Abstract
ReDuNing injection, prepared from a combination of Gardenia Jasminoides fruits, Lonicera japonica flower buds, and the Artemisia annua aerial part, is extensively used for treatment of viral upper respiratory tract infections in China. Iridoids, organic acids, and flavonoids are likely important compounds of the herbal injection because of their reported pharmacological properties. This study was designed to characterize pharmacokinetics and disposition of the major circulating herbal compounds in rats that received the injection intravenously. ReDuNing injection was found to contain 19 iridoids (content levels 0.01–27.93 mM), 16 organic acids (0.04–19.06 mM), and 11 flavonoids (<0.08 mM). After dosing the injection, the iridoids geniposide, secologanic acid, secoxyloganin, genipin-1-β-gentiobioside, geniposidic acid, sweroside, and shanzhiside and the organic acids chlorogenic acid, quinic acid, cryptochlorogenic acid, and neochlorogenic acid were found to be the major circulating compounds, with mean elimination half-lives of 0.2–0.9 hour; the other plasma compounds were at low exposure levels. These major circulating compounds exhibited small apparent volumes of distribution (0.03–0.34 l/kg). Most of the iridoids were eliminated predominantly via renal excretion of the unchanged compounds, whereas the organic acids were eliminated via methylation and sulfation and were excreted into urine as the unchanged and metabolized compounds. The methylated metabolites also underwent subsequent conjugations before hepatobiliary and renal excretion. In vitro data suggested that the metabolism of the organic acids in rats also occurred in humans. The current pharmacokinetic research could serve as a crucial step in identifying the chemical basis responsible for the therapeutic action of ReDuNing injection.
Introduction
ReDuNing is an herbal injection, which was approved in 2005 by the China Food and Drug Administration (China FDA), for the treatment of viral upper respiratory tract infections associated with high fever, chills, headache, myalgia, and cough with phlegm. Each milliliter of the injection is prepared from a combination of 0.60 g of Gardenia jasminoides fruits (Zhizi), 0.75 g of Lonicera japonica flower buds (Jinyinhua), and 1.25 g of the Artemisia annua aerial part (Qinghao), yielding an herb-to-injection ratio of 2.6:1. The injection is available as a sterile and nonpyrogenic dosage form for intravenous administration at a dosage of 20 ml once daily for three days. ReDuNing therapy appears to have a low incidence of side effects (Xu et al., 2009). Recently, ReDuNing injection has been also used as an add-on therapy in the conventional treatment of hand, foot, and mouth disease in children infected with coxsackievirus type A16 or enterovirus type 71 (Li et al., 2014). Despite its extensive use in clinics, little is known about the chemical basis responsible for the therapeutic action of ReDuNing injection.
Pharmacologically active constituents existing in adequate abundance in an herbal medicine and exhibiting favorable pharmacokinetic profiles are most likely to form the chemical basis responsible for the medicine’s therapeutic action. ReDuNing injection has been reported to contain iridoids, organic acids, and flavonoids (Li et al., 2015a). These compounds are important because of their reported antiviral, anti-inflammatory, and antioxidative properties as pure isolates (Shang et al., 2011; Liu et al., 2013). Although bioanalytical assays were developed to measure the concentrations of some iridoids and organic acids in human and rat plasma after dosing ReDuNing injection (Ni et al., 2015; Wang et al., 2015), pharmacokinetic information about ReDuNing injection is still limited. This hinders our understanding of the chemical basis responsible for the therapeutic action of ReDuNing injection. The current study was designed to assess the systemic exposure to ReDuNing compounds in rats that received the herbal injection intravenously and to investigate the pharmacokinetics and elimination of the major circulating herbal compounds.
Materials and Methods
A detailed description of the materials and methods is provided in the Supplemental Materials and Methods, which are available online.
ReDuNing Injection and Its Component Herbs.
Samples of six lots of ReDuNing injections (China FDA drug ratification number, GuoYaoZhunZi-Z20050217) and samples of its component herbs G. Jasminoides fruits, L. japonicae flower buds, and A. annua aerial part were obtained from Jiangsu Kanion Pharmaceutical Corporation (Lianyungang, Jiangsu Province, China).
