**Clinical Evaluation of Purified Shilajit on Testosterone Levels in Healthy Volunteers**

**Abstract and Figures**
Purified Shilajit, an Ayurvedic rasayana, was rigorously evaluated in a randomized, double-blind, placebo-controlled clinical study involving healthy male volunteers aged 45 to 55 years. Administered at a dose of 250 mg twice daily over 90 consecutive days, the results showed a significant increase (P < 0.05) in total testosterone, free testosterone, and dehydroepiandrosterone (DHEAS) compared to the placebo group. The levels of gonadotropic hormones, LH and FSH, were well-maintained throughout the study.

**HPLC Analysis**
High-Performance Liquid Chromatography (HPLC) chromatograms of Shilajit (PrimaVie™) using an RP-C 18 column indicate the presence of critical bioactive compounds, including fulvic acids and dibenzochromoproteins. The analysis was conducted under ambient conditions using Waters HPLC equipment, ensuring precise measurements of these components.

**Introduction**
Purified Shilajit (PS) is a revered substance in Ayurveda recognized for its numerous health benefits, particularly in addressing male reproductive disorders. This natural exudate, often referred to as the "sweat of mountains," is rich in minerals and organic compounds formed through centuries of decomposition and synthesis in the pristine environment of the Himalayas.

**Study Design and Methodology**
Conducted at J. B. Roy State Ayurvedic Medical College and Hospital in Kolkata, India, the clinical trial involved 96 healthy volunteers, who were randomly divided into two groups: one receiving PS and the other a placebo. The study adhered to ethical guidelines and utilized the St. Louis University ADAM questionnaire to assess testosterone deficiency.

**Results**
The treatment group exhibited a notable increase in testosterone and free testosterone levels, particularly on day 90 of the study, demonstrating the efficacy of PS in enhancing male androgenic hormone levels. DHEAS levels also showed a significant increase, reinforcing Shilajit's role in testosterone synthesis.

**Conclusion**
This study confirms that purified Shilajit can effectively increase testosterone levels and support overall male health, particularly for those experiencing andropause. The maintenance of LH and FSH levels underscores its influence on the hypothalamo-pituitary-gonadal axis, promoting hormone balance and vitality in men aged 45 to 55.

**Background**

Accelerated bone loss associated with aging and estrogen withdrawal is mediated in part by increased oxidative stress and inflammation.

**Objective**

Investigate dietary supplementation with a standardized aqueous extract of shilajit with clinically demonstrated antioxidant, anti-inflammatory, and collagen-promoting activity on attenuating bone lossin postmenopausal women with osteopenia.

**Design**

Sixty postmenopausal women aged 45 – 65 years with osteopenia were randomized to receive 1 of 3 treatments daily for 48 weeks: (1) placebo, (2) 250 mg shilajit extract, or (3) 500 mg shilajit extract. Bone mineral density (BMD) of the lumbar spine (LS) and femoral neck (FN) were measured at weeks 0, 24, and 48, and circulating markers of bone turnover(CTX-1, BALP, RANKL, OPG), oxidative stress(MDA, GSH), and inflammation (hsCRP) at weeks 0, 12, 24, and 48.

**Results**

BMD of both the LS and FN progressively decreased in women receiving placebo but was dose-dependently attenuated with shilajit extract supplementation, resulting in significantly increased percentage changes from baseline in BMD at 24- and 48-weeks in both supplemented groups compared to placebo (p < 0.001). CTX-1, BALP, and RANKL decreased, whereas OPG increased, in both groups supplemented with the shilajit extract, but not in the placebo group, resulting in significantly decreased or increased percentage changes from baseline, respectively. MDA was significantly decreased (p < 0.001) and GSH was significantly increased (p < 0.001) in both supplemented groups compared to placebo from week 12 for the duration of the study. Progressive reductions in hsCRP were observed in both supplemented groups, resulting in significantly decreased percentage changes from baseline in supplemented women compared to placebo (p < 0.001).

**Conclusion**

Daily supplementation with this shilajit extract supports BMD in postmenopausal women with osteopenia in part by attenuating the increased bone turnover, inflammation and oxidative stress that coincides with estrogen deficiency in this population at increased risk for osteoporosis and bone fractures.

**Introduction**

Bone is a living tissue that is continually renewed as old bone is broken down and new bone is made (i.e., remodeling) to maintain structural integrity throughout life. Osteoclasts dissolve or resorb bone, whereas osteoblasts make bone and suppress the activity of osteoclasts. Bone mass and density accumulates from birth through young adulthood but declines with advancing age as bone resorption exceeds bone formation, the main cause of microarchitectural deterioration. This rate of decline is especially high in women after menopause and reduced bone mineral density (BMD) increases risk of osteoporosis and fracture. Postmenopausal bone loss is associated with increased bone turnover as most markers of bone formation and resorption are elevated after menopause. Prospective studies suggest the increase in bone resorption begins approximately 2 years before and is maximal one year after the final menstrual period (Sowers et al., 2013).

