress

[0099] Oxidative stress analysis was conducted using the DCF ( 2 ' , 7 ' - dichlorof luorescin) assay, a method used to detect reactive oxygen species (ROS ) in cells . The level of oxidative stress in hepatocytes exposed to HEM was assessed with and without protective treatments . The DCF assay measures the fluorescence emitted by cells when DCFH-DA ( a non- f luorescent precursor ) is oxidised, forming the fluorescent compound DCF in the presence of ROS . By quanti fying the fluorescence intensity, we were able to evaluate the extent of oxidative damage in the cells . The graph shows the fold change in DCF fluorescence relative to the basal level (without stimulation with 750 pM H2O2) . Combining silymarin with HFM did not prevent the increase in oxidative stress induced by HFM ( Figure 4 ) . In contrast , krill oil , whether administered alone or in combination with silymarin and / or choline , maintained oxidative stress levels comparable to control conditions ( in the absence of HFM) . These findings corroborate previous observations and demonstrate that krill oil ef fectively mitigates oxidative damage and helps preserve hepatocyte function under metabolic stress , whether administered alone or together with silymarin and choline .

[0100] Example 8 :

[0101] Fat uptake

[0102] To determine the di f ferential ef fects of di f ferent treatments on lipid accumulation and resolution, THLE5B cells were treated with HEM and various compounds for 48 hours , after which they were stained with Oil Red 0.

[0103] The quanti fication results were normalised by the number of live cells remaining after treatment ( Figure 5 ) . While not statistically signi ficant , treatment with the individual compounds ( silymarin and krill oil ) resulted in the greatest fat uptake , suggesting an early and potent lipogenic response . In contrast , combined treatment with silymarin, krill oil and choline showed a slight reduction in fat uptake compared to HFM alone , suggesting a potential ef fect in limiting lipid accumulation during the initial exposure period ( see Figure 6 ) . While these results are preliminary, they suggest that the combined treatment could be beneficial in attenuating lipid accumulation and hepatocellular stress under metabolic overload .

[0104] Example 9 : Organoid viability

[0105] To more accurately assess the effects of compounds beyond what is observed in monolayer cultures, viability was evaluated in 3D organoids, which more closely mimic native architecture and cell interactions (see Figure 7) . Liver organoids were generated by isolating primary hepatocytes from mouse livers through enzymatic digestion with collagenase IV. The cells were then embedded in Matrigel, a supportive extracellular matrix, and cultured in conditions that promote 3D organoid formation. The organoids were then expanded, harvested, counted and re-seeded for treatment with silymarin, krill oil or a combination of the two, with or without a high-fat medium (HFM) , for 48 hours. Viability was then assessed using the CellTiter-Glo 3D assay, which measures ATP as an indicator of metabolically active cells. The luminescent signal reflects the number of viable cells within the organoids (Figure 8) . These results were comparable to those observed in 2D cultures, confirming consistent effects of the compounds across both models. Treatments involving krill oil and the two mixtures (silymarin and krill oil, and silymarin

[0106] + krill oil + choline) maintained similar viability levels, regardless of the presence of HFM. In contrast, treatment with silymarin alone combined with HFM resulted in a significant decrease in viability. This suggests that krill oil, whether used alone or in combination with silymarin and choline, may help counteract the effects of high-fat induced stress. Silymarin alone appears to be less effective in this context.

[0107] Example 10:

[0108] Long-term effects: colony formation In order to gain a better understanding of the long-term effects of krill oil, silymarin and their combinations on hepatocyte health, we evaluated their colony formation capacity. This assay evaluates the ability of cells to survive, proliferate and form colonies over time, offering insights into their regenerative potential beyond immediate viability measurements. THLE5B cells were initially plated and treated with the different compounds in DMEM for 48 hours. They were then re-plated at low density in normal DMEM medium to allow colony formation. After 12 days, the colonies were fixed, stained with crystal violet and counted to assess proliferative capacity. Colony formation analysis revealed a reduced number of colonies in the krill oil-treated group compared to the control group (treated with DMEM only) and the combination treatment group (treated with silymarin, krill oil and choline together) (Figure 9) . This suggests that, although krill oil alone may affect long-term proliferative capacity, combining it with silymarin appears to counteract this effect, promoting better cell survival and growth over time.

