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covers all the analysis methods which can be used to analyse bioactives in shilajit

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  3. 3. INTRODUCTION  Shilajit is perhaps the most potent rejuvenator and anti aging block buster ever known to the mankind. Attributed with many magical properties, shilajit is found predominately in the Himalayan region bordering India, China, Tibet and parts of central Asia. The existence and use of Shilajit was a closely guarded secret of the Yogis of Himalayas for many centuries. The Indian Yogis considered it as God's gift and a nectar of longevity. Ancient Indian scriptures mention of the wonderful powers of this elixir in healing most of the ailments of body and mind. An ancient Vedic Hindu text, called the Charaka Samhita written in 200 B.C. states that there is no curable disease in the universe, which is not effectively cured by Shilajit when it is administered at the appropriate time.  The resinous and nutrient-rich biomass has been touted for millennia by Ayurveda’s Materia Medica as the best carrier of energy and nutrition into the human body.
  4. 4. CONTINUE….  Modern science has recently proven this by identifying fulvic and humic acids, which are found in abundance in Shilajit, as the main substances responsible for energy production within the cell.Science is just beginning to understand the implications of the rich nutrients in the Shilajit.  Shilajit is actually a blackish brown exudation found in the serene surroundings of Himalayas. It is also found in most of the sedimentary rocks especially in Afghanistan, Bhutan, China, Nepal, Pakistan, USSR, Tibet as well in Norway, where they are gathered from steep rock faces at attitudes between 1000 and 5000 m.  Shilajit contains 85+ minerals in ionic form, Vitamins, Fulvic acid and very important phytonutrients. Thefulvic acid in shilajit is in it's most natural and purest form. Fulvic acid alone can transport the minerals through the thick cell walls. Shilajit has 85+ minerals apart from fulvic acid and can instantly supply them to the cells. It can effectively prolong cell life and keep it healthy for a very long time. So, yogis considered it Amrita or elixir of life.
  5. 5. CONTINUE…..  Nepali Shilajit is preferred because of its balance of ingredients, dynamic activity, and natural pH balance. Isagenix uses a pharmaceutical-grade of Shilajit.  The Shilajit is subjected to rigorous testing and comes with a Certificate of Analysis certifying its purity.  As per the references available to Mineralogy, is oxygenated hydrocarbon of diverse types, which is amorphous is nature luster is that of Black that has a melting pint of 90-1000 degree Celsius and burns with bright flame.  It is soluble in turpentine oil. It is a result of high degree of coalification that grade into Kerogen shale and eventually to petroleum.  Shilajit is liquidifies and gets converted into liquid state which is rich in rich in humic acid due to vegetal substance resulting from roots.  In nature it takes place in regions of coalification subjugated by abundant vegetation like Himalayan region of India
  6. 6. ORIGIN OF SHILAJIT  Many researchers claim that Shilajit exuding from the rocks of mountains is basically derived from vegetative source. Several shlokas of Susruta Samhita & Rasarangini also maintain this point of view. According to Sushruta, in the months of May-June the sap or juice of plants comes out as gummy exudation from the rocks of mountains due to strong heat of sun and Rasarangini. Dwarishtarang also claim that the Shilajit is an exudation of latex gum- resin etc. of plants which comes from the rocks of mountains in presence of scorching heat. But the exact source of the origin of Shilajit is still under controversy.  Early work on Shilajit showed that it is mainly composed of humus- the characteristic constituent of soils- together with other organic components.  Some workers think that Euphorbia royleana Boiss. plants are responsible for origin of Shilajit, because this plant is very rich latex.
  7. 7. CONTINUE….  The chemical analysis of Shilajit by researchers at Bananas Hindu University in India revealed that humification of some resin/latex bearing plants is the most likely source of Shilajit.  The recent discoveries suggest that the humification of resin-bearing plants was responsible for the major organic mass of Shilajit. And chemical analysis showed that about 80% of the humus components are present in Shilajit.  Another recent research claims that the mosses like species Barbula, Fissidenc Minium, Thuidium and species of Liverworts like Asterella, Dumortiera Marchantia, Pellia, Plagiochasma andStephenrencella-Anthoceros were present in the vicinity of Shilajit exuding rocks and these bryophytes are responsible for formation of Shilajit. The bryophytes reveal occurrence of minerals and metals in their tissue such as copper, silver, zinc, iron, lead etc, which are similar to the elements present in Shilajit.  The composition of Shilajit is influenced by factors such as the plant- species involved, the geological nature of the rock, local temperature profiles, humidity and altitude.
