A system for the production of food colors from Phellinus gilvus (Schwein.) Pat. with biochemical characterization and therapeutic anticancer and antioxidant potential

Abstract

A system (100) for the biochemical characterization and evaluation of the therapeutic potential of P. gilvus, comprising: a) an extract preparation unit (102) configured to prepare a methanolic extract from P. gilvus samples; b) a biochemical characterization unit (104) configured to analyze bioactive compounds in the extract; c) a biological activity evaluation unit (106) configured to evaluate the therapeutic properties of the extract; and d) a food colorant production unit (108) configured to process P. gilvus samples into food colorant independently of extract production.

Description

FIELD OF THE INVENTIONThe present disclosure relates to a system for biochemical characterization and evaluation of the therapeutic potential of Phellinus gilvus. More particularly, the present invention relates to a system for the preparation of an extract from P. gilvusand for evaluating its biochemical characterization and therapeutic potential as an anti-cancer agent, said system being further configured to recover pigments from the fruiting body of P. gilvus.BACKGROUND OF THE INVENTIONMedicinal fungi, including several species of Phellinus, have been documented in the anticenetric literature. The genus Phellinus comprises 439 Taxa worldwide, with 107species being known from India. Several species of Phellinus, including P. gilvus, have traditionally been used medicinally worldwide but require scientific validation. These fungi contain numerous bioactive compounds, including terpenoids, polyketides, steroids, flavonoids, polysaccharides, alkaloids and polyphenols, which contribute to their medical properties.Advanced analytical techniques such as high resolution liquid chromatography-mass spectrometry (HR-LCMS), gas chromatography-mass spectrometry (GC-MS) and Fourier transform infrared spectroscopy (FT-IR) are essential for the identification and characterization of these bioactive compounds. Despite the growing interest in fungal bioactive components for disease control and therapeutic applications, the evaluation of potential of P. gilvusas a natural food dye with anti-cancer activity has not been made yet.In view of the foregoing discussion, it will be apparent that there is a need for a system for biochemical characterization and therapeutic assessment of P. gilvusthat enables its development as a functional food ingredient with medical benefit.SUMMARY OF THE INVENTIONThe present disclosure relates to a system for biochemical characterization and evaluation of the therapeutic potential of P. gilvus. This invention relates to a comprehensive system for characterization of P. gilvus and uses its dual potential as food dye and anti-cancer agents. The system comprises separate units for extract preparation, biochemical characterization, evaluation of biological activity, food dye production and gross cultivation, thus allowing efficient analysis of bioactive compounds and the development of functional food dyes with therapeutic properties.An object of the present disclosure is to provide a system for biochemical characterization and evaluation of the therapeutic potential of P. gilvus. The system comprises: a) an extract processing unit for preparing methanolic extract from P. gilvussamples; b) a biochemical characterization unit configured to analyze bioactive compounds in the extract; c) a biological activity evaluation unit configured to evaluate the therapeutic properties of the extract; and d) a food dye preparation unit configured to process P. gilvussamples into food dyes independent of extract preparation.Another object of the present disclosure is to provide a system for biochemical characterization and evaluation of the therapeutic potential of P. gilvus.Another object of the present disclosure is to provide a system for the efficient preparation and characterization of P. gilvusextracts to identify bioactive compounds having anti-cancer and anti-oxidant properties.Another object of the present disclosure is to employ various characterisers to assess the HR LCMS, GCMS / MS and FTIR analyses performed on an extract of P. gilvus.Another object of the present disclosure is to provide a system configured for recovering the food dye from P. gilvus.In order to further clarify the advantages and features of the present disclosure, the invention will be explained in more detail with reference to specific embodiments that are illustrated in the accompanying drawings. This drawing shows only typical embodiments of the invention and is therefore not to be considered as limiting its scope. The invention will be described and explained