DESC:FIELD OF THE INVENTION
The present invention relates to formulations of Cyanidin-3-Glucoside (C3G) from purple corn and method of extracting C3G from purple corn. More specifically, the invention relates to phyto-liposomal formulations of C3G which enhance the bioavailability and efficacy of C3G. The method of extraction of C3G described in the present invention uses minimal solvents, and milder processing rendering the process eco-friendly, energy efficient and cost effective.
BACKGROUND
Purple corn (Zea mays L.) is a special corn variety and is a rich source of anthocyanins, especially cyanidin-3-glucoside anthocyanins along with other phytochemicals. A few other phytochemicals include pelargonidin-3-glucoside, and peonidin-3-glucoside. It originated in South America, mainly Peru, and cultivated also in Ecuador, Bolivia, and Argentina. It has been used for the preparation of traditional drinks and desserts, generating important quantities of residues, and its disposal involves high economic expense. Anthocyanins represent an important class of compounds that have a therapeutic and nutritional potential and also are used as food colorants. Anthocyanins provide red to a blue color to various foods and nutraceuticals and are a great alternative to replace synthetic colorants.
Purple corn cob is an economical source of anthocyanins. Purple corn cobs are often discarded as waste. There have been various approaches to extract anthocyanins from the whole cob to extract anthocyanins for use as colorants and nutraceuticals.
There are factors such as pH value, temperature and solvents that influence the color and stability of anthocyanins. In given acidic medium, four anthocyanin structures exist in equilibrium: flavylium cation, quinonoidal base, carbinol pseudobase and chalcone. The relative contents of these structures vary with pH and anthocyanin structure. It has been established that the high stability of anthocyanins is achieved in an acidic medium however, they may become unstable when they are extracted from a natural source. Part of their color may be lost due to formation of brownish degradation products and insoluble compounds during extraction. there is a need of method to extract stable anthocyanins from the purple corn cob and to develop formulations that maintain their stability and enhance their bioavailability and efficacy. Prior approaches to extract anthocyanins involve extraction with methanol or ethanol containing a small amount of acid for the purpose of preventing degradation of non-acylated compounds. Use of strong acid may cause partial hydrolysis of the acyl moieties on anthocyanins making them unsuitable for the extraction from purple corn [1].
Liposomes are small spherical vesicles composed of a lipid bilayer that can encapsulate a various bioactive compounds, including anthocyanins and present a solution to encapsulate anthocyanins without changing their structure. However, the bioavailability, efficacy and stability of C3G anthocyanins is limited with existing liposomes.
The present invention overcomes the gaps in the prior art regarding extraction of anthocyanins from purple corn by using no solvents, milder process conditions, and using organic acids. The formulations of cyanidin-3-glucoside described in the present invention have enhanced stability, bioavailability and efficacy. The formulations comprising the liposomal C3G anthocyanins described herewith have high encapsulation efficiency ,stability and efficacy.
SUMMARY
One aspect of the invention is a method of extracting C3G from purple corn cob wherein the method comprises the steps of;
a. Pulverizing purple corn cob to obtain a coarse powder;
b. Adding purified water to the coarse powder in step a wherein the purified water comprises citric acid to obtain a reaction mixture ;
c. refluxing the mixture in step b. to obtain a condensed mixture;
d. filtering the condensed mixture to obtain a filtrate enriched with C3G.
In one aspect, the purple corn cob is a whole corn cob. In one aspect, the pulverization in step a) is done at 40-75 °C. In one aspect ,the ratio of citric acid to water in step b) is 1:2 to 1:10 (wt/wt). In one aspect ,the duration for steeping coarse powder in purified water comprising citric acid is 2-3 hours in step b. In one aspect, the purified water is added to the coarse powder obtained in step b in a water bath and wherein the temperature of water bath is maintained at a temperature range of 25-75° C.
In one aspect, the filtrate obtained in step d. is further distilled to obtain a concentrate of total anthocyanins including C3G. wherein the anthocyanins amount ranges from 40-100 mg/ml.
In one aspect, the filtrate obtained in step d. is enriched with Chromium.
In one aspect, the filtrate obtained in step d. is enriched in corn starch, arabinoxylan, cellulose, or beta-glucan or a combination thereof. In one aspect, Tween-80, trehalose, or lecithin or a combination thereof are added to the dried filtrate of C3G obtained in step d. to make a stabilized formulation.
One aspect of the invention is a stabilized formulation of C3G extracted from purple corn cob, wherein the formulation comprises Tween-80, trehalose, or lecithin or a combination thereof.
In one aspect, the formulation comprises bioactive agents Bakuchi seed extract and Turmeric extract for psoriasis management.
In one aspect, the formulation comprises bioactive agents Bakuchi seed extract in the range of 3-10% (w/w) and Turmeric extract in the range of 7-15% (w/w).
In one aspect ,the formulation reduces pro-inflammatory cytokines (TNF-a, IL-17, IL-6, NF-kB, IL-4, TGF-ß) and enhances IL-10 levels.
In one aspect, the formulation effectively reduces the level of one or more oxidative stress markers selected from GST or GSH or SOD or CAT thereby preventing excessive free radical damage in psoriatic conditions.
In one aspect, the formulation is a topical formulation and is in a form selected from creams, gels, ointments, or serums.

DETAILED DESCRIPTION
Prior approaches to extract anthocyanins from purple corn involve use of high amount of solvents like ethanol or methanol, strong acids like HCL. A reference [1] reports maximum yield (5.90 mg/g dw) of anthocyanins was obtained with the combination of 80% (v/v) methanol and 1% (v/v) citric acid concentration.
Moreover, the extracted anthocyanin has a very low stability and is susceptible to degradation by oxidation.
The formulations of anthocyanins in general are highly hygroscopic and form lumps due to aggressive moisture retention from air. The present invention provides anthocyanin, especially C3G formulations that overcome these gaps.
Advantages of the Invention
• Scalable and Cost effective with minimum number of extraction steps
• Free from usage of petroleum derived products as harmful solvents like Dichloromethane (DCM), Methanol (MeOH), Hexane, and other chemical solvents that have toxic effects for human health are not used to extract C3G from purple corn.
• Use of Eco friendly solvents like Water
• Lower Extraction time
• No Need of Effluent Treatment as no toxic liquid discharge at the end of extraction process. Since, pure water is distilled from the in-process slurry and re-used (sustainable and preserving natural resources for fresh water).
• Low carbon foot print along with energy (power) saving. A separate distillation unit for distilling out pure water from the process is not used.
• Stable C3G formulations at room temperature and ambient temperatures. Enhanced stability & bioavailability of various actives derived from purple corn apart from C3G.
Brief Description of The Drawings
Figure 1shows QNMR_SPECTRAL REPORT For C3G Product from Purple Corn.
Internal calibrant used was benzoic acid.
Figure 2 shows 1H NMR Spectrum
Figure 3 shows the glucose levels over a period with treatment of purple corn extract/ C3G composition.
Figure 4. shows the skin tissue section from histopathology studies on 3 animals (A. animal 1, B. Animal 2. C. Animal 3)
Definitions
As used herewith the term “Phytoliposomal” refers to a lipid based vesicular system for encapsulating anthocyanin compounds including cyanidin-3-glucosides with one or more components. These components include bioactive molecules, functional molecules, antioxidants such as Ar-turmerone, spirulina extract, vitamins. Phytoliposome can be used interchangeably with phytosome ,a carrier-mediated vesicular system (phyto-liposome)

Phytosomes are delivery systems that are structurally related to liposomes and are prepared through the attachment of individual ingredients of herbal extracts to phosphatidyl choline, resulting in a formulation having higher solubility and enhanced absorption resulting in better pharmacokinetic and pharmacodynamic properties compared to the conventional herbal extracts.
As used herewith “Sunflower Lecithin” or lecithin derived from sunflower” comprises PC 16.2%, Phosphatidylinositols (PI) 16.5%, Phosphatidylethanolamine (PE) 5.3%.

