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Humic acid from Shilajit: A physico-chemical and spectroscopic characterization

March 2010
Journal of the Serbian Chemical Society 75(3)

DOI:10.2298/JSC1000006A

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Authors:

Suraj Agarwal

T John Group of Institutions

Md. Khalid Anwer

Prince Sattam Bin Abdulaziz University

Asgar Ali

Jamia Hamdard

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Shilajit is a blackish–brown exudation, consisting of organic substances, metal ions and minerals, from different formations, commonly found in the Himalayan region (1000–3000 m) from Nepal to Kashmir. Shilajit can also be collected throughout the mountain regions in Afghanistan, Bhutan, China, Bajkal, throughout Ural, Caucasus and Altai mountains also, at altitudes between 1000 to 5000 m. The major physiological action of shilajit has been attributed to the presence of bioactive dibenzo-α-pyrones together with humic and fulvic acids, which act as carrier molecules for the active ingredients. In this work, the aim was to extract humic acid from Shilajit from various sources and characterised these humic acids based on their physicochemical properties, elemental analysis, UV/Vis and FTIR spectra, X-ray diffraction pattern and DSC thermograms. The spectral features obtained from UV/Vis, FTIR, XRD and DSC studies for samples of different origins showed a distinct similarity amongst themselves and in comparison to soil humic acids. The surfactant properties of the extracted fulvic acids were investigated by determining the effect of increasing concentration on the surface tension of water. The study demonstrated that humic acids extracted from shilajit indeed possessed surfactant properties.

XRD Pattern of humic acid extracted from the rock Shilajit.

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DSC Spectra of humic acid extracted from the Pioneer Shilajit.

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J. Serb. Chem. Soc. 75 (3) 413–422 (2010) UDC *Shilajit:547.992:541.1.004.12:

JSCS–3974 543.42.004.12

Original scientific paper

doi: 10.2998/JSC090316006A 413

Humic acid from Shilajit – a physico-chemical and

spectroscopic characterization

SURAJ P. AGARWAL1, M. D. KHALID ANWER1,3*, RAJESH KHANNA2,

ASGAR ALI1 and YASMIN SULTANA1

1Dept. of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New

Delhi-110062, 2Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad,Uttar

Pradesh-201010, India and 3College of Pharmacy, Al-kharj, King Saud University, K.S.A.

(Received 16 March, revised 22 June 2009)

Abstract: Shilajit is a blackish–brown exudation, consisting of organic substan-

ces, metal ions and minerals, from different formations, commonly found in the

Himalayan region (1000–3000 m) from Nepal to Kashmir. Shilajit can also be

collected throughout the mountain regions in Afghanistan, Bhutan, China,

Bajkal, throughout Ural, Caucasus and Altai mountains also, at altitudes be-

tween 1000 to 5000 m. The major physiological action of shilajit has been

attributed to the presence of bioactive dibenzo-α-pyrones together with humic

and fulvic acids, which act as carrier molecules for the active ingredients. In

this work, the aim was to extract humic acid from Shilajit from various sources

and characterised these humic acids based on their physicochemical properties,

elemental analysis, UV/Vis and FTIR spectra, X-ray diffraction pattern and DSC

thermograms. The spectral features obtained from UV/Vis, FTIR, XRD and

DSC studies for samples of different origins showed a distinct similarity

amongst themselves and in comparison to soil humic acids. The surfactant

properties of the extracted fulvic acids were investigated by determining the

effect of increasing concentration on the surface tension of water. The study

demonstrated that humic acids extracted from shilajit indeed possessed surf-

actant properties.

Keywords: Shilajit; humic acid; FTIR spectra; DSC; XRD; surfactant properties.

INTRODUCTION

Shilajit, also known as salajit, shilajatu, mumie or mummiyo, is a blackish–

–brown exudate coming out from layer of rocks in many mountain ranges, espe-

cially in the Himalayas and Hindukush ranges of the Indian subcontinent.1 It is

also found in Russia, Tibet, Norway and other countries, where it is collected in

small quantities from steep rock faces at altitudes between 1000 and 5000 m. Shi-

* Corresponding author. E-mails: -manwer@ksu.edu.sa; mkanwer2002@yahoo.co.in

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414 AGARWAL et al.

lajit samples from different regions of the world, however, vary in their physiolo-

gical properties.2

It mainly consists of palaeohumus (around 80–85 %) and organic compounds

derived from vegetation fossils that were compressed under layers of rocks for

hundreds of years and underwent significant metamorphosis due to the prevalent

high temperature and pressure conditions.5

Extensive research has been performed to determine the exact chemical na-

ture of Shilajit. Earlier work on shilajit showed that its major organic constituents

included benzoic acid, hippuric acid, fatty acids, resin and waxy materials, gums,

albuminoids and vegetable matter with benzoic acid being the active substan-

ce.6,7 Extensive research in the eighties showed that the major organic mass of

Shilajit was comprised of humus (60–80 %) along with other components, such

as benzoic acid, hippuric acid, fatty acids, ichthyol, ellagic acid, resin, triterpenes,

sterol, aromatic carboxylic acid, 3,4-benzocoumarins, amino acids and phenolic

lipids.8 The major physiological action of Shilajit was found to be due to the pre-

sence of bioactive dibenzo-α-pyrones along with humic and fulvic acids, which

acted as the carrier molecules for the active substances.9–11 Recently, the physic-

co-chemical, spectral and thermal properties of shilajit and its humic substances

were reported, which further confirmed its humic nature.12–15 Elemental analysis

and spectroscopic techniques, such as UV/Vis, FTIR and X-ray diffraction and

DSC analyses have been widely used for the characterization of humic acids ob-

tained from lignite, charcoal, soil, sewage sludge and compost.16,17 In this study,

these methods were applied for the first time to humic acids extracted from Shi-

lajit from different sources.

