Toxicity of daphnane-type diterpenoids from Genkwa Flos and their pharmacokinetic profile in rat

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Toxicity of daphnane-type diterpenoids from Genkwa Flos and their pharmacokinetic profile in rat Yan-Yan Chen, Jian-Ming Guo, Ye-Fei Qian, Sheng Guo, Chun-Hua Ma, Jin-Ao Duan ∗ Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing, PR China

a r t i c l e

i n f o

Article history: Received 9 April 2013 Received in revised form 14 May 2013 Accepted 20 June 2013 Keywords: Daphnane-type diterpenoids Genkwa Flos Toxicity Pharmacokinetic profile

a b s t r a c t Daphnane-type diterpenoids (DDs) are the main types of plant diterpene orthoesters known and have remarkable biological activities. However, the in vivo toxicity and pharmacokinetic profile of DDs remains unkonwn. The aim of this study was to investigate the toxicity and pharmacokinetic profile of DDs from Genkwa Flos (Thymelaeaceae). The toxicity of diterpenoids was evaluated after oral administration of total diterpenoids extract from Genkwa Flos to rats, and the blood concentration of diterpenoids was analyzed by ultra performance liquid chromatography tandem triple-quadrupole mass spectrometry (UPLC–TQ-MS). The diterpenoids were confirmed to be the toxic components of Genkwa Flos. The pharmacokinetic profile of these diterpenoids was quite different due to their different structures. Although the contents of yuanhuafine and yuanhuapine were low in the extract, the blood concentrations were extremely high. In contrary, the contents of genkwanine F and Wikstroemia factor M1 in the extract were much higher, but they could not be detected in the blood. This result implied that yuanhuafine and yuanhuapine but not genkwanine F and Wikstroemia factor M1 were the potentail toxic components of Genkwa Flos in vivo. This paper shows for the first time the toxicity of diterpenoids from Genkwa Flos was correlated with their blood concentration and when DDs were used for medicinal purposes, their contents in herb as well as their blood concentrations should be considered. © 2013 Elsevier GmbH. All rights reserved.

Introduction Herbal medicines are widely used around the world, which have a history of several thousands of years for the prevention, diagnosis and treatment of diseases. Since early times, toxicity has been recognized as an intrinsic property of herbal medicines. The intrinsic toxicity of herbal plants generally results from the toxic chemical constituents in herbs (Efferth and Kaina, 2011). The best strategy to minimize the risk posed by toxic herbs is zero exposure. However, it is sometimes inevitable because the herbs are beneficial and necessary for a specific treatment due to the lack of alternatives. So the dosage is the key issue for both therapeutic efficacy and safety. Ideally any herbal medicine, especially those containing toxic components should be used under well-controlled conditions. As a well-known traditional Chinese medicine (TCM), Genkwa Flos (GF), the dried flower buds of Daphne genkwa Sieb. et Zucc. (Thymelaeaceae), has been used for the diuretic, antitussive, expectorant, abortifacient, and antitumor purposes for centuries (Medicine, 2006). In Shen Nong’s Herbal Classic, China’s oldest

∗ Corresponding author at: Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine, Nanjing 210046, PR China. Tel.: +86 25 85811116; fax: +86 25 85811116. E-mail address: [email protected] (J.-A. Duan).

pharmacy monograph, it was classified as “Xiapin” (low grade) which means mild toxicity existed. Even in the Pharmacopeia of the People’s Republic of China (Committee, 2010), it is recorded and described as slightly toxic. There is evidence that excessive and chronic use of GF will finally result in serious damage to liver, lung, kidney, brain and heart (Xiang et al., 2006; Yang et al., 1989), and it was publicly known to have irritation to mucous and skin (Xia, 2005). Recently, a metabonomic approach was applied to evaluate GF-induced hepatotoxicity and the mechanism was considered to be related to the disturbance of amino acid metabolism, gut microflora and bile acid biosynthesis (Geng et al., 2013). Previous phytochemical studies have indicated that GF contains different types of chemical components, including flavonoids, diterpenoids and coumarins (Akhtar et al., 2006; Hong et al., 2010; Li et al., 2010; Zhan et al., 2005), and among which, daphnane-type diterpenoids (DDs) are main active constituents. DDs are believed to be derived from a tigliane precursor and have an orthoester motif, though a very large number of DDs were identified, they occurred only in the plant families of Thymelaeaceae and Euphorbiaceae (Evans and Soper, 1978). DDs not only have remarkable biological activities such as antitumor (Badawi et al., 1983; Jo et al., 2012), antifertility (Hu et al., 1985; Wang et al., 1981; Ying et al., 1977), antihyperglycemic (Carney et al., 1999), antiviral (Allard et al., 2012), anti-bladder-hyper-reflexia (Appendino and Szallasi, 1997), anti-HIV (Asada et al., 2011; Huang et al., 2012)

