Inhibiting viability of triple-negative breast cancer cells

Abstract

Aspects of the present disclosure are directed to inhibiting viability of triple-negative breast cancer (TNBC) cells. According to an aspect of the present disclosure, a composition for use in inhibiting viability of TNBC cells contains 1,8-cineole as an active ingredient, and an excipient. In an embodiment, the 1,8-cineole is contained in one or more nutraceuticals. As a result, the composition may be administered to TNBC patients following fewer regulatory hurdles, owing to the minimal toxicity concerns associated with nutraceuticals. In another embodiment, the one or more nutraceuticals contain a first extract formed of one or more of Elettaria cardamomum and Salvia Rosmarinus, and the composition also contains a second extract formed of one or more of Piper nigrum and Piper longum. In another embodiment, the composition contains one or more other active ingredients selected from flavonoids, terpenoids, or alkaloids. In another embodiment, the excipient is a nutraceutically acceptable excipient.

Description

INHIBITING VIABILITY OF TRIPLE-NEGATIVE BREAST CANCER CELLSPriority Claim

[0001] The instant patent application is related to and claims priority from the Germany utility model application entitled, “RECOVEREEZ FORTE BASED COMPOSITION FOR TARGETING TRIPLE-NEGATIVE BREAST CANCER CELLS”, Serial No.: 20 2024 101 390.5, Filed: 20 March 2024, which is incorporated in its entirety herewith to the extent not inconsistent with the description herein.Background

[0002] Technical Field

[0003] Embodiments of the present disclosure relate generally to inhibiting viability of breast cancer cells, and more specifically to inhibiting viability of triple-negative breast cancer (TNBC) cells.

[0004] Related Art

[0005] Cancer, a multifaceted disease characterized by uncontrolled cell growth and proliferation, poses a significant global health challenge. Among its various forms, breast cancer stands out as one of the most prevalent malignancies, affecting millions of individuals worldwide. Despite advancements in treatment modalities, the management of breast cancer remains complex, particularly in the context of triple-negative breast cancer (TNBC), which lacks expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2).

[0006] Current therapeutic strategies for breast cancer primarily target hormone receptors (ER and PR) or HER2 overexpression, allowing for the development of hormone therapy and HER2- targeted therapies. However, TNBC, characterized by its aggressive behavior and limited treatment options, presents a formidable clinical challenge. Traditional chemotherapeutic agents, such as anthracy clines and taxanes, remain the mainstay of treatment for TNBC, albeit with limited efficacy and significant toxicity.

[0007] In recent years, targeted therapies have emerged as promising approaches for combating TNBC. For instance, inhibitors of the phosphatidylinositol-3-kinase (PI3K) / AKT / mammalian target of rapamycin (mTOR) signaling pathway, cyclin-dependent kinase 4 / 6 (CDK4 / 6) inhibitors, and histone deacetylase (HD AC) inhibitors have shown potential in preclinical and clinical studies. Additionally, immune checkpoint inhibitors, such as programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) inhibitors, have demonstrated efficacy in subsets of TNBC patients.

[0008] Despite these advancements, challenges persist in effectively managing TNBC, including intrinsic and acquired resistance to therapy, tumor heterogeneity, and the lack of actionablemolecular targets. Moreover, the identification of therapeutic agents with enhanced efficacy and reduced toxicity remains a critical unmet need in TNBC research.

[0009] Traditional cytotoxic agents, including anthracyclines (e.g., doxorubicin) and taxanes (e.g., paclitaxel), are commonly used in TNBC treatment regimens. However, these agents exhibit limited efficacy and significant adverse effects, highlighting the need for more targeted and less toxic therapies.

[0010] While targeted therapies, such as inhibitors of the PI3K / AKT / mTOR pathway and CDK4 / 6 inhibitors, hold promise for TNBC treatment, their clinical utility is limited by factors such as tumor heterogeneity and resistance mechanisms.[Oil] Immune checkpoint inhibitors, particularly PD-1 and PD-L1 inhibitors, have shown encouraging results in subsets of TNBC patients. However, response rates to immunotherapy vary, and predictive biomarkers for patient selection remain elusive. Furthermore, immune-related adverse events pose challenges in the clinical management of TNBC patients receiving immunotherapy.

[0012] Document entitled, “Evaluation of in vitro anticancer activity of 1,8-Cineole-containing n- hexane extract of Callistemon citrinus (Curtis) Skeels plant and its apoptotic potential” by Sowndarya Sampath et al., available at ScienceDirect, suggests inhibiting viability of cancer cells in general using 1,8 cineole. But this document does not disclose inhibiting viability of TNBC cells.

[0013] Document entitled, “Anti-Breast Cancer Activity of Essential Oil: A Systematic Review” by Mohammad Adam Mustapa et al., available at Applied Sciences Journal, suggests inhibiting viability of TNBCs using diterpenes from Cyphostemma juttae. This document also suggests potent of 1 ,8 cineole from the oils of L. nobilis fruits (which are nutraceutical) against breast cancer cell lines MCF7 and T47D, however breast cancer cell lines MCF7 and T47D are not TNBC cells / cell lines as known in the relevant art.

[0014] In light of the limitations of existing therapies, there is a pressing need for therapeutic agents that can inhibit viability of TNBCs, selectively target TNBC cells while minimizing off-target effects, and overcoming resistance mechanisms.

[0015] Aspects of the present disclosure are directed to inhibiting viability of TNBCs, selectively target TNBC cells while minimizing off-target effects, and overcoming resistance mechanisms.Brief Description of the views of Drawings

[0016] Example embodiments of the present disclosure will be described with reference to the accompanying drawings briefly described below.

[0017] Figure 1A is a graph depicting IC50 concentrations / values (in percentage) of 1,8-cineloe(referred to as Cl 8 or C-18) required to inhibit cell viability by 50% in three triple-negative breast cancer (TNBC) cell lines, MDA MB 231, MDA MB 453, and MDA MB 468 cells following 24hrs, 48hrs, and 72hrs of treatment, as measured by MTT assay.

[0018] Figure IB is a graph depicting percentage cell viability of MDA MB 231, MDA MB 453, and MDA MB 468 cells, following 24hrs, 48hrs and 72hrs treatment with 12% C18, as determined by MTT assay.

[0019] Figure 1C is a graph depicting percentage cell viability in MDA MB 231, MDA MB 453, and MDA MB 468 cells for different concentrations of Cl 8.

[0020] Figure ID is a graph depicting IC50 concentrations / values (in percentage) of Recovereez Forte (RF) required to inhibit cell viability by 50% in three triple-negative breast cancer (TNBC) cell lines, MDA MB 231, MDA MB 453, and MDA MB 468 cells following 24hrs, 48hrs, and 72hrs of treatment, as measured by MTT assay.

[0021] Figure IE is a graph depicting percentage cell viability of MDA MB 231, MDA MB 453, and MDA MB 468 cells, following 24hrs, 48hrs and 72hrs treatment with 24pg / ml of RF, as determined by MTT assay.

[0022] Figure IF is a graph depicting percentage cell viability in MDA MB 231, MDA MB 453, and MDA MB 468 cells for different concentrations of RF.

[0023] Figure 2A is a graph depicting apoptosis (in percentage) for corresponding IC 50 concentrations of Recovereez Forte (RF) and C18 in MDA MB 231 cells for different treatment periods.

[0024] Figure 2B is a graph depicting apoptosis (in percentage) for corresponding IC 50 concentrations of Recovereez Forte (RF) and Cl 8 in MDA MB 453 cells for different treatment periods.

[0025] Figure 2C is a graph depicting apoptosis (in percentage) for corresponding IC 50 concentrations of Recovereez Forte (RF) and Cl 8 in MDA MB 468 cells for different treatment periods.

[0026] Figure 3A-3E are images depicting histopathology of liver tissues for normal and EAC- induced mice models.

[0027] Figure 3F-3J are images depicting histopathology of kidney tissues for normal and EAC- induced mice models.

[0028] Figure 4A-4E are images depicting histopathology of liver tissues for normal and DAL- induced mice models.

[0029] Figure 4F-4J are images depicting histopathology of kidney tissues for normal and DAL- induced mice models.

[0030] FIG. 5A is a graph depicting the effect of Recovereez Forte (RF) on tumor volume inDalton’s Lymphoma Ascites (DAL) model.

[0031] FIG. 5B is a graph depicting the effect of Recovereez Forte (RF) on mean survival time (MST) and percentage increase in life span (%ILS) in tumor-bearing mice in both the Dalton’s Lymphoma Ascites (DAL) and Ehrlich Ascites Carcinoma (EAC) models.

[0032] 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

[0033] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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 inventionbelongs. The compositions, methods, and examples provided herein are illustrative only and not intended to be limiting.

[0038] Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.

[0039] 1. Terminology

[0040] Elettaria cardamomum: Commonly 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.

[0041] Salvia rosmarinus: Commonly known as rosemary, is a generally erect, rounded, evergreen shrub with aromatic, needle-like, gray-green leaves and tiny, two-lipped, pale blue to white flowers. Rosemary shrubs widely grown in hills of Tamil Nadu, India, have been used in relation to the subject invention.

[0042] Piperine extract: Refers to a standardized extract containing the active alkaloid piperine, derived from the fruit of the plant Piper nigrum (black pepper) and / or the plant Piper longum L. (long pepper), with thermogenic properties and also acts as a bioavailability enhancer. Black pepper and long pepper plants widely grown in Kerala, India, have been used in relation to the subject invention.