  8. 8. MAJOR BIOACTIVE COMPOUNDS IN SHILAJIT  Generally, native shilajit contains two classes of organic compounds - 1. Humic substances - Humic substances are the major organic constituents of native shilajit, present in an amount of about 80-85% therein, and have molecular weights ranging from several thousands for humic acids (HAs), and up to several million for polymeric humins (HMs), to only a few hundred for its fulvic acid (FAs) component. These substances also are found in soils and sediments distributed over the earth's surface, occurring in almost all terrestrial and aquatic environments. Humic substances are produced by the interactions of plants, algae, and mosses (bryophtes), with microorganisms, by a process known as humification. Humification of latex- and resin-bearing plants is primarily responsible for the production of the water-soluble humic substances.
  9. 9. 2. NON - HUMIC SUBSTANCES - The non-humic substances of shilajit are low molecular weight (Mw) compounds of plant and microbial origin, occurring in and around shilajit bearing rocks. The remaining non- humic organic masses in shilajit comprise a mixture of low Mwaromatic, aliphatic alicyclic, and heterocyclic (N-and S-containing) compounds. Of particular biological interest are low Mw oxygenated dibenzo-α-pyrones (DBP) and hydroxyacetophenones (HAPs). These basically include - a. Dibenzo-alpha pyrones, phospholipids, triterpenes and phenolic acids of low molecular weight b. Trace elements (Fe, Ca, Cu, Zn, Mg, Mn, Mo, P)  The low MW bioactive organic compounds, e.g. oxygenated dibenzo- α - pyrones (or equivalent biphenyl carboxylates) are the major entities. The medium MW fulvic acids (FA), act as carrier molecules to the bioactive substances during their systemic transport. The trace elements contribute to the healthful properties.
  10. 10.  The biological effects of shilajit are believed to be due to the two distinct classes of bioactive compounds: Dibenzo- α - pyrones DBPs, both mono- and bis-compounds thereof, in free and metal-ion conjugated forms; and fulvic acids (FAs) from shilajit-humic substances, which function as a carrier for the bioactive DBPs.  Differences in the biological effects of native shilajit can be attributed to qualitative and quantitative variations of both bioactive organic compounds and the fulvic acids in Shilajit samples from different locations.  Large amounts of contaminants, e.g. high Mw polymeric quinones, humins (HMs), and inorganic substances can be present. Furthermore, shilajit rhizospheres are always heavily infested at its periphery with a large array of microorganisms, some of which are producers of mycotoxins. Thus, the potential risk of ingesting shilajit in its native form, or only after rudimentary purification, with no control or defined standards, is quite apparent.
  12. 12. MATERIALS & METHODS USED  In this method, shilajit rock sample was obtained from Dabur Research Foundation, Ghaziabad, India.Some modification in the earlier reported methods was done & Ion exchange resins were used for the extraction (Ms Thermax Ltd., India, Ion Exchange India Ltd.). 1. Extraction of fulvic acid from Shilajit - The method consists of successive extraction of raw Shilajit with hot organic solvents of increasing polarity (chloroform, ethyl acetate and methanol) to remove the bioactive components. The residue needs to be dissolved in 0.1 N NaOH with intermittent shaking in presence of nitrogen. The suspension is then filtered and the filtrate is acidified to a pH of less than 3 to precipitate the HA. The filtrate thus obtained is shaken with macroporous ion-exchange resin in order to adsorb the FA, which is then eluted using 0.1 N aqueous sodium hydroxide solution. The FA obtained in alkali is passed through hydrogen saturated cation exchange resin in order to exchange the sodium ions with hydrogen ions. The final FA solution is concentrated and freeze dried to obtain amorphous FA.