EXPERIMENTAL

Materials and methods

An authentic sample of rock Shilajit (RS) was obtained from Dabur Research Foun-

dation, Ghaziabad, India. Dried Shilajit extracts were also obtained from three different com-

mercial sources in India, viz., Pioneer Enterprises (PE) – Mumbai, Natural Remedies (NR) –

– Bangalore and Gurukul Kangri (GK) – Haridwar. The humic acid was extracted from all the

samples of Shilajit and characterised based on their physico-chemical properties and their ele-

mental analysis. 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. The spec-

tral properties were compared with a humic acid standard from Sigma Aldrich.

Extraction of humic acid from Shilajit

Finely powdered shilajit was successively extracted18 with 500 ml each of hot organic

solvents of increasing polarity, i.e., chloroform, ethyl acetate and methanol, to remove the bio-

active components, specifically oxygenated dibenzo-α-pyrones. The so-obtained extracted

Shilajit was taken and dispersed in 0.10 M aqueous sodium hydroxide with intermittent shak-

ing under nitrogen at room temperature for 24 h. The suspension was filtered to remove humin

(insoluble in water at all pH values) and the filtrate was acidified with dilute HCl to a pH of

less than three. The solution was allowed to stand at room temperature (25 °C) overnight. The

humic acid, which separated out as a coagulate, was filtered, dried and pulverized.

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CHARACTERIZATION OF HUMIC ACID OF SHILAJIT 415

Elemental analysis

The C, H, N and S contents were determined by packing the fulvic acid powder in tin

boats after careful weighing (Balance – Mettler Toredo, MX5) with the aid of a CHNS ana-

lyzer (Vario EL-III). The obtained values are expressed as dry weight of powder, in mass %.

UV/Vis Spectroscopy

The UV/Vis spectra of various HA extracted from shilajit of different origins were ob-

tained on a Shimadzu, 1601 UV/Vis spectrophotometer by dissolving the various 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 was determined for

the various samples.

Fourier transform infrared spectroscopy (FTIR)

The FTIR spectra of HA samples were recorded on a Win-IRrez (Bio-Rad, Hercules,

CA, USA) using the potassium bromide (KBr) disc technique. The samples (2 mg) were mixed

with potassium bromide (about 100 mg) in a clean glass pestle and mortar and compressed to

obtain a pellet. The base line was corrected and scanning was performed from 4000–400 cm-1.

Powder X-ray diffraction

Powder X-ray diffraction patterns of powdered samples of HA were obtained using a

Panalytical X-ray diffractometer, PW3719. All the samples were treated according to the fol-

lowing specifications: target/filter (monochromator), Cu; voltage/current, 40 kV/50 mA; scan

speed, 4 °/min.

Differential scanning calorimetry (DSC)

A Perkin–Elmer Pyris 6 instrument was used for recording DSC thermograms of the HA

samples obtained from different shilajit sources. Samples (2–8 mg) were 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.

Scanning electron microscopy

Scanning electron micrographs of the powdered samples were obtained using a Joel JSM-

840 scanning electron microscope with a 10 kV accelerating voltage. The surface of samples

for SEM was made electrically conductive in a sputtering apparatus (Fine Coat Ion Sputter

JFC-1100) by evaporation of gold.

Surfactant properties

The surfactant properties of the humic acids were investigated by determining the effect

of increasing the concentration of humic acid on the surface tension of water. The surface ten-

sion of the solutions was determined by the drop-weight method using a stalagmometer. Solu-

tions of fulvic acids in the concentration range 0–1.4 % w/v were prepared. Each solution was

separately sucked into the stalagmometer and allowed to drop slowly from it. The drop rate

was adjusted to approximately 2–3 drops/min. and the weight of 10 drops was measured.

RESULTS AND DISCUSSION

Extraction of humic acid from shilajit

The yields obtained at the different stages of the earlier reported method and

the improved method for the extraction of humic acid from shilajit are compared

in Table I. The yields of HA extracted from shilajit from Dabur, Gurukul Kangri,

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416 AGARWAL et al.

Natural Remedies and Pioneer Enterprises were 2.5, 9.2, 8.7 and 8.7 %, respec-

tively. These are high proportion compared with those reported in the literature.18

The maximum yield of HA was obtained from the Gurukul Kangri shilajit.