[0043] Synergistic: Interaction or combination of two or more components, substances, ingredients to produce a combined effect greater than the sum of their separate effects.

[0044] 1,8 -cineole: Also known as eucalyptol, is an oxygenated monoterpene with a chemical formula of CioHisO. 1,8-cineole is referred to as C18 or C-18 in the present disclosure.

[0045] [3-cyclodextrins (|3-CDs): Cyclic carbohydrates made of seven glucopyranose units joined by a-l,4-glycosidic bonds in a ring.

[0046] Carboxymethyl cellulose (CMC): An anionic, water-soluble derivative of cellulose, a linear polysaccharide of anhydro-glucose.

[0047] 2. Overview

[0048] Aspects of the present disclosure are directed to inhibiting viability of triple-negative breast cancer (TNBC) cells. According to an aspect of the present disclosure, a composition for use in inhibiting viability of TNBC cells contains 1,8-cineole as an active ingredient and an excipient.

[0049] In an embodiment, the 1 ,8-cineole is contained in one or more nutraceuticals. As a result, the composition may be administered to TNBC patients following fewer regulatory hurdles, owing to the minimal toxicity concerns associated with nutraceuticals.

[0050] In another embodiment, the one or more nutraceuticals contain a first extract formed of one or more of Elettaria cardamomum and Salvia Rosmarinus, and the composition also contains a second extract formed of one or more of Piper nigrum and Piper longum.

[0051] In yet another embodiment, the first extract contains Elettaria cardamomum essential oil and Salvia rosmarinus essential oil in equal proportions by weight, the second extract contains 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 where the Elettaria cardamomum essential oil contains the 1, 8-cineole at a concentration of 45-53% (v / w), and the Salvia rosmarinus essential oil contains the 1, 8-cineole at a concentration of 45-53% (v / w).

[0052] In yet another embodiment, the Elettaria cardamomum essential oil and the Salvia rosmarinus essential oil contain one or more other active ingredients, where the one or more other active ingredients include one or more of flavonoids, terpenoids, and alkaloids.

[0053] In yet another embodiment, the nutraceutically acceptable excipient is [3-cyclodextrin, and the [3-cyclodextrin is in a molar amount equal to a sum of moles of the Elettaria cardamomum essential oil, the Salvia rosmarinus essential oil, and the Piperine Oleoresin, the Elettaria cardamomum essential oil contains a first set of the one or more other active ingredients, where the first set includes one or more of limonene, linalool, a-terpinenyl acetate, and a-terpineol, and the Salvia rosmarinus essential oil contains a second set of the one or more other active ingredients, where the second set includes one or more of rosmarinic acid and carnosic acid.

[0054] In yet another embodiment, the composition is a sustained-release formulation to enhance bioavailability and therapeutic efficacy.

[0055] In yet another embodiment, the composition inhibits the viability by one or more of apoptosis, modulating reactive oxygen species (ROS) levels, and inhibiting cell proliferation pathways, and the inhibition is in a range of 66.98 to 88.64%.

[0056] According to another aspect of the present disclosure, a method of treatment of triple negative breast cancer (TNBC) contains administering a therapeutically effective dose of the composition.

[0057] In an embodiment, the method also contains monitoring therapeutic response through one or more of tumor volume measurement, imaging or biomarker analysis.

[0058] In another embodiment, the therapeutically effective dose is administered in a mouse model in a range of 200 mg / kg to 400mg / kg. According to another aspect of the present disclosure, a method of preparing a composition for use in inhibiting viability of triple negative breast cancer (TNBC) cells includes (i) dissolving an excipient in a solvent to prepare a first solution, (ii) adding an oil mixture to said first solution to prepare a second solution, said oil mixture comprising one or more of Elettaria cardamomum essential oil and Salvia rosmarinus essential oil, each of theElettaria cardamomum essential oil and the Salvia rosmarinus essential oil containing 1,8-cineole as an active ingredient, and (iii) emulsifying said second solution to form an emulsion.

[0059] In an embodiment, the excipient is selected from one or more of P-cyclodextrin, carboxymethyl cellulose, Tween 80, and PEG 400, and the oil mixture includes the Elettaria cardamomum essential oil, the Salvia rosmarinus essential oil and Piperine Oleoresin.

[0060] In yet another embodiment, the oil mixture includes the Elettaria cardamomum essential oil and the Salvia rosmarinus essential oil in equal proportions by weight, and the 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, where the Elettaria cardamomum essential oil contains the 1 , 8-cineole at a concentration of 45-53% (v / w) and the Salvia rosmarinus essential oil contains the 1, 8-cineole at a concentration of 45-53% (v / w), and the P-cyclodextrin is in a molar amount equal to a sum of moles of the Elettaria cardamomum essential oil, the Salvia rosmarinus essential oil, and the Piperine Oleoresin.

[0061] In yet another embodiment, the method includes spray drying the emulsion to form a dried powder.

[0062] 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.

[0063] 3. Composition

[0064] 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 ingredient is 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.

[0065] In an embodiment of the present disclosure, the composition contains one or more nutraceuticals which contain 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.

[0066] In an embodiment of the present disclosure, the nutraceuticals include extracts formed of Elettaria cardamomum or Salvia Rosmarinus or both. In an example embodiment, the extracts are Elettaria cardamomum essential oil and Salvia rosmarinus essential oil. The Elettariacardamomum essential oil can be obtained from one or more of fruits, pods, and seeds of Elettaria cardamomum. The Salvia Rosmarinus essential oil can be obtained from one or more of leaves and twigs Salvia Rosmarinus. The Elettaria cardamomum essential oil and the Salvia rosmarinus essential oil (containing the active ingredient 1,8-cineole) are 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 and the Salvia rosmarinus essential oil can be formulated in any form (such as powder, liquid, suspension, etc.). In an example embodiment, the Elettaria cardamomum essential oil is obtained from cardamom plants grown in Idukki district of Kerala, India.

[0067] Use of such nutraceuticals containing 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 nutraceuticals have minimal or no concerns of toxicity.

[0068] In another embodiment of the present disclosure, the composition also contains one or more extracts (piperine extracts) formed of Piper nigrum or Piper longum or both. In an example embodiment, an extract is Piperine Oleoresin. Piperine Oleoresin can be obtained from fruits of Piper nigrum. Piperine Oleoresin 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 piperine extract boosts the bioavailability and the safety and efficacy profile of the active ingredient 1,8-cineole.

[0069] 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.) and Salvia rosmarinus essential oil includes polyphenols (for example, rosmarinic acid and carnosic acid) 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.

[0070] In yet another embodiment of the present disclosure, the composition also contains one or more excipients. The excipients help increase the solubility of active ingredient(s) (which may be hydrophobic and may thus exhibit low aqueous solubility) by forming micelles or inclusion complexes, thus increasing the membrane permeability of the active ingredient(s), facilitatingabsorption and bioavailability. The excipients also help improve the stability of the active ingredient(s). In an example embodiment, an excipient is a nutraceutically acceptable excipient selected from one or more of [3-cyclodextrin, Carboxymethyl cellulose, Tween 80 and polyethylene glycol 400 (PEG 400).

[0071] In an example embodiment, the composition can be in the form of capsules (made of soft gel, hard gelatin, hydroxypropyl methylcellulose, etc.), tablets (including in the form of chewable, effervescent, fast-dissolving, extended-release, etc.), powder, syrup, intravenous solutions, suspensions, emulsions, liposomes, micelles, microcapsules, and nanoparticles.

[0072] In an embodiment, the composition (Recovereez Forte (RF)) is in the form of powder, and the composition contains the Elettaria cardamomum essential oil and the 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%.

[0073] In an example embodiment, every 500 mg of the composition, that is in the form of powder, contains in weight by percentages, 40% of the Elettaria cardamomum essential oil, 40% of Salvia Rosmarinus essential oil, and 2% of piperine extract, such that the total amount of cineole is in the range of 88-92mg.

[0074] In another embodiment, the composition is in the form of an emulsion, and the composition contains one or more of carboxymethyl cellulose, Tween 80, and PEG 400 as an excipient.

[0075] 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, and the inhibition is in a range of 66.98 to 88.64%.

[0076] In an example embodiment, the composition is a sustained-release formulation to enhance bioavailability and therapeutic efficacy.

[0077] The description is continued below with respect to the method of preparation of the composition.

[0078] 4. Preparation of Composition

[0079] Present disclosure discloses a method of preparation of a composition containing 1, 8-cineole as an active ingredient, for use in inhibiting viability of triple negative breast cancer (TNBC) cells.

[0080] According to an aspect of the present disclosure, the method of preparing the compositionincludes dissolving an excipient in a solvent to prepare a first solution. In an example implementation, the excipient is selected from one or more of [3-cyclodextrin, carboxymethyl cellulose, Tween 80, and PEG 400, and the solvent is selected from one or more of distilled water and ethanol. The method also includes adding an oil mixture to the first solution to prepare a second solution. The oil mixture includes one or more of Elettaria cardamomum essential oil and Salvia rosmarinus essential oil. In an example implementation, the oil mixture contains the Elettaria cardamomum essential oil, the Salvia rosmarinus essential oil and Piperine Oleoresin. The method further includes emulsifying the second solution to form an emulsion.

