Novel plant based composition for the treatment of skin

Abstract

A pharmaceutical composition for treatment of psoriasis is disclosed in the present invention. The pharmaceutical composition is a plant extract rich in bioactive constituents and applied topically for psoriasis treatment without any adverse effects. The present invention also provides a method for obtaining the extract and identifies various uses of the extract as a medicament in both human and veterinary therapy. Specifically, a 1.4% concentration of the bioactive extract has been shown to prevent the formation of psoriasis plaques when applied topically and 3.7% extract was found to clear psoriatic scales after 8 days of application topically Fig. 1A

Description

NOVEL PLANT BASED COMPOSITION FOR THE TREATMENT OF SKIN RELATED DISORDERSFIELD OF THE INVENTION

[0001] The present invention is related to a plant extract. More specifically, the present invention is related to a plant extract which is rich in bioactive constituents, and a method of obtaining the plant extract, and various uses of the extract as a medicament in human and veterinary therapy.BACKGROUND OF THE INVENTION

[0002] Plant extracts have long been recognized for their pharmacological activity, particularly due to the presence of specific constituents such as coumarins and furanocoumarins. Thus, it is considered valuable to explore methods for extracting bioactive constituents from Limonia, Eugenia, and Cinnamon plants and assess their pharmacological potential.

[0003] Limonia plants, belonging to the Rutaceae family, are tropical and equatorial, commonly found in regions like India, Sri Lanka, Myanmar, and South America. Korean Patent No. KR1020080012542 A, titled "Cosmetic composition for pore-minimizing containing Limonia acidissima extract," utilizes extracts from the Limonia genus.

[0004] Psoriasis is a chronic, non-contagious autoimmune disease that affects the skin and joints. It commonly causes red, scaly patches on the skin. The scaly patches caused by psoriasis are called psoriatic plaques. These plaques, characterized by rapid skin accumulation, often appear silver-white. While they commonly occur on the elbows and knees, they can affect any area, including the scalp and genitals, typically located on the extensor aspect of joints.

[0005] In the United States, psoriasis ranks as the most prevalent autoimmune disease, affecting approximately 7.5 million Americans, or about 2.2 percent of the population. Globally, it affects around 125 million people, constituting 2 to 3 percent of the total population. Studies have shown that between 10 and 30 percent of individuals with psoriasis also develop psoriatic arthritis. Psoriasis prevalence in African Americans is 1.3 percent compared to 2.5 percent of Caucasians.

[0006] The economic burden of psoriasis is significant, with direct and indirect healthcare costs are approximately $11.25 billion annually, with work loss accounting for 40 percent of thisburden. On average, 60 percent of psoriasis patients miss 26 days of work annually due to their illness.

[0007] The treatments for psoriasis mainly include topical applications, phototherapy, and systemic administration of medications. In cases of severe psoriasis and psoriatic arthritis, systemic antibiotics may be prescribed, typically in pill or an injection form. There are many medications and synthetic drugs available for treating psoriasis, with the FDA approving three new treatment options for patients. These include Taclonex Scalp, a novel topical ointment designed specifically for scalp psoriasis; the Xtrac Velocity Excimer Laser System, which emits a high-intensity ultraviolet light beam effective for moderate to severe psoriasis; and adalimumab, a drug initially sanctioned for psoriatic arthritis but now also approved for moderate to severe psoriasis. With time, psoriasis can develop resistance to a particular therapy, necessitating periodic changes to prevent tachyphylaxis and minimize the risk of adverse reactions.

[0008] There are many limitations of existing treatments. There is a need to develop safe and effective medications for psoriasis and related skin disorders from natural sources. Therefore, the present invention involves identifying herbal extracts and oils in a unique combination that can be formulated as a topical application for psoriasis treatment without adverse effects.SUMMARY OF THE INVENTION

[0009] Aspects of the present invention provide a pharmaceutical composition for the treatment of skin related disorders, including but not limited to psoriasis and psoriatic plaques.

[0010] In one aspect of the present invention provides an apparatus and a method for obtaining a bioactive extract for the treatment of skin related disorders, including but not limited to psoriasis and psoriatic plaques.

[0011] In one aspect of the present invention, a method of obtaining bioactive extract comprising: putting ingredients including dried bark of Limonia acidissima tree of sizes measuring 50mm x 20mm x 20mm, dried bark of Cinnamon sizes measuring 200mm x 5mm x 5mm, and Cloves in a porcelain reactor; sealing the porcelain reactor airtight with a sealant, along with attaching a condenser vessel connected to a porcelain collector; heating the porcelain reactor slowly to a temperature of 250 - 450°C using a heat source, and maintaining the temperature for 4 to 5 hours under non-aqueous near-vacuum conditions for producingvapours; condensing the vapours produced during heating under non-aqueous near-vacuum conditions to produce a condensed liquid; and distilling the condensed liquid, and extracting bioactive extract into a porcelain collector, where the yield of the extraction is 5%.

[0012] In one aspect of the present invention, the method further involves chemical characterization and validation of the bioactive extract, the chemical characterization includes identifying physical characteristics of the bioactive extract, where the bioactive extract is a 'Dark Reddish Brown Liquid' with an odor reminiscent of a sooty smell, has a specific gravity of 1.05, and a boiling temperature exceeding 400°C.

[0013] In one aspect of the present invention, the method further involves Antipsoriatic Activity Test for evaluating a potential effect, which is performed by at least selected from Cell Proliferation Assay and Human IL-6 ELISA.

[0014] In one aspect of the present invention, the bioactive extract is effective against HaCaT cell (Keratinocytes) proliferation, which manifests as plaques in psoriasis, and capable of treating psoriatic plaques.

[0015] In one aspect of the present invention, the bioactive extract has an effect on IL-6 production by HaCaT cells, with higher concentrations of the extract leading to a greater inhibition of IL-6 release.

[0016] In one aspect of the present invention, the bioactive extract inhibits S100 A8 and SI 00 A9 antimicrobial proteins, which are responsible for initiating psoriasis in the human body.

[0017] In one aspect of the present invention, the bioactive extract inhibits an expression of an inflammatory cytokine TNFa.

[0018] In one aspect of the present invention, the bioactive extract is formulated as a topical application for the treatment of psoriasis.

[0019] In one aspect of the present invention, the bioactive extract is versatile, finding application as a medicament in both human and veterinary therapy, showcasing its broad therapeutic potential.

[0020] Further details and specification of the invention are described in the detailed description and accompanying figures, providing comprehensive insights into the operation and functionalities of the present invention.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The above-mentioned and other features and advantages of this present disclosure, and the manner of attaining them, will become more apparent and the present disclosure will be better understood by reference to the following description of embodiments of the present disclosure taken in conjunction with the accompanying drawings, wherein:

[0022] Fig.l illustrates a method of extracting a bioactive extract in accordance with an embodiment of the present invention;

[0023] Fig.2a illustrates a HPLC Chromatogram of the bioactive extract in accordance with an embodiment of the present invention;

[0024] Fig.2b illustrates an another HPLC Chromatogram of the bioactive extract in accordance with an embodiment of the present invention;

[0025] Fig.2c illustrates an another HPLC Chromatogram of the bioactive extract in accordance with an embodiment of the present invention;

[0026] Fig.2d illustrates an another HPLC Chromatogram of the bioactive extract in accordance with an embodiment of the present invention;

[0027] Fig.2e illustrates an another HPLC Chromatogram of the bioactive extract in accordance with an embodiment of the present invention;

[0028] Fig.3 illustrates a LC-MS Spectra of the bioactive extract in accordance with an embodiment of the present invention;

[0029] Fig.4 illustrates Cell Proliferation Assay of the bioactive extract at 24 hour exposure in accordance with an embodiment of the present invention;

[0030] Fig.5 illustrates Cell Proliferation Assay of the bioactive extract at 48 hour exposure in accordance with an embodiment of the present invention;

