Enhanced composition for inhibiting viability of triple-negative breast cancer (TNBC) cells and preparation thereof

Abstract

According to an aspect of the present disclosure, a composition for use in inhibiting viability of triple negative breast cancer (TNBC) cells contains 1,8-cineole as an active ingredient and β-cyclodextrin as a carrier, where the β-cyclodextrin encapsulates the 1,8-cineole forming a nanoformulation. According to another aspect of the present disclosure, a method of preparing a composition for use in inhibiting viability of TNBC cells includes forming a first solution by dissolving β-cyclodextrin in a solvent, forming a second solution by adding Elettaria cardamomum essential oil to the first solution, where the Elettaria cardamomum essential oil contains 1,8-cineole as an active ingredient. The method also includes sonicating the second solution to form a homogenized second solution, centrifuging the homogenized second solution to form a pellet, deep freezing the pellet, and lyophilizing the deep frozen pellet to form a nanoformulation, where the nanoformulation contains the 1,8-cineole encapsulated by the β-cyclodextrin.

Description

ENHANCED COMPOSITION FOR INHIBITING VIABILITY OF TRIPLE-NEGATIVE BREAST CANCER (TNBC) CELLS AND PREPARATION THEREOFPriority Claim

[0001] The instant patent application is related to and claims priority from the below two Germany utility model applications, which are incorporated in their entirety herewith to the extent not inconsistent with the description herein:

[0002] A. Entitled, “RECOVEREEZ FORTE BASED COMPOSITION FOR TARGETING TRIPLE-NEGATIVE BREAST CANCER CELLS”, Serial No.: 20 2024 101 390.5, Filed: 20 March 2024; and

[0003] B. Entitled, “A COMPOSITION FOR TREATING TRIPLE-NEGATIVE BREAST CANCER (TNBC) AND APPARATUS FOR SYNTHESIZING THEREOF”, Serial No.: 202024 102 879.1, Filed: 01 June 2024.Background

[0004] Technical Field

[0005] Embodiments of the present disclosure relate generally to inhibiting viability of breast cancer cells, and more specifically to enhanced composition for inhibiting viability of triple-negative breast cancer (TNBC) cells and preparation thereof.

[0006] Related Art

[0007] Breast cancer ranks as the second leading cause of cancer-related deaths among women worldwide. Triple Negative Breast Cancer (TNBC) constitutes 15-20% of cases and lacks targeted therapy. Tumor growth and treatment resistance are closely linked to a breakdown in apoptosis, a natural cell death process. Overcoming these obstacles is crucial for developing improved treatments and outcomes for TNBC patients.

[0008] Utilizing delivery systems to administer multiple medications concurrently shows promise for achieving synergistic anticancer effects. However, anticancer drugs face challenges like uncontrolled release, cardiac toxicity, short plasma half-life, harm to normal cells, limited permeation, and the development of drug resistance in cancer cells. Carbohydrate nanocarriers exploit the enhanced permeability and retention (EPR) effect to selectively target cancer cells, enhancing medication uptake.

[0009] Cyclodextrins (CDs), particularly those derived from carbohydrates like P-cyclodextrin (P- CD), have emerged as efficient drug delivery systems. P-CD is widely used in the pharmaceutical industry due to its non-toxic, biocompatible, and biodegradable nature. Its cone-shaped structure with multiple hydroxyl groups enables encapsulation of water insoluble drugs and stabilizes water-soluble medications, addressing issues like controlled release and drug stability.

[0010] Certain natural compounds extracted from herbs and spices exhibit immunomodulatory effects, showing potential in treating various diseases, including cancer. Despite their potential, the biological mechanisms behind these properties are not well understood. 1,8-cineole, a monoterpene found in eucalyptus, possesses diverse properties like anti-inflammatory, antimicrobial, and analgesic activities.[Oil] Aspects of the present disclosure relate to an enhanced composition for Triple Negative Breast Cancer (TNBC) and preparation thereof.Brief Description of the views of Drawings

[0012] Example embodiments of the present disclosure will be described with reference to the accompanying drawings briefly described below.

[0013] Figure 1 A depicts hydrodynamic size distribution of [3-CD.

[0014] Figure IB depicts hydrodynamic size distribution of [3-CD @ CO NF.

[0015] Figure 1C depicts the zeta potential of [3-CD.

[0016] Figure ID depicts the zeta potential of [3-CD @ CO NF.

[0017] Figure 2 depicts ultraviolet -visible spectroscopy (UV-Vis) spectra of [3-CD @ CO NF, CO, and [3-CD.

[0018] Figure 3 depicts photoluminescence spectroscopy analysis of [3-CD @ CO NF, CO, and [3- CD.

[0019] Figure 4 depicts Fourier transform infrared (FT-IR) spectra for functional group analysis of |3-CD@CO NF, CO, and [3-CD.

[0020] Figure 5A depicts morphology of [3-CD@CO NF observed by field emission-scanning electron microscopy (FE-SEM).

[0021] Figure 5B depicts the elemental composition of [3-CD@CO NF determined by energy- dispersive X-ray spectroscopy (ED AX).

[0022] Figure 6A depicts the size and shape of [3-CD@CO NF observed by high-resolution transmission electron microscopy (HR-TEM).

[0023] Figure 6B depicts the average size distribution of [3-CD @ CO NF.

[0024] Figure 7A depicts 2D surface topography of [3-CD@CO NF analyzed using atomic force microscopy (AFM).

[0025] Figure 7B depicts 3D surface topography of [3-CD @ CO NF analyzed using AFM.

[0026] Figure 8 A depicts in vitro drug release profile of Recovereez Forte DR and [3-CD @ CO NF at pH 7.4.

[0027] Figure 8B depicts in vitro drug release profile of Recovereez Forte DR and [3-CD@CO NF at pH 5.4.

[0028] Figure 9A depicts Higuchi model release kinetics for Recovereez Forte DR at pH 7.4.

[0029] Figure 9B depicts Higuchi model release kinetics for Recovereez Forte DR at pH 5.4.

[0030] Figure 9C depicts Higuchi model release kinetics for [3-CD@CO NF at pH 7.4.

[0031] Figure 9D depicts Higuchi model release kinetics for [3-CD@CO NF at pH 5.4.

[0032] Figure 10 is a table summarizing in vitro drug release kinetics models.

[0033] Figure 11 is a graph depicting cell viability of MD A- MB -231 cancer cells treated with Recovereez Forte DR and [3-CD@CO NF.

[0034] Figure 12A depicts morphology of MDA-MB-231 cancer cells treated with Control and Recovereez Forte DR at concentrations of 62.5, 125, and 250 pg / ml.

[0035] Figure 12B depicts morphology of MDA-MB-231 cancer cells treated with Control and [3- CD@CO NF at concentrations of 62.5, 125, and 250 pg / ml.

[0036] Figure 13A depicts cell death analysis of MDA-MB-231 cancer cells by AO / EtBr dual staining treated with the Recovereez Forte DR and the [3-CD@CO NF at a concentration of 70 pg / ml.