Phosphatidylinositols (PI) represent another class of GPLs that have been found to be involved with lipid and cell signalling, as well as regulation of membrane proteins and membrane trafficking.
Phosphatidylethanolamine (PE) is a multifunctional phospholipid required for mammalian development that is essential for a variety of cellular processes. PE is a non bilayer forming phospholipid containing a small polar head group diameter in proportion to its fatty-acid chains.

As used herewith “cyanidin-3-glucoside” (7084-24-4) refers to anthocyanin compounds found in black bean, purple corn, purple potato, colourful berries and has strong antioxidant potential and numerous other properties like anti-inflammatory activity.
As used herewith ,the term “C3G extract”, “purple corn extract” refers to C3G extract derived from purple corn cob by the method encompassed in the invention.
As used herewith, the term “stabilized formulation of C3G extract” refers to a prepared composition designed to preserve the functional integrity, potency, and efficacy of cyanidin-3-glucoside (C3G) over an extended period under diverse environmental conditions, including elevated temperatures, light exposure, oxidative stress, microbial growth, and hydrolytic challenges. The terms “phyto-liposomal formulation” , “stabilized formulation of C3G extract” , “stabilized C3G extract” refers to stable formulation of C3G extract derived from purple corn cob or purple corn whole cob and are used interchangeably herewith.
“PSO-care formulation” or stabilized formulation of C3G extract comprising Bakuchi seed extract, turmeric extract refers to a stabilized formulation of C3G extract for treatment and management of psoriatic conditions/psoriasis.
As used herewith, the term, Supercritical Fluid Extraction (SCFE) extract” is a highly refined and concentrated bioactive product obtained through the use of supercritical CO2 extraction technology. This process ensures selective extraction and preservation of thermolabile, bioactive compounds, delivering high efficacy and purity while maintaining the structural integrity of the active components.
As used herewith, the term "Verify Solvent Condensation" in this procedure involves confirming that water vapor is condensed at a rate of 5 liters/hour, supported by proper cooling and real-time monitoring systems. This ensures the integrity of the reaction environment, safety, and consistent product quality, making it a critical in-process check for maintaining operational efficiency.
As used herewith, the term “purple corn raw material” refers to purple corn cob, purple corn whole cob ,purple corn starch, arabinoxylan, cellulose, glucan derived from purple corn.
Embodiments:

The current invention encompasses stabilized formations of anthocyanin, Cyanidin-3-glucoside (C3G) extracted from purple corn. The current invention also encompasses a method of extracting cyanidin-3-glucoside from purple corn.
One embodiment of the invention is a formulation of cyanidin-3-glucoside derived from purple corn and one or more bioactive compound selected from Ar-turmerone, citral, eugenol, gingerol, zingiberene, lycopene , lutein , lycopene, zeaxanthin , chlorophyll , Beta - carotene ,phycocyanin , fuco-xanthin , bakuchiol ,psoralen ,iso psoralen , psoralidin ,methoxsalen , astaxanthin , omega 3,6,7,9 fatty acids.
In one embodiment cyanidin-3-glucoside is derived from purple corn cob.
In one embodiment cyanidin-3-glucoside comprises a plurality of anthocyanins or anthocyanidins.
In one embodiment the C3G formulations are stabilized by phytoliposomal technology. In one embodiment the stabilized formulations of C3G from purple corn comprise 10-30% C3G or C3G equivalent anthocyanin.
In one embodiment the stabilized formulations of C3G comprise C3G, Tween 80, Trehalose, lecithin. In one embodiment the lecithin is derived from sunflower.
In one embodiment of the invention, trehalose acts as a preservative by reducing the microbial load. In one embodiment of the invention trehalose is an encapsulating agent for C3G anthocyanins derived from purple corn. In one embodiment, trehalose coats the liposomes of C3G anthocyanins resulting in improved stability. In one embodiment, trehalose improves drug delivery across cell membrane. In one embodiment, trehalose imparts changes to the morphology of liposome layer of the C3G formulation imparting higher stability and improved drug delivery. In one embodiment, trehalose imparts thermal stability for heat sensitive molecules like C3G, leading to stability of the formulation at room temperature.
In one embodiment, the lecithin derived from sunflower contains phosphatidyl choline (PC), phosphatidyl inositol (PI), phosphatidyl ethanolamine (PE), and essential fatty acids (Omega 6, Omega 9).
In one embodiment of the invention, the PI, PE along with PC enhance bioavailability. In one embodiment of the invention, the PI, PE along with PC enhance efficacy.

In one embodiment the C3G formulations comprise cyanidin-3-glucoside derived from purple corn and one or more bioactive compound along with starch, glucan, arabinoxylan, cellulose or beta-glucan derived from purple corn or a combination thereof. In one embodiment, the starch, glucan, arabinoxylan, cellulose or beta-glucan components derived from purple corn are water soluble.

In one embodiment, the stabilized formulations of C3G exhibit optimum retention of naturally available protein, vitamin and minerals from purple corn raw material .In one embodiment the stabilization is done by Phyto-liposomal technology (PLT). In one embodiment, PLT adds additional stability & enhances bioavailability of various actives derived from Purple corn apart from C3G.

In one embodiment the phyto-liposomal formulations of C3G from purple corn are stable at room temperature. In one embodiment the C3G formulations are stable at temperatures ranging from -5 °C to 60 °C. In one embodiment the C3G formulations are stable when exposed to air
In one embodiment , the formulation of C3G encompassed by the invention has Stable Brick red to Purplish red colour.
In one embodiment the C3G formulation has 5-10 times bioavailability of Anthocyanin C3G due to application of phyto-liposomal technology.

In one embodiment the invention, the C3G formulations have hybrid properties of Liposomes, Phytosomes, Nanosomes (Phospholipid +purple corn Protein) and Niosomes.
In one embodiment the C3G formulation is water soluble. In one embodiment the water soluble C3G formulation comprises 10-15% maltodextrin.
In one embodiment , the C3G formulations is non water soluble. In one embodiment the non water soluble C3G formulation does not comprise maltodextrin.
In one embodiment , the C3G formulation has anti-cancer activity. In one embodiment, the cancer is selected from lung cancer, gastric cancer, colon cancer, cervical cancer, breast cancer, pancreatic cancer, prostrate cancer.
In one embodiment the C3G formulation has anti-diabetic activity. In one embodiment the C3G formulation inhibit activity of alpha-glucosidase. In one embodiment the C3G formulation inhibit the activity of alpha-amylase.
In one embodiment the C3G formulation has anti-inflammatory activity. In one embodiment the C3G formulation has anti-oxidant activity. In one embodiment the C3G formulation inhibits the activity of tyrosinase.
In one embodiment the stabilized formulation of C3G can be formulated in forms selected from tablets ,gummies ,lozenges, syrup, BFS (Blow Fill Seal) , Sachets (Powder or Granules), effervescent tablets, Ointments, oil or water base suspension, oil or water based emulsions, suppositories, gels (Types: Colloidal, Hydro, Organo, Xero) ,Creams ,Injectables.
In one embodiment the stabilized formulation of C3G can be formulated in food and beverage compositions such as , without limitation, from tea bags ,face packs, health drinks, sublingual strips, Jams, Ice creams, Confectionary, Desserts (biscuits, cakes, cookies, custards, gelatins, ice creams, pastries, pies, puddings, macaroons, sweet soups, tarts, fruit salad ) , snacks.

