Hypericum japonicum Thunb. ex Murray: Phytochemistry, Pharmacology, Quality Control and Pharmacokinetics of an Important Herbal Medicine

Hypericum japonicum Thunb. ex Murray is mainly distributed throughout Asia, Oceania and North America and is used as an important herbal medicine. H. japonicum contains many valuable secondary metabolites, such as flavonoids, phloroglucinols and xanthones and has hepatoprotective, anti-tumor, antibacterial, antiviral, and antioxidant activities and effects on the cardiovascular system and immunity. Coupled with phytochemical and pharmacological research, a series of analytical methods have been developed to evaluate the quality of H. japonicum based on its bioactive components. A pharmacokinetics study involved the absorption of two main flavonoids of H. japonicum in rats. This review aims to present an up-to-date and comprehensive overview of the phytochemistry, pharmacology, quality control and pharmacokinetics of H. japonicum, which should be useful for the greater development of H. japonicum, especially in the development of new drugs and therapeutics for various diseases.


Introduction
Hypericum japonicum Thunb. ex Murray (Hypericaceae) is an annual herb mainly distributed throughout Asia, Oceania and North America [1,2]. In China, it is widely distributed in Liaoning Province, Shandong Province and other provinces in southern China where it grows in rice fields, ditches, marshes, grasslands and waste places where the altitude is below 2800 m [2,3].
H. japonicum called "Tian-Ji-Huang" or "Di-Er-Cao" in Chinese was first listed as a herbal medicine in "Sheng Cao Yao Xing Bei Yao" published during the Qi Dynasty. It has been used in Traditional Chinese Medicine for a long time for relieving internal heat or fever, hemostasis and detumescence [4]. In addition, it has also been used as a medicinal herb in Asian countries, such as Japan, South Korea, Thailand, Nepal, India, Vietnam and Philippines [2]. H. japonicum has been studied due to its high value in traditional medicine. The results of modern pharmacological studies have shown that H. japonicum could be used for the treatment of bacterial diseases, infectious hepatitis, acute and chronic hepatitis, gastrointestinal disorders, internal hemorrhages and tumors, which generally matches its traditional uses [4,5]. So far, no studies on the toxicity of H. japonicum have been reported.
Recently, different classes of chemicals such as flavonoids, xanthonoids, chromone glycosides, phloroglucinol derivatives and lactones have been found in this plant. Among these isolated components, some single flavonoid compounds such as quercitrin, isoquercitrin and quercetin-7-O-α-L-rhamnose are shown to have a variety of bioactivities in vivo or in vitro, and thereby are thought as the bioactive components of H. japonicum. Hence, quality control based on these bioactive components to ensure the effects of H. japonicum materials and its related products is urgent and necessary. However, the quality control of H. japonicum is still not listed in the Chinese Pharmacopoeia (2010 edition) and other official pharmacopoeias. A number of studies have thus attempted to develop accurate, sensitive and selective analytical methods for qualitative and quantitative evaluation of H. japonicum.
Aiming to provide beneficial information for modern uses and scientific studies of H. japonicum, this review summarizes and evaluates the available phytochemical and bioactive properties of H. japonicum reported by the literature. Besides, the research progress in the quality evaluation and pharmacokinetics of H. japonicum are also presented.

Volatile Oil
The volatile oil extracted from the roots and the aerial parts (stems and leaves) of H. japonicum were analyzed by GC-MS, and thirty-two and forty-three constituents were identified, respectively. As the results show, there was obvious chemical variability in the volatile oil composition: the contents of six chemotyppes including hydrocarbons, alcohols (phenols), aldehydes/ketones, acids, esters and amines were 31.92%, 11.47%, 9.95%, 0.56%, 40.03% and 4.13% in the roots, respectively, and 62.16%, 8.12%, 2.72%, 1.24%, 18.96% and 5.75% in the aerial parts, respectively [25]. The main volatile components that represented more than 5% of the total volatile oils are summarized in Table 2.

Metallic Elements
Fifteen rare earth elements in wild H. japonicum materials were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). The results showed that the concentrations of La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb, Tm, Lu and Y ranged from 6 to 14522 ng/g, and among them the concentrations of La, Ce and Nd were higher than 2,000 ng/g [26]. In addition, the contents of other five metallic elements, Zn, Cu, Pb, Cr and Cd were measured by flame atomic absorption spectrophotmetry (FAAS) [27].