[0037] Figure 13B depicts a bar diagram representing cell death percentages in different groups (i.e., control group, the Recovereez Forte DR (70 pg / ml) and the [3-CD@CO NF (70 pg / ml).

[0038] Figure 14A depicts ROS generation of MDA-MB-231 cancer cells by DCFH-DA staining treated with the Recovereez Forte DR and the [3-CD@CO NF.

[0039] Figure 14B depicts fluorescent intensity of DCFH-DA dye observed in different groups under excitation and emission at 480 nm and 530 nm.

[0040] Figure 15A depicts a fluorescence microscopy image depicting changes in the Mitochondrial Membrane Potential (MMP) of MDA-MB-231 cancer cells using rhodamine- 123 fluorescence staining at a concentration of 70 pg / ml.

[0041] Figure 15B depicts fluorescence intensity of rhodamine-123 measured using a Spectramax multimode reader under excitation and emission at 485 nm and 530 nm.

[0042] Figure 16 depicts the pharmacokinetic profile of Recovereez Forte DR and [3-CD@CO NF in normal Swiss albino mice.

[0043] In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number. In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.Detailed Description

[0044] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment(s) illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

[0045] It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

[0046] Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0047] The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more compositions or elements or structures or components preceded by “comprises... a” does not, without more constraints, preclude the existence of other compositions or elements or other structures or other components or additional compositions or additional elements or additional structures or additional components.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The compositions, methods, and examples provided herein are illustrative only and not intended to be limiting.

[0049] Example embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.

[0050] 1. Terminology

[0051] [3-cyclodextrin (|3-CD): Cyclic carbohydrates made of seven glucopyranose units joined by a-l,4-glycosidic bonds in a ring.

[0052] Cardamom essential oil: An essential oil extracted from the seeds of Elettaria cardamomum, containing various bioactive compounds including 1,8-cineole. Elettaria cardamomum iscommonly known as green or true cardamom, is a herbaceous, perennial plant in the ginger family, native to southern India. Cardamom is the dried fruit of Elettaria cardamomum. Cardamom plants widely grown in Kerala, India, have been used in relation to the subject invention.

[0053] 1,8 -cineole: Also known as eucalyptol, is an oxygenated monoterpene with a chemical formula of CioHisO. 1,8 -cineole is also referred to as CO or cineole oil in the present disclosure.

[0054] Nanoformulation (NF): A formulation in which size of the particles is in nanometer range, as is well known in the relevant arts.

[0055] [3-CD@CO NF: A nanoformulation in which 1,8-cineole (CO) is encapsulated within [3- cyclodextrin (|3-CD).

[0056] Recovereez Forte (RF) DR: Recovereez Forte (RF) DR is a composition containing Elettaria cardamomum essential oil and Salvia rosmarinus essential oil in equal proportions by weight, Piperine Oleoresin at a concentration of 1-3 percent of the combined weight of the Elettaria cardamomum essential oil and the Salvia rosmarinus essential oil, and P-cyclodextrin in a molar amount equal to the sum of the moles of the Elettaria cardamomum essential oil, the Salvia rosmarinus essential oil, and the Piperine Oleoresin. The Elettaria cardamomum essential oil in the composition contains 1, 8-cineole in a range of 45-53% and the Salvia rosmarinus essential oil in the composition contains 1, 8-cineole in a range of 45-53%.

[0057] 2. Overview

[0058] Aspects of the present disclosure are directed to enhanced composition for inhibiting viability of triple-negative breast cancer (TNBC) cells and preparation thereof.

[0059] According to an aspect of the present disclosure, a composition for use in inhibiting viability of triple negative breast cancer (TNBC) cells contains 1,8-cineole as an active ingredient and P- cyclodextrin as a carrier, where the P-cyclodextrin encapsulates the 1,8-cineole forming a nanoformulation.

[0060] In an embodiment, the 1 , 8-cineole is contained in Elettaria cardamomum essential oil, where the Elettaria cardamomum essential oil contains the 1, 8-cineole at a concentration of 27-44 % (v / w).

[0061] In another embodiment, the composition contains 1 ml of the Elettaria cardamomum essential oil for every 500 mg of the P-cyclodextrin.

[0062] In yet another embodiment, size of particles in the nanoformulation is in a range of 65 - 110 nm.

[0063] In one more embodiment, hydrodynamic size of the particles is about 135 nm.

[0064] In yet another embodiment, zeta potential of the particles is about -2.26 mV.

[0065] According to another aspect of the present disclosure, a method of preparing a compositionfor use in inhibiting viability of triple negative breast cancer (TNBC) cells includes forming a first solution by dissolving P-cyclodextrin in a solvent, forming a second solution by adding Elettaria cardamomum essential oil to the first solution, where the Elettaria cardamomum essential oil contains 1,8 -cineole as an active ingredient. The method also includes sonicating the second solution to form a homogenized second solution, centrifuging the homogenized second solution to form a pellet, deep freezing the pellet, and lyophilizing the deep frozen pellet to form a nanoformulation, where the nanoformulation contains the 1,8-cineole encapsulated by the P- cyclodextrin.

[0066] In an embodiment, the forming of the first solution includes stirring a mixture of the P- cyclodextrin and the solvent for a duration of 1 hour at 800 rpm and 60 °C, the forming of the second solution includes stirring a mixture of the Elettaria cardamomum essential oil and the first solution for 3 hours at 900 rpm and at room temperature, the sonicating includes applying ultrasonic energy for 10 minutes, the centrifuging includes rotating the sonicated second solution, using a cooling centrifuge, for 10 min at 10,000 rpm and at 4 °C, the deep freezing includes keeping the pellet at -80 °C, and the lyophilizing includes maintaining the pellet at a pressure of 15 pascals and at a temperature below -80 °C.

[0067] In another embodiment, the solvent is distilled water and the dissolving entails dissolving 500 mg of the P-cyclodextrin for every 100 ml of the distilled water, and the adding entails adding 1 ml of the Elettaria cardamomum essential oil for every 500 mg of the P-cyclodextrin, where the Elettaria cardamomum essential oil contains the 1, 8-cineole at a concentration of 27-44 % (v / w).

[0068] In yet another embodiment, size of particles in the nanoformulation is in a range of 65 - 110 nm.

[0069] In yet another embodiment, hydrodynamic size of the particles is about 135 nm, and zeta potential of the particles is about -2.26 mV.

[0070] Several aspects of the present disclosure are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the disclosure can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well known structures, materials, or operations are not shown in detail to avoid obscuring the features of the disclosure. Furthermore, the features / aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.

[0071] 3. Composition

[0072] Present disclosure discloses a composition, containing 1,8-cineole as an active ingredient, for use in inhibiting viability of triple negative breast cancer (TNBC) cells. An active ingredientis a chemical compound that produces chemical or biological effect. Viability refers to the ability of cancer cells (including TNBC cells) to grow and proliferate successfully, and inhibiting viability of TNBC cells refers to hindering and / or preventing the viability of TNBC cells.