Menopause is a critical stage in a woman's lifetime to discuss bone health with healthcare providers since most fractures caused by osteoporosis occur in postmenopausal women (Siris et al., 2004; Sornay-Rendu et al., 2005; Pasco et al., 2006). An International Osteoporosis Foundation survey conducted in 11 countries showed denial of personal risk by postmenopausal women, lack of dialogue about osteoporosis with their doctor, and restricted access to diagnosis and treatment before the first fracture, resulting in under-diagnosis and under-treatment of the disease, indicating a significant awareness gap in this population (International Osteoporosis Foundation: How fragile is her future?2000). Reducing the population burden of fractures also requires attention to women with osteopenia because more than half of fragility fractures occur in this population (Pasco et al., 2006). In postmenopausal women with osteopenia, low BMD, increased bone turnover markers (BTM), and prior fracture are associated with an increased risk of fracture in the subsequent 10 years (Sornay-Rendu et al., 2005).

Aging is associated with chronic inflammation, which is amplified by estrogen deficiency in postmenopausal women, and proinflammatory cytokines promote bone loss (Pfeilschifter et al., 2002; Pacifici, 1996, 2008; Weitzmann and Pacifici, 2005). Estrogen suppresses the production of proinflammatory, pro-resorptive cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-8, IL-6, IL-17 as well as the receptor activator of nuclear factor kappa-B ligand (RANKL), whereas it has been shown to increase the production of antiresorptive cytokines such as transforming growth factor-β and interferon-γ as well as the production of osteoprotegerin (OPG) (Faienza et al., 2013; Azizieh et al., 2017; Hofbauer et al., 1999). The RANKL/OPG ratio is pivotal in the regulation of osteoclast differentiation, activation, and survival, so plays an essential role in the balance between bone formation and resorption (Simonet et al., 1997). This ratio can be increased during inflammatory conditions to favor bone resorption (Weitzmann, 2013). OPG protects against excessive bone resorption by binding to RANKL and preventing it from binding to its receptor, RANK, thereby functioning as a RANKL decoy receptor. Enhancement of OPG secretion by osteoblastic cells likely plays a major role in the antiresorptive action of estrogen on bones (Hofbauer et al., 1999). Also, TNF-α and IL-1β have been shown to inhibit the expression of collagen in cultured osteoblasts (Ding et al., 2009; Beresford et al., 1984), which may contribute to diminished bone formation since collagen is the most abundant protein in bone and provides the scaffolding for mineral deposition.

Trabecular bone loss accelerates during perimenopause, which coincides with the amplification of inflammation triggered by estrogen withdraw (Pacifici, 2008; Ershler and Keller, 2000). C-reactive protein (CRP), has been measured as a marker of chronic, low-grade systemic inflammation associated with aging and chronic disease states. Higher circulating levels of high-sensitivity CRP (hsCRP) has been associated with lower BMD in healthy premenopausal and postmenopausal women (Ginaldi et al., 2005); furthermore, serum levels of alkaline phosphatase, a BTM, was higher, suggesting increased bone turnover, in the subjects with higher hsCRP. CRP has also been directly associated with bone fracture risk. In healthy people over 40 years old, CRP levels were a strong predictor of non-traumatic fractures (Koh et al., 2005). In elderly Asian women with substantially lower circulating CRP levels than Caucasian counterparts, higher CRP was a significant predictor of osteoporotic fracture (Schett et al., 2006). Median hsCRP levels in this population were 0.16 in the lowest tertile, 0.36 mg/l in the middle tertile, and 1.14 mg/l in the highest tertile.

Oxidative stress coincides with pathological conditions characterized by increased inflammation (e.g., osteoporosis) as well as physiologic events such as aging and estrogen deficiency (Nakamura et al., 2011; Sendur et al., 2009; Lean et al., 2005). Evidence from epidemiological and animal studies indicate that increased oxidative stress associated with aging contributes to bone loss (Almeida et al., 2007). High levels of ROS promote osteoclastic bone resorption (Manolagas and Parfitt, 2010; Bax et al., 1992) and reduce osteoblast activity and differentiation (Garrett et al., 1990; Bai et al., 2004; Lee et al., 2006), thus favor bone loss. Reduced glutathione (GSH) is substantially lower in bone marrow of rodents after ovariectomy and normalized by administration of estrogen (Romagnoli et al., 2013). Administration of antioxidants that increase GSH levels blunted ovariectomy-induced bone loss, whereas a specific inhibitor of GSH synthesis caused bone loss. Moreover, estrogen was shown to increase levels of GSH in osteoclast-like cells in vitro and treatment of osteoclasts with a GSH precursor prevented osteoclast formation and proinflammatory signaling, but ROS exposure or inhibition of GSH synthesis had the opposite effect (Romagnoli et al., 2013). Treatment of osteoblast-like cells with GSH or a GSH precursor increased OPG levels to reduce the ratio of RANKL/OPG in favor of bone formation (Lee et al., 2006); diminished GSH due to its oxidation increased the RANKL/OPG ratio to favor osteoclastic bone resorption. Thus, evidence suggests oxidative stress mediates bone loss, particularly with estrogen deficiency since estrogen supports GSH levels and antioxidant defenses.

A woman's risk for developing osteoporosis and fragility fractures is determined by various factors, both modifiable and non-modifiable. Good nutrition and an active lifestyle are modifiable factors essential to optimizing bone health. Women should take preventative actions to reduce these risks, especially given the awareness and treatment gap. Nutraceuticals exhibiting relevant biological activities (i.e., antioxidant, anti-inflammatory) and safety is anticipated to be beneficial in combating accelerated bone loss that accompanies estrogen deficiency shortly after menopause.