One embodiment is a method of extracting C3G from purple corn cob wherein the method comprises the steps of;
a. Pulverizing purple corn cob to obtain a coarse powder;
b. Adding purified water to the coarse powder in step a wherein the purified water comprises citric acid to obtain a reaction mixture ;
c. refluxing the mixture in step b. to obtain a condensed mixture
d. filtering the condensed mixture to obtain a filtrate enriched with C3G
In one embodiment, the purple corn cob is a whole corn cob. In embodiment, the whole corn cob includes the seed (kernel).
In one embodiment, 99.9% of the coarse powder obtained from the pulverized purple corn cob in step a. passes through a 1mm sieve.
In one embodiment, the coarse powder obtained from the pulverized purple corn cob in step a. passes through a 1mm sieve.

In one embodiment, the pulverization in step a) is done at 40-75 °C.
In one embodiment, the ratio of citric acid to water in step b) is 1:2 to 1:10 (wt/wt).
In one embodiment, the duration for steeping coarse powder in purified water comprising citric acid is 2-3 hours.
In one embodiment, purified water is added to a coarse powder obtained in step b,in a water bath , wherein the temperature of water bath is maintained at a temperature range of 25-75° C.
In one embodiment, refluxing the mixture in step c. involves subjecting it to chilled water circulation. In one embodiment, the temperature maintained during chilled water circulation is -5° to 10 °C.
In one embodiment, the filtrate obtained in step d. is further distilled to obtain a concentrate of total anthocyanins including C3G. wherein the anthocyanins amount ranges from 40-100 mg/ml
In one embodiment, the filtrate obtained in step d. is enriched with Chromium.
In one embodiment, the Chromium is Chromium (III).
In one embodiment, condensation rate in step c. is maintained in the range of 1-10 litres/hour to achieve optimal reaction conditions. In one embodiment, the solvent condensation rate in step c. is 5 litres/hour. This ensures proper control over the reflux process, preventing water loss and maintaining the required solvent volume in the reaction system.
In one embodiment, the condensation output is measured every 15–30 minutes to confirm a steady flow rate.
In one embodiment, the filtrate obtained in step d. is enriched in corn starch, arabinoxylan, cellulose, or beta-glucan or a combination thereof.
In one embodiment, Tween-80, trehalose, or lecithin or a combination thereof are added to the dried filtrate of C3G obtained in step d. to make a stabilized formulation.
In one embodiment lecithin is derived from sunflower.
In one embodiment, the enriched components corn starch, arabinoxylan, cellulose, or beta-glucan replace bulking agents such as maltodextrin by 40-50%. In one embodiment, the components corn starch, arabinoxylan, cellulose, or beta-glucan are derived from purple corn replace bulking agents such as maltodextrin completely.

One embodiment of the invention is a method of making stabilized formulation of C3G derived from purple corn wherein Tween-80, trehalose, lecithin are added to the dried filtrate of C3G anthocyanins from step d. In one embodiment the method comprises the step of drying followed by sieving resulting in stabilized formulation of C3G derived from purple corn.
In one embodiment, the stabilized formulation is a phyto-liposomal formulation of C3G derived from purple corn cob.
In one embodiment, the drying is done by rotary evaporator drying.
In one embodiment the stabilized formulation of C3G is further formulated by adding bioactive compounds selected from one or more of Ar-turmerone, citral, eugenol, gingerol, zingiberene, lycopene, lutein, lycopene, zeaxanthin, chlorophyll , Beta - carotene ,phycocyanin , fuco-xanthin , bakuchiol ,psoralen ,iso psoralen , psoralidin ,methoxsalen , astaxanthin , omega 3,6,7,9 fatty acids.
In one embodiment, the stabilized formulation is subjected to drying after formulating with one or more bioactive compounds.
In one embodiment, the dried stabilized formulation has a moisture content of 2-3%.
In one embodiment, the stabilized formulation is a dark purplish red colored powder.
In one embodiment, the stabilized formulation is enriched in Chromium. In one embodiment , the chromium is Cr III.
One embodiment of the invention is a stabilized formulation of C3G extracted from purple corn cob, wherein the formulation comprises Tween-80, trehalose, or lecithin or a combination thereof.
In one embodiment of the invention, the stabilized formulation comprises bioactive agents Bakuchi seed extract and Turmeric extract
In one embodiment of the invention , the turmeric extract is an SCFE extract (super critical fluid extract).
In one embodiment, the bakuchi seed extract is an SCFE extract.
In one embodiment of the invention, the stabilized formulation comprises bioactive agents Bakuchi seed extract in the range of 3-10% (w/w) and Turmeric extract in the range of 7-15% (w/w).
In one embodiment of the invention, the stabilized formulation comprises bioactive agents Bakuchi seed extract and Turmeric SCFE extract and is used for psoriasis treatment. In one embodiment, the psoriasis treatment includes managing the psoriasis
In one embodiment, the stabilized formulation reduces pro-inflammatory cytokines (TNF-a, IL-17, IL-6, NF-kB, IL-4, TGF-ß) and enhances IL-10 levels.
In one embodiment the stabilized formulation effectively reduces the level of one or more oxidative stress markers selected from GST or GSH or SOD or CAT thereby preventing excessive free radical damage in psoriatic conditions.
In one embodiment, the formulation is a topical formulation and is in a form selected from creams, gels, ointments, or serums.

