A PHARMACEUTICAL COMPOSITION FOR INHIBITING SMOOTHENED PROTEIN (SMO) IN HEDGEHOG SIGNALING PATHWAY AND METHOD THEREOF

Abstract

The present invention discloses a pharmaceutical composition and method for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway. The composition comprises 75% w/v 1-phthalanone and 25% w/v cyclohexanone, 2',3',3'-trimethyl-2-(3-methyl-buta-1,3-dienyl), derived from Polyalthia longifolia leaves. The method involves collecting, drying, and extracting the leaves with methanol, followed by purification and concentration of the active compounds. The final extract is combined with a pharmaceutically acceptable carrier for administration. The composition and method effectively inhibit Smo activity by binding to the protein, potentially offering a therapeutic approach for Hedgehog pathway-related diseases. The extraction and formulation processes are optimized to preserve the active compounds and ensure efficacy. FIG.1

Specification

Description:TECHNICAL FIELD
[001] The present invention pertains to the field of pharmaceutical compositions, particularly focusing on compositions for inhibiting smoothened protein (SMO) in hedgehog signaling pathway and method thereof. More particularly, the present invention relates to the therapeutic application in cancer biology. It offers an alternative source of small drug molecules (1-phthalanone and cyclohexanone, 2', 3', 3'-Trimethyl -2-(3-methyl-buta-1, 3-dienyl)) from leaf extract of Polyalthia. These molecules bind with Smoothened protein of the Hedgehog Pathway and inhibit the aberrant activation of the pathway thereby preventing tumorigenesis.
BACKGROUND OF THE INVENTION
[002] Cancer remains one of the leading causes of death worldwide, often characterized by its resistance to conventional treatments such as chemotherapy and radiotherapy, particularly in cases involving aggressive tumors like those associated with the brain. One key driver of many cancers is the aberrant activation of the Hedgehog (Hh) signaling pathway, which plays a crucial role in the regulation of cell growth and differentiation during embryonic development. In adults, inappropriate reactivation of this pathway can lead to tumorigenesis, most notably through the overexpression of Smoothened (Smo) protein, a key component of the pathway.

[003] The Smoothened protein is central to the regulation of the Hedgehog pathway, acting as a critical modulator that can activate downstream components either through canonical means, involving the Gli transcription factors, or through non-canonical mechanisms. Aberrant activation of Smo leads to uncontrolled cell proliferation, contributing to cancer development, and Smo has thus become a primary therapeutic target for developing cancer inhibitors.

[004] Several existing drugs, such as Cyclopamine, Anta XV, GDC-0449, and NVP-LDE225, have been developed to inhibit Smo. However, these drugs often suffer from significant drawbacks, including the development of drug resistance and undesirable side effects, limiting their efficacy in clinical applications. This has prompted a need for the discovery of novel inhibitors of Smo that are both effective and safe for use in cancer therapy.

[005] Phytochemicals, which are bioactive compounds found in plants, have emerged as a promising source for drug development due to their natural abundance, cost-effectiveness, and potential efficacy.

[006] In light of the above, there is a need for pharmaceutical compositions comprising novel inhibitors of the Smoothened (Smo) protein derived from natural sources, particularly phytochemicals.