TABLE I. Comparison of the yields of humic acid from Shilajit obtained from different sources

Shilajit Yield of fulvic acid, %

Reported method Improved method

I II III Mean ± SD I II III Mean ± SD

RS 1.2 1.0 1.4 1.2 ± 0.2 2.2 2.8 2.6 2.5 ± 0.3

GK 7.5 7.8 7.3 7.5 ± 0.3 9.2 8.9 9.5 9.2 ± 0.3

R 5.8 6.2 6.9 6.3 ± 0.6 8.1 8.9 9.1 8.7 ± 0.5

PE 6.9 6.4 7.1 6.8 ± 0.4 9.3 8.8 8.5 8.9 ± 0.4

Physical characteristics

The physical characteristics of the humic acids extracted from Shilajit of dif-

ferent origin are listed in Table II. The extracted HA from shilajit of different

origins exhibit very similar physico-chemical characteristics, indicating no quail-

tative variation in the Shilajit samples and in their extraction procedure.18. Slight

variations are to be expected and were seen in these HA samples. All the HA

samples were brownish black in colour and had a typical characteristic odour and

taste. The pH of 2 % aqueous solutions ranged from 3.46 to 3.86. The ratio of the

absorbance at 465 and 665 nm (E4/E6) has been widely used by soil scientist for

characterization purposes. The E4/E6 ratio for all the examined HA samples ran-

ged from about 3.0 to 4.0, which are consistent with those reported in the litera-

ture.17

Elemental analysis

Elemental analysis of humic substances is generally used to establish their

nature and origin.20 As shown in Table III, a comparison of carbon, hydrogen, ni-

TABLE II. Comparison of the physical characteristics of humic acid from Shilajit of differen

origins

Characteristic Humic acid (RS) Humic acid (GK) Humic acid (NR) Humic acid (PE)

ature Dark brown

powder Dark brown

powder

Colour Dark Brown Dark Brown Dark Brown Dark Brown

Odour Characteristic Characteristic Characteristic Characteristic

Taste Characteristic Characteristic Characteristic Characteristic

H of 2 % aq.

solution 3.86 3.77 3.46 3.68

Absorbance at

465 nm (E4) 0.513 0.542 0.284 0.222

Absorbance at

665 nm (E6) 0.144 0.180 0.072 0.072

4/E6 ratio 3.56 3.01 3.94 3.08

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CHARACTERIZATION OF HUMIC ACID OF SHILAJIT 417

trogen and sulphur contents of the humic acids extracted from Shilajit of different

origins with those of soil humic acids21 and the Sigma Aldrich standard humic

acid revealed that the content of C, H, N and S were very low in the case of the

humic acids extracted from the pioneer shilajit. The carbon, hydrogen, nitrogen

and sulphur contents also varied significantly among the samples of humic acids.

These differences may be due to differences in the origin, different isolation tech-

niques and error in sampling and analysis. The C/N ratio also varied among the

samples of humic acids.

TABLE III. Elemental analysis of humic acids extracted from shilajit of different origins

Source of humic acid % C % H % N % S C/N ratio

Rock Shilajit (Dabur) 36.46 5.15 3.03 0.70 12.0

Shudh Shilajit (Gurukul Kangri) 45.36 5.92 2.31 0.39 19.63

Shilajit extract (Natural Remedies) 51.48 5.89 3.27 0.81 15.73

Shilajit extract (Pioneer Enterprises) 27.44 2.90 1.24 0.26 22.10

Sigma Aldrich (Std. HA) 42.28 4.25 0.57 0.81 73.09

UV/Vis Spectra

The UV/Vis spectra of the various samples of humic acids extracted from Shi-

lajit of different origin were recorded in water from 200 nm to 800 nm are shown

in Fig. 1. The samples did not exhibit any sharp maxima but exhibited a slight hump

near 260–280 nm, which is characteristic of humic substances.19 As discussed

previously, 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

Fig. 1. UV/Vis Spectra of humic acid extracted from Shilajit of different origins:

a) RS, b) GK, c) NR and d) PE.

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418 AGARWAL et al.

substances.22 The variation in the hump observed with the different samples of

Shilajit could be attributed to variations in the concentrations of aromatic com-

pounds, which in turn is characteristic of the difference in the humification process.

FTIR Spectra

The FTIR spectra (Fig. 2) of the extracted humic acids were characterised by

relatively few broad bands. All the humic acid samples exhibited 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, respecti-

vely. Sharp bands were 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 de-

formation of carboxyl groups, respectively.19

Fig. 2. FTIR Spectra of humic acid extracted from Shilajit of different origins:

a) RS, b) GK, c) NR, d) PE and e) Laurentian humic acid.

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 (Fig. 3) exhibited very small diffuse peaks

with a few intense peaks, implying its non-crystalline nature. This behaviour is

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CHARACTERIZATION OF HUMIC ACID OF SHILAJIT 419

consistent with the behaviour observed in the case of humic substances from other

sources.23,24

Differential scanning calorimetry (DSC)

The humic acid of pioneer Shilajit exhibited no sharp endothermic peak, in-

dicating that it does not have any defined melting point (Fig. 4). A shallow endo-

therm 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.24

Fig. 3. XRD Pattern of humic acid extracted from the rock Shilajit.

