Description:Technical field of the invention
[0002] The present invention relates to a method of preparation shilajit extract with enhanced purity. More particularly, the invention relates to an optimized method of preparation of shilajit extract with high purity and enhanced levels of bioactive components.
Background of the invention
[0003] Shilajit is a natural substance found predominantly in the Himalayan region. Shilajit is also known by the names of salajit, shilajatu, mimie, or mummiyo is a blackish-brown powder or an exudate obtained from high mountain rocks. Shilajit is known to have formed for centuries by the gradual decomposition of certain plants by the action of microorganisms. Shilajit is a potent and very safe dietary supplement, restoring the energetic balance and potentially able to prevent several diseases.
[0004] Shilajit is commonly used in Ayurvedic medicine. Shilajit is an effective and safe supplement exhibiting a positive effect on health and well-being. Consumption of shilajit has several advantages such as prevention of cognitive diseases including Alzheimer’s, increased levels of testosterone, treats chronic fatigue syndrome, slows ageing, reduces altitude sickness, treats anemia, infertility, improves cardiovascular health, improves bone density in females.
[0005] Shilajit has a unique phytocomplex rich in fulvic acid. Shilajit is composed mainly of humic substances, including fulvic acid, that account for around 60% to 80% of the total nutraceutical compound along with oligoelements.
[0006] Raw or unpurified shilajit contain several impurities like heavy metals (lead, mercury, arsenic, etc.), mycotoxins, and high molecular weight polymeric quinones. The impurities pose potential health risks. Purification of shilajit is crucial before consumption. Upon purification of shilajit, toxicity is reduced, purity is enhanced and the quality of shilajit is standardized.
[0007] Shilajit purification and extraction involves harvesting raw shilajit from rocks and dissolving in water, filtering to remove insoluble impurities like rock fragments and plant debris, multiple filtration to remove impurities, evaporation to obtain viscous residue, extraction with organic solvents to obtain soluble and insoluble fractions. The process often includes extracting with organic solvents, followed by precipitation and concentration of specific components like fulvic acids.
[0008] The extraction of shilajit, a natural resinous substance, from mountain rocks has potential disadvantages related to quality, purity, and consistency. Raw or unprocessed shilajit contain contaminants like heavy metals and mycotoxins, which require purification. Additionally, the quality and composition of shilajit vary significantly based on its origin and extraction methods, making it difficult to ensure consistent quality and safety.
[0009] The Patent Application No. US6869612B2 entitled “Process for preparing purified shilajit composition from native shilajit” discloses a purified shilajit composition native shilajit. The composition has an abundance of bioactive components, particularly, 0.3%, preferably 0.4-1%, by weight, oxygenated dibenzo-α-pyrones and at least 60%, preferably 65-70%, by weight of fulvic acids of low-to-medium molecular weight ({overscore (M)}n of 700-2000) with an E4/E6 ratio of 8-10 at λ465-665 nm, and whose 2% aqueous solution has a pH of ≧7. Personal care, pharmaceutical and nutritional use formulations of the purified shilajit composition also are described. The pharmaceutical or nutritional formulation comprises the composition in an amount of about 0.1 to 60% by weight. The skin care or protection formulation in the form of a lotion, cream or gel, the composition is present in an amount of about 0.1-5%. The pharmaceutical formulation is formulated in the form of a tablet, syrup, elixir or capsule. The nutritional formulation comprises about 0.5 to 30% of the composition. The skin care or protection formulation comprises a cosmetically acceptable carrier and cosmetic adjuvant comprising sunscreens, antioxidants, preservatives, perfumes, oils, waxes, propellants, waterproofing agents, emulsifiers, thickeners, humectants and emollients. The composition is devoid of humic acid, humins and polymeric quinones.
[0010] The Patent Application No. IN202421078724A entitled “A method for preparation of Asphaltum punjabianum resin with increased bioavailability” discloses a method for the preparation of Asphaltum punjabianum (shilajit) resin with increased bioavailability, 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 process involves dissolving natural himalayan shilajit rocks in purified water and filtering through mesh, followed by centrifugation to remove insoluble particles. The solution undergoes sonication to enhance its bioavailability and is then purified using column filtration. The aqueous layer is carefully collected, and the purified shilajit is dried under vacuum to form a semisolid resin. Shilajit resin containing 71-85% fulvic acid, demonstrates 90% bio-absorption and significantly enhances the bioavailability of other nutrient molecules like iron.
[0011] Shilajit consumption without preliminary purification leads to risks of intoxication given the presence of mycotoxin, heavy metal ions, polymeric quinones, and free radicals. The compositions of unpurified shilajit contain detectable heavy metals levels including lead, mercury, and arsenic. Additionally, the percentage of fulvic acid and other bioactive components being bioavailable is limited. Hence, there is a need for a standardized method for the preparation of shilajit which aids in obtaining shilajit with higher percentage of bioactive active, improved bioavailability and purity.