[0081] The description is continued below with respect to the step-by-step procedure, in an example implementation.

[0082] 4.1 Example Implementation

[0083] 4.1.1 Preparation of the Oil Mixture

[0084] Preparation of the oil mixture includes combining the Elettaria cardamomum essential oil and the Salvia rosmarinus essential oil in equal proportions by weight in a beaker, adding the Piperine Oleoresin at 1-3% of the combined weight of the Elettaria cardamomum essential oil and the Salvia rosmarinus essential oil, and stirring (for example, using a magnetic stirrer) until homogeneity is achieved / ob served.

[0085] The Elettaria cardamomum essential oil contains the 1, 8-cineole at a concentration of 45- 53% (v / w), and the Salvia rosmarinus essential oil contains the 1, 8-cineole at a concentration of 45-53% (v / w).

[0086] 4.1.2 Preparation of B-cyclodextrin Solution (the first solution)

[0087] Preparation of the [3-cyclodextrin solution includes dissolving [3-cyclodextrin, in a 1 : 1 molar ratio (i.e., wherein the [3-cyclodextrin is in a molar amount equal to a sum of moles of the Elettaria cardamomum essential oil, the Salvia rosmarinus essential oil, and the Piperine Oleoresin) to the oil mixture for encapsulating the oil mixture, in distilled water or ethanol, and heating gently to 40-50°C under continuous stirring until fully dissolved.

[0088] For the purpose of this preparation, the molecular weight of [3-CD is considered as 1135 g / mol, and the approximate molecular weight of the first solution is assumed to be -150 g / mol. For example, for a 20 g oil mixture, approximately 150 g of [3-cyclodextrin and 500 mF of distilled water are used.

[0089] 4.1.3 Formation of Inclusion Complex (adding the oil mixture to the first solution)

[0090] Formation of the inclusion complex includes adding the oil mixture, dropwise, to the [3- cyclodextrin solution, and stirring continuously to form the second solution. The formation further includes emulsifying the second solution (for example, using an ultrasonic homogenizer for 5-10 minutes at room temperature to ensure uniform dispersion of the oil mixture within the the [3-cyclodextrin solution) to form the emulsion.

[0091] In cases where the composition is in the form of powder, the process further includes the below steps.

[0092] 4.1.4 Spray Drying

[0093] Spray drying process includes feeding the emulsion into a spray dryer’s feed tank, by maintaining inlet temperature of the spray dryer between 150-180°C and outlet temperature of the spray dryer between 80-100°C, setting airflow rate and feed rate according to the specifications of the spray drying machine, and maintaining the feed rate between 5-10 mL / min.

[0094] The spray drying process further includes atomizing the emulsion into fine droplets using the spray dryer’s nozzle, after the emulsion is fed into the spray dryer’s feed tank.

[0095] The resulting powder is collected in the spray dryer's collection chamber.

[0096] 4.1.5 Collection and Storage of Powdered Formulation

[0097] The encapsulated powder is collected and, if necessary, further dried in a desiccator or vacuum oven at 40°C for 1-2 hours to remove residual moisture. The final product is stored in an airtight container protected from light and moisture to maintain stability.

[0098] 4.1.6 Key Parameters Monitored

[0099] Encapsulation efficiency is determined by measuring the amount of oil mixture encapsulated vs. free oil mixture using gas chromatography (GC). GC is also used to verify the amount of 1,8- cineole. The stability of the encapsulated powder is assessed under different storage conditions, including variations in temperature and humidity.

[0100] The description is continued below with respect to testing of the composition.

[0101] 5. Testing of the Composition

[0102] 5.1.1 Testing In-Vitro Anti-Cancer Activity of 1,8-cineole Against TNBC Cell Lines Using MTT Assay

[0103] Anti-cancer activity of 1,8-cineole against TNBC cell lines MDA MB 231, MDA MB 453 and MDA MB 468 was evaluated using an MTT assay.

[0104] MDA MB 231, MDA MB 453, and MDA MB 468 cell lines (source: National Centre for Cell Science, NCCS, Pune) were grown in medium supplemented with fetal bovine serum (FBS) (source: from #RM10432, Himedia). Doxorubicin (1 pM, Sigma) was included as a positive control. The cells were cultured in Leibovitz’s L- 15 medium supplemented with 10% FBS and 1% antibiotic-antimycotic solution under 5% CO2, 18-20% O2 at 37°C in a CO2 incubator, with respective passage numbers of 51, 35, and 61 for MDA MB 231, MDA MB 453, and MDA MB 468 cell lines. Cells were seeded at a density of 20,000 cells per well in a 96-well plate and allowed to adhere for 24 hours before treatment. C18 was administered at 1%, 1.5%, 3%, 6%, and 12%(w / v), while Doxorubicin (1 pM / mL) served as a positive control. Following incubation for 24, 48, and 72 hours, MTT reagent (0.5 mg / mL) was added, and the plates were further incubated in the dark for 3 hours. The MTT reagent was then removed, DMSO (100 pL / well) was added for solubilization, and absorbance was measured at 570 nm using an ELISA reader. Cell viability (%) (in terms of [Mean absorbance of treated cells / Mean absorbance of untreated cells] X 100) was calculated, and IC50 values were determined using linear regression analysis (Y = Mx + C, where Y = 50). The experimental results demonstrated a dose-dependent and time-dependent reduction in TNBC cell viability, as noted below for each of the cell lines.

[0105] MPA MB 231 cell line:

[0106] Average cell viability values (in percentages), based on 3 individual independent experiments, for C18 against the MDA MB 231 cells after a treatment period of 24 hrs, 48 hrs and 72 hrs are as shown below in Table-1, Table-2 and Table-3 respectively:Table- 1Table -2Table-3

[0107] MPA MB 453 cell line:

[0108] Average cell viability values (in percentages), based on 3 individual independent experiments, for Cl 8 against the MDA MB 453 cells after a treatment period of 24 hrs, 48 hrs and 72 hrs are as shown below in Table -4, Table-5 and Table-6 respectively:Table -4Table-5Table-6

[0109] MDA MB 468 cell line:

[0110] Average cell viability values (in percentages), based on 3 individual independent experiments, for C18 against the MDA MB 468 cells after a treatment period of 24 hrs, 48 hrs and72 hrs are as shown below in Table -7, Table-8 and Table -9 respectively:Table-7Table-8Table-9

[0111] Figure 1A is a graph depicting IC50 concentrations / values (in percentage) of 1,8-cineloe (Cl 8) required to inhibit cell viability by 50% in three triple-negative breast cancer (TNBC) cell lines, MDA MB 231, MDA MB 453, and MDA MB 468 cells following 24hrs, 48hrs, and 72hrs of treatment, as measured by MTT assay. From Figure 1A, it may be observed that C18 exhibits significant cytotoxicity with low IC50 values of 1.86+0.05%, 1.16+0.10%, and 0.28+0.06% in MDA MB 231; 2.82+0.03%, 1.73+0.07%, and 0.79+0.04% in MDA MB 453; and 2.11+0.06%, 1.35+0.09%, and 0.83+0.06% in MDA MB 468, for 24hrs, 48hrs, and 72hrs treatments, respectively. The results indicate that MDA MB 231 is the most sensitive to C18 as indicated by lower IC50 values.

[0112] Figure IB is a graph depicting percentage cell viability of MDA MB 231, MDA MB 453, and MDA MB 468 cells, following 24hrs, 48hrs and 72hrs treatment with Cl 8 at a concentration of 12% (w / v). It is understood that the graph is plotted for Cl 8 concentration of 12% (i.e., highest concentration used in the tests) only for illustrative purposes.

[0113] Figure 1C is a graph depicting percentage cell viability in MDA MB 231, MDA MB 453,and MDA MB 468 cells for different concentrations of Cl 8. Figure 1C also depicts the corresponding values for Dox-luM. It is understood that the graph is plotted for the treatment period of 24hrs only for illustrative purposes. The data shows a decrease in cell viability was with increasing Cl 8 concentrations, indicating a dose-dependent cytotoxic effect.

[0114] 5.1.2 Testing In-Vitro Anti-Cancer Activity of Recovereez Forte Against TNBC Cell Lines Using MTT Assay

[0115] Anti-cancer activity of Recovereez Forte (RF) against TNBC cell lines MDA MB 231, MDA MB 453 and MDA MB 468 was evaluated using an MTT assay.

[0116] MDA MB 231, MDA MB 453, and MDA MB 468 cell lines (source: National Centre for Cell Science, NCCS, Pune) were grown in medium supplemented with fetal bovine serum (FBS) (source: from #RM10432, Himedia). Doxorubicin (1 pM, Sigma) was included as a positive control. The cells were cultured in Leibovitz’s L- 15 medium supplemented with 10% FBS and 1% antibiotic-antimycotic solution under 5% CO2, 18-20% O2 at 37°C in a CO2 incubator, with respective passage numbers of 49, 32 and 59 for MDA MB 231, MDA MB 453, and MDA MB 468 cell lines. Cells were seeded at a density of 20,000 cells per well in a 96-well plate and allowed to adhere for 24 hours before treatment. RF was administered at 1.5, 3, 6, 12 and 24pg / ml (in powder form), while Doxorubicin (1 pM / mL) served as a positive control. Following incubation for 24, 48, and 72 hours, MTT reagent (0.5 mg / mL) was added, and the plates were further incubated in the dark for 3 hours. The MTT reagent was then removed, DMSO (100 pL / well) was added for solubilization, and absorbance was measured at 570 nm using an ELISA reader. Cell viability (%) (in terms of [Mean absorbance of treated cells / Mean absorbance of untreated cells] X 100) was calculated, and IC50 values were determined using linear regression analysis (Y = Mx + C, where Y = 50). The experimental results demonstrated a dose-dependent and time-dependent reduction in TNBC cell viability, as noted below for each of the cell lines.