[0031] Fig.6 illustrates ELISA IL 6 Standard Curve in accordance with an embodiment of the present invention;

[0032] Fig.7 illustrates IL 6 Response-Stimulated and Non-stimulated with LPS in accordance with an embodiment of the present invention;

[0033] Fig.8 illustrates different concentrations of extract to IL 6 response in accordance with an embodiment of the present invention;

[0034] Fig.9 illustrates SI 00 A8 / A9 Response-Stimulated and Non-stimulated with LPS in accordance with an embodiment of the present invention;

[0035] Fig.10 illustrates S100 A8 / A9 Response-Doxycyclin (Standard / Control) in accordance with an embodiment of the present invention;

[0036] Fig.11 illustrates ELISA test of different concentrations of the extract (SI 00 A8 / A9) response in accordance with an embodiment of the present invention;

[0037] Fig.12 illustrates different concentrations of Doxycyxlin and TNF-a relative expression in accordance with an embodiment of the present invention;

[0038] Fig.13 illustrates different concentrations of the bioactive extract and TNF-a relative expression in accordance with an embodiment of the present invention;

[0039] Fig.14 illustrates different concentrations of the bioactive extract and S100 A8 relative expression in accordance with an embodiment of the present invention;

[0040] Fig.15 illustrates different concentrations of the bioactive extract and S100 A9 relative expression in accordance with an embodiment of the present invention;

[0041] Fig.16 illustrates erythema scoring on C57BL mice after treatment with different doses of the bioactive extract in accordance with an embodiment of the present invention;

[0042] Fig.17 illustrates induration / epidermal thickness score on C57BL mice after treatment with different doses of the bioactive extract in accordance with an embodiment of the present invention;

[0043] Fig.18 illustrates desquamation scoring on C57BL mice after treatment with different doses of the bioactive extract in accordance with an embodiment of the present invention;

[0044] Fig.19 illustrates cumulative scores of erythema, induration and desquamation after treatment with different doses of the bioactive extract in accordance with an embodiment of the present invention; and

[0045] Fig.20 shows images of dermatological changes over time in accordance with an embodiment of the present invention;DETAILED DESCRIPTION OF THE INVENTION

[0046] Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the invention that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the invention is intended to be illustrative, and not restrictive. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention.

[0047] In this document, the terms "comprises," "comprising," or “including” or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a system, method, article, device or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such system, method, article, device, or apparatus. An element proceeded by "comprises ...a" does not, without more constraints, preclude the existence of additional identical elements in the process, product, method, article, device or apparatus that comprises the element.

[0048] The present invention provides a pharmaceutical composition for the treatment of skin related disorders, including but not limited to psoriasis and psoriatic plaques.

[0049] The pharmaceutical composition is bioactive extract obtained from dried bark of Limonia acidissima tree, dried bark of Cinnamon, and Cloves.

[0050] In one embodiment of the present invention, an apparatus and a method is provided for obtaining the bioactive extract. The method typically involves three steps: Extraction from the plant source, Chemical Characterization of the extract, and evaluation of the antipsoriatic activity of the bioactive extract, which are described hereinafter in details.

[0051] 1. Extraction method from the Plant source:

[0052] Fig.l illustrates an apparatus 10 and a method of extracting bioactive extract. The apparatus includes a porcelain reactor 12, a condenser vessel 14, a porcelain collector 16, a distillatory 18 and a heat source 20. The ingredients including, but are not limited to, the dried bark of a 14-year-old Limonia acidissima tree, reduced to sizes measuring 50mm x 20mm x 20mm, dried bark of Cinnamon measuring 200mm x 5mm x 5mm, and Cloves. Carefully weighed, 10 parts of Limonia bark, 0.3 parts of Cinnamon bark, and 0.15 parts of Clove buds are transferred into a porcelain reactor 12. The loaded porcelain reactor 12 is then sealed airtight with a sealant, along with a condenser vessel 14, further sealed with a porcelain collector 16. The reactor 12 is slowly heated to a temperature of 250 - 450°C, and the temperature is maintained for 4 to 5 hours under non-aqueous near-vacuum conditions. The vapours produced during heating under vacuum are condensed using a chilling unit, and the resulting extract is collected into the porcelain collector 16. The yield of the extraction process is 5%.

[0053] In one embodiment of the present invention, the extraction method is outlined in the following steps. Initially, the bark of Limonia trees older than 14 years is scraped carefully using mechanical scrapers and is collected in waterproof bags for storage. Subsequently, this bark is cut into uniform-sized smaller parts, approximately measuring 50mm x 20mm x 20mm, utilizing a mechanical cutter. The plant material undergoes thorough cleaning using vacuum pumps and air blowers to eliminate soil and dust particles. Following this, the cleaned material is subjected to drying under ambient conditions for approximately 24 hours in a hot air tray dryer. Once dried, the material is collected into stainless steel trays for further processing.

[0054] In the next step, the dried inner bark of Cinnamomum verum is obtained commercially and then cut into smaller parts measuring approximately 200mm x 5mm x 5mm using a mechanical cutter. These pieces are then stored for subsequent use.

[0055] Additionally, in another step, the aromatic flower buds of Eugenia caryophyllata are procured commercially and stored for further use.

[0056] In a subsequent step, a ratio of 10 parts of Limonia bark to 0.3 parts of Cinnamon bark and 0.15 parts of Clove buds is carefully weighed and then transferred into the porcelain reactor 12. The loaded porcelain reactor 12 is sealed airtight using a sealant, along with a condenser vessel 14, which is further sealed with a porcelain collector 16. The reactor 12 is then slowly heated to 450°C using a heat source 20, and the temperature is maintained for 4 to 5 hours under non-aqueous near-vacuum conditions. The vapors resulting from heating under vacuumare condensed using a chilling unit, and the extract is collected into the porcelain collector 16. The yield of this extraction process is 5%.

[0057] 2. Chemical Characterization of the extract and extract method validation:

[0058] Following the extraction method described above, the collected extract in the porcelain collector 16 undergoes characterization through various methods.

[0059] A) Physical Characteristics of the extract of the present invention are as follows:

[0060] The Table 1 below represent the Characteristics of the extract. The extract is a 'Dark Reddish Brown Liquid' with a characteristic odor reminiscent of a sooty smell. It has a specific gravity of 1.05 and a boiling temperature exceeding 400°C. Solubility tests reveal that the extract is insoluble in water but soluble in a variety of organic solvents including Acetonitrile, Ethanol, Acetone, Methanol, N-Butanol, Ter. Butanol, Iso Propyl Alcohol, Ethyl Acetate, Dichloro Methane, Chloroform, 1,4, Dioxane, Dimethylformamide, Dimethylsulfoxide. Partially Soluble in Toulene, Diethyl ether and Di isoprpyl ethyl ether. Additionally, the extract exhibits a pH of 5.10.

[0061] Table 1 below shows physical characteristics of the Bioactive ExtractTable 1

[0062] B) Chromatogram - HPLC

[0063] In one embodiment of the present invention, the extract is characterized by the High- performance liquid chromatography (HPLC) Chromatogram.

[0064] Chemical Properties:Chromatogram - HPLC MethodMobile Phase-A: 0.1 % Orthophopshoric Acid in Water Mobile Phase-B: Acetonitrile:water (70:30) Column: Inertsil ODS -150*4.6-3 Micron Column Temperature: 35 C Diluent: ACN+H2O (1 :1)Gradient (T / %B) : (0 / 5,100 / 95,120 / 95,120.1 / 5,125 / 5)Flow: 0.5mL / min Run Time: 125min Injection volume: 20pL Wavelength: 210nm

[0065] Fig.2a-2e illustrates the HPLC Chromatogram of the bioactive extract. A selective and sensitive high-performance liquid chromatography with UV detector (HPLC-UV) method was developed for analyzing the extract. The HPLC system utilized was the Shimadzu LC-2010HT with a UV detector, and data processing and chromatographic integration were conducted using 'LC Solution software Version 1.21'. Two mobile phases were employed: Mobile Phase-A consisted of 0.1% Orthophosphoric Acid in Water, while Mobile Phase-B comprised Acetonitrile and water in a ratio of 70:30. The column used was Inertsil ODS-150*4.6-3 Micron, maintained at 35°C. Dilution was performed with a mixture of acetonitrile and water in a 1 : 1 ratio, with a flow rate of 0.5 ml / min over a runtime of 125 min. UV detection was carried out at 210 nm. The HPLC analysis revealed 81 peaks eluted between 2.5 min and 115.1 min at various retention times.

