Analytical Study of Shilajatu

Introduction

Shilajatu is a mineral drug explained in Rasashastra under the group of Maharasa.¹ Its origin is said to be from the mountains of Himalayas.² It is also known as ‘Salajit’, ‘Shilajatu’, ‘Mumie or Mummiyo’, which is a pale brown to blackish-brown exudate, of variable thickness and consistency coming out from strata of rocks in several mountain ranges of the world, especially the Himalayan ranges of the Indian subcontinent.³

Characteristics of Shilajatu is said to be a juice like laksha that is exudated in the hills in the month of Jyeshta and Ashada (mid May to mid July) due to penetrating sunlight falling on the peaks.⁴ Shilajatu is soft as sludge and looks like shellac.⁵ Shilajatu is found in different regions and based on the geographic regions and place, the constituents tend to vary. Thus, samples were collected from two different sources, one from Nepal and another from India and both the samples were subjected to analytical studies to determine the changes in the composition of Shilajatu.

Identification test of Grahya (approved/ best variety) Shilajatu

If a Shilajatu burns without emitting smoke when placed on fire, forms the shape of Lingaakara on subjecting to heat and when placed in water, forms a streak of line before dissolving, it is said to be of good variety.¹ When dropped into water, it disperses into fine strains and slowly sets at the bottom.⁵

Shilajit humus consists of organic and mineral matter, and 5% trace elements.⁶ It is said to contain humus formed by profound alteration of organic matter in natural environments, along with other components such as benzoic acid, hippuric acid, albuminoids, triterpenes, sterol, aromatic carboxylic acid, 3,4-benzocoumarins, amino acids and phenolic lipids, etc.³

Materials and Methods

Collection of Shilajatu

Shilajatu was collected from two sources. First sample was collected from India while the second sample was collected from Nepal.

Sample from Nepal was collected since Shilajatu available in that region is known to be of superior quality. Another sample was procured from the local market as the objective of the study was to analyze the difference between samples of varied origin.

Analytical study

• Physico chemical analysis
• Phyto chemical test 
• Instrumental analysis - Energy Dispersive X-ray Spectroscopy (EDAX)

Physico chemical analysis

The standard protocol mentioned for analysis in Protocol of testing of Ayurveda, Siddha and Unani drugs as outlined by Central Council for Research in Ayurvedic Sciences (CCRAS) was carried.⁷

Phytochemical tests of Shilajatu⁸

Test for Alkaloids

a. Dragendroff's test: To 2-3 mL filtrate, few drops of Dragendroff's reagent were added for an orange brown precipitate to be formed.

b. Mayer’s test: To 2 mL of test solution, few drops of Mayer’s reagent were added to form a precipitate.

c. Wagner’s test: To 2 mL of test solution, few drops of Wagner’s reagent were added to get a reddish brown precipitate.

Tests for Carbohydrates

a. Benedict's test: Equal volumes of Benedict's reagent and test solution were mixed in the test tube. It was heated on a water bath for five minutes. The solution should appear green, yellow or red depending on quantity of reducing sugar.

b. Fehling's test: One mL Fehling's A and one mL Fehling's B solutions were mixed, boiled for a minute and then an equal quantity of test solution was added. Then it was heated in water bath for 5-10 min. An appearance of yellow colour initially and then a red precipitate should be appreciated.

Test for Protein

Biuret test: To 3 mL of test solution, 4% of NaOH was added along with few drops of 1% CuSO₄ solution. Appearance of violet or pink colour confirms the test.

Precipitation test: The test solution shows a white colloidal precipitate with the following reagents: (a) Absolute alcohol (b) 5% mercuric chloride solution (c) 5% copper sulphate solution (d) 5% lead acetate (e) 5% ammonium sulphate

Test for Tannins and Phenolic Compounds

a. Bromine water: To 2 mL of test solution, a few drops of bromine water were added, for decoloration of bromine water to occur

b. Lead acetate test: To 2 mL of the test solution, few drops of lead acetate solution were added to form a white precipitate.

c. Acetic acid solution: To 2 ml of the test solution, few drops of acetic acid solution were added, to form a red coloured solution.

Test for Glycosides

a. Borntrager's test for anthraquinone glycoside: To 3 mL extract, dilute sulphuric acid was added, boiled and then filtered. To the cold filtrate, an equal volume of chloroform was added and shaken well. The organic solvents were separated and ammonia was added. Ammoniacal layer turned pink.

