Description:Field of the Invention
The present invention relates to a method of determining the content purity of shilajit by quantification of fulvic acid in a sample of shilajit by UV Spectrophotometry.

Background of the Invention
Shilajit is a multi-component natural occurring mineral substance used in ayurveda and siddha systems of medicine. Its source can be traced to the mountainous regions, where the hilly tribes first identified its beneficial use. Shilajit is referred to as ‘rasayana’/‘rasayanam’ in ayurveda and siddha literature which means rejuvenator because it prevents ailment and enhances the quality of life.
Shilajit is a sticky and coherent material with a shiny and polished surface, easily soluble in water. Shilajit is composed of three primary chemical units namely, (1) low and medium molecular weight non-humic organic compounds comprising free and conjugated dibenzo-α-pyrones. (2) Medium and high molecular weight dibenzo-α-pyrones-chromoproteins, containing trace metal ions and colouring matter such as carotenoids and indigoids and (3) metallo-humates like fulvic acids and fusims with dibenzo-α-pyrones. Shilajit samples from various regions of the earth have similar physical properties and qualitative chemical composition, but they vary vividly in percentage ratio of chemical units. Solubility in water demonstrates that nearly 30–50% of the weight of shilajit passes into the supernatant liquid, and the remains includes mineral and plant residues in quantities depending on the purity of shilajit.
Amongst the numerous active chemical units of shilajit, fulvic acid and humic substances are important. Humic` substances can be fractionated into three components: humic acid, fulvic acid, and insoluble humin. A standard method based on acid precipitation is gaining acceptance for the quantification of humic acid, as it precipitates at pH < 2 and can thus be quantified by gravimetric measurements. However, the fulvic acid component remains in solution at all pH levels. Various methods, including acid precipitation, barium chloride precipitation, and spectrophotometric measurement, have been developed to quantify humic acid. Among these, the acid precipitation method is widely accepted for separating and quantifying humic acid.

Summary of the Invention
In one general aspect, the present invention relates to method of determining the content purity of fulvic acid in a sample of shilajit by UV Spectrophotometry; wherein the method comprises:
a) Preparing a standard stock solution of fulvic acid in a phosphate buffer of pH 7;
b) Preparing diluted calibrated concentration solutions from the above standard stock solution ranging from 5 mg/L to 180 mg/L;
c) Measuring absorbance of the above calibrated concentration solution at wavelengths 350 nm, 370 nm, 400 nm with UV spectrophotometer and plotting the graph with concentration against absorbance values;
d) Preparing a solution of the shilajit sample to tested in an alkali solution; acidifying the solution to pH less than 2 with an acid solution to precipitate humic acid and filtering the solution;
e) Adjusting the pH of the above filtrate with an alkali solution to pH 7; optionally diluting the filtrate with a phosphate buffer of pH 7;
f) Measuring absorbance of the sample at wavelengths 350 nm, 370 nm, 400 nm with the UV spectrophotometer;
g) Calculating corresponding fulvic acid concentration form the calibration curve prepared as above.

Detailed Description of the Invention
There is no practical and cost-effective method currently available for determining fulvic acid content. The present invention provides a spectrophotometric evaluation method for determining the fulvic acid content in commercial shilajit products. Based on the assumption that the optical properties of fulvic acids are independent of their sources, a calibration curve was established showing a linear relationship between varying concentrations of a standard fulvic acid and their UV/vis absorption at multiple wavelengths. The concentrations of fulvic acid in various commercial products were then obtained by measuring the samples' UV/vis absorption and applying the established calibration curve. The calculated fulvic acid concentrations were in reasonable agreement with the values obtained from carbon analysis of the test samples after appropriate corrections.
The method of the invention provides a quantitative correlation between UV/vis absorption and the concentrations of fulvic acid isolated from different sources, rather than focusing on subtle spectral differences between various humic substances. It operates on the assumption that the overall optical properties of humic substances are consistent, regardless of their origin. By measuring the optical absorption of fulvic acids at multiple wavelengths, potential interferences from most inorganic salts and contaminants, which may absorb at some wavelengths but not others is eliminated.

Methodology:
Standard of Fulvic Acid, Reagent Grade NaOH and KOH was used for the experiment. A Millipore system provided ultrapure water. A Shimadzu spectrophotometer and 1 cm plastic cuvettes were used for all absorbance measurements.

Weighed accurately 5g of shilajit sample in a 250ml conical flask. Added 50mL of 0.1N sodium hydroxide (NaOH) and heated the solution to warm. The contents were filtered and the supernatant liquid was collected in a 500ml beaker. Repeated the above extraction procedure until the 0.1 NaOH solution was colourless. All the extracted and filtered solution was collected in a 500ml beaker. The filtrate was acidified with 30% HCl, till the pH is about 2.0 to precipitate humic acid. The precipitate was separated and the filtrate was neutralized with 20-30% NaOH to adjust the pH to 7.0.

Example 1:
1000 mg/L fulvic acid stock standard solutions was prepared in 20 mM, pH 7 phosphate buffer. Five commercially available liquid and solid humic substance samples were obtained from different origins. For isolation of fulvic acid, the pH of these samples was adjusted to less than 2 to precipitate humic acid. After removal of humic acid precipitates and insoluble solids, the supernatants were adjusted to pH 7 and followed by dilution and absorbance was measured at wavelengths 350 nm, 370 nm, 400 nm.

Preparation of Calibration Curve:
Calibration curves were obtained by serial dilution of pH 7 fulvic acid standard stock solutions followed by absorbance measurement at the respective wavelengths. The calibrated concentrations ranged from 5 mg/L to 180 mg/L and R² obtained >0.999.
After appropriate dilution, serial solutions with decreasing fulvic acid concentrations were prepared for each of the five commercial fulvic acid samples. Since the calibration for fulvic acid is similar for pH 7 at all measured wavelengths, we only present the absorbance at 350 nm, 370 nm, 400 nm, for each solution at pH 7. The concentration of each fulvic acid sample was calculated by averaging the concentrations obtained from five serial solutions at multiple wavelengths according to the respective dilution factors.

Results:
Fulvic acid calibration using standard fulvic acid and correlation between fulvic acid concentrations obtained from UV spectrophotometry indicate that the spectrophotometric method can be applied as a simple alternative method for the quantification of fulvic acid. Our results also suggest that although the structure, configuration, functional moieties, molar mass, and intra and inter-molecular interactions may be significantly different, the overall optical behavior of fulvic acids from different sources is similar. Additionally, due to the absence or minimum absorption of most inorganic salts in the wavelengths 350 nm to 400 nm range, no removal of inorganic salts is required.

, C , Claims:1. A method of determining the content purity of fulvic acid in a sample of shilajit by UV Spectrophotometry; wherein the method comprises:
a) Preparing a standard stock solution of fulvic acid in a phosphate buffer of pH 7;
b) Preparing diluted calibrated concentration solutions from the above standard stock solution ranging from 5 mg/L to 180 mg/L;
c) Measuring absorbance of the above calibrated concentration solution at wavelengths 350 nm, 370 nm, 400 nm with UV spectrophotometer and plotting the graph with concentration against absorbance values;
d) Preparing a solution of the shilajit sample to tested in an alkali solution; acidifying the solution to pH less than 2 with an acid solution to precipitate humic acid and filtering the solution;
e) Adjusting the pH of the above filtrate with an alkali solution to pH 7; optionally diluting the filtrate with a phosphate buffer of pH 7;
f) Measuring absorbance of the sample at wavelengths 350 nm, 370 nm, 400 nm with the UV spectrophotometer;
g) Calculating corresponding fulvic acid concentration form the calibration curve prepared as above.