Compositions and methods for preparing biologically active triterpenoid cream ball formulations

Abstract

The invention relates to a composition for oral or topical human administration and a preparation method thereof. The present invention relates to pentacyclic triterpenoid compounds wherein said triterpenoid compounds are incorporated into lipid vesicles, preferably cream globules, surrounded by three layers of membranes. The triterpenoid is preferably betulinic acid, and / or betulinol, and / or lupenol, and / or oleanolic acid, and / or ursolic acid, most preferably betulinic acid. The incorporation of the triterpenoid compound into the milk fat globules or lipid vesicles at a high payload of at least 10 to at most 50 mg / ml is preferably accomplished with high energy emulsification techniques, most preferably ultrasound treatment. The composition is present as a liquid emulsion formulation in a preferred embodiment. In another preferred embodiment, the composition is a powder, which can be prepared from a liquid embodiment of the composition by, for example, spray drying or freeze drying techniques. The composition can be preferably used as a medicament, a food supplement or a nutraceuticals for the treatment or prevention of cancer, type 2 diabetes, obesity and cardiovascular diseases.

Description

Technical Field

[0001] This invention relates to specific formulations of bioactive pentacyclic triterpenoid compounds such as betulinic acid and betulin, particularly for use as pharmaceuticals or nutritional products, and more particularly for the treatment or prevention of cancer. Background Technology

[0002] Plant-derived molecules form the basis of many important chemotherapy drugs for cancer and other diseases, both directly and as lead compounds [1,2], and constitute a huge potential for new drugs [3]. Pentacyclic triterpenoids are the largest class of natural plant secondary metabolites with profound beneficial biological effects and pharmacological potential. These compounds have a 30-carbon skeleton containing five rings, five six-membered rings (ursane- and oleanane- subtypes) or four six-membered rings with one five-membered ring (lupinane subtype).

[0003] Betulinic acid, a phytochemical compound, is a member of the lupin-type pentacyclic triterpenoids and has demonstrated established anticancer activity in in vitro and animal experiments. Betulin, another bioactive member of this class of compounds, is a direct analog of betulinic acid, the only difference being that it lacks a carboxyl group at the C-28 (carbon) atom of the lupin skeleton. Lupin is the third member of the pentacyclic triterpenoids with a lupin skeleton and also has profound anticancer and anti-inflammatory biological effects [4]. Oleanolic acid and ursolic acid (two isomer analogs of oleanane and ursane-type pentacyclic triterpenoids) have established beneficial biological effects, especially anticancer activity [5]. The chemical structures of these bioactive pentacyclic triterpenoids are as follows: Figure 1 As shown.

[0004] Betulinic acid, betulin, lupeol, oleanolic acid, and ursolic acid are abundant in many plant species. For example, the outer bark of the birch (Birchia) contains a large amount of betulin, up to 30% of the dry weight of the bark[6], and about one-tenth of that amount of betulinic acid[7]. In particular, betulinic acid is known for its broad activity against all prevalent cancer types in vitro[8] and for its preclinical in vivo inhibition of tumor growth in major cancer types[9]. Other activities of this compound have also been identified, such as its anti-inflammatory effects, its anti-HIV effects, and its beneficial effects on type 2 diabetes[10-12].

[0005] Importantly, betulinic acid showed no cytotoxicity to non-cancerous healthy cells in vitro. Furthermore, no signs of toxicity were observed at concentrations that effectively limited cancer growth in animal studies, up to 500 mg / kg body weight [9,13], indicating a favorable therapeutic index for its clinical use.

[0006] Mechanistically, the anticancer properties of betulinic acid have been precisely located in different actions, pathways, and molecules that are partially intertwined. The selective cytotoxicity of betulinic acid to cancer cells can be explained by its specific mechanistic action on metabolic pathways that are adapted to divide cancer cells and make them vulnerable compared to healthy cells, making these pathways selective targets for drug intervention

[14] . For example, betulinic acid has been shown to inhibit the activity of stearoyl-CoA-desaturase (SCD-1), an enzyme whose proper function in cellular lipid metabolism is essential for cancer cell division and tumor growth

[15] . Similarly, the effect of betulinic acid on glucose metabolism in cancer cells

[16] , which is altered compared to healthy cells (the so-called Warburg effect

[17] ), may contribute to the selectivity of the compound against cancer cells and, more broadly, to multipharmacology

[18] .

[0007] In summary, these bioactive pentacyclic triterpenoids, particularly betulinic acid, are attractive candidates for combating the aforementioned conditions. Due to their lack of toxicity to healthy tissues, their preclinically established synergistic and initiating effects with standard-care cancer drugs [19-23] and their proposed roles in the prevention of cancer [24-26] and other diseases such as hepatotoxicity

[27] , type 2 diabetes

[28] , and diabetes-related diseases [29,30], especially betulinic acid, also constitutes a promising nutritional product or food supplement in addition to being a candidate adjuvant cancer drug.

[0008] However, despite their favorable activity characteristics and lack of toxicity, the preclinical development of these pentacyclic triterpenoids intended for clinical use is strongly hampered by significant adverse characteristics.

[0009] First, the bioactive triterpenoids described in this article are extremely hydrophobic and therefore have poor water solubility, which makes providing sufficient bioavailability of these compounds a huge challenge.

[0010] Secondly, the molar concentrations required for strong biological effects are quite high (compared to, for example, some established cancer drugs), such as in vitro in the range of 10-20 μM. In cancer cell line studies [8], the importance of designing formulations that not only enable good solubility of triterpenoids but also incorporate these compounds with high effective loads

[31] .

[0011] To address the poor water solubility of triterpenoids of interest, there are essentially two strategies available in the art, which have been detailed in numerous studies to date. Semi-synthetic derivatives with higher solubility than their natural analogs [11,32-35] have been sought, or drug carriers and delivery systems with high solubility and effective bioavailability for the compounds have been developed.

[0012] However, despite the identification of several interesting derivatives, none have been used clinically as pharmaceuticals or nutritional products. Therefore, given that the water solubility of triterpenoid derivatives in the prior art has only seen modest improvements, and considering the abundant availability of natural compounds in plant species (e.g., for betulinic acid and betulin in birch), natural pentacyclic triterpenoids must still be considered the primary candidates for development for human use.

[0013] 1.1 Ideal characteristics of triterpenoid formulations

[0014] A prerequisite for using natural pentacyclic triterpenoids, particularly betulinic acid and betulin, is the development of drug delivery systems that enable their efficient dissolution. Importantly, any formulation that addresses the two unfavorable characteristics of the pentacyclic triterpenoids addressed in this paper—their poor solubility and the required high in vivo bioavailability—by efficiently incorporating the triterpenoids at high concentrations should, in addition to having a high effective loading of the compounds, enable them to be efficiently delivered, taken up, and absorbed in the intestines after oral administration [36,37].

[0015] Oral administration is the preferred route for triterpenoids when used as adjuvants in combination therapy for cancer [19,20], and clearly when used as dietary supplements or nutritional products for anticancer chemoprevention

[25] , chemoprevention of other disease states, and to achieve other beneficial health effects.

[0016] Therefore, ideally, a drug carrier should meet three requirements. First, the triterpenoid compound should dissolve efficiently within the carrier system at high drug loading levels, where drug loading can be defined as the percentage of the encapsulated drug by mass of the drug carrier. Second, the carrier system should ensure efficient absorption of the encapsulated triterpenoid compound in the intestinal tract. Third, the triterpenoid delivery system itself should preferably be free of any excipients that could cause adverse toxic side effects, especially when pharmaceutically used in conjunction with standard care cancer chemotherapy or as a nutritional supplement.

[0017] In summary, developing such a delivery system is a huge challenge and a problem that needs to be solved for the current application of these promising pentacyclic triterpenoids in humans.

[0018] 1.2 Formulations of triterpenoid compounds in this art, such as those described for betulinic acid.

[0019] The field of drug delivery has been rapidly evolving over the past decade, particularly with the development of various organic nanoparticle-based systems designed to dissolve drugs that would otherwise be under-bioavailable. The ability of various nanoparticle-based drug carriers to dissolve and transport pentacyclic triterpenoids such as betulinic acid, betulin, lupeol, oleanolic acid, and ursolic acid, or their semi-synthetic derivatives, has been explored. Such novel formulations incorporating significant triterpenoids have been tested in various in vitro and in vivo model systems [38,39], and various delivery systems have been designed and tested, with the most important prior art discussed in this paper and summarized in Table 1.

[0020] Nanoscale delivery systems for betulinic acid include carbohydrate-based polymer nanoparticles such as cyclodextrin

[40] and chitosan

[41] , dendritic polymers

[41] , poly(lactic-co-glycolic acid copolymer) (PLGA) nanoparticles[42,43] and betulinic acid-containing PLGA nanoparticles copolymerized with polyethylene glycol (PEG)

[44] .

[0021] Lipid-based nanoparticles are another broad class of carriers used to deliver lipophilic anticancer compounds

[45] , and lipid carriers generally have fairly good loading capacity. Depending on the precise type of lipid carrier, they can often be scaled up from natural sources at a reasonable cost. In addition, lipid-based formulations are generally considered to be beneficial for the intestinal absorption of the lipophilic active pharmaceutical ingredient contained in the carrier

[46] , and in this category, betulinic acid has been tested in vitro and in vivo as a carrier encapsulated in liposomes [9,47] and in oil-in-water nanoemulsions

[48] .

[0022] Despite numerous efforts in the field of betulinic acid drug delivery, no reported formulation has stood out as advantageous to date. In general, there are several types of liposomes, which are the most commonly used lipid (nano) particles as drug carriers

[49] . Liposomes have good potential as drug carriers, but there are also disadvantages associated with liposomes

[50] , such as their moderate loading capacity for hydrophobic compounds and the high production cost of advanced types of liposomes.

[0023] For the delivery of bioactive triterpenoids in a pharmaceutical or nutritional context, the carrier should ideally encapsulate a sufficiently high amount of the active compound, and the formulation should have additional advantageous characteristics when administered orally. Studies have shown that liposomes may not adequately meet these prerequisites. For example, in an in vivo study testing betulinic acid, the maximum loading capacity of betulinic acid in large liposomes appeared to be 5 mg of betulinic acid per milliliter of liposomes [9].

[0024] This suggests that the weekly oral betulinic acid dose of 150 mg / kg body weight required for the antitumor effect observed in mice[9] would require a weekly dose of 12 gr betulinic acid for an 80 kg individual. Therefore, such a liposomal formulation (5 mg / ml betulinic acid) would need to be consumed at a rate of 2.4 Itr per week for human use. Such intake is not feasible. Patient (or customer) compliance would be very low, and in addition, such an extremely high daily dose of liposomal excipients is unhealthy. Proliposomes, which are recently developed carbohydrate carriers coated with powdered phospholipids (e.g., administered in pellet form) and reconstituted into liposomes upon the addition of water

[51] , would have the same problem: excessive excipient intake would be required to achieve adequate bioavailability of the active triterpenoid compound.

[0025] At lower in vivo concentrations, cancer prevention (i.e., chemoprevention) can be achieved, rather than the therapeutic effect of betulinic acid on cancer, and due to the high metabolic rate in mice, extrapolation from mouse studies to human use may be adjusted to a slightly lower human dose per kg body weight

[52] . However, the effective oral use of pentacyclic triterpenoid formulations for promoting human health may depend largely on the effective load of the active pharmaceutical ingredient in the formulation.

[0026] Therefore, the first priority in developing suitable formulations of triterpenoid compounds for oral administration is their high effective load, preferably at concentrations far exceeding the aforementioned 5 mg active ingredient / ml formulation, which is the highest liposomal effective load achieved in the prior art. This is a major problem to be solved in this field.

[0027] Furthermore, any oral formulation should ensure good intestinal absorption of the triterpenoid compound, and the excipients of the delivery system should be non-toxic and preferably permitted as food additives, such as those listed in the "Generally Recognized As Safe" (GRAS) FDA category, or even better, constitute natural foods or elements thereof. Liposome formulations are generally not considered the preferred drug carriers for oral delivery [53,54]. Liposomes and other triterpenoid formulations developed to date have weaknesses, as previously described and illustrated in the case of betulinic acid, and the most prominent literature is summarized in Table 1. Therefore, there is a continued need for suitable formulations of bioactive triterpenoid compounds that allow for oral administration in feasible dosage forms and at doses of active compounds with high bioavailability.

[0028] One object of the present invention is to overcome one or more of the above-mentioned problems, as well as any related problems. Summary of the Invention

[0029] In one aspect, the present invention provides formulation compositions of microparticle carrier systems for the oral delivery of highly hydrophobic, bioactive pentacyclic triterpenoid compounds, such as betulinic acid, betulin, lupeol, oleanolic acid, and ursolic acid. The present invention also provides methods for their preparation.

[0030] The triterpenoid compound formulations of the present invention preferably consist of natural (food) components, particularly milk fat globules, as the main excipients, and can be used as nutritional products or medicines without adverse side effects for improving general health and for treating, chemopreventing or improving the state of prevalent diseases such as cancer, cardiovascular disease and type 2 diabetes.

[0031] High-energy solubilization and emulsification methods, preferably a combination of high-shear mixing and ultrasonic treatment, can be used in this invention to solubilize and load high concentrations of triterpenoid compounds into milk fat globules, which are preferably the main excipients in a microparticle drug carrier system.