SUMMARY OF THE INVENTION

[007] In the light of the disadvantages mentioned in the previous section, the following summary is provided to facilitate an understanding of some of the innovative features unique to the present invention and is not intended to be a full description. A full appreciation of the various aspects of the invention can be gained by taking the entire specification and drawings as a whole. Embodiments described herein disclose a pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway and a method thereof.
[008] According to an embodiment of the present invention, the pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway comprises of 75% w/v of 1-Pthalanone, and 25% w/v of cyclohexanone, 2', 3', 3'-Trimethyl-2-(3-methyl-buta-1, 3-dienyl).
[009] According to another embodiment of the present invention, the method for preparing a pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway, comprising the steps of collecting young, tender leaves of Polyalthia longifolia, washing and cleaning the collected leaves, air-drying the cleaned leaves at an ambient temperature below 50°C, grinding the dried leaves into a fine powder, performing continuous extraction of the powdered leaves with methanol in a soxhlet apparatus at a 1:5 ratio of leaf powder to methanol, repeating the extraction until all the extract is in the methanol fraction, evaporating the methanol solvent at 65°C using a rotary evaporator to obtain a concentrated extract, adding a 1:1 mixture of hexane and diethyl ether to the concentrated extract, concentrating the mixture in a rotary evaporator at 65°C to remove residual methanol, concentrating the mixture at 40°C under atmospheric pressure to completely evaporate methanol and concentrate the chalcones in the hexane-diethyl ether fraction, performing qualitative phytochemical analysis to confirm the presence of bioactive compounds, evaporating the hexane-diethyl ether solvent at 40°C to obtain the final leaf extract powder and combining the leaf extract powder containing 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl) with a pharmaceutically acceptable carrier to form the pharmaceutical composition.
[0010] According to another embodiment of the present invention, the 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl) in the pharmaceutical composition binds to Smo and inhibit its activity.
[0011] According to another embodiment of the present invention, the temperature during solvent evaporation does not exceed 300°C to preserve the active compounds.
[0012] According to another embodiment of the present invention, the pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway comprises a therapeutically effective amount of 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl), and a pharmaceutically acceptable carrier, wherein the 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl) bind to Smo and inhibit its activity.
[0013] According to an embodiment of the present invention, the composition is formulated for oral, topical, or parenteral administration.
[0014] Additional features and advantages of various embodiments will be set forth in part in the description that follows, and in part will be apparent from the description, or may be learned by practice of various embodiments. The objectives and other advantages of various embodiments will be realized and attained by means of the elements and combinations particularly pointed out in the description and appended claims.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
[0015] The detailed description is provided with reference to the accompanying figures. These and other features and advantages of the present invention will be readily appreciated, as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings wherein:
[0016] Fig. 1 is a flow chart illustrating a method for preparing a pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway in accordance with an embodiment of the present invention;
[0017] Fig. 2 illustrates canonical activation of the Hedgehog pathway;
[0018] Fig. 3 illustrates non-canonical activation of the Hedgehog pathway;
[0019] Fig. 4 illustrates the flow of research which led to identify and quantify the compounds which binds to the Smoothened protein resulting in the modulation of the Hedgehog pathway;
[0020] Fig. 5 is a sensogram graph of the BioLayer Interferometry assay, depicting the association and dissociation interaction between the analyte (extract) and the substrate (Smoothened protein);
[0021] Fig. 6 is a graph illustrating equilibrium dissociation constant (Kd) between the Smoothened protein and the Polyalthia longifolia leaf extract;
[0022] Fig. 7 illustrates a liquid chromatography and mass spectrometric analysis results;
[0023] Fig. 8 illustrates bands of the up regulated Smoothened protein on nitrocellulose membrane after Western Blot gene expression analysis;
[0024] Fig. 9 illustrates densitometry graph of the bands obtained after the Western blot;
[0025] Fig. 10 is a graph representing the percentage reduction of DPPH depicting the antioxidant activity of the extract at different concentrations;
[0026] Fig. 11 is a graph representing the percentage inhibition to hemolysis by extract at different concentrations;
[0027] Fig. 12 is a graph representing the percentage inhibition to ATPase by the extract at different concentrations;
[0028] Fig. 13 is a graph showing Fluorescence Spectroscopic scan; and
[0029] Fig. 14 represents the pharmacokinetic and ADMET results of the two molecules, 1-Pthalanone & cyclohexanone, 2', 3', 3'-Trimethyl -2-(3-methyl-buta-1, 3-dienyl), identified after BLI/ LCMS describing their drug likeness.
DETAILED DESCRIPTION
[0030] For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe them. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Such alterations and further modifications in the illustrated system, and such further applications of the principles of the disclosure as would normally occur to those skilled in the art are to be construed as being within the scope of the present disclosure.
