Description:TECHNICAL FIELD OF INVENTION

The present invention is related to the field of nutraceuticals. More specifically, it relates to a method for preparation of asphaltum punjabianum (shilajit) resin with increased bioavailability.

BACKGROUND OF THE INVENTION

The background information herein below relates to the present disclosure but is not necessarily prior art.

Shilajit, a natural biological material, enriched with healing properties, reducing fatigue, woks as a natural energizer, stamina and known for management of male reproductive disorders (Diswas et al. 2010; Martin D., 2022). This natural herb can grows in the Himalayan mountains, known for fulvic acid, humic acid, minerals and antioxidant substances that helps as an antiaging compound for cells, diminish the generation of tumors in the body, neutralize the cellular toxins and improve the bioavailability of other vitamins and minerals within the human by making them bioavailable to our body (Agarwal et al. 2007; Wilson et al. 2011; Kamgar et al. 2023).

Shilajit has reported for ~85 types of minerals in their ionic form that plays main role in maintaining the energy metabolism and cell physiology balance in the human body (Yaqoob et al. 2023). The minerals abundantly available in Shilajit are generally not like the marketed food supplements/minerals since these are in the ion forms and have been basically secreted in their minimal form by plants and condensed with the earthy matters as well as algae, cyanobacteria, bacteria, lichens etc. Hence, Shilajit constituents are easily and actively absorb by the cellular system of the human.

The abundant microbial communities broken down the complex organic materials of soils and in order to transport minerals, enzymes and nutrients to plants. From there, complex photosynthesis reactions produce the components from different areas of the plant. Sugars from complex carbohydrates flow throughout the plant for food (Kim et al. 2024). Some are returned to the roots where microorganisms are fed producing fulvic acids as a complex with minerals and nutrients, to begin the cycle again.

Shilajit contain the major active component fulvic acid which is a pure natural and organic extract, contains ~74 essential complexes and minerals dissolved with 42% solid acidic foods. It arises from the deposition of vegetation roots secretion, dates back to ~75 million years, corresponding to the upper Cretaceous period. Fulvic acid is a combination of many antioxidants, amino acids, antibiotics, antivirals, antifungal substances, biochemicals, enzymes, free radical scavengers, hormones, photochemicals, nutrients and other elements. Published embodiment and research revealed that fulvic acid improves the availability of minerals within the human body, regenerates and prolongs the dwelling time of essential nutrients in the cellular system of body, decreases the damage caused by toxic compounds, heavy metals, free radicals and their consumption improves the permeability to the digestive, circulatory and cell membranes. Fulvic acid also known as a vital protective agent and an influential natural electrolyte that can reinstate the electrical balance of damaged cells, neutralize toxins and eliminate food poisoning in minutes (Andrade et al. 2023; Elahi et al. 2024).

A European patent EP1387614B1 described the method for the preparation of purified Shilajit from native Shilajit rocks and indicated an abundant composition of bioactive components, Specially, 0.3%, preferably 0.4-1%, w/w, oxygenated dibenzo-alpha-pyrones and at least 60%, preferably 65-70%, by weight of fulvic acids and whose 2% aqueous solution has a pH of >=7. Further, they indicate its application in pharmaceutical, cosmetics and nutritional for the different formulation’s preparation.

The embodiment US 5405613 refers as a nutraceutical to use the shilajit in vitamin/mineral compositions and indicated its application in the restoration of energy balance as well as increases the bioenergetic field in mammalians. The inventors further disclosed that shilajit is substantially robust than any other reported vitamin, mineral, nutraceutical material or plant herb. also, this patent has disclosed its minimal amount application to increase the efficacy of other vitamins or minerals for the energy production.

Furthermore, US Patent 6440436 displayed that Shilajit is a vital composition of various bioactive components that are necessary for toxic agents’ inhibition, for clinical, for pharmaceutical and for personal as well as nutritional care. Also, patent revealed the unique properties of the components used as an additive in other nutritional supplements.