[0109] Conclusion from examples 4-10:

[0110] Overall, the findings suggest that silymarin alone does not provide substantial short-term protection against stress induced by a high-fat diet, as indicated by reduced cell viability, increased oxidative stress and lipid accumulation. However, in the long term, silymarin appears to enhance the proliferative capacity of hepatocytes, suggesting that prolonged exposure or higher concentrations may be required for it to exert its full protective effects. Krill oil and both combination treatments (silymarin and krill oil, and silymarin, krill oil and choline) exhibited protective effects in the short term . However, krill oil alone had a negative impact on long-term proli ferative capacity, a limitation that was not observed with the combination treatments . Notably, the combination of silymarin and krill oil — especially when supplemented with choline — consistently improved cell viability, reduced oxidative stress and lipid accumulation, and supported colony formation . These results highlight the superior, more balanced protective ef fects of the combined treatments in enhancing cellular resilience under lipotoxic conditions .

[0111] Example 11 :

[0112] Transepithelial transport in Caco-2 model

[0113] References are made to Examples 1 and 2 . All experiments follow the provided description and the conditions remain unaltered .

[0114] Table 5 : The following test products were used in a concentration of 2 % :

[0115] (Total amount is about 5g)

[0116] Table 6: The krill oil is specified as follows:

[0117] Silbinin concentration was determined in the supplementation medium at the beginning of the experiment , as well as in the basal compartment after a 24-hour incubation period . This was achieved by means of liquid chromatography with tandem mass spectrometry ( LC-MS / MS ) .

[0118] Table 7A: Silibinin concentration in the supplementation medium

[0119] Table 7B : Silibinin concentration in the basal compartment after 24h incubation

[0120] Table 7C : Pappvalues of silibinin in the di f ferent products .

[0121] The transepithelial transport of silibinin from products 1, 2,

[0122] 3 and 4, and from the Silybum marianum extract, was determined for the respective 2% digest solution.

[0123] The results of this study are presented in Figure 10. Product

[0124] 4 has been identified as the most effective option in the used Caco-2 model.

[0125] Figures :

[0126] Figure 1A: Transepithelial transport of silybin (resp. silibinin) using Caco-2 models. Silybin concentration in the basal compartment after 24 h incubation with the respective digestion solution (2%) .

[0127] Values are expressed in ng / ml, mean ± SD, n=6.

[0128] Figure IB: Transepithelial transport of silybin (resp. silibinin) using Caco-2 models. Silybin concentration in basal compartment after 24 h incubation with the respective digestion solution {2%) .

[0129] Values are expressed in ng / ml, mean ± SD, n=6.

[0130] Figure 2 : Representative dose-response curves of silymarin (A) and krill oil (B) after 48 hours of treatment in THLE-5B cells. The experiment was repeated three times. Table 8: Absolute values (mean ± standard deviation) corresponding to the data shown in Figure 2.

[0131] Figure 3: The viability of THLE-5B cells was assessed after treatment with control medium (DMEM) , high-fat medium (HFM) , silymarin, krill oil, a mixture of silymarin and krill oil, and a mixture of silymarin, krill oil and artificial choline, both alone and in combination with HFM, for 48 hours. Data are presented as mean ± SD. Analysis by ANOVA followed by Tukey's post hoc test; p<0.05. The experiment was repeated four times.

[0132] Table 9: Absolute values (mean ± standard deviation) corresponding to the data shown in Figure 3 Figure 4 : Oxidative stress levels in THLE5B cells after 48- hour treatment. Fluorescence-based quantification of oxidative stress in hepatocytes after 48 hours' treatment with control medium (DMEM) , high-fat medium (HFM) , silymarin, krill oil, a silymarin and krill oil mixture, and silymarin and krill oil with artificial choline, both alone and in combination with HFM. Data are presented as mean ± SD. ANOVA analysis followed by a Tukey post hoc test was performed; p<0.05. The experiment was repeated three times.

[0133] Table 10: Absolute values (mean ± standard deviation) corresponding to the data shown in Figure 4.

[0134] Figure 5: Representative images of cells treated with different compounds after 48 hours and stained with Oil Red 0. The images illustrate the variation in cell numbers according to the different treatments (lOx magnification) .

[0135] Figure 6: The fat uptake of cells exposed to high-fat conditions and treated with different compounds was assessed. Fat uptake was assessed in cells treated with a high-fat medium (HFM) , silymarin, krill oil, a mixture of silymarin and krill oil, and a mixture of silymarin, krill oil and artificial choline in combination with HFM. After staining, the Oil Red 0 dye was extracted from the cells and quantified spectrophotometrically . Data are presented as mean ± SD. ANOVA analysis was performed, followed by a Tukey's post hoc test (p<0.05) . The experiment was repeated three times.