  13. 13. 2. UV–vis spectroscopy - UV–vis spectra can be obtained on a Shimadzu 1601 UV/ vis spectrophotometer by dissolving the FA sample in water and recording the spectra in a 1 cm quartz cuvette (200–800 nm). Since humic substances usually yield uncharacteristic spectra at UV and visible wavelengths, E4/E6 ratio (ratio of the absorbance of the solution at 465 and 665 nm) can be determined by dissolving FA sample in 0.05 N NaHCO3 solution. 3. Fourier transform infrared spectroscopy (FT-IR) - For FT-IR analysis, 2 mg of dried FA sample needs to be mixed with 100 mg KBr and compressed into a pellet on an IR hydraulic press. The infrared spectrum is recorded on an FTS 40 (Bio-Rad, USA) FTIR instrument (wavelength 4000–450 cm-1). 4. Proton nuclear magnetic resonance (1H NMR) - Proton magnetic resonance (1H NMR) spectra can be recorded on Bruker model DRX-300 NMR spectrometer in DMSO-d6 and D2O using tetramethylsilane (TMS) as the internal standard.
  14. 14. 5. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) - Ultrahigh resolution mass spectra can be obtained using an Apex Qe 9.4T FT-ICR mass spectrometer. The FA sample is desalted before mass spectrometric analysis using a styrene divinyl benzene polymer type adsorber and methanol as extraction solvent. After extraction of the FA from the adsorber the final concentration is around 1 mg/ml. For electrospray measurements the methanol extract is diluted with an aliquot of Milli-Q water. The mass spectrum of the FA sample is performed in negative ion mode with a detection range between m/z 210 and 1000. Two million data points are used for data acquisition resulting in a resolution of about 300.000 at m/ z 400. The spectrum is calibrated internally with fatty acids. The elemental composition analysis can be performed with data processing software for automatic assignment of the peaks in the spectrum using a mass tolerance of 1 ppm.
  15. 15. RESULTS OBTAINED 1. Physico-chemical properties and UV–vis spectroscopy - FA obtained from the Shilajit sample is of yellowish brown in colour and is completely soluble in water. The yield of FA (5.2 g/100 g Shilajit) is much . The pH of a 2% aqueous solution is 3.04 and the E4/E6 ratio is 7.9. Increasing E4/E6 ratios is associated with lower molecular weights and inversely related to the degree of condensation of the aromatic structures in FA. Hence, the large value of the E4/E6 ratio reflects the low degree of aromatic condensation and infers the presence of relatively more aliphatic structure. 2. Fourier transform infrared spectroscopy - FT-IR analysis of FA extracted from Shilajit exhibits a broad band at about 3382cm-1 which can be attributed to stretch of hydrogen bonded OH group. A peak at 2930 cm-1 (stretch of aliphatic C-H) is present. Two bands can also be observed in the region of 1613 cm-1 (aromatic C=C double bond, H-bonded C=O of conjugated ketones) and 1411 cm-1 (O-H bending vibrations of alcohols or carboxylic acids). The band at 1081 cm-1 can be attributed to C-O stretching indicating the presence of polysaccharide or polysaccharide like compounds.
  16. 16. 3) Proton nuclear magnetic resonance - 1H NMR spectra of fulvic acid in DMSO-d6 contained various signals at chemical shifts, 0.89–2.17 ppm, suggesting the presence of aliphatic multiplet protons (methyl protons, methylene protons and protons present on aliphatic carbons which are two or more carbons away from aromatic rings or polar functional groups). Signal due to DMSO-d6 solvent was found at 2.50 ppm. A broad absorption signal at 3.45 ppm was found due to presence of moisture in DMSO-d6. A sharp signal at 5.49 FT-IR Spectra of fulvic acid extracted from Shilajit
  17. 17. ppm is attributed to OH groups in sugar, which strongly suggests the presence of carbohydrate molecules in the Shilajit fulvic acid. Signals in 6.56–7.92 ppm region are due to the presence of multiplet aromatic protons and the broad singlet at 8.75 ppm indicates the presence of N containing molecules. Protons in the aliphatic region are five times more numerous than in the aromatic region, suggesting aliphatic rich structure for Shilajit fulvic acid. 1H NMR spectra of fulvic acid in D2O contains various signals at chemical shifts, 0.89–4.39 ppm, suggest-ing the presence of aliphatic protons (methyl protons, methylene protons and protons present on aliphatic carbons, which are two or more carbons away from aromatic rings or polar functional groups). Signals of carbohydrate and N containing protons were absent due to exchange of protons with deuterium oxide. Signals in 6.59– 7.90 ppm region are due to presence of aromatic protons. Again, protons in the aliphatic region are five times greater than in the aromatic region, suggesting an aliphatic rich structure of Shilajit fulvic acid. CONTINUE….
