Capsules comprising ursolic acid and low-energy process of making the same

Abstract

The present disclosure relates to a method of preparing nanocapsules of ursolic acid. The method comprises mixing ursolic acid with a poly(ethylene glycol)-containing polymer at ambient temperature to form a mixture, adding an aqueous carrier to the mixture, and homogenizing the mixture with the aqueous carrier under high-shear conditions to obtain an emulsion comprising a hydrophobic core consisting of the ursolic acid, encapsulated by a shell consisting of the poly(ethylene glycol)-containing polymer, wherein the poly(ethylene glycol)- containing polymer is Poloxamer 407, and wherein the method is performed without heating and without organic solvents. The present disclosure also relates to a core-shell capsule prepared by the method of the present disclosure.

Description

CAPSULES COMPRISING URSOLIC ACID AND LOW-ENERGY PROCESS OF MAKING THE SAME CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of Singapore application no.10202403585S filed on 15 November 2024, the contents of it being hereby incorporated by reference in its entirety for all purposes.TECHNICAL FIELD

[0002] The present disclosure generally relates to a method of preparing nanocapsules of ursolic acid, and to core-shell capsules comprising ursolic acidBACKGROUND

[0003] Ursolic acid is a naturally occurring triterpenoid compound known for its antiinflammatory and anti -tumor properties and has been investigated extensively for pharmaceutical applications. In dermatology, ursolic acid has been reported to improve fine wrinkles, stimulate fibroblast activity, and enhance skin suppleness. However, ursolic acid exhibits very poor water solubility (approximately 0.12 pg / L), and therefore its direct incorporation into aqueous cosmetic or pharmaceutical formulations is challenging.

[0004] Encapsulation is a known strategy to transform lipophilic molecules into water-dispersible nanosized capsules, thereby expanding their application in water-based formulations and potentially improving skin penetration. Ursolic acid is widely present in natural sources such as the peels of fruits (e.g., olives), as well as in herbs and spices such as rosemary and thyme, which also highlights the sustainability of the ingredient. Nevertheless, there remains a lack of encapsulation methods that can produce nanosized vesicles of ursolic acid under conditions consistent with green chemistry principles, such as solvent-free and energy-efficient processes.

[0005] Encapsulation of ursolic acid has been described in the prior art; however, many reported methods involve the use of organic solvents and / or heating processes.

[0006] For example, Korean patent no. KR100427777B1 discloses the preparation of nanoliposomes of ursolic acid using heating and stirring at 90 °C tol05 °C. Materials 2021, 14, 4917 discloses nanoparticles of ursolic acid prepared using DMSO as solvent in combination with heating at 60 °C. International Patent Application Publication No. WO2023 / 132789A2 discloses encapsulation of a related triterpenoid, maslinic acid, using a heating process. These disclosures indicate that heating is generally employed for encapsulating high-melting-point triterpenoids such as ursolic acid. However, the use of organic solvents raises concerns regarding residual solvent removal and may limit applicability in food and cosmetic applications. In addition, heating-based processes are more energy-intensive and less sustainable compared to non-heating approaches.

[0007] It is therefore desirable to provide a method for preparing nanocapsules of ursolic acid and core-shell capsules that address at least one of the problems described hereinabove, or at least provide an alternative solution.SUMMARY

[0008] In accordance with a first aspect of the present disclosure, a method of preparing nanocapsules of ursolic acid is provided. The method comprises mixing ursolic acid with a polyethylene glycol)-containing polymer at ambient temperature to form a mixture; adding an aqueous carrier to the mixture; and homogenizing the mixture with the aqueous carrier under high-shear conditions to obtain an emulsion comprising a hydrophobic core consisting of the ursolic acid, encapsulated by a shell consisting of the poly(ethylene glycol)-containing polymer, wherein the poly(ethylene glycol)-containing polymer is Poloxamer 407, and wherein the method is performed without heating and without organic solvent.

[0009] In some embodiments, the method further comprises freeze-drying the emulsion to form nanocapsules of ursolic acid in powder form.

[0010] In some embodiments, the method further comprises adding a polyacid into the emulsion to crosslink the shell consisting of the poly(ethylene glycol)-containing polymer to obtain crosslinked nanocapsules of ursolic acid.