[0066] Observations on HPLC analysis revealed that peaks eluted from 2.5 minutes to 115.1 minutes, with a total of 81 peaks detected at different retention times (RT’s).

[0067] C) Spectrometry - LCMS

[0068] In one embodiment of the present invention, the extract is characterized by the Liquid chromatography-Mass Spectrometry (LCMS) analytical method.

[0069] Conditions:Mobile Phase-A: 0.1 % Formic Acid in WaterMobile Phase-B: Acetonitrile: water (70:30)Column: Inertsil ODS -150*4.6-3 MicronColumn Temperature: 35 CDiluent: ACN+H2O (1 :1)Gradient (T / %B): (0 / 10, 15 / 50, 35 / 95, 45 / 95)Flow: 0.8mL / minRun Time: 45minInjection volume: 20pLWavelength: 210nm

[0070] Observations on LCMS: In LCMS analysis found peaks are eluted from 1.7min to 33min, with different masses are observed in the range from 120-1000 M / Z.

[0071] Fig.3 illustrates LC-MS data of the bioactive extract. LC-MS analysis was conducted using Mariner Bio spectrometry equipped with a binary pump. Mass fragmentations were identified using the spectrum database for organic compounds in the SDBS application. Two mobile phases were utilized: Mobile Phase-A consisted of 0.1% Formic Acid in Water, while Mobile Phase-B comprised Acetonitrile and water in a ratio of 70:30. The column used was Inertial ODS-150*4.6-3 Micron, maintained at 35°C. Dilution was performed with a mixture of Acetonitrile and water in a 1 : 1 ratio, with a flow rate of 0.8 ml / min over a runtime of 45 minutes. UV detection was conducted at 210 nm. LC-MS analysis revealed peaks eluted from 1.7 minutes to 33 minutes, with various masses observed in the range of 120 m / z to 1000 m / z.

[0072] 3. Antipsoriatic activity of the bioactive extract:

[0073] a) Cell Proliferation Assay:

[0074] The aim of the study was to screen and evaluate the potential effect of the bioactive extract on HaCaT cell lines (Keratinocytes) in vitro. The HaCaT cell line was obtained from ATCC, and a cell titer Gio assay was conducted using a 96-well plate. The cells were seeded onto the plate and treated with 10 different concentrations of the bioactive extract for 24 and 48 hours. Following treatment, the media was aspirated, and each well was supplemented with 50 pL of serum-free media and 50 pL of CTG solution. The assay plate was then incubated at 37°C for 3 hours. After incubation, luminescence readings were taken using an Envision plate reader.

[0075] Results: It was observed that the compound's activity started immediately, suggesting a reduction in psoriasis scales. After 24 hours of treatment, approximately 25% of HaCaT cells were inhibited at around 3.5% concentration of the extract. Following 48 hours of treatment, over 75% of cell inhibition was observed with a concentration of 3.7% of the bioactive extract. These findings indicate that the extract is effective against keratinocytic proliferation, which manifests as plaques in psoriasis. Consequently, the extract is capable of treating psoriatic plaques.

[0076] Fig.4 illustrates the cell proliferation assay of the bioactive extract after 24 hours. As shown in the Fig.4, the screening aimed to assess the potential effect of phytochemicals on HaCaT cell lines (Keratinocytes) using the Cell Titer Gio Assay for cell proliferation. Results indicate that activity starts at 0.1% concentration of the extract. At 1.2% concentration, approximately 10% of HaCaT cells are inhibited, while exposure to 3.7% of the extract results in inhibition of around 17% of HaCaT cells.

[0077] Fig.5 illustrates the cell proliferation assay of the bioactive extract after 48 hours. The aim was to screen the potential effect of phytochemicals on HaCaT cell lines (Keratinocytes) using the Cell Titer Gio Assay for cell proliferation. Results indicate that after 48 hours of treatment, approximately 60% of HaCaT cells were inhibited at 1.2% concentration of the extract. Moreover, more than 75% of cell inhibition was observed with 3.7% concentration of the bioactive extract.

[0078] b) ELISA: Human IL-6 ELISA.IL-6 Product name: Human IL-6 ELISA KitDetection method: ColorimetricSample type: Cell culture supernatant, Serum, Plasma Assay type: Sandwich (quantitative)Sensitivity: < 3 pg / ml Range: 1.37 pg / ml - 1000 pg / ml

[0079] Overview: Human IL-6 ELISA Kit

[0080] Abcam’s IL-6 Human ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro assay designed for the quantitative measurement of Human IL-6 in serum and cell culture supernatants. This assay utilizes an antibody specific for Human IL-6 coated on a 96-well plate.Standards and samples are pipetted into the wells, allowing IL-6 present in the sample to bind to the immobilized antibody. The wells are washed, a biotinylated anti-Human IL-6 antibody is added. After washing to eliminate unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted into the wells. The wells are washed again, and a TMB substrate solution is added, resulting in color development proportional to the amount of IL-6 bound. Addition of the Stop Solution changes the color from blue to yellow, with the intensity of the color measured at 450 nm. Achieve higher sensitivity in just 90 minutes with the Human IL-6 ELISA Kit (ab 178013) from the Simple Step ELISA range.

[0081] Materials:Table 2 below represent the material used the Human IL-6 ELISA kit.Table 2

[0082] Method:

[0083] HaCaT cells were seeded and allowed to incubate for 24 hours prior to treatment. After 24 Hrs, the cells were pre-treated with compounds at concentrations of 100%, 10%, and 1%. Following the pre-treatment, the HaCaT cells were stimulated with LPS (10 pg / mL). Supernatants were then collected and screened using ELISA according to the instructions provided with the kit. The data obtained from the ELISA assay is represented as % inhibition. This protocol was adapted and followed based on the methods described by Jun Wang et al., 2019.

[0084] Results: Analysis of IL-6 ELISA Standard

[0085] Fig.6 shows IL 6 plotted against OD@450 for the ELISA standard and Table 3 represent concentration IL 6 according to the OD@450.

[0086] Table 3 below shows the analysis of IL-6 ELISA standardTable 3

[0087] Analysis of Data: Table 4 below represent the data related to the concentration and theirInterpolated Values.

[0088] Table 4 shows the Analysis of Data - quantitative measurement of Human IL-6 in serum, cell culture supernatants.Table 4

[0089] Results: The results of the IL-6 ELISA assay conducted on HaCaT cells, the results indicate the percentage of inhibition of IL-6 production by HaCaT cells after treatment with different doses of compounds.

[0090] Fig.7 illustrates ELISA Dose - IL 6 Response-Stimulated and Non stimulated with LPS. Dose -response is plotted with IL 6 in the range of 1.37 pg / ml - 1000 pg / ml on Y axis with LPS stimulation and without LPS stimulation on the X axis. IL 6 response to different doses was recorded A significant increase in IL-6 release observed between non-LPS versus LPS condition validates the assay.

[0091] Fig.8 illustrates the dose-response relationship between different concentrations of the extract and IL-6 response. The IL-6 levels, ranging from 1.37 pg / ml to 1000 pg / ml, are plotted on the Y-axis, while the doses of the extract are represented on the X-axis. The IL-6 response to various doses of the extract was recorded. It is evident from the graph that the activity initiates at a concentration of 3.7% of the extract and becomes more pronounced at a concentration of 11%.