  18. 18. 4) Fourier transform ion cyclotron resonance mass spectrometry The mass average for all assigned peaks obtained was m/z 395. The mass spectra showed the sinuous pattern with maxima at every 14 daltons which is typical for humic and other substances that differ by CH2 units. Generally, there are three homologous series at every nominal mass, two of which are compounds that contain only C, H, and O and one that contains two nitrogen atoms. The elemental compositions of nitrogen free compounds are found using the van Krevelen plot approach . The intensity weighted average O/C ratio is found to be 0.55. The relative amount of oxygen in Shilajit is very high. The van Krevelen plot showed numerousmolecular formulas with O/C > 0.8 possibly indicating the presence of carbohydrates. The intensity weighted average H/C ratio of 1.27 also is extraordinarily high in Shilajit. Calculating the aromaticity index AI, it indicated that relatively few substances contained aromatic sub-structures. The average molecular formula of the nitrogen free elemental compositions measured by FT-ICR-MS in electrospray negative ion mode was C182H23.0O10.0. C/N ratios were not quantified because the applied extraction method preferably isolates N poor compounds. However, this study demonstrates that FT-ICR-MS is a valuable technique in the field of organic geochemistry, yielding molecular information on thousands of compounds in humic substances of solid samples.
  19. 19. CONTINUE…. Thus by the application of FT-ICRMS for soil and sediment samples can give us idea on the molecular composition of complex organic. Matter which is an important prerequisite for the determination of new source and process biomarkers. It has been shown that a resolving power of more than 300000 which can only be achieved by Fourier Transform ion cyclotron resonance mass spectrometry can detect and identify all compounds of the fulvic acids mixture of shilajit extract.
  21. 21. MATERIALS & METHODS USED  Here, an authentic sample of rock Shilajit (RS) was obtained from Dabur Research Foundation, Ghaziabad, India. Scanning electron microscopy and spectral analysis, such as UV/Vis, FTIR, DSC and X-ray diffraction, were performed. The E4/E6 ratio was also determined. 1. Extraction of humic acid from Shilajit - Finely powdered shilajit is successively extracted18 with 500 ml each of hot organic solvents of increasing polarity, i.e., chloroform, ethyl acetate and methanol, to remove the bioactive components, specifically oxygenated dibenzo-α- pyrones. The so-obtained extracted Shilajit is taken and dispersed in 0.10 M aqueous sodium hydroxide with intermittent shaking under nitrogen at room temperature for 24 h. The suspension is thenn filtered to remove humin (insoluble in water at all pH values) and the filtrate is then acidified with dilute HCl to a pH of less than three. The solution is then allowed to stand at room temperature (25°C) overnight. The humic acid, which separated out as a coagulate, is filtered, dried and pulverized.
  22. 22. 2. Elemental analysis - The C, H, N and S contents is determined by packing the fulvic acid powder in tin boats after careful weighing.The obtained values are expressed as dry weight of powder, in mass %. 3. UV/Vis Spectroscopy - The UV/Vis spectra is obtained on a Shimadzu, 1601 UV/Vis spectrophotometer by dissolving the HA samples in water and recording the spectra in a 1 cm quartz cuvette in the wavelength range 200–800 nm. Since humic substances usually yield uncharacteristic spectra in the UV and visible, the E4/E6 ratio (ratio of the absorbance of the solution at 465 and 665 nm)19 is determined for the various samples. 4. Fourier transform infrared spectroscopy (FTIR) - The FTIR spectra of HA samples is recorded on a Win-IRrez (Bio-Rad, Hercules,CA, USA) using the potassium bromide (KBr) disc technique. The samples (2 mg) is mixed with potassium bromide (about 100 mg) in a clean glass pestle and mortar and compressed to obtain a pellet. The base line is corrected and scanning is performed from 4000–400 cm-1. 5. Powder X-ray diffraction - Powder X-ray diffraction patterns of powdered samples of HA is obtained using a Panalytical X-ray diffractometer, PW3719. All the samples is treated according to the following specifications: target/filter (monochromator), Cu; voltage/current, 40 kV/50 mA; scan speed, 4 °/min.
