AN ESTIMATION OF HUMIC SUBSTANCES IN AN AYURVEDIC HERBOMINERAL DRUG SHILAJATU (ASPHALTUM) AS PART OF PHYTO-PHARMACEUTICAL STANDARDIZATION

Abstract

Shilajatu an important Ayurvedic drug having several medicinal properties. If administered after proper purificatory procedure (shodhana), it has miraculous benefits. Market products are subjected to some processing, but the purity among samples vary, thereby therapeutic efficacy too. Sothere is need for standardization of purity assessment strategies which could help to scrutinize the good sample. Previous efforts towards standardization of the raw drug shilajatu aimed to identify plant source in formation of shilajatu, along with basic phyto-pharmaceutical parameters assessment. Present work attempts to see how different the data varies when the basic phyto-pharmaceutical parameters performed on processed sample. Also an additional assay of shilajatu, to quantify fulvic acid through UV spectrophotometry, and quantify humic acid through gravimetric method of analysis was carried out. Lignin decomposition logic has been attempted using UV & chromatographic assay. Chromatography was performed as per Certified Reference Material. The data may be used as a reference parameter for purity analysis of processed shilajatu. Results showed the shilajatu from Nepal had high purity and greater percentage of fulvic acid compared to that from India. This work is unique among efforts towards establishment of universal strategies in purity assessment of shilajatu. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

Shilajatu an important Ayurvedic drug having several medicinal properties. If administered after proper purificatory procedure (shodhana), it has miraculous benefits. Market products are subjected to some processing, but the purity among samples vary, thereby therapeutic efficacy too. Sothere is need for standardization of purity assessment strategies which could help to scrutinize the good sample. Previous efforts towards standardization of the raw drug shilajatu aimed to identify plant source in formation of shilajatu, along with basic phyto-pharmaceutical parameters assessment. Present work attempts to see how different the data varies when the basic phyto-pharmaceutical parameters performed on processed sample. Also an additional assay of shilajatu, to quantify fulvic acid through UV spectrophotometry, and quantify humic acid through gravimetric method of analysis was carried out. Lignin decomposition logic has been attempted using UV & chromatographic assay. Chromatography was performed as per Certified Reference Material. The data may be used as a reference parameter for purity analysis of processed shilajatu. Results showed the shilajatu from Nepal had high purity and greater percentage of fulvic acid compared to that from India. This work is unique among efforts towards establishment of universal strategies in purity assessment of shilajatu.

KEYWORDS: Shilajatu, Chromatography, UV spectrophotometry, Gravimetric method, humic acid, fulvic acid.

INTRODUCTION

Shilajatu (Mineral Pitch) is a herbo-mineral drug formed out from fissures of iron rich rocks during hot weather. Traditional uses of Shilajatu indicate its efficacy in treating diabetes and diseases of the urinary tract as well as edema, tumors, diseases causing emaciation, epilepsy and insanity. Modern science extends its indications to all the systems of the human body with a significant number of additions in the reproductive and nervous system (Robert, 2004). Lots of controversy exists, regarding the sources and availability of pure form of shilajatu. This article aims to analyze the difference between samples collected from Nepal and India in comparison to synthetic fulvic acid. This work is unique among efforts towards establishment of universal strategies in purity assessment of shilajatu.

Shilajatu in its raw form contains free radicals and may also contain mycotoxins and fungal toxins. The processing needs to remove the free radicals, Polymeric Quinone radicals, toxins, mycotoxins and inactive ingredients (Shilajit, Ayurwiki.info). Only the purified extract gives the desired benefits of shilajatu. So for the present work, shilajatu which had undergone processing with triphala qwatha and cow's milk following the method described in Ayurvedic Texts was obtained (Shastri Ambika datta, 2010). Samples collected were subjected to phyto-pharmaceutical assay comprising quantitative and qualitative estimation of various constituents present therein, and the obtained results were compared. Previous efforts towards standardization of the raw drug shilajatu aimed to identify plant source in the formation of shilajatu, along with basic phyto- pharmaceutical parameters assessment (Saileshnath saxena, 1995). Present work attempts to see how different the data varies when the basic phyto-pharmaceutical parameters were performed on processed sample. The UV spectro-photometry and Gravimetric Methods were specially designed for the qualitative analysis of shilajatu. Method used for estimation of maturity of organic compost served as pedestal for application of above two methods here; as shilajatu itself is a complex mixture of organic humic substance.