in more detail with reference to the accompanying drawings.BRIEF DESCRIPTION OF THE FIGUREThese and other features, aspects, and advantages of the present disclosure will become more fully understood when the following detailed description is read with reference to the accompanying drawings, in which like characters represent like parts throughout. The following applies here: FIG. 1 shows a block diagram of a system for biochemical characterization and evaluation of the therapeutic potential of P. gilvusaccording to an embodiment of the present disclosure.Those skilled in the art will also appreciate that the elements in the drawing are shown for simplicity and are not necessarily to scale. For example, the flowcharts illustrate the method using the key steps to improve understanding of aspects of the present disclosure. Also, as for the construction of the apparatus, individual or plural components of the apparatus may be represented by conventional symbols in the drawing. The drawing may only show the specific details relevant to understanding the embodiments of the present disclosure in order not to obscure the drawing with details readily apparent to those skilled in the art after the present description.DETAILED DESCRIPTION:In order to promote an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and will be described in an comprehensible manner. However, the scope of the invention is not limited thereby. Changes and further modifications of the illustrated system, as well as further applications of the principles of the invention, are possible, as would normally occur to a person skilled in the art.It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be limiting thereof.References throughout this specification to "one aspect," "another aspect," or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the phrases "in one embodiment," "in another embodiment," and similar phrases in this specification may or may not refer to the same embodiment.The terms "comprises," "comprising," or other variations thereof are intended to cover a non-exclusive inclusion, such that a process or method comprising a list of steps may include not only those steps, but also other steps not expressly listed or inherent in that process or method. Likewise, the phrase "comprises... for" one or more devices, subsystems, elements, structures, or components does not exclude, without further limitations, the existence of other devices, subsystems, elements, structures, components, or additional devices, subsystems, elements, structures, or components.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art. The systems, methods, and examples provided herein are for illustrative purposes only and are not to be considered limiting.Embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings.FIG. 1 shows a block diagram of a system ( 100) for biochemical characterization and evaluation of the therapeutic potential of P. gilvusaccording to an embodiment of the present disclosure.Referring to Figure 1, the system (100) comprises: a) an extract preparation unit (102) configured to prepare methanolic extract from P. gilvussamples; b) a biochemical characterization unit (104) configured to analyze bioactive compounds in the extract; c) a biological activity evaluation unit (106) configured to evaluate the therapeutic properties of the extract; and d) a food dye production unit (108) configured to process P. gilvussamples in food color independent of extract preparation.In one embodiment, the extract processing unit (102) is configured to: a) crush fresh fruit bodies from P. gilvuswith 70% methanol; b) sonicate the mixture at 20°C for 30 minutes; and c) centrifuge the mixture at 10,000 U / min for 10 minutes to separate and collect the supernatant.In one embodiment, the biochemical characterization unit (104) comprises: a) a high resolution liquid chromatography mass spectrometer (HR-LCMS) configured to identify bioactive compounds; b) a gas chromatography mass spectrometry / mass spectrometry (GC-MS / MS) device configured to analyze volatile compounds; and c) a Fourier transform infrared spectroscopy (FTIR) device configured to determine functional groups in the extract.In one embodiment, the biochemical characterization unit (104) is configured to identify bioactive compounds, including at least one of the following compounds: 1-phenylethylamine, fexaramine, cynaroside A, procaine, 2-dehydroecdyson, cucurbitacin L, N-stearoyl glutamic acid, fentanyl, 11(12)-EET-d11, N-stearoyl arginine, 1-hydroxyprevitamin D3 diacetate, tribenuronmethyl, and tetracenomycin D3.In one embodiment, the biological activity evaluation unit (106): a) comprises antioxidant assay equipment configured to perform 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays for evaluating radical scavenging activity; and b) cell culture equipment configured to perform 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays on normal and cancerous cell lines.In one embodiment, the cell culture equipment is configured to maintain a) the human embryonic kidney epithelial cell line (HEK293) for biocompatibility assays and b) the human breast cancer cell line (MDA-MB-231) for assays for anti-cancer activity.In one embodiment, the food dye production unit (108) is configured to: a) dry P. gilvus samples in an oven at a temperature between 50 and 60° C.; b) grind the dried sample into a fine powder; c) sieve the powder through a fine mesh sieve; and d) autoclave the fine powder at 121 ° C. under 15 psi pressure for 15-20 minutes.In one embodiment, the system (100) further comprises a storage unit (110) configured to store the food dye in a sealed container air to control moisture and prevent moisture ingestion.In one embodiment, the system (100) further comprises a mycelium culture unit (112) configured to a) prepare a substrate from hardwood strains, wheat, sawdust and rice straw; b) add calcium carbonate to the substrate in a predetermined ratio; c) sterilize the substrate by autoclaving at 121°C for 15-20 minutes under 15 psi pressure; d) inoculate the sterilized substrate with PDA broth mycelium; and e) incubated the inoculated substrate to obtain fruit bodies.In one embodiment, the gross culture unit (112) is configured to use 5 grams of calcium carbonate per 1 kilogram of substrate.The present invention provides a comprehensive system for biochemical characterization and evaluation of the therapeutic potential of P. gilvuswith applications as a food dye having anti-cancer properties. The system consists of several integrated units which cooperate to analyze and use this medical fungus. The extract preparation unit uses a methanolic extraction technique with ultrasound and centrifugation to isolate bioactive compounds from fresh fruit bodies. These extracts are then analyzed by the biochemical characterization unit using complex analytical techniques such as HR-LCMS, GC-MS / MS and FTIR to identify certain bioactive compounds such as 1-phenylethylamine, fexaramine and cynaroside A. The biological activity evaluation unit evaluates the therapeutic potential of the extract by DPPH tests for antioxidant activity and MTT tests using the cell lines HEK293 (normal) and MDA-MB-231 (breast cancer) and provides quantitative data on biocompatibility and anti-cancer efficacy. The food dye production unit processes the fungus samples separately by drying, grinding, sieving and autoclaving to produce a stable natural dye with obtained therapeutic properties. The system also comprises a breeder plant for controlled cultivation of P. gilvuson a specific substrate of hardwood strains, wheat, sawdust and rice straw supplemented with calcium carbonate. This culture approach ensures sustained supply of the fungus for both analytical studies and dye production. The entire system represents an integrated approach to exploiting the medical and commercial potential of P. gilvusand addresses the growing demand for natural food additives with health promoting properties and potential applications in cancer treatment.In one embodiment, the system for biochemical characterization and evaluation of the therapeutic potential of P. gilvus having food dye and anti-cancer properties comprises various integrated units and processes as described below.In one embodiment, the extract processing unit is used for the collection and processing of P. gilvusspecies. Specimens were collected between July and October 2024 in natural habitates at the Kolhapur district in Maharashtra, India. Field observations included the detection of morphological and ecological characteristics. Microscopic investigations of the fresh fruit bodies were carried out by means of cotton blue dyeing under a research microscope of the Lawrence & Mayo N-300M type. Identification was confirmed by comparing the observed characteristics with the standard literature. Fresh specimens were labeled, transported to the laboratory and dried for further analysis. For extract preparation, the extract processing unit crushes 1 gram of the fresh fruit body with 10 ml of 70% methanol. The mixture is sonicated for 30 minutes at 20°C and then centrifuged for 10 minutes at 10,000 U / min. The supernatant is collected and stored for further investigation.In one embodiment, the food dye production unit processed P. gilvus samples by first drying them in an oven at 50-60°C until they were fully dried. The dried samples were roughly ground with mortar and plunger and then processed into a fine powder with a mill. During milling, care was taken to avoid heat-induced discolorations or decompositions by keeping the powder cool. The fine powder was sieved with a fine mesh sieve to remove larger particles. The resulting fine powder was autoclaved at 121°C under 15 psi pressure for 15-20 minutes. The powder was then stored in an air container.In one embodiment, the substrate for the mycelium culture unit was prepared by mixing hardwood strains, wheat, sawdust and rice straw in equal proportions. 