  23. 23. 6. Differential scanning calorimetry (DSC) - A Perkin–Elmer Pyris 6 instrument is used for recording DSC thermograms of the HA samples. Sample (2–8 mg) is accurately weighed and heated in closed aluminium crimp cells at a rate of 10°C/min under a dynamic nitrogen atmosphere (flow rate 20 ml/min) over the 50–300 °C temperature range. 7. Scanning electron microscopy - Scanning electron micrographs of the powdered samples is obtained using a Joel JSM- 840 scanning electron microscope with a 10 kV accelerating voltage. The surface of samples for SEM are made electrically conductive in a sputtering apparatus. 8. Surfactant properties - The surfactant properties of the humic acids are investigated by determining the effect of increasing the concentration of humic acid on the surface tension of water. The surface tension of the solutions is determined by the drop-weight method using a stalagmometer. Solutions of fulvic acids in the concentration range 0–1.4 % w/v were prepared. The drop rate was adjusted to approximately 2–3 drops/min. and the weight of 10 drops was measured.
  24. 24. RESULTS OBTAINED 1. Physical characteristics - The HA samples are brownish black in colour and have a typical characteristic odour and taste. The pH of 2 % aqueous solutions ranges from 3.46 to 3.86. The E4/E6 ratio for the HA samples range from about 3.0 to 4.0. 2. Elemental analysis – The humic acid found in the shilajit is made up of the carbon, nitrogen, hydrogen & sulphur.The carbon, hydrogen, nitrogen and sulphur contents also varied significantly among the samples of humic acids collected from different countries. These differences may be due to differences in the origin, different isolation techniques and error in sampling and analysis. The C/N ratio also varied among the samples of humic acids. 3. UV/Vis Spectra - The UV/Vis spectra of humic acids extracted from Shilajit is recorded in water from 200 nm to 800 nm. The samples did not exhibit any sharp maxima but exhibited a slight hump near 260–280 nm, which is characteristic of humic substances. This hump is attributed to the absorption of radiation by the double bonds C=C, C=O and N=N of the aromatic or unsaturated components of humic substances.
  25. 25. 4) FTIR Spectra - The FTIR spectra of the extracted humic acids are characterised by relatively few broad bands. All the humic acid samples exhibits broad bands at about 3400, 1725 and 1630 cm-1, which can be attributed to hydrogen bonded OH groups, C=O stretching of COOH groups and C=C double bonds, respectively. Sharp bands can observed in the region of 2925, 1400 and 1050 cm-1, which can be attributed to the bending vibration of aliphatic C−H groups, the O−H bending vibrations of alcohols or carboxylic acids and the OH bending deformation of carboxyl groups, respectively.
  26. 26. 5) X-Ray diffraction pattern - The X-ray diffraction pattern in the 2θ range from 10 to 70° of humic acid extracted from rock a shilajit sample exhibited very small diffuse peaks with a few intense peaks, implying its non-crystalline nature.
  27. 27. 6) Differential scanning calorimetry (DSC) - The humic acid of pioneer Shilajit exhibited no sharp endothermic peak, indicating hat it does not have any defined melting point . A shallow endotherm could be observed near 100°C, which could be attributed to dehydration of the sample. On the other hand, it showed an exothermic peak near 331°C, which could be attributed to the thermal degradation of carbohydrates, dehydration of aliphatic structures and decarboxylation of carboxylic groups.
  28. 28. 7) Scanning electron microscopy - The scanning electron micrographs of humic acid extracted from rock Shilajit showed a loose spongy structure of humic acids with the particles tending to aggregate to each other.
  29. 29. 8) Surfactant properties - Increasing the concentration of extracted humic acids in water clearly leads to a decrease the surface tension. The decrease is initially gradual until a concentration of about 0.8 %, w/v, after which it rises slightly and then became almost constant. This could be due to the formation of micelle at this concentration. This demonstrates that humic acids extracted from Shilajit indeed possess surfactant properties.
  30. 30. Analysis of 3- hydroxydibenzo-α- pyrone from shilajit
  31. 31.  3-hydroxydibenzo-α-pyrone (3-OH-DBP), a key bioactive component of shilajit is converted, among other products, to another active DBP derivative, viz. 3,8-hydroxydibenzo-α-pyrone, 3,8(OH)2-DBP, in vivo, when its precursor is ingested. 3,8(OH)2-DBP is then involved in energy synthesis in the mitochondria in the reduction and stabilization of coenzyme Q10 in the electron transport chain. As the chemical synthesis of 3,8(OH)2-DBP is a complex, multi-step process and economically not readily viable, so there was a development of a process using microorganisms for bioconversion of 3-OH-DBP to 3,8(OH)2-DBP. The biotransformation of 3-OH-DBP is achieved using Aspergillus niger, which is directly involved in the humification process on sedimentary rocks leading to “shilajit” formation. A 60% bioconversion of 3-OH-DBP to 3,8(OH)2-DBP and to its aminoacyl derivatives can be achieved. The products can then be characterized and estimated by high performance liquid chromatography (HPLC), high performance flash chromatography (HPFC) and gas chromatography-mass spectrometry (GC-MS) analyses. Among the Aspergilluss pecies isolated and identified from native “shilajit”, A. niger is found to be the most efficient for this bioconversion.
