Energy and Health Benefits of Shilajit

Energy and Health Benefits of Shilajit

12 Energy and Health Benefits of Shilajit Sidney J. Stohs1, Kanhaiya Singh2, Amitava Das2, Sashwati Roy2, Chandan K. Sen2 1 CR EIGH TON UNIVERS ITY SCH...

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12 Energy and Health Benefits of Shilajit Sidney J. Stohs1, Kanhaiya Singh2, Amitava Das2, Sashwati Roy2, Chandan K. Sen2 1 CR EIGH TON UNIVERS ITY SCHOOL OF PHARMACY AND HEALTH PROFESSIONS, OMAHA, NE, U N I TE D ST AT ES ; 2 THE OHIO STATE UNIVERSITY WEXNER MEDI CAL CE NTER, COLUMBUS, OH, UNITED STATES

Introduction Shilajit is a resinous phytomineral exudate found in sedimentary rocks that has an extensive history of use in traditional folk medicine, including Ayurveda. It is a brown to black product that is extruded from layers of rocks in mountainous regions during the hottest months of the year. It is known by a variety of other names, including mumie, moomiyo, mummiyo, mumijo, silajatu, and salajeet. It is obtained from various mountainous regions of India, Tibet, China, Russia, Afghanistan, Nepal, and the former USSR (Caucasus, Ural, Altai, Sayan, Kazakhstan, Uzbekistan, Baykal, and Tajikistan) (Schepetkin et al., 2002; Agarwal et al., 2007; Stohs, 2014). Shilajit has had many applications in folk medicine with numerous anecdotal reports of therapeutic efficacy. Although there have been a limited number of well-designed, placebo-controlled human and animal studies establishing efficacy, various studies have confirmed the safety and efficacy of shilajit. Shilajit, as the proprietary product PrimaVie, received GRAS (generally recognized as safe) status in 2015. Shilajit has been used as an adaptogen and anabolic, and has been known for promoting both physical and mental energy (Acharya et al., 1988; Schepetkin et al., 2002; Ghosal, 2006; Agarwal et al., 2007; Wilson et al., 2011; Stohs, 2014). In the former USSR, it was used surreptitiously for many years to enhance performance of Olympic athletes and special military forces while reducing stress-related injuries and facilitating recovery (Bucci, 2000). Various studies indicate that it possesses antiinflammatory and antioxidant properties and functions as a chemoprotectant and immunomodulator. As a consequence of these broad properties, shilajit has historically been used to treat stomach disorders and ulcers, bone fractures, inflammatory joint conditions, impotence, nerve and cardiovascular disorders, diabetes, wounds, muscle and tendon strains, and urinary tract infections as well as being used to promote physical performance and Sustained Energy for Enhanced Human Functions and Activity. http://dx.doi.org/10.1016/B978-0-12-805413-0.00012-0 Copyright © 2017 Elsevier Inc. All rights reserved.

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energy. It is also used to promote general health and longevity. In the United States, it is used as a dietary supplement, either as a stand-alone product or in combination with other ingredients. This review will summarize the human and animal research supporting these health claims, with emphasis on well-controlled studies conducted and published in the past 10 years.

Chemistry The chemical composition of shilajit depends upon many environmental and geographical factors as well as whether the product has been processed and purified (Stohs, 2014; Raju, 2012; Agarwal et al., 2007; Ghosal, 2006; Schepetkin et al., 2003). The fulvic acid (Fig. 12.1) content has been shown to vary with the country and region of origin (Ghosal et al., 1991). Fulvic acid is a polyhydroxy polycarboxylic acid produced by the biodegradation of organic matter and has high chelating and complexing ability. High-quality products used in dietary supplements are standardized to contain at least 50% fulvic acids and equivalents (polymers and related structures) along with dibenzoa-pyrones (DBPs) and DBP chromoproteins. DBPs and DBP chromoproteins are usually present in greater than 10% (Raju, 2012; Stohs, 2014). More recently, five new diterpenoids, referred to as mumic acids AeE, have been isolated and structurally characterized using spectroscopic data and chemical derivatization (Kiren et al., 2014). High-quality products used in dietary supplements should have a water-soluble extraction value greater than 80%. As many as 40 or more total minerals have been reported in a polyphenolic complex in shilajit, with the majority of these in small or trace amounts (Frolova and Kiseleva, 1996). In processed shilajit, the sum of potassium, calcium, and magnesium generally make up over 90% of the total mineral content, with sulfur and sodium being the next most common minerals (Raju, 2012). Variations in color of shilajit are generally due to differences in the content of minerals such as iron, copper, and silver (Ghosal, 2006; Agarwal et al., 2007). OH OH

COOH

COOH

HOOC O OH HOOC COOH

OH

COOH O

FIGURE 12.1 Fulvic acid.