Antioxidant Activity
The antioxidant effect of H. japonicum is one of the most prominent effects due to its responsibility for many of the other activities. The aqueous extracts of H. japonicum have been demonstrated to have obvious antioxidant activity by molybdenum reduction, DPPH scavenging, β-carotene bleaching inhibition and lipid peroxidation inhibition methods with values of 37.28 ± 0.54 μg/mg, IC 50 = 77.7 ± 5.6 μg, 83.18% and 95.38%, respectively. By reducing the generation of hydroxyl radicals, the aqueous extract effectively reduced the oxidative damage of the DNA [28].

Hepatoprotective Activity
Total bilirubin (TBIL), alanine transaminase (ALT) and aspartate transaminase (AST) in serum are three markers for liver function. After intraperitoneal administration with ethanol and ethyl acetate extract of H. japonicum (0.2, 0.6 and 1.8 g raw materials/kg) to rats with acute liver injury induced by D-aminogalactose, the levels of ALT and AST in all treatment groups were reduced remarkably when compared to those in the model group [30]. The aqueous extract (4.5 g raw material/kg) exhibited an obvious effect by decreasing AST, ALT and TBIL levels in serum of mice with liver injury induced by CCl 4 , indicating its hepatoprotective effects [31]. When three flavonoids isolated from H. japonicum, namely quercitrin, isoquercitrin and quercetin-7-O-α-L-rhamnoside (0.25, 0.5 and 1.0 mg/kg), were used separately to treat the liver injury in rats induced by CCl 4 and D-aminogalactose, the levels of AST, ALT and TBIL were significantly reduced at the three dose levels as compared to the model group [32]. Furthermore, intraperitoneal treatment with quercetin-7-O-α-L-rhamnoside (0.25, 0.5 and 1.0 mg/kg) of rats with liver fibrosis induced by bile duct ligation showed a reduction in levels of type III procollagen, hyaluronic acid, laminin and TNF-α in serum and expression of smooth muscle actin-α in liver [33].

Anti-Cancerous Activity
The anti-cancerous effect is a noticeable bioactivity for H. japonicum reported in the last decade. H. japonicum extract significantly inhibited the proliferation of human tongue cancer cell line TSCCa in vitro in a dose-dependent manner by damaging the mitochondria and rough endoplasmic reticulum [34]. The extract of H. japonicum significantly inhibited the proliferation of CNE-2 cells and HepG2 cells in vitro by inducing cell cycle arrest, showing a dose-dependent response at the low concentrations of 25 mg/mL and 5 mg/mL, respectively [35][36][37]. Meanwhile, the aqueous extract of H. japonicum displayed a synergistic tumor-inhibiting effect with 5-FU in mice at 3, 6 and 12 g/kg/day, administered 24 h after the tumor inoculation, once daily for 10 days, indicting its usefulness in antitumor therapy [38]. Using a serum pharmacology method, BEL-7404 liver cancer cells were co-cultured with the collected serum containing four fraction of H. japonicum (ethanol extract, ethyl acetate extract, n-butanol extract and aqueous extract) at different dosage. All extracts showed inhibitory activity on the growth of BEL-7404 cell with inhibitory rates of 29.74%, 53.80%, 40.79% and 54.24%, respectively [39].

Antibacterial Activity
Antibacterial activity for aqueous extract of H. japonicum was studied by the disc diffusion method. The results demonstrated that both Gram positive and Gram negative bacteria, including Escherichia coli, Alcaligens faecalis, Bacillus subtilis, E. aerogenes, Klebsiella pneumonia, Shigella flexneri, Salmonella enterica ser. Typhi, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Xanthomonas vesicatoria, X. oryzae pv. oryzae and X. malvacearum were inhibited by the extract, except for P. aeruginosa, and the minimum inhibitory concentration (MICs) of the extract was 1 mg/mL against all the test cultures used, except E. aerogenes and P. aeruginosa. This indicated the extract had a broad spectrum antibacterial activity [28].
Isojacareubin, the xanthone from the aerial parts of H. japonicum had an effect on methicillin-resistant Staphylococcus aureus (MRSA) with MICs/MBCs ranging from 4/16 to 16/64 μg/mL. When it was used together with some conventional antibacterial agents, namely ceftazidime, levofloxacin and ampicillin, the values of 50% of the fractional inhibitory concentration indices (FICI 50 ) were 0.25, 0.37 and 0.37, respectively, indicating good anti-MRSA activities [40].