  32. 32. MATERIALS & METHODS USED 1. Reagents - 3-OH-DBP can be prepared synthetically in the laboratory and is >95% pure (λmax 209.7, 275.7, 300.6, 336.3 nm). Reagents for preparation of the fermentation medium should be of analytical reagent grade. The water used throughout the study should be purified with a Millipore system . All solvents used for HPFC should be of GR grade. 2. Fungal strain - A native Aspergillus niger strain should only be used. 3. Biotransformations - Biotransformation of a synthetic 3-OH-DBP can be carried out by fermentation by adopting the optimized conditions. 4. Conditions adopted for biotransformation - Aspergillus niger strain should be cultured in agar medium and incubated for 5 days at 29-30ºC. The cells should be washed with 5.0 ml sterilized distilled water and 2.0 ml of the spore suspension. The fermentation medium should have the following composition: KH2PO4, 0.01%, MgSO4.7H2O, 0.01%, KCl 0.01%; NaNO3 0.05% and trace quantity of FeSO4.7H2O in 100 ml of double distilled de-ionized water. Cell growth in the fermentation media can then be determined by estimating the dry cell weight after the fermentation period of 7 days.
  33. 33. 5) Isolation of transformed metabolites - The mycelia can then be separated from the FM after 7 days of fermentation. The cells are then washed with distilled water and then dried in hot air oven and powdered by a homogenizer. Thereafter, the mycelia were extracted with ethyl acetate. The marc was again suspended in bligh and dyer solvent (CHCl3- MeOH- H2O, 1:2:0.8, v/v/v) and disintegrated by an ultrasonicator. The suspensions is centrifuged for 10 min at 5000 rpm. The organic layer is dried over anhydrous sodium sulphate, filtered and the solvent was removed under reduced pressure. The residue is dissolved in methanol. The chemical metabolites present in the methanolic extracts can then be characterized and detected initially by HPLC. For further separation and for obtaining pure end-products and the residual substrate (if any), the extracts are then subjected to HPFC. Each of the fractions thus obtained, should be analyzed by HPLC for identification of the desired products and the residual substrate, by comparing with authentic standards. To determine the amounts of generated products and the residual substrate the peak areas obtained from the HPLC chromatogram is plotted in standard curves of these products.
  34. 34. 6) Isolation, characterization and quantitation of obtained products - HPLC is then carried with PDA detector and isocratic mobile phase consisting of acetonitrile- ortho phosphoric acid- water (32:1:67) with a flow rate of 0.6 ml/min using a C18 reverse-phase column attached with a guard column for separation. The photodiode array detector wavelength is set 240 nm.The end products can then be further purified using HPFC equipped with normal phase cartridge silica column .Mobile phase used is chloroform and methanol with a gradient run . Flow rate is 5 ml/min. Quantitative determination of the end-products and unreacted 3-OH-DBP can be done by comparing the area under the curve (AUC) values of the end-products with the AUC values obtained from standard curve of the components determined in the HPLC chromatogram. GC-MS can be carried out using a column (5% phenyl)- methyl polysiloxane (30 m x 0.25 mm i.d.). Carrier gas used should be ultra pure helium with constant flow rate: 1.2 ml / min. The injection port temperature is kept at 260ºC. The samples are then injected using split ratio 1:20. The transfer line temperature should be 260ºC and the injection volume is 0.5 µL. The Mass Spectrometer should be of the mass range 50- 650, Ionization potential 70 eV, Emission current 10 micro amps., Ion trap temperature 180ºC, Manifold temperature 40ºC, Background mass 45 m/z & RF dump value 650 m/z.
