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

3.1. Quantification of echinacoside A in Rat and Human Liver S9 Analysis

echinacoside A was examined using rat and human liver S9 fractions and quantified by HPLC-QQQ/MS at different time points. The remaining verses time profile of echinacoside A, in rat and human liver S9 fraction treatment, are shown in Figure 1. Using the ‘well-stirred model’ and formula calculation [27], the predicted value is shown in Table 1. Rat liver S9 metabolized ~75.4% of echinacoside A within 60 min. Human liver S9 metabolized ~49.8% of echinacoside A within 120 min. In the presence of NADPH, echinacoside A was metabolized more slowly, with a t1/2 = 114.92 min in humans. EH is the fraction of the drug being filtered by the liver in one pass, in which the drug is irreversibly removed (extracted). We found that the EH of rat was higher than that of humans. This result is reasonable, judging from t1/2 values, because rat liver has been reported to metabolize faster than human liver [28]. Compared with other compound reports using rat liver S9, GKB202 of the Antrodia cinnamomea mycelium (t1/2 = 3.68 min) was metabolized more rapidly than echinacoside A [29]. 

Figure 1. Echinacoside A remaining versus time profile in rat and human liver S9. Data is presented in mean values of triplicate measurements. 

The previous report showed that there was no-observed-adverse-effect of 3 g/kg body weight/day echinacoside A-enriched HE in 28-day oral feeding in Sprague–Dawley rats. The current study dosage input for liver cell metabolism gives a reasonable correlation, with a previous in vivo study, of echinacoside A compound pharmacokinetics (PK) [30,31]. In one recent in vivo report, the Tsai et al. group detected echinacoside A in rat plasma after oral administration of HE mycelia (50 mg/kg) at 60 min [32]. This can correlate with the current in vitro findings, in which we demonstrated, theoretically, that there is still ~24.6% remaining of the compound after rat liver S9 metabolism. In addition, approximately half of unmetabolized echinacoside A were found in the remaining human liver S9, demonstrating that complete compound structures could enter the bloodstream, which contributes to its effectiveness in neuroprotection. Whether or not the common metabolites provide, the biological function is still under investigation and will be reported in a separate study

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3.2. In Vitro Metabolite Identifification in Rat and Human

Ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was used to identify the metabolites in the positive and negative modes. A comparison of echinacoside A metabolites, in this study, with rat and human liver S9 metabolites is shown in Table 2. Twenty-four metabolites were found after echinacoside A incubation and were assigned as M1-M24 (Table S1). Five common metabolites were detected after incubation with rat and human liver S9. The mass errors were all ≤5 ppm. The top three abundant metabolites, based on peak area, were Gluthation conjugation + Demethylation (M6), Demethylation (M2), and Demethylation + Hydrogenation (M3). Alcohols Dehydration (M1), Demethylation (M2), Demethylation + Hydrogenation (M3), 2 × Hydroxylation (M4), Demethylation, and two Hydroxylations (M5) metabolites were both identified in rat and human liver S9. The proposed metabolic pathway and possible metabolites of echinacoside A are shown in Figure 2. 

Table 2. Common liver metabolites of echinacoside A, were detected in rat and human liver S9, with an UPLC-QTOF/MS. 

The parent compound of the echinacoside A formula was C25H36O6. The MS spectrum of echinacoside A was found at [M + H]+ = 433.2573 and [M + Na]+ = 455.2405 (Figure 3A). The MS/MS spectrum by the collision energy of 10 V is shown in Figure 3B that corresponds to the cleavage of the C-O ether bond ([C20H29O2] + = 301.2162 and [C20H27O]+ = 283.2052) (Figure 3C) [15,32]. The Alcohols Dehydration metabolites (M1) formula was C25H34O5, with echinacoside A losing an H2O [33,34]. The MS spectrum of M1 was found at [M + H]+ = 415.2472 and [M + Na]+ = 437.2255. At a collision energy of 10V, the main product ions were m/z 283.2036, 215.1701, 181.1249, 135.0817, 119.0856, 105.0709, and 69.0327 (Figure 4A). Two product ions were analogous to those of echinacoside A ions (m/z 283.2036 and m/z 119.0856), and the production of m/z 301.2162 disappeared. Dehydrated M1 could be used to indicate water loss in pentose fragments [35]. The Demethylation metabolites (M2) formula was C24H34O6, with echinacoside A losing methylene (-CH2) [36]. At a collision energy of 40 V, the MS spectrum of M2 was found at [M + H]+ = 419.2407. The main product ions were m/z 312.0488, 269.9469, 231.0432, 123.0552, and 179.0486 (Figure 4B). The production at m/z 269.9469, 179.0486 corresponded to cleavage of the methylene (-CH3) and plus H+ . The Demethylation + Hydrogenation metabolites (M3) formula was C24H36O6 with M2 reduction in alkene (+H2) [37–39]. The MS spectrum of M3 was found at [M + H]+ = 421.2569 and [M + Na]+ = 443.2355. At a collision energy of 40 V, the main product ions were m/z 353.2141, 277.2183, 147.1172, and 77.0054 (Figure 4C). The production at m/z 353.2141, 277.2183 were corresponding to Hydrogenation. The 2 × Hydroxylation metabolites (M4) formula was C25H36O8 with echinacoside A Hydroxylation of isopropyl side chain and cyclopentene [40–42]. The MS spectrum of M4 was found at [M + H]+ = 465.2461 and [M + Na]+ = 487.2315. At collision energy of 20V, the main product ions were m/z 297.1842, 255.1389, 177.1115, 133.0860, and 89.0597 (Figure 4D). The production at m/z 297.1842, and 89.0598 were corresponding to Hydroxylation. The Demethylation and two Hydroxylation metabolites (M5) formula was C24H34O8 with M11 losing a methylene (-CH2). The MS spectrum of M5 was found at [M + H]+ = 451.2413 and [M + Na]+ = 473.2158. At a collision energy of 40 V, the main product ions were m/z 321.1516, 178.1225, 101.0705, and 59.0601 (Figure 4E). The production at m/z 321.1516 corresponded to M4 cleavage of the methylene (-CH3) and plus H+ .