The embodiment P2017530980A has indicted that pure shilajit is actively endorse collagen synthesis, thereby strengthening and repairing the body muscles, and the skin, cartilage, connective and vascular tissue in mammals, including humans, It relates to improving bone and dental health and / or treating diseases for abovesaid items.

OBJECTIVE OF THE INVENTION

The primary objective of the present invention is to provide a method for preparation of asphaltum punjabianum (shilajit) resin that has ability to enhances the efficacy and bioavailability of other essential molecules in human body.

Another objective of the invention is to deliver a natural, room temperature based, large shelf life, concentrate, enriched with high concentration of fulvic acid having low-cost based preparing and safe for human.

Yet another objective of the invention is to provide a method of preparation of said purified shilajit in the form of semi-solid, 100% water soluble extract.
Yet another objective of the invention is to to indicate purified shilajit application in the management of male sexual disorders, enhanced energy and stamina and indicate its potential application for the treatment of vitality, enhancement of endurance and several other in human.

Further objective of the present invention is to provide a process of preparation of said Shilajit extract which shows versatility in the further application as food.

SUMMARY OF THE INVENTION

Accordingly, the following invention provides a method for preparation of asphaltum punjabianum (shilajit) resin with increased bioavailability. this shilajit is an enriched fulvic acid and having abundance of rich minerals and uses thereof that are safe and effective due to their high bioavailability. The invented Shilajit also have potency to enhance the bioavailability of other minerals and biomolecules. Invention contained the unique sonication, centrifugation and column-based filtration methodology. The invention displayed its complete safety during the general health promotion vial preclinical MTT assay. The purified form of Shilajit contains at least 72% but more preferably 71-75% of fulvic acids by wt/wt and whose 1% of aqueous solution has a pH of ≥6.6-7.1. Further, Caco-2 cell line (epithelial cells isolated from colon tissue) mediated the bioavailability evaluated the 90% bio-absorption and bioavailability of Shilajit as well as enhancement of other nutrient molecules like Iron Concludingly, the present invented item denoted its application in direct nutritional application and in the nutraceutical formulations. The process begins by collecting natural Himalayan Shilajit rocks (101), which are dissolved in pure water and filtered through specific mesh sizes to remove impurities (102). The filtered solution is centrifuged (103) to further clarify it and then subjected to sonication (104), which helps break down complex molecules and enhance solubility. The aqueous layer is then collected (105) and purified using column filtration (106) to remove contaminants and concentrate the bioactive components. The final product is dried under vacuum conditions (107) to yield the purified Shilajit resin (108).
The preparation method ensures that the product is safe, cost-effective, and has a long shelf life, The resulting Shilajit resin is 100% water-soluble and can be stored at room temperature, maintaining its stability and efficacy for extended periods. It has applications in promoting vitality, energy, stamina, and in the management of male sexual health disorders, making it a versatile and valuable ingredient in both food and nutraceutical products.

BRIEF DESCRIPTION OF DRAWING

This invention is described by way of example with reference to the following drawings where,

Figure 1 of sheet 1 illustrated the flow diagram of method for preparing asphaltum punjabianum (shilajit) resin,
Where,
101 denotes natural Himalayan shilajit rocks,
102 denotes the process of dissolving the rocks in pure water and filtering the mixture through the desired mesh sizes,
103 denotes centrifugation,
104 denotes sonication,
105 denotes the collection of aqueous layer,
106 denotes column filtration,
107 denotes drying.
108 denotes purified shilajit resin.

Figure 1 of sheet 2 illustrated the comparative cell toxicity assay,
Where,
201 denotes negative control,
202 denotes positive control,
203 denotes the present purified shilajit resin,
204 denotes another market brand,

Figure 3 of sheet 2 illustrated the bar chart of comparative % fulvic acid bioavailability observed in Caco-2 cells.

Figure 4 of sheet 3 illustrated the bar chart of comparative % iron bioavailability observed in Caco-2 cells.