[0092] Based on Fig. 7 and Fig.8, the following conclusions can be drawn:

[0093] Fig.7 demonstrates a significant increase in IL-6 release between the non-LPS and LPS conditions, which validates the assay's effectiveness in detecting changes in IL-6 levels in response to stimuli. This observation indicates that the assay is capable of accurately measuring IL-6 production in HaCaT cells under different experimental conditions.

[0094] Fig.8 shows a dose-response relationship between different concentrations of the extract and IL-6 levels. A clear trend is observed, where IL-6 response increases with increasing doses of the extract. This finding suggests that the extract has an effect on IL-6production by HaCaT cells, with higher concentrations of the extract leading to a greater inhibition of IL-6 release.

[0095] In summary, the results from Fig.7 and Fig.8 indicate that the assay is reliable and sensitive in detecting changes in IL-6 levels, and that the extract exhibits a dose-dependent effect on IL-6 production in HaCaT cells.

[0096] Human Calprotectin ELISA (S100A8 / S100A9)

[0097] Materials:Sensitivity: 35 pg / mlRange: 32.77 pg / ml - 8000 pg / ml Sample type: Cell culture supernatant Detection method: Colorimetric Assay type: Sandwich (quantitative) Reacts with: Human

[0098] Method: Human Calprotectin ELISA Kit is designed for the quantitative determination of Calprotectin in cell culture supernatants, plasma, and serum samples. This assay employs an antibody specific for human Calprotectin coated on a 96-well plate. Standards and samples are pipetted into the wells and Calprotectin present in a sample is bound to the wells by the immobilized antibody. The wells are washed, and biotinylated anti-human Calprotectin antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Calprotectin bound. The Stop Solution changes the colour from blue to yellow, and the intensity of the color is measured at 450 nm.

[0099] Table 5 below shows the Quantitative determination of Calprotectin in cell culture supernatants, plasma, and serum samplesTable 5

[0100] Table 6 shows the Analysis of Calprotectin:Table 6

[0101] Results: The results are shown in the Fig. 9, Fig.10, and Fig.11.

[0102] Fig-9 illustrates the dose-response relationship for SI 00 A8 / A9 response, comparing samples stimulated with LPS and those without LPS stimulation. The Y-axis represents S100 A8 / A9 levels, ranging from 32.77 pg / ml to 8000 pg / ml, while the X-axisrepresents the presence or absence of LPS stimulation. The assay, designed for the quantitative determination of Calprotectin in cell culture supernatants, plasma, and serum samples, measures absorbance at 450 nm. Fig-9 demonstrates a significant increase in S100 A8 / A9 release observed between non-LPS versus LPS condition which validates the assay.

[0103] Fig.10 depicts ELISA Dose - S100 A8 / A9 Response-Doxycyclin (Standard / Control). The Y-axis represents S100 A8 / A9 levels, ranging from 32.77 pg / ml to 8000 pg / ml, while the X-axis represents the doses of Doxycycline. Fig.10 illustrates how the dose (concentration) of Doxycycline influences the levels of SI 00 A8 / A9, demonstrating the dose-response relationship between the two variables.

[0104] Fig.ll illustrates the dose-response relationship for S100 A8 / A9 response to Doxycycline (Standard / Control). The Y-axis represents SI 00 A8 / A9 levels, ranging from 32.77 pg / ml to 8000 pg / ml, while the X-axis represents the doses of the extract. The effect of the extract on SI 00, antimicrobial proteins, which are responsible for initiating psoriasis in the human body, is quantitatively evaluated. The graph indicates that a response can be observed starting at a concentration of 1.4% of the bioactive extract.

[0105] Conclusion: There was a significant increase in SI 00 A8 / A9 release observed between the non-LPS and LPS conditions, which validates the efficacy of the assay. Additionally, at 1% compound levels, inhibition was observed with the extract in comparison to the LPS-treated control. However, it's noteworthy to consider that the increase in S100 A8 / A9 levels at high compound concentrations could potentially be attributed to non-specific binding effects.

[0106] Gene Expression:

[0107] In another embodiment, the PCR Biomarker Study is performed for the gene expression. The Table 7 and Table 8 represent the reagents and material used.

[0108] Reagents:

[0109] Table 7 below shows the PCR Biomarker Study - SuppliersTable 7

[0110] Materials:

[0111] An “RNeasy” mini kit is used for RNA isolation and purification from a variety of sample types, including cells, tissues, blood, and other biological fluids. There are almost 50 reactions are observed.

[0112] Table 8 below shows the RNeasy mini - componentsTable 8

[0113] Principle:

[0114] In Two-step RT-qPCR, RNA transcripts are quantified by reverse transcribing them into cDNA first, and then qPCR is subsequently carried out. TaqMan RT-qPCR uses a nucleic-acid probe complementary to an internal segment of the target DNA. The probe is labelled with two fluorescent moieties. The emission spectrum of one overlaps the excitation spectrum of the other, resulting in “quenching” of the first fluorophore by the second. The probe is present during the PCR and if product is made, the probe is degraded via the 5'- nuclease activity of Taq -polymerase that is specific for DNA hybridized to template. The degradation of the probe allows the two fluorophores to separate, which reduces quenching and increases intensity of the emitted light. Further, fluorescent labelling enables the collection of data as PCR progresses.

[0115] Method:

[0116] A. RNA isolation: Using RNeasy mini kit

[0117] For Cells: To harvest cells, collect a maximum of 1 x 10A7 cells per sample and wash them with IX DPBS to eliminate any residual media. Next, lyse the cell pellet by adding 600 pl of RLT buffer. Utilize a needle and syringe for disruption and homogenization to obtain the cell lysate.

[0118] For Tissues: Fresh, frozen, or RNA later stabilized tissue samples (up to 30 mg, varying based on tissue type) are disrupted in RLT buffer and homogenized to obtain the tissue lysate.

[0119] Further, add an equal volume (600 pl) of 70% ethanol to the cell and tissue lysate and mix well by pipetting. Do not centrifuge. Transfer up to 700 pl of the sample, including any precipitate, to an RNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge the RNeasy Mini spin column for 15 seconds at >8000 x g at 4°C. Discard the flow- through. Add 700 pl RW 1 buffer to the RNeasy spin column and centrifuge for 15 s at >8000 x g at 4°C. Discard the flow-through. Add 500 pl RPE buffer to the RNeasy spin column and centrifuge for 15 s at >8000 x g at 4°C. Discard the flow-through. Add another 500 pl RPEbuffer to the RNeasy spin column and centrifuge for 120 s at >8000 x g at 4°C. Place the RNeasy spin column in a new 2 ml collection tube (supplied) and centrifuge at >8000 for 60 s to dry the membrane. Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 50 pl RNase-free water directly to the spin column membrane and centrifuge for 60 s at >8000 x g at 4°C to elute the RNA. Quantify in Nanodrop, select the option "RNA". Load 2 pl of RNase-free water to set the Blank, then load 2 pl of samples and measure the RNA concentration. RNA can be stored at -80°C.

[0120] cDNA synthesis: The cDNA synthesis is performed where a High-Capacity cDNA reverse Transcription Kit is used and 20 pl reaction is concluded. Based on RNA concentration, samples will be diluted to 2 pg / 10 pl.

[0121] 2X Reverse Transcription Master Mix will be prepared as mentioned below in the Table 9:

[0122] Table 9 below shows the HIGH-CAPACITY CDNA REVERSE TRANSCRIPTION WITH RNASE -used materialsTable 9

[0123] Further, Mix the 10 pl diluted RNA samples with the 10 pl Reverse Transcriptase Master Mix, briefly centrifuge proceed for PCR run. The Table 10 below represents steps for the PCR run.