Examples:
Example 1: Extraction of C3G from Purple corn cob
The extraction of anthocyanins from whole purple corn cob (Purple corn cob sourced from Purple Lifesciences farm located at Marrigudem village, Tipparthy Mandal, Nalgonda District, Telangana, 508247) includes dried cob and seeds begins with pulverization to produce a coarse powder of purplish-grey color. This is followed by a reflux extraction process using citric acid and purified water under controlled temperatures and stirring conditions. The supernatant is filtered, concentrated, and stabilized through the incorporation of bioactive agents such as corn starch, arabinoxylan, cellulose, and beta-glucan. Drying techniques, including vacuum tray drying and rotary evaporation, are employed to obtain a stable purplish-red concentrate. Advanced stabilization is achieved using Phyto-Liposomal technology, which incorporates sunflower lecithin, Tween 80, and trehalose. The final product, a therapeutically valuable stable anthocyanin powder, is sieved and processed to meet quality standards. Detailed steps with specific parameters for temperature, pressure, and duration performed to ensure reproducibility and high-quality outcomes.
Raw Material Preparation:
Pulverized whole purple corn cobs into a coarse powder using a milling machine.
Parameters: Mesh size: 60, Temperature: 45–75°C, Duration: 3–4 hours
Reflux Extraction:
Ingredients:Purple corn whole cob powder: 6.3 kg, Citric acid anhydrous: 1.32 kg
Purified water: 2.0 kg
Procedure: Mix components in a round bottom flask with an overhead stirrer. Reflux at 45–65°C for 30–60 minutes. Maintain chilled water circulation at -5 to -10°C.
In-process Check: Verify solvent condensation
Verifying solvent condensation refers to the process of monitoring and confirming that water, used as the solvent, is effectively cooled and condensed back into its liquid state during reflux. This includes ensuring that the condensation rate is maintained at 5 liters/hour to achieve optimal reaction conditions.
Condensation Rate Verification (5 Liters/Hour):
Target Rate: 5 liters/hour (±5% tolerance to account for minor variations).
Measurement Interval: The condensation output was measured every 15–30 minutes to confirm a steady flow rate.
System Calibration: Graduated collection cylinders or a flowmeter were used to monitor and validate the condensation rate in real time.
Process Adjustment: If the rate falls below the target, the cooling temperature or the heating intensity were adjusted to restore proper balance.
Filtration:
The light purplish-red supernatant was filtered using a nylon filter cloth (2–5 mm pore size) maintained at 40 ± 2°C.
Concentration: The solution was concentrated using a rotary evaporator. Parameters: Water bath temperature: 40°C, Pressure: 2 bar, Stirrer speed: 45–50 RPM, Chiller temperature: -5 to -10°C
Outcome: Purplish-red concentrate with 65–70% solvent removed
Stabilization via Mixing
Ingredients: Corn starch: 0.32 kg, Arabinoxylan: 0.12 kg, Cellulose: 0.23 kg, Beta-glucan: 0.045 kg
Procedure: Mix with the concentrated solution at 40–55°C for 30 minutes at 150 RPM. Ensure complete dissolution of stabilizing agents.
Outcome: Dark purplish-red solution.
Drying and Sieving:
A vacuum tray dryer to dry the slurry solution was used.
Parameters: Temperature: 85–120°C,Tray capacity: 0.7 kg, Duration: 2.5–3 hours
the resulting powder was sieved through a 60-mesh screen.
Observation: Initial product is unstable.
Phyto-Liposomal Technology:
Ingredients: Tween 80: 18 g , Sunflower lecithin: 2.5 g, Trehalose: 15 g, Purified water: 0.6 kg
The powder obtained after drying and sieving was combined , thereafter it was processed using a rotary evaporator at 45–85°C for 2.5–3 hours.
Outcome: Enhanced stability of the product.
Final Drying:
The Phyto - liposomal thick solution was dried using a rotary evaporator and vacuum tray dryer.
Parameters: Rotary evaporation temperature: 65–95°C, Duration: 2.5–3 hours
Outcome: Stable, dark purplish-red anthocyanin powder.
Final Processing: The stable powder was milled using a multimill and sieve through a vibrosifter (60 mesh). Optional: Employ spray drying for additional refinement.
Final Product: Obtain a therapeutically valuable, stable anthocyanin powder with synergistic effects.
Example 2: Effect of Purple corn extracted C3G on STREPTOZOTOCIN (STZ)-NICOTINAMIDE (NAD) -Induced Diabetic Rodents
The effects of different treatment regimens on STREPTOZOTOCIN (STZ)-NICOTINAMIDE (NAD) -induced diabetic rodents was evaluated. glucose levels at various intervals to assess the efficacy of the treatments were measured. The results indicate a significant reduction in glucose levels in treated groups compared to the disease control group.
Materials and Methods:
Animals: Rodents were selected for this study. The animals were housed in a controlled environment with a 12-hour light/dark cycle and given access to food and water ad libitum.
Induction of Diabetes: Diabetes was induced in rodents by administering streptozotocin STZ-NAD at an appropriate dose. The blood glucose levels of the rodents were monitored after three days to confirm the induction of diabetes.
Experimental Design: The animals were divided into five groups:
• Group 1 (Normal): Non-diabetic control rodents
• Group 2 (Disease Control): STZ-NAD induced diabetic rodents without treatment
• Group 3 (Standard): STZ-NAD induced diabetic rodents treated with a standard anti-diabetic drug
• Group 4 (Purple corn extract or C3G (low dose)): STZ-NAD induced diabetic rodents treated with a low dose (LD) of the test compound
• Group 5 (Purple corn extract or C3G (High dose)): STZ-NAD induced diabetic rodents treated with a high dose (HD) of the test compound
Glucose Monitoring: Blood glucose levels were measured at different time points:
• After the 3rd day of induction
• After the 7th day of treatment
• After the 14th day of treatment
• After the 21st day of treatment
Results: The glucose levels recorded at different time points are as follows in Table 1:
Group Grouping Glucose (mg/dl) After 3rd Day of Induction Glucose (mg/dl) After 7th Day of Treatment Glucose (mg/dl) After 14th Day of Treatment Glucose (mg/dl) After 21st Day of Treatment
1 Normal 81 87 80 79
2 Disease Control 290 305 355 376
3 Standard 256 195 180 124
4 Purple corn extracted C3G (low dose) 295 119.8 133.4 131.2
5 Purple corn extracted C3G (High dose) 274 113.8 118.8 117.8