Chapter 12  Energy and Health Benefits of Shilajit 189

The physiological and pharmacological effects of shilajit are attributed to the DBPs, DBP chromoproteins (DBPs conjugated to proteins), fulvic acid, and various polymeric forms of fulvic acid (Ghosal, 2006; Sharma et al., 2003; Schepetkin et al., 2003; Raju, 2012). Some individuals believed that the primary effects of shilajit were due to the ability of fulvic acid constituents to chelate the minerals associated with the product and facilitate cellular penetration (Agarwal et al., 2007; Carrasco-Gallardo et al., 2012). The overall mineral content of shilajit is small. At the doses given, it is doubtful that significant amounts of minerals are absorbed and penetrate cells, since the vast majority of minerals that are present occur in exceedingly small amounts. For example, in a typical shilajit dose of 200 mg, the total mineral content will be 2e3 mg, with about 90% being potassium, calcium, and magnesium. To put this in perspective, the typical daily recommended intake for calcium is 1000e1200 mg, whereas the daily values for magnesium and potassium are 400 mg and 3000 mg, respectively. Because of the relative rarity of the material, its overall complex nature, the processing required to prepare the final product, and the difficulty in standardizing the finished product, counterfeiting and adulteration are major problems (Schepetkin et al., 2003; personal experience of author). As a consequence, consumers are cautioned to use products from known and reputable manufacturers and suppliers.

Safety Studies Various studies in animals and humans have demonstrated the safety of shilajit, which has led to its receiving a self-affirmed GRAS designation. The acute lethal dose at which 50% of animals die (LD50) of a purified shilajit in rats was found to be 1000 mg/kg when given intraperitoneally (IP) and was greater than 2000 mg/kg when shilajit was given orally (Acharya et al., 1988). No internal organ histological or morphological changes were observed in rabbits or mice given 100 and 500 mg/kg shilajit (mumie) orally in water for 30 days (Kelginbaev et al., 1973). Shilajit (mumie) was given to rats at doses of 200 and 1000 mg/kg for 90 days (subchronic toxicity study) and produced no adverse effects on liver, kidneys, heart, blood cells, or nervous and endocrine systems (Anisimov and Shakirzyanova, 1982). Furthermore, shilajit (mumie) did not cause any embryotoxic or teratogenic effects in pregnant rats (Anisimov and Shakirzyanova, 1982) or mice (Al-Hamaidi et al., 2003). The LD50 of fulvic acids, which had been isolated from shilajit, was 1268 mg/kg when given orally to rats (Ghosal, 2006), indicating low toxicity. In an unpublished toxicity study (Raju, 2012), “purified” (processed and standardized) shilajit administered to rats at doses of 200 mg/kg and 400 mg/kg orally for 90 days did not produce any hepatic, renal, hemopoietic, or behavioral effects, and at an oral dose of 2000 mg/kg was well tolerated. In addition, no significant changes in weights of vital organs were observed as compared to control animals. Furthermore, doses of 10, 30, and 100 mg/kg of processed shilajit given to mice did not produce any metaphase

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chromosomal aberrations in bone marrow (Raju, 2012), indicating that the shilajit was not genotoxic. In a placebo-controlled, double-blind, randomized study in arthritic dogs, the twice daily administration of 500 mg purified shilajit for 5 months resulted in no changes in physical parameters or serum biomarkers (Lawley et al., 2013). Biomarkers of liver [bilirubin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT)], kidney (blood urea nitrogen and creatinine), heart, and muscle functions (creatine kinase) were assessed, while physical parameters measured included heart rate, body temperature, respiration rate, and body weight. Velmurugan et al. (2012) evaluated the safety of shilajit by administering groups of rats 500, 2500, or 5000 mg/kg daily for 90 days. The histology of all organs was normal except for what the authors referred to as “negligible changes” in liver and intestine at the highest dose. The weights of all organs were normal as compared to the control animals. Several studies have examined the human safety of shilajit. In a placebo-controlled study involving 20 healthy subjects given 2000 mg of processed shilajit daily for 45 days in capsule form, no significant changes in blood pressure, heart rate, or body weight were observed (Sharma et al., 2003). In addition, shilajit had no effect on blood glucose, urea, creatinine, uric acid, total protein, albumin, albumin/globulin ratio, alkaline phosphatase, ALT, or AST. Shilajit exhibited no evidence of systemic toxicity under these conditions. The administration of processed shilajit at a dose of 100 mg twice a day to 28 male subjects for 90 days had no significant effect on renal profile parameters, including urea, albumin, total protein, globulin, uric acid, bilirubin, alkaline phosphatase, ALT or AST (Biswas et al., 2009). Small but significant decreases in fasting blood glucose and creatinine levels were observed in shilajit-treated subjects. The results indicate that under these conditions, shilajit produced no evidence of systemic toxicity. In an unpublished safety study involving 43 healthy human volunteers (Raju, 2012), processed shilajit was given at a dose of 250 mg twice a day for 90 days. No changes in kidney or liver function tests were observed. Shilajit treatment decreased fasting blood sugar, uric acid, and erythrocyte sedimentation rate while increasing percent hemoglobin and platelet count. In a double-blind, placebo-controlled study, Sharma et al. (2003) administered 2000 mg of processed shilajit or placebo per day for 45 days to human subjects. Twenty subjects received the shilajit, whereas 10 subjects received the placebo. Significant decreases in serum cholesterol, low density lipoprotein, very low density lipoprotein, and triglycerides were observed in response to shilajit as compared to the placebo group. Improved antioxidant status in the form of increases in superoxide dismutase, vitamin C, and vitamin E was observed. High density lipoprotein also increased in shilajit-treated subjects. In summary, various studies with shilajit (mumie) in both animals and humans have demonstrated a very high degree of safety.