[0124] Table 10 shows the Time and Temperature Conditions in various stepsTable 10

[0125] According to an embodiment of the present invention, cDNA can be stored at - 20 °C.

[0126] B. Real Time -qPCR: Using TaqMan™ Fast Advanced Master Mix, 20 pl reaction

[0127] According to an embodiment of the present invention, for Real Time -qPCR the mixture is prepared as mentioned below in Table 11.

[0128] Table 11 shows the Master Mix Preparation MethodTable 11

[0129] The mixture is dispensed into the plate wells, and the plate is sealed with an optical adhesive film using a sealing adaptor. The sealed plate is then centrifuged for 20 seconds at 1000 rpm to eliminate any air bubbles. Then, place the plate in ABI Prism 7500 Thermo Cycler and run at TaqMan settings as represented below in Table 12.

[0130] Table 12 shows the Time and Temperature Conditions in various stepsTable 12

[0131] According to another embodiment of the present invention, the data is exported into excel and analyzed by using AACT method.

[0132] Results: The result are observed as presented in the Table 13 and Table 14.

[0133] Table 13 shows the TNF-a Comparative Analysis of Extract, Dexamethasone, Doxorubicin and Doxycycline Through Gene expression studies

[0134] 1. TNF-a: Results are depicted in the Table 13 below.Table 13

[0135] According to another embodiment of the present invention, Doxycycline is used as a positive control in this experiment. In Fig.12, the different concentrations of Doxycycline and their effect on relative TNFa expression are illustrated. Fig.12 shows the relative expression of TNFa on the Y-axis and the doses of Doxycycline on the X-axis. A 10-fold difference in relative TNFa expression, indicative of increases in inflammation, is observed between unstimulated and stimulated (LPS) conditions. Doxycycline serves as a positive control in this experiment.

[0136] Fig-13 illustrates the effect of different concentrations of the Bioactive Extract on relative TNFa expression. The Fig.13 shows the relative expression of TNFa on the Y-axis and the doses of the Bioactive Extract on the X-axis. A substantial difference in relative expression is observed between stimulated and non-stimulated conditions. Additionally, it is evident that at all concentrations, the Bioactive Extract inhibited the expression of the inflammatory cytokine TNFa.

[0137] Observations from Fig.12 and Fig.13 indicate a significant difference in TNF- a expression between unstimulated and stimulated (LPS) conditions, with a 10-fold variation observed. Additionally, across all concentrations, the Bioactive Extract consistently inhibited TNF-a expression, suggesting its potential anti-inflammatory effect.

[0138] 2. S100 A8 AMP: Results are depicted in the Table 14 below.

[0139] Table 14 shows the SI 00 A8 - Comparative Analysis of Extract,Dexamethasone, Doxorubicin and Doxycycline Through Gene expression studiesTable 14

[0140] Fig.14 depicts the effect of different concentrations of the Bioactive Extract on relative S100 A8 expression. The Fig.14 represents the relative expression of S100 A8 on the Y-axis against the doses of the Bioactive Extract on the X-axis. A significant 20-fold difference is observed between stimulated and unstimulated conditions. Furthermore, inhibition of SI 00A8 expression is noticeable starting at a concentration of 1.4% of the Bioactive Extract. These findings suggest a potential role of the Bioactive Extract in modulating S100 A8 expression, which is crucial in the initiation of psoriasis on the human body.

[0141] Fig-15 depicts the effect of different concentrations of the Bioactive Extract on relative S100 A9 expression. The Fig.15 represents the relative expression of S100 A9 on the Y-axis against the doses of the Bioactive Extract on the X-axis. A notable 8-fold difference is observed between stimulated and unstimulated conditions. Additionally, inhibition of SI 00 A9 expression is evident at all concentrations of the Bioactive Extract. These results suggest that the Bioactive Extract may effectively modulate S100 A9 expression, which, along with SI 00 A8, plays a significant role in the initiation of psoriasis on the human body.

[0142] According to another embodiment of the present invention, a significant 20-fold difference is observed between unstimulated and stimulated (LPS) conditions in S100A8 expression. Additionally, the Bioactive Extract demonstrated inhibition of S100A8 expression even at 1% compound levels. Doxycycline was employed as a positive control in this study.

[0143] 3. S100 A9 AMP Results:

[0144] According to another embodiment of the present invention, as shown in the Table 15 a significant 10-fold difference is observed between unstimulated and stimulated (LPS) conditions in SI 00 A9 expression. Moreover, the extract exhibited inhibition of SI 00 A9 expression even at 1% compound levels. Doxycycline served as a positive control in this experiment.

[0145] Table 15 shows the S100A9 Analysis - Comparative Analysis of Extract, Dexamethasone, Doxorubicin and Doxycycline Through Gene expression studiesTable 15

[0146] Fig-15 depicts the relative expression of S100 A9. The Fig.15 represents the relative expression of SI 00 A9 on the Y-axis against the doses of the Bioactive Extract on the X-axis. The effect on SI 00 A9, an antimicrobial protein responsible for the initiation of psoriasis on the human body, is quantitatively evaluated for dose-response results. A notable 8-fold difference is observed between stimulated and unstimulated conditions. Additionally, inhibition of SI 00 A9 expression is noticeable at all concentrations of the bioactive extract.

[0147] 4. IL-6:

[0148] According to another embodiment of the present invention, as shown in the Table 16 a marginal difference is observed between unstimulated and stimulated (LPS) conditions in IL-6 expression. Furthermore, the extract exhibited inhibition of IL-6 expression at 1% compound levels. Doxycycline was employed as a positive control in this study.

[0149] Table 16 shows the IL-6 Analysis- Comparative Analysis of Extract, Dexamethasone, Doxorubicin and Doxycycline Through Gene expression studies.Table 16

[0150] Acute Dermal Irritation / Corrosion of PRC0028 extract in New Zealand White Rabbits:

[0151] The obj ective of this study was to determine the degree of irritation or corrosion resulting from a single application of the test item PRC0028 to the skin of New Zealand White Rabbits. The animals were observed for 14 days for the presence of erythema and / or edema.

[0152] This study was conducted in accordance with OECD Guideline 404. The dermal irritation and corrosion potential of the test item PRC0028 were evaluated using New Zealand White Rabbits. Twenty-four hours prior to application, the dorsal area of the animals was clipped and the fur was removed. A total of 0.5 mL of the test item was applied to the shaved area (6 cm2). Initial testing was performed on one animal with exposure times of 3 minutes, 1 hour, and 4 hours. This animal was monitored for erythema and edema. The test was subsequently confirmed with two additional animals, each exposed for 4 hours. All animals were observed for 14 days for any signs of irritation or corrosion. Scores for erythema and edema were recorded during the observation period.

[0153] Throughout the observation period, all animals were monitored for mortality, morbidity, clinical signs, and the degree of irritation and / or corrosion following the single application of PRC0028. No abnormalities were observed in any of the clinical signs or lesions attributable to the test item.

[0154] The results demonstrated no mortality or signs of erythema or corrosion in any of the test item-treated animals. There were no treatment-related effects on food consumption or body weight. Gross and histopathological examinations revealed no treatment-related abnormalities in the skin.

[0155] Based on these findings, it is concluded that the test item PRC0028, when applied at a dose of 0.5 mL / site, does not cause irritation or corrosive effects on the skin of New Zealand White Rabbits following a 14-day observation period.

[0156] Objective of the Study: The purpose of this study was to assess the dermal irritation or corrosion potential of PRC0028 in New Zealand White Rabbits following a single dermal administration and a 14-day observation period.

[0157] Test System Details:— Species: New Zealand White Rabbits— Age at Treatment: 6-8 Weeks— Sex: Female (nulliparous and non-pregnant)— Number of Animals: 3— Cage Size: 800 mm x 746 mm x 1790 mm (LxWxH) SS316— Air Changes: At least 10 air changes per hour— Temperature: 22±3°C— Relative Humidity: 50-70%— Lighting: Artificial light with a 12-hour light / dark cycle

[0158] Dose Selection and Rationale: The dose selection for the study was based on the principles outlined in OECD Guideline 404, which addresses acute dermal toxicity. A dose of 0.5 mL of liquid was applied to the test site (6 cm2).