[0011] In accordance with a second aspect of the present disclosure, a core-shell capsule is provided. The core-shell capsule comprises a hydrophobic core consisting of ursolic acid, encapsulated by a hydrophilic shell consisting of a poly(ethylene glycol)-containing polymer crosslinked with a polyacid, wherein the poly(ethylene glycol)-containing polymer is Poloxamer 407, and wherein the weight ratio of the ursolic acid to the Poloxamer 407 ranges from 1:3.3 to 1:10.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Various embodiments of the present disclosure are described hereinbelow in the detailed description with reference to the following drawings:FIG. 1 is a graph showing a comparison of loading percentage of various feed ratio measured at room temperature and at 90 °C.FIG. 2A illustrates the particle size distribution of the redispersed powder of Control Sample 1, as measured by dynamic light scattering (DLS). As shown, a predominant peak is observed at approximately 749 nm, indicating the presence of particles with an average hydrodynamic diameter of about 749 nm.FIG. 2B illustrates the particle size distribution of the redispersed powder of Control Sample 2, as measured by DLS. As shown, a predominant peak is observed at approximately 488 nm, indicating the presence of particles with an average hydrodynamic diameter of about 488 nm.FIG. 2C illustrates the particle size distribution of the redispersed powder of Control Sample 3, as measured by DLS. As shown, a predominant peak is observed at approximately 759 nm, indicating the presence of particles with an average hydrodynamic diameter of about 759 nm.FIG. 2D illustrates the particle size distribution of the redispersed powder of Control Sample 4, as measured by DLS. As shown, a predominant peak is observed at approximately 659 nm, indicating the presence of particles with an average hydrodynamic diameter of about 659 nm.FIG. 3A illustrates the particle size distribution of the redispersed powder of Control Sample 9, as measured by DLS. As shown, a predominant peak is observed at approximately 372 nm, indicating the presence of particles with an average hydrodynamic diameter of about 372 nm.FIG. 3B illustrates the particle size distribution of the redispersed powder of Control Sample 10, as measured by DLS. As shown, a predominant peak is observed at approximately 197 nm, indicating the presence of particles with an average hydrodynamic diameter of about 197 nm.FIG. 3C illustrates the particle size distribution of the redispersed powder of Control Sample 11, as measured by DLS. As shown, a predominant peak is observed at approximately 389 nm, indicating the presence of particles with an average hydrodynamic diameter of about 389 nm.FIG. 4A illustrates the particle size distribution of the redispersed powder of Sample 1, as measured by DLS. As shown, a predominant peak is observed at approximately 432 nm, indicating the presence of particles with an average hydrodynamic diameter of about 432 nm.FIG. 4B illustrates the particle size distribution of the redispersed powder of Control Sample 5, as measured by DLS. As shown, a predominant peak is observed at approximately 302 nm, indicating the presence of particles with an average hydrodynamic diameter of about 302 nm.FIG. 4C illustrates the particle size distribution of the redispersed powder of Sample 2, as measured by DLS. As shown, a predominant peak is observed at approximately 431 nm, indicating the presence of particles with an average hydrodynamic diameter of about 431 nm.FIG. 4D illustrates the particle size distribution of the redispersed powder of Control Sample 6, as measured by DLS. As shown, a predominant peak is observed at approximately 281 nm, indicating the presence of particles with an average hydrodynamic diameter of about 281 nm.FIG. 4E illustrates the particle size distribution of the redispersed powder of Sample 3, as measured by DLS. As shown, a predominant peak is observed at approximately 360 nm, indicating the presence of particles with an average hydrodynamic diameter of about 360 nm.FIG. 4F illustrates the particle size distribution of the redispersed powder of Control Sample 7, as measured by DLS. As shown, a predominant peak is observed at approximately 329 nm, indicating the presence of particles with an average hydrodynamic diameter of about 329 nm.FIG. 4G illustrates the particle size distribution of the redispersed powder of Sample 4, as measured by DLS. As shown, a predominant peak is observed at approximately 435 nm, indicating the presence of particles with an average hydrodynamic diameter of about 435 nm.FIG. 4H illustrates the particle size distribution of the redispersed powder of Control Sample 8, as measured by DLS. As shown, a predominant peak is observed at approximately 447 nm, indicating the presence of particles with an average hydrodynamic diameter of about 447 nm.FIG. 5A shows an SEM image of particles of the redispersed powder of Control Sample 1. FIG. 5B shows an SEM image of particles of the redispersed powder of Control Sample 3. FIG. 5C shows an SEM image of particles of the redispersed powder of Control Sample 2. FIG. 5D shows an SEM image of particles of the redispersed powder of Control Sample 4 FIG. 5E shows an SEM image of particles of the redispersed powder of Sample 4.FIG. 5F shows an SEM image of particles of the redispersed powder of Sample 5. The scale bar represents 100 nm and is consistent across all the SEM images in FIGs. 5A to 5F.DESCRIPTION

[0013] The following description sets forth exemplary methods, parameters, and the like. The embodiments are described in sufficient detail to enable those skilled in the art to practise the invention. Other embodiments may be utilized, and structural and logical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0014] Features that are described in the context of an embodiment may correspondingly be applicable to the same or similar features in the other embodiments. Features that are described in the context of an embodiment may correspondingly be applicable to the other embodiments, even if not explicitly described in these other embodiments. Furthermore, additions and / or combinations and / or alternatives as described for a feature in the context of an embodiment may correspondingly be applicable to the same or similar feature in the other embodiments.