[0032] 2.1 Areas that need to be addressed

[0033] The bioactive pentacyclic triterpenoid compound of the present invention has the following chemical structure: Figure 2 As shown. 1, especially betulinic acid and betulinol, and even more so betulinic acid, have great potential as medicinal or nutritional compounds for therapeutic, chemopreventive and ameliorative states of epidemic and severe diseases, as well as well supported by a large number of preclinical in vitro and animal studies

[55] . For human use, however, the field still lacks important properties to be met, which is the main reason for the lack of clinical testing of bioactive triterpenoids.

[0034] First, the pharmacologically active doses required for these triterpenoids are typically relatively high (e.g., compared to most established cancer drugs). Inferences from animal studies suggest that high doses are useful for achieving effective triterpenoid bioactivity [9]. Such high doses are possible because they are not toxic to healthy cells and tissues [9]. Therefore, it is of paramount importance that, for clinical use, the formulation composition and its manufacturing process should be able to dissolve efficiently and achieve very high triterpenoid incorporation concentrations in drug delivery formulations.

[0035] Furthermore, ideally, the triterpenoid carrier should be effectively absorbed in the gastrointestinal tract and after oral administration, so that the triterpenoid is bioavailable at high in vivo doses.

[0036] Furthermore, as an important third requirement, the excipients of the drug delivery system should ideally be biocompatible, preferably constitute food-grade components, and have no harmful effects at the necessary high dosages. More preferably, the excipients themselves impart additional health benefits.

[0037] Finally, the high stability of the formulation and its cost-effective production are highly desirable beneficial properties for preferred formulations.

[0038] The invention disclosed herein is the result of a comprehensive study to discover solubilizing and carrier systems that satisfy all of the above-mentioned key points, thereby providing a solution to the needs of the art.

[0039] This study tested various candidate drug delivery compositions and possible formulations of the pentacyclic triterpenoids described herein, particularly betulinic acid. Besides liposomes, drug carrier platforms have been highly established to date for compounds that are particularly hydrophilic but also lipophilic, and various other organic, especially lipid-based, carrier systems have been analyzed. Furthermore, many different methods for successfully dissolving, assembling, and incorporating triterpenoids into these candidate drug carrier systems were analyzed.

[0040] 3.1 Administration Compositions of Triterpenoids Assembled from Milk Fat Globules

[0041] This invention discloses a microparticle formulation comprising a bioactive pentacyclic triterpenoid compound of interest and milk fat globules, preferably assembled using a high-energy emulsification method, particularly ultrasonic treatment. The formulation of this invention is a highly advantageous composition for the high-dose dissolution and encapsulation of these bioactive pentacyclic triterpenoid compounds, and their subsequent non-toxic oral delivery, resulting in the expected high bioavailability. The triterpenoid compound is preferably betulinic acid or betulinine, with betulinic acid being the most preferred embodiment. Compositions of lipophilic pentacyclic triterpenoid compounds dissolved in and assembled with milk fat globules appear to be advantageous drug carriers and delivery systems in all the aforementioned desirable respects.

[0042] It is noteworthy that the high effective loading of triterpenoids in this delivery system is a result of both the combined specificity of the bioactive triterpenoids and the combined specificity of the milk fat globules. In summary, this combined property enables the efficient dissolution of triterpenoids at high loading levels in milk fat globule-based triterpenoid carriers.

[0043] 3.2 Overview of Milk Components and Structure of Milk Fat Globules

[0044] Milk is a unique energy source for evolutionary development, a structural building block for molecules, and a protector of newborns. Animal milk, especially cow's milk, and its derivatives are common foods worldwide. Essentially, milk is a colloidal, multiphase, and multidispersed system containing protein particles and lipid vesicles. The main proteins in milk are casein, which is organized in micelles [56,57] and whey protein

[58] . Regarding the lipids in milk, milk can be considered an oil-in-water emulsion.

[0045] Before any processing, milk contains 2% to 5% lipids

[59] , which are organized in milk fat globules, which have a complex structure that is only found in milk. The diameter of milk fat globules in raw milk ranges from 0.1 to 15 μm

[60] . Milk fat globules consist of a triglyceride core contained in a three-layered lipid membrane, which is assembled during the secretion of milk fat globules from mammary epithelial cells

[61] .

[0046] More specifically, the physiological process typically supported is that the core of the triglyceride is first encased in the cytoplasm by a monolayer of polar lipids and proteins. Subsequently, during the secretion of the milk fat globule, a bilayer of membrane derived from the secretory epithelial cells is added, which contains glycosylated and non-glycosylated proteins, glycerophospholipids and sphingolipids, enzymes and cholesterol. During the secretion process, a protein interstitial layer is also formed between the monolayer and bilayer lipid membranes. These 10-50 nm thick multilayered membranes together constitute the three-layered milk fat globule membrane (MFGM)

[60] .

[0047] 3.3 Emulsifier function of components in milk fat globule membrane

[0048] Importantly, the amphiphilic properties of polar lipids in MFGMs, together with proteins in the membrane, are crucial for preventing the aggregation of triglycerides in the core of the milk fat globule, thereby ensuring the oil-in-water emulsion properties of the emulsion

[62] . Thus, the membrane components act as key emulsifiers to retain milk fat globules in the emulsion, and thus polar lipids and proteins in MFGMs play different roles

[63] .

[0049] Homogenization is a process typically achieved on industrially processed consumer milk from bovine sources to increase its fat stability by inducing casein micelles to adhere to the milk fat globule membrane, significantly reducing the average size of the milk fat globules and increasing its emulsifying properties [64-67].

[0050] 3.4 Aspects of Triterpenoid Compounds - Milk Fat Globule Preparations

[0051] This invention discloses a solubilization and carrier system for a target triterpenoid compound in a microparticle (or microvesicle) composition that is fully bound to and integrated into the milk fat globule.

[0052] The triterpenoid compound formulations of the present invention have lipid vesicle properties and, in a preferred embodiment, can be contained in an emulsion with high viscosity.

[0053] Furthermore, the triterpenoid-milk fat globule composition of the present invention can also be present in various suitable forms, or can be processed into various suitable forms, such as an emulsion further diluted in, for example, a dairy-based beverage, an ointment or cream suitable for topical application, or in powder form after spray drying, freeze drying or similar techniques, which powder can then be used, for example, in food supplements or beverages.

[0054] Depending on the initial dairy source used for the preparation of the fat globules—either raw milk or one of several possible post-processed dairy products, such as homogenized milk—the compositions of the present invention may contain a certain amount of additional dairy components. In particular, casein micelles and / or whey proteins may be present in a certain amount, or these dairy components may be completely or almost entirely absent from the compositions of the present invention.

[0055] In some embodiments of the invention, these additional milk-derived components may be intentionally added to the milk fat globules used to prepare the triterpenoid composition, as they can impart beneficial properties. For example, casein micelles adhering to the milk fat globules can further improve the emulsifying properties of the milk fat globule membrane, and whey proteins may also help to further improve the stability of the emulsifier

[68] .

[0056] 3.5 Compared with nanocarrier systems, the effective drug loading in triterpenoid-milk fat globule formulations

[0057] Over the past few decades, the development of drug delivery systems has expanded dramatically and fundamentally changed the field. In particular, colloidal carriers based on lipids or polymers have attracted much attention and have been designed in many different types. Nanocarriers are defined as having a diameter of <100 nm, but particles up to several 100 nm (or even 500 nm) are often also referred to as nanoscale

[69] , which have several advantageous features compared to micron-sized delivery systems.

[0058] The high surface area to volume ratio of nanocarriers improves their solubility, bioavailability

[65] , and stability, while their small size helps prevent them from being cleared by macrophages of the reticuloendothelial system via immune mechanisms

[70] . Because such nanoparticles can exhibit prolonged circulation time in cancer, and—according to the tumor microenvironment—a passive targeting mechanism known as the enhanced penetration and retention (EPR) effect can favorably induce higher deposition of carrier-containing drugs in tumors

[71] .

[0059] Nanocarriers have been developed in many different forms and can be classified according to the source of their excipients (natural or synthetic), which are organic or inorganic (the latter being gold nanoparticles for diagnostics), the basis of the chemical matrix (lipid-based or polymer-based), and their physicochemical particle composition (solid particles or core-shell-like vesicles with an aqueous core) [71-73].

[0060] Typically, and as understood by those skilled in the art, lipid-based compositions are logically envisioned as suitable drug (nano)carriers for strongly hydrophobic active pharmaceutical ingredients, such as pentacyclic triterpenoids. However, various polymer-based (nano)carriers also have the capacity to accommodate hydrophobic compounds

[74] .

[0061] Lipid-based colloidal nanocarriers can be classified into (1) liposomes, (2) solid lipid nanoparticles (SLN), (3) nanostructured lipid carriers (NLC), and (4) nanoemulsions [74,75]. Historically, liposomes, based on core-shell lipid types (i.e., an aqueous core contained in one or more lipid bilayers), have been the most studied in this field since their invention in the 1960s. Nano-sized liposomes can be classified as nanocarriers, but there are also (much) larger liposomes (up to >10 μm).

[0062] In the mid-1990s, solid lipid nanoparticles (SLNs) and later-developed nanostructured lipid carriers (NLCs) were designed to overcome several drawbacks of liposomes

[74] . SLNs have a hydrophobic core of solid lipids, such as triglycerides, waxes, or fatty acids, stabilized with emulsifiers such as phospholipid monolayers and / or Tween or bile salts, which act as coatings and surfactants. The next step was to develop NLCs by replacing a portion of the solid lipid core of SLNs with liquid lipids, which created space for further enhancement of drug loading of hydrophobic active pharmaceutical ingredients (APIs) and improved long-term stability by preventing API repulsion of the lipid core.

[0063] Oil-in-water (O / W) nanoemulsions are very similar to SLNs and NLCs, but consist entirely of liquid lipids surrounded by an emulsified monolayer of phospholipids. In terms of drug loading properties, liposomes are primarily suited for transporting hydrophilic APIs (in their aqueous core), while the lipid bilayer of liposomes may also contain hydrophobic APIs

[74] . The properties of SLNs, NLCs, and O / W nanoemulsions make them suitable only for transporting lipophilic bioactive compounds.

[0064] The intense focus on small nanoparticles as drug delivery over the past two decades can be partly considered an overhype [76-78], especially since micron-scale delivery systems also have advantages in some respects, depending on the intended use. Importantly, due to their small particle size, nanoscale particles have a much lower loading capacity than larger carriers such as large liposomes or (large) emulsions

[79] .

[0065] In fact, regarding the transport of triterpenoids, betulinic acid is incorporated into large liposomes with a much higher effective load than small nanoliposomes [9]. The most important betulinic acid formulations published in the literature are summarized in Table 1.

[0066] Therefore, contemporary biases against nanoscale delivery systems highlight the potential for hindering the discovery of optimal pharmaceutical formulation systems for (oral) delivery of pentacyclic triterpenoids. The limited availability of technical expertise from companies and academic research groups is also known to create another bias that impedes the discovery of optimal formulation methods.

[80]

[0067] The invention disclosed herein is the result of an in-depth analysis of the formulation problem to be solved, particularly regarding the chemical characteristics of triterpenoids of interest, combined with extensive empirical research.

[0068] The milk fat globule-triterpenoid drug carrier of the present invention is capable of containing a very high triterpenoid payload. This can be explained by a unique combination of the properties of the milk fat globule as an excipient on the one hand, and the properties of the triterpenoid compound on the other hand, most preferably, the triterpenoid compound being betulinic acid or betulinol.

[0069] As shown in the experimental section:

[0070] • By using the ultrasonic treatment techniques described and shown in Examples 1 and 2, betulinic acid and betulin can be effectively dissolved and incorporated into milk fat globules-betulinic acid (or betulin) formulations.

[0071] The milk fat globules-betulinic acid (or betulin) formulation of the present invention contains a high effective load of betulinic acid (or betulin), as shown in Example 2.

[0072] • Incorporation of betulinic acid (or betulin) alters the properties of the milk fat globule composition, as shown in Examples 3 and 4.

[0073] Furthermore, the milk fat globule-betulinic acid formulation exhibits high stability, as shown in the data in Example 5.

[0074] 3.6 The characteristic of triterpenoids and milk fat globules jointly promoting high levels of drug loading.

[0075] On the one hand, the special characteristics of milk fat globules, and on the other hand, the bioactive pentacyclic triterpenoid compounds of the present invention, preferably betulinic acid and betulinol, together form the basis of the advantageous properties of milk fat globule-triterpenoid compound formulations. Generally, milk fat globules are known to contain lipophilic bioactive substances [81-83].

[0076] As disclosed in this invention, it appears that loading pentacyclic triterpenoid drugs into milk fat globules is extremely effective, especially when done by a suitable loading method.

[0077] The relatively large size of (bovine) milk fat globules (average 4 μm, but may be smaller after possible prior homogenization processes) provides a relatively large volumetric load for lipophilic drugs in the triglyceride core. Furthermore, the milk fat globule membrane, a complex trilayer composed of different phospholipids and proteins, possesses excellent emulsifying properties, and its size and composition make it equally well-suited for accommodating large amounts of hydrophobic bioactive components.

[0078] The unexpectedly efficient and high triterpenoid loading capacity of the compositions of the present invention reveals a perfect match between the properties of the milk fat globule and the properties of the pentacyclic triterpenoid. Several specific characteristics of the triterpenoid, preferably betulinic acid or betulinol, can be theorized and are highly likely to contribute to a remarkably efficient physicochemical interaction between the milk fat globule and the triterpenoid, as described below. First, it is known that lupene-type pentacyclic triterpenoids, such as betulinic acid and lupelinol as shown in the literature, are miscible with the major structural outer leaf membrane phospholipids (glycerophosphatidylcholine and sphingomyelin) in certain molar ratios

[84] . Second, consistent with this finding, betulinic acid is known as a structural element in the lipid bilayer of liposomes, like cholesterol [9], and there are indeed strong indications of a direct interaction between phospholipids and pentacyclic triterpenoids [84-86]. Third, betulinic acid can be dissolved in nanoemulsions containing triglycerides

[48] . Therefore, these characteristics of pentacyclic triterpenoids, especially betulinic acid, are likely the basis for the efficient assembly of milk fat globule-triterpenoid particles and the high effective loading of betulinic acid into milk fat globules, located in the membrane and core.

