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Abstract

Shilajit has a longstanding use as an anti-aging and memory enhancing drug. It is known to have excellent anti-bacterial effects and is believed to be effective for cognitive enhancement, but is difficult to standardize because of the lack of quality control standards. This study, for the first time, proposes a quality control standard using a simultaneous analytical method for the drug’s multi-compound content using high-performance liquid chromatography-ultraviolet detection (HPLC-UV) as an aid for the internationalization of Mongolian Shilajit. Phenolic compounds 1-6 were isolated from Mongolian Shilajit extract using bioassay-guided isolation, and the isolated compounds were evaluated for cognitive-related anti-Alzheimer’s disease (AD) activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), and advanced glycation end-product (AGE) formation assays. The isolated compounds showed good effects for each activity. In addition, the isolated compounds were successfully quantified using a validated quantitative HPLC analysis method. As a result, the isolated compounds were suggested as standard marker compounds for Mongolian Shilajit. Also, we proved that the original material of Mongolian Shilajit is a lichen named Xanthoparmelia somloensis (Gyel.) Hale using HPLC-UV, ultra-high-performance liquid chromatography-electrospray ionization/hybrid linear trap-quadruple-orbitrap-high-resolution mass spectrometry (UHPLC-ESI/LTQ-HRMS).

Keywords

Alzheimer’s disease HPLC-UV Mongolian Shilajit phenolics simultaneous analysis

Shilajit is a natural multi-complex material.¹ For, centuries, it has been obtained as a product mixed with rock minerals through the process of accumulation and decomposition of materials such as plants, fungi, and lichens.² Therefore, its components vary depending on various conditions such as the mineral content of the rocks, surrounding plants, humidity, and bacteria.^3
-5 In general, Shilajit contains hippuric acid and benzoic acid.⁴ Because the raw material of Shilajit contains lower plants, such as lichen, moss, and liverwort, as well as animal urine and feces, these compounds do not vary.^5,6

Shilajit has been reported to have anti-inflammatory, anti-oxidant, memory enhancement, anti-Alzheimer’s disease (AD), and anti-diabetes activities.⁵ Nevertheless, little is known about its constituents and biological activities, which limits its international use.⁷ First, quality control standards are difficult because the standard marker compound of Shilajit has not been established.^7

-10 Secondly, there is a lack of scientific research on Shilajit.⁵ Thirdly, the focus is on research of Indian Shilajit. Shilajit is found in mountainous areas, such as the Himalayas, Altai, and the Urals, hence the chemical properties of the drug differ from mountain to mountain.¹¹ Mongolian Shilajit is known for its anti-bacterial effects that are attributable to its rich benzoic acid content.^12,13

Various plants, mosses, lichens, and liverworts are associated with Shilajit.⁵ We have collected the lichen, Xanthoparmelia somloensis (Gyel.) Hale around Mongolian Shilajit. Through high-performance liquid chromatography-ultraviolet detection (HPLC-UV) and ultra-high-performance liquid chromatography-electrospray ionization/hybrid linear trap-quadruple-orbitrap-high-resolution mass spectrometry (UHPLC-ESI/LTQ-HRMS), the relationship between Mongolian Shilajit and X. somloensis (Gyel.) Hale was established.

For the first time, this study proposes the standardization of quality of Mongolian Shilajit through isolation of standard marker compounds from Mongolian Shilajit extract, simultaneous analysis through HPLC-UV, validation, cognitive-related anti-Alzheimer’s disease (AD) assay, and identification of origin. The purpose of this study was to establish a quality control standard for Mongolian Shilajit.

Results and Discussion

Identification of Compounds 1‐6 Isolated From Mongolian Shilajit

Compounds 1-6 were identified as syringaldehyde (1), benzoic acid (2), phthalic acid (3), hippuric acid (4), urolithin B (5), and (+)-usnic acid (6) using ¹H and ¹³C –NMR spectroscopy, UHPLC-ESI/LTQ-Orbitrap-HRMS and by comparison with results of previous studies.^{14

-23} After identification of compounds 1-6, HPLC-UV analysis was performed to determine the major compounds of Mongolian Shilajit extract (Figure 1). However, compounds 5 and 6 were not detected at any of the UV wavelengths.

Figure 1

HPLC chromatograms of Mongolian Shilajit extract (1) and standard mixtures (2) (A) compound 4 (hippuric acid); (B) compound 1 (syringaldehyde); (C) compound 3 (phthalic acid); (D) compound 2 (benzoic acid).

Identifying the Origin of Mongolian Shilajit

To standardize Mongolian Shilajit, we first identified the substance from which it originated. Mongolian Shilajit extract, X. somloensis extract, and standard mixtures were analyzed by HPLC-UV. Peaks of compounds 1, 2, and 4 were identified in both the Mongolian Shilajit extract and X. somloensis extract. UHPLC-ESI/LTQ-Orbitrap-HRMS analysis of both extracts showed that compounds 1, 2, and 4 were present in both. As a result, X. somloensis was found to be the original material of Mongolian Shilajit.

DPPH Free Radical-Scavenging Activity of Mongolian Shilajit Extract and Fractions

The accumulation of active oxygen induces oxidative stress, resulting in cell damage, which could lead to several chronic diseases, including AD, whereas the anti-oxidant defense systems of the body protect against these chronic diseases.²⁴ Therefore, the potential anti-oxidant activity of the test samples was determined through analysis of DPPH activity, which is widely used to assay free radical-scavenging activity.²⁵ The extract, fractions, and compounds 1-6 of Mongolian Shilajit were tested for DPPH free radical-scavenging activity; the results are summarized in Table 1. The FSC₅₀ values of the positive control in the DPPH free radical-scavenging activity assay were determined to be adequate compared to previously reported results.²⁵ The EtOAc and n-BuOH fractions had high scavenging activity, whereas the Mongolian Shilajit extract and CH₂Cl₂, and water fractions showed mild activity. Compounds 5 and 6 showed good scavenging activity, suggesting they could be used as ROS inhibitors whereas compounds 1 and 4 showed only mild activity (Table 1).