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Please cite this article in press as: Chen, Y.-Y., et al., Toxicity of daphnane-type diterpenoids from Genkwa Flos and their pharmacokinetic profile in rat. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.06.012

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2

yuanhuafine R=CH3

yuanhuacine R=Ph yuanhuadine R=CH3

yuanhuatine R=Ph yuanhuapine R=CH3

genkwanine N R=COPh Wikstroemia factor M1 R= CO(CH=CH)2(CH2)4CH3

genkwanine F R=CO(CH=CH)2(CH2)4CH3

(3β,12α,13α)-3,12-dihydroxypimara -7,15-dien-2-one (IS)

Fig. 1. Chemical structure of diterpenoids and IS.

and neurotrophy (He et al., 2002b), but also exhibit toxicity such as cytotoxic (Zhan et al., 2005), pesticidal (Sakata et al., 1971) and irritant (Evans et al., 1992). DDs are a class of important natural compounds with a nonnegligible toxicity, however, as far as we are concerned, there have been few papers published on the in vivo toxicity and pharmacokineitcs studies for DDs. Since the relationship between the toxicity and toxic chemical components of herbal medicines as well as their pharmacokineitc profile has been paid more and more attention, this study was undertaken to investigate the toxicity and pharmacokinetic profile of DDs from GF in rats by evaluating the toxicological effects and determinating the blood concentration of each diterpenoid. Materials and methods Plant materials and chemicals The dried flower buds of Daphne genkwa Sieb. et Zucc. were collected from Liu’an city, Anhui province, China. The material was authenticated by the corresponding author, and the voucher specimen (No. 110326) was deposited at the Herbarium in Jiangsu Key Laboratory for High Technology Research of TCM Formulae, Nanjing University of Chinese Medicine. Yuanhuacine, yuanhuadine, yuanhuafine, yuanhuatine, yuanhuapine, genkwanine F, genkwanine N and Wikstroemia factor M1 and (3␤,12␣,13␣)-3,12dihydroxypimara-7,15-dien-2-one (internal standard, IS) were isolated and purified in our laboratory. On the basis of UV, NMR and MS analysis, their structures were confirmed, and their purities determined using UPLC–PDA-MS were over 98.0%. Their structures are presented in Fig. 1. Acetonitrile and methanol (HPLC grade) were purchased from Merck (Darmstadt, Germany) and deionized water was purified by an EPED super purification system (Eped, Nanjing, China). Other chemicals and solvents used in this study were of analytical grade (Nanjing Chemical Plant, Nanjing, China). Preparation of total diterpenoids extract from GF (TDG) One thousand grams of GF was soaked in petroleum and extracted three times by decocting with petroleum (1:10, 1:10, and then 1:8, w/v) for 2 h per time. The extracts were combined and petroleum was removed under reduced pressure, the residue was then dissolved and precipitated with dehydrated ethanol, stored at room temperature till cold. After the precipitation was