[0079] Triterpenoid drug loading in the milk fat globule microvesicle structure can therefore occur in the lipid core and the three membrane layers, such as... Figure 2 As shown.

[0080] Importantly, while nanoscale carrier systems offer certain advantages (as discussed above), microscale milk fat globule carrier systems better address the key essential requirement for efficient oral delivery of triterpenoid compounds with high bioavailability. This is because, as... Figure 2 As shown, there is a significant difference between the loading capacity of milk fat globules for hydrophobic compounds and the loading capacity of nanoscale carrier systems, particularly (nanoscale) liposomes with their aqueous cores that cannot carry hydrophobic bioactive compounds.

[0081] 3.7 Other advantageous features of the triterpenoid-milk fat globule composition

[0082] In addition to their optimal drug loading characteristics, milk fat globules also possess other features that facilitate the oral administration of drugs and nutritional products.

[0083] When incorporated into milk fat globule delivery systems, triterpenoids are expected to have good bioavailability after oral administration (as shown in Table 1). Milk and its components are increasingly considered excipients for drug delivery systems

[87] . Although the exact digestion of milk fat globules in the gastrointestinal tract has not been fully elucidated, it is well known that all the nutrients in milk, including milk fat globules, are finely and well digested and absorbed after oral ingestion

[88] . As will be understood by those skilled in the art, any bioactive compounds incorporated into milk fat globules will only be released, absorbed and become bioavailable after intestinal digestion and ingestion of milk fat globules. There are strong indications that milk fat globule membrane components can enhance the digestion of triglycerides

[89] and the bioavailability of bioactive compounds

[90] .

[0084] Furthermore, the formulations of the present invention were found to be stable, which is consistent with the literature, as milk fat globule membrane materials have the ability to stabilize emulsions

[91] .

[0085] In addition to the beneficial properties of milk fat globule membrane components in terms of digestibility and stability, milk fat globule membrane components (polar lipids, proteins, and glycoproteins) have been extensively demonstrated to confer beneficial health effects

[92] . Clearly, as a natural food component, milk fat globules will not cause any adverse reactions when used as a carrier system. Finally, based on commonly available milk components, the milk fat globule-triterpenoid compound formulations of the present invention can be manufactured at a relatively low cost. Invention Details

[0087] This invention relates to compositions comprising at least one triterpenoid compound, preferably a pentacyclic triterpenoid compound, wherein the pentacyclic triterpenoid compound is incorporated into milk fat globules or membrane-bound lipid vesicles, wherein the membrane comprises three layers:

[0088] - Phospholipid monolayer; and

[0089] - Phospholipid bilayer.

[0090] Both the triglyceride core and the three-layer membrane of the milk fat globule or lipid vesicle can be used as lipid compartments, which can be assembled with and incorporated into bioactive pentacyclic triterpenoid compounds.

[0091] The milk fat globule or lipid vesicle is surrounded by a three-layer membrane, wherein the three layers therefore comprise:

[0092] - Phospholipid monolayers, also known as lipid monolayers, are typically found within the three-layered membrane of milk fat globules; and / or

[0093] - Phospholipid bilayer, also known as lipid bilayer.

[0094] In one embodiment, a pentacyclic triterpenoid compound or a combination of two or more different pentacyclic triterpenoid compounds is contained in a plurality of fat globules.

[0095] The (spherical) lipid vesicles or spheres according to the invention have a core containing triglycerides. The core can be considered as a portion of the lipid vesicle or sphere enclosed or surrounded by (outer) three or more membranes. The core may contain, for example, at least 5 wt.% triglycerides relative to the weight of the lipid vesicle or sphere.

[0096] Therefore, the core may be surrounded or encapsulated by an (inner) monolayer containing (polar) phospholipids and / or proteins; and / or an (outer) bilayer containing (polar) phospholipids and / or proteins. Proteins may be, in particular, partially glycosylated and partially non-glycosylated, and / or lipids may be glycerophospholipids and / or sphingolipids. The monolayer and / or bilayer may also contain cholesterol, for example, at least 1% by weight relative to the weight of the particle or sphere.

[0097] In addition, the interstitial layer of proteins can exist between a monolayer and a bilayer. The (spherical) three-layer membrane, namely a monolayer, a bilayer, and an optional intermediate layer, can be 10-50 nm thick. This can be regarded as the envelope or membrane of lipid vesicles or spheres.

[0098] The lipid vesicles or lipid globules of the present invention are preferably milk fat globules. Therefore, the present invention preferably relates to milk fat globules containing at least one triterpenoid compound.

[0099] Therefore, the present invention essentially relates to compositions comprising pentacyclic triterpenoids, wherein the pentacyclic triterpenoids are incorporated into lipid globules or lipid vesicles having membranes, wherein the membranes are trilayers comprising a phospholipid monolayer; and phospholipid bilayers.

[0100] The fat globules used in the compositions according to this disclosure, preferably bovine fat globules, can be obtained by using fresh (raw) non-skimmed milk that still contains its full fat content (i.e., cream) as the source. The fat fraction of the milk containing the fat globules can be separated using conventional dairy processing procedures. Thus, essentially, the density difference between the milk fat and the (skimmed) fat fraction achieves the passive separation of the lighter fat fraction containing the fat globules, so that the fat globules rise to the top when the whole milk (containing all fat) is kept undisturbed for a time interval of, for example, 24 hours. The cream separation can be actively accelerated using a centrifuge, also known as a (milk) separator, by applying centrifugal force using common diary techniques. After separation, if necessary, the cream fraction can be washed with water or a saline solution to (further) remove casein and whey protein.

[0101] Instead of raw milk as the primary source of fat globules, homogenized milk can be used, which contains fat globules of reduced average size. This is achieved through a homogenization process applied to (raw) milk to prevent creaming (i.e., fat globules rising to the top due to the aforementioned density difference). Therefore, fat globules obtained from homogenized milk will have a significantly reduced average diameter: from an average of 2-12 μm to an average of <2 μm. The precise fat content in the harvested cream fraction can be analyzed using conventional dairy techniques such as the Gerber fat assay, and thus, the fat globule fraction can be analyzed.

[0102] With the continuous development of dairy technology, the complex separation of milk fat globule membranes is now possible

[93] . Therefore, it is feasible to prepare compositions according to the invention by using a portion of the separated milk fat globule membrane as a basic component in combination with triglycerides, which are the source of the fat core as the carrier of the lipid vesicle, and the pentacyclic triterpenoid compounds of the present invention. The obvious disadvantage of this artificially composed triterpenoid-containing lipid vesicle with a three-layer membrane is clearly its more laborious preparation and associated (much higher) cost compared to naturally separated milk fat globules, which are the basic source of the vesicles according to the invention.

[0103] Therefore, a preferred composition comprising a pentacyclic triterpenoid compound according to the present disclosure comprises one pentacyclic triterpenoid compound, or a combination of two or more different pentacyclic triterpenoid compounds, which are contained in a plurality of (natural) milk fat globules.

[0104] The lipid vesicles or microspheres (preferably milk fat globules) contained in the compositions of this disclosure may have a (median or average) diameter of 0.1 to 15 μm, preferably 1 to 10 μm, more preferably 2 to 6 μm, as determined by laser diffraction. In this disclosure, the median diameter is preferably determined by laser diffraction measurement, for example, laser diffraction measurement of the corresponding lipid vesicles (one or more) or microspheres (one or more) contained in the composition (or a sample thereof), wherein the D50 percentile value of such measurement represents the median diameter. The D50 value can be described as the value of the particle size at 50% of the cumulative volume-weighted particle size distribution determined by laser diffraction. Alternatively, the average diameter (i.e., the mean diameter) can be determined by laser diffraction measurement, for example, laser diffraction measurement of the corresponding lipid vesicles or microspheres contained in the composition (or a sample thereof), preferably determined by calculating the volume-weighted average (i.e., the average diameter). The average particle size diameter (primary symbol D[4,3], also known as the De Brouckere average diameter) is calculated as D[4,3] = (∑n i .d i 4 ) / (∑n i .d i 3 In other words, the average diameter in this paper preferably refers to the sum of the fourth powers of all measured particle diameters (1-n particles, d).i 4 Indicate (i th (particle diameter) 4 In the molecule, divide by the sum of the cubes of all measured particle sizes (1-n particles, d i 3 Indicate (i th (particle diameter) 3 Laser diffraction data are inherently volume-weighted because the diffraction of light is proportional to the volume of the particles; therefore, laser diffraction provides volume-weighted results. Technicians are very familiar with laser diffraction measurements used to determine the average or average particle size. This can be done, for example, with a Bettersizer S3 Plus instrument (Bettersize Instruments Ltd, Dandong, China) or a Mastersizer TM3000 instrument (Malvern Panalytical), according to the manufacturer's instructions. In this measurement, a laser beam is guided through the sample, and the scattered light is analyzed to determine the diameter distribution of the particles. The diffraction pattern provides information about the diameter of the present particles, allowing the calculation of the average or average diameter. Slightly reduced lipid globule size and a narrower size distribution can have the advantage of better maintaining the stability of the formulation. On the other hand, a larger average size of lipid globules (or lipid globule-based vesicles) can advantageously add to the final triterpenoid payload in the formulation. Therefore, a trade-off may exist between large lipid globules (or their derived vesicles) on the one hand and smaller lipid globules on the other. Depending on the specific application and the associated end product, large fat globules (with a wide size distribution) or fat globules with a reduced average size, such as those obtained using the aforementioned homogenization process, may be preferred. Slightly smaller fat globules with a narrower size distribution are expected to have a generally preferred combination of characteristics.

[0105] Pentacyclic triterpenoids can be triterpenoids selected from lupinane-type pentacyclic triterpenoids, oleanane-type pentacyclic triterpenoids, or ursane-type pentacyclic triterpenoids.

[0106] Preferably, the pentacyclic triterpenoid compound according to this disclosure is betulinic acid, and / or betulinol, and / or lupeol, and / or oleanolic acid and / or ursolic acid. More preferably, the pentacyclic triterpenoid compound is betulinic acid and / or betulinol, and most preferably, the compound is betulinic acid.

[0107] Support for the compositions based on the disclosures provided herein has been primarily achieved in experiments using betulinic acid. However, given the highly relevant chemical structures of other preferred pentacyclic triterpenoid compounds of interest, milk fat globule pharmaceutical compositions are likely also suitable as advantageous drug carrier systems for these compounds. Therefore, experimental support for the latter compounds (betulin, lupeol, oleanolic acid, and ursolic acid) will likely be obtained in further elaboration of the experiments.

[0108] The compositions of the present invention, containing triterpenoid compounds and lipid vesicles or microspheres, preferably milk fat globules, as a carrier system, are preferably contained in a liquid emulsion formulation comprising a plurality of said lipid vesicles, microspheres, or milk fat globules, for example at least 10. 4 10 5 10 6 10 7 10 8 10 9 10 10 10 11 10 12 10 13 / ml formulation.

[0109] The liquid emulsion formulations according to this disclosure preferably contain at least 10, 15, 20, 25, 30, 35, 40, 50, 60, 70 mg / ml (pentacyclic) triterpenoids, wherein preferably at least 50, 60, 70, 80, 90 wt.% are surrounded by a three-layer membrane and / or incorporated into fat globules or lipid vesicles (relative to the total weight of the triterpenoids in the formulation).

[0110] It is evident that, by using milk fat globules obtained from the cream fraction of milk as the source excipient for preparation, the initial composition according to this disclosure will have a liquid form. The viscosity of this liquid formulation is increased by incorporation of triterpenoid compounds, as in Example 4 and Figure 7 As shown. This is considered a beneficial effect because it will improve the stability of the formulation. In part, viscosity may also be affected by the preparation method, as ultrasonic treatment affects the size of the fat globules and may also affect the film composition.

[0111] Alternatively, and as another preferred embodiment, the composition according to this disclosure comprising a (pentacyclic) triterpenoid compound incorporated into lipid vesicles, preferably milk fat globules, may be in the form of a powder formulation. The composition comprising a pentacyclic triterpenoid compound in powder form is preferably obtained by spray drying or freeze-drying processes applied to the aforementioned liquid formulation.

[0112] Spray drying involves converting fluid materials into dry particles using a gaseous thermal drying medium. This method is based on conventional dairy product preparation.

[0113] Freeze-drying (or lyophilization) is an alternative drying method in which water is removed from a fluid material by freezing it, and ice is removed by placing the product under a vacuum, allowing ice to be converted directly from a solid to vapor. Both drying methods have interrelated advantages and disadvantages, particularly concerning product quality, including potential structural changes, procedural complexity, and equipment cost.

[0114] After drying, the subsequent powder form of the triterpenoid compositions of the present invention can be used for oral consumption (i.e., oral administration) after being dissolved (rehydrated) in beverage or pharmaceutical liquid or syrup products. Alternatively, the powder form embodiments of the compositions of the present invention can be used to prepare edible (food) products and / or dietary supplements, for example, in the form of energy, protein, and granola bars or baked goods such as biscuits, cakes, pastries, sauces, and dips. Oral administration of the powder embodiments of the present invention after combining the powders of the present invention into tablet or capsule formulations is also conceivable, although this option is less advantageous from the perspective of gastrointestinal absorption.