DETAILED DESCRIPTION OF THE INVENTION

As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

The present invention is related to a method for preparation of asphaltum punjabianum (shilajit) resin with increased bioavailability. In the present embodiment is a kind of unique solution to extract the broad-spectrum Shilajit extract with high fulvic acid content. Also, the extract has highest bioavailability and can enhance the bio absorption and bioavailability of other active minerals and ingredients. Hence, this embodiment can give a most bioavailable Shilajit for the versatile application in various food, nutraceuticals, and health application.

The principle of present embodiment is to create the conditions at room temperature to increase the exposure of all the Shilajit content from raw Shilajit to osmotic pressure that cause water to quickly interact, dissolved all the active portion by the ultrasonic sound waves. Further, the water-soluble metabolites will be taken out by column vessels filled with Stationary phase materials such as activated charcoal, silica gel, resin or Sephadex beads etc. Further, purified water is used as mobile phase. At the end all the harvest diluents will be collected and excess of water was evaporated under the moderate temperature with vacuum. End product is a semisolid having highest bioavailability as well as efficiency to enhance the bioavailability of other human required molecules. This has been proven with preclinical cell line assay.
Methodology:
Indigenous Shilajit rocks were collected from Himalayan Mountains (101), carefully. During selection of rocks, uniformity is physical characteristics such as rocks colour, pH, altitudes etc. were strictly followed. Further the material was soaked and mixed well with the 20%-75% volume of distilled water or pure water (102), or both, in the automatic blender or in the beaker and hold it for 2H-24H at room temperature.

Then, mixture was sonicated in the sonication tank at 12 kHz to 400 kHz for 5 min-2h, then hold for 5min-2h and again repeated the same process. Further, the sonicated material was centrifuged (103) at 3000RPM-12000RPM for 3 min-60 min to sediments the insoluble particles. The aqueous part was taken carefully in the cleaned or sterilized pot. Sedimented part was collected again and re-saturated with the distilled water or pure water or both, with the volume of 25%-200% and blended or mixed for 5-15 min. Mixed material was hold for 1h-15H at room temperature and was sonicated (104) in the sonication tank at 20 kHz to 100 kHz for 5 min-2h, then harvested in the vessel centrifuge for 5-12 min at 4000RPM-10000 RPM for 2-20min to separate the insoluble material. Further, the aqueous (105) as well as interphase layer was collected very carefully to avoid any type of sedimented materials. Next, all the filtrate were mixed together and posturized it 1H-5H at 121℃ and 15 psi to decontaminate the solution.

After pasteurization, filtrate was hold to adapt the room temperature and then passed though the high-pressure filtration assembly with 0.4μm filter. Filtrate was further passed though the column-based purification system, a more advanced technique that can further refine the substance, removing impurities and concentrating its beneficial components. This method uses a column filled with a stationary phase to separate Shilajit into its constituent parts based on their chemical properties.

Column (106) purified solution was then hold for vacuum air drying (107) to remove the maximum water portion. It has been done with the mild temperature range 20℃-37℃ with vacuum of 2Kpa to 50 Kpa. A flow of present invention is displayed in the Figure 1.

The obtained gel type material or semisolid concentrate material has further stored, in the cleaned or sterilized or both, in vessel or container made from wood, glass, steel or food grade plastic. The storage temperature of pure shilajit extract (108) concentrate is -20℃ to 37℃.

Quantification of Pure shilajit extract actives Fulvic acid was estimated by National Accreditation Board for Testing and Calibration Laboratories (NABL) accredited laboratory, Averin, Hyderabad, India. The in-house protocol (data not shown) was followed with the injection volume of 20μl for 10min with process channel 2998_chl_259nm@1.2nm and plotted against the standard fulvic acid. The results revealed the highest concentration of (fulvic acid 75% wt/wt).