MATERIALS AND METHODS

* Phytochemical analysis

* Powder microscopy

1. Sudda shilajatu of punjab, obtained from the pharmacy of IPGT & RA, Jamnagar, India (Sample I).

2. Sudda shilajatu from Singh Darbar Dawakhana, Kathmandu, Nepal (Sample II).

Shilajatu found in Nepal is considered as the best and is also acclaimed for a highly potential medicinal herbo-mineral drug.

Phytochemical assay of Shilajatu

Shilajatu Sample I & II were analyzed by using qualitative and quantitative parameters at Pharmaceutical chemistry laboratory of IPGT & RA, Gujarat Ayurveda University, Jamnagar.

RESULTS AND DISCUSSION

Organoleptic Parameters

The characters of the sample are tabulated in Table 1.

Microscopy of Shilajatu (Sample I and II)

Samples were subjected to microscopic analysis after staining the samples with and without water. The Microscopic features are tabulated in Table 2. Images of powder microscopy of sample I & II are shown in Figure 1, 2.

Physico - chemical parameters

Sample I & II were evaluated for physico- chemical parameters like water and methanol soluble extractive and pH. The water and methanol soluble extractive in sample II measured 61.03% and 26.31% respectively and that of sample I measured 25.2% and 18.2% respectively. The pH was 5 in both, acidic by nature.

Qualitative tests

The water extract of the samples were analyzed for different functional groups. Results of qualitative tests are shown in Table 3.

High Performance Thin layer chromatographic study (HPTLC)

Methonolic extract of Sample I & II was fixed through TLC. Findings of TLC shown in Figure 3 Later HPTLC was carried out. Methanolic extract of Sample I & II and Ful vie acid (standard) were spotted on pre-coated silica gel GF 6O254 aluminium base plate by Camag Linomate V sample applicator fitted with a 100 μ? Hamilton syringe, 10 ml of 25% ammonium Hydroxide : n-propanol (7:3v/v) was used as a mobile phase. The development distance was 6.4 cm (development time 30 min). After development, densitometric scanning was performed with a camag TLC scanner III in reflectance absorbance mode at 254 nm and 366 nm under control of win CATS software (V 1.2.1 camag) (Figure 4). The slit dimensions were 6 mm χ 0.45 mm and the scanning speed was 20 mm s"1. Visual observation under densitometer showed 2 spots. However the chromatogram showed two prominent spots at hRf at 0 and 80 in sample I and one spot at hRf 80 in sample II, one prominent spot at hRf 80 in Fulvic acid (standard), in short wave UV 254 nm. Result implies both the samples contained fulvic acid. Sample II showed high purity level whereas sample I had constituents other than fulvic acid too which impede its purity.

UV spectrophotometry

There is no any accepted method to quantify fulvic acid, which is practical and cost effective. Hence it was aimed to evaluate and develop a spectro-photometric fulvic acid quantification protocol for future laboratory analysis of shilajatu & other humic substances. UV spectrophotometry was carried out to evaluate the quantity of fulvic acid present in the Sample I & II compared to the reference standard fulvic acid.