Chapter 12  Energy and Health Benefits of Shilajit 191

Research Studies For thousands of years, shilajit (moomiyo, mummiyo, and mumie) has been used in folk medicine in India and Northern Asia (Schepetkin et al., 2002; Agarwal et al., 2007; Wilson et al., 2011). It has also been used as a performance-enhancing agent in the former USSR for many years as well as the treatment of various human maladies. Most research regarding moomiyo (mumie, shilajit) involving sports performance in the former USSR has not been published. An ever-increasing number of studies have been conducted in animals that examine the physiological/pharmacological effects and mechanisms of action of shilajit. A growing number of studies in animals and in vitro systems using standardized materials have been conducted. However, the number of peer-reviewed scholarly publications in the scientific literature involving human subjects remains small. Much of the early literature have involved anecdotal reports, poorly controlled studies, studies involving products of unknown composition, and publication of results in obscure journals (Schepetkin et al., 2002; Goshal, 2006; Agarwal et al., 2007; Wilson et al., 2011). This review summarizes published human, animal, and in vitro research studies as well as a number of unpublished research reports involving well-designed studies.

Human Studies A number of human studies have examined the effects of shilajit on energy production, testosterone and spermatogenesis, and muscle adaptation. Biswas et al. (2009) evaluated the spermatogenic activity of shilajit. Thirty-five infertile (oligospermic) male subjects were given 100 mg processed shilajit in capsule form twice a day for 90 days. Significant increases in normal (18.9%) and total (61.4%) sperm count and sperm motility (12.4% e17.4%) were observed in the 28 subjects who completed the study. A significant decrease in semen malondialdehyde levels was also observed, indicating that shilajit exhibited antioxidant activity. Furthermore, in addition to the increase in sperm count, shilajit treatment significantly increased serum testosterone (23.5%) and folliclestimulating hormone (FSH) (9.4%) levels. In a randomized, placebo-controlled, double-blind study involving healthy male subjects (45e55 years), the effects of purified shilajit on serum testosterone levels were examined (Pandit et al., 2016). Seventy-five subjects (38 treated; 37 control) completed the study. Treated subjects received 250 mg shilajit twice daily for 90 days, which resulted in significant increases in serum total testosterone (31.0%), free testosterone (51.1%), and dehydroepiandrosterone (37.3%). No significant changes were observed in the gonadotropic hormones FSH and luteinizing hormone. These results supported and confirmed the beneficial androgenic effects of purified shilajit. An unpublished pilot study involving six healthy human volunteers examined energy production and physical activity (Raju, 2012). The subjects were given 200 mg processed shilajit once daily for 15 days. Treatment with shilajit significantly increased energy

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production and physical exercise (Harvard step test). In addition, the energy production was confirmed based on increases in adenosine triphosphate (ATP), ATP/adenosine diphosphate (ADP) ratio, coenzyme Q10 (CoQ10), total adenine nucleotides, adenylate energy charge, and uric acid levels in whole blood. This study would have benefited from an adequate number of subjects. Nutrition-dependent skeletal muscle adaptation involves the proper control of regulatory processes through an array of gene expression changes. Proper balance between protein synthesis and protein degradation is important in maintaining the skeletal muscle mass in mature individuals (Goodman et al., 2011). Preserving muscle mass during mechanical unloading during orthopedic and related injuries is an important clinical issue, and therapies aimed at increasing the rate of protein synthesis may serve as important interventions. Protein synthesis is the cumulative result of translational efficiency and translational capacity (McCarthy and Esser, 2010). Translational capacity is the amount of protein synthesized from one unit of RNA, while translational capacity is reflected by the amount of ribosomes present per unit of the tissue. A study was conducted to determine the effects of a purified and standardized shilajit in skeletal muscle adaptation in 16 overweight (average BMI of 28.9) individuals (Das et al., 2016). Subjects consumed shilajit at 250 mg orally twice daily after a baseline visit for 8 weeks followed by supplementation for 4 weeks with exercise. Global gene expression profile using Affymetrix GeneChip Human Transcriptome Array 2.0 identified a cluster of 17 extracellular matrixerelated probe sets that were significantly upregulated in muscles following 8 weeks of supplementation as compared to baseline. This cluster of genes included tenascin XB, decorin, myoferlin, collagen (types I, III, V, VI, and XIV), elastin, fibrillin I, and fibronectin I. The upregulation was confirmed by using real time polymerase chain reaction. Supplementation did not alter lipid profiles, blood glucose levels, or muscle damage markers, including creatine kinase and serum myoglobin (Das et al., 2016). The results indicated that supplementation with shilajit promoted skeletal muscle adaptation via upregulation of this set of related genes and therefore may be beneficial as a fitness and sports performance supplement. Data mining through Ingenuity Pathway Analysis tool (IPA) (Ingenuity Systems, www.ingenuity.com) identified novel biological pathways affected by administering shilajit orally in experimental animals. The analysis of generated major pathways suggested that the administration of shilajit improves both the translational efficiency and capacity of the tissue by targeting eukaryotic initiation factor 2 (eIF2) signaling (Correctedelog (P-value) ¼ 9.25), eIF4-p70S6K signaling (Correctedlog (P-value) ¼ 3.61), mechanistic target of rapamycin (mTOR) signaling (Correctedlog (P-value) ¼ 3.09), and regulating cellular junctional proteins (Correctedlog(P-value) ¼ 2.36) (Fig. 12.2, Table 12.1). mTOR signaling is one of the major protein synthesis pathways involved in increasing the translational efficiency of the cells (You et al., 2015). Previous studies have also shown that this increase in protein translation is through the phosphorylation of substrates such as eIF 4E binding protein 1 (4E-BP1) and p70 ribosomal protein S6 kinase (p70S6k), which then