Test for Steroids

a. Salkowski reaction: To 2 mL of test solution, 2 mL chloroform and 2 mL concentrated sulphuric acid were added and shaken well. The layer of chloroform appears red and the layer of acid shows fluorescence of greenish yellow.

b. Libermann- Burchard reaction: Two mL of test solution was mixed with chloroform. 1-2 mL of acetic anhydride was added and two drops of concentrated sulphuric acid were added from the side of the test tube. First red, then blue and finally green color appeared.

Test for Saponin

a. Foam test: The extract of the drug was shaken with water robustly. A stable appearance of foam was observed.

Test for Flavonoids

a. Shinoda test: 5 mL of 95% ethanol/ t-butyl alcohol, and a few drops of concentrated HCl and 0.5 g magnesium turnings were added to the extract. There was appearance of orange, pink, red to purple colour.

b. Sulphuric acid: Flavones and flavanols dissolved on addition of 66% to 80% sulphuric acid to form a deep yellow coloured solution.

Mucilage

a. Powdered drug material showed red colour with ruthenium red

b. The powdered drug swelled in water

Energy Dispersive X-ray Spectroscopy (EDAX)⁹

Energy Dispersive X-ray Spectroscopy (EDAX) is the test utilized to analyze the chemical composition of materials. The abscissa of the EDAX spectrum indicates the ionization energy and the ordinate indicates the counts. The higher the counts of a particular element, the higher will be its presence at that point or area of interest.

Method: Onto the sample, a beam of high energy charged particles is focused. An electron from a higher binding energy electron level falls into the core hole and an X-ray with the energy of the difference of the electron level binding energies is emitted. The EDAX gives a spectrum that displays the peaks correlated to the elemental composition of the test sample.

Results

Elemental constituents of samples of Shilajatu (India and Nepal) are presented in Figure 1.

Discussion

The characteristic odour of Shilajatu was appreciated in both the samples which is attributed to the presence of hippuric acid in Shilajatu. Luster of Shilajatu of Nepal sample was earthy with granular and irregular form as the sample was crude and raw. The sample collected from pharmacy was fine grained with glossy and shiny luster as the Shilajatu was extracted from the crude raw ore.

The pH of both the tested samples from India and Nepal were 6.32 and 6.90, respectively which is almost neutral. The pH of the samples was slightly acidic as the extraction of Shilajatu is done by immersing in Triphala kwatha and the pH of kwatha ranges from 3.2 to 3.3.10

Specific gravity is the ratio of density of substance to the density of test substance. The specific gravity of the Shilajatu samples were 1.001 and 1.00077. It shows the density of the sample.

Loss on drying indicates the moisture content present in the drug. The loss on drying in the samples showed a difference of mere 1.4%.

The total ash indicates the unburnt inorganic material present in the drug. It determines the pre-concentration of trace substances. Total ash was 8.825 and 25.65, respectively in the Indian and Nepal samples, which suggest that the inorganic matter was more in the Nepal sample as it has more elements, and also the quantity of magnesium and chlorine were higher.

Acid insoluble ash indicates the percentage of siliceous impurities present in it and also indicates the genuineness of the product. Values of the samples were 0 and 1.3. The sample from Nepal had silica present in it, hence the percentage of acid insoluble ash was more.

Water-soluble ash gives information about the percentage of water-soluble inorganic matter. The values recorded were 7.3% and 12%, respectively for Indian and Nepal samples.

Phytochemical evaluation of aqueous extracts of Shilajatu samples showed presence of protein, tannin, steroid and saponin in both the samples and presence of flavonoids in the sample from Nepal, in concurrence with the previous studies.¹¹ Carbohydrate was not detected in previous works, while both our study samples exhibited the presence of carbohydrates.

EDAX provides information on the elemental composition of the drug. The sample from India had C, O, Na, Mg, S, Cl and K elements, whereas the sample from Nepal showed C, N, O, Na, Mg, Al, Si, Cl, K and Ca. Silica, nitrogen, calcium and aluminum were the other elements found more in the Nepal sample.

Shilajatu found in Nepal is considered the best and is also acclaimed as a highly potent medicinal herbomineral drug.¹²

^Conclusion

The characteristic odour of Shilajatu is present in both samples; change in luster is because one was an extracted sample and the other a crude sample. The pH of the samples was slightly acidic as the extraction of Shilajatu is done by immersing in Triphala kwatha which is acidic in nature. Phytochemical evaluation of aqueous extracts of Shilajatu samples showed the presence of carbohydrates, protein, tannin, steroid, saponin and mucilage in both the samples and presence of flavonoids in the Nepal sample. EDAX showed presence of more elements in the Nepal sample. Thus, based on the above analysis the sample from Nepal seems to be better.

Conflict of Interest

None