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

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.


Introduction
The Himalayas and Hindukush of the subcontinent contain shilajit which is a rich source of humic substances (HS) extracted from rocks in many parts of the world.Shilajit is also known as Asphaltum Punjabianum and is used in traditional medicine in different countries including Pakistan, Russia, India, Afghanistan, and Nepal etc as a refreshing and revitalizing agent.It is formed from plants and organic compounds that have been compressed by the layers of rocks at a high temperature and pressure under the action of microorganisms The remaining 20%-40% of shilajit are various kinds of mineral matter, and 5% are trace elements [1][2][3].Shilajit, its primary components, and prospective applications based on fulvic acid characteristics.Shilajit is a phytocomplex made up primarily of humic compounds.One of these, fulvic acid, is well known for its antiinflammatory, antioxidant, and memory-enhancing qualities.Primarily it is composed of humus (60%-80%) along with other components such as benzoic acid, hippuric acid, fatty acid, sterol, albuminoids, aromatic carboxylic acid, amino acids, ellagic acid, resin and waxy materials [4,5].Similarly, shilajit typically constitutes HS in which the fulvic acid acts as the bioactive component that is known to have immunomodulatory and psychoactive properties [6].
Shilajit has been used as an ancient medicine in different regions of the world under the indigenous systems and it possesses the ability to resorb tumours and pimples reported in Avicenna in Canon Medicinae 4. The therapeutic effect of shilajit is reported for the treatment of peptic ulcer, as an anti-inflammatory and antioxidant, anti-arthritic, cancer treatment, memory improving, and neuroprotective compound [7][8][9][10].Two different types of chemicals are responsible for the biological effects of shilajit.The first group consists of low molecular weight, bioactive organic compounds such as oxygenated dibenzo-α-pyrones (DBPs) as major entities, phenolic lipids, tirucallane triterpenes, and small tannoids (such as conjugate procyanidins and gallotannoids).Second group is the medium molecular weight compound such as fulvic (15%-20%), which acts as carrier molecules to the bioactive substances, therefore, significant changes in native shilajit's biological effects, i.e. the way that this health supplement is often marketed.In shilajit samples collected from various locations, qualitative and quantitative differences of both groups were observed [1].
Traditional herbo-medicine like shilajit is gaining more attention and interest due to its effective therapeutic effects in the treatment of common diseases like anaemia, diabetes, neuroprotective agents for alzhemimer's diseases, chronic pain, osteoporosis, and digestive issues [11].The antibacterial effects and cytotoxic potential were also reported [12].The difference in the chemical nature of shilajit from different regions of the world is responsible for its varying physico-chemical properties and its efficacy.The chemical composition of shilajit is affected by the ecological makeup of the mountain's rocks, variations in local humidity, temperature and plant species, for which organic chemicals such as fulvic acid, 3,4-benzocoumarins, resin, fatty acid, and benzoic acid are responsible.Shilajit extract contains a variety of organic components in variable amounts depending on the country, which affects the chemical and physical composition [12,13].
However, the physico-chemical characterization of Karakoram shilajit from different origins have not been reported in literature.Therefore, the present study deals with the determination of physico-chemical properties and spectral characterization of Karakoram shilajit from four different sites of district Skardu, Pakistan.Furthermore, detailed investigation with Osteoblast-like cell and angiogenic potential was revealed.Thus, opening new frontiers for the shilajit based composite fro biomedical applications.As the SKS and KSC exudation in the coldest place compared to KGS and KPSS, all the samples showed slight variation in their chemical compositions.Owing to the unique chemical and structural properties of shilajit, it may be useful for medicinal applications.

Purification of raw shilajit
Small rocks of raw shilajit were purchased from four different commercial sources i.e., siachen khaplu shilajit (SKS), kharmang pari saspolo shilajit (KPSS), kharmang ghandus shilajit (KGS), and kharmang shilajit center (KSC) of district Skardu, Pakistan.Shilajit extraction was done by some modifications.Briefly, shilajit was washed with hydrofluoric acid by centrifugation at 9000 g-force for 10 min.Following this, shilajit was washed twice with ethanol after discarding supernatant.Subsequently, pure shilajit was collected and dried for preliminary characterization.