[0015] In the context of various embodiments, the articles “a”, “an” and “the” as used with regard to a feature or element include a reference to one or more of the features or elements.

[0016] In the context of various embodiments, the term “about” or “approximately” as applied to a numeric value encompasses the exact value and a reasonable variance, e.g. within 10% of the specified value.

[0017] As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0018] By “comprising” it is meant including, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0019] By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0020] The present disclosure relates to a method of preparing nanoparticles of ursolic acid, and a core-shell capsule comprising the ursolic acid. In particular, the present disclosure relates to a method of preparing nanoparticles of ursolic acid without heating and without the use of organic solvents, and a core-shell capsule comprising the ursolic acid with crosslinked structure to provide pH sensitivity.

[0021] In various embodiments, the method comprises mixing ursolic acid and a polyethylene glycol)-containing polymer at ambient temperature to form a mixture. This isfollowed by adding an aqueous carrier to the mixture and homogenizing the mixture with the aqueous carrier under high-shear conditions to obtain an emulsion comprising a hydrophobic core consisting of the ursolic acid, encapsulated by a shell consisting of the polyethylene glycol)-containing polymer, wherein the polyethylene glycol)-containing polymer is Poloxamer 407, and wherein the method is performed without heating and without organic solvent.

[0022] As used herein, the term “poly(ethylene glycol)-containing polymer” refers to any polymer that comprises poly(ethylene glycol) (PEG) or poly(ethylene oxide) (PEO) segments in its structure. Such polymers may include, but are not limited to, PEG homopolymers of various molecular weights; block copolymers comprising PEG segments, such as poloxamers (eg., PEO-PPO-PEO triblock copolymers), PE G-poly (lactic acid) copolymers, PEG-polycaprolactone copolymers, and PEG-poly(lactic-co-glycolic acid) copolymers, graft or random copolymers having PEG side chains; PEG derivatives such as PEG diacrylate, PEG dimethacrylate, and PEG urethane crosslinked polymers; and PEGylated lipids or other PEGylated polymers.

[0023] In some embodiments, the poly(ethylene glycol)-containing polymer is a triblock copolymer. In certain embodiments, the poly(ethylene glycol)-containing polymer is Poloxamer 407 (also known under the trade name Pluronic F-127), a triblock copolymer of polyethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO). The Poloxamer 407 acts as a surfactant and its hydrophobic PPO block interacts with the hydrophobic ursolic acid, while its hydrophilic PEO blocks extend into water, forming a stable oil-in-water emulsion comprising polyethylene (PEG) shell, where ursolic acid droplets are encapsulated within the PEG shell to form the nanocapsules of ursolic acid.

[0024] Any suitable method of mixing the ursolic acid with the polyethylene glycol)-containing polymer may be employed, provided that the resulting mixture allows for the formation of nanocapsules upon homogenization with an aqueous carrier. Suitable methods ofmixing include, but not limited to, simple dry blending, mechanical stirring or pre-dispersion of the components prior to homogenization. In some embodiments, the mixing is carried out by blending a solid ursolic acid with a solid poly(ethylene glycol)-containing polymer. In some embodiments, the ursolic acid and the poly(ethylene glycol)-containing polymer are mixed in a weight ratio ranging from 1 : 3.3 to 1:10.

[0025] In various embodiments, the aqueous carrier is added to the mixture containing the ursolic acid and the poly(ethylene glycol)-containing polymer prior to homogenization. Suitable aqueous carriers may include, but not limited to, water-based carriers such as water, including deionized water, distilled water and purified water. In some preferred embodiments, the aqueous carrier is a water-based carrier.

[0026] In some embodiments, the aqueous carrier is added in an amount ranging from 0% to 90% by weight, based on the combined total weight of the mixture and the aqueous carrier that is subjected to homogenization.