Summary of the invention
[0012] The present invention overcomes the drawbacks of existing methods to obtain purified shilajit extract. The present invention discloses a method of preparation of shilajit extract with enhanced purity.
[0013] The method for the purification of shilajit resin comprises steps of weighing shilajit resin and grinding, adding ethanol and fermenting for 24 to 48 hours, hot water bath for 24 hours and collecting the supernatant, decantation and multiple filtrations of the supernatant, centrifuging the filtered supernatant and collecting the obtained supernatant from centrifugation, concentrating the supernatant, freeze drying the concentrated supernatant to obtain purified shilajit.
[0014] Further, the method for extraction of purified shilajit comprises steps of weighing purified shilajit and grinding, adding water and citric acid, subjecting to extraction by placing in hot water bath to obtain supernatant and remnant, separating and collecting the supernatant, collecting the remnant and subjecting the remnant to extraction, separating and collecting the supernatant obtained from extracting the remnant, mixing the obtained supernatants, centrifuging the mixed supernatants, collecting the supernatant comprising purified shilajit extract, concentrating the purified shilajit extract and spray drying.
[0015] The method disclosed in the present invention aids in obtaining shilajit extract with high concentrations of bioactive components. Also, the obtained shilajit extract is highly pure, bioavailable and free from contaminants. The parameters of the method are optimized and standardized to obtain high concentrations of bioactive components which are bioavailable.
Brief description of the drawings
[0016] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.
[0017] FIG 1a illustrates a flowchart for the method of purification of shilajit resin.
[0018] FIG 1b illustrates a flowchart for the method of extraction of purified shilajit resin.
[0019] FIG 2a illustrates the HPLC chromatogram of fulvic acid.
[0020] FIG 2b illustrates the HPLC chromatogram of the purified shilajit extract
[0021] FIG 3a tabulates the levels of metal contaminants in the purified shilajit extract.
[0022] FIG 3b tabulates the levels of heavy metal contaminants in the purified shilajit extract
Detailed Description of the Invention
[0023] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.
[0024] The term “Bioavailability” refers to the extent a substance or drug becomes completely available to its intended biological destination.
[0025] The present invention discloses a method of preparation of shilajit extract with enhanced purity. It is to be noted that the terms “shilajit”, “shilajit extract” and “purified shilajit extract”are synonymously used throughout the description.
[0026] The present invention discloses a method of preparation of shilajit extract with enhanced purity. The method for the purification of shilajit resin comprises steps of weighing shilajit resin and grinding, adding ethanol and fermenting for 24 to 48 hours, hot water bath for 24 hours and collecting the supernatant, decantation and multiple filtrations of the supernatant, centrifuging the filtered supernatant and collecting the obtained supernatant from centrifugation, concentrating the supernatant, freeze drying the concentrated supernatant to obtain purified shilajit. Further, the method for extraction of shilajit comprises steps of weighing purified shilajit and grinding, adding water and citric acid, subjecting to extraction by placing in hot water bath to obtain supernatant and remnant, separating and collecting the supernatant, collecting the remnant and subjecting the remnant to extraction, separating and collecting the supernatant obtained from extracting the remnant, mixing the obtained supernatants, centrifuging the mixed supernatants, collecting the supernatant comprising purified shilajit extract, concentrating the purified shilajit extract and spray drying. The method disclosed in the present invention aids in obtaining shilajit extract with high concentrations of bioactive components. Also, the obtained shilajit extract is highly pure, bioavailable and free from contaminants. The parameters of the method are optimized and standardized to obtain high concentrations of bioactive components which are bioavailable.
[0027] FIG 1a illustrates a flow chart for the method of purification of shilajit resin. The method (100a) for purification of shilajit resin begins at step (101a) by weighing 40g of shilajit resin. The weighed shilajit resin is subjected to grinding. The ground shilajit resin is transferred to a 500ml conical flask. At step (102a), the ground shilajit is subjected to aerobic fermentation by adding ethanol at a concentration of 70%. The fermentation is aerobic, at temperatures ranging from 74oC to 76oC for a duration of 24 hours to 38 hours.
[0028] At step (103a), upon fermentation, the conical flask comprising shilajit is placed in a hot steam bath for a duration of 24 hours at a temperature of 85oC. The temperature is frequently monitored. Monitoring temperature is crucial as increase in temperature leads to degradation of bioactive components such as fulvic acid and decrease in temperature leads to loss of stability. The optimization of temperature is vital for keeping stability.
[0029] At step (104a), the conical flask is removed from the hot water bath and the supernatant is collected by decantation. Upon collecting the supernatant, the supernatant is subjected to multiple stages of filtration. The supernatant is initially filtered using a muslin cloth. Subsequently, the supernatant is subjected to coarse filtration using a stainless-steel mesh ranging from 100 microns to 200 microns followed by fine filtration using a filter press of size ranging from 5 microns to 10 microns. Additionally, the supernatant is optionally treated with activated carbon at a w/v concentration of 0.8% at a temperature ranging from 80°C to 83°C for a duration of 90 minutes. Upon multiple filtration, shilajit resin is said to be purified and free from impurities.
[0030] At step (105a), the obtained filtered supernatant is subjected to centrifugation at a speed of 11,500 rpm for a duration of 20 minutes. Upon centrifugation, the supernatant is collected. At step (106a), the supernatant comprising purified shilajit is subjected to concentration. The supernatant comprising purified shilajit is concentrated by vacuum evaporation. Vacuum evaporation is achieved at a temperature ranging from 60°C to 70°C and under controlled pressure conditions of 600 to 750 mm of Hg. The purified shilajit is concentrated to a percentage of up to 95%.
[0031] At step (106a), the obtained purified shilajit is collected in a closed container. The obtained purified shilajit is further subjected to freeze drying at temperature of -30°C for a duration of 4 to 6 hours. Subsequently, the purified shilajit is further subjected to primary drying at a temperature of -20°C, at pressure ranging from 0.1mbar to 0.2 mbar for a duration of 24 to 48 hours and secondary drying at a temperature of 25°C at a pressure of 0.01 mbar for a duration of 6 to 12 hours. Upon freeze drying, the purified shilajit powder is stored under dry conditions.
[0032] According to an embodiment of the invention, the present invention discloses a method (100b) for the extraction of purified shilajit resin. FIG 1b illustrates a flowchart for the method of extraction of purified shilajit resin. The method (100b) begins at step (101b) by weighing 40g of purified shilajit resin. Further subjecting the weighed shilajit resin to grinding and transferring the ground shilajit resin to a 500ml conical flask. At step (102b), distilled water at a volume of 320ml and citric acid at a volume of 32ml and at a concentration of 10% is added to the conical flask comprising ground shilajit.