  35. 35. RESULTS OBTAINED  The HPLC chromatogram showed the presence of polar metabolites which can be identified as 3,8(OH)2-DBP (tR 4.684 min; λmax 219.1, 237.9, 280.4, 305.4, 355.4 nm) and its 3-O-aminoacylconjugates (tR 3.53 min; λmax 205, 245.5, 290.5, 335 nm, tR at 3.81 min λmax at 221.5, 255.8, 293.0 nm) and also some unreacted 3-OH-DBP (tR 11.41 min; λmax 209.7, 275.7, 300.6, 336.3 nm). The PDA spectrum of the product 3,8(OH)2-DBP is also identical with that of standard 3,8(OH)2- DBP. On subsequent acid hydrolysis of these products, two amino acids viz. glycine and arginine can be detected. The unreacted 3-OH-DBP can then be detected.  When the organic solvent extractives are separated by HPFC and further analyzed by HPLC ,fractions 6-7 of HPFC can then be identified to be 3-OH-DBP. For determination of the amounts of the products, the total areas obtained from the HPLCs were plotted in the standard curves of these compounds.
  36. 36.  To confirm the structures of the compounds, the end- products can then be analyzed by GC-MS using corresponding compounds as synthetic markers . The gas chromatogram of the reaction products showsresidual 3-OH-DBP as its silyl derivative, at tR 15.860 min with m/z values of 284(M+) and fragment ion peaks at 269(M-CH3)+, 241[M- (CH3+CO)]+ and the desired 3,8(OH)2- DBP, as its silyl derivative (2), at tR 19.615 with m/z values of 372(M+) and fragment ion peaks at 357(M-CH3)+, 329[M- (CH3+CO)]+.  3-O-Glycyl-8-hydroxydibenzo-α-pyrone (HPLC:tR 3.8 min) (6), as TMS derivative, is then subjected to EI-MS analysis. The mass spectrum shows molecular-ion (M+) peak at m/z 429 and fragment ion peaks at m/z 299 (due to 3-O·-8-O-trimethylsilyl dibenzo-α-pyrone moiety) and at m/z 130 (trimethylsilyl glycyl moiety). Based on these observations, a probable mechanism of biotransformation of 3-OH-DBP byAspergillus niger into 3,8(OH)2DBP and 3-O-glycyl-8-hydroxy- DBP is envisaged . In the first phase of the reaction, Aspergillus niger generates hydroxyl radicals which attack the hydroxyl group at C-3 position of 3-OH-DBP and causes abstraction of H· leading to the formation of a semiquinone radical which is resonance stabilized . Then, there is attack by ·OH radical, generated by Aspergillus niger, at the C-8 site of DBP. Rearrangement of this leads to formation of 3,8 (OH)2-DBP which is then transformed by aminoacylation
  37. 37. DIRECT COMPARISON OF 3-OH-DBP and 3,8-(OH)2 -DBP It can be done by HPTLC & HPLC. 1. HPTLC Conditions : HPTLC can be performed using pre-coated silica gel 60 F254 aluminium TLC plates. Solutions of marker 3-OH-DBP and 3,8- (OH)2-DBP, are applied and the plates were developed in a twin trough chamber with chloroform:methanol 9:1 (v/v) as mobile phase. Densitometric evaluation of the plates is performed at λ 240 nm by means of Camag TLC Scanner 3 in absorption mode. The scanned data is processed by means of Camag winCATS software, version 1.3.4. The plates are subsequently scanned to determine the UV reflectance spectra of each spot between 200 and 400 nm to identify the two bioactives of shilajit. 2. HPLC conditions for DBP analyses : HPLC analysis of DBPs is performed using a system comprised with PDA detector and Empower software with a NovaPak pre- packed column (RP C18, particle size 4 μm, 3.9 x 150 mm) fitted with a reverse phase guard column and acetonitrile:water:o- phosphoric acid 32:67:1 (v/v/v) as mobile phase, with a run time of 15 minutes and flow rate 1 ml/min in an isocratic mode. Detection is done at λ 240 nm.
  38. 38. MAJOR USES OF APPLICATION OF SHILAJIT  Shilajit is prescribed to treat genitourinary disorder, jaundice, gallstone, digestive disorders, enlarged spleen, epilepsy, nervous disorder, chronic bronchitis, anemia.  Shilajit is given along with milk to treat diabetes. Shilajit has also aphrodisiac property.According to Ayurveda, shilajit arrests the process of aging and produces rejuvenation which are two important aspects of an Ayurvedic rasayana.  Shilajit is useful for treating kidney stones, oedema, piles, internal antiseptic, adiposity, to reduce fat and anorexia. Shilajit is prescribed along with guggul to treat fractures. It is believed that it goes to the joints and forms a callus quickly. The same combination is also used to treat osteoarthritis and spondylitis.