5 g calcium carbonate were added per kg of substrate. Culture glasses containing 30 g of substrate and culture bags containing 50 g of substrate were prepared. These were autoclaved at 121°C and a pressure of 15 psi for 15-20 minutes. After cooling, the glasses and bags were inoculated with 50 day old PDA broth mycelium. Incubation was in a cool, dry and dark environment to promote fruit body development. In the culture study, the growth properties of mycelium on different media and substrates were evaluated. PDA and SDA media were used, with PDA promoting more consistent mycelium growth. For liquid culture, potato dextrose broth and yeast mannitol broth both showing favourable growth were used. Substrate tests showed that individual components such as wheat, sawdust or rice straw did not promote optimum growth. However, the combination of hardwood strains, wheat, sawdust and rice straw allowed optimum aeration and aided mycelium development.In one embodiment, the biochemical characterization unit is configured to analyze the methanolic extract using high resolution liquid chromatography mass spectrometry (HR-LCMS). The extract was analyzed with an Agilent Technologies HR LCMS system equipped with electrospray ionization (ESI). Chromatographic separation was achieved with a PepMap RSLC C18 column (2 μm, 100 Å, 50 cm) and a precolumn (Acclaim PepMap 100, 100 μm×2 cm, Nanoviper). The solvent system comprised solvent A (water) and solvent B (acetonitrile) with a 102 minute gradient program. The flow rate was set to 300 μl / min. Mass spectrometric analysis was performed in both positive and negative ionization modes. Data acquisition and compound identification were performed using the Compound Discoverer 2.1 software, ChemSpi, and PubChem 2. The functional groups in the extract were determined by Fourier transform infrared spectroscopy (FT-IR). Spectral data were recorded on an Alpha Bruker instrument in the range 4000 to 400 cm -1. Comparative spectral analysis was performed using the Essential FTIR program and the reference spectra were evaluated using the British Wiley Library. Gas chromatography-mass spectrometry / mass spectrometry (GC-MS / MS) was carried out using the system Shimadzu TQ 8050 Plus HS 20. Helium served as carrier gas. The furnace temperature of the column was set at 50°C and injection was performed in split mode at 250°C. Flow control was performed with a linear velocity mode of 36.5 cm / sec and a column flow rate of 1.01 ml / min. A temperature gradient program was used: start at 50°C for 2 minutes, ramp at 5°C / min to 180°C (held for 2 minutes), then to 250°C (held for 2 minutes) and finally to 260°C (held for 2 minutes). The temperature of the GC ion source was maintained at 200°C and the interface temperature at 270°C. The solvent shutdown time was 3.00 minutes. The detector gain was 1.17 kV and the scan range ranged from m / z 45 to m / z 500. The acquisition was carried out in the Q3 scan mode with a scan speed of 1666 and an event time of 0.3 seconds.In one embodiment, the biological activity evaluation unit is configured to evaluate the biocompatibility and anti-cancer potential of P. gilvusextract by MTT assay on the cell lines HEK293 and MDA-MB-231. The cells were cultured in T-25 flasks, trypsinized and centrifuged at 300 × g to obtain pellets. The cell suspension was adjusted to 10,000 cells per 200 μl in DMEM-HG and sown in 96-well plates. After incubation at 37°C in 5% CO 2 for 24 hours, the medium was removed and the cells treated with P. gilvusextract in concentrations of 31.55, 62, 125, 250, 500 and 1000 μg / ml. After a further 24 hours of incubation, MTT solution (0.5 mg / ml) was added and the plates incubated for three hours. The medium was then aspirated, 100 μl of DMSO added to dissolve the formazan crystals, and absorbance was measured at 570 nm and 630 nm using a BK-EL10A microplate reader. The percent viability was calculated using the following equation (1):Antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. A stock solution containing 100 mg / ml P. gilvusextract was prepared and sonicated for 10-15 minutes. From this stock solution, various concentrations (20-100 mg / ml) were mixed with 3 ml of 1 mM DPPH in methanol. After shaking and incubation in the dark for 30 minutes, the absorbance was