[0136] Table 11: Absolute values (mean ± standard deviation) corresponding to the data shown in Figure 6.

[0137] Figure 7 : Organoid viability. Representative images of mouse organoids treated with different compounds after 48 hours (5x magnification) .

[0138] Figure 8 : Viability of organoids treated with control medium (DMEM) , high-fat medium (HFM) , silymarin, krill oil, a mixture of silymarin and krill oil, and a mixture of silymarin, krill oil and artificial choline, both alone and in combination with HFM for 48 hours. Data are presented as mean ± SD. Analysis by ANOVA followed by Tukey's post hoc test; p<0.05. The experiment was repeated twice.

[0139] Table 12: Absolute values (mean ± standard deviation) corresponding to the data shown in Figure 8. Organoids viability %

[0140] Figure 9: Colony formation after treatment with different compounds in DMEM. (A) Representative images of colonies formed after incubating cells treated with different compounds for 48 hours for 12 days. (B) Colonies quantification. Data are presented as mean ± SD; ANOVA followed by Tukey's post hoc test ; p<0.05.

[0141] Table 13: Absolute values (mean ± standard deviation) corresponding to the data shown in Figure 8.

[0142] Figure 10: The apparent transport coefficient of silibinin in the different products is shown in accordance with Example 10. Values are given in ng / mL, mean ± SD (n = 6) .

Claims

Claims1. A composition comprising:(a) milk thistle extract containing silymarin, and(b) krill oil, wherein a weight ratio (w / w) of silymarin to the krill oil is 1 : 2 to 1 : 40, in particular 1 : 5 to 1 : 30.

2. The composition of claim 1 comprising:(a) milk thistle extract containing silymarin, and(b) krill oil, wherein a weight ratio (w / w) of silymarin to the krill oil is 1 : 8 to 1 : 20, in particular 1 : 10 to 1 : 15.

3. The composition of claim 1 comprising:(a) milk thistle extract containing silymarin, and(b) krill oil, wherein a weight ratio (w / w) of silymarin to the krill oil is 1 : 8 to 1 : 10, 1 : 10 to 1 : 20, 1 : 13 to 1 :17, 1 : 20 to 1 : 35 or 1 : 25 to 1 : 30.

4. The composition of claims 1 to 3 comprising:(a) milk thistle extract containing silymarin, and(b) krill oil, wherein a weight ratio (w / w) of silymarin to the krill oil is 35 mg - 160 mg : 450 mg - 1200 mg, in particular 35 mg - 160 mg : 450 mg - 700 mg, in particular 51 mg : 570 mg, in particular 40 mg - 80 mg : 1000 mg - 1350 mg, or80 mg - 150 mg : 1000 mg - 1350 mg, in particular 40 mg -80 mg : 400 mg - 800 mg or 80 mg - 150 mg : 00 mg - 800 mg .

5. A composition according to any of claims 1 to 4 wherein the krill oil contains at least the compounds:Total phospholipids >= 56 g / 100 gTotal omega 3 fatty acids >= 27 g / 100 g Choline >= 7 g / 100 g6. A composition according to any of claims 1 to 5 comprising an additional choline salt.

7. A composition according to any of claims 1 to 6 wherein the milk thistle extract contains 65-85% by weight of silymarin .

8. A composition according to any of claims 1 to 7 for use in the treatment or prophylaxis of a liver disease or disorder in a subject.

9. A composition according to any of claims 1 to 7 for use in the treatment or prophylaxis of a bile disease or disorder in a subject.

10. A composition of any of claims 1 to 8 for use in the treatment or prophylaxis of a liver disease or disorder in a subject selected from the group consisting of hepatitis, Steatotic Liver Disease (SLD) , Metabolic Dysfunction-associated Steatotic Liver Disease (MASLD) , Metabolic Dysfunction-associated Steatohepatitis (MASH) , Alcoholic Liver Disease (ALD) or cirrhosis.11 . A composition of claims 1 to 8 for use in the treatment or prophylaxis of Metabolic Dys function-associated Steatohepatitis (MASH) .12 . The composition for use according to any of claims 9 to 11 wherein the composition is a drug or a dietary supplement or a balanced diet .13 . Pharmaceutical preparations containing a composition for use according to any of claims 9 to 11 together with suitable carrier substances , in particular in the form of drops , capsules , soft capsules or gel capsules or infusions , preferably for oral applications .14 . Pharmaceutical preparations containing a composition for use according to any of claims 9 to 11 together with excipients and additives .

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