Specificity to undertake UV spectrophotometry:

Humic substances are formed by the microbial degradation of dead plant matter, such as lignin. These can be divided into three main fractions: humic acids, fulvic acids, and humin (Húmate fertilizer) {Shilajatu has been found to consist of a complex mixture of organic humic substances and plant and microbial metabolites come about in the rock rhizospheres of its natural habitat. The active principle of shilajatu is fulvic acid and it seems to have that unique capacity to dilate and permeate the thick cell walls to transmit the minerals into the cells, thereby overcome tiredness, lethargy, and chronic fatigue (Rudramani Shilajatu). Moreover shilajatu is not only about Fulvic acid. It contains more than 85 minerals and nutrients which can be instantly transported to the cells by fulvic acid {Rudramani Shilajatu). Based on the ratio of fulvic acid with humic acid, purity and action potential of shilajatu can be ascertained.

Aimed to establish a quantitative correlation between UV/viz absorption and the concentrations of fulvic acid isolated from different sources. Based on the assumption that the overall optical properties of humic substances are very similar regardless of their origins, this was undertaken. (Daqing Gan, 2007) An index was generated to quantify fulvic acid in a sample with both humic acid (HA) and fulvic acid (FA). If FA is more than HA, then the sample is good; or vice versa. The reactive moiety is equal to FA. This was based on the principle that, in agricultural science composting is believed to yield humus like substances through various biochemical process. This influences the quality of soil organic matter (Carmen, 2004). Shilajatu is also a complex form of organic humic substances. As humic substances are formed by the microbial degradation of dead plant matter, such as lignin. Conversion of lignin and other components into humic substances like humic acids, fulvic acids, and humin is known by Humification Index (HI). HI is used as process controlled parameter, while in shilajatu the humification process will be completed naturally (not induced). Hence HI will predict the quality of shilajatu. Thus parameter is used to determine the quality of naturally collected sample rather than only measuring selected FA/HA from decomposed mass. Knowing the ratios of HA to FA helps us predict HI, because FAs (more aliphatic and richer in carboxiylic acid, phenolic and quinone groups) are more soluble and reactive than HAs (more aromatic and insoluble when carboxylate groups are protonated at low pH). (Carmen, 2004) Quinones in FA have stronger antioxidant capacity and contain higher levels of phenolics than others which have potential antioxidant and chemo-protective agents. (Flua-Bin- Ii et al, 2011) This action is needed for neurodegenerative diseases like DPN.

The alkaline extract of sample I & II was prepared. After suitable dilution of one gram of sample with 50ml of 0.5 Normal NaOH (sodium hydroxide), it was kept overnight. Next day the supernatant was collected, and was scanned through 200-800 nm in a Shimadzu UV-visible double beam recording spectrophotometer (UV-160A) and the absorbance in spectra were recorded based on reference to standard range. Three standard wavelengths 260 nm, 280 nm, 472 nm were selected (Radoslaw Zbytniewski, 2005). The UV-visible spectrums of the alkaline extract of both samples were recorded Results observed under UV-visible spectrum has been presented in Table 4. It shows two absorption peaks at 472 nm and 664 nm, negative peaks at 280 nm, 260 nm. There is no absorption in the visible region. The following absorbance ratios was used for calculation (Kononova, M., 1968, Gieguzy nska ?, 1998J-

Q2/6(HA) = Absorbance Ratio of 260/664, denotes the relation between non-humified and strongly humified material. No decay started.

Q4/6(FA) = Absorbance Ratio 472/664, is often called the humification index. Typical values of the Q4/6 ratio for humified material are usually <5. (Gieguzy nska E, 1998)

Q2/4(lignin & others) = Absorbance Ratio 280/472, reflects the proportion between the lignins and other materials at the beginning of humification, and the content of materials at the beginning of transformation.

Low Q2/6 or Q4/6 ratios reflect a high degree of aromatic condensation and indicate a higher level of organic material humification. (Carmen, 2004)

As per index sample II contained good amount of FA (3.823) in comparison to sample I (3.071). The standard sample showed only 1.666 of fulvic acid. Table 4.