Chemicals and Reagents.
Reference standards of iridoids, organic acids, and flavonoids (purity ≥98% by high-pressure liquid chromatography) were obtained from Tauto Biotech (Shanghai, China), Shanghai Nature Standard R&D and Biotech (Shanghai, China), BioBioPha (Kunming, Yunnan Province, China), and Sigma-Aldrich (St. Louis, MO).
Rat Studies.
All animal care and experimental procedures were in compliance with government guidelines (The Ministry of Science and Technology of China, 2006) and were approved by an institutional animal care and use committee at Shanghai Institute of Materia Medica (Shanghai, China). Male Sprague-Dawley rats (200–230 g) were obtained from the Sino-British SIPPR/BK Laboratory Animal (Shanghai, China). Three rat studies were performed by giving a single 30-minute intravenous infusion of ReDuNing injection at 2 ml/kg via the tail veins. The rat dose was derived from the label daily dose for ReDuNing injection (20 ml/person) according to the dose normalization by body surface area (Reagan-Shaw et al., 2008).
In the first rat study, blood samples (∼80 μl) were collected from four rats, into heparinized tubes, before and at 0.083, 0.25, 0.5, 0.58, 0.75, 1, 1.5, 2.5, 4.5, 6.5, 8.5, and 24 hours after starting the infusion. The plasma fractions were prepared by centrifuging the blood samples. In the second rat study, urine samples were collected from four rats before and at 0–4, 4–8, and 8–24 hours after starting the infusion. In the third rat study, bile samples were collected from four rats before and at 0–2, 2–4, 4–6, 6–8, and 8–24 hours after starting the infusion.
In Vitro Metabolism Studies.
To characterize the metabolism of the herbal compounds in rats and to facilitate the prediction of the metabolism in humans, the metabolic capacities of cytochrome P450 (P450), carbonyl reductase (CR), catechol-O-methyltransferase (COMT), UDP-glucuronosyltransferase (UGT), and sulfotransferase (SULT) for ReDuNing compounds were assessed using rat liver microsomes and cytosol, as well as human liver microsomes and cytosol. The metabolic reactions were repeated by adding glutathione (GSH) into the incubations. Also, interplays of COMT with UGT and with SULT were also evaluated using the methods described by Li et al. (2015b).
Determination of Plasma Protein Binding.
Fractions of unbound compounds to rat plasma proteins (fu) were assessed by an ultrafiltration method (Guo et al., 2006).
Liquid Chromatography/Mass Spectrometry-Based Assays.
A Waters Synapt G2 high-definition time-of-flight mass spectrometer (Manchester, UK), interfaced via a Zspray/LockSpray electrospray ionization source with a Waters Acquity UPLC separation module (Milford, MA), was used for analysis of unchanged and metabolized herbal compounds in ReDuNing injection, the component herbs, rat biosamples, and in vitro metabolism study samples.
Data Processing.
The pharmacokinetic parameters were estimated by noncompartmental analysis using Kinetica software (version 5.0; Thermo Scientific, Philadelphia, PA). All data are expressed as mean ± standard deviation.