[0115] Generally, compared with liquid triterpenoid formulations, powdered triterpenoid compositions according to this disclosure may have the following advantages: extended shelf life, fewer restrictions on storage conditions, and / or less troublesome transportation conditions.

[0116] In a preferred embodiment, a composition comprising the triterpenoid compound of the present invention, such as a composition contained in a triple-layered lipid vesicle or milk fat globule, is for oral administration and / or contained in a food composition, beverage composition, food supplement composition, dietary composition, nutritional composition, or pharmaceutical composition.

[0117] Therefore, the formulations according to the invention can be used for direct oral administration and consumption in the form of a beverage, can be mixed into another beverage before oral administration, or can be used to prepare baked or otherwise prepared food products for use as dietary supplements or nutritional products.

[0118] Alternatively, in another preferred aspect, formulations comprising triterpenoid compounds, such as those contained in the three-layered lipid vesicles or milky fat globules according to this disclosure, can be applied in the form of ointments or creams suitable for topical application to the skin, for example, for the treatment of melanoma or skin diseases or for cosmetic purposes. One aspect of the triterpenoid compositions according to this disclosure can be used for ocular application, in the form of ophthalmic ointments or creams applied to the eyelids.

[0119] Since the excipients of the present invention are natural and food-derived, and are known to have optimal gastrointestinal uptake and absorption, the triterpenoid formulations of the present invention are likely to provide favorable bioavailability of the triterpenoid compounds when administered orally.

[0120] Due to the nature of its excipients, the topical application of the triterpenoid compound-milk fat globule formulations of the present invention is expected to provide sufficient skin bioavailability of the triterpenoid compounds, enabling them to produce beneficial bioactive effects on skin-related diseases and / or improve dermatological conditions.

[0121] As described in detail in the foregoing sections of this application, pentacyclic triterpenoid compounds of interest, particularly betulinic acid, betulinol, lupeol, oleanolic acid, and / or ursolic acid, have known biological effects that make the compounds suitable for therapeutic use, either alone or in combination with other chemotherapeutic compounds, particularly for the treatment of cancer. Thus, pentacyclic triterpenoid compounds, preferably betulinic acid, can be used therapeutically to reduce tumor growth, improve cancer-related health deterioration, and / or can be used as an active pharmaceutical ingredient to enhance the effects of other chemotherapeutic agents for cancer, for example, by combating (limiting) drug resistance. Many other beneficial effects of the triterpenoid compounds described herein are known, as detailed above. Therefore, other diseased health conditions may also benefit from the treatment of any of the pentacyclic triterpenoid compounds described herein, particularly betulinic acid, contained in the compositions of this disclosure. In particular, the known selective action of betulinic acid on cancer cells, reducing cancer cell proliferation, makes the pentacyclic triterpenoid compound compositions of the present invention also well-suited for use as a (chemopreventive) measure to prevent the development of cancer. Such uses are conceivable in the form of dietary (food) supplements or nutritional products. The use of the compositions described herein as dietary supplements is particularly anticipated for improving general (not necessarily disease-related) health; for example, the compositions of the invention can be used for therapeutic purposes, such as combating obesity, as supported by the literature, but are also anticipated, as deduced from preclinical in vitro and in vivo techniques, for the prevention, improvement, or treatment of one or more of the following conditions and states affecting health: type 2 diabetes, cardiovascular disease and / or its earlier stages, skin diseases, chronic inflammation, and motor injuries associated with Parkinson's disease. The proven efficacy of betulinic acid and other pentacyclic triterpenoids described herein against various cancer types, including those with the highest prevalence in men and women (lung cancer, colorectal cancer, breast cancer, prostate cancer, cervical cancer, or melanoma), in in vitro and preclinical animal studies makes the use of the compositions described herein as pharmaceutical or food supplements (or nutritional products) most preferred for combating or preventing cancer.

[0122] Therefore, compositions comprising the pentacyclic triterpenoid compounds of the present invention are provided for therapeutic purposes, such as for the treatment or prevention of cancer, type 2 diabetes, obesity, metabolic syndrome, cardiovascular disease, skin diseases, (chronic) inflammation, or Parkinson's disease and / or related motor injuries, preferably for the treatment or prevention of cancer. Cancer may be lung cancer, colorectal cancer, breast cancer, prostate cancer, cervical cancer, or melanoma.

[0123] In the comprehensive study leading to this invention, it was surprisingly discovered that, by using high-energy emulsification preparation techniques, particularly a combination of ultrasonic treatment and high-shear homogenization, drug carrier systems composed of milk fat globules, which are natural lipid vesicles with a three-layer membrane and a triglyceride core, are capable of taking up, incorporating, and encapsulating triterpenoid compounds with unexpectedly high effective loads, especially compared to other known (lipid-based) drug carrier formulations of the triterpenoid compounds of this invention (as detailed in Table 1).

[0124] In addition to the composition itself, the present invention also provides a method for preparing a (pentacyclic)triterpenoid compound composition, namely, a composition contained in (milk) fat globules or lipid vesicles according to the present invention, wherein the method comprises the following steps:

[0125] a) Provides a composition comprising lipid vesicles having a trilayer membrane comprising a monolayer of phospholipids and a bilayer of phospholipids, preferably milk fat globules themselves;

[0126] b) By mixing the composition provided in a) with one or more different (pentacyclic) triterpenoid compounds, preferably by applying high-shear mixing and / or ultrasonic treatment and / or high-pressure homogenization techniques, a (pentacyclic) triterpenoid composition of the present invention comprising one or more (pentacyclic) triterpenoid compounds incorporated into lipid vesicles, preferably milk fat globules, is provided, thereby achieving the incorporation of triterpenoid compounds into lipid vesicles, preferably milk fat globules, and the assembly of milk fat globules or lipid vesicles incorporating triterpenoid compounds.

[0127] In a preferred embodiment, in step b), the preparation method therefore includes a combination of one, two, or three high-energy emulsification and homogenization methods, such as ultrasonic treatment, high-shear mixing, and high-pressure homogenization, with ultrasonic treatment being the most preferred. By mixing and agitating the particles and / or triterpenoids, these individual or combined techniques greatly facilitate the efficient and rapid incorporation of triterpenoids into the lipid vesicles, preferably milk fat globules, of the present invention at high effective loadings, and these techniques themselves are considered ideal for the complete incorporation of triterpenoids into lipid vesicles, preferably milk fat globules.

[0128] The high-shear mixer used in this paper is a high-performance dispersing instrument that applies shear force based on the rotor-stator principle. The high circumferential rotational speed of the rotor, ranging from, for example, 3000 rpm to 35000 rpm, combined with the stator, provides the necessary shear force to decompose and mix the components to be mixed, thereby, for example, emulsifying liquids and / or dispersing powders and other materials into liquids. Therefore, the high-shear mixer used in this paper can be a device that uses a rotor for mixing, and this device typically consists of a rotor and a stator, wherein the rotor is capable of rotating to generate shear force. As mentioned above, rotor speeds ranging from, for example, 3000 rpm to 35000 rpm can be used.

[0129] High-shear mixing is particularly useful and can therefore be used specifically as a first dispersion step in combination with subsequent preparation steps involving ultrasonic treatment and / or high-pressure homogenization, preferably ultrasonic treatment.

[0130] In the (ultra)sonic processing, as preferably applied in step b, an ultrasonic transducer can be used to provide ultrasonic vibration. Ultrasonic processing is a process that uses sound waves to break down and mix components. This method is typically performed using an ultrasonic transducer, which is an instrument that generates sound waves and directs them to the components to be mixed. Different types of ultrasonic transducers exist, such as horn (also called probe) transducers, piezoelectric transducers, and langevin transducers

[94] . In this invention, preferably by using a horn (probe) type ultrasonic generator, the circulation of ultrasonic-induced pressure leads to the formation of microscopic vacuum bubbles in the solution. The bubbles continuously burst in a process called cavitation, which causes powerful vibrational waves with high energy that exert various physicochemical effects on the particles in the emulsion and cause triterpenoids to be enhanced and accelerated into the fat globules or fat globule-based vesicles of the compositions of this invention.

[0131] Alternatively or alternatively, step b) may include the use of high-pressure homogenization techniques, which means applying at least 800 bar to 4000 bar to force the (mixed) composition through a nozzle-type orifice having a diameter between 10 and 10000 nm, or using an associated high-pressure homogenization instrument.

[0132] Alternatively or additionally, step b) may include heating the (mixed) composition to a temperature of 30-90 degrees Celsius, and / or step b) may be performed at such a temperature.

[0133] At least one triterpenoid compound of the present invention can be advantageously incorporated into milk fat globules, which are combined with casein micelles and / or whey proteins (of natural milk origin), which can adhere to the milk fat globules, thereby providing a more uniform and stable emulsion.

[0134] The addition of natural milk proteins to milk fat globules or milk fat globul-based lipid vesicles is preferably performed prior to the preparation of the compositions of the present invention, but can alternatively be done after preparation. Depending on the treatment of the milk fat globule source used, as discussed earlier herein, milk proteins may still be present in the milk fat globule emulsion used to prepare the compositions. As described in the preceding sections of this application, various variants of casein and whey proteins are present in milk and their ability to improve emulsification processes is known in the literature, thus advantageously aiding in the preparation and stabilization of formulations.

[0135] Alternatively, to further improve the stability of the composition of the present invention, a non-dairy stabilizer, preferably carrageenan, which is a mixture of natural sulfated polysaccharides extracted from red edible seaweed and approved for food applications (European Food Additives No. E407), may be added together with or separately from the milk protein before or after the preparation of the composition.

[0136] In this document and its claims, the verb “comprising” and its variations are used in their non-limiting sense to indicate inclusion of the item following the word, but not exclusion of items not specifically mentioned. Furthermore, the indefinite article “a” or “an” reference to an element does not preclude the possibility of more than one element, unless the context explicitly requires the presence of exactly one element. The indefinite article “a” or “an” therefore generally means “at least one”.

[0137] The following examples illustrate different implementations of the present invention. Attached Figure Description

[0138] Figure 1 :

[0139] This invention relates to the chemical structures of five pentacyclic triterpenoid compounds.

[0140] Five pentacyclic triterpenoid compounds with profound pharmacological effects have been proposed in pharmaceutical formulations in this patent application.

[0141] Betulinic acid, belonging to the lupin subclass, has the IUPAC name (3β)-3-Hydroxylup-20(29)-en-28-acid and the chemical formula C30H48O3. Betulin also belongs to the lupin subclass, with the IUPAC name (3β)-Lup-20(29)-en-3,28-diol and the chemical formula C30H50O2. Lupinol also has a lupinane scaffold, with the IUPAC name (3β)-Lup-20(29)-en-3-ol and the chemical formula C30H50O. Oleanolic acid has an oleanane scaffold, with the IUPAC name (3β)-3-hydroxyoleanolic-12-en-28-acid and the chemical formula C30H48O3. Ursolic acid comes from the ursane subclass, and its IUPAC name is (3β)-3-hydroxyurs-12-ene-28-acid, with the chemical formula C30H48O3.

[0142] Figure 2 :

[0143] The efficient assembly mechanism of milk fat globules (MFGs) containing pentacyclic triterpenoids and the triterpenoid compounding under high effective loading Additives.

[0144] Figure 2 The thickness of the middle film is not proportional; the lower half of the manufacturing process does not contain triterpenoids.

[0145] The incorporation of hydrophobic triterpenoids is highly likely to occur within the three-layered membrane of milk fat globules (MFGs) and the triglyceride MFG core. Compared to smaller carrier systems (such as large liposomes or nano-sized liposomes), which are described as similar in size to MFGs, there is ample space for introducing bioactive triterpenoids into the MFG carrier. The membrane surface area of ​​200 nm diameter nanoparticles is 400 times smaller than that of MFG vesicles with a diameter of 4 μm, and the core contents of such MFGs are... The core volume of nanoparticles is 8,000 times larger. Furthermore, the aqueous core of liposome vesicles cannot be used to load hydrophobic compounds.

[0146] The relative contributions of the membrane and core to the loading of triterpenoids in the MFG are not precisely known, which makes it possible for the distribution of triterpenoids loaded in the core of the MFG to be 20% to 75%, respectively, in a nanoliposome vesicle. The total drug loading is greater than that of a single MFG vesicle 500 to 1600 times lower. Its extremely high drug loading capacity is a major advantage of the MFGas (oral) drug delivery system. When contained in an emulsion, even a much higher nanoparticle density (per volume unit) is not expected to significantly compensate for the much lower drug loading per nanoparticle.

[0147] Figure 3 :

[0148] RP-HPLC analysis of pentacyclic triterpenoid milk fat globule (MFG) formulations according to the present invention.

[0149] Figure A: Manufacturing betulinic acid preparations. Figure B: Manufacturing betulinic acid preparations.

[0150] The figures above (A and B) show representative RP-HPLC elution curves of samples taken from betulinic acid or betulin-milk fat globule formulations, with triterpenoid effective loads increasing at 10, 20, 30, and 50 mg / ml, demonstrating the efficient incorporation of triterpenoids within this concentration range.

[0151] Figures below (A and B): Peak area analysis of linear regression of chromatographic elution curves for four samples (n=4; standard deviation, SD, error bars indicated at each payload concentration) of the formulation with elevated triterpenoid concentrations, as shown. Strong linearity of drug incorporation was observed within this payload range. Four samples (n=4) were collected from different locations in the formulation without premixing to test for possible heterogeneous triterpenoid incorporation (or distribution) inherent in the formulation during preparation and / or instability evolving during storage, such as aggregation and / or phase separation (demulsification).