[0033] At step (103b), the conical flask comprising shilajit is subjected to extraction and is transferred to a hot water bath. The hot water bath is maintained at a temperature of 75oC. The conical flask is kept at the hot water bath for a duration of 7 hours. Upon completion of 7 hours, the conical flask comprising shilajit is subjected to cooling to obtain a supernatant and a remnant.
[0034] At step (104b), the obtained supernatant is separated and collecting using a muslin cloth by squeezing. At step (105b), the remnant is collected from the conical flask and is subjected to extraction. To the remnant, water is added at a ratio of 1:5. The remnant is kept in a hot water bath at a temperature of 75oC and for a duration of 5 hours. The extraction process is repeated with the remnant. Upon completion of extraction, the remnant is allowed to cool.
[0035] At step (106b), the supernatant obtained by extracting the remnant is separated and collected. The separation of supernatant is achieved by squeezing the muslin cloth. At step (107b), the obtained supernatants from the extraction steps are subjected to mixing. The mixed supernatants are subjected to centrifugation at a speed of 9200 rpm for a duration of 13 minutes to obtain a supernatant comprising shilajit extract and a pellet.
[0036] At step (108b), the obtained supernatant comprising shilajit extract is collected and quantified. The supernatant comprising shilajit extract is subjected to concentration and spray drying. The supernatant comprising shilajit extract is concentrated by vacuum evaporation at a temperature ranging from 60°C to 70°C and under controlled pressure conditions of 600 to 750 mm of Hg. The concentrated shilajit extract is subjected to spray drying with conditions of inlet temperature ranging from 140°C to 160°C and outlet temperature ranging from 75°C to 90°C. Upon completion of spray drying, fine-free flowing powder of shilajit is obtained and is stored in a cool and dark place.
[0037] The shilajit extract obtained from the method (100a & 100b) disclosed in the present invention exhibits a purity of >98%. The purified shilajit extract comprises fulvic acid at a concentration range of 55% to 65% and humic acid at a concentration of 35%. Furthermore, the shilajit extract comprises trace amounts of elements iron, zinc, magnesium, calcium which complements the bioactive components. The purified shilajit extract obtained from the method (100a & 100b) disclosed is free from microbial contaminants, aids in enhancing energy and stamina, provides cognitive support and exhibits anti-ageing properties. The method disclosed in the present invention is efficient, cost effective and scalable.
[0038] The present invention discloses a method of preparation of shilajit extract with enhanced purity. The method employs optimized choice of pressure and temperature conditions directly impacting the stability and integrity of key active moieties like fulvic acid, dibenzo-alpha-pyrones, and mineral complexes. Additionally, low-pressure drying maintains the colloidal mineral matrix, essential for the adaptogenic and rejuvenating properties of the shilajit extract. Optimized processing parameters ensures retention of the metallo-organic conjugates, which are responsible for electron transfer, chelation, and antioxidant activities.
[0039] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.
Example 1: Evaluation of fulvic acid in the purified shilajit extract by high pressure liquid chromatography (HPLC)
[0040] The present invention discloses a method of preparation of shilajit with enhanced purity. The embodiment further describes the evaluation of bioactive fulvic acid in purified shilajit extract by HPLC. The shilajit extract sample for HPLC was prepared by accurately weighing 500mg of shilajit extract in a flask and dissolving the extract in 90 ml of 50% aqueous methanol and 5mL=l of 5N HCl. The obtained solution was further sonicated for a duration of 5 minutes and kept on hot water at a temperature ranging from 85oC ± 5oC bath for a duration of 10 minutes with intermittent shaking. The volume was made up to 100 ml with 50% aqueous methanol to obtain 1mg/ml solution of shilajit. HPLC was run for standard fulvic acid sample and shilajit samples under conditions of C18 column, 0.1% formic acid/acetonitrile gradient with 254 nm detection.
[0041] FIG 2a illustrates the HPLC chromatogram of fulvic acid. The chromatogram indicates the obtained peaks corresponding to fulvic acid with retention times of 2.284 and 3.220. FIG 2b illustrates the HPLC chromatogram of the purified shilajit extract. The chromatogram indicates the obtained peaks corresponding to purified shilajit extract with retention times of 2.337 and 3.219. The chromatograms indicate the presence of high concentrations of bioactive components such as fulvic acid, humic acid in the shilajit extract. Hence, the presence and concentration of bioactive fulvic acid, humic acid are verified by HPLC evaluation.
Example 2: Evaluation of presence of heavy metals and minerals in the purified shilajit extract
[0042] The evaluation of presence of heavy metals and minerals in the purified shilajit extract was performed by ICP-MS (Inductively Coupled Plasma Mass Spectrometry). FIG 3a tabulates the levels of metal contaminants in the purified shilajit extract. FIG 3b tabulates the levels of heavy metal contaminants in the purified shilajit extract. The levels of heavy metal contaminants and metal contaminants are well within the limits of toxicity. The evaluation indicates that the obtained purified shilajit extract is pure, safe from the ill effects caused by heavy metal contamination.
Example 3: Evaluation of encapsulation of purified shilajit extract
[0043] The present invention discloses a method of preparation of purified shilajit extract. According to an embodiment of the invention, the obtained purified shilajit extract from the process is encapsulated with excipients. The encapsulation matrix comprises bioactive component shilajit extract at a concentration of 80%, microcrystalline cellulose as a bulking agent at a concentration of 15%, ascorbic acid as an antioxidant at a concentration of 0.2%, colloidal silicon oxide as an anti-caking agent at a concentration of 0.8% and a capsule shell made of hydroxypropyl methylcellulose. The encapsulation matrix comprises silicon dioxide at a concentration of 0.2% and maltodextrin at a concentration of 2% as stabilizers. The encapsulated shilajit extract is highly stable enabling protection from moisture, oxygen and sunlight. The encapsulated matrix comprising shilajit extract is highly compatible and facilitates faster dissolution. The encapsulated matrix maximizes the dose of bioactive shilajit extract and minimizes excipients to avoid unnecessary additives.
[0044] The purified shilajit extract obtained from the method (100a & 100b) disclosed in the present invention is formulated as sachets, effervescent tablets comprising water soluble shilajit powder, semi-solid shilajit resin extract capsule, shilajit liposomal gel and emulsion using lecithin and phospholipids, tablets, shilajit liquid extract used in functional beverages. The method (100a & 100b) aids in preserving bioactive fulvic acids, humic substances, and trace elements without thermal degradation. The method (100a & 100b) disclosed in the present invention is efficient, cost effective and scalable.