[0159] Route of Administration and Justification: Approximately 24 hours before testing, the fur was removed by closely clipping the dorsal area of the animals. Care was taken to avoid skin abrasion, and only animals with healthy, intact skin were used. The test item was applied to the shaved area for dermal exposure. A dose of 0.5 mL of liquid was applied to the test site. The study design of Acute Dermal Irritation on New Zealand Albino Rabbits shown in Table 17.

[0160] Table: 17 shows the Study Design - Acute Dermal Irritation on New Zealand Albino Rabbits.Table 17

[0161] Results

[0162] Clinical signs and Mortality: No treatment-related clinical signs were observed in any of the test item-treated animals throughout the experimental period. Additionally, no mortality was recorded during the study. Summarized data for clinical signs and mortality for all animals are presented in Table XX and Table XX, respectively. Detailed individual animaldata on clinical signs and mortality are provided in the Table 18 (Clinical Signs) and Table 19 (Mortality).

[0163] TABLE -18: shows the individual animal clinical signsTable 18TABLE - 19'. shows the individual animal mortality / morbidityTable 19

[0164] Detailed Clinical Examination: No treatment-related signs or symptoms were observed during the detailed clinical examination of all animals throughout the treatment period. A summary of the detailed clinical examination results is presented in Table 20.

[0165] Table 20 shows the individual animal detailed clinical examinationTable 20

[0166] Body Weight: The body weight of each animal was recorded throughout the observation period. The changes observed did not indicate any abnormalities. Summarized and individual body weight data are presented in Table 21.

[0167] TABLE 21 shows the individual animal body weight (kg) / weekTable 21

[0168] Food Consumption: No significant treatment-related changes in food consumption were observed in the rabbits following the application of the test item.

[0169] Necropsy and Gross Pathology Examination: No changes were observed in the animals following the application of the test item at the given observation period, even with a 0.5 mL dose. Therefore, the animals were not subjected to necropsy.

[0170] Conclusion: PRC0028 was evaluated for irritation or corrosion following dermal application, with both initial and confirmatory exposures for different time periods. The results are as follows:— No erythema or edema was observed at the site of application.— Alopecia was noted at the application site during the 14-day observation period.

[0171] Based on these results, PRC0028 was found to be non-irritant and noncorrosive.

[0172] Table 22 below represents summary of Erythema and Eschar formation.Table 22

[0173] Table 23 below represents summary of Edema formation.Table 23

[0174] Table 24 below represents grading of erythema and eschar formation.Table 24

[0175] Table 25 below represents Grading of Edema Formation.Table 25

[0176] Effect of PRC0028 on C57BL / 6 mice by Imiquimod induced psoriasis:

[0177] The study titled "Effect of PRC0028 on C57BL / 6 mice by Imiquimod induced psoriasis" was conducted to evaluate the therapeutic effects and safety profile of the investigational drug PRC0028 in C57BL mice. The experiment involved eight groups of female mice, each treated with different formulations to compare the effectiveness of PRC0028 against standard treatment and controls. The groups included a normal control, disease control with no treatment, a standard treatment group using Clobetasol propionate cream (0.025% w / w), and three groups treated with varying concentrations of PRC0028 extract (1.4%, 3.7%, and 11%). Additionally, there was a prophylactic group receiving 1.4% PRC0028 extract and a vehicle control group treated with paraffin liquid.

[0178] Psoriasis-like lesions were induced in the mice through daily topical application of IMQ cream over eight days. Following this induction, treatments were applied according to group assignments. The study measured various parameters, including the Psoriasis Area and Severity Index (PASI), which assessed erythema, skin thickness, and desquamation. Body weights and histopathological evaluations of skin and spleen samples were also conducted. The samples were stained with hematoxylin and eosin for microscopic examination to identify any signs of gross pathology. Additionally, the study evaluated the levels of inflammatory biomarkers IL-6 and TNF-a, which are crucial in understanding the drug's impact on inflammation associated with psoriasis.

[0179] The data collected from these observations were statistically analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test, with results expressed as Mean ± SEM. A p-value of < 0.05 was considered statistically significant, ensuring the reliability of the findings.

[0180] Objective of the Study: The objective of the study is to evaluate the therapeutic effects and safety profile of the investigational drug PRC0028 in treating psoriasis using C57BL mice. It aims to compare PRC0028's efficacy against standard treatment and controls.

[0181] Test System and Details: Table 26 below represents Test System and Details.Table 26

[0182] EXPERIMENTAL PROCEDURE:

[0183] Dose Selection and Rationale: Doses were selected based on information provided by the sponsor. A dose volume of 0.5 mL was applied every 12 hours. Details of the study design and dose specifics are outlined below in Table 27.Table 27

[0184] Route of Administration: The test item was administered through topical application.

[0185] Test Item Preparation and Dose Formulation: The test items were supplied by the sponsor at the desired concentrations. A dose volume of 0.5 mL was applied to the skin surface every 12 hours for a duration of 8 days.

[0186] Observations:

[0187] PSORIASIS AREA AND SEVERITY INDEX (PASI)

[0188] To assess a score of the severity of inflammation of the affected skin, an objective scoring system was developed based on the Clinical Psoriasis Area and SeverityIndex (PASI). This system was used to monitor and grade the severity of psoriasis-like lesions. Parameters were scored individually on a scale from 0 to 4: 0 - Absent; 1 - Mild; 2 - Moderate; 3 - Severe; 4 - Very Severe. The cumulative score, which includes erythema, scaling, and thickening, served as a measure of the severity of inflammation, as detailed in Table 28.

[0189] Table 28 below represents PASI Scoring outline.Table 28

[0190] Erythema Score: Erythema was evaluated at three key time points: before induction, after induction, and after treatment. The scores indicated that erythema was significantly higher when animals were treated with imiquimod, which induced psoriasis-like symptoms. Following treatment with the standard (Clobetasol propionate cream 0.025% w / w), graded test drug (1.4%, 3.7%, and 11% extracts), and vehicle (liquid paraffin), erythema scores showed a reduction in both the standard and test drug groups. In contrast, the prophylactic group did not develop erythema compared to the disease control group. The vehicle showed minimal reduction in erythema scores relative to the disease control group. The normal control group, which did not receive induction, did not exhibit any signs of erythema. Summarized erythema scoring data are presented in Table 29.Table 29

[0191] Key References- SD: Standard deviation, N - No. of animals, SD: Standard Deviation, G- Groups; D- Day; Gl: Normal Control, G2: Disease Control, G3: Standard, G4 : 1.4% extract G5 : 3.7% extract ,G6 : 11% extract, G7 : Prophylactic, G8 : vehicle control.

[0192] Fig. 16 shows erythema scoring after treatment. The Fig.16 illustrates erythema scores on C57BL mice following an 8-day treatment with different doses of the bioactive extract (test samples), the standard treatment, normal control, disease control, and the prophylactic group. The prophylactic group received a 1.4% concentration of the bioactive extract along with 5% imiquimod.

[0193] Erythema was evaluated at three time points: before induction, after induction, and after treatment. The scores indicated that erythema was elevated when the animals were treated with imiquimod, which induced psoriasis-like symptoms. Following treatment with the standard (Clobetasol propionate cream 0.025% w / w), graded test drug (1.4%, 3.7%, and 11% extracts), and vehicle (liquid paraffin), the results showed a reduction in erythema in both the standard and test drug groups. The prophylactic group did not develop erythema compared to the disease control group. The vehicle showed minimal reduction in erythema scores compared to the disease control group. The normal control group exhibited no signs of erythema, as induction was not performed on this group.