Shilajit is a natural exudate from high mountain rocks, especially the Himalayas, derived from the gradual decomposition of plant materials by microbes and containing humic substances, including fulvic acid, as well as minerals, has been used traditionally for centuries in Ayurvedic medicine to enhance vitality and help treat a number of ailments in part due to purported anti-inflammatory and antioxidant activity (Lean et al., 2003). However, the composition of shilajit, which influences biological activities, can vary by region and clinical research supporting its therapeutic potential is limited, although available evidence supports its general safety and anti-inflammatory/-oxidant properties (Lean et al., 2003; Stohs, 2014; Tripathi et al., 1996; Azizi et al., 2018). Dietary supplementation with a unique, standardized, aqueous extract of shilajit has been shown in clinical trials to be safe and positively regulate genes for collagen and the extracellular matrix in multiple tissues of healthy adults (Cesur et al., 2019; Das et al., 2016). It has also been shown to reduce markers of inflammation and oxidative stress as well as improve GSH levels in type 2 diabetics (Das et al., 2019), a condition characterized by chronic inflammation and oxidative stress, and risk factor for osteoporosis. Therefore, we evaluated oral supplementation with this purified shilajit in women within 5 years of the last menstruation on markers of bone turnover, inflammation, oxidative stress, and BMD as a promising nutraceutical for supporting musculoskeletal health.

**Subjects and methods**

This 48-week prospective, randomized, double-blind, placebo-controlled, parallel group intervention study was conducted at Nizam's Institute of Medical Sciences (NIMS), Hyderabad, India, after receiving approval from the Institutional Ethics Committee (IEC-NIMS; No. EC/NIMS/1978/2017; ESGS No. 518/2017), and adheres to CONSORT guidelines. The study was conducted following the Declaration of Helsinki (2013) and ‘Guidelines for Clinical Trials on Pharmaceutical Products in India – GCP Guidelines

**Results**

A total of 68 subjects were screened, out of which 60 eligible subjects were enrolled into the study. One subject dropped out of the study from the group supplemented with 250 mg shilajit extract daily before the first follow-up, citing logistical reasons. A total of 19 subjects in the placebo group and 20 subjects in each of the groups supplemented with the shilajit extract completed the 48-week treatment period (n = 59). Baseline characteristics of subjects enrolled in the study are presented 

**Conclusion**

Daily supplementation with this unique, aqueous extract of shilajit supports BMD in postmenopausal women with osteopenia in part by attenuating the increased bone turnover, inflammation and oxidative stress that coincides with estrogen deficiency in this population that is at increased risk for osteoporosis and fragility fractures. Thus, this dietary intervention may be a useful tool to discourage bone loss and combat fracture burden in aging, postmenopausal women. The bone health benefits

**Discussion**

Osteoporosis, the most common bone disease, is a major health concern, particularly for women. Preventing bone loss is important for women during and after menopause due to estrogen withdrawal coincident with increased inflammation, oxidative stress, and accelerated bone loss. Although effective osteoporosis treatments are available, a significant awareness and treatment gap exists in postmenopausal women. Osteoporosis is often referred to as a “silent disease” since bone loss occurs without

**Availability of data**

The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

**Author contributions**

UP – Protocol and study design, subject recruitment, follow-up of the subjects, interpretation of the data, drafting of the manuscript, approved the final version of the manuscript. CN – Protocol and study design, subject recruitment, follow-up of the subjects, acquisition of the data, interpretation of the data, drafting of the manuscript, approved the final version of the manuscript.

**Funding**

We acknowledge Natreon, Inc., USA, for providing investigational products  and kits for biomarkers.

**Trial registration**

This study was registered with Clinical Trials Registry – India (CTRI) with the registration number: CTRI/2018/04/013529 [Registered on: 27/04/2018] Trial Registered Retrospectively.

http://ctri.nic.in/Clinicaltrials/pmaindet2.php?trialid=23338&EncHid=&userName=shilajit

**Declaration of Competing Interest**

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

**Acknowledgments**

We are grateful to Dr. G. Radhika, Department of Gynecology, NIMS, for her clinical support. The authors also acknowledge Natreon, Inc., USA, for providing study medications and kits for biomarkers, Dr. I. Sravanthi, Ayurvedic physician for her expert advice, Mr. Muralidhar, the study coordinator, and Mr. Ravi Bhatt for review of statistical analyses.

**Shilajit: A Natural Phytocomplex with Potential Procognitive Activity**

**Abstract**

Shilajit is a natural substance found mainly in the Himalayas, formed for centuries by the gradual decomposition of certain plants by the action of microorganisms. It is a potent and very safe dietary supplement, restoring the energetic balance and potentially able to prevent several diseases. Recent investigations point to an interesting medical application toward the control of cognitive disorders associated with aging, and cognitive stimulation. Thus, fulvic acid, the main active principle, blocks tau self-aggregation, opening an avenue toward the study of Alzheimer's therapy. In essence, this is a nutraceutical product of demonstrated benefits for human health. Considering the expected impact of shilajit usage in the medical field, especially in the neurological sciences, more investigations at the basic biological level as well as clinical trials are necessary, in order to understand how organic molecules of shilajit and particularly fulvic acid, one of the active principles, and oligoelements act at both the molecular and cellular levels and in the whole organism.