Results and Discussion
Analysis of the chemical composition of ReDuNing injection was the first step in the pharmacokinetic research on the injection. The aims of the analysis were to understand which herbal compounds and what quantity were introduced into the bloodstream by dosing the injection and to understand the injection’s lot-to-lot variability. A total of 46 compounds (19 iridoids, 16 organic acids, and 11 flavonoids) were detected in ReDuNing injections (Fig. 1). The compounds could be graded according to their dose levels from a ReDuNing injection at the label daily dose of 20 ml/person. The iridoids geniposide (compound 6) and secologanic acid (compound 4) and the organic acids chlorogenic acid (compound 30), quinic acid (compound 25), cryptochlorogenic acid (compound 31), and neochlorogenic acid (compound 29) exhibited dose levels of >100 μmol/person (135–592 μmol/person); the injection exhibited small lot-to-lot variability for these compounds (3.1–7.9%), except for compound 4 at 26.4% (Supplemental Table 1). The sum of the dose levels of compounds 6 and 4 was 78.2% of the total dose of iridoids present in ReDuNing injection, whereas the sum of the dose levels of compounds 30, 25, 31, and 29 was 89.6% of the total dose of organic acids in the injection. The dose levels of secoxyloganin (compound 12), genipin-1-β-gentiobioside (compound 16), geniposidic acid (compound 3), sweroside (compound 2), shanzhiside (compound 9), isochlorogenic acid C (compound 35), caffeic acid (compound 24), isochlorogenic acid B (compound 33), and isochlorogenic acid A (compound 34) were in the range of 10–100 μmol/person (13–69 μmol/person); the lot-to-lot variability for these compounds was 6.0–16.1%. The remaining ReDuNing compounds (including all the detected flavonoids) were in the range of <10 μmol/person (0.0007–8 μmol/person); the lot-to-lot variability for these minor compounds was 6.3%–61.7%. Most of the ReDuNing iridoids originated from the component herbs Zhizi and Jinyinhua. However, compound 6 originated predominantly from Zhizi, whereas compound 4 originated predominantly from Jinyinhua. Most of the ReDuNing organic acids originated from Zhizi, Jinyinhua, and Qinghao. However, compounds 30 and 25 originated predominantly from Jinyinhua. Despite being minor constituents present in the component herbs, compounds 31 and 29 were also major ReDuNing organic acids, which, in part, were transformed from compound 30 during the preparation of the injection. Dawidowicz and Typek (2014) reported heating-induced conversion of chlorogenic acid into cryptochlorogenic acid and neochlorogenic acid in water.
Iridoids (both compound names and ID numbers, black), organic acids (purple), and flavonoids (brown) present in ReDuNing injection. (A) Stacked chromatograms of herbal compounds from a typical sample of ReDuNing injection by mass spectrometry–based monitoring of their ionized molecules. (B–D) Content levels (mg/g) of compounds in the component herbs Zhizi (G. Jasminoides fruits), Jinyinhua (L. japonicae flower buds), and Qinghao (A. annua aerial part), respectively. (E) Content levels (mM) of the compounds in samples of six lots of ReDuNing injection. (F) Compound dose levels (μmol/person) of the compounds from ReDuNing injection (lot number 141106) at the label daily dose of 20 ml/person. (G) Percentage doses of iridoids in the total dose of iridoids and percentage doses of organic acids in the total dose of organic acids in ReDuNing injection (lot number 141106). See Supplemental Table 1 for the compounds’ names and the associated detection and characterization information.