0.15 5.0 0.10 2.5

0.05

RhoGDI Signaling

Breast Cancer Regulation by Stathmin1

Tight Junction Signaling

Remodeling of Epithelial Adherens Junctions

Protein Ubiquitination Pathway

Sertoli Cell-Sertoli Cell Junction Signaling

mTOR Signaling

phagosome maturation

Regulation of eIF4 and p70S6K Signaling

eIF2 Signaling

Threshold 0.0

Ratio

-log(B-H p-value)

0.20

7.5

0.00

FIGURE 12.2 The significant canonical pathways generated from the genes upregulated using Ingenuity Pathway Analysis tool (IPA) (Ingenuity Systems, www.ingenuity.com) having a elog(P value) of 1.3. BeH multiple testing correction. P-value was used as the scoring method for significant pathways sorting.

Table 12.1 Significant Canonical Pathways and Genes Which Were Upregulated After Supplementation With Purified and Standardized Shilajit Using Ingenuity Pathway Analysis Tool (IPA) Ingenuity Canonical Pathways

Llog(BeH P-value)

eIF2 signaling

9.25

Regulation of eIF4 and p70S6K signaling Phagosome maturation

3.61 3.46

mTOR signaling

3.09

Sertoli celleSertoli cell junction signaling Protein ubiquitination pathway

2.72

Remodeling of epithelial adherens junctions Tight junction signaling

2.36

Breast cancer regulation by Stathmin1 RhoGDI signaling

2.72

1.98 1.6 1.38

Molecules Involved RPL11, RAF1, EIF3C, RPLP1, RPS3A, RPS27, EIF4E, RPS4X, RPL27 A, EIF1, RPS20, RPS9, AKT3, RPS2, RPS3, RPL31, RPL34, RPL3, RPS10, EIF2S2, RPL10 A, RPL27, RPL26L1, RPS15 A, RPL13 A, RPSA, RPL38 RAF1, EIF3C, EIF1, RPS20, RPS3A, RPS27, RPS9, PPP2R5B, RPS10, AKT3, RPS15 A, RPS2, RPS3, RPS4X, EIF4E, EIF2S2, RPSA TUBA1B, CTSK, YKT6, HLA-A, PRDX1, TUBB4B, RAB7A, TUBB, ATP6AP1, GPAA1, TSG101, DYNLRB1, ATP6V0A1, TUBB6, CTSB MAPKAP1, EIF3C, RPS20, RPS3A, RPS27, STK11, RPS9, PPP2R5B, RAC1, RPS10, AKT3, RPS15 A, RPS2, RPS3, PRKD3, EIF4E, RPS4X, RPSA TUBA1B, RAF1, TJP2, TUBB4B, ACTB, RAC1, CTNNA1, JAM2, YBX3, NOS3, TUBB, TUBB6, PRKAR1B, PRKACA, AKT3, ACTG1 USP21, FZR1, CRYAB, HLA-A, HSPA9, HSPD1, HSPA8, TRAF6, PSMC1, PSMD10, HSP90AB1, CUL2, UBE2V1, PSMB1, PSMA4, ANAPC5, SMURF2, UBA1, UBC, PSMC3 TUBA1B, TUBB6, TUBB4B, ACTB, CTNNA1, RAB7A, TUBB, ACTG1, MAPRE3 TJP2, YKT6, MYL6, ACTB, PPP2R5B, RAC1, CTNNA1, JAM2, YBX3, GPAA1, PRKAR1B, PRKACA, AKT3, ACTG1 TUBA1B, RAF1, TUBB4B, PPP2R5B, RACK1, RAC1, TUBB, TSG101, GNAI2, CALM1 (includes others), PPP1R10, TUBB6, PRKAR1B, PRKACA, PRKD3 GNAI2, PAK6, CFL1, MYL6, WASF2, ACTB, RACK1, CD44, RAC1, RDX, ARHGDIA, ACTG1, MYL12A