, Claims:We Claim:
1. A method of preparation of preparation of purified shilajit extract, the method comprising:
a) a method of purification of shilajit resin, the method (101a) comprising steps of:
i. weighing 40g of shilajit resin and subjecting the weighed shilajit resin to grinding and transferring the ground shilajit resin to a 500ml conical flask;
ii. subjecting the ground shilajit to fermentation by adding ethanol at a concentration of 70%;
iii. placing the conical flask comprising fermented shilajit in hot steam bath for a duration of 24 hours;
iv. subjecting the supernatant collected after the fermentation to decantation and multiple filtration;
v. subjecting the obtained filtered supernatant to centrifugation at a speed of 11,500 rpm for a duration of 20 minutes and collecting the obtained supernatant after centrifugation;
vi. subjecting the obtained supernatant comprising purified shilajit to concentration by vacuum evaporation up to 95%; and
vii. collecting the obtained purified shilajit in a closed container and subjecting the purified shilajit to freeze drying.
b) a method of extraction of purified shilajit, the method (101b) comprising steps of:
i. weighing 40g of purified shilajit resin and subjecting the weighed shilajit resin to grinding and transferring the ground shilajit resin to a 500ml conical flask;
ii. adding 320ml of distilled water and 32ml of citric acid to the conical flask comprising ground purified shilajit;
iii. subjecting the conical flask comprising purified shilajit mixture to extraction in hot water bath at a temperature of 75oC for a duration of 7 hours and cooling to obtain a supernatant and a remnant;
iv. separating and collecting the supernatant using muslin cloth by squeezing;
v. collecting the remnant shilajit from the conical flask and subjecting to extraction by adding water at a ratio of 1:5 and keeping in hot water bath at a temperature of 75oC for a duration of 5 hours followed by cooling;
vi. separating and collecting the supernatant obtained from the remnant extraction using muslin cloth by squeezing;
vii. subjecting the obtained supernatants to mixing and centrifuging the supernatants to obtain purified shilajit extract;
viii. collecting the obtained supernatant comprising purified shilajit extract and subjecting the obtained shilajit extract to concentration and spray drying.