The results indicated that the purple corn extracted C3G at both low and high doses significantly reduced glucose levels compared to the disease control group. The high-dose treatment (Purple corn extract or C3G (High dose)) was more effective in maintaining lower glucose levels over time. The standard treatment also exhibited a substantial reduction in glucose levels. The untreated disease control group showed a progressive increase in glucose levels, confirming the efficacy of the purple corn extract in managing diabetes.
Thus, the purple corn extracted C3G reduces blood glucose levels in STZ-NAD induced diabetic rodents. The findings suggest its potential use as an anti-diabetic treatment.
Example 3: Stability of the Purple Corn Extracted C3G
The stability of C3G extracted from purple corn cob was assessed under accelerated conditions over a six-month period by analysing sensory, physicochemical, nutritional, elemental, microbiological, and vitamin content parameters at different time intervals (0th day, 1st month, 3rd month, and 6th month).
Sensory Parameters
Color: The extract maintained its purple hue throughout, indicating the stability of anthocyanins.
Odor & Taste: Both remained characteristic, confirming no significant degradation or undesirable changes.
Texture: The extract retained its powder form without aggregation, ensuring ease of handling and formulation.
Physicochemical Properties
Anthocyanin Pigment (mg/l): Showed slight degradation from 540 mg/l (0th day) to 515 mg/l (6th month), indicating good stability of bioactive compounds.
Moisture Content (%): Remained relatively stable (10.3% to 10.25%), preventing microbial spoilage and structural changes.
Water Activity: Decreased slightly from 0.29 to 0.26, further supporting microbial stability.
Ash Total (g/100g): Minor fluctuation observed (21.5 to 21.2), indicating consistent mineral content.
Tapped and Untapped Density (g/ml): Slight reduction observed (0.87 to 0.84 for tapped, 0.65 to 0.62 for untapped), maintaining flow properties.
Crude Fat, Fiber & Protein (g/100g): Stable across time points, confirming no significant compositional loss.
Total Carbohydrates (g/100g): Slightly reduced (53.5 to 52.9), with no major impact on nutritional profile.
Energy Content (kcal/100g): Minimal changes (285 to 282 kcal/100g), ensuring its caloric stability.
Heavy Metals and Trace Elements (mg/kg or µg/100g)m : Arsenic, Cadmium, Lead, Mercury, Chromium, Copper, Iron, Manganese, Nickel, Selenium, Zinc: Levels remained within safe limits, with negligible fluctuations over six months.
Microbiological Stability
Total Plate Count: Slight reduction (1.6 × 104 to 1.4 × 104 cfu/g), indicating stable microbial integrity.
Pathogens (E. coli, S. aureus, Yeasts & Molds, Pseudomonas aeruginosa, Salmonella): Remained undetectable or within safe limits, ensuring microbial safety.
Aflatoxins (B1, B2, G1, G2, Sum): Consistently below 0.5 µg/kg, confirming toxin stability.
Amino Acid Profile (g/100g or mg/100g)
Key amino acids (Alanine, Arginine, Aspartic Acid, Glutamic Acid, Histidine, Leucine, Lysine, Phenylalanine, Threonine) showed minimal degradation, ensuring protein stability.
Vitamin Stability (mg/100g or g/100g)
Vitamin B1, B3, and C: Minor reduction observed (Vitamin B1: 0.09 to 0.08, Vitamin B3: 7.4 to 7.3, Vitamin C: 0.09 to 0.09), indicating good retention under accelerated conditions.
Fatty Acid Composition (g/100g)
Monounsaturated, Polyunsaturated, and Saturated Fatty Acids: Maintained stable levels, confirming lipid integrity and preventing rancidity.
This six-month accelerated stability study demonstrated that purple corn extract maintained its sensory, physicochemical, nutritional, and microbiological stability, with minimal degradation of anthocyanins, vitamins, and amino acids. The extract remained safe and suitable for long-term formulation in nutraceutical, pharmaceutical, and functional food applications.
This stability data indicates high shelf-life and suitability for various applications.
The detailed values are mentioned in Table 2.
Table 2
Accelerated Stability
Parameter Unit 0th Day 1st Month 3rd Month 6th Month
Colour IR Purple Purple Purple Purple
Odour IR Characteristic Characteristic Characteristic Characteristic
Taste IR Characteristic Characteristic Characteristic Characteristic
Texture IR Powder Powder Powder Powder
Anthocyanin Pigment mg/l 540 533 521 515
Ash Total g/100g 21.5 21 21.3 21.2
Tapped Density g/ml 0.87 0.86 0.87 0.84
Untapped Density g/ml 0.65 0.63 0.63 0.62
Crude Fat g/100g 2.4 2.35 2.3 2.25
Crude Fiber g/100g 1 0.98 0.94 0.96
Crude Protein g/100g 12 11.95 11.9 11.85
Energy kcal/100g 285 281 283 282
Moisture g/100g 10.3 10.15 10.2 10.25
Total Carbohydrates g/100g 53.5 53.3 53.6 52.9
Water Activity 0.29 0.28 0.28 0.26
Arsenic (As) mg/kg 0.11 0.08 0.09 0.08
Cadmium (Cd) mg/kg 0.15 0.14 0.15 0.12
Chromium (Cr) µg/100g 290.5 287 289.5 289
Copper (Cu) mg/kg 19.4 19.1 19.2 19.1
Iron (Fe) mg/100g 22.1 21.5 21.9 21.8
Lead (Pb) mg/kg 0.3 0.31 0.28 0.27
Manganese (Mn) mg/kg 17.8 17.1 17.7 17.65
Mercury (Hg) mg/kg 0.01 0.01 0.01 0.01
Nickel (Ni) mg/kg 1.8 1.69 1.78 1.77
Selenium (Se) µg/100g 9.9 9.6 9.7 9.6
Zinc (Zn) mg/100g 2.4 2.38 2.4 2.34
Aflatoxin B1 µg/kg 0.5 0.5 0.5 0.5
Aflatoxin B2 µg/kg 0.5 0.5 0.5 0.5
Aflatoxin G1 µg/kg 0.5 0.5 0.5 0.5
Aflatoxin G2 µg/kg 0.5 0.5 0.5 0.5
Sum of Aflatoxins µg/kg 0.5 0.5 0.5 0.5
Total Plate Count cfu/g 1.6x10^4 1.5x10^4 1.4x10^4 1.4x10^4
Escherichia coli cfu/g <10 <10 <10 <10
Staphylococcus aureus cfu/g <10 <10 <10 <10
Yeasts & Moulds cfu/g <10 <10 <10 <10
Pseudomonas aeruginosa /g Not Detected Not Detected Not Detected Not Detected
Salmonella /25g Not Detected Not Detected Not Detected Not Detected
Alanine g/100g 0.75 0.73 0.72 0.7
Arginine g/100g 0.37 0.39 0.38 0.37
Aspartic Acid g/100g 1.29 1.28 1.26 1.3
Glutamic Acid g/100g 3.64 3.63 3.65 3.58
Histidine g/100g 0.2 0.17 0.18 0.19
Leucine g/100g 0.58 0.53 0.56 0.55
Lysine g/100g 0.33 0.32 0.31 0.3
Phenylalanine g/100g 0.27 0.29 0.28 0.3
Threonine mg/100g 0.27 0.26 0.25 0.24
Vitamin B1 mg/100g 0.09 0.07 0.08 0.08
Vitamin B3 mg/100g 7.4 7.1 7.2 7.3
Vitamin C g/100g 0.09 0.08 0.08 0.09
Monounsaturated Fatty Acids g/100g 0.75 0.7 0.72 0.73
Polyunsaturated Fatty Acids g/100g 0.95 0.94 0.93 0.94
Saturated Fatty Acids g/100g 0.7 0.67 0.68 0.69
The formulation addresses the following key stability challenges of C3G
ThermalStability:
C3G, being thermolabile, requires protection against high temperatures. The formulation ensures stability in the 65°C to 80°C range, far beyond the typical storage requirement of below -20°C.
Photo-Stability:
C3G is photosensitive and prone to degradation under light. The formulation mitigates light-induced damage by forming protective barriers.
Oxidative Stability:
C3G's susceptibility to oxidation is addressed through the inclusion of antioxidants and excipients that reduce oxygen exposure.
Microbial Stability:
Prevents microbial growth in formulations, ensuring safety and extended shelf life.
Hydrolytic Stability:
Protects the glycosidic bond of C3G from water-induced hydrolysis in aqueous formulations.
Example 4: A Stabilized formulation of C3G (PSO_CARE) extracted from purple corn for Psoriasis Management.
A stabilized formulation for psoriasis care or treatment comprising C3G extract has been developed using bioactive plant-based compounds, including purple corn extract/C3G Turmeric SCFE (Super critical fluid extraction) extract and bakuchi seed extract, known for their therapeutic effects on psoriasis and other inflammatory skin conditions. The addition of bakuchi seed extract enhances collagen synthesis, promotes skin renewal, and offers natural retinoid-like effects without irritation. The formulation is scientifically optimized for enhanced skin absorption and prolonged stability.
The synergistic effects of SCFE extracts on GST, GSH, SOD, and CAT highlight their potential in managing oxidative stress and inflammatory disorders like psoriasis.
Active Ingredients:
Cyanidin-3-Glucoside (C3G): 1% (w/w)
A potent antioxidant that reduces oxidative stress by neutralizing free radicals and stabilizing oxidative stress markers such as GST (Glutathione S transferase), GSH (Reduced Glutathione), SOD (Superoxide Dismutase), and CAT (Catalase).
Bakuchi Extract: 5% (w/w)
Contains bioactive compounds such as psoralen, known for their anti-
inflammatory and skin-healing properties, particularly effective in managing
psoriatic conditions.
Turmeric Extract: 10% (w/w)
Rich in curcuminoids and aromatic turmerone, providing anti-inflammatory and
antioxidant effects to mitigate oxidative stress and cytokine-mediated damage.
Excipients:
Tween 20 (Polysorbate 20): 84% (w/w)
A surfactant and stabilizing agent that ensures uniform dispersion of active
ingredients, enhances solubility, and protects thermolabile and photosensitive
components in the formulation.
4.1 The accelerated stability study was conducted under controlled conditions (40°C and 75% RH) and analyzed for:
Chemical Stability:
Anthocyanin Pigment: Retained 90% of its initial concentration after six months, ensuring the antioxidant efficacy of the product.
Curcuminoids: Maintained potency, providing sustained anti-inflammatory benefits.
Bakuchi Seed Extract: Stability confirmed through phytochemical retention, ensuring consistent bioactivity for skin regeneration.
Hexanal Levels: Remained below 1.0 mg/kg, confirming minimal lipid oxidation and product freshness.
Peroxide Value: Stayed below 0.2 meq/kg, ensuring stability and resistance to oxidative degradation.
Physical Stability:
Visual Appearance: Maintained its golden yellow liquid consistency, with no phase separation or discoloration.
Odor: Retained its characteristic profile, indicating no degradation of aromatic components.
Microbiological Stability: Escherichia coli, Staphylococcus aureus, Yeasts & Molds, Pseudomonas aeruginosa, and Salmonella remained undetected, confirming its microbiological safety.
Total Plate Count: Below industry-standard limits, ensuring product purity.
The accelerated stability study confirms that PSO Care, enriched with bakuchi seed extract, remains stable and effective over time.
4.2 ACUTE DERMAL TOXICITY (OECD 402)
Acute dermal toxicity was performed according to OECD Guideline for Testing of Chemicals – No. Test: 402, Acute Dermal Toxicity-Fixed Doses Procedure.
The acute dermal toxicity test stage was divided into two, i.e., the preliminary test (dose search), and then proceed to the main test stage. In stage 1, a preliminary test uses one experimental animal, the evaluation steps will be performed as per the guidelines. If there was no information regarding the test material, it was recommended to start testing with a dose of 200 mg/kg BW, but if there was enough information, it could be started with a dose of 1000 mg/kg BW (OECD, 2017).
In this study, an initial dose of 1000 mg/kg BW of test substance was used. The test material was applied to the specified skin, covered with sterile gauze, and wrapped with a non-irritating plaster to ensure the test material remained in contact with the skin for 24-hour and there was no chance of the test material being ingested by the test animal. At the end of the exposure period, the plaster was removed and the remaining test material was rinsed using distilled water. Then observations was carried out during the 0, 24, 48, and 72-hours after exposure to the test material, and continued until the 14-day. Observations include clinical assessment, weight loss, observation of skin anatomy pathology, and death. If the test animal experiences symptoms of severe toxicity or death, then evaluation continues using a lower dose with a new test animal. Evaluation will be stopped if the lowest dose test showed death or symptoms of severe toxicity (Solikhah et al., 2022). The highest dose that does not cause death or does not show signs of severe toxicity will be used in the main evaluation phase (Puspitasari et al., 2021).
In the main stage, the same procedure was carried out as per guidelines. The evaluation wasbe carried out with a predetermined dose with the addition of two new experimental animals for respective treatment and control groups. In this study, the treatment group was be applied topically with test groups, and the control group wase given a placebo in the same amount as the treatment.
Clinical Observation
Observations was performed on both groups clinically immediately after the exposure period. Special attention to hourly checks is carried out in the first 6-hour and monitored for 24-hour, then continued once every day until the 14-day. Observations carried out included weighing the rat's body weight, visual clinical observations, and the death of test animals. No mortality or abnormal behavior was reported.
Observation of Skin Anatomy Pathology
Anatomical pathology observations was carried out on the treated skin areas, carried out at 24, 48, and 72-hours after the exposure period for the appearance of irritation symptoms such as erythema and edema.
Table 3: Draize dermal irritation scoring system.
Score Translation
0 No erythema or oedema
1 Very inappreciable oedema or erythema
2 Small oedema with raised skin at the edges of the area
3 Moderate to severe erythema or oedema
4 Severe erythema or oedema