[0073] The composition contains P-cyclodextrin (P-CD) as a carrier to enhance drug delivery by enhancing stability, solubility, and bioavailability. The P-CD helps increase the solubility of active ingredient(s) (which may be hydrophobic and may thus exhibit low aqueous solubility) by forming micelles or inclusion complexes with 1,8 -cineole (CO), thus increasing the membrane permeability, facilitating absorption and bioavailability.

[0074] In an embodiment of the present disclosure, the P-CD encapsulates the 1,8-cineole forming a nanoformulation (i.e., P-CD@CO NF), in which size of the particles (i.e., particles in which 1,8- cineole is encapsulated by P-CD) is in nanometer range. The encapsulation of the 1,8-cineole in the hydrophobic cavity of P-CD protects the 1,8-cineole from degradation and oxidation, prolongs shelf life and ensures sustained release. In an example embodiment, the size of particles is in a range of 65 - 110 nm, with the average size of the particles being 88 nm. In another example embodiment, the hydrodynamic size of the particles is about 135 nm, and the zeta potential of the particles is about -2.26 mV.

[0075] In an embodiment of the present disclosure, the composition contains a nutraceutical which contains the 1,8-cineole. A nutraceutical, as is well known in the relevant arts, refers to biologically active component(s) of food in a nonfood matrix / substance.

[0076] In an embodiment of the present disclosure, the nutraceutical includes an extract of Elettaria cardamomum. In an example embodiment, the extract is Elettaria cardamomum essential oil. The Elettaria cardamomum essential oil can be obtained from one or more of fruits, pods, and seeds of Elettaria cardamomum. The Elettaria cardamomum essential oil (containing the active ingredient 1,8-cineole) is obtained through at least one of the methods of extraction such as steam distillation, solvent extraction, hydro-distillation, microwave extraction, supercritical fluid extraction, ultrasound-assisted extraction, cold press extraction, enfleurage, accelerated solvent extraction, vacuum distillation, etc. The Elettaria cardamomum essential oil can be formulated in any form (such as powder, liquid, suspension, etc.). In an embodiment, the Elettaria cardamomum essential oil contains the 1, 8-cineole at a concentration of range of 27-44 % (v / w). In an example embodiment, the Elettaria cardamomum essential oil contains the 1 , 8-cineole at a concentration of 29 % (v / w).

[0077] Use of such a nutraceutical containing the 1,8-cineole, in the composition for use in inhibiting viability of TNBC cells, has an advantage in that the composition may be administered on TNBC patients following fewer regulatory hurdles. Also, such a nutraceutical has minimal or no concerns of toxicity.

[0078] In an embodiment, for every 500 mg of the -cyclodextrin, the composition contains 1 ml of the Elettaria cardamomum essential oil.

[0079] In yet another embodiment of the present disclosure, the composition further contains one or more other active ingredients containing phytochemicals selected from flavonoids, terpenoids, or alkaloids. In an example embodiment, the Elettaria cardamomum essential oil includes monoterpenes (for example, a-Terpineol, Limonene, a-Pinene, Camphene, Sabinene, Borneol, etc.) as one or more other active ingredients. The other active ingredients, in combination with 1,8-cineole, provide synergistic anticancer effects such as arresting cell cycle to inhibit proliferation, downregulating proinflammatory cytokines (TNF-a, IL-6, and NF-KB), enhancing ROS modulation etc.

[0080] In an example embodiment, the composition inhibits the viability by one or more of apoptosis, modulating reactive oxygen species (ROS) levels, and inhibiting cell proliferation pathways.

[0081] In an example embodiment, the composition is a sustained-release formulation to enhance bioavailability and therapeutic efficacy.

[0082] The description is continued below with respect to the method of preparation of the composition.

[0083] 4. Example Preparation of Composition

[0084] Present disclosure discloses a method of preparation of a composition for use in inhibiting viability of triple negative breast cancer (TNBC) cells, where the composition contains 1,8-cineole as an active ingredient and [3-CD as a carrier, and where the [3-CD encapsulates the 1,8-cineole forming a nanoformulation.

[0085] According to an aspect of the present disclosure, the method of preparing the composition includes forming a first solution by dissolving P-cyclodextrin in a solvent. In an example implementation, the solvent is distilled water. The method also includes forming a second solution by adding Elettaria cardamomum essential oil to the first solution, where the Elettaria cardamomum essential oil contains 1,8-cineole as an active ingredient. In an embodiment, the Elettaria cardamomum essential oil contains the 1, 8-cineole at a concentration of range of 27-44 % (v / w). In an example embodiment, the Elettaria cardamomum essential oil contains the 1, 8- cineole at a concentration of 29 % (v / w).

[0086] The method further includes sonicating the second solution to form a homogenized second solution. As known in the relevant arts, sonicating generally entails applying ultrasonic energy (for example, ultrasound waves) to disperse or homogenize particles in a liquid. The sonication results in reduced particle size (and thus enhances encapsulation efficiency).

[0087] The method additionally includes centrifuging the homogenized second solution to form a pellet. As known in the relevant arts, centrifuging includes application of centrifugal force for separating substances of different densities and removing moisture. The method further includes deep freezing the pellet. As known in the relevant arts, deep freezing includes rapid cooling to subzero temperatures to prevent degradation and improve stability before further processing. The method in addition includes lyophilizing the deep frozen pellet to form a nanoformulation (for example, in the form of powder), in which the P-CD encapsulates the 1,8-cineole. As known in the relevant arts, lyophilizing (freeze-drying) is a dehydration process which includes removing ice / water from a frozen substance, typically using a vacuum, forming powder.

[0088] The description is continued below with respect to the step-by-step procedure, in an example implementation, using an example apparatus / system containing a stirrer capable of rotating at varying speeds, a temperature-controlled heating system, a sonicator, a deep freezer, and a lyophilizer.

[0089] The first solution is formed (for example, in a vessel) by dissolving 500 mg of the -CD in 100 ml of distilled water. The forming of the first solution includes stirring (for example, using a magnetic stirrer) the mixture of the P-cyclodextrin and the distilled water for a duration of 1 hour at 800 rpm and 60 °C. Then the second solution is formed by adding 1 ml of the Elettaria cardamomum essential oil (containing 1,8-cineole in the range of 25-53 % (v / w)) to the first solution, and stirring (for example, using a magnetic stirrer) the mixture of the Elettaria cardamomum essential oil and the first solution for 3 hours at 900 rpm and at room temperature. The second solution is sonicated using Bandelin Sonoplus sonicator at cycle 6 (for example, at 20 kHz) for 10 min to obtain to form the homogenized second solution. The homogenized second solution is centrifuged at 10,000 rpm for 10 min at 4 °C using a cooling centrifuge (for example, REMI CPR-30), to form a pellet. The pellet is deep frozen by keeping at -80 °C in a deep freezer (for example, Thermo scientific, Forma FDE series) overnight (generally, 8 hrs). The deep frozen pellet is then lyophilized (freeze drying using Penguin Classic method), using a lyophilizer, by maintaining condenser temperature below -80 °C and the vacuum at 15 pascals to obtain P- CD@CO NF powder.