[0152] Figure 4 :

[0153] The microstructure of the betulinic acid-milk fat globule formulation according to the present invention and the control formulation were compared using an optical microscope.

[0154] (A) A control formulation of betulinic acid dispersed in bovine skim milk, lacking milk fat globules, and (B) a composition according to the invention, comprising betulinic acid at an effective loading of 32 mg / ml, incorporated into milk fat globules, as shown in bright-field microscopy at 100x magnification. The dispersion and emulsification procedures were identical for the preparation of (A) and (B). In (A), distinct rod-shaped undissolved betulinic acid deposits were observed, which were completely absent in the betulinic acid composition contained within the milk fat globules.

[0155] Figure 5 :

[0156] Particle size distribution analysis of the triterpenoid milk fat globule composition of the present invention, measured by laser diffraction.

[0157] Laser diffraction experiments were performed using a Bettersizer S3 Plus particle size analyzer (Bettersize Instruments Ltd, Dandong, China). Preparations without triterpenoids (control) or containing triterpenoids (betulin or betulin) were compared.

[0158] Figure A: Left, comparing the betulinic acid-milk fat globule composition (betulinic acid effective load, 32 mg / ml) with a control milk fat globule formulation without betulinic acid but prepared using the same procedure (high shear mixing, sonication); Right, comparing the composition of betulin incorporated into the milk fat globules (betulinic acid effective load, 20 mg / ml) with a control milk fat globule formulation without betulinic acid but prepared using the same procedure (high shear mixing, sonication).

[0159] Figure B: The same experiment as in Figure [A] is represented by a pie chart to better visualize the differences in granularity distribution: indicating the % of particles for each granularity category.

[0160] Figure 6 :

[0161] Characteristics of triterpenoid-milk fat globule formulations analyzed by acoustic spectroscopy.

[0162] As shown, acoustic spectroscopic experiments were performed on a control milk fat globule formulation without betulinic acid and the composition of the present invention containing betulinic acid incorporated into milk fat globules (MFG). When the control milk fat globule formulation did not contain betulinic acid, it underwent the same preparation procedure as the MGF betulinic acid formulation, namely high-shear mixing and sonication. Measurements were performed at a controlled temperature. A significant difference in attenuation spectra was observed when betulinic acid was incorporated into the MFG carrier.

[0163] Figure 7 :

[0164] Viscosity distribution of betulinic acid-milk fat globule formulation at a defined temperature.

[0165] The viscosity of three betulinic acid-milk fat globule compositions with increased betulinic acid loading was tested together with a control formulation (empty MFG) without loaded milk fat globules. Viscosity was measured under tightly controlled temperature conditions because viscosity and temperature are strongly interdependent. The betulinic acid loading of the formulations is represented in the figures (15, 25, and 35 mg / ml). Figure A depicts all three betulinic acid formulations with the control. Figure B depicts only the 15 mg / ml formulation with the "empty" control to better visualize the dynamic effects and viscosity enhancement due to the incorporation of betulinic acid, which also occurs at low betulinic acid concentrations.

[0166] Figure 8 :

[0167] Stability of betulinic acid-milk fat globule formulations after centrifugation and long-term storage.

[0168] Figure A. Effect of centrifugation on emulsion stability by measuring centrifugation-induced disintegration of the betulinic acid-milk fat globule composition compared to a control emulsion. The betulinic acid-containing composition assembled in milk fat globules at a payload of 35 mg / ml according to this disclosure was centrifuged at 3400 g for 50 min (tube 1). The control emulsion was similarly centrifuged, and an O / W emulsion containing untreated milk fat globules derived from homogenized milk fat globules with a particle size distribution of 0.2-2 μm was used (tube 2), or an untreated heterogenized cream derived from relatively large milk fat globules with a particle size distribution of 1-10 μm was used (tube 3). Figure B. Shelf life stability of the milk fat globule-betulinic acid formulation according to the invention after storage at 4°C for 3 months. Compositions incorporating betulinic acid into milk fat globules (MFG) according to the invention are shown in different variations. Tube 1: MFG derived from homogenized milk with an effective betulinic acid loading of 25 mg / ml; Tube 2: MFG derived from homogenized milk with an effective betulinic acid loading of 40 mg / ml; Tube 3: MFG derived from raw milk with an effective betulinic acid loading of 25 mg / ml. Detailed Implementation

[0169] Example 1

[0170] Preparation of the triterpenoid milk fat globule composition according to the present invention and the source of milk fat globules.

[0171] The compositions according to the invention are prepared using a basic scheme. Several variations have been performed with regard to the precise source and content of the excipients used and the details of the preparation scheme. Essentially, the milk fat globules used as the source of the composition are derived from homogenized milk fat, such as that commercially supplied by various dairy companies, or from raw (fresh) milk, such as that supplied by (biological) dairy farms. Raw milk cream is collected (or “skimmed”) according to the basic dairy process briefly outlined above to obtain the milk fat globules contained in the cream fraction.

[0172] Both milk fat globule sources can then be used in the preparation of the compositions of the present invention, taking into account the shelf life of the source. Compositions prepared using milk fat globules from both sources, as described above, each have different average globule sizes and are similar in terms of the effective load of pentacyclic triterpenoids incorporated into the milk fat globule drug carrier system.

[0173] Formulation preparation, methods and materials

[0174] Dairy cream formulations containing 20 to 100 mL volumes of high-density milk fat globules (at least 30% milk lipid, v / v) are used as the excipient source for the compositions of the present invention. High-purity isolated pentacyclic triterpenoid compounds (>98%, as confirmed by mass spectrometry) are added in powder form at concentrations ranging from mg triterpenoid / mL excipient, for example 10, 20, 30, 40, or 50 mg / mL. For the control compositions, no triterpenoid compounds are added, but the same preparation protocol is followed as for the triterpenoid compositions (in the pair of compositions to be compared).

[0175] For most formulations, the application includes a first dispersion step (equipment: UltraTurrax Displer, model T25, from IKA) involving high-shear mixing at 15,000–20,000 rpm for 2–5 minutes to complete the initial coarse dispersion of the triterpenoid powder in the entire contents of the excipient volume used. However, for some other preparations, the first high-shear mixing step is omitted because it appears to be optional if a second sonication stage is performed according to the most preferred steps and parameter settings.

[0176] During the second preparation stage, an ultrasonic treatment procedure was followed, which appeared essential to ensure proper and complete encapsulation and incorporation of the pentacyclic triterpenoid compound into the milk fat globules. Therefore, ultrasonic treatment was used in the preparation of all formulations according to the invention, as it would otherwise seem impossible to achieve complete incorporation of the triterpenoid compound under high payloads. The ultrasonic treatment was performed using an ultrasonic probe-type processor (also a manufactured tip, horn, or polar ultrasonic instrument). The equipment used was an ultrasonic processor of model FS150-N for small-volume preparation and an ultrasonic processor of model FS1800-N for larger volumes (Zhengzhou TCH Instrument Co., Ltd., Zhengzhou, China), with adjustable maximum output power of 80 watts and 1800 watts and a frequency of 20 kHz, respectively. The following sequence of ultrasonic treatment cycles and parameter settings were used for the 80-watt ultrasonic instrument (FS150-N), using… The probe was used. Five sonication cycles were applied with increasing power, starting at 35% power intensity and then cycling at 70%, 80%, 85%, and 90% power intensity. Each cycle lasted 5 minutes, and within each cycle, sonication was performed with 10-second sonication pulses, alternating with 5-second rest periods between pulses (a total of 20 cycles of 10-second pulses / 5 minutes). The primary reason for using this setup was to avoid excessively raising the temperature above 60°C in the composition being prepared. While increased temperature can aid in the emulsification process and the embedding of triterpenoids into the fat globules, excessively high temperatures can adversely cause protein denaturation in the fat globule membrane. Therefore, it was chosen to maintain a safe temperature range. The temperature was monitored during sonication between and within cycles. Thus, the applied cycle and pulse settings ensured that the temperature of the formulation never rose above 60°C. The applied sonication probe tip was immersed to a depth of 25% below the surface of the total formulation height. After the sonication process, allow the formulation to acclimate to room temperature, and then store it at 6-8°C until use.

[0177] As those skilled in the art will understand, it is anticipated that the use of high-pressure emulsification technology instead of ultrasonic treatment can similarly achieve complete incorporation of pentacyclic triterpenoids into milk fat globules. Similarly, in some embodiments of the compositions of the present invention, it is conceivable that the combination of ultrasonic treatment and high-pressure emulsification technology during preparation can advantageously facilitate the scale-up process required for the production of large-volume formulations

[94] .

[0178] Regarding the preparation methods used in this invention, it is worth noting that in any publication in which milk fat globules are used to incorporate active biological compounds, the encapsulation method does not in any sense involve the use of sonication or another high-energy emulsification technique, such as high-pressure emulsification or high-shear mixing. Instead, only simple co-incubation (i.e., stirring at 25°C) is applied

[83] , or organic solvents are used to aid the incorporation process [81, 82].

[0179] As shown in Example 2, and possibly according to Figure 2 The assembly principle shown is followed, and the preparation method described herein successfully incorporates triterpenoids, preferably betulinic acid and betulin, into milk fat globules with high effective loading. The unsuccessful emulsification and incorporation of triterpenoids into milk fat globules using several tested low-energy emulsification methods (e.g., stirring at 40°C) also highlights the importance of the high-energy emulsification technology applied herein.

[0180] Example 2

[0181] RP-HPLC and microscopy revealed the efficient incorporation of triterpenoids into milk fat globules.

[0182] Preparation method of triterpenoid milk fat globule composition sample

[0183] A two-phase liquid-liquid extraction (LLE) method was developed using methanol (MeOH) and isooctane as immiscible organic solvents to separate pentacyclic triterpenoids from their sample matrix (milk fat globule components). Briefly, samples of the triterpenoid-milk fat globule formulation were collected from at least four different locations within a formulation (contained in a 50 mL storage vial) using a sampling spatula and weighed. A two-phase liquid extraction system containing MeOH to dissolve the triterpenoids (betulin or betulin) and isooctane to dissolve the matrix lipids from the milk fat globules was constructed. For this purpose, equal volumes (7 mL) of MeOH and isooctane were taken, and 3% (by volume) of water was added to advantageously alter the liquid-liquid equilibrium between the two organic solvents. The sample removed from the formulation was added to the two-phase LLE system. The formulation sample was selected to be approximately 200 mg. After obtaining a series of samples whose triterpenoid content must be quantitatively compared by HPLC, with most weights slightly varying, the volumes of MeOH and isooctane were adjusted, if necessary, to obtain an equivalent mg ​​formulation / ml two-phase LLE organic solvent system. The samples were vigorously mixed in the LLE system using vortexing. The LLE system was then heated to 40°C and incubated for 30 minutes to aid sample disintegration. Then, to further improve sample dissolution, the LLE system containing the samples was vigorously mixed at 10,000 rpm using a high-shear mixer (Ultraturrax, model T25, a high-shear mixer operating on a rotor-stator principle), and then undisturbed at 40°C for 2 hours to re-establish phase separation between the MeOH and isooctane solvent layers.

[0184] From the potential MeOH phase containing dissolved triterpenoids, 1.2 mL of sample was taken and centrifuged at 10,500 rpm for 5 min to remove any possible debris, followed by 300 μL of sample for HPLC analysis. Quantitative RP-HPLC analysis of the formulation sample thus prepared for HPLC, combined with standard addition experiments, compared to a range of triterpenoid (betulinic acid or betulin) standard samples, revealed that the sample preparation procedure described herein resulted in an average loss of 30% of triterpenoid content (data not shown).

[0185] RP-HPLC analysis of triterpenoids, method

[0186] RP-HPLC analysis was performed using a chromatographic system consisting of a Waters Alliance 2695 separation module connected to a Waters 996 photodiode array detector. The stationary phase was a C-18 column (Waters...). C18 (5 μm, 4.6 x 250 mm). The injection volume was 20 μl at a controlled temperature of 30 °C, and the flow rate was 1.5 mL / min. For separation, elution was performed in isocratic mode using a mobile phase of acetonitrile-water in a 91:9 (v / v) ratio. The detection wavelength of the PDA detector was set to 210 nm. The chromatographic peaks of betulinic acid and / or betulin were confirmed by comparison of their retention times with >98% purity betulinic acid and betulin standards (Sigma-Aldrich) dissolved in methanol.

[0187] RP-HPLC analysis of triterpenoid-milk fat globule formulations: results

[0188] HPLC chromatography of a sample prepared from a combination of betulinic acid from milk fat globules and betulinol from milk fat globules effectively detected triterpenoid compounds in the formulation. Figure 3 Figures A and B above show representative elution curves. Note the difference in retention times between betulinic acid and betulinol, and show the linearity of triterpenoid incorporation, even at high payloads. Figure 3 A and B (see diagram below). Different findings suggest that betulinic acid and betulin are completely incorporated into the milk fat globules. Emulsions of betulinic acid or betulin in milk fat globules are extremely stable. Figure 8 A and B) were homogenized and emulsified with betulinic acid (or betulin), as shown in Figures 33A and 33B below, illustrating only the small standard deviation between samples prepared from four different locations in the emulsion. It is noteworthy that random sampling at different points in the emulsion was performed after at least one week of undisturbed shelf resting, without any pre-mixing (dispersion) of the emulsion. Furthermore, when sampling was performed from the upper lipid fraction of the emulsion after vigorous centrifugation (see Example 5 and...), Figure 8 A, centrifugation as shown in tube 1), the results were similar, with almost identical triterpenoid concentrations observed in samples taken from different points throughout the whole lipid fraction of the emulsion, also indicating a stable and homogeneous distribution of triterpenoids throughout the formulation (data not shown). However, as expected, higher concentrations of triterpenoids were detected in the HPLC samples from the upper lipid fraction compared to the uncentrifuged emulsion, because the aqueous lower fraction separated (and analyzed) after centrifugation ( Figure 8 A, tube 1) contains no triterpenoids (data not shown).