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promote translation initiation by enhancing the formation of the elF4F complex and recruiting the 43S preinitiation complex to the 50 cap of almost all mRNAs (Haghighat et al., 1995; Hara et al., 1997). The rate of protein synthesis is also determined by the number of ribosomes available. Wnt/beta-catenin signaling pathway works independently from the mTOR pathway in the regulation of ribosomal biogenesis (Armstrong and Esser, 2005). The IPA guided network prediction of the genes upregulated in the shilajit group compared to placebo identified several genes involved in protein synthesis pathway (Fig. 12.3, Table 12.2). The upstream trigger (alpha catenin) activates an array of transporter proteins (SNX, S100A6, and TCOF1), translation regulators (YBX, LARP1), and enzymes (MYO19, LYZ, CSTB, and GART), which, in combination, help in the anabolism of structural proteins. In addition, ribosomal proteins (MRPS21, MRPL23, TCOF1, TBRG4, ASAP2, and AP3D1) were also upregulated in shilajit -administered mice compared to the placebo, supporting the fact that shilajit helps in maintaining the structural muscle mass by controlling both translation efficiency and translation capacity. A number of earlier studies that examined the health benefits of mumie (shilajit) in human subjects were published in Russian. These studies examined the effects of mumie on suppurative wounds (Muratova and Shakirov, 1968; Tazhimametov et al., 1987), peripheral nervous system diseases (Koziovskaia, 1968), bone fractures (Kelginbaev et al., 1973) and bone regeneration (Suleimanov, 1972), postoperative trepan cavities of the middle ear (Psakhis and Aizenberg, 1976), and benign prostatic hyperplasia (Andriukhova, 1997).

Animal Studies What may have been the first study to examine the effects of shilajit (mumie) on energy metabolism was conducted in rats (Shvetskii and Vorobeve, 1978). The study demonstrated that shilajit enhanced energy, protein, and nucleic acid metabolism. Bhattacharyya et al. (2009a,b) conducted a series of studies examining the effects of shilajit and its constituent DBPs on mitochondria and energy production. The treatment of mice IP with 20 mg of a mixture of the DBPs resulted in the detection of the DBPs and their redox products in hepatic mitochondria, the site of ATP and energy production (Bhattacharyya et al., 2009a). Furthermore, CoQ10 was augmented in plasma and organs relative to the control animals, and in vitro erythrocyte membrane lipid peroxidation was inhibited when rats were treated orally with 3,8-DBP. The results suggest a mechanism regarding how shilajit supports the energy-synthesizing ability of mitochondria, and provides at least a partial explanation for the physical performance and relief from fatigue reported in response to shilajit. In a subsequent study, Bhattacharyya et al. (2009b) examined the effects of shilajit on energy status in mice. When mice were forced to swim daily for 7 days and received either the placebo or 30 mg shilajit/kg orally per day for the last 4 days, the forced

Chapter 12  Energy and Health Benefits of Shilajit 195

MRPL23 TBRG4 ASAP2

MRPS21 TCOF1 FANCI

AP3D1 GART ANXA2

Cyclin D

FSCN1*

TNFAIP2 CTSB

MYO19

Gsk3

Collagen type I Wnt

LARP1 ILF3

TJP2

Arp2/3 CTNNA1

SHKBP1

SNX9

Alpha catenin

YBX3

LYZ

DCUN1D1

YBX2 ASB3/GPR75-ASB3

S100A6 TSNAX

FXR1

FAM3D DNPEP

FIGURE 12.3 Ingenuity Pathway Analysis tool (IPA)egenerated network of upregulated genes after shilajit supplementation suggesting the proteins involved in increasing translation efficiency and capacity.

swimming exercise resulted in an 82% decrease in muscle ATP levels. However, treatment with shilajit nearly doubled the ATP in muscle of mice forced to swim. Smaller effects of shilajit were observed with respect to ATP in blood and brain. CoQ10 administration resulted in a protection of muscle ATP similar to shilajit. In mice treated with a combination of shilajit and CoQ10, the muscle ATP levels were 2.44 times higher than untreated animals forced to swim. The primary biochemical function of CoQ10 is to aid in mitochondrial synthesis of ATP. The results support the contention that shilajit can increase energy, relieve fatigue, and support endurance. The effects of a processed and standardized shilajit (25, 50, and 100 mg/kg/day for 21 days) on various stress factors in rats forced to swim 15 min per day for 21 days were assessed (Surapaneni et al., 2012). The product contained 0.43% DBPs, 20.45%

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Table 12.2 Names and Cellular Localization of Proteins Involved in Increasing Translation Efficiency and Capacity After Shilajit Supplementation Symbol

Entrez Gene Name

Location

Family

Alpha catenin ANXA2 AP3D1

Alpha catenin Annexin A2 Adaptor-related protein complex 3 delta 1 subunit Actin-related protein complex ArfGAP with SH3 domain, ankyrin repeat and PH domain 2 Ankyrin repeat and SOCS box containing 3 Collagen I Catenin alpha 1 Cathepsin B Cyclin D1 Defective in cullin neddylation 1 domain containing 1 Aspartyl aminopeptidase Family with sequence similarity 3 member D Fanconi anemia complementation group I Fascin actin-bundling protein 1 FMR1 autosomal homolog 1 Phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase Glycogen synthase kinase Interleukin enhancer binding factor 3