Table 1

DPPH FSC₅₀ of Mongolian Shilajit Extract, Fractions, and Compounds 1-6.

Sample	FSC₅₀ ^a (µg/mL)
Extract	289.5 ± 11.3 ***
n-Hexane fraction	>500
CH₂Cl₂ fraction	254.7 ± 14.3 ***
EtOAc fraction	23.3 ± 6.2 *
n-BuOH fraction	89.1 ± 12.9 **
Water fraction	380.7 ± 10.6 ***
L-ascorbic acid^b	8.3 ± 0.1 ***

Compound	FSC₅₀ ^a (µM)
1	110.3 ± 7.5 **
2	>500
3	>500
4	175.0 ± 4.6 **
5	80.9 ± 1.0 ***
6	51.2 ± 4.5 **
L-ascorbic acid^b	1.16 ± 0.1 ***

Data are the mean ± S.D (n = 3).

^aFSC₅₀ calculated from the least-squares regression line of logarithmic concentrations plotted against residual activity.

^bL-ascorbic acid was used as the positive control for DPPH free radical-scavenging; *P < 0.05, **P < 0.01, and *** P < 0.001 compared to the control.

Inhibitory Activity of the Extract, Fractions, and Compounds 1-6 of Mongolian Shilajit on AChE, BChE, and BACE1 Activity and AGE Formation

AD has several causes, including excessive degradation of the neurotransmitters ACh and BCh, resulting in decreased levels as a result of increased levels of the enzymes AChE and BChE, which ultimately lead to the development of neurological diseases.^26
-28 In addition, BACE1 is an enzyme that produces Aβ protein, which constitutes the senile plaques found in the brains of patients with AD.²⁹ Moreover, AGE plays a role in the loss of neuronal dysfunction in neurodegenerative diseases.³⁰ Therefore, we assessed the potential involvement of the inhibition of AChE, BChE, and BACE1 activity and AGE formation by the Mongolian Shilajit extract, fractions, and compounds 1-6 in the prevention of AD, and the results are summarized in Table 2. The IC₅₀ values of the positive control in inhibiting AChE, BChE, and BACE1 activity and AGE formation were similar to previously reported values.^18,19,31,32 The Shilajit extract and n-BuOH fraction inhibited AChE activity (IC₅₀ 422.83 ± 6.11 and 240.28 ± 8.58 µg/mL, respectively, whereas the n-BuOH fraction and extract showed greater inhibition of BChE activity (IC₅₀ 55.85 ± 1.01, 59.36 ± 0.76 µg/mL, respectively) than the other fractions. The CH₂Cl₂ and water fractions exhibited mild activity (IC₅₀ 197.52 ± 6.42, 110.44 ± 2.89 µg/mL, respectively). The extract and n-BuOH and water fractions showed greater inhibition of BACE1 activity (IC₅₀ 54.83 ± 11.41, 56.64 ± 1.54, and 158.70 ± 14.97 µg/mL, respectively) than the other fractions. Finally, the n-BuOH fraction (IC₅₀ 141.77 ± 9.38 µg/mL) most potently inhibited AGE formation, followed by the water and EtOAc fraction (IC₅₀ 209.69 ± 10.12 and 214.42 ± 7.10 µg/mL). The n-BuOH fraction showed good inhibitory efficacy against the activity of AChE, BChE, and BACE1 activity and AGE formation.

Table 2

IC₅₀ of Mongolian Shilajit Extract, Fractions, and Compounds 1-6 Against AChE, BChE, BACE1 Activity, and AGE Formation.

Sample	IC₅₀ ^a (µg/mL)
Sample	AChE	BChE	BACE1	AGE formation
Extract	422.8 ± 6.1 ***	59.4 ± 0.8 ***	54.8 ± 11.4 **	>500
n-Hexane fraction	ND^e	>500	495.9 ± 19.4 *	>500
CH₂Cl₂ fraction	ND^e	197.5 ± 6.4 **	>500	>500
EtOAc fraction	>500	>500	ND^e	214.4 ± 7.1 **
n-BuOH fraction	240.3 ± 8.6 **	55.8 ± 1.1 ***	56.6 ± 1.5 **	141.8 ± 9.4 **
Water fraction	>500	110.4 ± 2.9 ***	158.7 ± 15.0 ***	209.7 ± 10.1 **
Berberine^b	0.14 ± 0.01 ***	0.12 ± 0.01 ***	-	-
Quercetin^c	-	-	6.9 ± 0.2 ***	-
AG hydrochloride^d	-	-	-	118.5 ± 4.7 ***

Compound		IC₅₀ ^a (µM)
1	>500	450.3 ± 6.6 **	34.9 ± 1.7 **	65.7 ± 0.6 ***
2	>500	ND^e	309.3 ± 3.5 *	>500
3	ND^e	ND^e	133.4 ± 13.9 *	108.5 ± 9.7 **
4	227.7 ± 11.1 **	129.5 ± 6.7 **	269.2 ± 19.4 *	170.8 ± 5.9**
5	ND^e	118.2 ± 4.4 **	35.6 ± 2.8 ***	50.8 ± 6.3 ***
6	611.0 ± 10.4 **	173.8 ± 9.4 *	43.1 ± 3.9 ***	>500
Berberine^b	0.11 ± 0.01 ***	1.16 ± 0.01 ***	-	-
Quercetin^c	-	-	10.7 ± 0.2 ***-	-
AG hydrochloride^d	-	-	-	169.4 ± 6.8 **

Data are the mean ± S.D (n=3).