[0031] 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 a process or method. Similarly, one or more devices or sub-systems or elements or structures or components preceded by "comprises... a" does not, without more constraints, preclude the existence of other devices, sub-systems, elements, structures, components, additional devices, additional sub-systems, additional elements, additional structures, or additional components. Appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.
[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The system, methods, and examples provided herein are only illustrative and not intended to be limiting.
[0033] In the following specification and the claims, reference will be made to a number of terms, which shall be defined to have the following meanings. The singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.
[0034] Embodiments described herein disclose a pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway and a method thereof.
[0035] According to an embodiment of the present invention, the pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway comprises of 75% w/v of 1-Pthalanone, and 25% w/v of cyclohexanone, 2', 3', 3'-Trimethyl-2-(3-methyl-buta-1, 3-dienyl).
[0036] Fig. 1 is a flow chart illustrating a method for preparing a pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway in accordance with an embodiment of the present invention.
[0037] According to another embodiment of the present invention, the method for preparing a pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway, comprising the steps of collecting young, tender leaves of Polyalthia longifolia at 102, washing and cleaning the collected leaves at 104, air-drying the cleaned leaves at an ambient temperature below 50°C, grinding the dried leaves into a fine powder at 106, performing continuous extraction of the powdered leaves with methanol in a soxhlet apparatus at a 1:5 ratio of leaf powder to methanol, repeating the extraction until all the extract is in the methanol fraction at 108, evaporating the methanol solvent at 65°C using a rotary evaporator to obtain a concentrated extract at 110, adding a 1:1 mixture of hexane and diethyl ether to the concentrated extract at 112, concentrating the mixture in a rotary evaporator at 65°C to remove residual methanol at 114, concentrating the mixture at 40°C under atmospheric pressure to completely evaporate methanol and concentrate the chalcones in the hexane-diethyl ether fraction at 116, performing qualitative phytochemical analysis to confirm the presence of bioactive compounds 118, evaporating the hexane-diethyl ether solvent at 40°C to obtain the final leaf extract powder 120 and finally at 122 combining the leaf extract powder containing 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl) with a pharmaceutically acceptable carrier to form the pharmaceutical composition.
[0038] According to another embodiment of the present invention, the 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl) in the pharmaceutical composition binds to Smo and inhibit its activity.
[0039] According to another embodiment of the present invention, the temperature during solvent evaporation does not exceed 300°C to preserve the active compounds.
[0040] According to another embodiment of the present invention, the pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway comprises a therapeutically effective amount of 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl), and a pharmaceutically acceptable carrier, wherein the 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl) bind to Smo and inhibit its activity.
[0041] According to an embodiment of the present invention, the composition is formulated for oral, topical, or parenteral administration.
[0042] WORKING EXAMPLES AND EXPERIMENTS
[0043] The Sonic Hedgehog (Hh) signaling pathway plays a critical role in vertebrate embryonic development. Aberrant activation of this pathway in adults can lead to various cancers, many of which are resistant to conventional radio- and chemotherapy. This resistance necessitates the identification of novel Hh pathway inhibitors suitable for clinical trials.
[0044] One of the key regulators of the Hh pathway is the Smoothened protein (Smo). Its de-repression and subsequent internalization can lead to aberrant pathway activation. This activation occurs through both canonical (Gli-dependent) and non-canonical (Gli-independent) mechanisms. In the canonical pathway, Smo activates Gli proteins, while in the non-canonical pathway, it triggers the activation of small GTPases like RhoA and Rac1.
[0045] Smo has been recognized as a promising drug target, but existing Smo inhibitors, such as Cyclopamine, Anta XV, GDC-0449, and NVP-LDE225, have limitations. These limitations include drug resistance and significant side effects, highlighting the need for the identification of alternative Smo inhibitors with improved efficacy and safety profiles.
[0046] The present invention describes the potential of 1-phthalanone and cyclohexanone, 2',3',3'-trimethyl-2-(3-methyl-buta-1,3-dienyl) as therapeutic agents targeting Smo. These compounds, derived from Polyalthia longifolia leaf extracts, have been shown to bind to Smo, thereby inhibiting its internalization and preventing aberrant activation of the Hh pathway. This binding was confirmed through biolayer interferometry (BLI) studies.
[0047] In addition to their Smo-inhibitory activity, these compounds also demonstrate favorable drug-likeness properties based on ADMET (absorption, distribution, metabolism, excretion, and toxicity) analysis. Furthermore, Polyalthia longifolia leaf extracts exhibit antioxidant and ATPase activity, suggesting potential additional therapeutic benefits.