Fig. 4. DSC Spectra of humic acid extracted from the Pioneer Shilajit.

Scanning electron microscopy

The scanning electron micrographs (Fig. 5) of humic acid extracted from rock

Shilajit of Dabur showed a loose spongy structure of humic acids with the par-

ticles tending to aggregate to each other.

Surfactant properties

As can be seen in Fig. 6, increasing the concentration of extracted humic

acids in water clearly led to a decrease the surface tension. The decrease was ini-

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420 AGARWAL et al.

tially gradual until a concentration of about 0.8 %, w/v, after which it rose slight-

ly and then became almost constant. This could be due to the formation of mi-

celle at this concentration. This demonstrates that humic acids extracted from Shi-

lajit indeed possess surfactant properties. The value of 0.8 %, w/v, for the critical

micelle concentration (CMC) is in agreement with the reported value of 0.7 %,

w/v, for humic acids extracted from soil.26

Fig. 5. Scanning electron micrographs of humic acid from rock shilajit; a) 500×; b) 1500×.

Fig. 6. Effect of humic acid concentration

on the surface tension of water.

CONCLUSIONS

Humic acids from the various samples of shilajit were characterised and their

physico-chemical and spectral properties compared. Such results are presented for

the first time herein. The spectral features obtained from UV/Vis, FTIR, XRD and

DSC studies for samples of different origins showed a distinct similarity amongst

themselves and in comparison to soil humic acids. The surfactant properties of

humic acids were investigated by determining the effect of increasing concen-

tration of humic acids on the surface tension of water. The study demonstrated

that the humic acids extracted from shilajit indeed possessed surfactant properties.

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CHARACTERIZATION OF HUMIC ACID OF SHILAJIT 421

Acknowledgements. Our thanks go to the Hamdard National Foundation, Jamia Ham-

dard, New Delhi, for a fellowship and the financial aid given for the project. The authors are

also grateful to Dr. G. N. Qazi, vice chancellor, Jamia Hamdard, for providing the facilities.

ИЗВОД

ХУМИНСКА КИСЕЛИНА ИЗ ШИЛAЏИТА – ФИЗИЧКО–ХЕМИЈСКА И

СПЕКТРОСКОПСКА КАРАКТЕРИЗАЦИЈА

SURAJ P. AGARWAL1, M. D. KHALID ANWER1,3, RAJESH KHANNA2, ASGAR ALI1 и YASMIN SULTANA1

1Dept. of Pharmaceutics, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi-110062,

2Dabur Research Foundation, 22, Site IV, Sahibabad, Ghaziabad,Uttar Pradesh-201010, India и

3College of Pharmacy, Al-kharj, King Saud University, K.S.A

Шилаџит је црно-мрк ексудат, који се састоји од органских супстанци, металних јона и

минерала, различитог састава. Широко је распрострањен у хималајској регији (на висинама

1000–3000 m) од Непала до Кашмира. Шилаџит се такође може наћи у планинским регијама

Авганистана, Бутана, Кине, Бајкала, као и на Уралу, Кавказу и Алтају, на висинама између

1000 и 5000 m. Основна физиолошка активност шилаџита приписана је присуству биоак-

тивних дибензо-

-пирона поред хуминске и фулвинске киселине, које делују као носећи

молекули активних састојака. У овом раду, циљ је био да се екстрахују хуминске киселине

из шилаџита различитог порекла и да се оне окарактеришу на основу физичко–хемијских

својстава, елементалне анализе, UV/Vis и FTIR спектара, дифрактограма X-зрака и DSC тер-

мограма. Спектралне карактеристике узорака различитог порекла добијене UV/Vis, FTIR и

XRD методама, као и информације добијене DSC техником, показале су очигледну међу-

собну сличност узорака и сличност са хуминским киселинама из земљишта. Испитивана је и

површинска активност екстрахованих фулвинских киселина одређивањем утицаја њихове

концентрације на површински напон воде. Испитивања су показала да су екстраховане

хуминске киселине заиста површински активне.

(Примљено 16. марта, ревидирано 22. јуна 2009)

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Full-text available

Dec 2022

Background: Shilajatu is one among the most used and important drugs in Ayurveda. However, a big percentage of Shilajatu samples available in market is not genuine. The analytical standards of Shilajatu are still not in Ayurvedic pharmacopoeia or any authoritative publication. So, the characterization and standardization of Shilajatu is required. Moreover, Shilajeet is vaguely being used by the layman and even professionals. It’s therapeutic potential needs to be specifically evaluated. Aims: To compare pharmaceutico-analytical characteristics of Shilajatu collected from Himachal Pradesh with that samples obtained from standard commercial sources and their in-vitro antioxidant study. Methodology: The collection of samples from the site near Rampur Bushahr, Himachal Pradesh will be done in favourable season (in the month of April to June and September-October) by personal visits. The samples of Shilajatu from two sites shall be taken for this study. Two market samples shall also be bought from two standard commercial sources. All the samples shall be taken in triplicate to ensure data accuracy. Minimum 4 sources samples in triplicate, so total minimum 12 raw samples shall be taken. Further analysis of raw samples then pharmaceutical processing (Shodhana) and again analysis of processed samples shall be done. In analysis organoleptic, Physico-Chemical, elemental analysis and estimation of Humic acid and Fulvic acid shall be done of minimum 12 Raw samples and 12 processed samples (total minimum 24 samples). In-Vitro antioxidant study shall be done for both sources, processed samples. Observations & Results: The observations noted while collection, analysis, processing of all samples shall be documented and presented in the form of data, photographs, tables, chart, etc. as applicable. Further the processed Shuddha Shilajeet shall be subjected to an In-Vitro antioxidant study. Findings will be analyzed for interpretation of results. Conclusion: A comparative data of samples of Shilajatu, collected from site of natural occurrence and standard commercial sources, with respect to pharmaceutical work, analysis and In-Vitro antioxidant study will be generated.