^aIC₅₀ calculated from the least-squares regression line of logarithmic concentrations plotted against residual activity.

^bBerberine was used as the positive control for AChE and BChE inhibition.

^cQuercetin was used as the positive control for BACE1 inhibition.

^dAG hydrochloride was used as the positive control for the inhibition of AGE formation.

^eND, not detectable; *P < 0.05, ** P < 0.01, and *** P < 0.001 compared with the control.

Compounds 1-6 were tested for AChE, BChE, and BACE1 activity and AGE formation, and the results are summarized in Table 2. Compound 4 showed the greatest inhibitory effects against AChE and BChE activity. Compound 4 has an N-acylglycine unit and N-acylglycine can play a potential role in the nervous system.³³ Compound 5 exhibited good efficacy against BChE and BACE1 and inhibited significant AGE formation, in that order. Compound 5 is a dibenzo-α-pyrone. This is known to be able to inhibit β-amyloid aggregation.³⁴ In addition, compound 1 showed inhibitory effects on BACE1 and inhibited significant AGE formation, whereas compound 3 showed greater inhibitory effects against AGE formation than the other compounds. Compounds 1, 3, and 5 showed better inhibition of AGE formation than the positive control AG hydrochloride. Compound 6 showed greater inhibition of BACE1 activity than the other compounds. Compound 6 is a dibenzofuran. Benzofuran is the basic structure of galantamine, an acetylcholinesterase inhibitor. Recently, benzofuran has been shown to have the ability to suppress BACE1, in addition to acetylcholinesterase inhibition,³⁵ whereas the effect of compound 2 was mild. These results suggest that these bioactive compounds have the potential as inhibitors for the treatment of AD.

Validation of HPLC Analysis

Compounds 1-4 are benzenoids, so they are detected at similar wavelengths by HPLC-UV. Compounds 5 and 6 were not detected at all by HPLC-UV. Thus, we have selected compounds 1-4 as the marker compounds of Mongolian Shilajit.

Specificity

Optimal ultraviolet wavelength screening was conducted using HPLC-PDA between 210 and 400 nm, and a wavelength of 232 nm was selected in the PDA spectrum. Using the developed HPLC analytical method, the 4 major compounds of Mongolian Shilajit extract were separated without interference.

Linearity

The linearity of the 4 compounds was measured at five final concentrations within 2.5 to 50 µg/mL. In all calibration curves, the correlation coefficient (r ²) of compounds 1-4 was 0.99 or higher. The results are shown in Figure 2.

Figure 2

Calibration curves and correlation coefficients of compounds 1-4 (n = 3).

Limit of Detection (LOD)/Limit of Quantification (LOQ)

The LOD values of compounds 1, 2, 3, and 4 ranged from 0.27 to 0.75 µg/mL, and LOQ values of compounds 1, 2, 3, and 4 ranged from 0.82 to 2.27 µg/mL (Table 3).

Table 3

Calibration Curves and Linear Range of Compounds 1-4.

Compound	Calibration equation^a	CorrelationCoefficient(r ²)^b	LinearRange(μg/mL)	LOD(μg/mL)	LOQ(μg/mL)
1	y = 5077.3 x +5071.8	0.9994	2.5‐50	0.38	1.15
2	y = 3027.4 x – 1774.9	0.9997	2.5‐50	0.75	2.27
3	y = 1839.8 x +2075.3	0.9998	2.5‐50	0.48	1.45
4	y = 1252.9 x +1145.9	0.9996	2.5‐50	0.27	0.82

Each value is the mean of 3 determinations.

Compound 1: syringaldehyde; Compound 2: benzoic acid; Compound 3: phthalic acid; Compound 4 : hippuric acid.

^aY = peak area, X = concentration of standard (μg/mL).

^br² = correlation coefficient for five final concentrations in the calibration curve.

Intra—Inter Day Precision & Trueness

The precision and trueness of analysis were evaluated by 4 standard mixture solutions. Intra-day precision measurement was conducted in a day and repeated on 3 days. The intra-day precision of compounds 1, 2, 3, and 4 ranged from 0.81% to 2.09%, and the inter-day precision from 1.01% to 1.99%. The trueness of intra-day ranged from 100. 1% to 109.5%, and inter-day trueness from 100.6% to 110.5% (Table 4).

Table 4

Intra-Day and Inter-Day Precision and Trueness of Compounds 1-4.

Compound	Con. (μg/mL)	Precision (C.V.%)		Trueness (%)
Compound	Con. (μg/mL)	Intra-day	Inter-day	Intra-day	Inter-day
1	100	1.73	1.81	103.8	101.3
	60	1.11	1.23	104.6	102.2
	40	0.99	1.03	109.5	109.1
2	100	1.25	1.30	108.9	101.6
	60	1.84	1.71	103.8	102.3
	40	2.09	1.99	105.5	102.3
3	100	1.33	1.26	104.5	103.4
	60	1.25	1.28	102.5	102.0
	40	2.09	1.99	105.5	102.3
4	100	1.18	1.21	100.1	100.6
	60	1.04	1.14	104.6	102.7
	40	0.81	1.01	102.6	101.3

Each value was presented by calculating mean of triplication.

Compound 1: syringaldehyde; Compound 2: benzoic acid; Compound 3: phthalic acid; Compound 4: hippuric acid.