[0048] Phytochemicals are a promising source for new drug development due to their potential for cost-effective and efficacious formulations. Polyalthia longifolia, known for its abundance of chalcones and traditional use in Ayurveda and contemporary medicine, has been investigated in this study.
[0049] Brain cancers often arise from aberrant activation of the Sonic Hedgehog (Hh) pathway, leading to resistance to conventional cancer therapies and poor prognosis. The Smoothened protein (Smo) is a key regulator of the Hh pathway and a potential drug target. Aberrant Smo activation, through both canonical and non-canonical mechanisms, can drive the development of these cancers.
[0050] This study aimed to identify phytochemicals that can bind to Smo and prevent its internalization. Western blotting of the hexane:diethyl ether leaf extract of Polyalthia showed a concentration-dependent increase in binding with Smo antibodies, indicating that phytochemicals in the plant extract were bound to Smo. Biolayer interferometry (BLI) studies further confirmed this binding. Liquid chromatography-mass spectrometry (LC-MS) analysis following BLI showed that 1-phthalanone and cyclohexanone, 2',3',3'-trimethyl-2-(3-methyl-buta-1,3-dienyl) were bound to Smo. Additionally, 1-phthalanone showed excellent drug-likeness scores across various criteria in the virtual ADME (absorption, distribution, metabolism, and excretion) and pharmacokinetic evaluation.
[0051] Western blotting was carried out with MDA-MB-231 cell lines at the 56th passage and 90% confluence. The cells were treated with different concentrations of the extract (2.5-7.5%) and harvested after 24 hours. Total protein content was estimated by the Bradford method. Anti-Smo primary antibodies conjugated with anti-rabbit IgG-HRP were used to detect the presence of Smo in the cell lysate.
[0052] Biolayer interferometry (FortéBio Octet Red 96) was carried out by immobilizing Smo protein (10 µM) onto a streptavidin-conjugated amine-reactive second-generation (AR2G) sensor. The sensor was immersed in the plant extract, and the binding kinetics were determined for 20 cycles. The eluents from BLI were introduced into an LC-MS C18 column at a flow rate of 1 µL/min to identify the bound molecules. Acetonitrile was used at a flow rate of 2 mL/min to elute the bound molecules. MS analysis was performed by electrospray ionization (ESI) using an Impact HD (Bruker) ESI QTOF high-resolution mass spectrometer and a Dionex Ultimate 3000 (Thermo) micro-LC. The fragments obtained were identified using the NIST library. Of the 12 molecules eluted, only two (cyclohexanone, 2',3',3'-trimethyl-2-(3-methyl-buta-1,3-dienyl) and 1-phthalanone) were found to be bound and eluted at 10 and 11 minutes, respectively.
[0053] Three web servers-Molinspiration, SwissADME, and SEESAR Stardrop-were used to determine the ADME and PK properties. The pharmacokinetic properties included molecular weight (MW < 500 Da), number of hydrogen donors (nON ≤ 5), hydrogen acceptors (nOHNH ≤ 10), number of rotatable bonds (nrotb < 10), and partition coefficient (logP ≤ 5). ADMET properties included solubility coefficient (logS), gastrointestinal absorption (GIa), blood-brain barrier permeation (BBBp), skin permeability, various CYP enzyme inhibition, and synthetic accessibility (SynAcc).
[0054] Cyclohexanone, 2',3',3'-trimethyl-2-(3-methyl-buta-1,3-dienyl) exhibited pharmacokinetic properties well within the acceptable range. It had an MW of 206.32, nON of 0, nOHNH of 1, nrotb of 2, and logP of 3.88. The ADMET properties, such as gastrointestinal absorption, skin permeation, and blood-brain permeation, showed positivity. It also had good solubility and an easy synthetic accessibility value of 3.94.
[0055] 1-Phthalanone exhibited exceptional pharmacokinetic properties well within the acceptable range. It had an MW of 134.13, nON of 0, nOHNH of 2, nrotb of 0, and logP of 1.357. The ADMET properties, such as gastrointestinal absorption, skin permeation, and blood-brain permeation, indicated high absorption and permeation. It also had an outstanding solubility index with an easy synthetic accessibility value of 1.34.
[0056] Cyclopamine, a standard inhibitor used against Smo, is known to have below-average bioavailability. It has an MW of 411.62, nON of 3, nOHNH of 2, nrotb of 0, and logP of 5.88. Although it has high gastrointestinal absorption, it also has high blood-brain and skin permeation. It had a moderate solubility index with a high synthetic accessibility value of 6.3, implying difficulty in synthesis.
[0057] Cytotoxicity studies by MTT assay of the crude extract against MDA-MB-231 and HeLa cell lines yielded an IC50 of 7.1 µg/mL against MDA-MB-231 cell lines and 3.03 µg/mL against HeLa cell lines. The selectivity index was 2.23 and 0.12 for MDA-MB-231 cells and HeLa cells, respectively.
[0058] Further, the leaf extracts also showed good antioxidant (above 70%) and ATPase (26.34%) activities at minimal concentrations. Reduced hemolysis (up to 46%) was observed with increasing concentrations of the extract (up to 7.5%).
[0059] The findings recommend that the crude extract of Polyalthia containing 1-phthalanone and cyclohexanone, 2',3',3'-trimethyl-2-(3-methyl-buta-1,3-dienyl) can be used as a potential anticancer agent. Fig. 2 illustrates canonical activation of the Hedgehog pathway and Fig. 3 illustrates non-canonical activation of the Hedgehog pathway.
[0060] How to Make and Use the Invention
[0061] The crude extract of Polyalthia longifolia containing 1-phthalanone and cyclohexanone, 2',3',3'-trimethyl-2-(3-methyl-buta-1,3-dienyl) can be utilized as a potential anticancer agent in various forms, including nutraceutical or herbal formulations.
[0062] Method