Assessment of Acute Dermal Toxicity
Determination of the LD50 of test preparations was based on the Global Harmonized System of Classification and Labeling of Chemicals. The LD50 value was calculated (Table 3).
Histopathological studies
The tissue samples collected at the end of toxicity studies for histopathological analysis. The tissues will be immediately.
blotted, dried and fixed in formalin (10%), then dehydrated in acetone and embedded in paraffin wax for taking sections (4 µm thickness) with the help of microtome. The sections of the tissues will be then stained with haematoxylin-eosin and processed for photo microscopic examination using digital microscope
Results : In the skin irritation test, no erythema or oedema was seen in both the control animals and the test animals after 3 days of experimentation. Observations carried out included weighing the visual clinical observations, and the death of test animals. No mortality or abnormal behavior was reported. No change meant that the manner in which the animals behaved after acclimatization did not alter when the skin was shaved and test substances were applied.
Observation of Skin Anatomy Pathology
Scoring was found to be O using Draize dermal irritation scoring system. No erythema or oedema was observed over the 14-day study period in both the control and PSO- CARE (Bakuchiol and Purple Corn Anthocyanin) treated animals.
Fig 4. A. Animal 1: Revealed normal skin histology with the presence of hair follicles and a few sweat glands and sebaceous glands. Epidermis, dermis, subcutaneous fats and muscular layers are seen without any abnormality.
Fig 4. B. Animal 2: Revealed normal skin histology with the presence of hair follicles and a few sweat glands and sebaceous glands. Hair follicles are in high number when compared to skin 1. Epidermis, dermis, subcutaneous fats and muscular layers are seen without any abnormality.
Fig 4. C. Animal 3: Revealed normal skin histology with the presence of hair follicles and a few sweat glands and sebaceous glands. Epidermis, dermis, subcutaneous fats and muscular layers are seen without any abnormality. Distribution of adipocytes surrounding the hair follicles noticed in a few areas.

4.3 Curative effects of Stabilized C3G formulation from purple corn cob for Psoriasis (PSO CARE) on imiquimod-induced psoriasis model
Protocol: Adult wistar rats were procured from Mass Biotech, Chengalpattu. IAEC Approval was obtained from SRM College of Pharmacy, SRM Institute of Science and Technology, Kanchipuram. Rats of 8–11?weeks old were used in the study. In all the experimental groups, animals were smoothly and clean shaven along their back part. The total study duration was conducted for a period of 21 days with 7 days of Psoriasis induction and 14 days of treatment. Psoriasis in mouse was induced using imiquimod-induced psoriasis, the animal’s dorsal part was shaved and commercially available 5% (IMQ) cream (Aldara; 3?M Pharmaceuticals) with dose of 62.5?mg of imiquimod was topically applied for 7 consecutive days and translating in a daily dose of 3.125?mg of the active compound. The treatment was done with Pso Care formulation for a period of 14 days.
After 14?days skin biopsies were taken immediately, fixed in 10% formalin and embed in paraffin. The tissue section (4?µm thick) was taken and stained with haematoxylin and eosin & Masson’s Trichrome staining.
4.4 Scoring severity of skin inflammation
Anti psoriatic activity of the given test formulations was evaluated on the basis of PASI (Zero, Slight, Moderate, Very marked) score along with the histopathology of skin [3]
Table 4

S.No

Groups Initial grading after induction of psoriasis using Imiquimod (PASI)
Final grading after induction of psoriasis using Imiquimod (PASI) after 14 days
1 Normal control None None
2 Diseased Control Very Marked Very Marked
3 Standard control Very Marked Marked
4 Low dose Very Marked None

5
Intermediate dose
Very Marked
None
6 High dose Very Marked None

4.5 ANTI OXIDANT ACTIVITY OF PSO CARE
The DC group exhibited significantly increased oxidative stress markers (GST, GSH, SOD, and CAT) compared to the control, while PSO CARE treatment (D1, D2, D3) helped restore antioxidant balance in a dose-dependent manner. GST, GSH, SOD, and CAT levels decreased in the PSO CARE-treated groups, suggesting a potential protective effect against oxidative damage. D2 and D3 doses showed the most effective normalization, highlighting PSO CARE’s therapeutic potential in psoriasis management.
D1- low dose (200mg/kg)
D2-intermediate dose (300mg/kg)
D3-high dose (400mg/kg)
(The above listed dose amount is as per body weight)
Table 5 :GST (Glutathione S Transferase)
Groups
GST Conc ng/mg GST Conc pg/mg
Conc ug/mg Stdev Conc pg/mg Stdev Sem
Normal 0.338 0.01 338 11.0 6.35
Neg. Control 0.585 0.18 585 177.3 102.35
Standard 0.359 0.01 359 6.1 3.51
Test (Low Dose) (D1) 0.472 0.01 472 5.7 3.28
Test (Intermediate Dose) (D2) 0.448 0.01 448 9.8 5.67
Test (High Dose) (D3) 0.376 0.01 376 14.4 8.29