[0027] In various embodiments, the step of homogenizing is performed using a pressurized homogenizer or a high-shear mixer. Suitable high-shear mixers include, but not limited to, rotor-stator high-shear mixer, inline high-shear mixers and ultra-high shear inline mixers, or equivalent devices capable of generating high-shear conditions sufficient to reduce particle size into the nanometer range An exemplary high-shear mixer is an Ultra-Turrax T18 / T25 rotorstator homogenizer or an equivalent rotor-stator mixer. Suitable pressurized homogenizers include high-pressure homogenizers operating at pressures ranging from 50 bar (5MPa) to 1500 bar (150 MPa).

[0028] In various embodiments, the step of homogenizing is performed at a speed of 3,000 to 25,000 rpm.

[0029] In various embodiments, the step of homogenizing is performed under high-shear conditions corresponding to an energy density of less than 4.5 xlO10J / m1, preferably less than 3.0 xl010J / m3, ormore preferably 1.5 xlO10J / m3.

[0030] In various embodiments, the step of homogenizing is performed under high-shear conditions for a duration ranging from 8 to 12 minutes, preferably 10 minutes.

[0031] The method of the present disclosure may optionally include freeze-drying the emulsion to form nanocapsules of ursolic acid in powder form. Any conventional method of freeze-drying may be employed without departing from the scope of the present disclosure. For example, the emulsion obtained according to the present method may be subjected to freezing at a temperature ranging from -20 °C to -30 °C, and drying under a vacuum. The choice of freeze-drying parameters, such as freezing rate, shelf temperature, chamber pressure, or cycle duration, may be varied as needed, provided that the resulting dried nanocapsules retain the nanocapsule structure.

[0032] In various embodiments, the method further comprises adding a polyacid into the emulsion to crosslink the shell consisting of the poly(ethylene glycolj-containing polymer to obtain crosslinked nanocapsules of ursolic acid.

[0033] The polyacid useful for crosslinking PEG-containing shells or nanocapsules may include, but is not limited to, polyacrylic acid, poly(methacrylic acid), poly(maleic acid) or poly(itaconic acid) In some embodiments, the polyacid is polyacrylic acid

[0034] The carboxyl groups of the polyacid form hydrogen bonds or covalent linkages with hydroxyl groups of the PEG segments in the poly(ethylene glycolj-containing polymer shell, thereby stabilizing the nanocapsules and providing a crosslinked outer shell. Such crosslinking enhances structural integrity and confers pH-responsive release properties.

[0035] In various embodiments, the poly(ethylene glycolj-containing polymer contains an ethylene glycol unit, and the molar ratio of acid group of the polyacid to the ethylene glycol unit of the poly(ethylene glycolj-containing polymer ranges from 2: 1 to 0.01 : 1.

[0036] In some embodiments, the emulsion prepared by the method of the present disclosure comprises nanocapsules of ursolic acid having a particle size ranging from 1 nm to 1,000 nm.

[0037] In some embodiments, the nanocapsules of ursolic acid are present in the powder form, having a particle size ranging from 1 nm to 1,000 nm.

[0038] The method of the present disclosure is a low-energy process that employs solid Poloxamer 407 (Pluronic F-127) as a surfactant and water as an aqueous carrier to form nanocapsules of ursolic acid. In contrast to conventional approaches, the method of the present disclosure does not require the use of any pre-heating step nor organic solvents. As such, the method avoids the energy consumption associated with thermal treatment and eliminates concerns regarding residual organic solvents that may limit applicability in food, cosmetic, or pharmaceutical formulations. By relying solely on a non-ionic PEG-containing surfactant and water, the method provides a straightforward and reproducible route to stable nanocapsules of ursolic acid Accordingly, the method follows the principles of green chemistry and offers a more sustainable and environmentally friendly approach for encapsulation of poorly soluble active compounds such as ursolic acid.

[0039] In a second aspect of the present disclosure, a core-shell capsule is provided. The core-shell capsule comprises a hydrophobic core consisting of ursolic acid, encapsulated by a hydrophilic shell consisting of a poly(ethylene glycol) -containing polymer crosslinked with a polyacid, wherein the poly(ethylene glycol)-containing polymer is Poloxamer 407, and wherein the weight ratio of the ursolic acid to the Poloxamer 407 ranges from 1 :3.3 to 1 : 10.

[0040] In various embodiments, the polyacid is polyacrylic acid.

[0041] In various embodiments, the poly(ethylene glycol)-containing polymer contains an ethylene glycol unit, and the molar ratio of acid group of the polyacid to the ethylene glycol unit of the poly(ethylene glycol)-containing polymer ranges from 2: 1 to 0.01 : 1.

[0042] In some embodiments, the core-shell capsule is in the form of an aqueous emulsion and has a particle size ranging from 1 nm to 1,000 nm.