[0063] Young, tender leaves of Polyalthia longifolia are collected, washed, and air-dried at an ambient temperature below 50°C. The dried leaves are ground into a fine powder and subjected to continuous extraction with methanol in a soxhlet apparatus, using a 1:5 ratio of leaf powder to methanol. The extraction process is repeated until all the extract is transferred to the methanol fraction. The methanol is then evaporated at 65°C using a rotary evaporator, resulting in a thick, green, resinous liquid. A 1:1 mixture of hexane and diethyl ether is added to this liquid, and the mixture is concentrated in a rotary evaporator at 65°C to remove residual methanol. The temperature is further reduced to 40°C under atmospheric pressure to completely evaporate the methanol and concentrate the chalcones in the hexane:diethyl ether fraction. Qualitative phytochemical analysis, following the procedure described by S. B. Gokhale and C. K. Kokate (2008), is performed to confirm the presence of bioactive compounds, including alkaloids, carbohydrates, glycosides, flavonoids, and chalcones. Finally, the hexane:diethyl ether solvent is evaporated at 40°C to obtain the final leaf extract powder.
[0064] The preferred method for preparing the pharmaceutical composition involves the following key steps:
[0065] Solvent Extraction: Utilizing a mixture of diethyl ether and hexane for the extraction process aids in the effective partitioning and isolation of the target compounds (1-phthalanone and cyclohexanone, 2',3',3'-trimethyl-2-(3-methyl-buta-1,3-dienyl)) from the Polyalthia longifolia leaf extract.
[0066] Powder Formulation: After the removal of the solvents, the resulting powder containing the active compounds can be readily dissolved in water or other suitable solvents, facilitating the preparation of various pharmaceutical formulations, including solutions, suspensions, or emulsions, for diverse administration routes (e.g., oral, topical, or parenteral).
[0067] The extract containing 1-Pthalanone &cyclohexanone, 2', 3', 3'-Trimethyl -2-(3-methyl-buta-1, 3-dienyl) shows good antioxidant, anti hemolytic and ATPase activity in addition to it showing anticancer activity by MTT assay.
[0068] Experimental Results
[0069] Fig. 4 illustrates the flow of work to extract the Polyalthia longifolia leaves with 1:1 hexane and diethyl ether after which, binding and regulation was measured using BLI and western blotting. Other bioassays such as antioxidant, hemolysis, and ATPase were performed to gauge its pharmacologic sensitivity. At the same time, insilico ADMET analysis gave its drug-likeness index.
[0070] Fig. 5 is a sensogram graph of the BioLayer Interferometry assay, depicting the association and dissociation interaction between the analyte (extract) and the substrate (Smoothened protein).
[0071] Fig. 6 is a graph illustrating equilibrium dissociation constant (Kd) between the Smoothened protein and the Polyalthia longifolia leaf extract.
[0072] Fig. 7 illustrates a liquid chromatography and mass spectrometric analysis results reveals the compounds, 1-phthalanone and 2', 3', 3'-Trimethyl -2-(3-methyl-buta-1, 3-dienyl) -cyclohexanone, binding to the Smoothened protein.
[0073] Fig. 8 illustrates bands of the up regulated Smoothened protein on nitrocellulose membrane after Western Blot gene expression analysis. Lane 1 shows the lysate proteins from the cells treated with standard drug Paclitaxel while lane 2 and lane 3 represent the proteins from the untreated and extract-solvent treated cells. The lanes 4, 5 and 6 of the blot, shows the proteins expressed in the cells treated with plant extract of the concentration 2.5%, 5% and 7.5% respectively. The 40 kd marker is visible in lane 7.
[0074] Fig. 9 illustrates densitometry graph of the bands obtained after the Western blot. The densitometry analysis of the bands of different concentrations shows that the protein in lane 6 with 7.5% extract shows 3 times more expression than the protein in lane 4 with 2.5% of the crude extract.
[0075] Fig. 10 is a graph representing the percentage reduction of DPPH depicting the antioxidant activity of the extract at different concentrations.
[0076] Fig. 11 is a graph representing the percentage inhibition to hemolysis by extract at different concentrations.
[0077] Fig. 12 is a graph representing the percentage inhibition to ATPase by the extract at different concentrations.
[0078] Fig. 13 is a graph showing Fluorescence Spectroscopic scan wherein prescan is represented by the graphs in blue and red; fluorescence graph of Polyalthia longifolia leaf extract is represented by green which shows a sharp fluorescence of 318 a.u. at 506.5.; and
[0079] Fig. 14 represents the pharmacokinetic and ADMET results of the two molecules, 1-Pthalanone & cyclohexanone, 2', 3', 3'-Trimethyl -2-(3-methyl-buta-1, 3-dienyl), identified after BLI/ LCMS describing their drug likeness.
[0080] Overall, the present invention offers a significant advancement in the development of composition for Smo inhibitors. The composition comprising 1-phthalanone and cyclohexanone, 2',3',3'-trimethyl-2-(3-methyl-buta-1,3-dienyl), derived from Polyalthia longifolia leaf extracts, effectively bind to Smo, inhibiting its internalization and preventing aberrant activation of the Hh pathway. This targeted approach offers a potential solution to the limitations of existing Smo inhibitors, which often suffer from drug resistance and undesirable side effects. Additionally, the favorable drug-likeness properties, antioxidant activity, and ATPase activity of these compounds suggest potential multifaceted therapeutic benefits.
[0081] 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 disclosure and are not intended to be restrictive thereof. While specific language has been used to describe the disclosure, any limitations arising on account of the same are not intended.
[0082] The figures and the foregoing 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, the order 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 the acts need to be necessarily 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. , Claims:1. A pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway comprising:
75% w/v of 1-Pthalanone, and
25% w/v of cyclohexanone, 2', 3', 3'-Trimethyl-2-(3-methyl-buta-1, 3-dienyl)