[0117] MDA MB 231 cell line:

[0118] Average cell viability values (in percentages), based on 3 individual independent experiments, for RF against the MDA MB 231 cells after a treatment period of 24 hrs, 48 hrs and 72 hrs are as shown below in Table- 10, Table- 11 and Table- 12 respectively:Table- 10Table- 11Table- 12

[0119] MPA MB 453 cell line:

[0120] Average cell viability values (in percentages), based on 3 individual independent experiments, for RF against the MDA MB 453 cells after a treatment period of 24 hrs, 48 hrs and 72 hrs are as shown below in Table-13, Table-14 and Table-15 respectively:Table- 13Table- 14Table- 15

[0121] MDA MB 468 cell line:

[0122] Average cell viability values (in percentages), based on 3 individual independent experiments, for RF against the MDA MB 468 cells after a treatment period of 24 hrs, 48 hrs and 72 hrs are as shown below in Table-16, Table-17 and Table-18 respectively:Table- 16Table- 17Table- 18

[0123] Figure ID is a graph depicting IC50 concentrations / values (in percentage) of RF required to inhibit cell viability by 50% in three triple-negative breast cancer (TNBC) cell lines, MDA MB 231, MDA MB 453, and MDA MB 468 cells following 24hrs, 48hrs, and 72hrs of treatment, as measured by MTT assay. From Figure ID, it may be observed that RF exhibits significant cytotoxicity with low IC50 values of 6.99+1.22 pg / mL, 3+0.09 pg / mL, and 1.63+0.08 pg / mL in MDA MB 231 ; 7.58+0.89 pg / mL, 2.24+1.20 pg / mL, and 0.17+0.06pg / mL in MDA MB 453; and 15. 17+3. 14pg / mL, 3.17+0.40pg / mL, and 0.87+0.05pg / mL in MDA MB 468, for 24hrs, 48hrs, and 72hrs treatments, respectively.

[0124] Figure IE is a graph depicting percentage cell viability of MDA MB 231, MDA MB 453, and MDA MB 468 cells, following 24hrs, 48hrs and 72hrs treatment with RF at a concentration of 24pg / ml. It is understood that the graph is plotted for RF concentration of 24pg / mL (i.e., highest concentration used in the tests) only for illustrative purposes.

[0125] Figure IF is a graph depicting percentage cell viability in MDA MB 231, MDA MB 453, and MDA MB 468 cells for different concentrations of RF. Figure IF also depicts the corresponding values for Dox-lpM. It is understood that the graph is plotted for the treatment period of 24hrs only for illustrative purposes. The data shows a decrease in cell viability was with increasing RF concentrations, indicating a dose-dependent cytotoxic effect. It is understood that the graph is plotted for the treatment period of 24hrs only for illustrative purposes.

[0126] In an embodiment, the compositions for targeting triple -negative breast cancer (TNBC) cells primarily consist of Recovereez Forte (RF) as the active ingredient, formulated with pharmaceutically acceptable carriers. These carriers may include solvents such as dimethyl sulfoxide (DMSO), ethanol, or phosphate-buffered saline (PBS), tailored to ensure optimal solubility and stability of RF. Recovereez Forte (RF) is present in the compositions at concentrations ranging from 0.1% to 5% (w / v), with specific percentages depending on the carrier and formulation requirements.

[0127] In one embodiment, the composition may comprise RF dissolved or suspended in a solvent such as DMSO, with RF concentrations ranging from 0.5% to 4% (w / v). Alternatively, RF may be encapsulated within liposomes, micelles, or nanoparticles, with concentrations ranging from 0.1% to 2% (w / v). These formulations offer versatility in delivery and enhance RF's bioavailability and targeted delivery to TNBC cells. The compositions are prepared through a methodical process, involving the mixing of RF with the pharmaceutically acceptable carrier under sterile conditions to ensure product quality and safety. Sterilization techniques such as filtration or autoclaving may be employed to eliminate microbial contaminants, thereby meeting regulatory standards for pharmaceutical products.

[0128] The cytotoxicity studies involved seeding monolayer cell cultures with adjusted cell counts of 1.0 x 105cells / ml using respective media containing 10% FBS. After 24 hours, the supernatant was removed, and different concentrations of test substances were added to the wells.

[0129] 5.2 In vitro screening of Apoptosis Activity of Recovereez Forte and 1,8-cineole Against TNBC Cell Lines using Apoptosis Assay

[0130] In vitro screening of apoptosis activity of Recovereez Forte and 1,8-cineole against TNBC cell lines MDA MB 231, MDA MB 453 and MDA MB 468 was evaluated using annexin V staining method followed by flow cytometry analysis. MDA MB 231, MDA MB 453, and MDA MB 468 cell lines (source: National Centre for Cell Science, NCCS, Pune) were grown in medium supplemented with fetal bovine serum (FBS) (source: from #RM10432, Himedia). The cells were cultured in Leibovitz’s L-15 medium supplemented with 10% FBS and 1% antibiotic-antimycotic solution under 5% CO2, 18-20% O2 at 37°C in a CO2 incubator, with respective passage numbers of 52, 36 and 62 for MDA MB 231, MDA MB 453, and MDA MB 468 cell lines. Doxorubicin (1 pM / mL) (source: Cat No: D1515, Sigma) served as a positive control. RF and C18 was administered at their respective IC50 concentrations for 24h, 48h and 72h as detailed in Table-19 below.Table- 19

[0131] Following incubation, cells were harvested using trypsin-EDTA, washed with Dulbecco's Phosphate Buffered Saline (D-PBS) (source: #TL1006, Himedia), and resuspended in Annexin V binding buffer. FITC Annexin V (source: Cat No: 51-65874X, BD Biosciences) was added to the cell suspension and incubated in the dark at room temperature (25 °C) for 15 minutes. Then, Propidium Iodide (PI) (source: Cat No. 51-662 HE, BD Biosciences) was added to stain the cells to distinguish necrotic cells from cells undergoing apoptosis. The stained cells were then analyzed using flow cytometry. The experimental results demonstrated time -dependent apoptotic induction by Cl 8 and RF in TNBC cells are as noted below for each of the cell lines.

[0132] MPA MB 231 cell line:

[0133] The results of Annexin V / PI expression study in MDA MB 231 cell line after treatment periods of 24 hrs, 48 hrs and 72 hrs (with corresponding IC 50 concentrations) are as shown below in Table-20, Table -21 and Table-22 respectively:Table-20Table-21Table-22

[0134] MDA MB 453 cell line:

[0135] The results of Annexin V / PI expression study in MDA MB 453 cell line after treatment periods of 24 hrs, 48 hrs and 72 hrs (with corresponding IC 50 concentrations) are as shown belowin Table-23, Table -24 and Table-25 respectively:Table-23Table-24Table-25

[0136] MPA MB 468 cell line:

[0137] The results of Annexin V / PI expression study in MDA MB 468 cell line after treatment periods of 24 hrs, 48 hrs and 72 hrs (with corresponding IC 50 concentrations) are as shown below in Table-26, Table -27 and Table-28 respectively:Table-26| C-18 1.35% | 5.77 | 67.42 | 23.76 | 3.05 |Table-27Table-28

[0138] Figure 2A is a graph depicting apoptosis (in percentage) for corresponding IC 50 concentrations of Recovereez Forte (RF) and C18 in MDA MB 231 cells for different treatment periods. It is understood that apoptosis percentage includes the summation of late apoptosis percentage and early apoptosis percentage. The results are compared to an untreated control and a Doxorubicin (1 pM) positive control.

[0139] Figure 2B is a graph depicting apoptosis (in percentage) for corresponding IC 50 concentrations of Recovereez Forte (RF) and Cl 8 in MDA MB 453 cells for different treatment periods. It is understood that apoptosis percentage includes the summation of late apoptosis percentage and early apoptosis percentage. The results are compared to an untreated control and a Doxorubicin (1 pM) positive control.

[0140] Figure 2C is a graph depicting apoptosis (in percentage) for corresponding IC 50 concentrations of Recovereez Forte (RF) and Cl 8 in MDA MB 468 cells for different treatment periods. It is understood that apoptosis percentage includes the summation of late apoptosis percentage and early apoptosis percentage. The results are compared to an untreated control and a Doxorubicin (1 pM) positive control.

[0141] The observations suggested that the given test compounds significantly enhanced the apoptosis in human breast cancer cells on time dependent manner.

[0142] The compound, RF caused 23%, 32% and 45% apoptosis and 0.07%, 6.86% and 20.89% of Necrosis was observed on MDA MB 231 cells after the treatment period of 24, 48 and 72hours respectively.

[0143] The compound, RF caused 50.69%, 74.49% and 85.63% apoptosis and 5.23%, 4.38% and 4.19% of Necrosis was observed on MDA MB 453 cells after the treatment period of 24, 48 and 72hours respectively.