Quantitative HPLC Analysis of Isolated Compounds

The isolated compounds 1-6 showed an effect in each active experiment. However, only compounds 1-4 were detected simultaneously by HPLC-UV. Thus, in this study, we developed a standard extraction method that established quality control standards based on 1-4. The 4 major compounds were extracted using different solvent systems and extraction times. The optimal extraction solvent system for these 4 compounds was 50% methanol, which further showed an optimal extraction time range of 30‐120 min. The results showed that an extraction time of 120 minutes was superior to the others (30, 60, and 90 minutes). The contents of the bioactive compounds 1-4 were 180, 10, 10, and 60 µg/g, respectively. The content analysis using HPLC-UV compares to previously reported values.^36,37 These findings suggest that the developed HPLC analytical method for these 4 compounds was successfully conducted and quality control standards criteria were established for Mongolian Shilajit.

Conclusions

This study conducted standard marker compound isolation from Mongolian Shilajit extract, cognitive-related AD activity assay, development of a simultaneous analysis method for multi-compounds content using HPLC-UV, and origin identification to establish the standard quality control criteria for Mongolian Shilajit. The Mongolian Shilajit extract and fractions were evaluated for anti-oxidant and anti-AD activity. Among them, the n-BuOH fraction showed the greater anti-oxidant and anti-AD activity. Syringaldehyde, benzoic acid, phthalic acid, hippuric acid, urolithin B, and (+)-usnic acid were identified in the extracts. Hippuric acid (4) showed good inhibition of AChE and BChE activity, whereas syringaldehyde (1) inhibited BACE1 activity and AGE formation. Moreover, syringaldehyde (1), phthalic acid (3), and urolithin B (5) showed significant inhibitory effects on AGE formation. Benzoic acid (2) showed mild inhibitory activity against BACE1, whereas urolithin B (5) and (+)-usnic acid (6) exhibited DPPH free radical-scavenging activity and inhibition of BACE1. The isolated compounds 1-6 showed activity in the various AD tests. This indicated that Mongolian Shilajit extract was effective for the treatment of AD. In addition, we established an HPLC method for the simultaneous analysis of compounds 1-4 as quality control standards of Mongolian Shilajit. These results suggest the isolated compounds as standard marker compounds of Mongolian Shilajit. Furthermore, we have identified through HPLC and UHPLC-ESI/LTQ-Orbitrap-HRMS that X. somloensis is the original material of Mongolian Shilajit. Overall, the results present quality control standards for Mongolian Shilajit.

Materials and Methods

Plant and Raw Materials

X. somloensis and Mongolian Shilajit samples (Figure 3) were both collected from the Khovd aimag - Ховд аймагin, Mongolia in April 2020. They were authenticated by Professor Wan Kyunn Whang, and Professor Enkhjargal Enkhtaivan of the Mongolian Academy of Sciences Institute of General and Experimental Biology in Mongolia.

Figure 3

Mongolian shilajit, X. Somloensis (Gyel.) Hale.

Instruments and Reagents

Ethanol (EtOH), methanol (MeOH), n-hexane, dichloromethane (CH₂Cl₂), ethyl acetate (EtOAc), n-butanol (n-BuOH), and distilled water were used as solvents for extraction, fractionation, and open column chromatography. The open column chromatography was conducted using Sephadex LH-20 (25, 100 µm; Pharmacia, Stockholm, Sweden), MCI CHP 20P gel (Supelco, St. Louis, MO, USA), and octadecylsilane (ODS) gel (400, 500 mesh; Waters, Milford, MA, USA) columns. Dimethyl sulfoxide-d₆ (DMSO-d₆ ), MeOH-d₄ (CD₃OD), and chloroform-d (CDCl₃; Sigma Aldrich Co., St. Louis, MO, USA) were used as solvents for nuclear magnetic resonance (NMR) imaging. The proton (¹H)- and carbon (¹³C)-NMR spectra were recorded at 600 and 150 MHz, respectively, using a JEOL ECZ600R spectrometer (JEOL, Tokyo, Japan). Chemical shifts are presented as parts per million (ppm) on the δ scale, and coupling constants (J) are shown in Hertz. Electron ionization-mass spectrometry (EI-MS) was performed using UHPLC-ESI/LTQ-HRMS with an Ultimate 3000 rapid separation LC system (Thermo, Darmstadt, Germany). HPLC was conducted using Empower Pro 2.0 software, a Waters 2695 system pump with a Waters 2489 photodiode array detector (Waters, Milford, MA, USA), and a Nouryon Kromasil C₁₈ separation column (4.6 × 250 mm, 5 µm, Bohus, Sweden). HPLC-grade solvents, such as acetonitrile, distilled water, acetic acid, and formic acid were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The DPPH free radical-scavenging activity and anti-AD inhibitory assays were performed using an Infinite^® F200 PRO spectrophotometer (Männedorf, Zürich, Switzerland). Other solvents and reagents, including DMSO, L-ascorbic acid, DPPH, sodium phosphate buffer, sodium azide, electric-eel AChE, horse serum BChE, acetylthiocholine iodide, butyrylthiocholine chloride, 5,5-dithiobis [2-nitrobenzoic acid] (DTNB), bovine serum albumin (BSA), glucose, fructose, berberine chloride, and aminoguanidine (AG) hydrochloride were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). BACE1 FRET assay kits (β -Secretase) were purchased from PanVera Co. (Madison, WI, USA).