Review on Shilajit Used as Pakistani Traditional Medicine

Article

Full-text available

Nov 2021

Shilajit contains a blackish-brown exudation and a mineral-rich complex organic compound. Its source can be obtained from mountainous ranges of the world, where the hilly tribes first identified its beneficial use such as the Himalayan region from Gilgit to Skardu in Pakistan. This review article focuses on the potential applications of shilajit used in Pakistan’s traditional medicine. The major physiological action of Shilajit has been attributed to the presence of bioactive dibenzo-α-pyrones (DBPs) along with fulvic acids (FA) and humic acid (HA), which act as carrier molecules for the active ingredients. For many years, shilajit is extensively used as a part of the ayurvedic drug for the treatment of various ailments such as anaemia, viral infection, diabetes, wound healing, liver disability and allergic disorders. Also, shilajit can settle the body’s immune system because it has anti-inflammatory properties. Keywords: Shilajit; Herbomineral Drug; Fulvic Acid; Traditional Medicine

Biocarbon Meets Carbon—Humic Acid/Graphite Electrodes Formed by Mechanochemistry

Article

Full-text available

Dec 2019

Humic acid (HA) is a biopolymer formed from degraded plants, making it a ubiquitous, renewable, sustainable, and low cost source of biocarbon materials. HA contains abundant functional groups, such as carboxyl-, phenolic/alcoholic hydroxyl-, ketone-, and quinone/hydroquinone (Q/QH2)-groups. The presence of Q/QH2 groups makes HA redox active and, accordingly, HA is a candidate material for energy storage. However, as HA is an electronic insulator, it is essential to combine it with conductive materials in order to enable fabrication of HA electrodes. One of the lowest cost types of conductive materials that can be considered is carbon-based conductors such as graphite. Herein, we develop a facile method allowing the biocarbon to meet carbon; HA (in the form of a sodium salt) is mixed with graphite by a solvent-free mechanochemical method involving ball milling. Few-layer graphene sheets are formed and the HA/graphite mixtures can be used to fabricate HA/graphite hybrid material electrodes. These electrodes exhibit a conductivity of up to 160 S·m−1 and a discharge capacity as large as 20 mAhg−1. Our study demonstrates a novel methodology enabling scalable fabrication of low cost and sustainable organic electrodes for application as supercapacitors.

JOURNAL OF NATURAL REMEDIES

Article

Full-text available

Sep 2024

Shilajeet: Classical Ayurveda Texts to Current Research - A Review

Copper Speciation in Wine Growing-Drain Waters: Mobilization, Transport, and Environmental Diffusion

Article

Full-text available

Jan 2024

Copper (Cu) has been used to treat vines for a long time, which has led to its accumulation in vineyard soils. In the present work, the mobilization of copper from these soils and its transport, and diffusion outside the plots by drain water were investigated. For this, the distribution of copper between the dissolved and colloidal phases, and within the colloidal phase, of these waters was determined using an investigation strategy based on the coupling between a size separation technique, asymmetric flow field-flow fractionation, and several detectors. First, the total copper concentrations in water from different drains were monitored over a period of 2 years: Cu was mainly found in the fraction of < 450 nm. Then, the distribution of copper on the size continuum was more closely studied in water from one of the drains, sampled over a winter period. Between 45 and 75% of Cu was found in the 2–450 nm colloidal fraction. The <450 nm colloidal phase of the drain waters was found to be mainly composed of humic acids (~15 to 60 mg L−1) and clay-rich particles (~100 to 650 mg (Al) L−1). These particles also contained (hydr)oxides of iron and manganese. The concentrations of Fe and Mn were approximately 100 to 200 times lower than those of Al. The majority of humic acids had an apparent molar mass of ≤ 10 kDa. They were distributed along the size continuum: (i) in a population with an average size of ~20 nm, probably consisting of supramolecular entities, and (ii) associated with clay-rich particles with a size of ~120–200 nm. Copper was found to be complexed with humic acids and associated with clays via clay-humic complexes. Copper mobilization from the soil to the water and its transport to the drain water appeared governed by the soil humidity level and the rainfall.