**Introduction**

Shilajit also known in the north of India as salajit, shilajatu, mimie, or mummiyo is a blackish-brown powder or an exudate from high mountain rocks, especially in the Himalayans mountains between India and Nepal, although it has been also found in Russia, Tibet, Afghanistan, and now in the north of Chile, named as Andean Shilajit [1]. Shilajit has been known and used for centuries by the Ayurvedicmedicine, as a rejuvenator and as antiaging compound. There are two important characteristics of a rasayana compound in the ancient Indian Ayurvedic medicine: that is, to increase physical strength and to promote human health [2]. The health benefits of shilajit have been shown to differ from region to region, depending on the place from which it was extracted [3, 4].

**Origins of Shilajit**

Considering its unique composition as a phytocomplex, very rich in fulvic acid, researchers hypothesize that Shilajit is produced by the decomposition of plant material from species such as Euphorbia royleana and Trifolium repens [4, 5]. This decomposition seems to occur through centuries, and on this basis, shilajit is considered a millenary product of nature. However, further studies have identified that several other plant organisms may generate shilajit, such as molds as Barbula, Fissidens, Minium, and Thuidium and other species like Asterella, Dumortiera, Marchantia, Pellia, Plagiochasma, andStephenrencella-Anthoceros [4].

**Molecular Composition of Shilajit**

Shilajit is composed mainly of humic substances, including fulvic acid, that account for around 60% to 80% of the total nutraceutical compound plus some oligoelements including selenium of antiaging properties [6, 7] (Figure 1). The humic substances are the results of degradation of organic matter, mainly vegetal substances, which is the result of the action of many microorganisms. Components are divided operationally in humins, humic acid, and fulvic acids according to their solubility in water at different pH levels. Humins are not soluble in water under any pH condition. Humic acid is soluble in water under alkaline conditions and has a molecular weight of 5–10 kDa. Fulvic acid is soluble in water under different pH conditions, and because of its low molecular weight (around 2 kDa), it is well absorbed in the intestinal tract and eliminated within hours from the body [8, 9]. It is likely that the curative properties attributable to shilajit are provided by the significant levels of fulvic acids that shilajit contains, considering that fulvic acid is known by its strong antioxidant actions [9] and likely has systemic effects as complement activator [10]. Recent studies on the composition of Andean Shilajit in Chile have evidenced an ORAC index between 50 and 500 Trolox units/g of material, which is substantially higher than Noni and blueberries (Quinteros et al., unpublished data). In this context, shilajitseems to be a powerful antioxidant phytocomplex.

Shilajit, its main components, and potential uses based on properties of fulvic acid. This phytocomplex known as shilajit is mainly composed of humic substances. One of them, fulvic acid, is known by its properties such as antioxidant, anti-inflammatory, and memory enhancer. Novel investigations indicate that fulvic acid is an antiaggregation factor of tau protein in vitro [1], which projects fulvic acid as a potential anti-Alzheimer's disease molecule.

Other molecules present in shilajit preparations are eldagic acid, some fatty acids, resins, latex, gums, albumins, triterpenes, sterols, aromatic carboxylic acids, 3,4-benzocoumarins, amino acids, polyphenols, and phenolic lipids [3, 6, 11]. Certainly its molecular composition varies from region to region. Newer investigations based on high-performance size exclusion chromatography (HP-SEC) show that shilajit contains specific molecular species of polysaccharides and lignins [10]. As humic components, humins, humic acids, and fulvic acids are found in all shilajitpreparations, being the last one, fulvic acids, the biologically active compound, along with dibenzo-α-pyrones, which acts as carrier of other substances [3]. 

** Traditional Uses of Shilajit**

Shilajit is an important, known component of the ayurvedic medicine given its characteristics as a rasayana. In this context, health benefits such as an increase in longevity, rejuvenating, and arresting aging roles have been attributed to it [3]. Traditionally, shilajit is consumed by people from Nepal and the North of India, and children usually take it with milk in their breakfast. The Sherpas claim to have shilajit as part of their diet; they constitute a population of strong men with very high levels of a healthy longevity. Our laboratory has found evidence on the high activity of the Andean form of shilajit in improving cognitive disorders and as a stimulant of cognitive activity in humans [1] (Table 1).

Table 1.

Morphometric study of primary cultured rat hippocampal cells exposed to Shilajit and the Brain Up-10 formulae that contain Shilajit plus complex B vitamins (Vit B6, B9, and B12).

Control

Shilajit**

Brain Up-10*

Neuronal cells per field

367 ± 23

345 ± 42

396 ± 16.0

Percentage of cells with neuronal processes

18.0 ± 2.1

26.0 ± 3.2**

43.0 ± 3.1**

Fraction of axon-like processes

0.22

0.29

0.41

Processes length (μm)

17.4 ± 7.2

26.0 ± 4.5**

39.6 ± 8.0**

Hippocampal cells were grown in Petri dishes in the presence of either 10 mg/mL Shilajit or the formulation of Brain Up-10 [30] plus vitamins of the B complex. In the control, cells were grown in culture medium without Shilajit or the formulation. Mean of 5 determinations (n = 5) (significance of differences with respect to control, **P < 0.001).