Extraction, Fractionation, and Isolation of Mongolian Shilajit

Dried and powdered Mongolian Shilajit (1.0 kg) was extracted with MeOH (5 L × 3) at 28 ℃. The solution was filtered, and the dry extract (340.5 g) was obtained after the solvent was removed in vacuo. This was suspended in water and sequentially partitioned with n-hexane, CH₂Cl₂, EtOAc, and n-BuOH (each, 5 L × 3 times), and the yield of the various solvent fractions was obtained (5.20 g, 12.24 g, 9.76 g and 145.74 g, and that of the water fraction was 142.52 g). Among the fractions, the n-BuOH fraction showed the strongest inhibitory effect in the 4 AD inhibitory assays conducted; therefore, we further analyzed this fraction using open column chromatography. The n-BuOH fraction (145.74 g) was also subjected to MCI gel column chromatography with step gradient elution with 5% to 80% MeOH, and 5 sub-fractions were obtained. Sub-fraction 1 (10.1 g) was separated using ODS column chromatography with 5% MeOH, and sub-fractions 1‐1 (3.2 g) to 1‐2 (5.1 g) were obtained. Sub-fraction 1‐1 was separated using Sephadex LH-20 column chromatography with 20% MeOH to obtain compound 1 (37.2 mg). Recrystallization of sub-fraction 1‐2 in 50% EtOH led to the isolation of compound 2 (120.1 mg). Sub-fraction 2‐2 (5.2 g) was obtained from sub-fraction 2 (11.7 g) using ODS column chromatography with a 10% MeOH solvent system. Sub-fraction 2-2-1 (3.4 g) was separated from sub-fraction 2‐2 using MCI gel column chromatography with 10% MeOH. Sub-fraction 2-2-1 was recrystallized in 10% EtOH to obtain compound 3 (29.1 mg). Sub-fraction 3 (18.4 g) was obtained from sub-fraction 3‐1 to 3‐4 using MCI gel column chromatography using a 10% MeOH solvent system. Among the fractions, sub-fraction 3‐4 (3.6 g) was separated from sub-fraction 3-4-2 (1.7 g) using Sephadex LH-20 with 20% MeOH. Sub-fraction 3-4-2 was subjected to MCI gel column chromatography with 10% MeOH to obtain compound 4. Compound 4 (30.1 mg) was recrystallized after using Sephadex LH-20 from 10% EtOH. Sub-fraction 5 (16.9 g) was separated to obtain sub-fraction 5‐2 (7.2 g) using a Sephadex LH-20 column with 5% MeOH, and further separation using Sephadex LH-20 with 5% MeOH yielded sub-fraction 5-2-3 (3.1 g) and 5-2-4 (1.4 g). Sub-fraction 5-2-3 was subjected to Sephadex LH-20 with 10% EtOH to obtain compound 5 (4.6 mg). Sub-fraction 5-2-4 was separated using Sephadex LH-20 with 10% EtOH to yield compound 6 (7.1 mg).

UHPLC-ESI/LTQ-Orbitrap-HRMS Conditions

The molecular weights of compounds 1-6 were determined using UHPLC-ESI/LTQ-Orbitrap-HRMS. One mg of isolated compounds 1-6 in 1 ml of MeOH was filtered through a 0.45 µm syringe filter. This was diluted to 0.25 to 5 ug/mL and used for UHPLC-ESI/LTQ-orbitrap-HRMS analysis for mass determination. A Hypersil GOLD column (C₁₈, 2.1 × 50 mm, 1.9 µm) was used for the analysis. The mobile phase was run on a gradient schedule with solvent A (water, 0.1% formic acid, v/v) and solvent B (acetonitrile, 0.1% formic acid, v/v). Solvent B was increased from 10% to 50% for 18 minutes and then from 50% to 100% for 5 minutes, maintained for 3 minutes, decreased from 100% to 10%, and maintained for 10 minutes. The flow rate of the mobile phase was 0.3 mL/min, the injection volume was 10 µL, the column temperature was maintained at 30 °C, and UV detection was not used. The optimal UHPLC-ESI/LTQ-Orbitrap-HRMS analysis conditions were as follows: heater and capillary temperatures, 300°C and 360 °C, respectively, auxiliary and sheath flow rate, 10 and 45 L/h, respectively; S-lens RF level, 50.0 V; spray capillary voltage, 3.0 kV; full MS AGC target, 3e⁶; and full MS resolution, 35,000.

HPLC-UV Analysis

Sample preparation

One mg of powdered Mongolian Shilajit and X. somloensis were dissolved in 50% MeOH (1 ml) and filtered through a 0.45 µm syringe filter. For simultaneous content analysis, samples were sonicated with MeOH and EtOH at different concentrations (30%, 50%, 70%, and 100%) and extraction times (30, 60, 90, and 120 minutes). All samples were filtered through a 0.45 µm syringe filter.

Preparation of standard solution

One mg of isolated compounds 1-4 was dissolved in 1 ml of MeOH and prepared as a standard solution of 1000 mg/L. This was diluted to 2.5‐750 ug/mL and used for HPLC-UV analysis to establish and validate the analysis conditions.

Analytical conditions

For the analysis of compounds 1-4 and other samples, the mobile phases A and B consisted of water containing 1% acetic acid and acetonitrile, respectively, and were run on the following gradient schedule. Mobile phase B was maintained for 20 minutes, increased from 25% to 40% for 6 minutes, and maintained for 9 minutes. The flow rate of the mobile phase was 0.7 mL/min, and the injection volume was 10 µL. The column temperature was maintained at 28 °C, whereas the detector was set at a UV absorbance wavelength of 232 nm.

Validation

To verify the reproducibility and trueness of the HPLC-UV method, specificity, linearity, limit of detection (LOD) and limit of quantitation (LOQ), precision, and trueness were evaluated through the recovery.

Specificity

Optimal ultraviolet wavelength screening was conducted using HPLC-PDA under UV 210‐400 nm.

Linearity

The stock standard solution (compounds 1, 2, 3, and 4) was manufactured with 5 levels of concentrations of standard mixtures (0.25, 1000 µg/mL). The calibration curves of the 4 standards were calculated using linear least-squares regression

Limit of detection and limit of quantitation (LOD, LOQ)

These was determined as the quantifiable concentration of the compounds having a signal-to-noise ratio of ≥3.3 and ≥10, respectively.