Characterization and medicinal applications of Karakoram shilajit; angiogenesis activity, antibacterial properties and cytotoxicity

Article

Full-text available

Oct 2023

Shilajit is a natural substance found in the Himalayan region from Nepal to Pakistan. It is a decomposition product of Royle's spurge, white clover, and different species of molds. The decomposition takes place over a time span of centuries by the action of microorganism. In the present study, shilajit samples from four different origins including siachen khaplu shilajit (SKS), kharmang pari saspolo shilajit (KPSS), kharmang ghandus shilajit (KGS), and kharmang shilajit center (KSC) of district Skardu, Pakistan were investigated. These samples were characterized using scanning electron microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), and UV-visible spectroscopy (UV/vis). SEM revealed a notable difference in the shape and size of collected samples. All samples were found to possess crystalline nature, which is confirmed from XRD. The presence of multi-components and complex silicates confirmed the presence of humic substances (HS) in shilajit. A slight disparity in physiological properties of four samples were revealed due to geographical variations and ecological conditions, which determine the natural synthesis of shilajit. All samples exhibited antibacterial effects against Gram negative bacteria; Escherichia coli (E. coli). About 76 %, 98 %, and 100 % of bacteria were killed by SKS, both KPSS and KGS, and KSC samples, respectively. The cell viability analysis revealed that the KPSS (66%) and KGS (53 %) were cyto-compatible as compared to the SKS (23%) and KSC (25%) samples. The Chick Chorionic Allantoic Membrane (CAM) assay was used to observe the angiogenic potential for SKS, KSC, and KGS samples. Hence, shilajit sample could be a potential candidate for the medicinal applications and offer a new approach to biomedical applications.

Characterization of humic acids produced from fungal liquefaction of low-grade Thar coal

Article

May 2021
PROCESS BIOCHEM

Fungal liquefaction of coal is a promising technology to convert low rank coal into value-added products such as humic acids (HAs) that are extensively used as fertilizer. Use of Pakistani Thar lignite for humic acid extraction can promote agriculture industry in Pakistan. In this study, fifty-five fungal strains were isolated from Thar lignite and screened for their lignite solubilization efficiency. Isolates M13 and MI exhibited highest capacity for lignite solubilization and HA was extracted with 67.4 ± 4.3 percent (w/w) yield. Elemental analysis showed increased nitrogen (0.72–1.39 percent) and oxygen (41.7–48 percent), and decreased carbon (51.8–46.1 percent) and sulphur (1.18–0.73 percent) contents in bio-extracted HA (bHA) compared to chemically extracted HA (cHA). UV/Vis spectroscopy showed increased E4/E6 value from 5.6 ± 0.39 (cHA) to 6.28 ± 0.35 (bHA) indicating reduced molecular mass and aromaticity and increased bioactivity of molecule. FTIR analysis also confirmed these results. HPLC analysis of bHA and cHA showed typical choromatogram of humic acids. Enzyme activity staining indicated the presence of fungal cellulase and xylanase on SDS-PAGE gel. The degradation of lignite macromolecules by lignin degrading fungi is a complex process dependent on alkaline substances, chelators, surfactants and extracellular fungal enzymes.

Hazardous or Advantageous: Uncovering the Roles of Heavy Metals and Humic Substances in Shilajit (Phyto-mineral) with Emphasis on Heavy Metals Toxicity and Their Detoxification Mechanisms

Article

Full-text available

Feb 2024
BIOL TRACE ELEM RES

Shilajit is a phyto-mineral diffusion and semi-solid matter used as traditional medicine with extraordinary health benefits. This study provides a comprehensive data on Shilajit with emphasis on heavy metal profile, associated toxicities, and metal detoxification mechanisms by humic substances present in Shilajit. Data was searched across papers and traditional books using Google Scholar, PubMed, Science Direct, Medline, SciELO, Web of Science, and Scopus as key scientific databases. Findings showed that Shilajit is distributed in almost 20 regions of the world with uses against 20 health problems as traditional medicine. With various humic substances, almost 11 biological activities were reported in Shilajit. This phyto-mineral diffusion possesses around 65 heavy metals including the toxic heavy metals like Cu, Al, Pb, As, Cd, and Hg. However, humic substances in Shilajit actively detoxify around 12 heavy metals. The recommended levels of heavy metals by WHO and FDA in herbal drugs is 0.20 and 0.30 ppm for Cd, 1 ppm for Hg, 10.00 ppm for As and Pb, 20 ppm for Cu, and 50 ppm for Zn. The levels of reported metals in Shilajit were found to be lower than the permissible limits set by WHO and FDA, except in few studies where exceeded levels were reported. Shilajit consumption without knowing permissible levels of metals is not safe and could pose serious health problems. Although the humic substances and few metals in Shilajit are beneficial in terms of chelating toxic heavy metals, the data on metal detoxification still needs to be clarified.