[0090] The P-CD @ CO NF powder obtained through the method noted above contains particles in which 1,8-cineole is encapsulated by P-CD, where the particles are in nanometer range.

[0091] Though the example embodiment above refers to 500 mg of the P-CD, 100 ml of distilled water, and 1 ml of the Elettaria cardamomum essential oil, it is understood that the constituents (P-CD, distilled water and Elettaria cardamomum essential oil) can be used in other quantities / volumes, in ratios noted above, as would be apparent to a skilled practitioner from the present disclosure.

[0092] In an embodiment, the vessel includes a heating element configured to maintain the temperature of 60 °C. In an embodiment, the means for adding Elettaria cardamomum essential oil contains a calibrated syringe or pipette for accurate measureing and dispensing of the 1,8- cineole into the P-CD solution.

[0093] It is understood that, in alternative embodiments, other equivalent apparatus / system (and corresponding settings / processes) can be used.

[0094] The description is continued below with respect to physicochemical and morphological analyses of -CD @ CO NF powder.

[0095] 5.1 Physicochemical and Morphological Analyses of P-CD @ CO NF Powder

[0096] Physicochemical and morphological analyses were performed for P-CD @ CO NF powder, prepared with the Elettaria cardamomum essential oil containing the 1, 8-cineole at a concentration of 29 % (v / w).

[0097] Particle size distribution (hydrodynamic size distribution) analysis and zeta potential (surface charge) analysis of the P-CD @ CO NF were performed using Zetasizer Nano ZS90 system (Malvern Instruments, Malvern, UK).

[0098] The hydrodynamic size distribution analysis for the P-CD and the P-CD @ CO NF revealed that the P-CD exhibited a hydrodynamic size of 93.3 nm (Fig. 1A), while the P-CD@CO NF displayed an increased size of 135.0 nm (Fig. IB). As known in the relevant arts, the hydrodynamic size (for example, found using dynamic light scattering analysis) indicates diameter of a particle in an aqueous solution. The increased size confirmed successful encapsulation of the CO within the P-CD (P-CD matrix).

[0099] The zeta potential analysis showed a zeta potential of -3.54 mV for the P-CD (Fig. 1C) and -2.26 mV for the P-CD@CO NF (Fig. ID). As known in the relevant arts, zeta potential is a measure of effective electric charge on a nanoparticle's surface, quantifying the charges and indicating the stability of the nanoformulation. The observed decrease in zeta potential validates the formation of the P-CD @ CO NF. A zeta potential within the range of -30 mV to +30 mV is commonly deemed adequate to establish a strong repulsive force, promoting enhanced physical colloidal stability. This attribute can be advantageous for optimizing the effectiveness of drug delivery systems.

[0100] Spectroscopy analysis was performed on the P-CD@CO NF, using UV-visible spectroscopy, photoluminescence (PL) spectroscopy, and Fourier Transform Infrared (FT-IR) spectroscopy to confirm the encapsulation of the CO within the P-CD.

[0101] Figure 2 depicts Ultra violet-visible spectroscopy analysis of the P-CD @ CO NF, the CO, and the P-CD using an UV-Visible Spectroscopy (SHIMADZU - UV1800, Japan). As known inthe relevant arts, UV-Visible Spectroscopy is a technique used to analyze the absorption of ultraviolet and visible light by a material. As depicted in Figure 2, the UV -visible spectroscopy analysis demonstrated a maximum absorption peak at 252 nm for the P-CD @ CO NF, when compared to 249 nm absorption peak for the CO. The P-CD showed no absorption peak. The observed shift in the absorption peak of the P-CD@CO NF relative to the CO confirms the successful loading of the CO into the P-CD matrix (i.e., encapsulation of the CO within the P-CD).

[0102] Figure 3 depicts Photoluminescence spectroscopy analysis of the P-CD @ CO NF, the CO, and the P-CD using a spectrofluorometer (Jasco FP-8300). As known in the relevant arts, Photoluminescence (PL) spectroscopy is a technique that measures the emission of light from a material after it absorbs photons. As depicted in Figure 3, the P-CD @ CO NF and the CO reported prominent fluorescence peaks at 300 nm and 602 nm, whereas the P-CD shows no absorption peaks as well as any major fluorescence peaks. Similar fluorescence peaks for the P-CD@CO NF and the CO, confirm the successful encapsulation of the CO in the P-CD.

[0103] Figure 4 is a graph depicting functional group analysis of the P-CD @ CO NF, the CO, and the P-CD using Fourier Transform Infrared Spectroscopy (FT-IR) (Thermo Nicolet iS5). As known in the relevant arts, FT-IR is a technique used to obtain an infrared spectrum of absorption or emission of a solid, liquid, or gas, enabling the identification of functional groups in a molecule. As depicted in Figure 4, the FT-IR characteristics peaks of the P-CD@CO NF when compared to peaks of the P-CD and the CO show the presence of similar functional groups at different peak formation with slight peak shift. The FT-IR peaks ranging from 3454.80 cm'1to 3347.35 cm'1confirms the presence of the OH stretching functional group in both the P-CD and the P-CD @ CO NF. Similarly, the rise in peaks at 2088.94 cm'1to 2060.09 cm'1indicates the presence of 0=0 stretching group between the P-CD and the P-CD@CO NF groups. The FT-IR peaks ranging from 1642 cm'1to 1639.66 cm'1correspond to the H-OH bending vibrational band of absorbed water, and peaks recorded at 701.44 cm'1to 630.76 cm'1were due to C-O-C stretching groups between P-CD and P-CD @ CO NF. Further, the characteristic peaks of CO were also observed in P- CD@CO NF. The FT-IR peaks ranging from 2983.17 cm'1to 2977.40 cm1, 1399.51 cm'1to 1371.39 cm'1, and 1049.03 cm'1denotes the stretching vibration of aliphatic C-H groups, bending of C=O, and C-0 stretching groups, respectively, confirming the interaction between the CO and the P-CD @ CO NF. This characteristic band shifting confirms the successful loading of the CO in the P-CD @ CO NF.

[0104] Figure 5 A depicts the morphology of the P-CD @ CO NF observed by Field Emission- Scanning Electron Microscopy (FE-SEM), and Figure 5B depicts elemental groups present in the P-CD@CO NF by Energy Dispersive X-ray Analysis (EDAX). The FE-SEM was determined using CARL ZEISS-SIGMA 300 with energy dispersive spectroscopy (EDS). As known in therelevant arts, FE-SEM is a technique that provides detailed surface morphology and topography of nanoparticles using a focused electron beam and Energy Dispersive X-ray Spectroscopy (EDAX) is an analytical technique used with electron microscopy to determine the elemental composition of a sample. As depicted in Figure 5A, the morphology observed by FE-SEM shows both cuboidal and spherical shapes of the P-CD@CO NF. As depicted in Figure 5B, the EDAX spectrum presented firm peaks of carbon and oxygen, with a mass percentage of 53.60 ± 0.37 and 46.40 ± 0.77, respectively.