[0194] Epidermal Thickness (Induration): Epidermal thickness or induration of shaved skin was measured before induction, after induction, and after treatment. The scoring of epidermal thickness showed that, the thickness was high due to the scaling formed during psoriasis induction. The thickness of normal animal was equivalent to 1mm and after induction, the skin thickness was visibly increased and was measured with the help of vernier calipers. The normal control group and prophylactic group did not show any difference during the study period and remained same as before induction. The epidermal thickness after induction of psoriasis was high in all other groups. The treatment groups have shown a significant decrease in thickness after treatment with the standard and test drug at different graded doses. There has been no change in the thickness of disease control and vehicle treated group. The summarized table for Induration / epidermal thickness was represented in Table 30.Table 30

[0195] Key References- SD: Standard deviation, N - No. of animals, SD: Standard Deviation, G- Groups; D- Day; Gl: Normal Control, G2: Disease Control, G3: Standard, G4 : 1.4% extract G5 : 3.7% extract ,G6 : 11% extract, G7 : Prophylactic, G8 : vehicle control.

[0196] Fig. 17 shows the induration / epidermal thickness scores. The Fig.17 represents induration scoring on C57BL mice following an 8-day treatment with different doses of the bioactive extract (test samples), the standard treatment, normal control, disease control, and the prophylactic group. The prophylactic group received a 1.4% concentration of the bioactive extract along with 5% imiquimod.

[0197] Epidermal thickness or induration of the shaved skin was measured before induction, after induction, and after treatment. The scoring revealed that epidermal thickness increased due to scaling from psoriasis induction. The normal animal skin thickness was approximately 1 mm; after induction, the thickness visibly increased and was measured using vernier calipers. Both the normal control and prophylactic groups showed no significant change in thickness throughout the study period and remained consistent with measurements before induction. In contrast, epidermal thickness was significantly higher in all other groups following psoriasis induction. The treatment groups exhibited a significant decrease in thickness after treatment with the standard and test drugs at various doses. No changes in thickness were observed in the disease control and vehicle-treated groups.

[0198] Desquamation (Scaling): Desquamation was formed because of the accumulation of keratocytes due to psoriasis induction after the application of imiquimod for7 days. Silver colored scales had appeared on the exposed skin surface of C57BL / 6 mice exposed to imiquimod application. The animals from groups except G1 and G7 has shown the scale formation and the scoring was given based on the severity of scale formation after the induction of psoriasis. There has been reduction in the scaling after the treatment with the standard drug and the test drug. Due to prophylactic treatment, there has been no change in the skin of animals from group 7 when compared with the disease control group. The scaling was significantly reduced in treatment groups when compared with the disease control. The scaling of the skin from group 8, vehicle treated animals remained the same as that of the disease control group. The scaling of G3(Std); treatment groups, G4 & G5, has shown a complete recovery from scaling when compared with the disease control. There has been a decrease in the scaling of G6 treated animals when compared with the disease control. The summarized table for Desquamation scoring was represented in Table 31.Table 31

[0199] Key References - SD: Standard deviation, N - No. of animals, SD: Standard Deviation, G- Groups; D- Day; Gl: Normal Control, G2: Disease Control, G3: Standard, G4 : 1.4% extract G5 : 3.7% extract ,G6 : 11% extract, G7 : Prophylactic, G8 : vehicle control.

[0200] Fig. 18 shows desquamation / scaling after treatment. The Fig.18 represents desquamation scoring on C57BL mice following an 8-day treatment with different doses of the bioactive extract (test samples), the standard treatment, normal control, disease control, and the prophylactic group, which received a 1.4% concentration of the bioactive extract along with 5% imiquimod.

[0201] Desquamation, resulting from the accumulation of keratinocytes due to psoriasis induction after 7 days of imiquimod application, was observed as silver-colored scales on the exposed skin of C57BL / 6 mice. All groups except G1 (normal control) and G7 (prophylactic) exhibited scale formation, and scoring was based on the severity of scaling following psoriasis induction.

[0202] Treatment with the standard drug and the test drugs resulted in a reduction in scaling. Prophylactic treatment in group G7 showed no significant change in skin condition compared to the disease control group. Scaling was significantly reduced in the treatment groups compared to the disease control group. In group G8 (vehicle-treated animals), scaling remained consistent with the disease control group. Groups G3 (standard), G4, and G5 (test drugs) showed complete recovery from scaling compared to the disease control, while group G6 (another test drug) also demonstrated a reduction in scaling compared to the disease control.

[0203] Table 32 below represents summary of cumulative score.Table 32

[0204] Key References - SD: Standard deviation, N - No. of animals, SD: Standard Deviation, G- Groups; D- Day; Gl: Normal Control, G2: Disease Control, G3: Standard, G4 : 1.4% extract G5 : 3.7% extract ,G6 : 11% extract, G7 : Prophylactic, G8 : vehicle control

[0205] Fig.19 represents PASI scoring, which includes the cumulative scores for erythema, induration, and desquamation. This scoring assesses the intensity of redness, thickness, and scaling associated with psoriasis. The figure shows that the prophylactic groupeffectively prevented erythema, scaling, and induration. Additionally, the 1.4% and 3.7% concentrations of the extract significantly reduced psoriasis, outperforming the standard Clobetasol propionate in terms of efficacy.

[0206] Biochemical Analysis: The biochemical parameters were analyzed using the serum samples separated from centrifugation by ERBA Biochemical analyzer. The elevated levels of IL-6 and TNF-a were also be evaluated.

[0207] Histopathology: Formalin fixed skin samples were subjected to histopathological examination. Tissue processing was done to dehydrate in ascending grades of alcohol, clearing in xylene and embedded in paraffin wax. Paraffin wax embedded tissue blocks were sections at 5 pm thickness with the Rotary Microtome. All the slides were stained with Hematoxylin & Eosin (H & E) stain. The prepared slides were examined under microscope by Pathologist to note histopathological lesions, if any. Severity of the observed lesions were recorded as NAD =No Abnormality Detected, 1 Mild changes (+1), Moderate changes (+2), Severe changes (+3) and extremely severe [4] and distribution was recorded as focal, multifocal and diffuse.

[0208] Following parameters were evaluated:Keratin1) Munro abscess - Number and size of abscess2) Hyperkeratosis - Severity3) Parakeratosis - SeverityEpidermis1) Epidermal hyperplasia - (measured in inter follicular epidermis) - Acanthosis.2) Lack of granular Layer3) Length and clubbing of rete ridges formation - severity4) Thinning above papillaeDermis1) Dermal inflammation and infiltration of inflammatory cells lymphocytes / neutrophils - Severity2) Papillary congestion / hemorrhages / angiogenesis3) Dermal FibrosisDermal FibrosisDermal Inflammation

[0209] Observations: The levels of IL-6 and TNF-alpha were measured to assess the inflammatory response in the treatment groups. There were no significant changes in IL-6 and TNF-alpha levels in the test item-treated animals when compared to the vehicle control group. This indicates that the treatment did not induce any notable inflammatory response as measured by these cytokines. The summarized data for IL-6 and TNF-alpha levels are presented in Table 33 and Table 34.

[0210] Table 33 below represents summary of il-6 before and after induction of psoriasis.Table 33

[0211] Key References: SD: Standard Deviation, G- Groups; D- Day; Gl : Normal Control, G2: Disease Control, G3: Standard, G4: 1.4% extract, G5: 3.7% extract, G6: 11% extract G7: Prophylactic, G8: vehicle control.