Precision and trueness

Standard mixtures (40, 60, and 100 µg/mL) were used for intra and inter day precision analysis. Precision (coefficient of variation [C.V%]) and trueness (%) were calculated using the linear range of the standard curves of the 3 different concentrations (40, 60, and 100 µg/mL) within 1 day or 3 consecutive days. Intra-inter day trueness was marked as the observed concentration value relative to the true concentration. The intra-inter day precisions was marked as the relative standard deviation (RSD).

Bioactivity assay

Anti-oxidant activity

DPPH Free Radical-Scavenging Activity

DPPH free radical-scavenging activity was measured using methods described in previous studies,^25,38 with modifications. All test samples were dissolved in 10% DMSO at 5 different concentrations (10, 500 µg/mL for the extract and fractions and 100‐1000 µM for the compounds). The assay mixture consisted of 180 µL 0.1 mM DPPH in EtOH and 20 µL of the test samples. The mixture was shaken for 10 s using a vortex mixer and incubated at 37 °C for 20 minutes. The reaction was performed in a 96-well plate, and the activity was measured using a UV-Visible (VIS) spectrophotometer at 517 nm with L-ascorbic acid as a positive control.²⁵ The inhibitory activity was calculated using the following formula: (Ac − As/Ac) ×100, where Ac and As are the absorbance values of the control and sample, respectively. The half-maximal free radical-scavenging activity concentration (FSC₅₀) values of triplicate measurements of the samples and L-ascorbic acid, as the control, were calculated and expressed as the mean ± standard deviation (SD).

Anti-AD Activity

Cholinesterase (ChE) inhibitory activity

The cholinesterase (ChE) inhibitory activity was measured as previously reported,^17,39 with modifications, using AChE and BChE. All test samples were dissolved in 10% DMSO at 5 different final concentrations (10, 800 µg/mL for the extract and fractions and 10‐500 µM for the compounds). The assay mixture consisted of 20 µL of the test samples, 140 µL of 100 mM sodium phosphate buffer at pH 8.0, and 20 µL of 0.36 U/mL of either AChE or BChE. The mixture was incubated in a 96-well plate at room temperature for 15 minutes; then, 10 µL each of 0.5 mM DTNB and either ACh or BCh was added, followed by 15 minutes incubation. Subsequently, the mixture was analyzed at 412 nm using a spectrophotometer. Berberine, a typical ChE inhibitor, was used as a positive control.³¹ Inhibitory activity was calculated using the following formula: (Ac − As/Ac) ×100, where Ac and As represent the change in absorbance for the control and sample, respectively, after 15 minutes. The half-maximal inhibitory concentration (IC₅₀) values of triplicate measurements of the samples and berberine, as the control, were calculated and expressed as the mean ± SD.

BACE1 inhibitory activity

BACE1 activity was measured using a kit according to the manufacturer’s recommended protocol. All test samples were dissolved in 50 mM sodium acetate (pH 4.5) at 5 different final concentrations (25, 250 µg/mL for the extract and fractions and 25‐250 µM for the compounds). The assay mixture containing 10 µL of the test samples and 10 µL 1.0 U/mL BACE1 substrate (75 µM Rh-EVNLDAEFK-Quencher in 50 mM ammonium bicarbonate) was shaken for 5 s in a vortex mixer. To start the reaction, 10 µL BACE1 enzyme was added to the assay mixture in a blank 384-well plate, followed by incubation for 60 minutes at room temperature; then 10 µL BACE1 stop solution (50 mM sodium acetate at pH 4.5) was added. The fluorescence was subsequently measured using a spectrofluorometer at excitation and emission wavelengths of 545 and 585 nm, respectively, with quercetin as a positive control.³² The inhibitory activity was calculated using the following formula: (Ac − As/Ac) ×100, where Ac and As represent the change in fluorescence of the control and sample, respectively, after 60 minutes. The IC₅₀ values of triplicate measurements of the samples and quercetin were calculated as the mean ± SD.

Inhibitory activity against AGE formation

Inhibition of AGE formation was measured as described in previous studies,³² with modifications. All test samples were dissolved in 10% DMSO to 5 different final concentrations (10, 500 µg/mL for the extract and fractions and 10‐100 µM for the compounds). The assay mixture contained the following constituents: BSA (10 mg/mL), substrate as 0.4 M fructose and glucose, and 50 mM phosphate buffer (pH 7.4) with 0.02% sodium azide. The assay mixture was incubated in a black 96-well plate at 60 °C for 48 hours, and the fluorescence was measured using a spectrofluorometer at excitation and emission wavelengths of 350 and 450 nm, respectively. AG hydrochloride was used as a positive control.³² The inhibitory activity was calculated using the following formula: (Ac − As/Ac) ×100, where Ac and As represent the fluorescence of the control and sample, respectively. The IC₅₀ values of triplicate measurements of the samples and AG hydrochloride were calculated and expressed as mean ± SD.

Statistical Analysis

All assays were performed in triplicate, and the data, which are presented as the mean ± SD, were analyzed using one-way analysis of variance. Differences in the results were considered statistically significant at P < 0.05, P < 0.01, and P < 0.001.

Footnotes

Acknowledgments

This research was supported by the Chung-Ang University Research Scholarship Grants in 2019.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the Chung-Ang University Research Scholarship Grants in 2019.

ORCID iD

SukJin Lee

References

Aziz

Khaliq

Ur-Rehman

et al. Phytochemical screening and biological studies of Shilajit (Asphaltum). Int J Phytomedicine. 2017;9(1):15.doi:10.5138/09750185.1883

Carrasco-Gallardo

Guzmán

Maccioni

. Shilajit: a natural phytocomplex with potential procognitive activity. Int J Alzheimers Dis. 2012;2012:1-4.doi:10.1155/2012/674142

22482077

Lawley

Gupta

RC.