To investigate the systemic exposure to ReDuNing compounds, rats received a 30-minute intravenous infusion of the herbal injection. As shown in Fig. 2, a total of 39 unchanged herbal compounds (19 iridoids, 16 organic acids, and 4 flavonoids) were detected in the plasma samples of the rats after starting the infusion. The iridoids geniposide (compound 6), secologanic acid (compound 4), secoxyloganin (compound 12), and geniposidic acid (compound 3) and the organic acids chlorogenic acid (compound 30), quinic acid (compound 25), cryptochlorogenic acid (compound 31), and neochlorogenic acid (compound 29) exhibited high levels of systemic exposure; the iridoids sweroside (compound 2), genipin-1-β-gentiobioside (compound 16), and shanzhiside (compound 9) were at lower levels. The chemical structures of these major circulating ReDuNing compounds are shown in Fig. 2. The plasma flavonoids detected were at quite low levels. A total of 40 unchanged ReDuNing compounds were detected in the rat urine samples, whereas only 24 such compounds were detected, all at low levels, in the bile samples (Fig. 2). Renal excretion of unchanged compound was found to be the main elimination pathway for the major circulating iridoids compounds 6, 12, 16, 2, and 9, with fractions of doses excreted into urine (fe-U) of 64.0–87.5%, whereas the fe-U of the iridoid compound 3 was 334.8%. As shown in Supplemental Table 2B, an in vitro metabolism study using rat liver microsomes suggested that P450-mediated and NADPH-dependent oxidative ester cleavage of compound 6 probably took place in rats to yield compound 3. Unlike for these iridoids, renal excretion was a minor elimination pathway for the iridoid compound 4 (fe-U, 7.3%), and the same was true for biliary excretion (fraction of dose excreted into bile, fe-B; 0.1%). A reduced metabolite of compound 4 (M4R) was detected in rat plasma, urine, and bile samples (Supplemental Fig. 1 and Supplemental Table 2A); the metabolism was probably mediated by carbonyl reductase (CR) in rat liver cytosol fortified with NADPH (Supplemental Table 2B) and could be inhibited by quercetin. The organic acid compound 25 was also primarily eliminated unchanged via renal excretion (fe-U, 89.7%). The fe-U values of the other organic acids, compounds 30, 31, and 29 (31.9–44.2%), were lower than that of compound 25. Several metabolites of these compounds were detected in the rats (Supplemental Fig. 1 and Supplemental Table 2A). Because compounds 30, 31, and 29 were isomers with the same molecular mass 354 daltons, characterization of the parent compound and metabolite relationship was achieved by in vitro metabolism study using purified compounds as substrates (Supplemental Table 2B). As a result, the detected plasma metabolites were methylated metabolites of compound 30 (M30M-1 and M30M-2), 31 (M31M-1 and M31M-2), and 29 (M29M-1 and M29M-2) and their sulfated metabolites (M30S-2, M31S, and M29S). These metabolites were excreted into urine and, all to a lesser extent, into bile. In addition, the methylated metabolites were subsequently glucuronized to yield M30M-G, M31M-G, and M29M-G (excreted into urine and bile) or conjugated with GSH to yield M30M-GSH-1, M30M-GSH-2, M31M-GSH, and M29M-GSH (all excreted only into bile). Figure 3 shows the proposed metabolic pathways of compounds 6, 4, 30, 31, and 29 in rats intravenously receiving the ReDuNing injection. The in vitro metabolism studies using human liver microsomes and cytosol suggested that the metabolism of compounds 6, 4, 30, 31, and 29 in rats probably occurred similarly in humans (Supplemental Table 2B).
Systemic exposure to and excretion of unchanged iridoids (compound ID numbers, black), organic acids (purple), and flavonoids (brown) in rats receiving a 30-minute intravenous infusion of ReDuNing injection (lot number 141106) at 2 ml/kg and chemical structures of the major circulating herbal compounds. (A–C) ReDuNing compounds (shown as compound ID) ranked according to their content levels in ReDuNing injection (Fig. 1E). See Supplemental Table 1 for the compounds’ names. The symbol × (in red) denotes that the compound was not detected.
Proposed metabolic pathways for the iridoids (in black) geniposide (compound 6) and secologanic acid (compound 4) and the organic acids (in purple) chlorogenic acid (compound 30), cryptochlorogenic acid (compound 31), and neochlorogenic acid (compound 29) in rats intravenously receiving ReDuNing injection. The metabolite ID is used to indicate a compound being a metabolite, showing its parent compounds, type of metabolism, and metabolite isomer. For instance, M30 in M30M-1 denotes that the compound is a metabolite of chlorogenic acid (compound 30). G, glucuronidation; Glc, b-Dglucopyranosyl; Glu, glucuronosyl; GSH, glutathionylation; M, methylation; 1, first eluted metabolite isomer; R, reduction; S, sulfation.