Table 6 :GSH (Reduced Glutathione)
Groups
GSH Conc ng/mg GSH Conc pg/mg
Conc ug/mg Stdev Conc pg/mg Stdev Sem
Normal 0.214 0.01 214 13.7 7.88
Neg. Control 0.337 0.05 337 51.4 29.67
Standard 0.225 0.01 225 12.2 7.02
Test (Low Dose) 0.266 0.06 266 56.0 32.34
Test (Intermediate Dose 0.246 0.02 246 23.6 13.65
Test (High Dose) 0.221 0.02 221 22.9 13.22

Table 7: CAT (Catalase)
Groups
CAT µM CAT nmol/mg
CAT µM Stdev CAT nmol/mg Stdev Sem
Normal 0.378 0.01 378 8.9 5.13
Neg. Control 0.607 0.03 607 33.6 19.41
Standard 0.394 0.02 394 20.3 11.70
Test (Low Dose) 0.492 0.12 492 116.2 67.11
Test (Intermediate Dose 0.461 0.05 461 45.6 26.34
Test (High Dose) 0.393 0.02 393 15.3 8.82

Table 8: SOD (Superoxide Dismutase
Groups
SOD Conc ng/mg SOD Conc pg/mg
SOD Conc ng/mg Stdev Conc pg/mg Stdev Sem
Normal 0.187 0.03 187 29.0 16.74
Neg. Control 0.347 0.03 347 28.0 16.18
Standard 0.212 0.01 212 6.1 3.51
Test (Low Dose) 0.283 0.08 283 78.8 45.50
Test (Intermediate Dose 0.248 0.01 248 8.5 4.93
Test (High Dose) 0.197 0.00 197 3.2 1.86

4.6 ELISA Analysis in tissue sample
The DC group showed significantly elevated pro-inflammatory cytokines (TNF-a, IL-17, IL-6, NF-kB, IL-4, TGF-ß), while PSO CARE treatment (D1, D2, D3) effectively reduced these markers, indicating its anti-inflammatory potential. Additionally, IL-10 (anti-inflammatory cytokine) increased with PSO CARE, confirming its role in immune modulation. These results suggest that PSO CARE alleviates psoriasis-related inflammation and restores immune balance.
MARKER ANALYSIS
Table 9: TNF-Alpha
Groups
TNF-Alpha ng/mg TNF-Alpha pg/mg
Conc ug/mg Stdev Conc pg/mg Stdev Sem
Normal 0.280 0.03 280 34.9 20.17
Neg. Control 0.656 0.17 656 172.9 99.81
Standard 0.315 0.01 315 11.0 6.36
Test (Low Dose) 0.520 0.06 520 62.4 36.02
Test (Intermediate Dose 0.387 0.03 387 28.7 16.60
Test (High Dose) 0.355 0.04 355 39.9 23.05
Table 10: IL-6
Groups
IL-6 ng/mg IL-6 pg/mg
Conc ug/mg Stdev Conc pg/mg Stdev Sem
Normal 0.292 0.04 292 44.6 25.76
Neg. Control 0.431 0.06 431 64.7 37.36
Standard 0.299 0.02 299 18.3 10.59
Test (Low Dose) 0.375 0.07 375 67.9 39.20
Test (Intermediate Dose 0.335 0.04 335 43.0 24.84
Test (High Dose) 0.310 0.04 310 38.0 21.96

Table 11: TGF-beta
Groups
TGF-beta ng/mg TGF-beta pg/mg
Conc ug/mg Stdev Conc pg/mg Stdev Sem
Normal 0.292 0.04 174 35.0 20.22
Neg. Control 0.431 0.06 518 126.8 73.19
Standard 0.299 0.02 193 24.9 14.36
Test (Low Dose) 0.375 0.07 380 36.8 21.26
Test (Intermediate Dose 0.335 0.04 300 6.7 3.84
Test (High Dose) 0.310 0.04 219 42.0 24.25

Table 12: NFkb
Groups
Nfkb ng/mg Nfkb pg/mg
Conc ug/mg Stdev Conc pg/mg Stdev Sem
Normal 0.323 0.03 323 28.2 16.29
Neg. Control 0.490 0.04 490 43.7 25.21
Standard 0.341 0.04 341 41.9 24.17
Test (Low Dose) 0.425 0.04 425 39.7 22.93
Test (Intermediate Dose 0.370 0.05 370 48.2 27.83
Test (High Dose) 0.352 0.04 352 37.2 21.50

Table 13: IL-10
Groups
IL-10 ng/mg IL-10 pg/mg
Conc ug/mg Stdev Conc pg/mg Stdev Sem
Normal 0.415 0.03 415 32.3 18.66
Neg. Control 0.662 0.04 662 37.4 21.62
Standard 0.439 0.04 439 37.8 21.83
Test (Low Dose) 0.513 0.06 513 59.3 34.24
Test (Intermediate Dose 0.468 0.01 468 11.2 6.44
Test (High Dose) 0.456 0.04 456 37.1 21.40
Table 14: Il-4
Groups
IL-4 ng/mg IL-4 pg/mg
Conc ug/mg Stdev Conc pg/mg Stdev Sem
Normal 0.416 0.04 416 37.6 21.71
Neg. Control 0.688 0.06 688 56.9 32.84
Standard 0.433 0.02 433 18.5 10.69
Test (Low Dose) 0.593 0.08 593 81.3 46.96
Test (Intermediate Dose 0.498 0.07 498 70.9 40.95
Test (High Dose) 0.459 0.03 459 29.1 16.80

RTPCR Results
The RT-PCR results align with the ELISA findings, confirming that pro-inflammatory markers (TNF-a, IL-17, IL-6, IL-23, NF-kB, IL-4, and TGF-ß) were significantly elevated in the disease control (DC) group and reduced in (stabilized formulation comorising C3G, bakuchi extract, turmeric SCFE extract ) PSO CARE-treated groups (D1, D2, D3) in a dose-dependent manner. Additionally, IL-10 (anti-inflammatory cytokine) was suppressed in the DC group but increased with PSO CARE treatment, reinforcing its immunomodulatory potential. These results strongly validate PSO CARE's anti-inflammatory effects in psoriasis treatment.
RTPCR
Table 15 : ??-actin
Groups CT CT CT Average
G1 Normal 19.06 19.04 19.05
G2 Neg. Control 18.96 19.12 19.04
G3 Standard 19.12 19.06 19.09
G4 Test (Low Dose) 19.09 19.13 19.11
G5 Test (Intermediate Dose 19.12 19.09 19.11
G6 Test (High Dose) 19.83 19.02 19.43

Table 16: IL-4
Groups CT CT CT Average ?Ct ??Ct Fold change Stdev
G1 Normal 27.34 28.23 27.79 8.74 0.00 1.00 0.63
G2 Neg. Control 26.01 26.54 26.28 7.24 -1.50 2.83 0.37
G3 Standard 27.89 27.23 27.56 8.47 -0.27 1.20 0.47
G4 Test (Low Dose) 27.18 27.06 27.12 8.01 -0.73 1.65 0.08
G5 Test (Intermediate Dose 27.12 27.45 27.29 8.18 -0.56 1.47 0.23
G6 Test (High Dose) 25.78 26.14 25.96 6.54 -0.70 1.62 0.25

Table 17: Nfkb
Groups CT CT CT Average ?Ct ??Ct Fold change Stdev
G1 Normal 24.15 24.43 24.29 5.24 0.00 1.00 0.20
G2 Neg. Control 22.12 22.89 22.51 3.47 -1.78 3.42 0.54
G3 Standard 23.52 24.75 24.14 5.05 -0.20 1.14 0.87
G4 Test (Low Dose) 23.12 23.79 23.46 4.35 -0.90 1.86 0.47
G5 Test (Intermediate Dose 24.18 23.34 23.76 4.66 -0.59 1.50 0.59
G6 Test (High Dose) 22.26 22.97 22.615 3.19 -0.27 1.21 0.50