2. A method for preparing a pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway, comprising the steps of:
a. collecting young, tender leaves of Polyalthia longifolia;
b. washing and cleaning the collected leaves;
c. air-drying the cleaned leaves at an ambient temperature below 50°C;
d. grinding the dried leaves into a fine powder;
e. performing continuous extraction of the powdered leaves with methanol in a soxhlet apparatus at a 1:5 ratio of leaf powder to methanol, repeating the extraction until all the extract is in the methanol fraction;
f. evaporating the methanol solvent at 65°C using a rotary evaporator to obtain a concentrated extract;
g. adding a 1:1 mixture of hexane and diethyl ether to the concentrated extract;
h. concentrating the mixture in a rotary evaporator at 65°C to remove residual methanol;
i. concentrating the mixture at 40°C under atmospheric pressure to completely evaporate methanol and concentrate the chalcones in the hexane-diethyl ether fraction;
j. performing qualitative phytochemical analysis to confirm the presence of bioactive compounds;
k. evaporating the hexane-diethyl ether solvent at 40°C to obtain the final leaf extract powder; and
l. combining the leaf extract powder containing 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl) with a pharmaceutically acceptable carrier to form the pharmaceutical composition.

3. The method as claimed in claim 2, wherein the 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl) in the pharmaceutical composition bind to Smo and inhibit its activity.

4. The method as claimed in claim 2, wherein the temperature during solvent evaporation does not exceed 300°C to preserve the active compounds.

5. A pharmaceutical composition for inhibiting the Smoothened protein (Smo) in the Hedgehog signaling pathway as claimed in claim 1, comprising a therapeutically effective amount of 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl), and a pharmaceutically acceptable carrier, wherein the 1-phthalanone and cyclohexanone, 2',3',3'-Trimethyl-2-(3-methyl-buta-1,3-dienyl) bind to Smo and inhibit its activity.

6. The pharmaceutical composition as claimed in claim 1, wherein the composition is formulated for oral, topical, or parenteral administration.

Dated this 07th day of November 2024

Signature

Jinsu Abraham
Patent Agent (IN/PA-3267)
Agent for the Applicant

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