Table 1 summarizes the pharmacokinetic parameters of the major circulating herbal compounds in rats receiving the ReDuNing injection. Their plasma concentrations rose as the 30-minute infusion continued, with the maximum plasma concentrations (Cmax) being measured just before completion of the infusion (Supplemental Fig. 2). The ReDuNing compounds exhibited short elimination half-lives (t1/2), i.e., 0.3–0.9 hour for the iridoids geniposide (compound 6), secologanic acid (compound 4), secoxyloganin (compound 12), genipin-1-β-gentiobioside (compound 16), geniposidic acid (compound 3), sweroside (compound 2), and shanzhiside (compound 9) and 0.2–0.5 hour for the organic acids chlorogenic acid (compound 30), quinic acid (compound 25), cryptochlorogenic acid (compound 31), and neochlorogenic acid (compound 29). The mean total plasma clearance (CLtot,p) values of the iridoids and organic acids were 3–22% and 7–21% of rat cardiac plasma output (7.3 l/hour/kg; Toutain and Bousquet-Mélou, 2004), respectively, indicating the clearance of these compounds was not high. The mean distribution volumes at steady state (VSS) of iridoids (0.2–0.4 l/kg) and organic acids (0.3–0.4 l/kg) were close to the rat extracellular volume (0.3 l/kg; Davies and Morris, 1993). The iridoids and organic acids were poorly bound to rat plasma protein with fractions of compounds unbound to rat plasma proteins (fu) of 75.2–99.0% and 48.8–65.8%, respectively.
Pharmacokinetics of major circulating iridoids and organic acids in rats receiving a 30-minute intravenous infusion of ReDuNing injection
Chinese herbal medicines are often combinations of multiple herbs and have very complex chemical compositions. Pharmacokinetic research, serving as a compound sieve (Liu, et al., 2009), is a crucial step in identifying the chemical basis responsible for the therapeutic actions of herbal medicines. In the current study, 11 unchanged herbal compounds (seven iridoids and four organic acids) showed considerably high levels of systemic exposure with short t1/2 (<1 hour) in rats intravenously receiving ReDuNing injection. Also, several methylated and sulfated metabolites of the organic acids exhibited high levels of systemic exposure. Further investigation of these major circulating herbal compounds, including: (1) assessing their pharmacodynamic activities related to the injection’s therapeutic action, (2) exploring the translation of the compounds’ individual activities into the injection’s overall action, and (3) evaluating their pharmacokinetic compatibility (i.e., exhibiting limited pharmacokinetic interaction problems that can counteract the compounds’ synergistic or additive pharmacodynamic effects), will finally lead to understanding the chemical basis responsible for the therapeutic action of ReDuNing injections. The current pharmacokinetic study in rats also facilitates designing a future clinical pharmacokinetic study of ReDuNing injections and future systematic evaluation of potential pharmacokinetic herb–drug interactions related to the injection.
Authorship Contributions
Participated in research design: C. Li, Cheng.
Conducted experiments: Cheng, Du, Yu, Xu, F. Wang, L. Li, Olaleye, Yang, Chen, Zhong, Liu, J. Li.
Contributed new reagents: Xiao, Z. Wang.
Performed data analysis: C. Li, Cheng.
Wrote or contributed to the writing of the manuscript: C. Li, Cheng.
Footnotes
- Received May 18, 2016.
- Accepted September 1, 2016.
This work was supported by the National Science Fund of China for Distinguished Young Scholars [Grant 30925044], the National Science and Technology Major Project of China “Key New Drug Creation and Manufacturing Program” [Grants 2009ZX09304-002], and the National Basic Research Program of China [Grant 2012CB518403].
This work was previously presented as a poster presentation as follows: Cheng C, Du F, Yu K, Wang F, Xu F, Chen F, Li L, Zhong C, Li J, Wang Z et al. (2016) Pharmacokinetics and disposition of circulating iridoids and phenolic acids after intravenous administration of ReDuNing injection in rats. Abstract P-PKPD-16 13th Meeting of the Asia Pacific Federation of Pharmacologists, “New Paradigms in Pharmacology for Global Health”; 2016 1–3 Feb; Bangkok, Thailand. Asia Pacific Federation of Pharmacologists.
↵
This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- COMT
- catechol-O-methyltransferase
- fe-U
- fraction of dose excreted into urine
- GSH
- glutathione
- P450
- cytochrome P450
- SULT
- sulfotransferase
- t1/2
- elimination half-life
- UGT
- uridine 5′-diphospho-glucuronosyltransferase
- Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics
In this issue
Rat Pharmacokinetic Studies of ReDuNing Injection