The effects of mumie extract on cell proliferation and enzyme expression of human osteoblast-like cells (MG63)

Article

Full-text available

Sep 2019

Background: Mumie, as an inorganic and semi-solid herbal substance, could be obtained from crevice caves and is used for bone diseases in traditional medicine. This study investigated the effects of this substance on the expression of bone alkaline phosphatase (BALP) enzyme as well as proliferation and mortality rates of MG63 human osteoblast-like cells. Materials and methods: The MG63 cells were cultured and the effect of 100, 200 and 300 μg/ml of mumie extract on cell viability were compared with zoledronic acid and estradiol valerate as positive controls, as well as with MG63 cells alone as the negative control group. The activity rate of the BALP enzyme was also assessed. Results: During 48 hours of the study period, the concentrations of 100 and 200μg/ml of mumie extract increased the proliferation rate and decreased the mortality rate of MG63 cells significantly; however, the concentration of 300μg/ml decreased the proliferation rate and increased the mortality rate of the cells. Also, BALP enzyme expression was slightly affected by 100 and 200 μg/ml of mumie extract whilst it was significantly decreased by the concentration of 300 μg/ml. Conclusion: This study showed that mumie extract has an increasing effect on proliferation rate and a decreasing effect on the mortality rate of osteoblast cells in low concentrations; however, the higher concentrations of this substance could be toxic and effect inversely

Physico-chemical, spectral and thermal characterization of shilajit, a humic substance with medicinal properties

Article

Full-text available

Jan 2008

In the present study, shilajit samples from various sources were characterized based on their physico-chemical properties, UV, FTIR and 1H NMR spectra, X-ray diffraction pattern and DSC thermograms. All the samples showed a distinct similarity with respect to their physico-chemical as well as spectral and thermal characteristics. Being natural in origin, some variation can be expected and was seen in these samples too. The studies confirmed the complex multi-component and humic nature of shilajit.

Glass transition and crystallite melting in natural organic matter

Article

Full-text available

Oct 2005
ORG GEOCHEM

The presence of (CH2)n crystalline components in lipid extracts from soil and peat was detected by solid-state NMR and X-ray diffraction. The lipid fractions were extracted from geosorbents including the IHSS Pahokee peat and leonardite, a second peat and its humic acid. The existence of two peaks in the 30–33 ppm NMR shift region demonstrates the presence of semicrystalline carbon in the lipid fraction, presumably in long aliphatic chains. X-ray diffraction patterns of the lipid fractions are similar to polyethylene, with the strongest reflections at 4.13 and 3.72 Å. The melting temperatures of the lipid fractions vary among the geosorbents and are in the same temperature interval with the glass transition reported in the literature for the IHSS peat and leonardite. After removing the fraction containing the crystalline components, the leonardite and Guanella Pass peat humic acid do not show any thermal events in their respective DSC thermograms. This finding suggests that, at least for the systems studied, the reported glass transition was actually the melting of the crystallites present in the lipid fraction.

Mast cell protecting effects of shilajit and its constituents

Article

Jan 1989

The effects of shilajit and the combined effects of its main constituents, fulvic acids (FAs), 4'-methoxy-6-carbomethoxybiphenyl (MCB) and 3,8-dihydroxy-dibenzo-α-pyrone (DDP), were studied in relation to the degranulation and disruption of mast cells against noxious stumuli. Shilajit and different combinations of FAs. MCB and DDP provided statistically significant protection to antigen-induced degranulation of sensitized mast cells, markedly inhibited the antigen-induced spasm of sensitized guinea-pig ileum, and prevented mast cell disruption induced by compound 48/80. The findings are appraised in view of the clinical use of shilajit in the treatment of allergic disorders in Ayurvedic medicine.

Humus, the epitome of Ayurvedio makshika

Article

Jan 1997

Ayurvedio makshika, a maharasa (rejuvenator, adaptogen), has been shown to be constituted of a large number of low Mr (mol. wt) humio intermediates, and medium and high Mr humio compounds. These results dispel a long standing misbelief that the bioactive ingredients of makshika constitute only inorganic minerals, viz. iron and chalco-pyrites. The stability of the makshika-humus core (str 5) appears to be due to complexation with transition metal ions which produce resonance stabilised rnetallo-organic species (str 6a,b and 7). The low Mr organic compounds of makshika, in their natural habitats, find ecological niche within the micropores of humus and thereby fend off weathering and other extranuous onslaughts for ages. Humus seems to be not one but of all maharasas̀ epitome. The general features of makshika and shilajit are compared in the light of their origin and biological significance.

Enhancement of the dissolution and diuretic effect of furosemide through a novel complexation with humic acid extracted from shilajit

Article

Nov 2007

Furosemide, a thiazide diuretic exhibits extremely low aqueous solubility. This study investigated the effects of complexation of furosaemide with humic acid extracted from shilajit on release rate and in vivo diuretic effect on male Wistar rats. Solid complexes of furosemide with humic acid extracted from shilajit were prepared by solvent evaporation and freeze drying methods in the molar ratio 1:1 and 1:2 (furosemide: humic acid). The complexes were characterized by differential scanning calorimetry, fourier transform infrared spectroscopy and scanning electron microscopy. The comparative release study of furosemide and complexes were carried out in phosphate buffer of pH 5.8. Solvent evaporated and freeze dried complex showed significant improvement in release rate as compared to pure furosemide. Maximum release was observed by the freeze dried complex in the molar ratio 1:2. The optimized complex (1:2 freeze dried) showed significant increase in diuresis in male Wistar rats as compared to pure furosemide. This study confirms that humic acid have a potential to increase the bioavailability of low bioavailable drugs.