Considering the actions of fulvic acid in preventing tau self-aggregation into pathological filaments, this compound appears to be of interest for prevention of Alzheimer's disease [1]. Other common traditional uses include its action in genitourinary disorders, jaundice, digestive disorders, enlarged spleen, epilepsy, nervous disorders, chronic bronchitis, and anemia [2]. Shilajit has been also useful for the treatment of kidney stones, edema, and hemorrhoids, as an internal antiseptic, and to reduce anorexia. Also, it has been claimed in India to be used as yogavaha [12, 13], that is, as synergistic enhancer of other drugs. Organic components of shilajitplay also a role in transporting different mineral substances to their cellular targets.

**Novel Investigations**

Preclinical investigations about shilajit indicate its great potential uses in certain diseases, and various properties have been ascribed, including (1) antiulcerogenic properties [14]; (2) antioxidant properties [15, 16]; (3) cognitive and memory enhancer [1, 10, 17]; (4) antidiabetic properties [18]; (5) anxiolytic [12]; (6) antiallergic properties and immunomodulator [2, 19, 20]; (7) anti-inflammatory [21]; (8) analgesic [16]; antifungal properties [22]; (9) ability to interact positively with other drugs [23]; (10) protective properties in high altitudes [24]; (11) neuroprotective agent against cognitive disorders [1, and Farias et al. unpublished clinical trials]. Unfortunately shilajit lacks systematic documentation and well-established clinical trials on its antioxidative and immunomodulatory actions in humans, and it is expected that considering the reported benefits evidenced from trials will be obtained in the near future [25]. 

**Patenting**

A few patents already exist that protect the use of shilajit in India and Nepal, such as US Patent 5,405,613—vitamin/mineral composition [26]; US Patent application number 20030198695—Herbo-mineral composition [27]; US Patent number 6,440,436—Process for preparing purified shilajitcomposition from native shilajit[28]; US Patent number 6,558,712—Delivery system for pharmaceutical, nutritional and cosmetic ingredients [29]. Other recent patent about a phytocomplex with vitamins added is WO 2011/041920 [30].

**Potential Risks**

Studies indicate the shilajit consumption without preliminary purification may lead to risks of intoxication given the presence of mycotoxin, heavy metal ions, polymeric quinones (oxidant agents), and free radicals, among others [3]. Therefore, a purified, ready-for-use preparation for human consumption must be used. However, recent studies indicate that several ayurvedic products including shilajit and other Indian manufactured products commercialized by the Internet may contain detectable heavy metals levels as lead, mercury, and arsenic [31]. This study showed the presence of heavy metals and other minerals, including gems, is associated with the belief that when mixed with shilajit or other herbal preparations they generate a better response from the body in a synergic manner. This is what is known as rasa-shastra in ayurvedic medicine. Rasashastra experts claim that if this is prepared, administered, and consumed properly, it is safe and has therapeutic advantages [31]. It is worth considering that recent clinical reports indicate cases of lead poisoning in patients who have used ayurvedic products against weakening [32, 33].

**Commentary and Discussion**

Shilajit has a comfortable position as the rasayana because of its excellence, well known in the Eastern culture, and now being introduced with great interest in the occidental world. The vast majority of published papers on this theme are from India, leaving this sector of the planet as an expert in their field, since this is a product that is extracted, marketed, and investigated in these latitudes. However, this generates a segmentation of shilajit, relegating it only to what has always been assumed: a natural product that is part of natural alternative medicine and not as a result of medical and biotechnology innovation worldwide. This is evidenced quite clearly by reviewing the literature today, and note that the journals where studies on shilajit are published (jobs are plentiful) are mainly reviewed in the Eastern. Given this, it is necessary that shilajit break the cultural paradigm and enter into the rest of the world by the hand of rigorous research at the molecular and cellular levels, which could elucidate the interactions of the active ingredients of the different shilajit preparations with biomolecules. This will facilitate our understanding of their mechanisms of action.

**Conclusion**

Shilajit is a potent and very safe dietary supplement, potentially able to prevent several diseases, but its main medical application now appears to come from its actions in benefit of cognition and potentially as a dietary supplement to prevent Alzheimer's disease. In essence, this is a nutraceutical product. Considering the expected impact of shilajit applications in the medical field, especially in neurological sciences, more investigations at the basic biological level are necessary, and certainly well-developed clinical trials, in order to understand how its active principles act at molecular and cellular levels.

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**Skin Transcriptome of Middle-Aged Women Supplemented With Natural Herbo-mineral Shilajit Shows Induction of Microvascular and Extracellular Matrix Mechanisms**

**Objective**

Shilajit is a pale-brown to blackish-brown organic mineral substance available from Himalayan rocks. We demonstrated that in type I obese humans, shilajit supplementation significantly upregulated extracellular matrix (ECM)–related genes in the skeletal muscle. Such an effect was highly synergistic with exercise. The present study (clinicaltrials.gov ) aimed to evaluate the effects of shilajit supplementation on skin gene expression profile and microperfusion in healthy adult females.

**Methods**

The study design comprised six total study visits including a baseline visit (V1) and a final 14-week visit (V6) following oral shilajit supplementation (125 or 250 mg bid). A skin biopsy of the left inner upper arm of each subject was collected at visit 2 and visit 6 for gene expression profiling using Affymetrix Clariom™ D Assay. Skin perfusion was determined by MATLAB processing of dermascopic images. Transcriptome data were normalized and subjected to statistical analysis. The differentially regulated genes were subjected to Ingenuity Pathway Analysis (IPA®). The expression of the differentially regulated genes identified by IPA® were verified using real-time polymerasechain reaction (RT-PCR).

