Identification of known chemicals and their metabolites from Alpinia oxyphylla fruit extract in rat plasma using liquid chromatography/tandem mass spectrometry (LC–MS/MS) with selected reaction monitoring
Introduction
Pharmacokinetic (PK) information is helpful in the design and interpretation of clinical studies and mechanism of action. Since herbal products often consist of more than one chemical constituent and the active constituents are often unknown, standard PK measurements that expected for synthetic or highly purified new drugs to demonstrate systemic exposure to a product in animals and/or humans may be difficult to obtain. However, when feasible, sponsors and investigators are encouraged to monitor the blood levels of known active constituents, representative markers, or major chemical constituents in a botanical drug product [1].
Alpinia oxyphylla (Zingiberaceae, Yizhi in Chinese), an herbaceous perennial plant, its capsularfruit is common used in traditional East Asian medicine for treatment of diarrhea, intestinal disorders and dieresis [2], [3], [4]. Moreover, Yizhi capsularfruit is usually used in compound formula, or fufang. Among them, the most famous is SuoQuan pills, which was first described in Chinese canonical medicine about 800 years ago for the treatment of different urinary incontinence symptoms including frequency, urgency and nocturia [5]. In this formula, Yizhi capsularfruit is used as the jun (emperor) herb [6]. Reports published recently have showed that Yizhi capsularfruits have anti-inflammatory activities [7], [8], anti-allergy [9], anti-ulcer [10] and neuro-protective roles [11], [12], etc. Natural product chemistry studies reveal that the capsularfruit contains sesquiterpenes, diarylheptanoids, flavonoids, volatile oil, steroids and their glycosides, etc. [13]. The content levels of flavonoids (e.g., tectochrysin, izalpinin, chrysin, apigenin-4′,7-dimethylether and kaempferide), diarylheptanoids (e.g., yakuchinone A, yakuchinone B and oxyphyllacinol) and sesquiterpenes (e.g., nootkatone) in the fruits and its pharmaceutical preparations have been reported by our group [5], [14]. We found that the alcoholic extract had anti-diarrheal activities and the Yizhi flavonoids might be the active constituents [15]. Low systemic exposure to parent yakuchinone A after oral administration of Suoquan pills to rats has been reported recently [16]. However, the fates of these constituents in the body after oral or intravenous administration remain largely unknown.
In order to understand the mechanism of action of Yizhi extract and corresponding pharmaceutical preparations, it is critical to know the chemicals and their metabolites present in plasma after oral administration of Yizhi extract. Besides the stated nine main constituents, there are more components occurring in the fruit. Unfortunately there are no well-established standards for all of the chemicals contained within Yizhi fruit and its extract. The absence of such standards represents a major challenge to investigate the metabolism and PK behavior of these chemicals and metabolites in animal or human plasma after oral administration of Yizhi extract. This apparent conundrum has been discussed in FDA guidance for botanical drug products [1] and marker compound(s) strategy was recommended for pharmaceutical practitioners. Recently, a new theory of “PK markers” was put forward by Li and co-workers [17], [18]. When bioactive constituents from an herb are measurable in a bio-sample and have favorable PK properties that could be used to substantiate systemic exposure to the herb, they are referred to as “PK markers” of the herb. These efforts on multi-component PK studies for herbal products will surely bring forth good fruits. A major goal of this study was to identify the known chemicals and their metabolites in rat plasma after oral administration of Yizhi extract. In the current study, mass spectrometry along with enzymatic, acidic and methanol treatments was used to identify Yizhi extract known chemicals and metabolites in rat plasma. Notably, we found that metabolic reduction of Yizhi diarylheptanoids occurred in rats. Yakuchinone B was reduced to yakuchinone A and then to oxyphyllacinol in a stepwise manner and subsequently glucuronidated by UDP-glucuronosyl transferase. The gained information is relevant to charactering the PK properties of these known chemicals in the future study.
Section snippets
Chemicals and materials
Reference standard of nootkatone (purity, 98%; similarly hereinafter) was purchased from Sigma Chemical Co. (St. Louis, MO). Yakuchinone A (98%), yakuchinone B (98%) and oxyphyllacinol (98%) were purchased from Chemfun Medical Technology (Shanghai) Co., Ltd. (Shanghai, China). Kaempherol was obtained from the National Institutes for Food and Drug Control (Beijing, China). Tectochrysin, izalpinin, chrysin, kaempferide and apigenin-4′,7-dimethylether were separated and identified from A. oxyphylla
Parent chemicals from Yizhi extract in rat plasma
After p.o. administration of Yizhi extract to rats, the parent chemicals of tectochrysin, chrysin, apigenin-4′,7-dimethylether (Fig. 1a), nootkatone (Fig. 1b), yakuchinone A/B and oxyphyllacinol were detected (Fig. 1c). On the contrast, the free forms of izalpinin and kaempferide were almost undetectable (Fig. 1b).
After p.o. ingestion of Suoquan pills or Suoquan capsules, the parent drugs of Yizhi flavonoids except for tectochrysin were almost undetectable, but the nootkatone, yakuchinone A and
Conclusions
In conclusion, low or trace plasma level of parent chemicals were measured after p.o. administration of Yizhi extract, Suoquan capsules and Suoquan pills to rats. Yizhi flavonoids and diarylheptanoids formed mainly monoglucuronide metabolites. Diglucuronide metabolites for chrysin, izalpinin and kaempferide were also detected. Demethylation of kaempferide to form kaempferol was observed but free kaempferol was undetectable because of rapid glucuronidation. Metabolic reduction of Yizhi
Competing interests
There are no competing interests to declare.
Acknowledgements
We are grateful to Dr. Li Li from Shanghai Institute of Materia Medica, China and Dr. Dan-Dan Tian from The Chinese University of Hong Kong, China for valuable comments and suggestions regarding this article. We are also grateful to Dr. Chen Cheng and Dr. Mei-Juan Li from Shanghai Institute of Materia Medica for screening relevant articles from reference lists.
This work was supported by Grants 812189 and 813196 from the Natural Science Fund of Hainan Province, Grants ZDZX2013008-2 and
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