Preliminary characterization
The morphology of samples was determined by field emission scanning electron microscopy coupled with energy dispersive spectroscopy (FESEM-EDS: Tescan-MIRA III).X-ray diffraction (XRD: Panalytical-PW3719) analysis of powdered samples was carried out at 2θ range of 20°-80°(step size: 0.02) using CuKα radiation.The ultraviolet-visible spectroscopy (UV-vis: Shimadzu-601) was performed by dissolving the shilajit in deionized water and recording the spectrum in a 1 cm 3 quartz cuvette by scanning from 200 to 400 nm.To determine functional groups in each sample, fourier transform infrared spectroscopy (FTIR: Nicolet Summit LITE) in a transmittance mode was done in the range of 4000-400 cm −1 with a scanned speed of 4 m s −1 .

Antibacterial activity
Antibacterial efficacy of the samples was checked against pathogenic Staphylococcus aureus (S. aureus) and Escherichia Coli (E.coli).Samples were UV sterilized for 1 h before test.Bacterial strains were cultured in sterilized nutrient broth media for 24 h at 37 °C and OD was set as 0.015 at 600 nm [14].20 mg of shilajit powder of each sample added in 2 ml bacterial culture and incubated at 37 °C.After 24 h, OD 600 of test samples and control was measured to determine the antibacterial efficacy of the sample.The test was performed in triplicate.

Cell viability
In-vitro cyto-compatibility assessment of shilajit was done using stem cells via WST-8 assay (Sigma-Aldrich).
The study was carried out using indirect assay.Mesenchymal stem cells were cultured at 37 °C in Dulbecco's modified eagle's medium (DMEM; Gibco) supplemented with 10 vol% fetal bovine serum (FBS; Sigma-Aldrich) and 1 vol% penicillin-streptomycin (Pen Strep; Sigma-Aldrich) in a humidified 5% CO 2 atmosphere.Mesenchymal stem cells Stem cells were obtained from the bone marrow of the patient presented at the Bone Marrow Transplant Unit at the CMH Rawalpindi (the extraction procedure was carried out according to the ethical guidelines and approval of the ethical review board at the CMH.Cells were washed with phosphate buffer saline (PBS; Gibco) after incubation and detached from the wall of cell culture flask using Trypsin (Sigma-Aldrich, St Louis, MO).Tissue culture plate was used as a control.Shilajit powders were incubated in DMEM for 24 h and extracts were kept in contact with the cells in a 24 well plate following the procedure mentioned in [15].
After incubation, the medium was removed from the wells, and plate was washed twice with PBS.Later, 1 ml medium containing 1 vol% WST-8 reagent was added in each well and the plate was incubated again at 37 °C for 2 h. 4 × 100 μl supernatant from each well was subsequently transferred into 96-well plate (Corning, Cambridge, MA) and absorbance was recorded at 450 nm on a microplate reader (PHOmo, Autobio Labtec Instruments Co. Ltd).The absorbance value of the control was considered as 100% viability.All measurements were performed in triplicate and average results were reported.

CAM assay
The angiogenic potential of shilajit for biological applications was determined via chorioallantoic membrane (CAM) assay.For this purpose, day 6 fertilized eggs were purchased from 'Punjab Poultry Research Institute, Rawalpindi' and placed in the incubator at 37 °C and 55%-65% of relative humidity.On day 7, eggs were taken out of incubator and 1 × 1 cm 2 of small squares were made on the top of the eggshells with sterilized saw blade.The sterilized sample were implanted inside the eggs and the eggs were closed with parafilm and adhesive tape.Samples were retrieved on day 14 and the eggs were sacrificed.