Goad

JT.

Canerdy

TD.

Kalidindi

. Anti-inflammatory and anti-arthritic efficacy and safety of purified Shilajit in moderately arthritic dogs. JVSAH. 2013;1(3).doi:10.15744/2348-9790.1.302

Ghosal

. Shilajit in Perspective. Narosa Publishing House; 2006.

Agarwal

SP.

Khanna

Karmarkar

Anwer

MK.

Khar

. Shilajit: a review. Phytother Res. 2007;21(5):401-405.doi:10.1002/ptr.2100

17295385

Ghosal

Lal

Singh

SK.

Goel

RK.

Jaiswal

AK.

Bhattacharya

. The need for formulation of Shilajit by its isolated active constituents. Phytother Res. 1991;5:211-216.doi:10.1002/ptr.2650050505

Schepetkin

IA.

Xie

Jutila

MA.

Quinn

. Complement-fixing activity of fulvic acid from Shilajit and other natural sources. Phytother Res. 2009;23(3):373-384.doi:10.1002/ptr.2635

19107845

Hiradate

Yonezawa

Takesako

. Isolation and purification of hydrophilic fulvic acids by precipitation. Geoderma. 2006;132(1-2):196-205.doi:10.1016/j.geoderma.2005.05.007

FC.

Evnas

RD.

Dillon

PJ.

Cai

. Rapid quantification of humic and fulvic acids by HPLC in natural waters. J Appl Geochem. 2007;22(8):1598-1605.doi:10.1016/j.apgeochem.2007.03.043

10.

Zhang

Liu

et al. Extraction and functional group characterization of fulvic acid from Hami lignite. ChemistrySelect. 2019;4:1448-1455.doi:10.1002/slct.201803291

11.

Konstantinov

Vladimirov

Grigoryev

AS.

Kudryavtsev

AV.

Perminova

IV.

Nikolaev

. Molecular composition study of Mumijo from different geographic areas using size- exclusion chromatography, NMR spectroscopy, and high-resolution mass spectrometry. Functions of Natural Organic Matter in Changing Environment. 2013:283-287.doi:10.1007/978-94-007-5634-2_52

12.

Galgóczy

Guba

Papp

Krisch

Vágvölgyi

Tserennadmid

. In vitro antibacterial effect of a mumijo preparation from Mongolia. Afr J Microbiol Res. 2011;5(22):3832-3835.doi:10.5897/AJMR11.798

13.

Sukhdolgor

Orkhonselenge

Davaadamdin

. Biochemical study of Mumijo in Uvs province, Mongolia. Mong J Chem. 2011;12:56-59.doi:10.5564/mjc.v12i0.173

14.

Yokozawa

Chen

CP.

Dong

Tanaka

Nonaka

GI.

Nishioka

. Study on the inhibitory effect of tannins and flavonoids against the 1,1-diphenyl-2 picrylhydrazyl radical. Biochem Pharmacol. 1998;56(2):213-222.doi:10.1016/S0006-2952(98)00128-2

9698075

15.

Kim

JO.

Kim

SJ.

Kim

JB.

Nam

WH.

Lee

JB.

Lee

. Comparison of ingredient and efficacy of Galgeun-tang (Gegen-tang) mix extract powder and decoction. J Physiol & Pathol Korean Med. 2019;33(1):39-47.doi:10.15188/kjopp.2019.02.33.1.39

16.

Ellman

GL.

Courtney

KD.

Andres

Featherstone

. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol. 1961;7(2):88-95.doi:10.1016/0006-2952(61)90145-9

13726518

17.

Ali

MY.

Jannat

Jung

HA.

Choi

RJ.

Roy

Choi

. Anti-Alzheimer's disease potential of coumarins from Angelica decursiva and Artemisia capillaris and structure-activity analysis. Asian Pac J Trop Med. 2016;9(2):103-111.doi:10.1016/j.apjtm.2016.01.014

26919937

18.

Kuk

EB.

AR.

et al. Anti-Alzheimer’s disease activity of compounds from the root bark of Morus alba L. Arch Pharm Res. 2017;40(3):338-349.doi:10.1007/s12272-017-0891-4

28093699

19.

Lee

YK.

Bang

HJ.

Whang

. Bioassay-guided isolated compounds from Morinda officinalis inhibit Alzheimer’s disease pathologies. Molecules. 2017;22(10):1638.

20.

Okoyama

Abe

Kurisaki

Wakita

. 13C and 27Al NMR study on the interaction in acidic aqueous solution between aluminium ion and iron, salicylic acid and phthalic acid: as model compounds with functional groups of fulvic acid. Anal Sci. 1997;13(Supplement):425-428.doi:10.2116/analsci.13.Supplement_425

21.

Rohloff

. Analysis of phenolic and cyclic compounds in plants using derivatization techniques in combination with GC-MS-based metabolite profiling. Molecules. 2015;20(2):3431-3462.doi:10.3390/molecules20023431

25690297

22.

Katherine

. NMR parameters of biologically important aromatic acids I. Benzoic acid and derivatives. J Magn Reson. 1969;2(3):361-376.doi:10.1016/0022-2364(70)90106-X

23.

Wang

Liu

Peng

Tong

Feng

. New pathways for the biodegradation of diethyl phthalate by Sphingobium yanoikuyae SHJ. Process Biochem. 2018;71(12):152-158.doi:10.1016/j.procbio.2018.05.010

24.

Sachin

SF.

Revannath

DD.

Gitaram

. Efficient and green protocol for the synthesis of hippuric acid. Int J Sci Res. 2020;9(1):1148-1150.doi:10.21275/ART20204235

25.