Antiviral Activity
Using a serum pharmacology method, the anti-HBV effect for different extracts (ethanol extract, ethyl acetate extract, n-butanol extract and aqueous extract) of H. japonicum were tested in vitro. The aqueous extract showed better anti-HBV activity than the other three extracts with inhibitory rates on HBeAg and HBsAg of 70% and 30%, respectively [39]. In an in vivo study, H. japonicum extract showed strong activity against duck hepatitis B virus at dosage of 6.5, 13.0 and 26.0 mg/kg, once a day, for 28 days [41]. In addition, the 75% ethanol extract of H. japonicum exhibited anti-influenza virus H3N2 effect in vivo after 10 g/kg oral administration in mice infected with the H3N2 virus [42].

Effects on Cardiovascular System
The bioactive compound quercetin-7-O-α-L-rhamnoside isolated from H. japonicum was tested for its coagulant activity via an in vitro experiment. This flavonol glycoside (10 −5 g/L) exhibited excellent effects on promoting the coagulation of activated partial thromboplastin time and prothrombin time in dose-dependent manner [7]. Oral administration of the aqueous extract of H. japonicum (11.25 g raw material/kg/day) in hyperlipidemic rats for 8 weeks, significantly reduced the serum levels of triglycerides, total cholesterol, low density lipoprotein cholesterol and atherosclerotic index, but increased the level of high density lipoprotein cholesterol. Meanwhile, the hematocrit, blood and plasma viscosity were markedly decreased in this study, indicating the aqueous extract of H. japonicum had an adjustment effect on hemorheology [43]. Furthermore, the contents of monocyte chemoattractant protein-1, lipoprotein associated phospholipase A2 and macrophage migration inhibitory factor in serum were also obviously decreased, indicating the inhibitory effect on the atherosclerosis process [44].

Effects on Immunity
The effect of H. japonicum extract on systemic immune functions was investigated by subcutaneous injection in rats with a dosage of 2 g raw material/kg, and the results showed that H. japonicum extract obviously increased the phagocytic rate of neutrophils (83.5% ± 5.1%) and enhanced the T lymphocyte ratio (68.8% ± 6.5%) in the peripheral blood of male rats [45]. In addition, the extract showed strong effects on improving the immune function, reducing the toxic effect of 5-fluorouracil and prolonging the survival time in the tumor-bearing mice. Therefore, it was considered to have immunoregulatory activity [38].