[0105] Figure 6A is a micrograph that depicts the size and shape of the P-CD@CO NF observed by High-Resolution Transmission Electron Microscopy (HR-TEM), and Figure 6B is a graph depicting average particle size distribution histogram. Figures 6A and 6B are observed using a High Resolution-Transmission Electron Microscopy (HR-TEM) (FEI- TECHNAI, G2Twin). As known in the relevant arts, HR-TEM is a high-magnification imaging technique that enables the visualization of nanoparticle morphology, crystallinity, and atomic structures at the nanometer scale. As depicted in Figure 6 A, the shape of P-CD@CO NF is cuboidal and roughly spherical shapes. The image analysis was used to quantify the size of P-CD@CO NF using ImageJ software. The particle size of the the P-CD@CO NF ranges from 65-110 nm, with an average particle size of 88 nm, based on the particle size distribution values in Figure 6B.

[0106] In this study, the fact that the hydrodynamic size (135.0 nm) (from Fig IB) is larger than the average particle size (88 nm) (from figure 6B) suggests the presence of a significant layer of water molecules and possibly ions associated with the surface of the P-CD@CO NF when they are in water (solution).

[0107] Figure 7A depicts a 2-D image of P-CD@CO NF obtained using atomic force microscopy and Figure 7B depicts a 3-D image of P-CD@CO NF obtained using atomic force microscopy. The images of Figures 7A and 7B reveal that the P-CD@CO NF have smooth surface topography, with Figure 7B also confirming the uniformity in size of the P-CD@CO NF with a height profile of 5 nm. The topography analyzed using an atomic force microscopy (AGILENT-5500).

[0108] Thus, the morphological analysis conducted through diverse microscopic techniques has consistently verified the encapsulation of CO within the synthesized [3-CD.

[0109] 6. Testing of Example Embodiments of the Composition

[0110] For the purpose of testing, P-cyclodextrin powder was purchased from Otto Chemicals, Mumbai. Cell culture reagents including heat-inactivated fetal bovine serum (FBS), DMEM medium, glutamine, penicillin-streptomycin, trypsin, Phosphate buffered saline (PBS), MTT reagent, DCFH- DA dye, rhodamine- 123, acridine orange, ethidium bromide, and ethanol were obtained from Himedia Chemicals, Mumbai. Swiss albino mice are procured from the central animal facility, Hanagal ShriKumareshwar college of Pharmacy, Bagalkot. The triple negative MDA-MB-231 cancer cell lines were obtained from the NCCS (National Centre for Cell Science) in Pune, India. The cancer cell line was nurtured in a DMEM medium accompanied by 10% FBS and 1% penicillin / streptomycin. The cell line was maintained in a 5% humidified CO2 incubator at 37 °C.

[0111] Also, for the purpose of this testing, [3-CD@CO NF powder, prepared with the Elettaria cardamomum essential oil containing the 1, 8-cineole at a concentration of 29 % (v / w), was used.

[0112] 6.1 In vitro release and kinetics studies of -CD@CO NF and Recovereez Forte DR

[0113] The in vitro release of the / ?-CD@CO NF and the Recovereez forte DR was measured in a PBS buffer with two different pH values of 7.4 and 5.4 using the dialysis method (cut-off MW: 3500). The [3-CD@CO NF and the Recovereez forte DR were dispersed in phosphate-buff ered saline (PBS) at pH 7.4 and 5.4 and stirred at 150 rpm at 37 °C for 48 h. At determined time intervals, 5 mL solution was collected, and 5 ml of buffer solution was poured back into the container to maintain the equilibrium of the buffer. The [3-CD@CO NF and the Recovereez forte DR concentrations were determined by calculating the absorbance at 250 nm with a Spectramax microplate multimode reader (Molecular Devices, USA). The amount of [3-CD@CO NF and Recovereez forte DR were calculated as a percentage and then plotted as a function of time (48 h). The following formula calculated the rate of drug release (%): amount of drug releaseDrug release (%) = - — - xlOO amount of drug on nanocarrier

[0114] The results obtained from in vitro drug release were fitted to several kinetic equations such as zero order, first order, Higuchi model, and Hixson-Crowell cube root law model to understand the in vitro release kinetics of [3-CD@CO NF and Recovereez forte DR. The equation was fitted to individual dissolution data at two different pH ranges with linear regression. The coefficient of correlation (R2) values was calculated by regression analysis.

[0115] The drug release profile of the [3-CD@CO NF and the Recovereez Forte DR was bi-phasic (burst and sustained release) and was measured under normal physiological conditions (PBS, pH 7.4) and acidic conditions (PBS, pH 5.4).

[0116] Figure 8 A depicts in vitro drug release profile of the Recovereez Forte DR and the [3- CD @CO NF at a pH of 7.4, Figure 8B depicts in vitro drug release profile of the Recovereez Forte DR and the [3-CD@CO NF at a pH of 5.4.

[0117] As depicted in Figure 8 A, the in vitro drug release profile of the [3-CD@CO NF and the Recovereez Forte DR at pH 7.4 shows an initial burst release of 35.64% and 15.20% respectively, followed by a sustained release up to 80.23% for the [3-CD@CO NF and 66.37% for the Recovereez Forte DR over 48 hours of incubation. It is understood that the graph is plotted for the treatment at pH 7.4 only for illustrative purposes.

[0118] As depicted in Figure 8B, the in vitro drug release profile of the P-CD @ CO NF and the Recovereez Forte DR at pH 5.4 shows a burst release of 31.92% for both the P-CD@CO NF and the Recovereez Forte DR. The sustained release reaches up to 90.36% for the P-CD@CO NF and 74.47% for the Recovereez Forte DR after 48 hours. It is understood that the graph is plotted for the treatment at pH 5.4 only for illustrative purposes.

[0119] The rise in the percentage of drug release under acidic conditions is attributed to the hydrolysis of the P-CD, facilitating the rapid diffusion of the P-CD@CO NF and the Recovereez Forte DR into cancer cells. The in vitro release investigation substantiates that the P-CD@CO NF enhances the release of the 1,8-cineole.

[0120] The release kinetic studies of the P-CD@CO NF and the Recovereez Forte DR were determined by fitting in the various kinetic models described below.Zero-order model: Q=Qo+Kot Higuchi model: Q=Knt1 / 2Hixson-Crowell cube root law: Q1 / 3-QO1 / 3=KHC t

[0121] Figure 9A is a release kinetics graph depicting the Higuchi model drug release profile for the Recovereez Forte DR at pH 7.4, Figure 9B is a release kinetics graph depicting the Higuchi model release kinetics for the Recovereez Forte DR at pH 5.4, Figure 9C is a release kinetics graph depicting the Higuchi model fit for the P-CD @ CO NF at pH 7.4, and Figure 9D is a release kinetics graph depicting the Higuchi model fit for the P-CD@CO NF at pH 5.4.