[0189] Furthermore, microscopic examination of the triterpenoid-milk fat globule (MGF) composition and its comparison with a control formulation of triterpenoids without MFG provide a strong additional indication that the amount of triterpenoid initially added during the preparation of the formulation according to the invention is completely incorporated into the MFG.

[0190] Bright-field microscopy of triterpenoid-milk fat globule compositions

[0191] Bright-field optical microscopy (100x magnification, using a Kern OBL137C832 microscope, Kern & Sohn GmbH, Germany) of a betulinic acid-milk fat globule formulation (with an effective loading of 32 mg / ml betulinic acid). Figure 4 The image in B shows the normal morphology of the fat globules, with no visible signs of crystallization or amorphous betulinic acid deposition.

[0192] On the other hand, the betulinic acid control formulation dispersed in skim milk according to the same emulsification process ( Figure 4 In A), there is a lack of milk fat globules, and the betulinic acid deposits are abundant and clearly visible as rod-shaped structures.

[0193] The importance of this type of microscopic particle characterization lies in direct observation without the need for complex interpretation issues, such as laser diffraction and acoustic spectroscopy results of complex emulsions, which often obscure and hinder clear conclusions.

[0194] Example 3

[0195] Particle size characterization of triterpenoid-milk fat globule compositions

[0196] Particle characterization using laser diffraction measurements, including size distribution.

[0197] The particle characteristics of the milk fat globule (MFG) control formulation and the MFG formulation of this invention containing a bioactive pentacyclic triterpenoid compound were analyzed by laser diffraction using a Bettersizer S3 Plus particle size analyzer (Bettersize Instruments Ltd, Dandong, China). The control formulation did not contain triterpenoid molecules, but the preparation procedure, including high-shear mixing and sonication, was performed identically to that of the formulation containing the triterpenoid compound.

[0198] In some cases, laser diffraction measurements can be difficult to interpret definitively. Figure 5 The experiments shown (the same data are presented in Figures A and B, but represented differently for clarity and interpretation) strongly suggest that the particle size of MFG increases with the incorporation of bioactive triterpenoids, as MFG particle sizes >5 μm are not noticeable in the control formulation, but are significant after incorporation of pentacyclic triterpenoids (betulinic acid or betulinine). However, differences other than MFG size between the control and triterpenoid formulations cannot be completely ruled out as a potential sole or partial cause of the observed effect, particularly the occurrence of MFG particle aggregates. However, all samples underwent the same pretreatment, including a short pre-ultrasonic treatment step prior to laser diffraction measurements to break down the aggregates, making the latter explanation unlikely.

[0199] Furthermore, compared to formulations containing only excipients and undergoing the same treatment but without triterpenoids, the volume-weighted average (i.e., average) particle size and median particle size obtained by laser diffraction in formulations containing betulinic acid were significantly increased (data not shown). This is related to... Figure 5 The results shown are consistent, indicating the particle-enlarging effect of triterpenoids. The volume-weighted average (i.e., average) particle diameter, denoted as D[4,3], can be obtained using the formula (∑n i .d i 4 ) / (∑n i .d i 3 The median particle size is synonymous with the D50 value, indicating the 50th percentile particle size in the size distribution.

[0200] Particle characterization measured by acoustic spectroscopy

[0201] The analysis of the acoustic attenuation principle of particles in concentrated emulsions involves an acoustic sensor that measures the attenuation of ultrasonic waves passing through the emulsion in the frequency range of 1 to 100 MHz, with a variable transmitter-detector distance. For this purpose, an acoustic-electro-acoustic spectrometer DT-1202 (Dispersion Technology Inc., USA) was used.

[0202] exist Figure 6 A clear mutual effect was observed, with the betulinic acid-milk fat globule formulation exhibiting lower acoustic attenuation than the "empty" control formulation below 35 MHz, while the opposite effect was observed above 35 MHz. The profound difference in acoustic attenuation curves between milk fat globules containing and without pentacyclic triterpenoids must be due to the incorporation of the compounds, given the similar preparation procedures.

[0203] The detailed interpretation of these results is not without complexity

[95] , as viscosity and particle composition, in addition to particle size, can also affect the attenuation spectrum, and the latter two parameters can also be altered by incorporating bioactive triterpenoids. Generally, lower acoustic attenuation in the low acoustic frequency range is associated with larger fat globule particles

[95] , indicating that incorporation of triterpenoids increases the size of the fat globules, which is consistent with results observed using laser diffraction. Figure 5 However, the viscosity changes (see...). Figure 7 This can at least partially explain the observed effects.

[0204] Example 4

[0205] Adding betulinic acid to enhance the viscosity of the preparation

[0206] Viscosity measurements of betulinic acid-milk fat globule formulations were performed using an A&D Company Ltd. (Tokyo, Japan) SV-10 sinusoidal vibration viscometer with a temperature sensor and viscosity measurement limits of 0.3–10000 mPa·s (millipascals per second). The apparatus employs a sinusoidal vibration method. Dynamic viscosity was measured continuously under strictly controlled temperature conditions. The formulation to be tested was placed in a sample cup within a water jacket with circulating water, allowing for precise temperature control. The refrigerated bath circulator used for this purpose was a Thermo Scientific Neslab RTE-201 model. Figure 7 The results shown indicate that the incorporation of betulinic acid significantly increases viscosity, including a strong positive correlation between the effective loading of betulinic acid in the formulation and the level of viscosity increase. The enhanced viscosity is likely to lead to increased formulation stability, which is another advantageous feature of the triterpenoid compositions disclosed herein. The viscosity enhancement is likely due to the incorporation of betulinic acid, a cholesterol-like substance, into the three-layer membrane of the milk fat globules. Cholesterol is known to make the membrane more rigid, thereby increasing viscosity

[96] .

[0207] Example 5

[0208] The composition of the present invention is stable.

[0209] For oral or topical application, and as those skilled in the art will understand, it is important that the formulation to be administered is stable to the homogeneous composition and also stable over time during storage. Stability was tested using two methods: centrifugation and a (simple) shelf-life storage test.

[0210] Accelerated stability testing was conducted by centrifugation at high g. For this purpose, the betulinic acid-milk fat globule (MFG) formulation was centrifuged at 3400 g for 50 minutes. The profound effects of incorporating betulinic acid into the MFG composition and its preparation method were observed. After centrifugation, the betulinic acid composition exhibited two-phase separation, with an aqueous layer at the bottom of the tube and a homogeneous lipid phase at the top. Figure 8 A, tube 1). In contrast, the untreated milk fat globule "source composition" obtained from homogenized milk or raw milk (containing relatively small or large MFGs, as previously described) showed significantly different phase separation after centrifugation, with a three-phase separation at the top of a transparent oil layer (A, tube 1). Figure 8 A, tubes 2 and 3. Notably, in tube 2, an O / W dairy emulsion containing homogeneous small-sized MFGs, second-phase separation was only detectable under translucent light. These results indicate that incorporating betulinic acid into the fat globules, along with its preparation process (primarily sonication), achieves increased (lipid particle) stability in the formulation, as no oil phase containing “released” fat from the fat globule core is observed. This can be explained by incorporating betulinic acid into the MFG membrane, thereby increasing membrane rigidity and its resistance to centrifugation-induced membrane disruption.

[0211] Shelf life stability was tested after various storage intervals. Figure 8 Figure B shows the results of storing three betulinic acid-milk fat globule formulations with different betulinic acid effective loads at 4°C for 3 months. In all cases where these tests were performed, the compositions disclosed herein remained highly stable without phase separation, which, if it occurred, would indicate undesirable flocculation and / or aggregation within the emulsion. A storage temperature of approximately 4°C is a generally acceptable and required condition for dairy (based) (food) products, and the apparent undisturbed emulsion stability after 3 months at this temperature can be considered an advantageous feature for the triterpenoid formulations of this invention.

[0212] Furthermore, similar stability was observed after one month in storage experiments at room temperature, with no bacterial growth in most test conditions (8 out of 9 test formulations: >85%) (data not shown). This clearly advantageous antimicrobial effect can be explained by the combined effect of an ultrasonic treatment procedure with known antimicrobial activity and the incorporation of betulinic acid (a compound with proven antimicrobial activity).

[0213] Example 6

[0214] Evaluation of the betulinic acid-milk fat globule composition of the present invention compared with betulinic acid formulations reported in the art.

[0215] As shown in Table 1, a comprehensive evaluation of the most prominent reported betulinic acid formulations with pharmaceutical potential was conducted. Birch acid was chosen for this overview because its chemical structure is very similar to other triterpenoids involved in this invention, resulting in strong hydrophobicity, and most published formulation studies have focused on betulinic acid.

[0216] Indicators for evaluating drug loading capacity (effective payload) and formulation excipients were assessed. Other listed characteristics were also evaluated, namely (1) the labor-intensive nature of preparation and associated cost-effectiveness of production, (2) potential toxicity and associated possible regulatory constraints, (3) assessment and / or estimation of formulation stability, and (4) estimated suitability for oral administration and nutritional product development. A comprehensive evaluation of these different betulinic acid formulations of the present invention, compared to compositions of the present invention comprising triterpenoid compounds (preferably betulinic acid and / or betulinic acid) incorporated into preferred milk fat globules, revealed that the compositions of the present invention possessed advantageous characteristics relative to other formulations in all aspects listed in Table 1, thus offering a beneficial advantage.

[0217]

[0218] References

[0219] 1.Mishra BB,Tiwari VK:Natural products:an evolving role in futuredrug discovery.Eur J Med Chem 46:4769-4807,2011.

[0220] 2.Newman DJ,Cragg GM:Natural products as sources ofnew drugs over thelast 25years.J Nat Prod 70:461-477,2007.

[0221] 3.Dehelean CA,Marcovici I,Soica C,Mioc M,Coricovac D,Iurciuc S,CretuOM,Pinzaru I:Plant-Derived Anticancer Compounds as New Perspectives in DrugDiscovery and Alternative Therapy.Molecules 26:1109,2021.

[0222] 4.Liu K,Zhang X,Xie L,Deng M,Chen H,Song J,Long J,Li X,Luo J:Lupeoland its derivatives as anticancer and anti-inflammatory agents:Molecularmechanisms and therapeutic efficacy.Pharmacol Res 164:105373,2021.

[0223] 5.Sun,Fang,Wang,Hu:Structure-activity relationships of oleanane-andursanetype triterpenoids.Botanical Studies 47:339-368,2006.

[0224] 6.Margaret M.O'Connel,Michael D.Bentley,Christopher S.Campbell,Barbara J.W.Cole:Betulin and Lupeol in bark from four white-barkedbirches.Phytochemistry 27:2175-2176,1998.

[0225] 7.Soica CM,Dehelean CA,Peev C,Aluas M,Zupko I,Kasa P,Jr.,Alexa E:Physico-chemical comparison ofbetulinic acid,betulin and birch bark extractand in vitro investigation of their cytotoxic effects towards skin epidermoidcarcinoma(A431),breast carcinoma(MCF7)and cervix adenocarcinoma(HeLa)celllines.Nat Prod Res 26:968-974,2012.

[0226] 8.Kessler JH,Mullauer FB,de Roo GM,Medema JP:Broad in vitro efficacyofplant-derived betulinic acid against cell lines derived from the mostprevalent human cancer types.Cancer Lett 251:132-145,2006.

[0227] 9.Mullauer FB,van BL,Daalhuisen JB,Ten Brink MS,Storm G,Medema JP,Schiffelers RM,Kessler JH:Betulinic acid delivered in liposomes reducesgrowth of human lung and colon cancers in mice without causing systemictoxicity.Anticancer Drugs 22:223-233,2011.

[0228] 10.Lou H,Li H,Zhang S,Lu H,Chen Q:A Review on Preparation ofBetulinicAcid and Its Biological Activities.Molecules 26:2021.

[0229] 11.Hordyjewska,Ostapiuk,Horecka,Kurzepa:Betulin and betulinic acid:triterpenoids derivatives with a powerful biological potential.Phytochem Rev18:929-951,2019.

[0230] 12.Mullauer FB,Kessler JH,Medema JP:Betulinic acid,a natural compoundwith potent anticancer effects.Anticancer Drugs 21:215-227,2010.

[0231] 13.Pisha E,Chai H,Lee IS,Chagwedera TE,Farnsworth NR,Cordell GA,Beecher CW,Fong HH,Kinghorn AD,Brown DM,Wani MC,Wall ME,Hieken TJ,Das GuptaTK,Pezzuto JM:Discovery ofbetulinic acid as a selective inhibitor of humanmelanoma that functions by induction of apoptosis.Nat Med 1:1046-1051,1995.

[0232] 14.Pavlova NN,Zhu J,Thompson CB:The hallmarks of cancer metabolism:Still emerging.Cell Metab 34:355-377,2022.

[0233] 15.Potze L,Di Franco S,Grandela C,Pras-Raves ML,Picavet DI,van VeenHA,van Lenthe H,Mullauer FB,van der Wel NN,Luyf A,van Kampen AH,Kemp S,EvertsV,Kessler JH,Vaz FM,Medema JP:Betulinic acid induces a novel cell deathpathway that depends on cardiolipin modification.Oncogene 35:427–437,2015.