Quality Control
So far, the quality evaluation of H. japonicum materials has been not listed in the Chinese Pharmacopoeia (2010 edition), even though it has been used as a traditional medicine for a long time. Much effort thus is made to establish a comprehensive method for ensuring the quality of H. japonicum. For the safety and efficacy of herbal medicines, the first step in assuring quality is correct identification [46]. Traditionally morphological and microscopic approaches have been used for H. japonicum identification [3]. However, these traditional methods are very limited for Hypericum herbs or processed products with similar macroscopic and anatomical characteristics. DNA markers have now become the popular means for the identification of plants because genetic composition is effective and unique for each individual and is less affected by age, physiological condition, environmental factors, harvest time, storage and processing conditions [47]. Internal transcribed spacer sequence (ITS) of nuclear ribosomal DNA (nrDNA) as a common DNA marker was applied to H. japonicum. A series of ITS sequences of H. japonicum from different research groups are reported in GenBank. However, there is still no literature on a molecular method developed for accurate identification of H. japonicum.
Qualitative and quantitative analysis for the quality control of H. japonicum has mostly focused on flavonoid components since flavonoids have been proven to be largely responsible for many of the biological activities of H. japonicum in pharmacological studies. Thin layer chromatography (TLC) has been applied to qualitative identification of H. japonicum by using the flavonoid components, isoquercitrin and quercitrin as chemical markers [48]. However, TLC quantification is thought to be relatively poor in sensitivity, resolution and reproducibility. High performance liquid chromatography (HPLC) coupled with ultraviolet spectroscopy (UV) and electrospray ionization (ESI)-mass spectrometry (MS) have become the common analytical techniques for separation and quantitation of chemical markers from complicated herbal medicine extracts. HPLC methods for the quantitation of specific chemicals in H. japonicum are summarized in Table 3. Isojacareubin, quercetin-7-O-α-Lrhamnoside, quercetin and quercitrin, each of which was recognized as characteristic and/or bioactive component, were used as individual chemical markers for quality evaluation of H. japonicum materials [49][50][51][52][53]. A lot of HPLC methods on the basis of multiple components with potential bioactivities have been developed for quantitation and presenting further useful information on this species. As the results show, the contents of flavonoids considered as the bioactive components in H. japonicum materials were found to vary [8,[54][55][56][57][58][59][60][61]. Various reasons, such as growth environment, growth periods, harvesting season, processing method, storage condition and period might cause variation in the chemical compositions of the various samples [8,56,[58][59][60].
Chemical fingerprints could show the complicated components of herbal medicines, not only the naturally occurring bioactive and characteristic components, but also their relative ratios [62]. Chemical fingerprint analysis which is recognized as a reliable means for the identification and qualification of herbal medicines, has been accepted by Food and Drug Administration (2000) [63], European Medicines Agency (2001) [64], and State Food and Drug Administration of China (2000) [65] and other authorities as a strategy for quality assessment of herbal medicines. A HPLC-diode array detector (DAD) fingerprint was applied to 56 batches of H. japonicum materials from six provinces in China. The results showed that the most relevant factor in secondary metabolites of H. japonicum was collection location, followed by harvesting time [62]. Moreover, the on-line LC-MS n technique, which provides a ready method for elucidation of compounds and more information in the fingerprint by reprocessing the mass spectrometry data using tandem MS, has become a powerful means in the quality control and quantitative analysis of complicated medicinal herbs. A HPLC-PAD-ESI-MS n method was successfully developed to evaluate the quality of H. japomicum from different origins by establishing chromatographic fingerprints, in which 20 common peaks of 10 batches of H. japomicum were identified [58]. Headspsce GC-MS (HSGC-MS) was used to establish the fingerprint of the volatile components from 11 batches of H. japonicum materials. The HSGC-MS fingerprint with 12 common peaks was characteristic and useful for quality control of H. japonicum materials [66]. Additionally, a high performance capillary electrophoresis (HPCE) fingerprint based on six main peaks was also established for quality control of H. japonicum materials [67].

Pharmacokinetics
A HPLC-DAD method was established and applied successfully to the pharmacokinetic study of quercitrin and isoquercitrin in rat plasma after oral administration of 80% ethanol extract of H. japonicum at a dose of 23.0 g/kg, equivalent to 48.3 and 62.1 mg/kg of quercitrin and isoquercitrin, respectively. The pharmacokinetic results suggested that the maximum concentrations of quercitrin and isoquercitrin occured after approximately 1.30 and 1.17 h, respectively. Quercitrin and isoquercitrin were still detectable in rat plasma for at least 24 h after oral administration of the extract. These findings provided useful evidence for evaluating the clinical efficacy of H. japonicum [68].

Conclusions
Pharmacological studies on flavonoids have been performed in vitro and in vivo in animals, while the pharmacological studies on other main bioactive components such as phloroglucinols and xanthones are rare. Though several pharmacological mechanisms related to biological activity have already been explained, the comprehensive pharmacological mechanisms of H. japonicum need to be elucidated. Based on phytochemical and pharmacological research, the flavonoids responsible for the good hepatoprotective, anti-tumor and antibacterial activities were selected as chemical markers to evaluate the quality of H. japonicum and its products. Meanwhile, various methods have been successfully applied to the simultaneous analysis of the bioactive compounds in H. japonicum. However, pharmacokinetics studies on the main components, especially the bioactive components are still largely lacking, therefore firm evidence for further clinical application is necessary in order to assess the therapeutic potential of H. japonicum and its pharmaceutical commodities.