measured at 517 nm. Methanol served as a blank. Radical scavenging activity was calculated using equation (2) below, where Ac is the absorbance of the control and As is the absorbance of the sample.The results of biochemical characterization and evaluation of the therapeutic potential of P. gilvus revealed that the methanolic extract of P. gilvuscontained several bioactive compounds with different therapeutic properties. Compounds such as 1-phenylethylamine, fexaramine, cynaroside A, procaine, 2-dehydroecdyson, cucurbitacin L, N-stearoyl glutamic acid, fentanyl, 11(12)-EET-d11, N-stearoyl arginine, 1-hydroxyprevitamin D3 diacetate, tribenuronmethyl and tetracenomycin D3 were identified in the extract using advanced analytical techniques. These compounds have demonstrated a wide range of biological activities including anti-cancer, antioxidant, antimicrobial, anti-inflammatory, antibacterial, neuroprotective, antifungal, antidiabetic, liver protecting, anti-leishmanic, anesthetic, ecdysteroidal, antiviral, anthelmintic, blood pressure regulating, antibiotic, stress inhibiting, HIV inhibiting, cardioprotective, antithrombotic, antihypertensive, opioid, antinociceptive and malarial effects.The antioxidant potential of the extract was detected by a concentration-dependent reaction. The extract achieved a free radical scavenging activity of up to 80%, indicating its effective free radical neutralizing ability. Cytotoxicity and biocompatibility tests with the human breast cancer cell line MDA-MB-231 and the normal human embryonic kidney epithelial cell line HEK293 additionally confirmed the biological relevance of the extract. The results showed that the extract, at concentrations between 500 μg / ml and 1000 μg / ml, showed significant cytotoxic effects on the cancer cells, resulting in over 50% cell death. At the same time, at 500 μg / ml, over 90% of the normal cells remained viable, and even at 1000 μg / ml, cell viability remained at 85%Profiling of the methanolic extract by high resolution liquid chromatography mass spectrometry (HR-LCMS) revealed the presence of 71 compounds in positive ionization mode. Of these, 11 compounds were identified as major, 21 as medium and 39 as minor, by frequency. The most frequently identified compound was 1-phenylethylamine at a frequency of 526496, while cucurbitacin D was the least frequently at 11928. 52 different compounds identified in this mode have been assigned a total of 36 different bio-activities. Cynaroside A with a frequency of 305. 96 and Somniferin at 40109 exhibited the highest number of biological activities including anti-cancer, antioxidant, antimicrobial, anti-inflammatory, antibacterial, neuroprotective, antifungal, antidiabetic, liver protective, anti-leishmanial, antistress, cardioprotective and anti-HIV activity.A total of 85 compounds were identified in the negative ionization mode. Of these, 4 compounds were present in large, 28 in medium and 53 in low frequency. N-stearoyl arginine was the most common compound in this mode at 538978 whereas bisthiosemi was the least common at 7656. In total, 23 different bio-activities were associated with 46 identified compounds. Among them, 2-O-protocatechuoylalphitolic acid, at a frequency of 87727, showed the highest number of bioactivitys, including anti-cancer, anti-oxidative, antimicrobial, anti-inflammatory, neuroprotective, antidiabetic, anti-aging, anti-ulcer, liver protecting and antiviral activity.The biocompatibility test clearly showed that the extract is harmless to normal human cell lines at the concentrations tested and high cell viability is ensured. The cytotoxicity assay on breast cancer cells confirmed the anti-cancer activity of the extract at higher concentrations and indicated a therapeutic potential for use. The antioxidant test reinforced these results since the extract had a radical scavenging activity of about 78-79% at concentrations of 80 and 100 mg / ml. It was also observed that the reducing power of the extract increases as the extract concentration increases.Overall, P. gilvuscontains a broad range of bioactive compounds and has multifunctional properties, including potent antioxidant and anti-cancer effects. The results emphasize their potential as a source of bioactive metabolites, support the traditional use of Phellinus species, and suggest their relevance to future pharmaceutical applications.The drawings and the foregoing description show examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be divided into multiple functional elements. Elements of one embodiment may be added to another embodiment. For example, the order of the processes described herein may be changed and is not limited to the manner described herein. Moreover, the actions of a flow chart need