2. TOPICS COVERED -
1. INTRODUCTION
2. ORIGIN OF SHILAJIT
3. MAJOR BIOACTIVE COMPOUNDS IN SHILAJIT
4. ANALYSIS OF FULVIC ACID FROM SHILAJIT
5. ANALYSIS OF HUMIC ACID FROM SHILAJIT
6. ANALYSIS OF 3-hydroxydibenzo-α-pyrone FROM
SHILAJIT
7. MAJOR USES & APPLICATIONS OF SHILAJIT
8. PRECLINICAL RESEARCH ON ACTIVITY OF SHILAJIT
9. CONCLUSION
REFERENCES
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. 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. 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. 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. 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. 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.
10. 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.
11. 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.
13. 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.
14. 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.
15. 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.
16. 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.
17. 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
18. 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….
19.
20.
21. 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.
22. 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.
25. 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.
26. 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.
27. 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.
28. 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.
29. 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.
30. 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.
31. 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.
32. 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.
33. 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.
35. 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.
36. 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.
37. 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.
38. 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.
39. 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.
40. 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
41. 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.
42.
43. 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.
44. 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
45. 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.
46. 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.
47. REFERENCES
[1] S. P. Agarwal, M. D. K. Anwer, and R. Khanna, “spectroscopic characterization
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[2] J. Of, T. H. E. Serbian, C. Society, and K. A. Salman, “Humic acid from Shilajit : A
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[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
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