Physical properties of shilajit
The preliminary inspection revealed that shilajit samples were different shades of brown and black in colour as shown in figure 1 (A, D, G, J) astringent in taste and had a pungent odor.The characteristic odor of shilajit has been attributed to the presence of a number of compounds including benzothiazole, 2-ethylhexanoic acid, pcresol, 2-ethylhexanoic acid, 3-4-ethylphenol, naphthalene, 2,4-bis (1,1-dimethylethyl) phenol, and 2,4dimethylquinoline.The pH of 2% aqueous solutions of SKS, KPSS, KGS, and KSC was obtained as 6.80, 8.72, 5.20, and 8.04, respectively.Similar physio-chemical characteristics were observed from a different origin of shilajit [13].

SEM analysis
FESEM images of the powdered shilajit samples are shown in figure 1(B), (E), (H), (K).The morphologies of shilajit particles exhibited a spongy structure of HS with the presence of internal space.SEM showed SKS, with a round and rough surface of agglomerated particles which is shown in figure 1(B).By contrast, figure 1(E) exhibits perfectly spherical-shaped morphology of KPSS nanoparticles.However, KGS particles depicted flakelike shape morphology (figure 1(H)) with agglomeration.The KSC sample (figure 1(K)) had irregular morphology and was also agglomerated due to nano-size.The variation in morphologies of four different samples due to the difference in ecological nature of shilajit.
The EDS reveals that all samples contained Na, Mg, Al, and Si (figure 1(F), (I), (L) except SKS (figure 1(C)).Furthermore, K was present in KGS and KSC (figure 1(I), (L)).Cl and Fe were only detected in KGS (figure 1(I)) whereas KSC exclusively contained Cu and Ag (figure 1(L)).All these elements have their own medicinal significance such as Cu and Ag are antibacterial, and Ca and Mg are healthy for bones etc [16][17][18][19].SEM analysis revealed that these organic materials were affixed to the minerals surfaces.Hence, the multi-elements found in the shilajit samples are all beneficial which supports the traditional use of shilajit in ayurvedic medicine.

XRD analysis
A typical XRD pattern of the rock shilajit samples from different origins are presented in figure 2. The sample largely exhibited a crystalline nature as evident from the presence of sharp diffraction peaks because of different kinds of complex silicates and carbide in its composition.For instance, silicon carbide, iron carbide, calcium carbide, and calcium aluminum silicate etc The vast and amorphous form was corroborated by the shilajit signal generated at 56°.A few peaks were found which could be attributed to the presence of lipid crystallites, clay particles and other micro-crystalline materials in shilajit samples [20].Although all the XRD patterns were relatively similar, there was a slight variation in the peak shifts.The crystallite size of KSC, KPSS, SKS and KGS are listed in the table 1.

UV-vis spectroscopy
The UV-vis spectra of the shilajit samples were studied.For each sample, 0.01 g shilajit was dispersed in 10 ml deionized water and pattern was recorded from 200 to 400 nm as shown in figure 3. The samples did not exhibit any sharp maxima, and the absorbance value decreased with increasing wavelength.A slight hump of four different samples appeared in a range of 220-290 nm, which is the characteristic of HS.The absorption peaks at 240-290 nm, indicating the presence of conjugated carbonyl groups with two double bonds in the humic acid molecules [21].However, the slight variation of hump of the SKS sample from KPSS, KGS, and KSC could be   attributed to different concentrations of aromatic compounds.It also represented the hydrazine carbonyl groups, unsaturated thiol, and aldehyde [22].

FTIR analysis
FTIR spectra of the samples were characterized by relatively few sharp bands as shown in Figure 4.This is expected since shilajit consists of a complex mixture of diverse materials.Indicating an extensive work done on the HS derived from organic fraction of soil, coal and peat etc, the sharp band of KGS, KPSS, KSC, and SKS were observed between 3671-3682 cm −1 can be attributed to hydrogen bond associated -OH stretching or -NH stretching [23].However, the bands in the range of 2902-2976 cm −1 can be attributed to the bending vibration of aliphatic C-H stretches.FTIR peaks observed between 1049-1234 cm −1 may correspond to the bending vibration of C-O stretches of alcohol, phenol and OH bending deformation of the carboxyl group, respectively.FTIR frequency range and functional groups present in the samples are mentioned in table 2. The spectra reflect the prevalence of oxygen-containing functional groups such as OH, COOH, and C-O [21,24].