2. The method as claimed in claim 1, wherein the fermentation of the shilajit resin is aerobic at a temperature range of 74oC to 76oC and for a duration of 24 hours to 38 hours.

3. The method as claimed in claim 1, wherein the temperature of the hot water bath for the purification of shilajit is monitored at 85oC.

4. The method as claimed in claim 1, wherein the multiple filtration is achieved by passing the supernatant through muslin cloth, coarse filtration using a stainless-steel mesh ranging from 100 microns to 200 microns, fine filtration using a filter press of size ranging from 5 microns to 10 microns and treating with activated carbon at a w/v concentration of 0.8% at a temperature ranging from 80°C to 83°C for a duration of 90 minutes.

5. The method as claimed in claim 1, wherein the purified shilajit is freeze dried at a temperature of -30°C for a duration of 4 to 6 hours.

6. The method as claimed in claim 1, wherein the purified shilajit and shilajit extract are concentrated using vacuum evaporation at a temperature ranging from 60°C to 70°C and under controlled pressure conditions of 600 to 750 mm of Hg.

7. The method as claimed in claim 1, wherein the citric acid is used at a concentration of 10%.

8. The method as claimed in claim 1, wherein the mixture of supernatants obtained after the extraction is centrifuges at a speed of 9200rpm for a duration of 13 minutes.

9. The method as claimed in claim 1, wherein the purified shilajit extract is spray dried at inlet temperature ranging from 140°C to 160°C and outlet temperature ranging from 75°C to 90°C

10. The method as claimed in claim 1, wherein the purified shilajit extract comprises bioactive components fulvic acid at a concentration range of 55% to 65% and humic acid at a concentration of 35%.