Chemical Studies on a Nepalese Panacea - Shilajit (I)

Article

Jan 1987

Shilajit is an asphalt-like substance found embedded in rocky sediments in the Himalayas in western Nepal at altitudes between 2500-5000 m. It is popularly used in Nepal as a tonic. Chemical analysis of shilajit revealed that two-thirds by weight of this medicinal material was extractable by warm 50% alcohol. Repeated crystallization of the hydroalcoholic extract has led to the isolation of crystals, which were subsequently identified as calcium benzoate. The antiseptic properties of benzoates may account for the antiseptic effects of shilajit in places where hygiene remains at a low level.

X-ray diffraction studies on the crystallinity and molecular weight of humic acids

Article

Aug 1971
SOIL BIOL BIOCHEM

Humic acids of different origins were investigated for crystallinity by means of X-ray diffraction techniques. Only the humic acid formed in a culture of Aspergillus flavus on a modified Czapek-Dox medium which contained 0·1 % phthalate was crystalline as found by electron microscopy and confirmed by X-ray diffraction techniques.Evidence showed that during the process of drying a rearrangement takes place in the humic acid which results in an ordered arrangement of the lattice structure. The nine lines of the powder diagram could be interpreted on the basis of a hexagonal unit cell with the dimensions a = 13·5 Å and c = 10·9 Å.On the strength of these findings a minimum molecular weight of M = 1393 was arrived at which is in good agreement with a series of molecular weights published for humic acids. By taking into account the effect of solvation a particle weight of 26,700 was calculated for humic acids suspended in water.

Humic acids from lignite. 1. Analytical characteristics and thermal degradation

Article

Oct 1975
FUEL

Francisco Martin

Humic acids (HA) extracted from a lignite with two alkaline reagents have been studied. In contrast to humic acids extracted from soil HA, the lignite acids show no differences in their physical and chemical analyses with the exception of the total carbonyl content. After acid hydrolysis the analytical characteristics do not change, but their behaviour on Sephadex indicates a molecular condensation. The parent material has similar characteristics to those reported in the literature and also shows no analytical differences after acid hydrolysis, but a number of i.r. absorption bands, due to kaolinite, the only clay mineral present, disappear after this treatment. By pyrolysis/gas chromatography both the lignite and the humic acids produce several low-b.p. compounds, the same in the three cases but in different amounts. By methylation it has been proved that a great part of such compounds are produced by radical reactions. Some, which do not increase on methylation, may indicate the presence of some longer chains in lignites.

Characteristics of humic substances in heathland and forested peat soils of the Wicklow Mountains

Article

Jan 2001

Humic substances are an extremely important soil component because they constitute a stable fraction of carbon and improve water-holding capacity, pH buffering and thermal insulation. To understand the role of humic substances (HS) in carbon dynamics, it is necessary to evaluate how concentrations and characteristics vary with location. To this end, we extracted, fractionated, quantified and characterised HS from six peat and peaty mineral soils from the Vartry catchment. The extraction procedure was an adaptation of the International Humic Substance Society method. We then isolated samples using XAD-8 resin columns and dialysis analysed them for water, ash, elements (carbon, hydrogen, nitrogen, oxygen), amino acids and carbohydrates, and by Fourier- transform infrared spectroscopy. Although the characterisation of the HS could not reveal details of chemical processes, we were able to identify a number of characteristics that pointed to differences between forested and non-forested sites. In general, the HS at forested sites indicated more humified organic matter and quite different decomposition processes. The results indicate a need to extend research into HS to allow a more detailed evaluation of the effect of forestation on soil organic matter dynamics.

The core structure of Shilajit humus

Article

Dec 1991
SOIL BIOL BIOCHEM

The nature of the building blocks and their alignments in the humus 'core' of shilajit were determined by mild and drastic degradations and by comprehensive spectroscopic analyses of the products. Mild hydrolysis of humic acids (HAs) from shilajit afforded two new dibenzo-alpha-pyrones, viz. 3-O-palmitoyl-8-hydroxydibenzo-alpha-pyrone (1) and 3-O-beta-D-glucosyl-8-hydroxydibenzo-alpha-pyrone (2), and two new tirucallane-type triterpenic acids, viz. 24(Z)-3-beta-hydroxy-tirucalla-8,24-dien-26-oic acid (3) and 24(Z)-3-beta-hydroxy-tirucalla-7,24-dien-26-oic acid (4). The resistant HAs (RHAs), obtained after mild hydrolysis, when subjected, separately, to KMnO4 oxidation and Zn dust distillation gave several aromatic carboxylic acids, polynuclear aromatic hydrocarbons, a simple dibenzo-alpha-pyrone (= 3,4-benzo-coumarin) and fluorene. These products, except the two last-named compounds, have been reported from similar degradations of soil-sediment humus indicating the inherent structural similarities of humus from two dissimilar sources. On the basis of the above and related observations, a partial structure of the shilajit humus core, involving oxygenated dibenzo-alpha-pyrones, is postulated. Additionally, the necessity of standardization of shilajit, a panacea in oriental medicine, on the basis of its active principles and carrier molecules (e.g. fulvic acids, FAs) is suggested.