[0043] In some embodiments, the core-shell capsule is in powder form and has a particle size ranging from 1 nm to 1,000 nm.

[0044] The core-shell capsules of the present disclosure exhibit a relatively high loading of about 15%, which is much higher than that achieved by certain commercial products, and a high encapsulation efficiency (EE%) in the range of 69 to 83%. These results demonstrate that a no-heating, low energy process is suitable for preparing nano-encapsulated ursolic acid using Pol oxamer 407 (Pluronic F-127) as a surfactant, while maintaining high encapsulation efficiency.

[0045] To facilitate a better understanding of the present disclosure, the following examples of specific embodiments are given. In no way should the following examples be read to limit or define the entire scope of the disclosure. One skilled in the art will recognize that the examples set out below are not an exhaustive list of the embodiments of this disclosure.EXAMPLESExample 1

[0046] Examples 1 to 4: Control Samples with Tween 65

[0047] Preparation of Control Sample 1

[0048] Ursolic acid and Tween 65 (also known as Polysorbate 65) were blended at 90 °C in a weight ratio of 1 :5. The resulting blend was allowed to solidify upon cooling, and the solid mass was broken into small pieces. 20 mL of deionized water was added to the mixture, and the mixture was homogenized using a high-shear mixer (Ultra Turrax T18 / T25 basic homogenizer) until a fine and homogeneous emulsion was obtained. The final emulsion was optionally freeze-dried to afford a waxy solid.Example 2

[0049] Preparation of Control Sample 2

[0050] Control Sample 2 was prepared in the same manner as described in Example 1, except that the weight ratio of ursolic acid to Tween 65 was adjusted to 1:3.3.Example 3

[0051] Preparation of Control Sample 3

[0052] Ursolic acid and Tween 65 were blended at 90 °C in a weight ratio of 1:5. The resulting blend was allowed to solidify upon cooling, and the solid mass was broken into small pieces. 20 mL of deionized water was added to the mixture, and the mixture was homogenized using the high-shear mixer until a fine and homogeneous emulsion of ursolic acid in Tween 65 / water was obtained. The emulsion formed comprises polyethylene (PEG) shell capsules which the PEG shell was formed from the PEG-like chains from the Tween 65.

[0053] Polyacrylic acid (PAA) (mw=5,000, 50% solution, PolyScience) was added to the emulsion, and further homogenized for about 3 minutes to crosslink the PEG shell of the capsules. In this example, 0.15 g of PAA was added to the emulsion containing 2 g of Tween 65, which converted a degree of about 10% crosslinking, calculated using molar ratio. The final emulsion was optionally freeze-dried to afford a waxy solid.Example 4

[0054] Preparation of Control Sample 4

[0055] Control Sample 4 was prepared in the same manner as described in Example 3, except that the weight ratio of ursolic acid to Tween 65 was adjusted to 1:3.3.Example 5

[0056] Examples 5 to 8: Control Samples with Poloxamer 407 (Pluronic F-127). prepared with heating

[0057] Preparation of Control Sample 5

[0058] Ursolic acid and Pluronic F-127 were blended at 90 °C in a weight ratio of 1 : 10. The resulting blend was allowed to solidify upon cooling, and the solid mass was broken into small pieces. 20 mL of deionized water was added to the mixture, and the mixture was homogenized using the high-shear mixer until a fine and homogeneous emulsion was obtained. The final emulsion was optionally freeze-dried to afford a powdered solid.Example 6

[0059] Preparation of Control Sample 6

[0060] Control Sample 6 was prepared in the same manner as described in Example 5, except that the weight ratio of ursolic acid to Pluronic F-127 was adjusted to 1:8.Example 7

[0061] Preparation of Control Sample 7

[0062] Control Sample 7 was prepared in the same manner as described in Example 5, except that the weight ratio of ursolic acid to Pluronic F-l 27 was adjusted to 1 :5.Example 8

[0063] Preparation of Control Sample 8

[0064] Control Sample 8 was prepared in the same manner as described in Example 5, except that the weight ratio of ursolic acid to Pluronic F-127 was adjusted to 1 :3.3 Example 9

[0065] Examples 9 to 11 : Control Samples with Pluronic F-127, prepared without heating

[0066] Preparation of Control Sample 9

[0067] Ursolic acid and Pluronic F-127 were blended at room temperature in a weight ratio of 1 :8. 20 mL of deionized water was added to the mixture, and the mixture was homogenized using the high-shear mixer for a short duration (about 10 min) until a fine and homogeneous emulsion was obtained. The final emulsion was optionally freeze-dried to afford a powdered solid.Example 10

