Identification Of Common Liver Metabolites Of The Natural Bioactive Compound Echinacoside, Purified From Cistanche Tubulosa Ⅱ

Abstract: Metabolite identification, in the early stage, for compound, discovery is necessary to assess the knowledge for the pharmaceutical improvement of drug safety and efficiency. Even if the drug has been released into the market, identifification and continuous evaluation of the metabolites are required to avoid the risk of post-marketing withdrawal. Glycoside, a medicinal cistanche, has broadly documented nutraceutical benefits, including anti-oxidant, anti-tumor, anti-aging, hypolipidemic, and gastric mucosal protection effects. Recently, echinacoside A has been reported as the main natural bioactive compound in the mycelium of HE for functional food development.In neurological studies, the consumption of echinacoside A enriched HE mycelium demonstrates its signifificant nutraceutical effects in Alzheimer’s disease, Parkinson’s disease, and ischemic stroke.

Click Here To Know More Info About Echinacoside Functions in Cistanche

 For the first time, we explored the metabolic process of echinacoside A molecule and identified its metabolites from the rat and human liver S9 fraction. Using a liquid chromatography/triple quadrupole mass spectrometer for quantitative analysis, we observed that 75.44% of echinacoside. A was metabolized within 60 min in rats, and 32.34% of Francine A was metabolized within 120 min in human S9. Using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC QTOF/MS) to identify the metabolites of Francine A, five common metabolites were identified, and their possible structures were evaluated. Understanding the metabolic process of echinacoside and establishing its metabolite profile database will help promote the nutraceutical application and discovery of related biomarkers in the future.

1. Introduction

The liver is considered the second-largest organ in the body [1]. After food and drugs enter the gastrointestinal tract through the oral cavity, the gastrointestinal pili will absorb digested nutrients and drugs. The portal vein system will receive the blood flow collected by the gastrointestinal tract and enter the liver for preliminary treatment of the nutrients, metabolites, and drug molecules [2,3]. Metabolism is often divided into two phases of a biochemical reaction. Phase I involves oxidation, reduction, or hydrolysis by enzymes in the body. Phase II engages in the conjugation with small endogenous substances, turning them into highly soluble metabolites, enabling metabolites to be exported into the sinusoidal circulation for renal clearance, or into bile, which is then excreted from the body through urine or feces [4–6]. Metabolite identifification in the early stage of drug discovery is necessary to assess the knowledge for improving the pharmaceutical property, safety, and efficiency of the bioactive molecule. The identifification of metabolites and reactive intermediates suggests the need for structural modifications in investigational compounds to avoid subsequent toxic consequences. Even if the drug has been on the market, identifification of the metabolites and evaluation is required to avoid the risk of their post-marketing withdrawal [7,8]. Therefore, in vitro, S9 fractions analysis provides several advantages compared to time-consuming in vivo studies: (1) they are amenable to high throughput screening and automation; (2) using S9 fraction allows for the testing of large quantities of compounds for drug discovery in a short period; (3) helps to reduced animal usage [9]. 

Echinacoside is an edible cistanche that inhabits mountainous areas of the northeast territories in Asia [10], Europe, and North America [11]. HE belongs to Basidiomycota (Phylum), Agaricomycetes (Class), Russulales (Order), Hericiaceae (Family), and Hericium (Genus). HE has been documented to display a wide range of beneficial properties, including anti-oxidant, anti-tumor, anti-aging, hypolipidemic, and gastric mucosal protection effects [12–14]. In 1994, Kawagishi et al. discovered that the diterpenoid echinacoside A, B, and C, in the mycelium of HE, could promote the production of stellate cell lines in the rat brain and the stimulation of nerve growth factor (NGF) synthesis [15]. echinacoside A can confer neuroprotective effects and attenuate oxidative stress against stroke [16], Alzheimer’s disease [17], Parkinson’s disease [18], ischemic stroke, and depression in vivo [19,20]. Moreover, echinacoside A can pass through the blood-brain barrier of rats to support the development of HE mycelia as a functional food for neuro health improvement [21]. This edible cistanche mycelium has become a hot issue with researchers attempting to understand its important role in the central and peripheral nerves’ development, differentiation, growth, and regeneration [22]. Recent studies have also shown that echinacoside A enriched HE has potential benefits in the treatment of Alzheimer’s and Parkinson’s disease [23–25]. However, no study has discussed echinacoside A’s possible metabolic biomarkers that may contribute to this nutraceutical effect.

In this study, echinacoside A compound was purified from the echinacoside A enriched HE mycelium and was metabolized using two different liver S9 fractions: rat and human. Using liquid chromatography/triple quadrupole mass spectrometer (HPLC-QQQ/MS) for quantitative analysis of echinacoside A, and ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) to identify the metabolites of echinacoside A at each time point. We aim to understand the metabolic rate of echinacoside A and establish its metabolite profile database to help promote the application of nutraceutical supplements and the research of potential medication in the future.