There have been 70 different secondary metabolites isolated from either HE sporophore or mycelium. These metabolites are identified as polysaccharides, polyketides, phenols, terpenoids, and bioactive molecules [43–45]. We speculated that these common metabolites, which we analyzed, might also be the precursors or intermediate products of the mycelium secondary metabolites mentioned before. Nevertheless, it has been reported that M1, M2, M3, M4, and M5 may represent key intermediates between echinacoside A and secondary metabolites for rat and human studies. Due to the increasing use of HE mycelium as a functional supplement, identifying these metabolites will also reveal useful biomarkers. Taking cholesterol as an example, the precursors or intermediate products of various substances, produced by the liver enzymes process, were metabolized to oxysterol, which is now understood as a potent metabolite in sterol metabolism and synthesis regulation [46]. 

Figure 3. (A) MS spectrum of echinacoside A with an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry. (B) MS/MS spectrum of echinacoside A with an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry. (C) The proposed structures of product ions, produced by echinacoside A, at a collision energy of 10 V. (a) [C20H29O2]+ = 301.2162, (b) [C20H27O]+ = 283.2052.  : Parent ion.

Figure 4. MS and MSMS spectrum of (A) Alcohols Dehydration (M1), (B) Demethylation metabolites (M2), (C) Demethylation + Hydrogenation metabolites (M3), (D) 2 × Hydroxylation metabolites (M4), (E) Demethylation and two Hydroxylations (M5) metabolite using an ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry.  : Parent ion. 

M2 and M3 were detected at 60 min for rat liver S9, whereas for human liver S9, M2 and M3 were detected at 10, 30, 60, and 120 min. We conjectured that M3 might be produced by two distinct metabolic pathways; one from echinacoside A to M3, and the other was derived from M1. In contrast, M4 and M5 were detected at 10, 30, and 60 min rat liver S9. In human liver S9, M4 was detected at 10, 30, 60, and 120 min. M5 was detected at 120 min. The time of appearance can explain that M5 metabolic pathway may be derived from M4. 

This study provides additional information for the HE mycelium clinical studies missing information, which is the metabolic action from the bioactive compound. In a recent randomized, double-blind placebo-controlled study, three 350 mg/g HE mycelia (containing 5 mg/g echinacoside A) capsules intervention, for 49 weeks, demonstrated higher Cognitive Abilities Screening Instrument, Mini-Mental State Examination, and Instrumental Activities of Daily Living scores and achieved a better contrast sensitivity, in patients with age > 50 years old and diagnosis of probable Alzheimer’s Disease, when compared to the placebo group [19]. By understanding echinacoside A metabolites profile, we can interpret that echinacoside A enriched HE mycelia improved neurocognitive disorder, which might be contributed mostly from the unmetabolized compound. As for echinacoside A, metabolites are well-tolerated and might be important in achieving neuro-nutritional benefits, which will be investigated in future studies. 

This is the first study to establish the mass spectrometry analysis of metabolic stability and identify of fifteen common metabolites of echinacoside A found in rat and human liver S9 fractions. The results from this study will help to understand the characteristics, safety, and efficiency of this unique bioactive compound. Our results indicate that echinacoside A’s clinical benefits may arise from the compound and its metabolites. In the future, we will analyze metabolites from blood, organs, and tissues to establish a complete metabolism database.Such work is expected to help functional research food, nutraceutical supplements, and precision medicine.

Supplementary Materials: The following are available online at 1, Table S1: Identity of echinacoside A metabolites in rat and human liver S9 with a UPLC-QTOF/MS. 

Author Contributions: Data interpretation, Y.-H.K., T.-W.L., J.-Y.L., Y.-W.C. and T.-J.L.; methodology; Y.-H.K., T.-W.L. and T.-J.L.; investigation conducted by T.-J.L. and C.-C.C.; raw materials provided by: Y.-H.K., T.-W.L., J.-Y.L., Y.-W.C., T.-J.L. and C.-C.C.; visualization: Y.-H.K.; original draft preparation was done by Y.-H.K., T.-W.L., T.-J.L. and C.-C.C.; writing—review and editing, Y.-H.K., T.-J.L. and C.-C.C. All authors have read and agreed to the published version of the manuscript. 

Funding: This research received no external funding. 

Data Availability Statement: The raw materials and compounds presented in this study are available from the corresponding author upon request. 

Conflicts of Interest: The authors declare no conflict of interest.

Sample Availability: Samples of the compounds are available from the authors. 

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