[0068] Preparation of Control Sample 10

[0069] Control Sample 10 was prepared in the same manner as described in Example 9, except that the mixture was homogenized in the high-shear mixer for a duration of about 20 minutes.Example 11

[0070] Preparation of Control Sample 11

[0071] Control Sample 11 was prepared in the same manner as described in Example 9, except that the mixture was homogenized in the high-shear mixer for a duration of about 30 minutes.Example 12

[0072] Examples 12 to 16: Samples with Pluronic F-127, prepared using a low-energy process without heating

[0073] Preparation of Sample 1

[0074] Ursolic acid and Pluronic F-127 were blended at room temperature in a weight ratio of 1 : 10. 20 mL of deionized water was added to the mixture, and the mixture was homogenized using the high-shear mixer for a short duration (about 10 min) until a fine and homogeneous emulsion was obtained. The final emulsion was optionally freeze-dried to afford a powdered solid.Example 13

[0075] Preparation of Sample 2

[0076] Sample 2 was prepared in the same manner as described in Example 12, except that the weight ratio of ursolic acid to Pluronic F-127 was adjusted to 1 :8. Also see Control Sample 9 described in Example 9.Example 14

[0077] Preparation of Sample 3

[0078] Sample 3 was prepared in the same manner as described in Example 12, except that the weight ratio of ursolic acid to Pluronic F-127 was adjusted to 1 :5.Example 15

[0079] Preparation of Sample 4

[0080] Sample 4 was prepared in the same manner as described in Example 12, except that the weight ratio of ursolic acid to Pluronic F-127 was adjusted to 1 :3.3.Example 16

[0081] Preparation of Sample 5

[0082] Ursolic acid and Pluronic F-127 were blended at room temperature in a weight ratio of 1 :3.3 20 mL of deionized water was added to the mixture, and the mixture was homogenized using the high-shear mixer for a short duration (about 10 min) until a fine and homogeneous emulsion was obtained. The final emulsion was optionally freeze-dried to afford a powdered solid. The emulsion formed comprises polyethylene (PEG) shell capsules which the PEG shell was formed from the PEG-like chains from the Pluronic F-127.

[0083] Polyacrylic acid (PAA) (mw=5000, 50% solution, PolyScience) was added to the emulsion, and further homogenized for about 3 minutes to crosslink the PEG shell of the capsules. In this example, 0.15 g of PAA was added to the emulsion containing 2 g of Pluronic F-127, which converted a degree of about 8% crosslinking, calculated using molar ratio. The final emulsion was optionally freeze-dried to afford a powdered solid.Example 17

[0084] HPLC loading measurement

[0085] One of the criteria for evaluating the encapsulation process is the encapsulation efficiency (EE%), which is defined as the percentage of drug that is successfully entrapped / loaded into the capsules or nanoparticles. The amount of the loaded ursolic acid in the nanocapsules of ursolic acid is measured using a high-performance liquid chromatography (HPLC).

[0086] Table 1 summarizes the feed ratios, measured loading and encapsulation efficiency (EE%) for Control Samples 1 to 4.

[0087] The capsules comprising Tween 65 as the surfactant used a preheating method which yielded a very high loading efficiency for both the non-crosslinked and crosslinked samples. This indicates the suitability of using Tween 65 as the surfactant in conjunction with preheating for encapsulation of the ursolic acid. This result is consistent with that disclosed in an earlier patent publication, WO2023132789A2, on core-shell capsule comprising maslinic acid Themethod involved a preheating process which helps to circumvent the challenge associated with the waxy nature of Tween 65, which is however inferior to non-heating process in terms of energy saving.

[0088] Table 1: Summary of the feed ratio, measured loading and encapsulation efficiency (EE%) for the Control Samples 1 to 4.

[0089] The feed ratio, measured loading and encapsulation efficiency (EE%) for the Control Samples 5 to 11 and the Samples 1 to 5 are summarized in Table 2.

[0090] Control Samples 5 to 8 are prepared under pre-heating conditions, which serve as the benchmark for the low-energy method of the present disclosure without heating. As shown in Table 2, the encapsulation efficiency for each of the Control Samples 5 to 8 is relatively high and is about 80%, 81%, 77% and 89%, respectively. Control Samples 9 to 11 were explored to investigate the effect from homogenization duration on the encapsulation. As can be seen, the EE% values for Control Samples 9 to 11 are 76%, 74% and 61%, respectively, indicating that longer homogenization duration is unnecessary to achieve good encapsulation efficiency. As such, the process with shorter duration is preferred, highlighting the low-energy process disclosed in the present disclosure.