BEST METHOD FOR PERFORMING OF THE INVENTION

A method for preparing Asphaltum Punjabianum resin, comprising the steps of collecting natural Himalayan Asphaltum Punjabianum (Shilajit) rocks (101), selected based on uniform physical characteristics including colour, pH, and altitude; dissolving the collected Shilajit rocks in pure water (102), with the water volume constituting 20% to 75% of the total mixture, and maintaining the mixture at room temperature for a duration of 2 to 24 hours; filtering the resulting mixture through mesh sizes of 20 to 100 micrometers (102) to remove coarse impurities; centrifuging the filtered solution (103) at speeds ranging from 6000 RPM to 14000 RPM for 3 minutes to 60 minutes to clarify the solution; subjecting the clarified solution to sonication (104) in a sonication tank at frequencies between 10 kHz and 200 kHz for 5 minutes to 10 hours, followed by a resting period of 5 minutes to 2 hours, and repeating the sonication process as necessary to enhance the solubility of bioactive components; collecting the aqueous layer (105) from the centrifuged and sonicated mixture; purifying the aqueous layer using column chromatographic filtration (106), wherein the column is filled with a stationary phase selected from activated charcoal, silica gel, resin, or Sephadex beads, and purified water is used as the mobile phase; drying the purified solution under vacuum conditions (107) at temperatures ranging from -20°C to 37°C and vacuum pressures of 2 kPa to 50 kPa to yield a purified Shilajit resin (108); and storing the purified Shilajit resin (108) in cleaned and sterilized vessels or containers made from wood, glass, steel, or food-grade plastic at temperatures between -20°C and 37°C to ensure stability and a long shelf life.

The purified Asphaltum Punjabianum (Shilajit) resin (108) is a semi-solid, gel-type, or partially liquid substance that is 100% aqueous soluble, exhibits a pale brown and clear appearance when dissolved in clean water, contains 72% to 82% fulvic acid by weight, and maintains a pH of 6.6 to 7.1 in a 1% aqueous solution. the purified Asphaltum Punjabianum (Shilajit) resin (108) is safe for consumption at desired doses and demonstrates high bioavailability of fulvic acid, ranging from 50% to 90%.

Cell Line Cytotoxicity Assay:

Caco-2 Human colorectal adenocarcinoma cell line was used for evaluation of cytotoxicity of Pure shilajit extract. The assessment was carried out by the MTT assay (calorimetric assay). The principle involved in the procedure is the capability of reducing MTT into coloured formazan by the viable cells, and the amount of formazan produced was a marker for cell viability.

Caco-2 cell lines were cultured in DMEM supplemented with 10% fetal calf serum and 1% antimycotica-antibiotic mixture culture plates at 37°C and 5% CO2-humidified incubator. Cells seeded at a density of 1 × 105cells/well in 96-well plate for 24h; then, cells were washed and incubated in fresh medium. Pure shilajit extract were added at equivalent concentration of 500 μg and 1000 μg to triplicate wells and kept for 24h, after which cells were washed three times with PBS. After washing, 20μL of MTT solution (5-mg/mLstock solution) was added to each well, and cells were then incubated for additional 4h.

The un-reacted MTT dye and medium were aspirated off, and 100μL of DMSO was added to each well to ensure solubilization of formazan crystals. The contents of the plates were mixed for 15min to achieve complete solubilization of the formazan crystals, and the measurement of optical density was carried out at 570nm with a micro plate spectrophotometer (MRX Micro plate Reader, Dynatech Laboratories Inc., Chantilly, VA, US) at 570nm. Toxicology comparison with control was displayed in the figure 2. Figure revealed that Pure shilajit extract treated cell lines are healthier as equivalent to negative control. Though, others (market brand) has toxic impact on cell line. It revealed that Pure shilajit extract purity and validate its safety.

Bioavailability Assay:

500μl cell suspension Caco-2 cell lines (Cell density 10x103 cells per well) were taken and maintained for 21 days to get the differentiated Caco-2 monolayer. During the experiment integrity of Tight junction of monolayer of Caco2 cell culture was confirmed by TEER measurement by Millicell –ERS system (Merck Millipore) with the TEER value of 1100 - 1350 Ω.cm2 respectively. Further, the cells were treated with the with 500ug/ml of the Pure shilajit extract except the control wells and incubated the plate for 18HR at 37°C in the presence of 5% CO2 in incubator. After the incubation period, plates were taken out from incubator and spent media was removed and well were washed twice with the 500ul of PBS buffer. Further, the monolayer was lyzed with the 500ul of 0.1% PBST buffer (purchased from Sigma Aldrich, Cat No: D8537-500ml) for 10mins and scraped the entire monolayer with the help of cell scraper and transferd the whole contents to sterile eppendorf tube. The tubes were centrifuged at 10000 rpm for 5min at 4 °C to harvest the supernatant for the HPLC analysis.