filtered, the ethanol was removed under reduced pressure and 16 g total diterpenoids extract was obtained. The contents of eight diterpenoids in total diterpenoids extract from GF (TDG) were measured quantitatively by external standard method using the same chromatography conditions as described above. The contents of yuanhuacine, yuanhuadine, yuanhuafine, yuanhuadine, yuanhuapine, genkwanine F, genkwanine N and Wikstroemia factor M1 in the extract were 7.24, 10.70, 1.97, 1.04, 1.31, 11.90, 4.03 and 23.31 mg/g, respectively. Animals Male Sprague-Dawley rats weighing 180–200 g were purchased from Shanghai Slac Laboratory Animal Co. Ltd., China. The animals were housed under controlled temperature (25 ± 1 ◦ C), relative humidity (40–70%), and a 12-h light/dark cycle for minimum of 7 days before use and fed with food and water ad libitum. All the procedures were in strict accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council. Oral toxicity Twenty-four rats were divided into four groups (n = 6) at random. After fasting over night, TDG solution (dissolved in physiological saline solution with 0.5% Tween Monostearate (Tween 80)) was orally administrated to rats at doses of 0.1, 0.25, 0.5 and 1 g/kg. In order to evaluate the toxicity, blood samples were analyzed. Blood samples were collected from each rat by retro-orbital puncture at a predetermined time interval of pre-dose, 0.17, 0.33, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h into the tubes containing EDTA-2K. Plasma was separated by centrifuging the blood samples at 13 000 rpm and finally stored by freezing at −80 ◦ C until analysis. LC–MS/MS analysis LC–MS/MS conditions Chromatographic analysis was performed on a Waters Acquity UPLC system (Waters Corp., Milford, MA, USA). A Thermo Syncronic C18 column (100 mm × 2.1 mm, 1.7 ␮m) was employed and the column temperature was maintained at 35 ◦ C. The mobile phase was composed of A (0.1% formic acid in water) and B (acetonitrile) using a gradient elution of 40–95% B at 0–7 min with a flow rate set at 0.40 ml/min. The auto-sampler was conditioned at 4 ◦ C and the injection volume was 5 ␮L. Mass spectrometry detection was

Please cite this article in press as: Chen, Y.-Y., et al., Toxicity of daphnane-type diterpenoids from Genkwa Flos and their pharmacokinetic profile in rat. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.06.012

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Table 1 Precursor/product ion pairs and parameters for MRM of compounds used in this study. Coumponds

Retention time (min)

MRM transitions (precursor → product)

Cone voltage (V)

Collision energy (eV)

Yuanhuacine Yuanhuadine Yuanhuafine Yuanhuatine Yuanhuapine Genkwanine F Genkwanine N Wikstroemia factor M1 IS

5.88 5.03 2.73 3.97 2.81 6.09 4.48 6.00 2.83

649 → 151 587 → 151 541 → 323 605 → 325 543 → 325 655 → 151 591 → 105 637 → 151 319 → 283

24 24 16 20 22 28 24 26 14

20 18 12 14 14 24 36 20 10

performed using a Xevo Triple Quadrupole MS (Waters Corp., Milford, MA, USA) equipped with an electrospray ionization (ESI) source. The ESI source was set in positive ionization mode. The parameters in the source were set as follows: capillary voltage 2.5 kV, source temperature 150 ◦ C, desolvation temperature 550 ◦ C, cone gas flow 50 l/h, desolvation gas flow 1000 l/h. The analyte detection was performed by using multiple reaction monitoring (MRM) mode. The cone voltage and collision energy were optimized for each analyte and selected values are given in Table 1. Dwell time was automatically set by the software. Preparation of standard solution and quality control (QC) samples The appropriate amounts of yuanhuacine, yuanhuadine, yuanhuafine, yuanhuatine, yuanhuapine, genkwanine F, genkwanine N and Wikstroemia factor M1 were separately weighed and dissolved in methanol as the stock solutions. Then, all the eight stock solutions were mixed and diluted with methanol to prepare a final mixed standard solution, giving a final concentration of 505 ng/ml for yuanhuacine, 520 ng/ml for yuanhuadine, 530 ng/ml for yuanhuafine, 515 ng/ml for yuanhuatine, 515 ng/ml for yuanhuapine, 460 ␮g/ml for genkwanine F, 475 ␮g/ml for genkwanine N, and 480 ␮g/ml for Wikstroemia factor M1 , respectively. The mixed stock solution was serially diluted with methanol to provide working standard solutions of desired concentrations. The internal standard solution was prepared to the concentration of 20 ng/ml in methanol. For the validation of the method, three concentrations of standard solution containing yuanhuacine (2.52, 50.5 and 505 ng/ml), yuanhuadine (2.60, 52.0 and 520 ng/ml), yuanhuafine (2.65, 53.0 and 530 ng/ml), yuanhuatine (2.58, 51.5 and 515 ng/ml), yuanhuapine (2.58, 51.5 and 515 ng/ml), genkwanine F (2.30, 46.0 and 460 ng/ml), genkwanine N (2.38, 47.5 and 475 ng/ml) and Wikstroemia factor M1 (2.40, 4.80 and 480 ng/ml) were used for preparing the QC samples. The standards and quality controls were extracted on each analysis day with the same procedures for plasma samples as described below. Samples preparation To a 200 ␮l portion of each plasma, 100 ␮l IS solution (20 ng/ml) and 500 ␮l methanol were added in an eppendorf tube to precipitate protein. The mixture was vortexed for 1 min and then centrifuged at 13,000 rpm for 10 min. The supernatant was transferred into another eppendorf tube and blown to dryness with nitrogen at 37 ◦ C. The residue was re-constituted in 100 ␮l 40% acetonitrile solutions, and centrifuged (13,000 rpm for 10 min). The supernatant was transferred to an autosampler vial and an aliquot of 5 ␮l was injected onto the UPLC–TQ-MS system for analysis. Validation of the methods The specificity of the method was evaluated by comparing the chromatograms of six different batches of blank plasma samples,