Antibacterial analysis
Antibacterial activity of shilajit samples was checked via OD 600 measurement against two bacterial strains, Gram positive (Staphylococcus aureus; S aureus) and Gram negative (Escherichia coli, E.coli).Testing on these two types of bacterial strains provides a better understanding of the effectiveness of the antibacterial agent against different bacterial cell wall structures.In addition, these both strains are readily available in culture collections and laboratories, making them easily accessible for research purposes.20 mg of each powder was incubated with 2 ml nutrient broth inoculated with S. aureus and E.coli, and OD 600 was set at 0.015 before incubation [27].After 24 H OD 600 was measured.All samples showed a remarkable decrease in OD value after being treated with E.coli, but no antibacterial activity was shown by shilajit samples against S.aureus.The results are presented in the form of bacteria killing percentage in figure 5. Experiment was carried out in triplicate for both bacterial strains and the average % value indicate that 76% of bacteria were killed by SKS sample, 98% of bacteria were killed by both KPSS and KGS samples while the best result was shown by KSC sample which completely inhibited the growth of bacteria as presented in figure 5.The 100% bacterial growth inhibition of the KSC sample could be attributed to the presence of Ag and Cu in its composition as detected in EDS (figure 1(L)).Hence, the findings of present study indicate that shilajit exhibits an appreciable antibacterial potential against E.coli which could be associated to the presence of fulvic and benzoic acidic components in shilajit.These components interact with the cell membrane and eventually lead to the cell-lysis.Moreover, Shilajit is believed to have immunomodulatory effects, that it can enhance the ability of immune system to combat bacterial infections [28][29][30].Some compounds in shilajit interfere with biofilm formation or they disrupt existing biofilms.However, the difference in percentage of bacteria killing is associated with variation in herbs and plants regional species as previously reported [12].
Shilajit also exhibits antioxidant properties due to the 3-hydroxy-and the 3,8-dihydroxy-DBPs.Moreover, the oxidative stress plays a role in bacterial infections because shilajit has a potential to act against harmful effects of reactive oxygen species.The antioxidant compounds present in shilajit act as scavengers, which neutralize the free radicals.Antioxidants in shilajit can stabilize these highly reactive molecules by donating electrons.Hence, prevent them from causing damage to cellular components [31].The changing concentration and antibacterial effect of fulvic acidic electrolytic value is a second suggested explanation of the analysed shilajit sample's antimicrobial activity.Fulvic acid enhances biomembrane permeability, which can sensitise cell membranes for improved assimilation or absorption of other active ingredients.This effect will interfere with the electrochemical gradient and osmolarity within the microbial cell, disrupt intracellular homeostasis, and cause lysis of the cell.As a polyelectrolyte, fulvic acid is described as 'much electric' [32].
In the case of S.aureus, it is suggested that antibacterial potential of the samples could be investigated by increasing the amount of shilajit powder.In this concern, further purification process of shilajit sample may enhance its antimicrobial properties.The antibacterial study can be expanded by using further strains such as Bacillus subtilis (Grampositive), Pseudomonas aeruginosa, and S. canrnasou [32].