HPLC analysis was performed on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) that was composed of two LC-20 AD pump, rheodyne injector, an SPD 20 diode array detector, CBA-20A controller, CT0-20AC column oven. The separation was carried out on a Shimadzu Shimpack C18 Reverse phase column (250 mm × 4.6 mm ID, 5 μm). Water as solvent A and acetonitrile as solvent B was used as mobile phase in gradient mode. The gradient profile was set as 0-10% B for 0–4 min, 10% B for 4–12 min, 10–80% B for 12–18 min, 80-10% B for 18–20 min. Flow rate was set at 0.8 ml/min. Injection volume was taken as 20μl. Column temperature was adjusted to 30°C and the detection wavelength was set at 215 nm. Peaks were assigned by comparing with the corresponding reference standard (Fulvic acid) retention time and UV spectra.

Prior to HPLC analysis, the freshly prepared HPLC mobile phase was passed through a 0.45 μm membrane filter and degassed using a sonicator. Sample containing solution was filtered through 0.22 μm membrane syringe-driven filter into a HPLC vial for HPLC analysis. Stock solutions of Fulvic acid standard was prepared by dissolving the standard in methanol (10.8mg/5ml) and performed HPLC analysis.
% Fulvic acid content was calculated by using the below formula:

Sample response/Std response x Std weight (mg)/Volume of solvent x Volume of Sample/Sample weight (mg) x Potency/100 * 100

Unknown concentration of Fulvic acid was calculated by using the below formula:
X=Sample area/Std area x Std conc. (mg/ml)

% Bioavailability of Fulvic acid in Caco2 cells was calculated by using the below formula:

% bioavailability= (Rate of bioavailability of fulvic acid of Pure shilajit extract /Known conc. of Fulvic acid/ml) x100

The bioavailability graph (Figure 3) has clearly pointed the highest bioavailability of Pure shilajit extract (contained 75% fulvic acid) against control as well as other brand and found 90% of bioavailability against the 100% pure fulvic acid.
Bio-efficacy Assay:

Another set of prepared CaCo2 cell line (as described for bioavailability assay) were treated with 100ug/ml of FeSO4 followed by adding the appropriate concentrations of the Pure shilajit extract (125μg, 250μg and 500 μg), other Shilajit brands except the control wells. FeSO4 alone was used as a Negative control and FeSO4+150ug/ml of Ascorbic acid was used as a std control for the study.

The experimental plates were then incubated for 18HR at 37°C in a 5% CO2 atmosphere. After that spent media was removed from wells and washed with the 500ul of Ice-cold saline (0.9% NaCl). Then, 500ul of Stop solution (140mM NaCl+10mM PIPES), for 1min was used and then two times washing was done with the 500ul of Removal solution (Stop solution+5mM Bathophenanthroline disulfonic acid). Further, the cells were Solubilized with the 100ul of 0.5N NaOH solution and measured the Pure shilajit extract mediated enhancement of Iron utilization by microplate absorbance Spectrophotometer (xMark, BioRad, USA). Briefly, 100ul of cell lysate were added to 100ul of Iron releasing solution (1.4M HCl+4.5% KMnO4 and heated the reaction mixture for 2hours at 60°C. Then 100ul of Iron detection reagent (6.5mM Ferrozine+2.5mM ammonium acetate+1M ascorbic acid) was added and incubated for 30mins at RT. Further, the plate reaction mixture absorbance of respective wells of 96well plate were recorded at the absorbance of 550nm.