[0144] The compound, RF caused 60.42%, 69.78% and 70.47% apoptosis and 3.11%, 6.47% and 45.85% of Necrosis was observed on MDA MB 468 cells after the treatment period of 24, 48 and 72hours respectively.

[0145] The compound C18 caused 19%, 51.68% and 43.66% apoptosis and 15.15%, 12.14% and28.31% of Necrosis was observed on MDA MB 231 cells after the treatment period of 24, 48 and 72hours respectively.

[0146] The compound C18 caused 45.77%, 75.13% and 83.65% apoptosis and 5.48%, 0.24% and 4.96% of Necrosis was observed on MDA MB 453 cells after the treatment period of 24, 48 and 72hours respectively.

[0147] The compound, C18 caused 66.98%, 70.47% and 88.64% apoptosis and 7.42%, 5.77% and 2.86% of Necrosis was observed on MDA MB 468 cells after the treatment period of 24, 48 and 72hours respectively.

[0148] The reference std, Doxorubicin caused 30.18%, 43.37% and 47.92% apoptosis and 8.06%, 15.81% and 20.29% of Necrosis was observed on MDA MB 231 cells after the treatment period of 24, 48 and 72hours respectively.

[0149] The reference std, Doxorubicin caused 29.1%, 67.96% and 84.14% apoptosis and 5.38%, 8.04% and 2.66% of Necrosis was observed on MDA MB 453 cells after the treatment period of 24, 48 and 72hours respectively.

[0150] The reference std, Doxorubicin caused 50.51%, 60.42% and 79.57% apoptosis and 8.46%, 2.27% and 3.46% of Necrosis was observed on MDA MB 468 cells after the treatment period of 24, 48 and 72hours respectively.

[0151] In summary, the observed Apoptosis / Necrosis study results revealed that RF and CF caused effective apoptotic potential on time dependent fashion on all Human breast cancer cells like the std drug, Doxorubicin. However, the observed apoptosis rate in MDA MB 453 and 468 cells is higher than that in the MDA MB 231 cells.

[0152] 5.3 In vivo Anticancer Activities of Recovereez Forte (RF) against Ehrlich Ascites Carcinoma (EAC) & Dalton’s Ascites Lymphoma (DAL) Cells in Swiss Albino Mice

[0153] In vivo anti-cancer activities of Recovereez Forte (RF) against EAC and DAL cells inoculated into Swiss albino mice were evaluated.

[0154] Anti-cancer activities of Recovereez Forte (RF) at concentrations of 200 mg / kg and 400 mg / kg respectively were studied against Ehrlich Ascites Carcinoma (EAC) and Dalton’s ascites lymphoma (DAL) cells in Swiss albino mice by monitoring parameters such as tumor size, tumor volume and body weight measurement, survival time of tumor bearing mice. Along with serum antioxidant (SOD, CAT, MDA) levels and Liver (AST, ALT) levels, histopathology of liver and kidney tissue, and hematological parameters, such as red blood cells, white blood cells, and hemoglobin content, were also measured. All the treatment groups were compared with 5- fluorouracil used as positive control group.

[0155] Experimental animal:

[0156] Swiss albino mice of 5 weeks to 7 weeks old, weighing 20 g to 30 g were procured from the central animal facility, Hanagal Shri Kumareshwar college of Pharmacy, Bagalkot. The mice were kept in iron cages with sawdust and straw bedding that was changed once a week regularly. Standard mouse diet and water ad libitum, under the standard conditions in accordance with CPCSEA (Committee for the purpose of control and supervision of experiments on animals) guidelines for the care and use of laboratory animals. The protocol used in this study for the use of mice as the animal model for research was approved by HKCP / IAEC, Clear / 1 / 2022- 23 / Ph. D6.

[0157] In-vivo Studies:

[0158] Swiss albino mice were acclimatized for 7 days in a controlled environment. The mice were then divided into 5 groups, with 6 animals in the normal control group and 8 animals in each of the remaining groups. All groups, except Group I (Normal Control), were injected with 0.2 ml (136 x 104 EAC cells) of EAC cells. After 24 hours, animals from Group III and Group IV were treated with the Recovereez Forte (RF) at concentrations of 200 mg / kg and 400 mg / kg, per organism (p.o.), respectively. Group V animals were treated with 5-FU (20 mg / kg, p.o.,) as a standard for 21 days. Changes in body weight were recorded for every seven days. The survival rate was also tracked and expressed as mean survival time (MST) and percent increase of life span (%ILS). On day 21st, the animals were anesthetized and tested for haematological parameters such as red blood cell (RBC) count, White blood cell (WBC) count and Haemoglobin (Hb) content. Also serum samples were evaluated for Aspartate Transaminase (AST), Alanine Transaminase (ALT) & antioxidant potential by observing the levels of Superoxide dismutase (SOD), Catalase (CAT) & Malondialdehyde (MDA). On the 21st day, all mice were sacrificed, and livers and kidneys were isolated for histopathological studies.

[0159] The treatment protocol for DAL cell inoculated model is summarized below in Table-29:Table-29

[0160] Blood was withdrawn from each mouse by retro orbital puncture blood collecting method. From the collected blood hematological parameters were checked, the remaining blood was centrifuged, and serum was used for the estimation of biochemical parameters viz., AST & ALT.

[0161] Hematological parameters: At the time of dissection, blood was collected from each group of experimental animals, and it was used for the estimation counts for red (RBC), white blood cells(WBC) and hemoglobin (Hb) content, were performed. The animals were anesthetized, and blood was collected for analysis.

[0162] RBC Count: Red Blood Cells (RBC) count is determined by diluting a blood sample with Hayem’s fluid, which prevents haemolysis and rouleaux formation, and stains the nuclei of RBCs. The diluted blood is then loaded into a hemacytometer and allowed to settle for 3-4 minutes. The cells are then viewed under a microscope at 40X magnification. The cells in 4 corners and 1 central square of 16 small squares each, i.e., a total of 80 small squares in the Neubauer’s chamber, are counted on both sides of the hemacytometer and an average is calculated. The total RBC count is then calculated as the average number of RBC X 106cells / pl.

[0163] WBC Count: White Blood Cells (WBC) count is determined by diluting a blood sample with Turk’s fluid, which destroys RBCs and stains the nuclei of WBCs. The diluted blood is then loaded into a hemacytometer and allowed to settle for 3-4 minutes. The cells are then viewed under a microscope at 10X magnification. The cells in 4 corners of 16 small squares each, i.e., a total of 64 small squares in the Neubauer’s chamber, are counted on both sides of the hemacytometer and an average is calculated. The total WBC count is then calculated as the average number of cells X 103cells / pl.

[0164] Hb Content: Haemoglobin (Hb) content is determined by converting Hb into acid hematin using dil. HC1 (0.1 N). This results in a brown solution, the intensity of which indicates the amount of acid hematin and thus the Hb concentration. The colour is compared to a brown-tinted glass filter to get the reading in gm / lOOml blood. The procedure involves drawing blood into a pipette, expelling it into a Sahli tube containing 0.1N HC1, and allowing it to stand for 8-10 minutes. During this time, the acid ruptures the red cells and releases haemoglobin, which is converted into hematin.

[0165] The tube is then removed from the comparator, distilled water is added, and the colour is checked against the standard glass. The reading on the Sahli tube gives the Hb content in gm / 100 ml blood.

[0166] Serum liver biochemical enzyme:

[0167] Alanine Transaminase (ALT): Alanine Transaminase (ALT or GPT) is an enzyme that facilitates the transfer of an amino group from alanine to 2-oxoglutarate, resulting in the formation of pyruvate and glutamate. The concentration of ALT is determined by measuring the rate of decrease of NADH at a wavelength of 340 nm. A serum sample of 25 pl was mixed with 500 pl of Working Reagent and loaded into a semi-autoanalyzer. A blank reading was taken using water instead of the serum sample. After 3 minutes of incubation, the preset absorbance was read using the semi autoanalyzer. The result, given as U / l of ALT in each sample, was calculated by repeating these steps for at least three replicates to obtain the mean and standard error mean of ALT.

[0168] Aspartate Transaminase (AST): Aspartate Transaminase (AST or GOT) is an enzyme that facilitates the transfer of the amino group from aspartate to 2-oxoglutarate, resulting in the formation of oxaloacetate and glutamate. The concentration of AST is determined by measuring the rate of decrease of NADH at a wavelength of 340 nm. A serum sample of 25 pl was mixed with 500 pl of Working Reagent and loaded into a semi-autoanalyzer. A blank reading was taken using water instead of the serum sample. After 3 minutes of incubation, the preset absorbance was read using the semi-autoanalyzer. The result, given as U / l of ALT in each sample, was calculated by repeating these steps for at least three replicates to obtain the mean and standard error mean of ALT.

[0169] Derived parameters

[0170] Body weight

[0171] Measurement of Mean survival time (MST) and % increase in Life span (%ILS): Five groups of mice (6 in the normal control group and 8 in each of the remaining groups) were used for the experiment. A total of 136 x 104 EAC cells were inoculated in each mouse on day zero. The treatment was initiated after 24 h of tumor cell inoculation and continued for 10 days. The weight changes in each mouse were recorded daily, and the increase in tumor weight was monitored. The host survival was recorded and expressed as the mean of survival time in days. The percent increase in the life span was calculated, using the following formula:

[0172] Antioxidant assays

[0173] At the end of the experimental period, the animals will be dissected, and liver will be excised. It was rinsed in ice-cold normal saline solution followed by cold 0.15 M Tris-HCl (pH 7.4), bottle dried and weighed. A 10% w / v homogenate will be prepared in 0.15 M Tris HC1 buffer and the homogenate will be centrifuged at 1500 rpm for 15 min at 4°C. The supernatant was collected and used for the estimation of Superoxide dismutase (SOD), Catalase (CAT), and Malondialdehyde (MDA).