Panyo

Matsunami

Panichayupakaranant

. Bioassay-guided isolation and evaluation of antimicrobial compounds from Ixora megalophylla against some oral pathogens. Pharm Biol. 2016;54(9):1522-1527.doi:10.3109/13880209.2015.1107106

26809027

26.

Capllonch

MC.

García-Raso

Terrón

Apella

MC.

Espinosa

Molins

. Interactions of d(10) metal ions with hippuric acid and cytosine. X-ray structure of the first cadmium (II)-amino acid derivative-nucleobase ternary compound. J Inorg Biochem. 2001;85(2-3):173-178.doi:10.1016/S0162-0134(01)00196-9

11410237

27.

Qiu

Zhou

Jin

et al. In vitro antioxidant and antiproliferative effects of ellagic acid and its colonic metabolite, urolithins, on human bladder cancer T24 cells. Food Chem Toxicol. 2013;59:428-437.doi:10.1016/j.fct.2013.06.025

23811531

28.

Rashid

MA.

Majid

MA.

Quader

. Complete NMR assignments of (+)-usnic acid. Fitoterapia. 1999;70(1):113-115.doi:10.1016/S0367-326X(98)00033-1

29.

Braca

Sortino

Politi

Morelli

Mendez

. Antioxidant activity of flavonoids from Licania licaniaeflora . J Ethnopharmacol. 2002;79(3):379-381.doi:10.1016/S0378-8741(01)00413-5

11849846

30.

Tang

Gao

Xing

et al. Quercetin prevents ethanol-induced dyslipidemia and mitochondrial oxidative damage. Food Chem Toxicol. 2012;50(5):1194-1200.doi:10.1016/j.fct.2012.02.008

22365892

31.

Bierer

LM.

Haroutunian

Gabriel

et al. Neurochemical correlates of dementia severity in Alzheimer’s disease: relative importance of the cholinergic deficits. J Neurochem. 1995;64(2):749-760.doi:10.1046/j.1471-4159.1995.64020749.x

7830069

32.

Mukherjee

PK.

Kumar

Mal

Houghton

. Acetylcholinesterase inhibitors from plants. Phytomedicine. 2007;14(4):289-300.doi:10.1016/j.phymed.2007.02.002

17346955

33.

Heinrich

Lee Teoh

. Galanthamine from snow drop-the development of a modern drug against Alzheimer’s disease from local Caucasian knowledge. J Ethnopharmacol. 2004;92(2-3):147-162.doi:10.1016/j.jep.2004.02.012

15137996

34.

Dash

Emran

TB.

Uddin

MMN.

Islam

Junaid

. Molecular docking of fisetin with AD associated AChE, ABAD and BACE1 proteins. Bioinformation. 2014;10(9):562-568.doi:10.6026/97320630010562

25352723

35.

Sasaki

Fukatsu

Tsuzuki

et al. Advanced glycation end products in Alzheimer’s disease and other neurodegenerative diseases. Am J Pathol. 1998;153(4):1149-1155.doi:10.1016/S0002-9440(10)65659-3

9777946

36.

Diuzheva

Carradori

Andruch

et al. Use of innovative (micro)extraction techniques to characterise Harpagophytum procumbens root and its commercial food supplements. Phytochem Anal. 2018;29(3):233-241.doi:10.1002/pca.2737

29143440

37.

Gokhan

Marcello

Azzurra

et al. Chemical characterization, antioxidant properties, anti-inflammatory activity, and enzyme inhibition of Ipomoea batatas L. leaf extracts. Int J Food Prop. 2017;20:1907-1919.doi:10.1080/10942912.2017.1357127

38.

Connor

Vaughan

CW.

Vandenberg

. N-Acyl amino acids and N-acyl neurotransmitter conjugates: neuromodulators and probes for new drug targets. Br J Pharmacol. 2010;160(8):1857-1871.doi:10.1111/j.1476-5381.2010.00862.x

20649585

39.

Zhang

. Effects of galantamine on β-amyloid release and beta-site cleaving enzyme 1 expression in differentiated human neuroblastoma SH-SY5Y cells. Exp Gerontol. 2010;45(11):842-847.doi:10.1016/j.exger.2010.06.008

20600777

Development of Simultaneous Analysis Method for Multi-Compounds Content of New Shilajit Using HPLC-UV and the Cognitive Enhancing Effect: Mongolian Shilajit

Abstract

Keywords

Results and Discussion

Identification of Compounds 1‐6 Isolated From Mongolian Shilajit

Identifying the Origin of Mongolian Shilajit

DPPH Free Radical-Scavenging Activity of Mongolian Shilajit Extract and Fractions

Inhibitory Activity of the Extract, Fractions, and Compounds 1-6 of Mongolian Shilajit on AChE, BChE, and BACE1 Activity and AGE Formation

Validation of HPLC Analysis

Specificity

Linearity

Limit of Detection (LOD)/Limit of Quantification (LOQ)

Intra—Inter Day Precision & Trueness

Quantitative HPLC Analysis of Isolated Compounds

Conclusions

Materials and Methods

Plant and Raw Materials

Instruments and Reagents

Extraction, Fractionation, and Isolation of Mongolian Shilajit

UHPLC-ESI/LTQ-Orbitrap-HRMS Conditions

HPLC-UV Analysis

Sample preparation

Preparation of standard solution

Analytical conditions

Validation

Specificity

Linearity

Limit of detection and limit of quantitation (LOD, LOQ)

Precision and trueness

Bioactivity assay

DPPH Free Radical-Scavenging Activity

Anti-AD Activity

Cholinesterase (ChE) inhibitory activity

BACE1 inhibitory activity

Inhibitory activity against AGE formation

Statistical Analysis

Footnotes

Acknowledgments

Declaration of Conflicting Interests

Funding

ORCID iD

References