Cytoplasm Plasma membrane Cytoplasm

Group Other Transporter

Cytoplasm Nucleus

Complex Other

Cytoplasm Other Plasma Membrane Cytoplasm Nucleus Nucleus

Transcription regulator Complex Other Peptidase Group Other

Cytoplasm Extracellular space

Peptidase Cytokine

Nucleus Cytoplasm Cytoplasm Cytoplasm

Other Other Other Enzyme

Cytoplasm Nucleus Cytoplasm

Group Transcription regulator Translation regulator

Extracellular space Cytoplasm Cytoplasm Cytoplasm Cytoplasm Other Cytoplasm Cytoplasm

Enzyme Other Other Enzyme Transporter Other Transporter Other

Nucleus Plasma membrane Extracellular space Nucleus Extracellular space Cytoplasm Nucleus

Transporter Kinase Other Transporter Group Translation regulator Transcription regulator

Arp2/3 ASAP2 ASB3/GPR75-ASB3 Collagen type I CTNNA1 CTSB Cyclin D DCUN1D1 DNPEP FAM3D FANCI FSCN1 FXR1 GART

Gsk3 ILF3 LARP1 LYZ MRPL23 MRPS21 MYO19 S100A6 SHKBP1 SNX9 TBRG4 TCOF1 TJP2 TNFAIP2 TSNAX Wnt YBX2 YBX3

La ribonucleoprotein domain family, member 1 Lysozyme Mitochondrial ribosomal protein L23 Mitochondrial ribosomal protein S21 Myosin XIX S100 calcium binding protein A6 SH3KBP1 binding protein 1 Sorting nexin 9 Transforming growth factor beta regulator 4 Treacle ribosome biogenesis factor 1 Tight junction protein 2 TNF alpha-induced protein 2 Translin associated factor X Wnt Y-box binding protein 2 Y-box binding protein 3

Chapter 12  Energy and Health Benefits of Shilajit 197

DPB-chromoproteins, and 56.75% fulvic acids. Shilajit reversed the forced swimminge induced increase in immobility, the decrease in climbing behavior, the decrease in plasma corticosterone levels, and the decrease in adrenal gland weight. Shilajit treatment also prevented forced swimmingeinduced mitochondrial dysfunction as evidenced by stabilizing electron transport chain enzymes and mitochondrial membrane potential. In an early study by Visser (1987), fulvic acids were shown to stimulate respiration in rat liver mitochondria and also increased oxidative phosphorylation when present in concentrations between 40 and 360 mg/L. These results are consistent with the previously mentioned animal studies and provide mechanistic information regarding the increased energy and higher ATP levels. Several studies have examined the antiinflammatory effects of shilajit. In a welldesigned series of studies on the effects of shilajit performed in various animals species, Acharya et al. (1988) demonstrated that shilajit at a dose of 200 mg/kg IP exhibited significant analgesic activity as compared to controls using the rat tail flick method. Shilajit, at a dose of 50 mg/kg IP, also decreased carrageenan-induced inflammation in the rat paw by approximately 75%. Shilajit given orally at doses of 50e200 mg/kg twice a day to rats resulted in a significant, dose-dependent decrease in the gastric ulcer index, thereby demonstrating antiulcerogenic activity. Shilajit did not have significant activity with respect to the CNS, blood pressure, or skeletal muscle, and no antihistaminic activity based on studies in dogs, frogs, and guinea pigs. Shilajit exhibited antiulcerogenic and antiinflammatory activity when given to rats at a dose of 100 mg/kg orally twice a day (Goel et al., 1990). An increase in the mucosal barrier was believed to be produced by shilajit based on the decreased gastric ulcer index and increased carbohydrate/protein ratio. In addition, shilajit administration decreased carrageenan-induced acute pedal edema, granuloma pouch, and adjuvant-induced arthritis in rats, indicating significant antiinflammatory activity. Ghosal (2006) suggested that the antiulcerogenic effect of fulvic acids and the biphenyls present in shilajit were due to protection of the gastrointestinal mucosa with less loss of mucosal cells. Shilajit has also been shown to attenuate acetic acid and formalin-induced writhing in mice, thus demonstrating its antiinflammatory activity (Malekzadeh et al., 2015). A dose-dependent increase in the analgesic effects of shilajit was demonstrated at doses of 0.75, 7.5, and 75 mg/kg. No significant differences were observed between 75 and 750 mg/kg shilajit and up to 4 mg morphine or 30 mg sodium diclofenac, which were used as positive controls. The antiinflammatory and antiarthritic effects of shilajit have been studied in moderately arthritic dogs in a randomized, placebo-controlled, double-blind study (Lawley et al., 2013). Ten animals received either 500 mg shilajit twice daily or placebo for 5 months. The animals receiving shilajit showed significant pain reduction by day 60 with maximum reduction in pain by day 150. The authors concluded that shilajit markedly improved the daily life of the animals. The study suffers from the fact that there were only five treated and five control animals.