[0174] Antioxidant Parameters:

[0175] Superoxide Dismutase (SOD):

[0176] Superoxide Dismutase (SOD) is an enzyme that catalyzes the conversion of superoxide into oxygen and hydrogen peroxide. The concentration of SOD is determined by the rate of decrease in Nitro Blue Tetrazolium, measured at 340 nm, in the presence of Phenazine Methosulphate and NADH.

[0177] The procedure involves the following steps: A mixture is prepared with 0.1ml of supernatant, 1.2 ml of sodium pyrophosphate buffer, 0.1 ml of Phenazonium methosulphate, 0.3 ml of Nitro Blue Tetrazolium, and 0.2 ml of NADH. The mixture is incubated for 90 seconds at 30°C.The reaction is stopped by adding 0.1ml of glacial acetic acid. The mixture is stirred vigorously and shaken with 4.0 ml of n-butanol and then centrifuged at 4000 rpm for 10 minutes. The absorbance of the organic layer is measured at 560 nm against a blank. The enzyme activity is defined as the enzyme concentration required to inhibit the absorbance of chromogen production by 50% in a control sample under the assay conditions. The SOD estimation is calculated using the formula:Control O.D - Experimental O.D x Df / (Control absorbance / 2) x 1 / Protein in mg = Units / mg protein, where O.D = optical density; Df = Dilution factor.

[0178] Catalase (CAT): Catalase (CAT) activity is measured by its ability to oxidize hydrogen peroxide (H2O2) into water and oxygen. The UV light absorption of the hydrogen peroxide solution can be measured between 230 to 250 nm, and the difference in absorbance per unit time indicates the catalase activity.

[0179] The procedure involves the following steps:

[0180] Liver and kidney tissues are homogenized in ice-cold tris-buffer to produce a 10% w / v homogenate. 0.1 ml of the supernatant is added to a test tube containing 1.9 ml of 50 mM phosphate buffer. To this mixture, 1.0 ml of freshly prepared 30 mM H2O2 is added and the change in absorbance is measured for 3 minutes at 240 nm at an interval of 30 seconds. A blank is prepared using distilled water instead of the homogenate. One unit of enzyme activity is defined as the enzyme concentration required to inhibit the change in absorbance by 50% in one minute in the control sample. The activity of catalase is expressed as pg / Moles of H2O2 metabolized / mg of protein / minl2. The catalase estimation is calculated using the formula:Absorbance x Volume of Reaction Mixture CAT = _ - _43.6 x Volume of Sample

[0181] Malondialdehyde (MDA): The process involves the reaction of oxygen free radicals with lipids in the membrane to create lipid peroxidase. This is then heated with Thiobarbituric Acid TBA) and acetic acid, producing a chromogen. The chromogen is extracted using a combination of n-butanol and pyridine, and the absorbance of the organic phase is measured at a wavelength of 532 nm. The procedure includes preparing various solutions and a standard graph using Malondialdehyde-bisdiethyl-acetal (TEP). The reactants are heated in an oil bath, and the resulting chromogen is extracted, and its absorbance measured.

[0182] For the sample procedure, tissue is homogenized and treated with sodium dodecyl sulphate, acetic acid, and thiobarbituric acid. The mixture is heated, cooled, and then mixed with n-butanoland pyridine. After centrifugation, the organic layer’s absorbance is measured. The tissue Malondialdehyde (MDA) level is measured from the standard curve and expressed as nmol / g wet tissue.

[0183] Formula for the estimation of MDA in tissue:Concentration of MDA in sample: x = y - 0.0162 / 0.065

[0184] Histopathology of liver and kidney tissues:

[0185] A portion of liver and kidney tissues were fixed overnight in a 10 % neutral formaldehyde solution. Tissue slices were subjected to haematoxylin and eosin staining and histologic study by light microscopy. Slides were coded and examined for the grading of histopathological alterations.

[0186] Statistical analysis:

[0187] Results were expressed as mean ± SEM (n=6). Experiments were evaluated statistically with one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test; p<0.05 was considered statistically significant compared to EAC / DAL control group.

[0188] Effect of Recovereez Forte (RF) in EAC model:

[0189] The effect of RF on change in body weight (EAC model) is as noted below in Table-30. All data were expressed as mean ± SEM (n=6). The parameters were analyzed by one way ANOVA followed by Dunnett’s test for multiple comparison, *p<0.05, **p<0.01 and ***p<0.001 is considered significant compared with EAC control group.Table-30

[0190] The effect of RF on MST & %ILS (EAC model) is as noted below in Table-31. All data were expressed as mean ± SD (n=6). The parameters were analyzed by one way ANOVA followed by Dunnett’s test for multiple comparison, *p<0.05, **p<0.01 and ***p<0.001 is considered significant compared with positive control group.Table-31

[0191] The effect of RF on RBC, WBC and Hb (EAC model) is as noted below in Table-32. All data were expressed as mean ± SEM (n=6). The parameters were analyzed by one way ANOVA followed by Dunnett’s test for multiple comparison, *p<0.05, **p<0.01 and ***p<0.001 is considered significant compared with EAC control group.Table-32

[0192] The effect of RF on AST and ALT (EAC model) is as noted below in Table-33. All data were expressed as mean ± SD (n=6). The parameters were analysed by one way ANOVA followed by Dunnett’s test for multiple comparison, *p<0.05, **p<0.01 and ***p<0.001 is considered significant compared with EAC control group.

[0193] The effect of RF on serum antioxidants (EAC model) is as noted below in Table-34. All data were expressed as mean ± SEM (n=6). The parameters were analyzed by one way ANOVA followed by Dunnett’s test for multiple comparison, ***p<0.001 is considered significant compared with EAC control groups.Table-34

[0194] The histopathology of liver and kidney tissues for EAC-induced mice models are illustrated in Figures 3A to 3J. The effect of Recovereez Forte at two separate doses - 200mg / kg and 400 mg / kg as well as 5-FU (20mg / kg) has been studied. The left panel depicts the overall tissue architecture and the condition of the liver (hepatocytes) or kidney (glomerulus) tissue, and the right panel depicts the vascular and structural integrity of the tissues.

[0195] Figure 3A is an image depicting healthy liver of untreated normal mice (i.e., Normal control group - liver parenchyma). The normal control group liver parenchyma shows intact perivenular hepatocytes, periportal hepatocytes and midzonal hepatocytes. The central vein and sinusoids appear within normal limits.

[0196] Figure 3B is an image depicting damaged liver of untreated control mice in the EAC model (i.e., EAC control group - liver parenchyma). The periportal, perivenular and midzonal hepatocytes show degenerative changes. The periportal zone shows mild inflammation. The central vein appears congested and sinusoids appear dilated.

[0197] Figure 3C is an image depicting therapeutic effect of RF (200 mg / kg) on the liver of EAC model mice (i.e., Recovereez Forte (200 mg / kg, p.o.,) - liver parenchyma). The periportal zone shows moderate to dense inflammation, but the central vein and sinusoids appear within normal limits. The periportal, perivenular, and midzonal hepatocytes show degenerative changes with moderate inflammation.

[0198] Figure 3D is an image depicting therapeutic effect of RF (400 mg / kg) on the liver of EAC model mice (i.e., Recovereez Forte (400 mg / kg, p.o.,) - liver parenchyma). The perivenular and midzonal hepatocytes show focal degenerative changes, while the periportal hepatocytes appear intact. The central vein and sinusoids appear congested.

[0199] Figure 3E is an image depicting effect of standard treatment with 5-FU (25 mg / kg), showing a liver structure similar to the normal control (i.e., 5-FU (25 mg / kg, p.o.,) - liver parenchyma). The perivenular hepatocytes, periportal hepatocytes, and midzonal hepatocytes appear intact, with the central vein and sinusoids remaining within normal limits.

[0200] Figure 3F is an image depicting healthy kidney of untreated normal mice (i.e., Normal control group - kidney). The normal control group kidney tissue shows normal glomerular cellularity, intact tubules, and blood vessels within normal limits. The interstitium also appears normal.

[0201] Figure 3G is an image depicting damaged kidney of untreated control mice in the EAC model (i.e., EAC control group - kidney). The glomerulus exhibits hypercellularity, and the tubules show moderate necrosis of epithelial cells. Mild congestion is observed in the blood vessels, while the interstitium shows mild inflammation.

[0202] Figure 3H is an image depicting therapeutic effect of RF (200 mg / kg) on the kidney of EAC model mice (i.e., Recovereez Forte (200 mg / kg, p.o.,) - kidney). The glomerulus retains normal cellularity, while the tubules exhibit moderate necrosis of epithelial cells. Mild congestion is observed in the blood vessels, but the interstitium remains within normal limits.

[0203] Figure 31 is an image depicting therapeutic effect of RF (400 mg / kg) on the kidney of EAC model mice (i.e., Recovereez Forte (400 mg / kg, p.o.,) - kidney). The glomerulus retains normal cellularity, the tubules remain intact, and the interstitium exhibits mild inflammation. Moderate congestion is observed in the blood vessels.