[0122] Values of the coefficient of correlation (R2) for the Higuchi model were the highest compared to other models for both P-CD @ CO NF and Recovereez Forte DR, as displayed in Figure 10. Based on the R2values, it can be concluded that the release of drugs from both formulations in different pH mediums follows the Higuchi model.

[0123] 6.2 Cell viability by MTT reagent

[0124] The triple negative breast cancer MDA-MB-231 cells (6000-8000 cells / well) were seeded into 96-well plates and incubated at 37 °C for 24 hours. Then, the confluent cells were treated with P-CD@CO NF and Recovereez forte DR at a dose ranging from 0 to 500 pg / ml. Untreated cells were used as control, and the cells treated with different groups were incubated in a humidified incubator with 5% CO2 at 37 °C for 48 hours. After 48 hours treatment, the old medium was discarded, and 100 pL of MTT solution was added to the wells. 96-well plate was further incubated for another 4 hours at 37 °C. After 4 hours of incubation, 100 pL of DMSO was added to each well to dissolve the formazan crystals and further incubated for 40 min in dark conditions at room temperature. The absorbance values of P-CD@CO NF and Recovereez forte DR were measured using a Spectramax Microplate Multimode Reader (Molecular Devices, USA) at 570 nm.OD TestCell viability (% ) x 100OD Control

[0125] The MTT colorimetric assay was employed to evaluate the cytotoxic impact of P-CD @ CO NF and Recovereez Forte DR on MDA-MB-231 cell lines. Figure 11 is a graph depicting cell viability analysis of MDA-MB-231 cancer cells against the Recovereez Forte and the P-CD@CO NF (data represent mean values ± SD, where n = 3). As may be observed from Figure 11, results demonstrate that the treatment of MDA-MB-231 breast cancer cells with the P-CD@CO NF exhibits enhanced cytotoxicity compared to the Recovereez Forte DR. The inhibitory concentration (IC50) values obtained were 70.33 ± 5.41 pg / ml and 128.25 ± 1.65 pg / ml for the P- CD@CO NF and the Recovereez Forte DR, respectively.

[0126] The observed reduction in cell viability induced by P-CD@CO NF suggests that the encapsulation of CO in the hydrophobic cavity of P-CD enhances the cytotoxic activity of CO when compared to Recovereez Forte DR. Figures 12A and 12B are images depicting the morphology of MDA-MB-231 cancer cells treated with different concentrations of Recovereez Forte DR and P-CD@CO NF, respectively. The scale bar represents 100 pm. Furthermore, the sustained release of CO from P-CD contributes to improved therapeutic efficacy against MDA- MB-231 cancer cells.

[0127] Thus, the MTT data underscores that the P-CD @ CO NF exhibits greater therapeutic potenial than the Recovereez Forte DR against MDA-MB-231 cancer cells, indicating the successful encapsulation of free CO within the cancer cells. Further, IC50 value obtained for P- CD@CO NF was used for other cytotoxic studies.

[0128] 6.3 Cell Death Analysis

[0129] The MDA-MB-231 cancer cells were treated with the P-CD@CO NF and the Recovereez forte DR at a concentration of 70 pg / ml. After 48 hours treatment, cancer cells were washed thrice with PBS, and then fixed with 100% icecold methanol. After fixation, 2 pl mixture of AO / EtBr (1:1 ratio) dye was added to the treated cells and kept in the dark for 40 min. Subsequently, the cancer cells were visualized under a fluorescence microscope (Life Technologies, USA).

[0130] Figure 13A depicts cell death analysis of MDA-MB-231 cancer cells by AO / EtBr dual staining treated with the Recovereez Forte DR and the P-CD@CO NF at a concentration of 70 pg / ml. The alterations in the morphological characteristics of MDA-MB-231 cancer cells following exposure to the P-CD @ CO NF and the Recovereez Forte DR, evaluated using AO / EtBr dual staining and observed through fluorescence. From Figure 13 A, it may be observed that MDA- MB-231 cancer cells treated with the Recovereez Forte DR exhibited early apoptotic features, as evidenced by the presence of both AO / EtBr dual fluorescence dye. In contrast, the treatment withthe P-CD@CO NF revealed late apoptosis in cancer cells, as indicated by condensed nuclei, membrane blebbing, and the presence of apoptotic bodies in MDA-MB-231 cancer cells.

[0131] Figure 13B depicts a bar diagram representing cell death percentages in different groups (i.e., control group, the Recovereez Forte DR (70 pg / ml) and the [3-CD@CO NF (70 pg / ml). Data represent mean values ± SD, where n = 3, and ***P < 0.001 when compared with the control. The scale bar represents 100 pm. The P-CD@CO NF significantly increased the apoptotic percentage of cell death at a concentration of 70 pg / ml, outperforming Recovereez Forte DR in inducing apoptosis.

[0132] 6.4 Reactive Oxygen Species (ROS) Assay

[0133] The MDA-MB-231 cancer cells were treated with the P-CD@CO NF and the Recovereez forte DR at a concentration of 70 pg / ml. After 48 hours treatment, cancer cells were washed thrice with PBS, and then fixed with 100% icecold methanol. After fixation, 2 pl of DCFH-DA fluorescent dye was added to the treated cells and kept in the dark for 40 min. Subsequently, the cancer cells were visualized under a fluorescence microscope (Life Technologies, USA). The fluorescent intensity was measured at 485 nm and 535 nm using a Spectramax microplate multimode reader (Molecular Devices, USA).

[0134] Figure 14A depicts ROS generation of MDA-MB-231 cancer cells by DCFH-DA staining treated with the Recovereez Forte DR and the P-CD@CO NF. Figure 14B is a bar graph depicting fluorescent intensity of DCFH-DA dye observed in different groups under excitation and emission at 480 nm and 530 nm, respectively. Data represent mean values ± SD, where n = 3, and ****P < 0.0001 when compared with the control. The scale bar represents 100 pm.

[0135] The elevated levels of reactive oxygen species (ROS) have the potential to induce cancer cell death which was observed by DCFH-DA staining. Fluorescence microscopy analysis revealed an amplified intracellular ROS level in cancer cells treated with the [3-CD@CO NF compared to those treated with Recovereez Forte DR at a concentration of 70 pg / ml. In contrast, the untreated control cells exhibited a negligible increase in intracellular ROS levels.

[0136] As may be observed from Figure 14B, the sustained release of CO from [3-CD contributed to the heightened intracellular accumulation of CO, leading to cell death through an augmentation in intracellular ROS generation.