% Iron bioavailability is calculated using below formula:

% Iron bioavailability = (Abs of treated cells/Abs of Untreated cells) x 100-100

Figure 4 clearly indicated that Pure shilajit extract can potentially enhanced the bioavailability of iron and hence it can clearly conceptualize that it has best bio-efficacy to enhance the bioabsorption of other nutrients, minerals, vitamin etc. which make it a unique compound for the application as a nutraceutical agent or an effective synergistic compound for any kind of nutraceuticals or health supplement manufacturing.

The method for preparing Shilajit resin (Pure shilajit extract) involves several key steps. Initially, the process includes filtration through mesh sizes ranging from 20 to 100. The filtrate is then sonicated in a sonication tank at frequencies between 10 kHz and 200 kHz for a duration of 5 minutes to 10 hours. Following this, centrifugation is performed at speeds of 6,000 RPM to 14,000 RPM for a period of 3 to 60 minutes, after which the mixture undergoes further purification via a column chromatographic method. The final product is characterized as a semisolid, gel-like, or partially liquid substance that can be stored in cleaned or sterilized vessels made of wood, glass, steel, or food-grade plastic at temperatures between -20℃ and 37℃. This purified Shilajit is 100% water-soluble, exhibiting a pale brown, clear appearance when dissolved in clean water, and contains 72-82% fulvic acid. Additionally, it is deemed safe for consumption at the recommended doses and demonstrates a high bioavailability of fulvic acid, ranging from 50% to 90%. Pure shilajit extract can serve as a primary ingredient or a substitute in the formulation of health or nutritional compositions, due to its bioabsorption properties and its ability to enhance the bio-efficacy of other molecules or compounds.

While various embodiments of the present disclosure have been illustrated and described herein, it will be clear that the disclosure is not limited to these embodiments only. Numerous modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art, without departing from the spirit and scope of the disclosure, as described in the claims.
, Claims:1. A method for preparation of asphaltum punjabianum resin with increased bioavailability, comprising the steps of;

collecting natural himalayan asphaltum punjabianum (shilajit) rocks (101) selected based on uniform physical characteristics, including color, pH, and altitude;

dissolving the collected shilajit rocks in pure water (102), wherein the volume of water constitutes 20% to 75% of the total mixture, and maintaining the mixture at room temperature for a duration of 2 to 24 hours;

filtering the resulting mixture through mesh sizes of 20 to 100 micrometers (102) to remove coarse impurities;

centrifuging the filtered solution (103) at speeds ranging from 6000 RPM to 14000 RPM for a duration of 3 minutes to 60 minutes to clarify the solution;

subjecting the clarified solution to sonication (104) in a sonication tank at frequencies between 10 kHz and 200 kHz for a period of 5 minutes to 10 hours, followed by a resting period of 5 minutes to 2 hours, and repeating the sonication process as necessary, to enhance solubility of bioactive components;

collecting the aqueous layer (105) from the centrifuged and sonicated mixture;

purifying the aqueous layer using column chromatographic filtration (106), wherein the column is filled with a stationary phase selected from activated charcoal, silica gel, resin, or Sephadex beads, and using purified water as the mobile phase;

drying the purified solution under vacuum conditions (107) at temperatures ranging from -20°C to 37°C and a vacuum pressure of 2 kPa to 50 kPa to yield a purified Shilajit resin (108); and

storing the purified asphaltum punjabianum (shilajit) resin (108) in cleaned and sterilized vessels or containers made from wood, glass, steel, or food-grade plastic at temperatures between -20°C and 37°C to ensure stability and a long shelf life;

2. The method for preparation of asphaltum punjabianum resin with increased bioavailability as claimed in claim 1, wherein the purified asphaltum punjabianum (shilajit) resin (108) as a semi-solid, gel-type, or partially liquid substance that is 100% aqueous soluble, exhibits a pale brown and clear appearance when dissolved in clean water, contains 72% to 82% fulvic acid by weight, and maintains a pH of 6.6 to 7.1 in a 1% aqueous solution.

3. The method for preparation of asphaltum punjabianum resin with increased bioavailability as claimed in claim 1, wherein the purified asphaltum punjabianum (shilajit) resin (108) is safe for consumption at desired doses and demonstrates high bioavailability of fulvic acid ranging from 50% to 90%.