[0204] Figure 3J is an image depicting effect of standard treatment with 5-FU (25 mg / kg), showing a kidney structure similar to the normal control (i.e., 5-FU (20 mg / kg, p.o.,) - kidney). The glomerulus exhibits normal cellularity, the tubules remain intact, the blood vessels show mild congestion, and the interstitium remains within normal limits.

[0205] Effect of Recovereez Forte (RF) in DAL model:The effect of RF on change in body weight (DAL model) is as noted below in Table-35. All data were expressed as mean ± SEM (n=6). The parameters were analyzed by one way NOVA followed by Dunnett’s test for multiple comparison, *p<0.05, **p<0.01 and ***p<0.001 is considered significant compared with DAL control group.Table-35

[0206] The effect of RF on MST and %ILS (DAL model) is as noted below in Table-36. All data were expressed as mean ± SD (n=6). The parameters were analyzed by one way ANOVA followed by Dunnett’s test for multiple comparison, *p<0.05, **p<0.01 and ***p<0.001 is considered significant compared with DAL control group.Table-36

[0207] The effect of RF on RBC, WBC and Hbg (DAL model) is as noted below in Table-37. All data were expressed as mean ± SEM (n=6). The parameters were analyzed by one way ANOVA followed by Dunnett’s test for multiple comparison, *p<0.05, **p<0.01 and ***p<0.001 is considered significant compared with DAL control group.Table-37

[0208] The effect of RF on tumor weight and tumor volume (DAL model) is as noted below in Table-38. All data were expressed as mean ± SEM (n=6). The parameters were analyzed by one way ANOVA followed by Dunnett’s test for multiple comparison, ***p<0.001 is considered significant compared with DAL control group.

[0209] The effect of RF on AST and ALP (DAL model) is as noted below in Table-39. All data were expressed as mean ± SEM (n=6). The parameters were analyzed by one way ANOVA followed by Dunnett’s test for multiple comparison, ***p<0.001 is considered significant compared with DAL control group.

[0210] The effect of RF on serum antioxidants (DAL model) is as noted below in Table -40. All data were expressed as mean ± SEM (n=6). The parameters were analyzed by one way ANOVAfollowed by Dunnett’s test for multiple comparison, ***p<0.001 is considered significant compared with dal control groups.Table-40

[0211] The histopathology of liver and kidney tissues for DAL-induced mice models are illustrated in Figures 4A to 4J. The effect of Recovereez Forte at two separate doses - 200mg / kg and 400 mg / kg as well as 5-FU (20mg / kg) has been studied. The left panel depicts the overall tissue architecture and the condition of the liver (hepatocytes) or kidney (glomerulus) tissue, and the right panel depicts the vascular and structural integrity of these tissues.

[0212] Figure 4A is an image representing the healthy liver of untreated normal mice (i.e., Normal Control Group). Arrow on the left-hand side image indicates section studied shows liver parenchyma with intact architecture. The periportal, perivenular and midzonal hepatocytes appear intact. Arrow on the right-hand side image indicates the central vein appears and sinusoids appear within normal limits.

[0213] Figure 4B is an image representing the damaged liver of untreated control mice in the DAL model (i.e., DAL- induced Group). Arrow on the left-hand side image indicates the section studied from liver parenchyma shows intact architecture. The periportal, perivenular and midzonal hepatocytes show moderate degenerative changes. The periportal zone shows moderate inflammation. Arrow on the right-hand side image depicts the central vein appears congested and sinusoids appear dilated.

[0214] Figure 4C is an image representing the therapeutic effect of RF (200 mg / kg) on the liver of DAL model mice (i.e., Recovereez Forte (RF) (200 mg / kg, p.o.,)). Arrow on the left-hand side image indicates the section studied from liver parenchyma shows intact architecture. The periportal, perivenular and midzonal hepatocytes show mild degenerative changes. The periportal zone shows mild inflammation. Arrow on the right-hand side image indicates the central vein appears and sinusoids appear within normal limits.

[0215] Figure 4D is an image representing the therapeutic effect of RF (400 mg / kg) on the liver of DAL model mice (Recovereez Forte (RF) (400 mg / kg, p.o.,)). Arrow on the left-hand side image indicates the section studied from liver parenchyma shows intact architecture. The perivenular andmidzonal hepatocytes show focal degenerative changes. The periportal zone appears intact. Arrow on the right-hand side image indicates the central vein appears and sinusoids appear congested.

[0216] Figure 4E is an image representing the effect of standard treatment with 5-FU (25 mg / kg) (5-FU (20 mg / kg, p.o..,)), showing a liver structure with moderate inflammation compared to the normal untreated controls. Arrow on the left-hand side image indicates section studied shows liver parenchyma with intact architecture. The periportal, perivenular and midzonal hepatocytes appear intact. The periportal zone shows moderate inflammation. Arrow on the right-hand side image indicates the central vein appears and sinusoids appear congested at places.

[0217] Figure 4F is an image representing the healthy kidney of untreated normal mice (i.e., Normal control group). Architecture remains intact. Arrow on the left-hand side image glomerulus shows normal cellularity. Arrow on the right-hand side image shows tubules remain intact. Blood vessels and interstitium are within normal limits.

[0218] Figure 4G is an image representing the damaged kidney of untreated control mice in the DAL model (DAL- Induced). Arrow on the left-hand side image indicates glomerulus shows hyper cellularity. Arrow on the right-hand side image shows tubules having moderate necrosis of epithelial cells. Blood vessels and interstitium are within normal limits.

[0219] Figure 4H is an image representing the therapeutic effect of RF (200 mg / kg) on the kidney of DAL model mice (Recovereez Forte (RF) (200 mg / kg, p.o.,)). Architecture remains intact. Arrow on the left-hand side image indicates glomerulus shows normal cellularity. Arrow on the right-hand side image shows tubules having mild to moderate necrosis of epithelial cells. Blood vessels also show moderate congestion and interstitium having mild inflammation.

[0220] Figure 41 is an image representing the therapeutic effect of RF (400 mg / kg) on the kidney of DAL model mice (Recovereez Forte (RF) (400 mg / kg, p.o.,)). Arrow on the left-hand side image indicates glomerulus shows normal cellularity. Arrow on the right-hand side image shows tubules having mild necrosis of epithelial cells. Blood vessels are within normal limits and interstitium shows moderate to dense inflammation.

[0221] Figure 4J is an image representing the effect of standard treatment with 5-FU (25 mg / kg) (5-FU (20 mg / kg, p.o.,)), showing a kidney structure similar to the normal control. Architecture remains intact. Arrow on the left-hand side image indicates glomerulus shows normal cellularity. Arrow on the right-hand side image shows tubules remain intact and blood vessels shows moderate congestion. Interstitium is within normal limits.

[0222] FIG. 5A is a graph depicting the effect of Recovereez Forte (RF) on tumor volume in Dalton’s Lymphoma Ascites (DAL) model. The tumor volume (mm3) was measured following oral administration of RF at doses of 200 mg / kg and 400 mg / kg, with 5-Fluorouracil (5-FU, 20 mg / kg) used as a standard control.

[0223] FIG. 5B is a graph depicting the effect of Recovereez Forte (RF) on mean survival time(MST) and percentage increase in life span (%ILS) in tumor-bearing mice in both the Dalton’s Lymphoma Ascites (DAL) and Ehrlich Ascites Carcinoma (EAC) models. The MST (in days) was recorded for RF-treated groups at doses of 200 mg / kg and 400 mg / kg, with 5-Fluorouracil (5-FU, 20 mg / kg) as a standard control.

[0224] Thus, the results showed that RF has a positive effect against EAC and DAL cells. An assessment was conducted by comparing these results with those obtained using the standard drug 5- Fluorouracil. Our study revealed that RF significantly (p<0.001) reduces cancer cell growth in both in vivo models (EAC & DAL). In EAC model, RF at both the doses (200 and 400 mg / kg) significantly (p<0.001 ) improved the MST in tumor bearing mice.

[0225] The RF was also found effective in increasing the MST in EAC inoculated mice. However, drastic fall in body weight was observed in mice treated with the higher dose (400 mg / kg) of RF fraction compared to their zero-day reading and EAC control group mice on last 12th day and 14th day data, though no visible signs of toxicity and change in vital functions were observed in any of treated animals. Prolongation of life span is a reliable criterion forjudging the anticancer efficacy of compounds (Hogland, 1982) and the RF 400 mg / kg meets this criterion. Previous studies showed that Cassia fistula (Gupta et al., 2000), Hygrophila spinosa (Mazumder et al., 1997), Solanum pseudocapsicum leaves (Badami et al., 2003), Careya arborea (Natesan et al., 2007) reduce the EAC induced mortality and increase the MST are considered as effective anticancer plants. A similar inference is possible here, as RF effectively reduces EAC induced mortality and elevates MST in tumor bearing mice. Myelosuppression and anemia have been frequently observed in ascites carcinoma (Price and Greenfield, 1958) and similar findings were also observed in our present study. In EAC control mice, elevated WBC count and reduced hemoglobin and RBC count was observed. Anemia (reduced hemoglobin) encountered in ascites carcinoma mainly occurs due to iron deficiency, either by hemolytic or myelopathic conditions, which finally lead to reduced RBC number. It is observed that RF 200 and 400 mg / kg treatment prevented the fall in hemoglobin content and ameliorated the normal values of RBC and WBC. Treatment with RF maintains the normal values of whole blood count, which supports its hematopoietic protecting activity. The mechanism behind the hematopoietic protection is beyond the scope of the present study. Induction of myelotoxicity in EAC mice is of immunological significance to meet the adverse situation developed by the introduction of foreign bodies in the blood.