Table 18: IL-10
Groups CT CT CT Average ?Ct ??Ct Fold change Stdev
G1 Normal 24.12 24.02 24.07 5.02 0.00 1.00 0.07
G2 Neg. Control 22.04 22.48 22.26 3.22 -1.80 3.48 0.31
G3 Standard 23.02 24.66 23.84 4.75 -0.27 1.21 1.16
G4 Test (Low Dose) 23.92 23.22 23.57 4.46 -0.56 1.47 0.49
G5 Test (Intermediate Dose 23.56 24.09 23.83 4.72 -0.30 1.23 0.37
G6 Test (High Dose) 22.56 22.28 22.42 3.00 -0.22 1.17 0.20

Table 19: IL-6
Groups CT CT CT Average ?Ct ??Ct Fold change Stdev
G1 Normal 23.77 23.93 23.85 4.80 0.00 1.00 0.11
G2 Neg. Control 22.12 22.67 22.40 3.36 -1.45 2.72 0.39
G3 Standard 24.23 23.23 23.73 4.64 -0.16 1.12 0.71
G4 Test (Low Dose) 23.02 23.26 23.14 4.03 -0.77 1.71 0.17
G5 Test (Intermediate Dose 23.34 23.67 23.51 4.40 -0.40 1.32 0.23
G6 Test (High Dose) 22.16 22.67 22.42 2.99 -0.37 1.29 0.36

Table 20: TNF Alpha
Groups CT CT CT Average ?Ct ??Ct Fold change Stdev
G1 Normal 24.23 24.78 24.51 5.46 0.00 1.00 0.39
G2 Neg. Control 23.03 23.26 23.15 4.11 -1.35 2.55 0.16
G3 Standard 24.56 24.34 24.45 5.36 -0.10 1.07 0.16
G4 Test (Low Dose) 24.12 23.56 23.84 4.73 -0.73 1.65 0.40
G5 Test (Intermediate Dose 24.18 24.02 24.10 5.00 -0.46 1.38 0.11
G6 Test (High Dose) 23.20 23.67 23.44 4.01 -0.09 1.07 0.33

Table 21: IL-17
Groups CT CT CT Average ?Ct ??Ct Fold change Stdev
G1 Normal 24.56 24.78 24.67 5.62 0.00 1.00 0.16
G2 Neg. Control 23.09 23.56 23.33 4.29 -1.34 2.52 0.33
G3 Standard 24.73 24.51 24.62 5.53 -0.09 1.06 0.16
G4 Test (Low Dose) 24.12 23.89 24.01 4.90 -0.73 1.65 0.16
G5 Test (Intermediate Dose 24.34 23.89 24.12 5.01 -0.61 1.53 0.32
G6 Test (High Dose) 23.23 23.78 23.51 4.08 -0.20 1.15 0.39

Table 22: IL-23
Groups CT CT CT Average ?Ct ??Ct Fold change Stdev
G1 Normal 23.89 23.94 23.92 4.87 0.00 1.00 0.04
G2 Neg. Control 22.23 22.56 22.40 3.36 -1.51 2.85 0.23
G3 Standard 24.36 23.34 23.85 4.76 -0.11 1.08 0.72
G4 Test (Low Dose) 23.12 23.45 23.29 4.18 -0.69 1.61 0.23
G5 Test (Intermediate Dose 23.36 23.58 23.47 4.37 -0.50 1.41 0.16
G6 Test (High Dose) 22.42 22.89 22.66 3.23 -0.12 1.09 0.33

References
1. Yang, Z., Fan, G., Gu, Z., Han, Y., & Chen, Z. (2007). Optimization extraction of anthocyanins from purple corn (Zea mays L.) cob using tristimulus colorimetry. European Food Research and Technology, 227(2), 409–415. doi:10.1007/s00217-007-0735-4
2. Yuying Hu., Xiangjian Liu., Fenglin Liu, Jingxian Xie., Qubo Zhu., Songwen Tan Trehalose in Biomedical Cryopreservation–Properties, Mechanisms, Delivery Methods, Applications, Benefits, and Problems. ACS Biomater. Sci. Eng. 2023, 9, 3, 1190–1204 https://doi.org/10.1021/acsbiomaterials.2c01225
3. Mysliwietz J, Thierfelder S. Antilymphocytic antibodies and marrow transplantation. XII. Suppression of graft-versus-host disease by T-cell-modulating and depleting antimouse CD3 antibody is most effective when preinjected in the marrow recipient. Blood.1992;80(10):261–7
,CLAIMS:1.A method of extracting C3G from purple corn cob wherein the method comprises the steps of;
a. Pulverizing purple corn cob to obtain a coarse powder;
b. Adding purified water to the coarse powder in step a wherein the purified water comprises citric acid to obtain a reaction mixture ;
c. refluxing the mixture in step b. to obtain a condensed mixture;
d. filtering the condensed mixture to obtain a filtrate enriched with C3G.
2. The method as claimed in claim 1, wherein the purple corn cob is a whole corn cob.
3. The method as claimed in claim 1, wherein the pulverization in step a) is done at 40-75 °C.
4. The method as claimed in claim 1, wherein the ratio of citric acid to water in step b) is 1:2 to 1:10 (wt/wt).
5. The method as claimed in claim 1, the duration for steeping coarse powder in purified water comprising citric acid is 2-3 hours in step b.
6. The method as claimed in claim 1, wherein the purified water is added to the coarse powder obtained in step b in a water bath and wherein the temperature of water bath is maintained at a temperature range of 25-75° C.
7. The method as claimed in claim 1 wherein refluxing the mixture in step c. involves subjecting it to chilled water circulation.
8. The method as claimed in claim 1, wherein the filtrate obtained in step d. is further distilled to obtain a concentrate of total anthocyanins including C3G. wherein the anthocyanins amount ranges from 40-100 mg/ml.
9. The method as claimed in claim 1, wherein the filtrate obtained in step d. is enriched with Chromium.
10.The method as claimed in claim 1, wherein the filtrate obtained in step d. is enriched in corn starch, arabinoxylan, cellulose, or beta-glucan or a combination thereof.
11. The method as claimed in claim 1, wherein Tween-80, trehalose, or lecithin or a combination thereof are added to the dried filtrate of C3G obtained in step d. to make a stabilized formulation.
12. The method as claimed in claim 11, wherein the stabilized formulation is subjected to drying after formulating with one or more bioactive compounds.
13. A stabilized formulation of C3G extracted from purple corn cob , wherein the formulation comprises Tween-80, trehalose, or lecithin or a combination thereof.
14. The formulation as claimed in claim 13. wherein the formulation comprises bioactive agents Bakuchi seed extract and Turmeric extract for psoriasis management.
15. The formulation as claimed in claim 14. wherein the formulation comprises bioactive agents Bakuchi seed extract in the range of 3-10% (w/w) and Turmeric extract in the range of 7-15% (w/w).
16. The formulation as claimed in claim 14, wherein the formulation reduces pro-inflammatory cytokines (TNF-a, IL-17, IL-6, NF-kB, IL-4, TGF-ß) and enhances IL-10 levels.
17. The formulation as claimed in claim 14, wherein the formulation effectively reduces the level of one or more oxidative stress markers selected from GST or GSH or SOD or CAT thereby preventing excessive free radical damage in psoriatic conditions.
18. The formulation as claimed in claim 14, wherein the formulation is a topical formulation and is in a form selected from creams, gels, ointments, or serums.