**Results**

Supplementation with shilajit for 14 weeks was not associated with any reported adverse effect within this period. At a higher dose (250 mg bid), shilajit improved skin perfusion when compared to baseline or the placebo. Pathway analysis identified shilajit-inducible genes relevant to endothelial cell migration, growth of blood vessels, and ECM which were validated by quantitative real-time polymerasechain reaction (RT-PCR) analysis.

**Conclusions**

This work provides maiden evidence demonstrating that oral shilajit supplementation in adult healthy women induced genes relevant to endothelial cell migration and growth of blood vessels. Shilajit supplementation improved skin microperfusion.

**Introduction**

Shilajit is a resinous blackish-brown sticky tar-like herbomineral exudate that seeps from sedimentary rocks of steep mountainous regions and has reported medicinal properties (1, 2). Although geographic and environmental factors determine the composition of shilajit (1, 3), chemical characterization of shilajit has revealed the presence of three major components as represented by dibenzo-α-pyrones (DBPs, also known as urolithins in free form as well as conjugated with chromoproteins), fulvic acid with DBP core nucleus, and humic acid (2, 3). Shilajit and its active constituents have been reported to possess an array of pharmacological properties including adaptogenic, antioxidant, anti-inflammatory, immunomodulatory, anti-diabetic, and neurological properties (4, 5).

Skin aging is characterized by wrinkles, dryness, laxity, thinning, irregular pigmentation, and loss of elasticity (6). Decrease in dermal thickness and vascularity is a hallmark of cutaneous aging (7). Aging is associated with decreased cutaneous perfusion (8). Dietary supplements show promise in preventing and managing serious health conditions. The present study was aimed at determining the effect of supplementing with a standardized shilajit extract on skin gene expression profile and related function.

**Materials and methods**

Shilajit (PrimaVie® Shilajit) capsules (125 mg referred to as S125, 250 mg referred to as S250) and placebo were provided by Natreon, Inc.. PrimaVie® Shilajit (U.S. patents: US 6,869,612, and 6,558,712) is a purified and standardized shilajit extract and contains at least 60% fulvic acid and equivalents with high levels of DBPs and DBP chromoproteins (9, 10, 18). Each capsule contained standard components including gelatin, microcrystalline cellulose, croscarmellose sodium, fumed silicon-dioxide, and magnesium stearate as excipients, which are of national formulary grade.

**Study subjects and experimental design**

Study protocols (clinicaltrials.gov NCT02762032) and materials were approved by the Western Institutional Review Board. Written informed consent was collected from all subjects before participation in the study. Female subjects aged between 30 and 65 were included in the study. Three groups (each with n = 15) of subjects were randomized (www.random.org). Supplement randomization was done at study visit 1 and distribution of the supplements were done at each study visit. During each visit, imaging and skin assessment were performed. Group 1 received placebo capsules; Groups 2 and 3 received 125 mg or 250 mg capsules of shilajit bid, respectively. Oral supplementation was continued for 14 weeks and six assessment visits were performed during the duration of study. The study design comprised six visits—visit 1: baseline; visits 2 through 6: after 2, 4, 8, 12, and 14 weeks of oral supplement of shilajit, respectively. During the study visit, the dietary and medical history and medications were recorded. Digital photographs of the face (left, right, and front views) were taken using a DSLR camera (Nikon D80 with 55–300mm lens) with neutral expressions without skin makeup. Noninvasive measurements such as transepidermal water loss (TEWL), hydration, elasticity, and dermascopic image were recorded with Dermalab (cyberDERM, Inc.) (11, 12). During visits 2 and 6, a skin biopsy of the left inner upper arm was taken. Review of adverse events and supplement count/compliance were performed. Any self-reported deviations were documented. Subjects using medications for cardiovascular disease–related disorders (hydrochlorothiazide, aspirin, steroids, ACE inhibitors, beta-blockers, and statins) were excluded from the study. Pregnant females and individuals receiving treatment for being immunocompromised were also not included in the study. The demographics of participating subjects are presented in Table 1.

Table 1.

Subject Demographics

Parameters

Values

Total Subjects

45

Age (years)

42.09 ± 1.17

Body weight (kg)

76.24 ± 2.75

Body mass index (kg/m2)

29.52 ± 1.42

Race

Caucasian

38

African American

6

Asian

1

**Dermascopic image processing**

A MATLAB (Mathworks Inc.) program code was developed for this study Supplemental data 1. Images from the dermascopic imaging system were processed to multicolor coded images which were used for the detection of skin microperfusion (13–15). Regions of interest (ROIs) were traced and signal intensity was computed. Two-dimensional (2-D) ‘trapz()’ MATLAB function algorithm was used to calculate the area under the curve (AUC) by integrating intensity units over area of interest, which is a measure of total energy over the ROI.

**Safety monitoring**

No adverse effect was reported that was directly related to the dietary supplement.

**Skin biopsy collection**

Biopsy site was the left inner arm. Biopsy specimens were taken with a 3 mm punch from the upper inner left arm at week 2 and week 14. Wound care materials and care instructions were provided to the subjects. Due to practical limitations, biopsies could not be collected on visit 1. Biopsy specimens were processed for GeneChip® analysis and mRNA expression using quantitative real-time polymerase chain reaction (RT-PCR).