[0137] 6.5 Mitochondrial Membrane Potential (MMP) Assay

[0138] The MDA-MB-231 cancer cells were treated with the [3-CD@CO NF and the Recovereez forte DR at a concentration of 70 pg / ml. After 48 hours treatment, cancer cells were washed thrice with PBS, and then fixed with 100% icecold methanol. After fixation, 2 pl of DCFH-DA fluorescent dye was added to the treated cells and kept in the dark for 40 min. Subsequently, the cancer cells were visualized under a fluorescence microscope (Life Technologies, USA). Thefluorescent intensity was measured at 485 nm and 535 nm using a Spectramax microplate multimode reader (Molecular Devices, USA).

[0139] Figure 15 A is a fluorescence microscopy image depicting changes in the Mitochondrial Membrane Potential (MMP) of MDA-MB-231 cancer cells using rhodamine- 123 fluorescence staining at a concentration of 70 pg / ml. The fluorescence images of the control cells indicated an absence of early signs of apoptosis, suggesting a maintained polarized mitochondrial membrane. Conversely, cells subjected to Recovereez Forte DR displayed early signs of apoptosis, characterized by reduced green fluorescence in the rhodamine-123 staining. Notably, cells treated with P-CD@CO NF exhibited a substantially greater alteration in MMP compared to other treatment groups.

[0140] Figure 15B is a bar diagram representing the fluorescence intensity of rhodamine- 123 measured using a Spectramax multimode reader under excitation and emission at 485 nm and 530 nm, respectively. Data represent mean values ± SD, where n = 3, and P < 0.0001 when compared with the control. The scale bar represents 100 pm.

[0141] 6.6 In vivo Pharmacokinetics Study

[0142] For in vivo pharmacokinetics studies, the Recovereez Forte DR and the / ?-CD@CO NF levels in blood were assessed in normal male Swiss albino mice procured from the central animal facility, Hanagal Shri Kumareshwar college of Pharmacy, Bagalkot. Each group consisted of 6 animals and 200 mg / kg, b / w of each drug (Recovereez Forte DR and / ?-CD@CO NF) were mixed in 5ml of distilled water, and administered orally using an oral gavage needle. After that blood samples were collected from the tail vein of mice at different time intervals. The collected blood samples were then centrifuged in cooling centrifuge (REMI CPR-30) at 10,000 rpm for 10 minutes at a temperature of 4 °C to isolate the plasma from blood of male Swiss albino mice. Subsequently, the plasma was placed in 96 well plate and inserted inside the multimode reader (Molecular Devices, USA) for analysis at excitation wavelength of 250 nm. All research and animal care procedures have been approved by the Institute’s Animal Ethical Committee, Annamalai University (IAEC proposal NO. AU-IAEC / 1368 / 9 / 23).

[0143] Oral bioavailability, assessed through pharmacokinetic studies, signifies the proportion of an orally administered dose that reaches the bloodstream, becoming available for a therapeutic response. In male Swiss albino mice given a single oral dose of 200 mg / kg body weight, plasma drug levels for the Recovereez Forte DR and the [3-CD@CO NF were measured using a multimode reader.

[0144] Figure 16 is a graph depicting the pharmacokinetic profile of the Recovereez Forte DR and the [3-CD@CO NF in normal Swiss albino mice. The graph depicts the mean concentration profiles (pg / ml) of the P-CD@CO NF and the Recovereez Forte DR in plasma over time. Table-1 provideskey pharmacokinetic parameters, such as Cmax, Tmax, AUCo— >-24, AUCo— ’■<«, Vd, Cl, and ti / 2. The plasma concentration profiles for the Recovereez Forte DR and the P-CD @ CO NF revealed Cmax, values of 39174.2 ± 1709.22 and 51494 ± 855.32, with corresponding Tmax values of 6h and 8h, respectively. Additionally, pharmacokinetic studies affirmed that the incorporation of P-CD improved the bioavailability of CO in the form of the P-CD @ CO NF.

[0145] Pharmacokinetics parameters of the Recovereez Forte DR and the P-CD@CO NF in normal Swiss albino mice are shown below in Table-1. Each value is the mean of six mice with standard deviation; *P < 0.2, **P < 0.01, and ***P <0.001, when the P-CD@CO NF compared with the Recovereez Forte DR. Tmax: maximum time, Cmax:maximum concentration, AUCo- >24: Area under curve 0 to 24 hours, AUCo^: Area under curve 0 to infinity, Va: Volume distribution, CL: Clearance, ti / 2: half time.Table- 1

[0146] 6.7 Serum Biochemical Analysis

[0147] For the biochemical analysis, six male Swiss albino mice were grouped into one group, and groups were denoted as I, II, III. Group I mice received a daily dose of normal saline for 14 days. Group II mice were administered Recovereez Forte DR (200 mg / kg, b / w, orally) for 14 days; Group III mice were treated with ?-CD@CO NF (200 mg / kg, b / w, orally) for 14 days. After completion of the respective treatment; the blood was collected and subjected to isolation of serum. The isolated serum was assessed for liver and kidney function parameters by using standard kits. Liver function was evaluated through parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Moreover, renal function was assessed by measuring creatinine and urea levels.

[0148] Table-2 presents the levels of liver function markers — ALT (Alanine Aminotransferase), AST (Aspartate Aminotransferase), and ALP (Alkaline Phosphatase) and kidney function markers — urea and creatinine in the serum of male Swiss albino mice after a 14-day treatment with various groups. Serum biochemical analysis revealed no significant adverse effects of oral administration of the Recovereez Forte DR and the P-CD @ CO NF on liver and kidney function markers in male Swiss albino mice. Thus, it is confirmed that P-CD enhances the bioavailability of CO without causing any adverse effects on liver and kidney function markers. Each value is the mean of six mice with standard deviation; *P < 0.2, **P < 0.01, and ***P < 0.001, when compared with the control group.Table -2

[0149] All statistical analyses were performed by analysis of variance method (ANOVA) method using GraphPad Prism (version 9.0). The values of p < 0.05 were considered to be significant.

[0150] Thus, the test results conclude that P-CD enhances the therapeutic and pharmacokinetic properties of CO compared to the Recovereez Forte DR. The P-CD @ CO NF underwent various characterization techniques (physicochemical and morphological analyses) to confirm the successful encapsulation of CO in P-CD. Subsequently, in vitro drug release studies confirmed that the P-CD @ CO NF exhibits a higher percentage of sustained drug release in two different mediums compared to the Recovereez Forte DR. Furthermore, the drug release profiles of the P- CD@CO NF and the Recovereez Forte DR follow the Higuchi model of release kinetics. Cell cytotoxicity assays revealed that the P-CD@CO NF are more potent in generating reactive oxygen species (ROS), inducing mitochondrial membrane depolarization, and increasing cell death compared to the Recovereez Forte DR. Pharmacokinetic analysis conducted in male Swiss albino mice demonstrated that the P-CD @ CO NF significantly enhance the bioavailability of CO compared to the Recovereez Forte DR. Serum biochemical data indicated negligible adverseeffects on liver and kidney function tests in male Swiss albino mice treated with both the [3- CD@CO NF and the Recovereez Forte DR.