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Ghosal (2006) summarized a number of early studies on the immunomodulatory effects of processed shilajit, which suggest that shilajit enhances the lytic potential of polymorphonuclear leukocytes. The administration of a 200e600 mg/dose of shilajit to mice resulted in significant morphological and phagocytotic changes in peritoneal macrophages (Baumik et al., 1993; Ghosal et al., 1995), demonstrating its immunomodulatory capabilities. The antiinflammatory effects of shilajit may at least in part be explained by these effects. The effects of shilajit on spermatogenesis and ovogenesis in rats were investigated (Park et al., 2006). Shilajit administration daily for 6 weeks resulted in a significant increase in sperm count. Ovulation was induced in seven out of nine female rats in the shilajit group as opposed to three out of nine in the control group, indicating a questionable result in the female rats. The antioxidant effects of shilajit have been demonstrated in several animal studies. Shilajit was shown to prevent lead-induced oxidative stress in a 6-week feeding study in chicks (Kumar et al., 2010). Shilajit was included in the diet at 100 ppm. Antioxidant status was assessed based on glutathione peroxidase activity, glutathione reductase activity, catalase activity, glutathione content, and lipid peroxidation (thiobarbituric acid reactive substances). Shilajit treatment (25, 50, and 100 mg/kg/day for 21 days) also attenuated swimming-induced oxidative stress as evidenced by decreases in nitric oxide and lipid peroxidation and increases in catalase and superoxide dismutase activities (Surapaneni et al., 2012). The effects of shilajit on brain edema, intracranial pressure, and neurologic outcomes following traumatic brain injury have been studied in rats (Khaksari et al., 2013). Rats were treated IP with 0, 150, or 250 mg/kg shilajit 1, 24, 48, and 72 h after trauma. Intracranial pressure was significantly reduced at 24, 48, and 72 h after trauma in the shilajit-treated animals, while brain water and Evans blue dye uptake were also significantly decreased as compared to control. Neurological outcomes also significantly improved in the treated rats. In a study in mice, processed shilajit administration (0.1 and 1.0 mg/kg IP) resulted in significant inhibition of the development of tolerance to morphine (10 mg/kg IP twice daily) after 6 days of treatment (Tiwari et al., 2001). Shilajit per se did not exhibit any analgesic activity in the mice. No explanation was provided for the observed effect. Several studies have examined the antidiabetic effects of shilajit in animals. Bhattacharaya (1995) showed that the oral administration of 50 mg/kg and 100 mg/kg of a process and standardized shilajit attenuated streptozotocin-induced diabetes in rats. It also increased pancreatic islet superoxide dismutase, leading to a decrease in free radical production and accumulation. Kanikkannan et al. (1994) observed that a processed shilajit (1.0 mg/kg subcutaneously) prevented streptozotoxin-induced diabetes in rats. Furthermore, shilajit potentiated the hypoglycemic action of insulin. The cardioprotective effects of shilajit (mumie) have been studied in rats (Joukar et al., 2014). Rats received 250 or 500 mg shilajit per day orally for 7 days. Isoproterenol (85 mg/kg) was injected subcutaneously to induce myocardial damage. Shilajit pretreatment provided

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significant protection against the cardiac damaging effects of the isoproterenol, including a reduction in the severity of cardiac lesions. The mechanisms involving protection were not clear to the authors. The ability of shilajit to protect against radiation-induced apoptosis in rat ovaries has been reported (Kececi et al., 2016). Rats were pretreated with shilajit or the vehicle and subject to irradiation. The animals were sacrificed 4 days after radiation exposure. Shilajit prevented the radiation-induced decreases in primordial, primary, preantral, and atretic follicles. Shilajit treatment also decreased the expression of p53, Bax, and caspase 3, thereby blocking the radiation-induced apoptotic pathway. Durg et al. (2015) examined the antiepileptic and antipsychotic activities of standardized shilajit in rats and mice. The animals were given 25 or 50 mg shilajit orally daily for 15 days. Seizures and psychotic behavior were then induced with isonicotinyl hydrazine (INH), pentylenetetrazole (PTZ), apomorphine, or electroshock. Shilajit pretreatment significantly decreased seizures induced by INH, PTZ, and electroshock, while shilajit significantly inhibited climbing and stereotypical behaviors induced by apomorphine. The authors suggested that the antiepileptic activity of shilajit may be due to enhancing the gamma aminobutyric acid (GABA) neurotransmitter (GABAergic) system, while the antipsychotic activity of shilajit may possibly be due to antidopaminergic and/or GABA-mimetic actions. The ability of shilajit to reduce alcohol withdrawal anxiety has been studied in mice (Bansal and Banerjee, 2016). Shilajit treatment significantly decreased ethanol intake and increased water consumption. The shilajit altered cortical-hypocampal dopamine in the mice but had no effect of GABA levels. In an early study, Schliebs et al. (1997) demonstrated that shilajit (40 mg/kg IP for 7 days) administration differentially affected cholinergic but not GABAergic or glutaminergic markers in rat brain as determined by brain slice histochemistry and autoradiography. The data suggested that shilajit preferentially affected cortical and basal forebrain cholinergic signal transduction cascade. An increase in the cholinergic signal transduction cascade could explain, at least in part, anecdotal reports of cognition and memory-enhancing effects of shilajit. Bhattarai et al. (2016) have examined the effects of shilajit on preoptic hypothalamic neurons in juvenile mice using a voltage clamp model. Shilajit induced a reproducible dose-dependent inward current, which persisted in the presence of tetrodotoxin, suggesting a postsynaptic action of shilajit, but was almost completely blocked by strychnine, a glycine receptor antagonist. The authors concluded that shilajit contains ingredients that influence hypothalamic neurophysiology through activation of strychnine-sensitive glycine receptoremediated responses postsynaptically. In a study involving rats, shilajit administration (400 mg/mL) exhibited an in vivo peripheral parasympathomimetic effect, which can provide at least a partial explanation for the reported effects on spermatogenesis, as well as the anecdotal reports on overall fertility and libido (Kaur et al., 2013). These conclusions were based on the administration of shilajit alone and in combination with acetylcholine, and they assessed changes in heart rate, blood pressure, respiratory rate, and neuromuscular transmission.