[0091] Samples 1 to 4 were prepared under a no-heating, low-energy process. In contrast, Control Samples 5 to 8 were prepared with heating, while the remaining process parameters were maintained unchanged. The encapsulation efficiencies of Samples 1 to 4 were relatively high, and were determined to be 69%, 76%, 74%, 83%, respectively, which demonstrate thatthe no-heating, low energy process is suitable for preparing nano-encapsulated ursolic acid using Pluronic F-127 as the surfactant with relatively high encapsulation efficiency. The loading percentages (%) measured by HPLC for Control Samples 5 to 8 and Samples 1 to 4 were plotted in FIG. 1, where both curves well matching each other, which confirms the disclosed low-energy method is a working process. Sample 5 is the sample prepared without heating using Pluronic F-127 as the surfactant with crosslinking structure, which reported a loading of about 15%. This is much higher than some commercial products such as Active Ursolic Acid Liposome with a loading of only about 0.2%.

[0092] Table 2: Summary of the feed ratio, measured loading and encapsulation efficiency (EE%) for Control Samples 5 to 11, and Samples 1 to 5.Example 18

[0093] Dynamic Light Scattering Characterisation

[0094] In order to further evaluate the properties of the formed nanocapsules of ursolic acid, a particle size analysis for the redispersed particle powder was conducted. The obtained powderfrom freeze drying is re-dispersed in water and the hydrodynamic size of the emulsion particle sizes are measured by dynamic light scattering (DLS).

[0095] FIGs 2A to 2D show the DLS size distributions of the redispersed particles of (A) Control Sample 1, (B) Control Sample 2, (C) Control Sample 3, and (D) Control Sample 4. At feed ratios of 1:5 and 1:3.3, the size distributions of the redispersed particles in the absence of crosslinking were centred at 749 nm (Control Sample 1) and 488 nm (Control Sample 2), respectively. With crosslinking, the size distributions of the redispersed particles for feed ratios of 1:5 and 1:3.3 were centred at 759 nm (Control Sample 3) and 659 nm (Control Sample 4), respectively. The particle sizes increased slightly after crosslinking, which is expected since the particles were first formed and subsequently subjected to crosslinking during homogenization.

[0096] FIGs 3A to 3C show the DLS size distributions of the redispersed particles of (A) Control Sample 9, (B) Control Sample 10, and (C) Control Sample 11. For samples processed for durations of 10 min, 20 min and 30 min, respectively, the peaks were centred at 372 nm (Control Sample 9), 197 nm (Control Sample 10) and 389 nm (Control Sample 11), respectively. The peaks of the particle size distributions do not follow a clear ascending or descending trend, where the peaks are within a broad range of 200 to 400 nm. The results demonstrate that a longer homogenization duration is unnecessary for producing smaller particle size distributions. The calculated energy densities for Control Samples 8, 9 and 10 are 1.50 x 1010J / m3, 3.00 x 1010J / m3and 4.50 x 1010J / m3, respectively.

[0097] FIGs. 4A to 4H show the DLS size distributions of the redispersed particles of (A) Sample 1, (B) Control Sample 5, (C) Sample 2, and (D) Control Sample 6, (E) Sample 3, (F) Control Sample 7, (G) Sample 4, and (H) Control Sample 8.

[0098] The size distribution of the redispersed particles of Sample 1 was centred at 432 nm, while that of the Control Sample 5 was centred at 302 nm. The size distribution of the redispersed particles of Sample 2 was centred at 431 nm, and that of Control Sample 6 was centred at 281 nm. The size distribution of the redispersed particles of Sample 3 was centred at360 nm, whereas that of Control Sample 7 was centred at 329 nm. The size distribution of the redispersed particles of Sample 4 was centred at 435 nm, and that of Control Sample 8 was centred at 447 nm. The results demonstrate that at lower feed ratio (i.e., 1:10 and 1:8), the peaks of the particle size distributions were slightly smaller when a heating process was employed. The peaks of the particle size distributions for samples prepared with heating and without heating at higher feed ratios (i.e., 1 :5 and 1 :3.3) were much closer to each other, being centred at 350 nm and 440 nm, respectively.