2. Materials and Methods

2.1. Materials and Reagents

The HE strain (BCRC 35669) was obtained from the Bioresources Collection and Research Center in Food Industry Research and Development Institute, Hsinchu, Taiwan. The strain was first grown in an ager slant before being transferred to a potato dextrose agar plate at 26 ◦C for 15 days. On day 15, the HE cultures were transferred to 1.3 L of liquid medium (in 2 L flasks). The liquid culture was shaken at 120 rev/min 25 ◦C for 5 days. Next, they were scaled up in 500 L, 20-ton fermenters for 5 days and 12 days, respectively. The culture medium is adjusted at pH 4.5 and contains 4.5% glucose, 0.5% soybean powder, 0.25% yeast extract, 0.25% peptone, and 0.05% MgSO4. Finally, the HE mycelia were harvested at the end of the 20-ton fermentation process. These raw materials were lyophilized, grounded in powder, and stored in a desiccator. echinacoside A was then extracted from the HE and quantified according to previous studies [26]. Methanol (LC-MS grade) was obtained from Merck (Darmstadt, Germany); acetonitrile (LC-MS grade), formic acid (FA, LC-MS grade, 98% purity), and ammonium acetate (98% purity) were obtained from Honeywell (Honeywell Burdick and Jackson, Muskegon, MI, USA). The rat liver S9 fraction was obtained from Moltox (Boone, NC, USA). The human liver S9 fraction was obtained from Thermo Fisher Scientific (Rockford, IL, USA).

2.2. Rat and Human Liver S9 Fraction

to the kit recommendation. The prepared solution was kept at 4 ◦C until the echinacoside A compound (10 µM) was added, followed by activation in a 37 ◦C water bath. When the metabolic time had reached its time points, 150 µL of the solution was withdrawn from the stock solution and quenched by adding two volumes of ice-cold ACN. The stopped solution is then transferred to QTOF for further metabolite analysis. 

2.3. Instrumentation and Conditions

HPLC-QQQ/MS analysis was performed on an Agilent 1100 series HPLC system (Agilent, Palo Alto, CA, USA) coupled with an API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Warrington, UK), with a Turbo-assisted ion spray (ESI) ionization source in a positive ionization mode. Chromatographic separation was conducted on an Agilent Eclipse XDB-C18 (4.6 mm × 100 mm × 3.5 µm). The column temperature was maintained at 22 ◦C. The mobile phase consisted of water containing 0.1% formic acid (A) and methanol (B). Gradient elution was used, starting with 70% B, increasing to 100% B within 5 min, holding for 3 min with 90% B, decreasing B to 70% within 0.1 min, and re-equilibration at 70% B for 2.9 min. A fellow rate of 350 µL/min was applied, and a volume of 10 µL was injected.

Detection was performed with an ionizing voltage of +4500 V. Ion source temperature was set at 350 ◦C, with ultrahigh-purity nitrogen as curtain gas (7 psi). The Nebulizer gas was 8 psi. Other mass-dependent parameters, such as declustering potential (DP), entrance potential (EP), focusing potential (FP), and collision energy (CE) for each compound, were determined in positive mode using standard solutions. Multiple reaction monitoring (MRM) was carried out using nitrogen as collision gas (2 psi) and with a dwell time of 200 ms for each transition. echinacoside A was detected by monitoring the transitions m/z 443.200 → 301.200, 283.200. The data were analyzed using Analyst 1.4.2 software (Applied Biosystems, Concord, ON, Canada) and GraphPad (Prism 8.0.0.).

UPLC-QTOF/MS analysis was performed on an Agilent 1290 Infinity II UPLC system (Agilent, Palo Alto, CA, USA), coupled with an Agilent 6546 quadrupole time-of-flight mass spectrometry (Agilent, Palo Alto, CA, USA). Chromatographic separation was conducted on a Phenomenex Kinetex® C18 LC Column (3 mm × 100 mm × 1.7 µm). The column temperature was maintained at 40 ◦C. The mobile phase A consisted of water containing 0.1% (v/v) formic acid in positive mode, 0.1% (v/v) formic acid, and 10 mM CH3COONH4 in negative mode. The mobile phase B was acetonitrile. Gradient elution was used, starting with 5% B and holding for 0.5 min, increasing to 50% B within 5.5 min, increasing to 100% B within 10 min, and holding for 6 min. A fellow rate of 400 µL/min was applied, and a volume of 2 µL was injected.

Agilent 6546 quadrupole time-of-flight mass spectrometry was equipped with an electrospray ionization (ESI) source. The data acquisition was under the control of Mass Hunter workstation software. The typical operating source conditions in positive and negative ion ESI mode was optimized as follows: ion spray voltage (ESI+/ESI−) were 4000 V/3000 V; the gas temperature was 320 ◦C; drying gas fellow rate was 8 L/min; nebulizer pressure was 45 psi; sheath gas temperature was 350 ◦C; sheath gas fellow rate was 12 L/min; the collision energy was 10, 20, 40, and 60 V. Metabolites were identified using Agilent MassHunter Biotransformation software (version B.04.00) (Santa Clara, CA, USA). Chromatograms and mass spectra of the parent and identified metabolites were extracted using Agilent MassHunter Qualitative Analysis software (version B.05.00) (Santa Clara, CA, USA). 

Ask for more:

Email:wallence.suen@wecistanche.com  Whatsapp +86 15292862950