In-vitro cytotoxicity assay
The cell viability was calculated by WST-8 assay.To investigate the viability of Mesenchymal stem cells and osteoblasts in shilajit extracts, WST-8 assay was carried out after 48 h of cultivation.The viability value of the control samples without adding any extracts was taken as a positive control (100% cell viability).After 48 h incubation, the viability of stem cells was determined as shown in the figure 6(a).for MSC stem cells.The KPSS sample showed 66% viable cells and 53% viability was observed for the KGS sample.However, the SKS and KSC samples showed only 23% and 25% cell viability, respectively.Cell viability of Shilajit for Osteoblasts is shown in figure 6(b) for all samples which shown an excellent cell viability having greater than 80%.The one-way analysis of variance (ANOVA) showed that the percentage cell viabilities of shilajit powders were significantly different from control at p < 0.05.
Shilajit has shown greater cell viability for osteoblasts as compared to stem cells.As shilajit is a source of mineral ingredients like Ca and Mg, which play a key role in promoting osteogenesis which induce Ca deposition and promote bone mineralization and osteogenesis [33].Moreover, Shajit has a potential to enhance bone formation through synthesis of nucleic acids, polysaccharides, proteins and hormones as they are needed for cell and tissue growth.Moreover, the findings of present study has revealed, in vitro treatment of osteoblasts with shilajit that enhanced osteoblast proliferation.As the analysis of shilajit in the present study showed that it is a great source of elements such as magnesium and calcium.The results also indicated that shilajit has an abundant potential for promoting osteoblasts proliferation.Thus, it can be further used in bone tissue engineering which is a beneficial approach towards the treatment of bone defects.
3.8.Angiogenic potential CAM assay was performed to study the potential of shilajit of different origins in supporting new blood vessels formation (Figure 7).The yellow circle on SKS, KSC and KGS show the position of the shilajit on the CAM.The KPSS sample spoiled the egg after three days of incubation, hence the egg was discarded and not shown here.It is suggested that the low amount of KPSS samples should be tested on fertilized eggs in future.The maximum number of new blood vessels were observed in the SKS (figure 7(A)) followed by KSC (figure 7(B)).The KGS sample (figure 7(C)) showed that very few blood vessels were formed.The difference in the number of blood vessels is due to the different chemical composition of shilajit based on their geographical location.In the EDX, Cu was detected in the KSC sample (figure 1(L)).Cu ions can enhance the angiogenesis due to its ability to stabilize the expression of hypoxia-inducible factor (HIF-1α) thus enhancing the blood vessels formation [34].Moreover, release of Cu stimulates the transforming growth factor-β (TGF-β) which is vital for the growth of new blood vessels formation [35].Other samples do not contain Cu in their composition; therefore, they did not show effective angiogenic activity.The experiment was carried out in triplicate and the statistical significance in the angiogenic potential of different samples was analyzed using ANOVA as provided in figure 7(D).The analysis revealed that the results were significantly different for SKS, KSC and KGS whereas the best result was obtained for the SKS sample, which showed no significance difference from the control.

Conclusions
The present study of shilajit samples from four different sources of district Skardu was characterized with respect to their physico-chemical, spectral and biological properties.The chemical elucidation of crude shilajit from four geographical sites revealed the presence of HS and several other organic constituents.Additionally, shilajit samples clearly showed the presence of metals/trace elements, with calcium dominating, followed by magnesium, potassium, sodium, iron, and aluminium.The main bioactive in shilajit is fulvic acid and humic acid, a potent organic electrolyte renowned for balancing plant and animal life by raising the electrical potential for cell healing.The antibacterial activity of KSC showed highest bacteria killing compared to KGS, KPSS and SKS.However, the maximum number of new blood vessels were observed in the SKS and KSC.The KPSS sample showed 66% viable cells and 53% viability was observed in KGS.
The further refinement of the chemical constituents of shilajit may enhance their potency in terms of bacterial inhibition, cytotoxic efficacy, and angiogenic potential to promote the creation of unique broadspectrum antibacterial, cytotoxic and angiogenic herbal formulation in the future for medicinal applications.An in vivo study of this herbomineral drug on cancer, and cartilage repair should be further performed.

Figure 2 .
Figure 2. XRD pattern of four different samples depicting crystalline nature.

Figure 4 .
Figure 4. FT-IR spectra of raw shilajit from four different sources.

Table 1 .
Result of crystallites size by XRD.

Table 2 .
FTIR frequency range and functional groups present in the samples.