not be performed in the order shown; nor do all actions necessarily need to be performed. Also, actions that are not dependent on other actions may be performed in parallel with the other actions. The scope of the embodiments is by no means limited by these specific examples. Numerous variations, whether or not explicitly stated in the specification, such as differences in structure, dimensions, and material use, are possible. The scope of the embodiments is at least as broad as recited in the following claims.Advantages, other advantages and solutions to problems have been described above with reference to specific embodiments. However, the advantages, merits, solutions to problems and any components that may result in an advantage, merit or solution being introduced or enhanced are not to be understood as critical, required or essential features or components of individual or all claims.REFERENCES100 A system for biochemical characterization and evaluation of therapeutic potential Von P. Gilvus. 102 Extract processing unit 104 Biochemical characterization unit 106 Biological activity evaluation unit 108 Food dye production facility 110 Storage unit 112 Gross culture unit

Claims

A system (100) for biochemical characterization and evaluation of the therapeutic potential of P. gilvus comprising: a) an extract preparation unit (102) configured to prepare a methanolic extract from P. gilvus samples; b) a biochemical characterization unit (104) configured to analyze bioactive compounds in the extract; c) a biological activity evaluation unit (106) configured to evaluate the therapeutic properties of the extract; and d) a food dye production unit (108) configured to process P. gilvus food color samples independent of extract preparation.The system (100) of claim 1, wherein the extract processing unit (102) is configured to: a) crush fresh fruit bodies of P. gilvus with 70% methanol, b) sonicate the mixture at 20°C for 30 minutes, and c) centrifuge the mixture at 10,000 rpm for 10 minutes to separate and collect the supernatant.The system (100) of claim 1, wherein the biochemical characterization unit (104) comprises: a) a high resolution liquid chromatography mass spectrometer (HR-LCMS) configured to identify bioactive compounds; b) a gas chromatography mass spectrometry / mass spectrometry (GC-MS / MS) device configured to analyze volatile compounds; and c) a Fourier transform infrared spectroscopy (FTIR) device configured to determine functional groups in the extract.The system (100) of claim 3, wherein the biochemical characterization unit (104) is configured to identify bioactive compounds including at least one of the following compounds: 1-phenylethylamine, fexaramine, cynaroside A, procaine, 2-dehydroecdyson, cucurbitacin L, N-stearoyl glutamic acid, fentanyl, 11(12)-EET-d11, N-stearoyl arginine, 1-hydroxyprevitamin D3 diacetate, tribenuronmethyl, and tetracenomycin D3.The system (100) of claim 1, wherein the biological activity evaluation unit (106) comprises: a) antioxidant assay equipment configured to perform 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays for evaluating radical scavenging activity; and b) cell culture equipment configured to perform 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays on normal and cancerous cell lines.The system (100) of claim 4, wherein the cell culture equipment is configured to maintain a) the human embryonic kidney epithelial cell line (HEK293) for biocompatibility assays; and b) the human breast cancer cell line (MDA-MB-231) for assays for anti-cancer activity.The system (100) of claim 1, wherein the food dye production unit (108) is configured to: a) dry P. gilvus samples in an oven at a temperature between 50-60°C; b) mill the dried samples to a fine powder; c) screen the powder through a fine mesh screen; and d) autoclave the fine powder at 121 °C for 15-20 minutes under a pressure of 15 psi.The system (100) of claim 1, further comprising a storage unit (110) configured to store the food coloring matter in an airtight container to maintain humidity under control and prevent moisture ingestion.The system (100) of claim 1, further comprising a mycelium culture unit (112) configured to a) prepare a substrate from hardwood strains, wheat, sawdust and rice straw; b) add calcium carbonate to the substrate at a predetermined ratio; c) sterilize the substrate by autoclaving at 121°C for 15-20 minutes under 15 psi pressure; d) inoculate the sterilized substrate with PDA broth mycelium; and e) incubated the inoculated substrate to obtain fruit bodies.The system (100) of claim 8, wherein the gross culture unit (112) is configured to use 5 grams of calcium carbonate per 1 kilogram of substrate.

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