The UV spectrum is much practical, rapid means of estimating the concentration of a fulvic acid solution. It estimates characteristic pH independent, exponential spectrum of a standard FA in the 200 - 800 nm wavelength range. Given the length and complexity of FA sample isolation and the likelihood of at least some sample loss on the chromatography columns involved, this simple spectro- photometric approach described here (Elham A, 2009) merits application for the analysis of fulvic acid solutions. The results of this work have potential use for the routine analysis of FA samples and for the certification and regulation of commercial shilajatu and its formulations.

Gravimetric method/Acid precipitation method

The acid precipitation method has been widely accepted for the separation and subsequent quantification of humic acid (Thurman EM, 1981). The quantification of humic substances is important because humic materials have a relatively high content of free radicals which play important roles in polymerization and redox reactions. This affects the mobility of metals (both those with nutrient value and those that are of concern because they are pollutants (Carmen, 2004). The humic acid (HA) precipitates at pH < 2 and thus can be quantified by gravimetric measurements. A comparison of appropriate methods of HA analysis favors HA precipitation from alkaline solution by addition of concentrated HCl followed by washing of the precipitate with water and oven drying at 110°C (Fataftah AK, 2001). The humic and fulvic acids were extracted into a strongly basic aqueous solution of sodium hydroxide. This was made acidic by adding normal hydrochloric acid (6N HCl) by adjusting the pH up to 2 and kept overnight. Precipitate obtained was filtered and was oven dried at 110 0 C. HA is precipitated from this solution, leaving the fulvic acids in solution. This measure implies to the weight of HA in sample I & II. This measure should be low to say if good sample. This is the operational distinction between humic and fulvic acids HA in sample II (- 9.000) was less compared to sample I(- 16.357).The standard showed -10.703 of HA. Table 5.

By this procedure, Sample I contained 0.320 gms, sample II 0.546 gms and standard contained 0.094gms of humic acid out of 1 gm of samples. This high amount of humic acid compared to standard may be due to the method followed for purification of sample I & II. Subjection of drugs to processing might have switched humification. Decomposition is controlled by climatic condition (Choppin 1985), induced naturally. So based on the level of humification the quantity of humic acid could be varied.

CONCLUSION

Humic substances are used in medical science with substantial benefits in improving the health status. Shilajatu is one such complex mixture of humic substance used widely for its strengthening and rejuvenative qualities. Market products are subjected to some processing, but the purity among samples vary, thereby therapeutic efficacy too. So there is need for standardization of purity assessment strategies, which could help to scrutinize the good sample. There are no universal standards established to quantify the humic substances in shilajatu. Knowing the ratio of FA and HA helps to predict the activity potential of shilajatu and its purity. Keeping this in mind the UV spectrophotometer and Gravimetric methods were specially designed for easy and cost effective qualitative analysis of shilajatu. Method used for estimation of maturity of organic compost served as pedestal for application of above two methods here. It is reported that both the samples meets the minimum standards as reported in API at the preliminary level. The shilajatu sample collected from Nepal showed better level of fulvic acid comparatively. The results of this work have potential for the routine analysis of FA samples and for the certification and regulation of commercial shilajatu and other fulvic acid containing drugs. Thus inference from this study may be used as reference method in further quality control researches.

ACKNOWLEDGEMENTS

Harisha CR, Head of Pharmacognosy Laboratory, for carrying out pharmacognostical works related to work and Prof. Prajapati PK, Director of Pharmacy, Department of Rasashastra, IPGT & RA, Gujarat Ayurveda University, Jamnagar for his guidance during the work.

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Cite this article:

Akarshini A M, Renuka, Shukla VJ, Baghel MS (2013), AN ESTIMATION OF HUMIC SUBSTANCES IN AN AYURVEDIC HERBOMINERAL DRUG SHILAJATU (ASPHALTUM) AS PART OF PHYTO PHARMACEUTICAL STANDARDIZATION, Global J Res. Med. Plants & Indigen. Med., Volume 2(5): 365-373

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Conflict of Interest: None Declared