plasma samples spiked with the analytes and IS, and plasma samples after an oral dosage. Blank plasma samples were analyzed for endogenous interference, followed by spiking with IS for the interference of IS. To evaluate linearity, calibration curve was prepared by spiking pooled blank plasma with an appropriate amount of working solution to produce the calibration curve points equivalent from 1.15–460 ng/ml to 1.32–530 ng/ml for the eight compounds and 20 ng/ml of IS. Calibration curve was calculated using weighted (1/x2 ) linear regression of internal ratios (analyte/IS peak area) vs. analyte concentrations. LLOQ was defined as the lowest plasma concentration in the calibration curve that can be quantitatively measured with a signal-to-noise ratio (S/N) at 5 and with a precision of 20% and accuracy of 80–120%. Six replicates of QC samples with three batches were assayed to calculate the precision and accuracy of this method on three validation days. The precision is expressed by relative standard deviation (RSD) between the replicate measurements. Accuracy is defined as relative error (RE) which is calculated using the formula RE % = [(measured value − theoretical value)/theoretical value] × 100. The extraction recoveries of analytes at three QC levels were evaluated by determining the peak area ratios of the analytes in the post-extraction spiked samples to that acquired from preextraction spiked samples. The matrix effects were measured by comparing the peak areas of the analytes dissolved in the pretreated blank plasma with that of pure standard solution containing equivalent amounts of the analytes. The stability of the analytes in rat plasma was assessed by analyzing QC samples at three concentration levels. The short-term stability was determined with QC samples stored for 24 h at room temperature. The long-term stability was tested after storage of samples at −80 ◦ C for one month. The freeze/thaw stability was examined at three freeze/thaw cycles between −80 ◦ C and room temperature. The post-preparative stability was assessed by left QC samples in the UPLC–MS auto-sampler for 24 h at 4 ◦ C.

Results Oral toxicity of TDG in rat Toxic symptoms after oral administration of TDG in rat In order to know the toxic symptoms caused by TDG, a single administration of TDG was given orally to rats at doses at 0.1, 0.25, 0.5 and 1 g/kg. The general behavior of rats was monitored for 24 h after oral administration of TDG. Death was considered as cessation of respiration and failure to respond to tactile stimuli. Five minutes after given 1 g/kg dose of TDG, all rats subsequently became completely immobile with pronounced respiratory distress and marked abdominal breathing, and then, the respiratory rate became progressively slower, until it ceased. Pronounced

Please cite this article in press as: Chen, Y.-Y., et al., Toxicity of daphnane-type diterpenoids from Genkwa Flos and their pharmacokinetic profile in rat. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.06.012

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B

100

10

time to death (h)

mortality(%)

A

80 60 40 20 0

8 6 4 2 0

0

0.25

0.5

0.75

dose (g/kg)

1

0

0.25

0.5

0.75

1

dose (g/kg)

Fig. 2. Relationship of dose and mortality (A) or time to death (B) after TDG administration in SD rats (n = 6).

convulsions were observed shortly before death. At the doses of 0.5 and 0.25 g/kg, similar symptoms were observed such as decreased motor activity and decreased respiratory rate before death. At the dose of 0.1 g/kg, immobile activity was observed, but the rats recovered within 30 min to an apparently normal state, and no adverse effects were observed during the subsequent 24 h observation period. Mortality and time to death at different TDG doses The mortality and death time is a magnitude frequently used for the toxicological evaluation of toxin. Here we studied the mortality and death time in four groups of rats treated with increasing doses of TDG (0.1, 0.25, 0.5 and 1 g/kg). No deaths were recorded at lower dose (0.1 g/kg). When orally given high, middle and low dose (1, 0.5 and 0.25 g/kg) of TDG, rats were all dead while the time to death were different (Fig. 2). The median time of death was 0.76 h, 6.5 h and 7.75 h at high, middle and low dose, respectively. Plots of TDG dose vs. time to death in SD rats showed curvilinear relationship with increasing TDG dose producing corresponding decrease in time to death (Fig. 2).