[0226] Previous studies showed that most of the plants reduce EAC induced myelotoxicity due to their immune boosting, antioxidant, and free radical scavenging activity (Manjula et al., 2010). A depleted endogenous antioxidant enzyme with enhanced free radicals’ generation and MDA is welldocumented in carcinogenesis (Szatrowski and Nathan, 1991). Many tumor cells have prooxidant status, which leads to generalize oxidative stress (Sun, 1990). In our present study, significant fall in hepatic SOD and catalase levels was observed in EAC inoculated mice. Similar set of animals also showed elevated MDA levels which further support oxidative in tumor bearing mice. Antioxidant molecules, such as GSH and vitamin E and C as well as antioxidant enzymes such as SOD, catalase, and glutathione peroxidase, have been long believed to have protective and anticancer activities by scavenging excess of free radicals. Antioxidants also play a protective role through differential regulation of transcriptional activator activities and redox modulation of gene expression, which is well documented. Previous studies have shown that antioxidants inhibit the growth of tumor cells by inhibiting cellular differentiation (Storz et al., 1990; Allen, 1993). Antioxidants alter the intracellular redox state, thereby enhancing the effects of cytotoxic therapy. It was reported that plant-derived extracts containing antioxidant principles showed cytotoxicity towards tumor cells (Jiau-Jian and Larry, 1977) and antitumor activity in experimental animals (Ruby et al., 1995).

[0227] The observed activities of the RF at 200 & 400 mg / g significantly (jxO.00 / ) in this study may be attributed to the antioxidant and antitumor principles present in the formulation. For studies on solid tumors, Dalton lymphoma inoculated mice were used to assess the antitumor activity of RF at dose of 200 & 400 mg / kg. After tumor transplantation regular and rapid growth in ascitic tumor volume was observed in the DAL control mice. Both, the doses of RF at 200 & 400 mg / kg significantly (jxO.OO! ) reduced development of tumor. RF at both the doses (200 and 400 mg / kg) was found to be effective in reducing tumor weight and volume. However, RF was found effective at 200 mg / kg, further increasing the dose at 400 mg / kg, significant (jxO.OO / ) reduction in activity was observed. The reduction in body weight may be due to ameliorative properties of RF.

[0228] Hence, inhibiting tumor progression and development supports the anticancer activity of both the studied by RF.

[0229] In conclusion, the study revealed that RF possess significant (p<0.001) antitumor activity in both EAC and DAL in vivo system. RF at dose of 200 & 400 mg / kg are effective against tumor development and progression. Therefore, RF can be considered as a potent anticancer agent.

[0230] 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.

[0231] 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.

[0232] 5. Conclusion

[0233] 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.

[0234] 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

What 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; and an excipient.

2. The composition of claim 1, wherein said 1,8-cineole is comprised in one or more nutraceuticals.

3. The composition of claim 2, wherein said one or more nutraceuticals comprising a first extract formed of one or more of Elettaria cardamomum and Salvia Rosmarinus', and wherein said composition further comprising a second extract formed of one or more of Piper nigrum and Piper longum.

4. The composition of claim 3, wherein said first extract comprising Elettaria cardamomum essential oil and Salvia rosmarinus essential oil in equal proportions by weight, wherein said second extract comprising Piperine Oleoresin at a concentration of 1-3 percent of the combined weight of said Elettaria cardamomum essential oil and said Salvia rosmarinus essential oil, and wherein said Elettaria cardamomum essential oil comprising said 1, 8-cineole at a concentration of 45-53% (v / w), and said Salvia rosmarinus essential oil comprising said 1, 8- cineole at a concentration of 45-53% (v / w).

5. The composition of claim 4, wherein said excipient is a nutraceutically acceptable excipient selected from one or more of P-cyclodextrin, Carboxymethyl cellulose, Tween 80 and PEG 400.

6. The composition of claim 5, wherein said Elettaria cardamomum essential oil and said Salvia rosmarinus essential oil comprising one or more other active ingredients, wherein said one or more other active ingredients comprising one or more of flavonoids, terpenoids, and alkaloids.

7. The composition of claim 6, wherein said nutraceutically acceptable excipient is P- cyclodextrin, and wherein said P-cyclodextrin is in a molar amount equal to a sum of moles of said Elettaria cardamomum essential oil, said Salvia rosmarinus essential oil, and said Piperine Oleoresin;wherein said Elettaria cardamomum essential oil comprising a first set of said one or more other active ingredients, wherein said first set comprising one or more of limonene, linalool, a- terpinenyl acetate, and a-terpineol; and wherein said Salvia rosmarinus essential oil comprising a second set of said one or more other active ingredients, wherein said second set comprising one or more of rosmarinic acid and carnosic acid.

8. The composition of claim 4, wherein said composition is a sustained-release formulation to enhance bioavailability and therapeutic efficacy.

9. The composition of claim 1, wherein said composition inhibits said viability by one or more of apoptosis, modulating reactive oxygen species (ROS) levels, and inhibiting cell proliferation pathways, and wherein said inhibition is in a range of 66.98 to 88.64%.

10. The composition of claim 4, wherein said composition is administered in vitro to one or more breast cancer cell lines selected from MDA MB 231, MDA MB 453, and MDA MB 468, wherein concentration of said 1,8-cineole is in a range of 1% to 12% (w / v).

11. The composition of claim 10, wherein a combination of said first extract and said piperine extract is present at a concentration ranging from 0.1% to 5% (w / v), and wherein said combination is solubilized in a solvent selected from one or more of: dimethyl sulfoxide (DMSO) at a concentration of 1% to 10% (v / v), ethanol at a concentration of 5% to 20% (v / v), and phosphate -buffered saline (PBS) at a concentration of 50% to 95% (v / v).

12. The composition of claim 4, wherein said composition is administered in vivo at a dosage ranging from 200 mg / kg to 400mg / kg, wherein said composition reduces tumor volume by 35.3% to 42.7%, and wherein said composition increases mean survival time (MST) by 60 to 87%.

13. A method of treatment of triple negative breast cancer (TNBC), said method comprising: administering a therapeutically effective dose of said composition of any of claims 1-12.

14. The method claim 13, further comprising monitoring therapeutic response through one or more of tumor volume measurement, imaging or biomarker analysis.

15. The method claim 13, wherein said therapeutically effective dose is administered in a mouse model in a range of 200 mg / kg to 400mg / kg.

16. A method of preparing a composition for use in inhibiting viability of triple negative breast cancer (TNBC) cells, said method comprising: dissolving an excipient in a solvent to prepare a first solution; adding an oil mixture to said first solution to prepare a second solution, said oil mixture comprising one or more of Elettaria cardamomum essential oil and Salvia rosmarinus essential oil, each of said Elettaria cardamomum essential oil and said Salvia rosmarinus essential oil comprising 1,8-cineole as an active ingredient; and emulsifying said second solution to form an emulsion.

17. The method of claim 16, wherein said excipient is selected from one or more of - cyclodextrin, carboxymethyl cellulose, Tween 80, and PEG 400; and wherein said oil mixture comprises said Elettaria cardamomum essential oil, said Salvia rosmarinus essential oil and Piperine Oleoresin.

18. The method of claim 17, wherein said oil mixture comprises said Elettaria cardamomum essential oil and said Salvia rosmarinus essential oil in equal proportions by weight, and said Piperine Oleoresin at a concentration of 1-3 percent of the combined weight of said Elettaria cardamomum essential oil and said Salvia rosmarinus essential oil, and wherein said Elettaria cardamomum essential oil comprising said 1, 8-cineole at a concentration of 45-53% (v / w), and said Salvia rosmarinus essential oil comprising said 1, 8- cineole at a concentration of 45-53% (v / w); and wherein said -cyclodextrin is in a molar amount equal to a sum of moles of said Elettaria cardamomum essential oil, said Salvia rosmarinus essential oil, and said Piperine Oleoresin.

19. The method of claim 18, further comprising spray drying said emulsion to form a dried powder.

20. A composition for targeting triple-negative breast cancer (TNBC) cells, comprising Recovereez Forte (RF) as an active ingredient; a pharmaceutically acceptable carrier, whereinRecovereez Forte (RF) is present in the composition at a concentration ranging from 0.1% to 5% (w / v).

21. The composition as claimed in claim 20, wherein the pharmaceutically acceptable carrier comprises a solvent selected from the group consisting of dimethyl sulfoxide (DMSO) present at a concentration of 1% to 10% (v / v), ethanol present at a concentration of 5% to 20% (v / v), and phosphate -buffered saline (PBS) present at a concentration of 50% to 95% (v / v).

22. The composition as claimed in claim 20, wherein Recovereez Forte (RF) is present in the composition at a concentration ranging from 1% to 3% (w / v).

23. The composition as claimed in claim 20, wherein Recovereez Forte (RF) is formulated as a powder, liquid solution, or suspension.

24. The composition as claimed in claim 20, wherein Recovereez Forte (RF) is obtained from natural sources or synthesized through chemical processes.

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