  39. 39. PRECLINICAL RESEARCH ON ACTIVITY OF SHILAJIT 1. Antiulcerogenic and antiinflammatory activity 2. Antioxidant activity 3. Memory enhancement and anxiolytic activity 4. Antistress activity 5. Antiallergic activity 6. Anti AIDS activity 7. Immunomodulatory activity 8. Antidiabetic activity 9. Learning augmentation
  40. 40. CONCLUSION  Fulvic acids are extracted from Shilajit using ion exchange resin.The large value of E4/E6 from UV–vis spectroscopy is associated with lower molecular weights, which is consistent with the average molecular weight (395 m/z) valuesestimated from FT-ICR-MS. This large value of E4/E6 ratio reflects the low degree of aromatic condensation and infers the presence of relatively more aliphatic structure that again is strongly supported by FT-ICR-MS. The FT-IR spectra of fulvic acid extracted from shilajit showed bands of hydrogen bonded OH groups and conjugated C=C double bonds as well as C-O stretching of polysaccharide or sugar like substances. 1H NMR spectra in DMSO-d6 and D2O contains various signals of aliphatic multiplet protons. A sharp signal of OH group in sugar was observed in DMSO-d6 but absent in D2O solvent due to exchange of protons with deuterium oxide. These observations strongly support the presence of carbohydrate molecules in the Shilajit fulvic acid.
  41. 41.  Humic acids characterised based on the physico-chemical and spectral properies. The spectral features obtained from UV/Vis, FTIR, XRD and DSC are used for the analysis.The surfactant properties of humic acids can then be investigated by determining the effect of increasing concentration of humic acids on the surface tension of water. The study demonstrated that the humic acids extracted from shilajit indeed possessed surfactant properties.  These DBPs are essential for the therapeutic activity of shilajit. In summary, it can be stated that the mechanism by which dibenzo-α-pyrones are produced in shilajit, can be simulated in situ using Aspergillus niger. The optimized conversion of 3-OH-DBP into another highly bioactive form 3,8(OH)2-DBP is a novel reaction catalyzed by the filamentous fungi Aspergillus niger. The DBP-converting factor is perhaps a metabolism related enzyme which by oxidation forms 3,8(OH)2-DBP; which acts as a substrate for further enzymatic reactions like conjugation with amino acids to form aminoacyl conjugates and render these compounds more water-soluble and hence more bioavailable. In certain cases, these mechanisms are operative in the fungi not only to detoxify the toxic substance, but also to make them beneficial for the organism.Thus, here methods like HPLC & GC-MS are used for its analysis.
  42. 42. REFERENCES [1] S. P. Agarwal, M. D. K. Anwer, and R. Khanna, “spectroscopic characterization Available online at www.shd.org.rs/JSCS/ Available online at www.shd.org.rs/JSCS/,” vol. 75, no. 3, pp. 413–422, 2010. [2] J. Of, T. H. E. Serbian, C. Society, and K. A. Salman, “Humic acid from Shilajit : A physico-chemical and spectroscopic characterization,” no. April, 2016. [3] “Elemental analysis of fulvic acids of shilajit using ultra high resolution mass spectrometry,” p. 300000. [4] S. P. Agarwal, R. Khanna, R. Karmarkar, K. Anwer, and R. K. Khar, “Shilajit : A Review,” vol. 405, no. December 2006, pp. 401–405, 2007. [5] M. Witt, “Spectroscopic characterization of fulvic acids extracted from the rock exudate Shilajit,” no. April 2016, 2008. [6] A. Islam, R. Ghosh, D. Banerjee, P. Nath, U. K. Mazumder, and S. Ghosal, “Biotransformation of 3-hydroxydibenzo-α-pyrone into 3, 8 dihydroxydibenzo-α- pyrone and aminoacyl conjugates by Aspergillus niger isolated from native ‘shilajit,’” Electron. J. Biotechnol., vol. 11, no. 3, pp. 1–10, 2008.
  43. 43. THANK YOU

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covers all the analysis methods which can be used to analyse bioactives in shilajit


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