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In Vitro Studies Various in vitro studies have been conducted to obtain information regarding possible mechanisms of action of shilajit. The antioxidant activities of 3-hydroxy-DBPs and the 3,8-dihydroxy-DBPs, which are believed to be active constituents of shilajit, were demonstrated in vitro using five free radical scavenging assays (Battacharyya et al., 2009a). KU812 cells incubated with fulvic acid affected the expression of genes involved in signal transduction, cytokineecytokine receptor interaction, and immune response pathways, as well as cell adhesion molecule and IgE receptor b subunit responses (Motojima et al., 2011). These results demonstrate the wide range of potential physiological effects that can be modulated by fulvic acids and help explain the immunomodulatory responses observed as a result of shilajit ingestion. Mouse brainstem slices were incubated with shilajit in the presence of various receptor antagonists and channel blockers in order to assess their ability to exhibit glycine- and GABA-mimetic actions on the brainstem substantia gelatinosa neurons of the trigeminal subnucleus caudalis (Yin et al., 2011). Shilajit induced inward currents in a concentration-dependent manner. The results indicated that shilajit has CNS-sedating ingredients and may provide at least a partial explanation regarding reports of skeletomuscular pain relief as well as the antiepileptic and antipsychotic effects. Incubation of rat corpus cavernosum strips with shilajit (400 and 800 mg/mL) resulted in a concentration-dependent relaxation of the strips and enhanced acetylcholinemediated relaxation, suggesting an increased blood flow to the groin and therefore beneficial effects with regard to spermatogenesis and increased testosterone production (Kaur et al., 2013).

Discussion and Summary In recent years, a rapidly growing number of animal and human studies have been published regarding the physiological/pharmacological effects of shilajit (mumie, moomiyo) as well as its mechanisms of action. Studies in both animals and humans indicate that shilajit has a wide margin of safety and is free of adverse effects at the doses that are commonly used. Most human studies have focused on energy production and the androgenic effects of shilajit, including testosterone production and spermatogenesis. Shilajit was also shown to facilitate muscle adaptation. Published human and animal studies have shown that shilajit increases spermatogenesis in infertile males. An unpublished human study has also provided support for the beneficial effects with respect to both ATP and CoQ10 production. Animal studies have supported these observations, demonstrating that shilajit enhances energy (ATP) production, relieves fatigue, and promotes endurance. These effects may be mediated by the ability of shilajit to stabilize electron transport chain enzymes and mitochondrial membranes. Animal studies have also shown that

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DBPs exhibited mitochondrial protective effects, which can further explain the beneficial effects with respect to physical performance and relief from fatigue. Extensive studies were reported to have been conducted within the USSR between 1960e1990 on the physical and mental revitalizing effects of shilajit. These studies were not published and were classified. Shilajit was purported to be widely used by USSR Olympic athletes, as well as military special forces, for its energy-producing and adaptogenic effects (Bucci, 2000; personal communication with Dr. N. Volkov). Various animal studies have shown that shilajit exhibits antioxidant, antiinflammatory and antiarthritic, immunomodulatory, and tissue-protective activities. In addition, antiepileptic and antipsychotic effects have been demonstrated in animals. This broad spectrum of effects has not been confirmed in human studies, in part because the studies have not been conducted. Animal studies have also examined the potential effects on various neurotransmitters and have demonstrated significant cholinergic and parasympathomimetic effects, which can explain the potential benefits with respect to cognitive function and enhanced fertility. Enhancement of the GABAergic system, binding to glycine receptors, and inhibition of dopaminergic actions have also been implicated in the antiepileptic and antipsychotic effects. Antiinflammatory and tissue protective effects may involve shilajitinduced decreased expression of signal transduction factors as p53, Bax, and caspase 3. Finally, a need exists for further studies, primarily in humans, with processed and standardized shilajit preparations. Consumers should keep in mind that products are marketed that do not conform to the chemical composition of “purified” shilajit and may be either adulterated or counterfeit. Therefore, caution must be taken with respect to the acquisition of shilajit, and only processed and standardized products should be consumed.

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