[0099] Despite the observation that the no-heating samples exhibited relatively larger particle sizes compared to the heating process at lower feed ratios, the particle sizes of the samples across all feed ratios were consistently small, being in the range of 350 nm to 450 nm, which confirms that the low-energy method of the present disclosure is suitable for producing nanosized capsules of ursolic acid.Example 19

[0100] SEM Characterisation

[0101] The morphological properties of the redi spersed particles were examined using SEM. The nanocapsules prepared from Tween 65 as the surfactant are as shown in FIGs. 5A to 5F.With feed ratios of 1 :5 (FIG. 5A, FIG. 5B) and 1 :3.3 (FIG. 5C, FIG. 5D), the particles appeared irregular in shape, with sizes ranging from 100 nm to 400 nm. The morphology did not show any significant differences as a result of crosslinking. The smaller particle sizes apparent under SEM are believed to be due to shrinkage occurring upon drying of the coating on the substrate. The results confirm that the method with preheating for Tween 65 based composition could generate very consistent nanocapsules of ursolic acid.

[0102] In contrast, the nanocapsules prepared from the Pluronic F-127 as the surfactant are as shown in FIG. 5E (non-crosslinked) and 5F (crosslinked). Sizes ranging from 100 to 200 nm were observed for both the non-crosslinked sample (Sample 4) and the crosslinked sample(Sample 5). The SEM images in FIG. 5E and FIG. 5F confirm that the nanosized capsules were constructed successfully with the low -energy process of the present disclosure.

[0103] Although embodiments of the invention have been shown and described, the invention is not limited to the described embodiments. Instead, it would be appreciated by those skilled in the art that various modifications and variations can be made to the embodiments of the invention without departing from the scope of the invention, the scoop of which is set forth in the following claims.

Claims

Claims1. A method of preparing nanocapsules of ursolic acid, the method comprising:mixing ursolic acid with a poly(ethylene glycol)-containing polymer at ambient temperature to form a mixture;adding an aqueous carrier to the mixture; andhomogenizing the mixture with the aqueous carrier under high-shear conditions to obtain an emulsion comprising a hydrophobic core consisting of the ursolic acid, encapsulated by a shell consisting of the poly(ethylene glycol)-containing polymer,wherein the poly(ethylene glycol)-containing polymer is Poloxamer 407, and wherein the method is performed without heating and without organic solvent.

2. The method of claim 1, further comprising:freeze-drying the emulsion to form nanocapsules of ursolic acid in powder form.

3. The method of claim 1, wherein the high-shear conditions correspond to an energy density of less than 4.5 xlO10J / m3.

4. The method of claim 1, wherein the step of homogenizing is performed for a duration ranging from 8 to 12 minutes.

5. The method of claim 1, wherein the weight ratio of the ursolic acid to the poly(ethylene glycol)-containing polymer ranges from 1:3.3 to 1:10.

6. The method of claim 1, further comprising:adding a polyacid into the emulsion to crosslink the shell consisting of the polyethylene glycol)-containing polymer to obtain crosslinked nanocapsules of ursolic acid.

7. The method of claim 6, wherein the polyacid is polyacrylic acid.

8. The method of claim 6, wherein the poly(ethylene glycol)-containing polymer contains an ethylene glycol unit, and the molar ratio of acid group of the polyacid to the ethylene glycol unit of the poly(ethylene glycol)-containing polymer ranges from 2: 1 to 0.01 : 1.

9. The method of claim 1, wherein the emulsion comprises nanocapsules of ursolic acid having a particle size ranging from 1 nm to 1,000 nm.

10. The method of claim 2, wherein the nanocapsules of ursolic acid in the powder fonn have a particle size ranging from 1 nm to 1,000 nm.

11. The method of claim 1 , wherein the aqueous carrier is a water-based carrier.

12. A core-shell capsule comprising a hydrophobic core consisting of ursolic acid, encapsulated by a hydrophilic shell consisting of a poly(ethylene glycol)-containing polymer crosslinked with a polyacid, wherein the poly(ethylene glycol)-containing polymer is Poloxamer 407, and wherein the weight ratio of the ursolic acid to the Poloxamer 407 ranges from 1:3.3 to 1:10.

13. The core-shell capsule of claim 12, wherein the polyacid is polyacrylic acid.

14. The core-shell capsule of claim 12, wherein the poly(ethylene glycol)-containing polymer contains an ethylene glycol unit, and the molar ratio of acid group of the polyacid to the ethylene glycol unit of the poly(ethylene glycol)-containing polymer ranges from 2:1 to 0.01:1.

15. The core-shell capsule of claim 12, wherein the core-shell capsule is in the form of an aqueous emulsion and has a particle size ranging from 1 nm to 1,000 nm.

16. The core-shell capsule of claim 12, wherein the core-shell capsule is in powder form and has a particle size ranging from 1 nm to 1,000 nm.

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