**GeneChip® probe array analyses**

GeneChip® analysis was done using Affymetrix Clariom™ D Assay as described previously (16–19) to identify sets of genes differentially expressed in the skin samples at different visits. Briefly, total RNA was isolated using the mirVana Isolation Kit as per the manufacturer’s protocol (Thermo Fisher Scientific) (18, 20, 21). RNA integrity was assessed using the Agilent 2100 Bioanalyzer. The isolated RNA was used to generate ss-cDNA using the GeneChip® WT PLUS reagent kit. Biotin-labeled ss-cDNA was hybridized, washed, and stained on the Affymetrix Fluidics Station 450 according to the manufacturer’s protocol and scanned with the Affymetrix GeneChip Scanner 3000 7 G (16–18) The expression data have been submitted to Gene Expression Omnibus (GEO) at NCBI (http://www.ncbi.nlm.nih.gov/geo/; series accession number GSE114170). Data files were generated and processed with Affymetrix software and Expression Console. Differentially expressed genes were identified using a two-class t test where significance level was set at p < 0.05 with Benjamini-Hochberg correction for false discovery rate (16, 18). Significantly differentially regulated coding genes were subjected to functional analysis using Ingenuity® Pathway Analysis as previously described (22).

**Validation of microarray results using quantitative RT-PCR**

For gene expression, total cDNA was synthesized using the SuperScript III First Strand Synthesis System (Thermo Fisher Scientific) (23). Candidate genes were verified by RT-PCR by using SYBR green-I and primers as previously described using β-actin as a housekeeping gene (20, 21, 24–27).

**Statistical analysis**

Data analysis was performed in a blinded fashion. Since fold-change values used for statistical analysis were highly skewed and non-normal, the values were transformed using natural logarithm. The transformed values were then used for all subsequent analyses. Paired two-tailed t tests were used to determine significant differences across baseline (or visit 2 for RT-PCR) and final visit (visit 6) for each subject in all the groups: Placebo, S125, and S250. Next, to detect the efficiency of the treatment with respect to the placebo, two-sample one-tailed t tests were used on the log-transformed fold-change values from the final visit in each group. p < 0.05 was considered statistically significant.

**Results**

**Shilajit supplementation increased skin microperfusion**

Dermascopic images were used to determine whether shilajit supplementation (Figure 1A) had any effect on skin blood microperfusion. Increased reddish hue of the skin as detected using MATLAB color-coded images indicated improved skin microperfusion (Figure 1B-E). Interestingly, oral Shilajit increased skin redness at a 250 mg dose. However, shilajit did not influence skin perfusion at a 125 mg dose (Figure 1).

Figure 1.

Shilajit improves skin microperfusion. A, Study design. B, Dermascopic images of the cheek. C, MATLAB multicolor coded dermascopic images. D, 3-D scatterplot of the Visible Bands of MATLAB processed dermascopic images. E, The sum of the area under the curve of red and green channels were plotted graphically. The intensity of the red and green channels was calculated from the multicolor images processed by MATLAB software from the raw dermascopic images. S125 represents shilajit 125 mg and S250 represents shilajit 250 mg. Data are mean ± SEM (n = 13). *p < 0.05 compared to the baseline visit. †p < 0.05 compared to placebo.

**Transcriptome profiling of skin following oral shilajit supplementation**

Skin samples were collected at visit 2 and visit 6. RNA extraction and target labeling were done, and GeneChip® data analysis was performed using Affymetrix Clariom™ D Assay (28) as described previously (16, 17) to determine the changes in the transcriptome of skin in response to oral shilajit supplementation. The high-resolution array design contains more than 6.0 million probes including coding transcripts and non-coding transcripts of which 70% cover exons for coding transcripts, and the remaining 30% cover exon-exon splice junctions and non-coding transcripts (28). A total of ~ 5000 annotated probe sets were differentially (p < 0.05) regulated following 14-week supplementation (250 mg bid) as compared to placebo (Figure 2).

Figure 2.

Heat map illustrating cluster of transcripts sensitive to shilajit supplementation (250 mg bid). Shilajit-sensitive transcripts were subjected to hierarchical clustering. A, Heat map illustrating cluster of transcripts that were upregulated upon shilajit supplementation. B, Heat map (top 100 candidates) demonstrating cluster of transcripts that were upregulated upon shilajit supplementation.

**Pathway analysis and validation using RT-PCR**

Ingenuity® Pathway Analysis (IPA®) is a powerful analysis and search tool that assists in evaluating the significance of “omics” data identifying novel mechanistic pathways. IPA analysis identified an extracellular matrix (ECM)–related cluster of probe sets that was significantly upregulated in visit 6 of the shilajit-supplemented group as compared to visit 6 of the placebo group (Figure 3). Among these upregulated genes, Col1A1, Col5A2, and Col14A1 were found to be significantly increased following shilajit supplementation as verified using quantitative RT-PCR (Figure 4). In addition, IPA analysis between visit 2 and visit 6 of the shilajit-supplemented group (250 mg bid) revealed upregulated genes involved in growth of blood vessels and movement of vascular endothelial cells upon shilajit supplementation (Figure 5A). TGFb1 and VEGFA path of vascularization was induced by shilajit supplementation (Figure 5B and C). These genes involved in the growth of blood vessels and endothelial cell migration included ITGA5, JAM3, LGALS1, LOX, MMP2, PDGFRB, PRKG1, RECK, SERPINF, SPARC, THBS2, TIMP1, TNN, and TIMP2. The expression of these genes was verified using quantitative RT-PCR (Figures 6 and 7) and was found to be upregulated in response to shilajit supplementation.