[0234] 16.Heiss EH,Kramer MP,Atanasov AG,Beres H,Schachner D,Dirsch VM:Glycolytic switch in response to betulinic acid in non-cancer cells.PLoS ONE9:e115683,2014.

[0235] 17.Epstein T,Gatenby RA,Brown JS:The Warburg effect as an adaptationof cancer cells to rapid fluctuations in energy demand.PLoS ONE 12:e0185085,2017.

[0236] 18.Reddy AS,Zhang S:Polypharmacology:drug discovery for thefuture.Expert Rev Clin Pharmacol 6:41-47,2013.

[0237] 19.Wang YJ,Liu JB,Dou YC:Sequential treatment with betulinic acidfollowed by 5-fluorouracil shows synergistic cytotoxic activity in ovariancancer cells.Int J Clin Exp Pathol 8:252-259,2015.

[0238] 20.Pandita A,Kumar B,Manvati S,Vaishnavi S,Singh SK,Bamezai RN:Synergistic combination of gemcitabine and dietary molecule induces apoptosisin pancreatic cancer cells and down regulates PKM2expression.PLoS ONE 9:e107154,2014.

[0239] 21.Gao Y,Jia Z,Kong X,Li Q,Chang DZ,Wei D,Le X,Huang S,Wang L,Xie K:Combining betulinic acid and mithramycin A effectively suppresses pancreaticcancer by inhibiting proliferation,invasion and angiogenesis.Cancer Res 2011.

[0240] 22.Yamai H,Sawada N,Yoshida T,Seike J,Takizawa H,Kenzaki K,Miyoshi T,Kondo K,Bando Y,Ohnishi Y,Tangoku A:Triterpenes augment the inhibitoryeffects of anticancer drugs on growth of human esophageal carcinoma cells invitro and suppress experimental metastasis in vivo.Int J Cancer 125:952-960,2009.

[0241] 23.Sawada N,Kataoka K,Kondo K,Arimochi H,Fujino H,Takahashi Y,MiyoshiT,Kuwahara T,Monden Y,Ohnishi Y:Betulinic acid augments the inhibitoryeffects ofvincristine on growth and lung metastasis of B16F10 melanoma cellsin mice.Br J Cancer 90:1672-1678,2004.

[0242] 24.Nakagawa-Goto K,Yamada K,Taniguchi M,Tokuda H,Lee KH:Cancerpreventive agents 9.Betulinic acid derivatives as potent cancerchemopreventive agents.Bioorg Med Chem Lett 19:3378-3381,2009.

[0243] 25.Fulda S:Betulinic Acid for cancer treatment and prevention.Int JMol Sci 9:1096-1107,2008.

[0244] 26.Cichewicz RH,Kouzi SA:Chemistry,biological activity,andchemotherapeutic potential ofbetulinic acid for the prevention and treatmentof cancer and HIV infection.Med Res Rev 24:90-114,2004.

[0245] 27.Zheng ZW,Song SZ,Wu YL,Lian LH,Wan Y,Nan JX:Betulinic acidprevention of d-galactosamine / lipopolysaccharide liver toxicity is triggeredby activation of Bcl-2 and antioxidant mechanisms.J Pharm Pharmacol 63:572-578,2011.

[0246] 28.Silva FS,Oliveira PJ,Duarte MF:Oleanolic,Ursolic,and BetulinicAcids as Food Supplements or Pharmaceutical Agents for Type 2Diabetes:Promiseor Illusion?JAgric Food Chem 64:2991-3008,2016.

[0247] 29.Ajala-Lawal RA,Aliyu NO,Ajiboye TO:Betulinic acid improves insulinsensitivity,hyperglycemia,inflammation and oxidative stress in metabolicsyndrome rats via PI3K / Akt pathways.Arch Physiol Biochem126:107-115,2020.

[0248] 30.Xie W,Hu W,Huang Z,Li M,Zhang H,Huang X,Yao P:Betulinic acidaccelerates diabetic wound healing by modulating hyperglycemia-inducedoxidative stress,inflammation and glucose intolerance.Burns Trauma 10:tkac007,2022.

[0249] 31.Castillo PM,Jimenez-Ruiz A,Carnerero JM,Prado-Gotor R:ExploringFactors for the Design of Nanoparticles as Drug Delivery Vectors.Chemphyschem19:2810-2828,2018.

[0250] 32.Liu HR,Ahmad N,Lv B,Li C:Advances in production and structuralderivatization of the promising molecule ursolic acid.Biotechnol J16:e2000657,2021.

[0251] 33.Nguyen HN,Ullevig SL,Short JD,Wang L,Ahn YJ,Asmis R:Ursolic Acidand Related Analogues:Triterpenoids with Broad Health Benefits.Antioxidants(Basel)10:2021.

[0252] 34.Gupta N:A Review on Recent Developments in the AnticancerPotential of Oleanolic Acid and its Analogs(2017-2020).Mini Rev Med Chem22:600-616,2022.

[0253] 35.Bildziukevich U,Ozdemir Z,Wimmer Z:Recent Achievements inMedicinal and Supramolecular Chemistry of Betulinic Acid and ItsDerivatives.Molecules 24:2019.

[0254] 36.Shultz MD:Two Decades under the Influence of the Rule of Five andthe Changing Properties of Approved Oral Drugs.J Med Chem62:1701-1714,2019.

[0255] 37.Ruiz-Picazo A,Lozoya-Agullo I,Gonzalez-Alvarez I,Bermejo M,Gonzalez-Alvarez M:Effect of excipients on oral absorption process accordingto the different gastrointestinal segments.Expert Opin Drug Deliv18:1005-1024,2021.

[0256] 38.Mierina I,Vilskersts R,Turks M:Delivery Systems for Birch-barkTriterpenoids and their Derivatives in Anticancer Research.Curr Med Chem 27:1308-1336,2020.

[0257] 39.Saneja A,Arora D,Kumar R,Dubey RD,Panda AK,Gupta PN:Therapeuticapplications ofbetulinic acid nanoformulations.Ann N YAcad Sci1421:5-18,2018.

[0258] 40.Wang HM,Soica CM,Wenz G:A comparison investigation on thesolubilization of betulin and betulinic acid in cyclodextrin derivatives.NatProd Commun 7:289-291,2012.

[0259] 41.Zadeh MT,Khamesipour A,Shafiee AM,Ebrahimi SH,Haji Molla HM,Mosaffa N,Ramezani A:Comparative analysis between four model nanoformulationsof amphotericin B-chitosan,amphotericin B-dendrimer,betulinic acid-chitosanand betulinic acid-dendrimer for treatment of Leishmania major:real-time PCRassay plus.Int J Nanomedicine14:7593-7607,2019.

[0260] 42.Kumar P,Gautam AK,Kumar U,Bhadauria AS,Singh AK,Kumar D,Mahata T,Maity B,Bera H,Saha S:Mechanistic exploration of the activities ofpoly(lactic-co-glycolic acid)-loaded nanoparticles ofbetulinic acid againsthepatocellular carcinoma at cellular and molecular levels.Arch PhysiolBiochem 128:836-848,2022.

[0261] 43.Kumar P,Singh AK,Raj V,Rai A,Keshari AK,Kumar D,Maity B,Prakash A,Maiti S,Saha S:Poly(lactic-co-glycolic acid)-loaded nanoparticles ofbetulinic acid for improved treatment of hepatic cancer:characterization,invitro and in vivo evaluations.Int J Nanomedicine13:975-990,2018.

[0262] 44.Saneja A,Kumar R,Singh A,Dhar DR,Mintoo MJ,Singh G,Mondhe DM,PandaAK,Gupta PN:Development and evaluation of long-circulating nanoparticlesloaded with betulinic acid for improved anti-tumor efficacy.Int J Pharm 531:153-166,2017.

[0263] 45.Garcia-Pinel B,Porras-Alcala C,Ortega-Rodriguez A,Sarabia F,PradosJ,Melguizo C,Lopez-Romero JM:Lipid-Based Nanoparticles:Application and RecentAdvances in Cancer Treatment.Nanomaterials(Basel)9:2019.

[0264] 46.Porter CJ,Trevaskis NL,Charman WN:Lipids and lipid-basedformulations:optimizing the oral delivery of lipophilic drugs.Nat Rev DrugDiscov 6:231-248,2007.

[0265] 47.Liu Y,Gao D,Zhang X,Liu Z,Dai K,Ji B,Wang Q,Luo L:Antitumor drugeffect of betulinic acid mediated by polyethylene glycol modifiedliposomes.Mater Sci Eng C Mater BiolAppl 64:124-132,2016.

[0266] 48.Cavazos-Garduno A,Ochoa Flores AA,Serrano-Nino JC,Martinez-SanchezCE,Beristain CI,Garcia HS:Preparation of betulinic acid nanoemulsionsstabilized by omega-3 enriched phosphatidylcholine.Ultrason Sonochem 24:204-213,2015.

[0267] 49.Nsairat H,Khater D,Sayed U,Odeh F,Al BA,Alshaer W:Liposomes:structure,composition,types,and clinical applications.Heliyon8:e09394,2022.

[0268] 50.Daraee H,Etemadi A,Kouhi M,Alimirzalu S,Akbarzadeh A:Applicationof liposomes in medicine and drug delivery.Artif Cells Nanomed Biotechnol 44:381-391,2016.

[0269] 51.Dhiman N,Sarvaiya J,Mohindroo P:A drift on liposomes toproliposomes:recent advances and promising approaches.J Liposome Res1-15,2022.

[0270] 52.Musther H,Olivares-Morales A,Hatley OJ,Liu B,Rostami HA:Animalversus human oral drug bioavailability:do they correlate?Eur J Pharm Sci 57:280-291,2014.

[0271] 53.Manconi M,Caddeo C,Manca ML,FaddaAM:Oral delivery of natural compounds by phospholipid vesicles.Nanomedicine(Lond)15:1795-1803,2

[0272] 54.He H,Lu Y,Qi J,Zhu Q,Chen Z,Wu W:Adapting liposomes for oral drugdelivery.Acta Pharm Sin B 9:36-48,2019.

[0273] 55.Amiri S,Dastghaib S,Ahmadi M,Mehrbod P,Khadem F,Behrouj H,Aghanoori MR,Machaj F,Ghamsari M,Rosik J,Hudecki A,Afkhami A,Hashemi M,LosMJ,Mokarram P,Madrakian T,Ghavami S:Betulin and its derivatives as novelcompounds with different pharmacological effects.BiotechnolAdv

[0274] 56.McMahon DJ,Oommen BS:Supramolecular structure of the casein micelle.J Dairy Sci 91:1709–1721,2008.

[0275] 57.Sadiq U,Gill H,Chandrapala J:Casein Micelles as an EmergingDelivery System for Bioactive Food Components.Foods 10:2021.

[0276] 58.Ma F,Wei J,Hao L,Shan Q,Li H,Gao D,Jin Y,Sun P:Bioactive Proteinsand their Physiological Functions in Milk.Curr Protein Pept Sci 20:759-765,2019.

[0277] 59.Jensen RG:The composition of bovine milk lipids:January1995 toDecember 2000.J Dairy Sci 85:295-350,2002.

[0278] 60.Singh H,Gallier S:Nature's complex emulsion:The fat globulesofmilk.Food Hydrocoll 68:81,2017.

[0279] 61.Lopez C:Milk fat globules enveloped by their biological membrane:Unique colloidal assemblies with a specific composition and structure.CurrOpin Colloid Interface Sci 16:391-404,2011.

[0280] 62.Braun K,Hanewald A,Vilgis TA:Milk Emulsions:Structure andStability.Foods 8:483,2019.

[0281] 63.He S,Tang H,Yi H,Xu W,Ma Y,Wang R:Properties of emulsions frommilk fat globule membrane and its components.Int J Food Prop 20:S1342-S1353,2017.

[0282] 64.Argov N,Lemay DG,German JB:Milk Fat Globule structure&function;nanosciece comes to milk production.Trends Food Sci Technol 19:2008.

[0283] 65.Ye A,Anema SG,Singh H:Changes in the surface protein ofthe fatglobules during homogenization and heat treatment of concentrated milk.JDairy Res 75:347-353,2008.

[0284] 66.Ye A,Cui J,Dalgleish D,Singh H:Effect of homogenization and heattreatment on the behavior of protein and fat globules during gastricdigestion ofmilk.J Dairy Sci 100:36-47,2017.

[0285] 67.Kielczewska K,Ambroziak K,Krzykowska D,Aljewicz M:The effect ofhigh-pressure homogenisation on the size of milk fat globules and MFGMcomposition in sweet buttermilk and milk.Int Dairy J 113:104898,2021.

[0286] 68.Zhou X,Sala G,Sagis LMC:Bulk and interfacial properties of milkfat emulsions stabilized by whey protein isolate and whey proteinaggregates.Food Hydrocolloids 109:106100,2020.

[0287] 69.Shi J,Kantoff PW,Wooster R,Farokhzad OC:Cancer nanomedicine:progress,challenges and opportunities.Nat Rev Cancer17:20-37,2017.

[0288] 70.Tang Y,Wang X,Li J,Nie Y,Liao G,Yu Y,Li C:Overcoming theReticuloendothelial System Barrier to Drug Delivery with a"Don't-Eat-Us"Strategy.ACS Nano 13:13015-13026,2019.

[0289] 71.Zhao B,Li X,Kong Y,Wang W,Wen T,Zhang Y,Deng Z,Chen Y,Zheng X:Recent advances in nano-drug delivery systems for synergistic antitumorimmunotherapy.Front Bioeng Biotechnol 10:1010724,2022.