[0151] Thus, it is concluded that the -CD@CO NF, prepared with the Elettaria cardamomum essential oil containing the 1, 8-cineole at a concentration of 29 % (v / w), showed better therapeutic efficiency compared to the Recovereez Forte DR prepared with the Elettaria cardamomum essential oil containing the 1, 8-cineole at a concentration of 45-53 % (v / w).

[0152] The -CD@CO NF prepared with Elettaria cardamomum essential oil containing lesser concentration of 1, 8-cineole has economic significance, as the composition will be cheaper with Elettaria cardamomum essential oil containing lesser concentration of 1, 8-cineole. The cost of Elettaria cardamomum essential oil increases with increased of concentration of 1, 8-cineole.

[0153] It is understood that, as the concentration of 1, 8-cineole increases in the Elettaria cardamomum essential oil used for the preparation of the -CD@CO NF, the therapeutic effect will further increase.

[0154] The drawings and the forgoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims.

[0155] Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any component(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all the claims.

[0156] 7. Conclusion

[0157] References throughout this specification to “one embodiment”, “an embodiment”, or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrases “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0158] While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of example only, and not limitation. Thus, the breadth and scope of the present disclosure should not be limited by any of the above -described embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

rece ve y e n erna ona ureau on ep em er\NO 2025 / 196687 PCT / IB2025 / 052931AMENDED CLAIMSWhat is Claimed is:

1. A composition for use in inhibiting viability of triple negative breast cancer (TNBC) cells, said composition comprising:1,8-cineole as an active ingredient; and5 [3-cyclodextrin as a carrier, wherein said [3-cyclodextrin encapsulating said 1,8-cineole forming a nanoformulation.

2. The composition of claim 1, wherein said 1,8-cineole is comprised in Elettaria cardamomum essential oil, and wherein said Elettaria cardamomum essential oil comprising said10 1, 8-cineole at a concentration of 27-44 % (v / w).

3. The composition of claim 3, wherein said composition comprising 1 ml of said Elettaria cardamomum essential oil for every 500 mg of said [3-cyclodextrin.15 4. The composition of claim 1, wherein size of particles in said nanoformulation is in a range of 65 - 110 nm.

5. The composition of claim 4, wherein hydrodynamic size of said particles is about 135 nm.

206. The composition of claim 4, wherein zeta potential of said particles is about -2.26 mV.

7. A method of preparing a composition for use in inhibiting viability of triple negative breast cancer (TNBC) cells, said method comprising:25 forming a first solution by dissolving [3-cyclodextrin in a solvent; forming a second solution by adding Elettaria cardamomum essential oil to said first solution, wherein said Elettaria cardamomum essential oil comprising 1,8-cineole as an active ingredient; sonicating said second solution to form a homogenized second solution;30 centrifuging said homogenized second solution to form a pellet; deep freezing said pellet; and lyophilizing said deep frozen pellet to form a nanoformulation, wherein said nanoformulation comprising said 1,8-cineole encapsulated by said [3-cyclodextrin.35 8. The method of claim 7, wherein:25AMENDED SHEET (ARTICLE 19)said forming of said first solution comprising stirring a mixture of said [3-cyclodextrin and said solvent for a duration of 1 hour at 800 rpm and 60 °C; said forming of said second solution comprising stirring a mixture of said Elettaria cardamomum essential oil and said first solution for 3 hours at 900 rpm and at room temperature;5 said sonicating comprising applying ultrasonic energy for 10 minutes; said centrifuging comprising rotating said sonicated second solution, using a cooling centrifuge, for 10 min at 10,000 rpm and at 4 °C; said deep freezing comprising keeping said pellet at -80 °C; and said lyophilizing comprising maintaining said pellet at a pressure of 15 pascals and at a10 temperature below -80 °C.

9. The method of claim 7, wherein: said solvent is distilled water and said dissolving comprising dissolving 500 mg of said [3- cyclodextrin for every 100 ml of said distilled water; and15 said adding comprising adding 1 ml of said Elettaria cardamomum essential oil for every 500 mg of said [3-cyclodextrin, wherein said Elettaria cardamomum essential oil comprising said 1, 8-cineole at a concentration of 27-44 % (v / w).

10. The method of claim 7, wherein size of particles in said nanoformulation is in a range20 of 65 - 110 nm.

11. The method of claim 10, wherein hydrodynamic size of said particles is about 135 nm, and wherein zeta potential of said particles is about -2.26 mV.25 12. A composition for Triple-negative breast cancer (TNBC) comprising:[3-cyclodextrin and cineole oil, wherein the [3-cyclodextrin is present at a concentration of 0.5% (w / v) and the cineole oil is present at a concentration of 0.29% (v / v).

13. The composition as claimed in claim 12, further comprising distilled water, wherein30 the [3-cyclodextrin is dissolved in the distilled water.

14. The composition as claimed in claim 12, wherein the cineole oil comprises 29% (v / v) of the total composition.35 15. The composition as claimed in claim 12, wherein the [3-cyclodextrin is present at a26AMENDED SHEET (ARTICLE 19)concentration of 500 mg / 100 ml of distilled water.

16. An apparatus for preparing [3-cyclodextrin loaded 1,8-cineole ([3-CD ©CO) nanofibers, comprising:5 a vessel suitable for containing [3-cyclodextrin (|3-CD) dissolved in distilled water; a magnetic stirrer configured to agitate the [3-CD solution at 800 rpm and 60 °C for 1 hour; a means for introducing cardamom essential oil into the [3-CD solution, wherein the cardamom essential oil comprises 1,8 cineole (CO);10 a magnetic stirrer configured to further stir the [3-CD and the cardamom essential oil mixture at 900 rpm for 3 hours; a sonicator configured to sonicate the [3-CD and the cardamom essential oil mixture at cycle 6 for 10 minutes; a deep freezer capable of maintaining a temperature of -80 °C; and15 a lyophilizer configured to freeze-dry the mixture at a vacuum of 15 pascals to obtain [3- CD@CO nanofibers.

17. The apparatus as claimed in claim 16, wherein the vessel includes a heating element configured to maintain the temperature of the [3-CD solution at 60 °C during agitation.2018. The apparatus as claimed in claim 16, wherein the means for introducing the cardamom essential oil comprises a calibrated syringe or pipette for accurate measurement and dispensing of the cardamom essential oil into the [3-CD solution.25 19. The apparatus as claimed in claim 16, further comprising a temperature-controlled environment for the vessel, maintaining a humidity level of 5% and a temperature of 37 °C during subsequent incubation.

20. The apparatus as claimed in claim 16, wherein the sonicator is configured to operate30 at a frequency of 20 kHz during the sonication step.27AMENDED SHEET (ARTICLE 19)

Images