**Shilajit supplementation did not adversely affect skin properties**

Digital macrophotography and dermascopic imaging were performed on both left and right cheeks using the DermaLab Combo® device to determine the effect of shilajit supplementation on the properties of the skin. Trans-epidermal water loss (TEWL), an index for skin barrier function, surface electrical capacitance for skin hydration, and skin elasticity for tissue stiffness were measured to assess the effect of shilajit supplementation on skin. Analysis of TEWL revealed that there was no difference in the skin integrity in the treated groups compared to the placebo group (Figure 8), indicating that the supplement intake did not affect the barrier function of the skin. Similarly, there was no significant difference in the hydration, elasticity, viscoelasticity, and retraction time (Figure 8), establishing that shilajit supplementation did not adversely affect the quality of the skin. Thus, shilajit supplementation was safely tolerated.

**Discussion**

Skin microcirculation has a major thermoregulatory function, a nutritional role, and implications in cutaneous aging (29). A decrease in dermal vascularity is commonly encountered during cutaneous aging (7). Compromised circulation in the skin may cause cosmetic defects like unattractive skin tone and discoloration (8). The aged skin suffers from compromised sympathetic reflex as manifested by inability to vasoconstrict or vasodilate in response to changes in environmental temperature (30). In the cosmetic market, nutraceuticals play a major role (31–34). However, the scientific literature on the mechanism of action of such off-the-shelf nutraceuticals is scanty (35–37). Human studies must test not only efficacy but also safety of nutraceuticals because use of inappropriate supplements is known to pose risk to human health (38, 39). In this work, supplementation of shilajit to middle aged women for 14 weeks did not result in any reported adverse effects. Furthermore, basic skin function tests demonstrated no adverse effect of shilajit on skin health in middle-aged women. Our findings are consistent with previous findings that have demonstrated the safety of shilajit (40, 41).

The dietary supplement L-arginine improved microperfusion of the rat skin (42). Consistently, transdermal delivery of L-arginine improved cutaneous blood flow in the feet of diabetic patients (43). Significant improvement of reddish coloration of the face under standardized testing conditions provided the first line of evidence suggesting that shilajit may improve skin microcirculation. Follow-up on this line of finding was performed by collection of skin biopsy and unbiased query of the human skin transcriptome. Additional validation of the findings of such screening was performed using quantitative polymerase chain reaction (PCR). Ingenuity pathway analyses of GeneChip® data identified genes related to endothelial cell migration and growth of blood vessels, being significantly induced in response to shilajit supplementation. Both of these functions are known to be directly responsible for skin angiogenesis (44). Statistical treatment of large data sets such as those acquired by GeneChip® include conservative measures to manage risks of false discovery (16, 18, 45). Using such conservative analyses and quantitative PCR analyses, it was noted that shilajit was effective in the induction of such genes. Angiogenesis is a well-regulated physiological process that includes blood vessel formation followed by timely regression (46, 47). Consistent with that notion, the current work reports the induction of angiogenic mediators as well as anti-angiogenic pathways that are necessary for regression. Interestingly, the application of rigorous quantitative PCR revealed that shilajit was more effective at the lower dosage studied (125 mg bid). Such dose–effect response is not uncommon, as reported previously (48–51).

ECM provides scaffold support as well as biochemical cues for skin cells to grow and is recognized as a major determinant of skin health (52). Age-related decay of ECM accounts for skin thinning and wrinkles (7). In the current study, upregulation of the ECM genes, Col1A1, Col5A2, and Col14A1 was statistically significant upon shilajit supplementation. Collagens constitute a major component of skin ECM and provide structural support as well as molecular signals to resident cells (27, 52). A recent study addressing the mechanism of action of shilajit in improving human skeletal muscle adaptation to exercise provided first evidence on shilajit-induced expression of ECM-related genes (18). Shilajit supplementation significantly increased ECM-related gene expression in overweight/class I obese human subjects. is evident across independent studies that when taken orally Of interest, exercise and shilajit were synergistic in shilajit modifies organ ECM composition. In the skeletal upregulating collagen and other ECM proteins (18). Thus, it muscle, ECM is known to play an essential role in the development, maintenance, and regeneration of the organ well as ECM-related genes is of particular significance (53, 54). The observation that shilajit induced genes involved because these two functional domains of tissue biology are in growth of blood vessels and endothelial cell migration as known to be directly related (55, 56).

**Conclusion**

The current study provides interesting insight into the mechanism of action of shilajit on human skin health. The findings of this work therefore lays the rationale for further mechanistic studies addressing shilajit-inducible signaling pathways that are common to both vascular and ECM function.

Supplementary Material

supplemental

NIHMS1059921-supplement-supplemental.pdf(54.6KB, pdf) 

Acknowledgment

Parts of this work were supported by an R01 NS085272 and research grant by Natreon Inc, NJ, USA.

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