[0290] 72.Dan N:Core-shell drug carriers:liposomes,polymersomes,andniosomes.Chapter 2 from book:Nanostructures for Drug Delivery.Page63-105,2017.

[0291] 73.Siepmann J,Faham A,Clas SD,Boyd BJ,Jannin V,Bernkop-Schnurch A,Zhao H,Lecommandoux S,Evans JC,Allen C,Merkel OM,Costabile G,Alexander MR,Wildman RD,Roberts CJ,Leroux JC:Lipids and polymers in pharmaceuticaltechnology:Lifelong companions.Int J Pharm558:128-142,2019.

[0292] 74.Lu H,Zhang S,Wang J,Chen Q:A Review on Polymer and Lipid-BasedNanocarriers and Its Application to Nano-Pharmaceutical and Food-BasedSystems.Front Nutr 8:783831,2021.

[0293] 75.Mehanna MM,Mneimneh AT:Formulation and Applications of Lipid-BasedNanovehicles:Spotlight on Self-emulsifying Systems.Adv Pharm Bull 11:56-67,2021.

[0294] 76.Sarah Faulkner.Are nanoparticles overhyped?2017.

[0295] Ref Type:Online Source:https: / / www.medicaldesignandoutsourcing.com / nanoparticles-an-industry-buzz word-meets-reality /

[0296] 77.Patra JK,Das G,Fraceto LF,Campos EVR,Rodriguez-Torres MDP,Acosta-Torres LS,Diaz-Torres LA,Grillo R,Swamy MK,Sharma S,Habtemariam S,Shin HS:Nano based drug delivery systems:recent developments and future prospects.JNanobiotechnology 16:71,2018.

[0297] 78.Müller RH.Nanoparticles-the hype and the decline?Why?2020.

[0298] Ref Type:Online Source:https: / / www.controlledreleasesociety.org / news / nanoparticles-hype-and-decline-why

[0299] 79.De Jong WH,Borm PJ:Drug delivery and nanoparticles:applicationsand hazards.Int J Nanomedicine 3:133-149,2008.

[0300] 80.Ditzinger F,Price DJ,Ilie AR,Kohl NJ,Jankovic S,Tsakiridou G,Aleandri S,Kalantzi L,Holm R,Nair A,Saal C,Griffin B,Kuentz M:Lipophilicityand hydrophobicity considerations in bio-enabling oral formulationsapproaches-a PEARRL review.J Pharm Pharmacol 71:464-482,2019.

[0301] 81.Nitin N,Alshehab M.Encapsulating Molecules in Fat Globules.WO2020 / 227030A1.2022.30-4-2020.RefType:Patent

[0302] 82.Alshehab M,Nitin N:Encapsulation and release of curcumin using anintact milk fat globule delivery system.Food Funct 10:7121-7130,2019.

[0303] 83.Repasky EA,Bankert RB.Use ofMilk Globules as Carriers forDrugs.US4994496A.1991.United States of America.9-9-1987.Ref Type:Patent

[0304] 84.Broniatowski M,Flasinski M,Wydro P:Investigation of theinteractions of lupane type pentacyclic triterpenes with outer leafletmembrane phospholipids--Langmuir monolayer and synchrotron X-ray scatteringstudy.J Colloid Interface Sci 381:116-124,2012.

[0305] 85.Chen Y,Sun R,Wang B:Monolayer behavior ofbinary systems ofbetulinic acid and cardiolipin:thermodynamic analyses of Langmuir monolayersand AFM study of Langmuir-Blodgett monolayers.J Colloid Interface Sci 353:294-300,2011.

[0306] 86.Abboud R,Charcosset C,Greige-Gerges H:Tetra-and Penta-CyclicTriterpenes Interaction with Lipid Bilayer Membrane:A Structural ComparativeStudy.J Membr Biol 249:327-338,2016.

[0307] 87.Salim M,Eason T,Boyd BJ:Opportunities for milk and milk-relatedsystems as'new'low-cost excipient drug delivery materials.Adv Drug Deliv Rev183:114139,2022.

[0308] 88.Singh H:Symposium review:Fat globules in milk and their structuralmodifications during gastrointestinal digestion.J Dairy Sci102:2749-2759,2019.

[0309] 89.Bach Korsholm KK,Heerup C,Rongaard Stange JT,Geng X,Drachmann N,Nordby P,Bekker JP,Ifaoui I,Mullertz A,Torp SP,Stampe OM,Thymann T:BovineMilk-Derived Emulsifiers Increase Triglyceride Absorption in Newborn Formula-Fed Pigs.Nutrients 13:2021.

[0310] 90.Gazquez A,Sabater-Molina M,Dominguez-Lopez I,Sanchez-Campillo M,Torrento N,Tibau J,Moreno-Munoz JA,Rodriguez-Palmero M,Lopez-Sabater MC,Larque E:Milk fat globule membrane plus milk fat increase docosahexaenoicacid availability in infant formulas.Eur J Nutr Online-ahead-of-print:2022.

[0311] 91.He S,Ye A:Formation and gastrointestinal digestion of beta-carotene emulsion stabilized by milk fat globule membrane.J Food Process Eng42:e13301,2019.

[0312] 92.Thum C,Roy NC,Everett DW,McNabb WC:Variation in milk fat globulesize and composition:A source ofbioactives for human health.Crit Rev Food SciNutr 63:87-113,2023.

[0313] 93.Wang C,Qiao X,Gao Z,Jiang L,Mu Z:Advancement on Milk Fat GlobuleMembrane:Separation,Identification,and Functional Properties.Front Nutr 8:807284,2022.

[0314] 94.Yao C,Zhao S,Liu L,Liu Z,Chen G:Ultrasonic emulsification:basiccharacteristics,cavitation,mechanism,devices and application.Front Chem SciEng 16:1560-1583,2022.

[0315] 95.Dukhin AS,Goetz PJ,Travers B:Use of ultrasound for characterizingdairy products.J Dairy Sci 88:1320-1334,2005.

[0316] 96.Chakraborty S,Doktorova M,Molugu TR,Heberle FA,Scott HL,DzikovskiB,Nagao M,Stingaciu LR,Standaert RF,Barrera FN,Katsaras J,Khelashvili G,BrownMF,Ashkar R:How cholesterol stiffens unsaturated lipid membranes.ProcNatlAcad Sci U S A 117:21896-21905,2020.

[0317] 97.Castor TP:Phospholipid nanosomes.Curr Drug Deliv 2:329-340,2005.

[0318] 98.Dehelean CA,Feflea S,Ganta S,Amiji M:Anti-angiogenic effects ofbetulinic acid administered in nanoemulsion formulation using chorioallantoicmembrane assay.J Biomed Nanotechnol 7:317-324,2011.

[0319] 99.Harwansh RK,Mukerjee PK,Biswas S:Nanoemulsion as a novel carriersystem for improvement ofbetulinic acid oral bioavailability andhepatoprotective activity.J Mol Liq 237:361,2017.

[0320] 100.Bravo-Alfaro DA,Ochoa-Rodríguez RL, -Ortega F,Luna-Barcenas G,García HS:Self-nanoemulsifying drug delivery system(SNEDDS)improves the oral bioavailability of betulinic acid.J Mol Liq364:119946,2022.

[0321] 101.Dutta D,Paul B,Mukherjee B,Mondal L,Sen S,Chowdhury C,Debnath MC:Nanoencapsulated betulinic acid analogue distinctively improves colorectalcarcinoma in vitro and in vivo.Sci Rep 9:11506,2019.

[0322] 102.Wu ZC,Liu XY,Liu JY,Piao JS,Piao MG:Preparation of Betulinic AcidGalactosylated Chitosan Nanoparticles and Their Effect on Liver Fibrosis.IntJ Nanomedicine 17:4195-4210,2022.

[0323] 103.Zadeh MT,Shafiee AM,Haji Molla HM,Khamesipour A,Mosaffa N,Ramezani A:Novel Nanosized Chitosan-Betulinic Acid Against ResistantLeishmania Major and First Clinical Observation of such parasite inKidney.Sci Rep 8:11759,2018.

[0324] 104.Sardar A,Gautam S,Sinha S,Rai D,Tripathi AK,Dhaniya G,Mishra PR,Trivedi R:Nanoparticles of naturally occurring PPAR-gamma inhibitor betulinicacid ameliorates bone marrow adiposity and pathological bone loss inovariectomized rats via Wnt / beta-catenin pathway.Life Sci309:121020,2022.

[0325] 105.Dai L,Liu R,Hu L,Wang J,Si CL:Self-assembled PEG-carboxymethylcellulose nanoparticles / a-cyclodextrin hydrogels for injectableand thermosensitive drug delivery.RSC Adv 7:2905,2017.

[0326] 106.Dai L,Cao X,Liu KF,Li CX,Zhang GF,Deng LH,Si CL,He J,Lei JD:Self-assembled targeted folate-conjugated eight-arm-polyethylene glycol-betulinicacid nanoparticles for co-delivery of anticancer drugs.J Mater Chem B 3:3754-3766,2015.

Claims

1. A composition comprising a pentacyclic triterpenoid compound, wherein the pentacyclic triterpenoid compound is incorporated into a milk fat globule or a membrane-bound lipid vesicle, wherein the membrane comprises three layers: - Phospholipid monolayer; and - Phospholipid bilayer.

2. The composition comprising a pentacyclic triterpenoid compound according to claim 1, wherein one pentacyclic triterpenoid compound or a combination of two or more different pentacyclic triterpenoid compounds is contained in a plurality of fat globules.

3. The composition comprising a pentacyclic triterpenoid compound according to any one of the preceding claims, wherein the milk fat globule or lipid vesicle has an average diameter of 0.1 to 15 μm, preferably 1 to 10 μm, more preferably 2 to 6 μm, as determined by laser diffraction.

4. The composition comprising a pentacyclic triterpenoid compound according to any one of the preceding claims, wherein the pentacyclic triterpenoid compound is betulinic acid, and / or betulinol, and / or lupeol, and / or oleanolic acid, and / or ursolic acid.

5. The composition comprising a pentacyclic triterpenoid compound according to any one of the preceding claims, wherein the pentacyclic triterpenoid compound is betulinic acid and / or betulinol.

6. The composition comprising a pentacyclic triterpenoid compound according to any one of the preceding claims, wherein the pentacyclic triterpenoid compound is betulinic acid.

7. The composition comprising a pentacyclic triterpenoid compound according to any one of the preceding claims, wherein the pentacyclic triterpenoid compound is contained in a liquid emulsion formulation, preferably, wherein the liquid emulsion formulation comprises at least 10, 15, 20, 25, 30, 35, 40, 50, 60, or 70 mg / mL of the pentacyclic triterpenoid compound, preferably at least 50, 60, 70, 80, or 90% by weight incorporated into fat globules or lipid vesicles.

8. The composition comprising a pentacyclic triterpenoid compound according to any one of claims 1-6, wherein the pentacyclic triterpenoid compound is contained in a powder formulation, the powder formulation preferably obtained by spray drying or freeze drying techniques applied to the liquid formulation according to claim 7.

9. A composition comprising a pentacyclic triterpenoid compound according to any one of the preceding claims, wherein the pentacyclic triterpenoid compound is for oral administration and / or is contained in a food composition, beverage composition, food supplement composition, dietary supplement composition, nutritional composition or pharmaceutical composition for oral administration.

10. The composition comprising a pentacyclic triterpenoid compound according to any one of claims 1-8, wherein the pentacyclic triterpenoid compound is for topical application to the skin, preferably as an ointment or cream for pharmaceutical or cosmetic use.

11. The composition comprising a pentacyclic triterpenoid compound according to any one of the preceding claims is used for therapeutic purposes.

12. The use of a composition comprising a pentacyclic triterpenoid compound according to any one of the preceding claims for the treatment or prevention of cancer, type 2 diabetes, obesity, metabolic syndrome, cardiovascular disease, skin disease, (chronic) inflammation or Parkinson's disease and / or related movement disorders, preferably for the treatment or prevention of cancer.

13. Use of a composition comprising a pentacyclic triterpenoid compound according to any one of the preceding claims for the treatment or prevention of cancer, wherein the cancer is lung cancer, colorectal cancer, breast cancer, prostate cancer, cervical cancer, or melanoma.

14. A method for preparing a composition comprising a pentacyclic triterpenoid compound according to any one of the preceding claims, the method comprising the steps of: a) Provides a composition comprising lipid vesicles having a trilayer membrane comprising a monolayer of phospholipids and a bilayer of phospholipids, preferably milk fat globules; b) By mixing the composition provided in a) with one or more different pentacyclic triterpenoid compounds, preferably by applying high-shear mixing and / or ultrasonic treatment and / or high-pressure homogenization techniques, a pentacyclic triterpenoid composition according to any one of the preceding claims is provided, thereby achieving the incorporation of the triterpenoid compound into the lipid vesicles, preferably milk fat globules, and the assembly of milk fat globules or lipid vesicles incorporating the triterpenoid compound.

15. The method of claim 14, wherein in step b), ultrasonic treatment is applied to agitate the fat globules or lipid vesicles and the pentacyclic triterpenoid compound, thereby dispersing the pentacyclic triterpenoid compound in the emulsion containing the fat globules or lipid vesicles and incorporating the pentacyclic triterpenoid compound into the fat globules or lipid vesicles.

16. The method according to any one of claims 14-15, wherein in step a) or b), milk protein, preferably casein, casein micelles and / or whey protein, is added.

17. The combination of the composition comprising a pentacyclic triterpenoid compound according to any one of claims 1-13 with milk protein, preferably casein, casein micelles and / or whey protein.

Images