[0212] Table 34 below represents MEAN TNF-a before and after induction of psoriasis.Table 34

[0213] Key References: SD: Standard Deviation G- Groups; D - Day; Gl : Normal Control, G2: Disease Control, G3: Standard, G4: 1.4% extract, G5: 3.7% extract, G6: 11% extract, G7: Prophylactic, G8: vehicle control

[0214] Table 35 represents Summary of Histopathological Observation.Table 35

[0215] Conclusion: Based on the PASI score erythema, scaling, IL-6, and TNF-a measurements, the study concludes that significant changes were observed in the PASI scoring for erythema, Induration, and desquamation parameters before and after psoriasis treatment. There has been an increase in the erythema, induration, and desquamation observed in animals treated with imiquimod 5% cream in comparison with the normal group. Animals from the prophylactic group have shown no increase in all the parameters and the results show that the prophylactic group remained almost identical to the normal control.

[0216] After treatment, a significant decrease in PASI score parameters, erythema, induration, and desquamation was observed in all the treatment groups except vehicle treated group when compared with the disease control. The immunomodulators, IL6 and TNF-a have shown a significant decrease in the treatment groups when compared with the disease control group. The vehicle treated group has not shown much variation in these parameters when compared with the disease control.

[0217] The recovery from psoriasis after treatment with PRC0028 at different doses when compared with the disease control and standard was that the prophylactic group has prevented the occurrence of psoriasis when applied at the early stage of psoriasis, after three days of induction. The treatment groups has shown that there has been a significant reduction in psoriasis after the application of 3.7% extract of PRC0028 for 8 days. The dilution of 1.4% PRC0028 has shown much better response to that of standard drug, clobetasol propionate cream 0.025%w / w.

[0218] Fig. 20 depicts images of dermatological changes over time from psoriasis study.

[0219] According to the embodiments of the present invention, the bioactive extract has been applied to patients suffering from psoriasis. After application for over two weeks, the psoriasis lesions have completely cleared without any relapse for over 5 years.

[0220] As described above, the present invention the bioactive extract is formulated as a topical application for psoriasis treatment without adverse effects.

[0221] As various changes can be made in the above-described subject matter without departing from the scope and spirit of the present invention, it is intended that all subject matter contained in the above description, or defined in the appended claims, be interpreted as descriptive and illustrative of the present invention. Many modifications and variations of the present invention are possible in light of the above teachings. Accordingly, the present description is intended to embrace all such alternatives, modifications, and variances which fall within the scope of the appended claims.

Claims

Claims:

1. A method of obtaining a bioactive extract, the method comprising: putting ingredients including dried bark of Limonia acidissima tree of sizes measuring 50mm x 20mm x 20mm, dried bark of Cinnamon sizes measuring 200mm x 5mm x 5mm, and Cloves in a porcelain reactor; sealing the porcelain reactor airtight with a sealant, along with attaching a condenser vessel connected to a porcelain collector; heating the porcelain reactor slowly to a temperature of 250 - 450°C using a heat source, and maintaining the temperature for 4 to 5 hours under non-aqueous near-vacuum conditions for producing vapours; condensing the vapours produced during heating under non-aqueous near-vacuum conditions to produce a condensed liquid; and distilling the condensed liquid, and extracting bioactive extract into a porcelain collector, where the yield of the extraction is 5%.

2. The method of claim 1, wherein further comprises chemical characterization and validation of the bioactive extract, the chemical characterization includes identifying physical characteristics of the bioactive extract, where the bioactive extract is a 'Dark Reddish Brown Liquid' with an odor reminiscent of a sooty smell, has a specific gravity of 1.05, and a boiling temperature exceeding 400°C.

3. The method of claim 1, wherein the bioactive extract is insoluble in water but soluble in a variety of organic solvents including Acetonitrile, Ethanol, Acetone, Methanol, N-Butanol, Ter. Butanol, Iso Propyl Alcohol, Ethyl Acetate, Dichloro Methane, Chloroform, 1,4, Dioxane, Dimethylformamide, and Dimethylsulfoxide, further partially Soluble in Toulene, Diethyl ether and Di isoprpyl ethyl ether.

4. The method of claim 1, wherein the bioactive extract is exhibits a pH of 5.10.

5. The method of claim 1, where the bioactive extract is characterized by a High- performance liquid chromatography (HPLC) Chromatogram.

6. The method of claim 1, wherein the bioactive extract is characterized by a Liquid chromatography-Mass Spectrometry (LCMS) analytical method.

7. The method of claim 1, wherein further the bioactive extract undergoes for Antipsoriatic activity test for evaluating a potential effect, which is performed by a method including at least selected from Cell Proliferation Assay and Human IL-6 ELISA.

8. The method of claim 1, wherein the bioactive extract is used for the treatment of skin related disorders.

9. The method of claim 1, wherein the bioactive extract is used for the treatment of psoriasis.

10. The method of claim 1, wherein a 1.4% concentration of the bioactive extract prevents formation of psoriasis plaques when applied topically.

11. The method of claim 1, wherein the bioactive extract is effective against HaCaT cell (Keratinocytes) proliferation, which manifests as plaques in psoriasis, and capable of treating psoriatic plaques.

12. The method of claim 1, wherein the bioactive extract has an effect on IL-6 production by HaCaT cells, with higher concentrations of the extract leading to a greater inhibition of IL- 6 release.

13. The method of claim 1, wherein the bioactive extract inhibits SI 00 A8 and SI 00 A9 antimicrobial proteins, which are responsible for initiating psoriasis in the human body.

14. The method of claim 1, wherein the bioactive extract inhibits an expression of an inflammatory cytokine TNFa.

15. The method of claim 1, wherein the bioactive extract is formulated as a topical application for the treatment of psoriasis.

16. A pharmaceutical composition for treating psoriasis, comprising: a bioactive extract obtained from ingredients including dried bark of Limonia acidissima tree of sizes measuring 50mm x 20mm x 20mm, dried bark of Cinnamon sizes measuring 200mm x 5mm x 5mm, and Cloves;wherein, the bioactive extract is obtained by heating the ingredients including dried bark of Limonia acidissima tree of sizes measuring 50mm x 20mm x 20mm, dried bark of Cinnamon sizes measuring 200mm x 5mm x 5mm, and Cloves in a porcelain reactor to a temperature of 250 - 450°C using a heat source, and maintaining a temperature for 4 to 5 hours under nonaqueous near-vacuum conditions for producing vapours, then condensing the vapours produced during heating under non-aqueous near-vacuum conditions to produce a condensed liquid, and the bioactive extract is collected into a porcelain collector, where yield of the extraction is 5%.

17. The pharmaceutical composition of claim 16, wherein the bioactive extract is effective against HaCaT cell (Keratinocytes) proliferation, which manifests as plaques in psoriasis, and capable of treating psoriatic plaques.

18. The pharmaceutical composition of claim 16, wherein the bioactive extract has an effect on IL-6 production by HaCaT cells, with higher concentrations of the extract leading to a greater inhibition of IL-6 release.

19. The pharmaceutical composition of claim 16, wherein the bioactive extract inhibits S100 A8 and S100 A9 antimicrobial proteins, which are responsible for initiating psoriasis in the human body.

20. The pharmaceutical composition of claim 16, wherein the bioactive extract inhibits an expression of an inflammatory cytokine TNFa.

21. The pharmaceutical composition of claim 16, wherein the bioactive extract is formulated as a topical application for the treatment of psoriasis.

22. The pharmaceutical composition of claim 16, wherein a 1.4% concentration of the bioactive extract is a prophylactic agent and prevents formation of psoriasis plaques when applied topically.

23. The pharmaceutical composition of claim 16, wherein a 3.7% concentration of the bioactive extract prevents formation of psoriasis plaques when applied topically.

24. The pharmaceutical composition of claim 16, wherein the bioactive extract at concentrations of 1.4% and 3.7% is safe for use as a topical treatment and is formulated as a topical application for the treatment of psoriasis.

25. The pharmaceutical composition of claim 16, wherein the bioactive extract at concentration of 1.4% clears psoriatic plaques within 8 days of application when applied topically.

26. The pharmaceutical composition of claim 16, wherein the bioactive extract at concentration of 3.7% clears psoriatic plaques within 8 days of application when applied topically.

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