Pharmacokinetics profile of TDG Method validation The chromatogram of a blank plasma sample showed no endogenous peaks interfering the analysis of yuanhuacine, yuanhuadine, yuanhuafine, yuanhuatine, yuanhuapine, genkwanine F, genkwanine N and Wikstroemia factor M1 , and IS (Fig. 3A). Due to the efficient sample treatment and high selectivity of MRM, matrix effect for these analytes was insignificant as demonstrated in Fig. 3B. Five of the eight analytes could be detected in rat plasma after oral administration of TDG using the developed method (Fig. 3C). The regression equation, correlation coefficients and linearity ranges for the eight analytes are shown in Table S1. The results showed that they all exhibited good linearity. The LLOQs for yuanhuacine, yuanhuadine, yuanhuafine, yuanhuatine, yuanhuapine, genkwanine F, genkwanine N and Wikstroemia factor M1 were 1.26, 1.30, 1.32, 1.29, 1.29, 1.15, 1.19 and 1.20 ng/ml, respectively. The precision and accuracy data for intra-day and inter-day analyses were shown in Table S2. The results showed that this

Fig. 3. MRM Chromatograms of eight diterpenoids and IS in plasma: (A) blank plasma, (B) blank plasma spiked with the eight analytes and IS, (C) plasma sample after oral administration of TDG.

Please cite this article in press as: Chen, Y.-Y., et al., Toxicity of daphnane-type diterpenoids from Genkwa Flos and their pharmacokinetic profile in rat. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.06.012

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Fig. 4. Semi-logarithmic plot of mean blood concentration vs. time of diterpenoids after oral administration of TDG.

LC–MS/MS method had satisfactory reproducibility with precision (RSD) less than 8.7% and accuracy (RE %) ranging from −5.8% to 12.1% within three QC samples. As shown in Table S2, extraction recoveries of analytes and IS were in the range of 76.4–90.3%. The matrix effect of blank plasma of analytes and IS was found to be within the acceptable range; all values were more than 74.2%. Stability of the analytes during the sample storing and processing procedures was fully evaluated by analysis of QC samples. The results were shown in Table S3. The results indicated that these analytes in rat plasma were all stable for one-month storage at −80 ◦ C, 24 h in the auto-sampler (4 ◦ C), 24 h at room temperature and three freeze–thaw cycles with accuracy in range of 79.8–102.4%. The time-course of diterpenoids in blood after oral administration of TDG The dosages of 1, 0.5, 0.25 and 0.1 g/kg for i.g were used to investigate the dynamic time-course of diterpenoids in blood. Blood samples were collected at 0.17, 0.33, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 10, 12 and 24 h and analyzed by LC–MS/MS for the toxin presence. It showed that yuanhuacine, yuanhuafine, yuanhuatine, yuanhuapine and genkwanin N could be detected at lethal dosage, and the blood concentration reached its peak when the rats dead, but when the dosage decreased to 0.1 g/kg, only yuanhuafine and yuanhuapine could be detected (Fig. 4).

below 10 ng/ml (Fig. S1). Therefore, these findings indicated that the time to death is closely related to the blood concentration of diterpenoids. As shown in Fig. 5, under the lethal doses (0.25, 0.5, 1 g/kg), the blood concentration of yuanhuacine, yuanhuafine, yuanhuatine, yuanhuapine and genkwanin N at the time of death was 7.91–296.03, 51.95–366.79, 2.97–26.00, 62.47–270.78 and 1.79–63.04 ng/ml, respectively. Based on the experimental results, a suggested safe dosage of TDG is 0.1 g/kg (or yunhuafine, yuanhuapine blood concentration below 10 ng/ml, other diterpenoids below 1.50 ng/ml). Toxic dosage of TDG would be above 0.25 g/kg, which corresponds to the five diterpenoids blood concentration above 20, 150, 12, 120 and 10 ng/ml. It suggested that the toxicity of diterpenoids is correlated with the blood concentration in rats. So caution has to be taken when using GF for medicinal purposes, and limits for diterpenoids and their blood concentrations should be established. From these results, the diterpenoids from GF were confirmed to be the toxic components after oral administration at four dosages. The pharmacokinetic profile of these diterpenoids was quite different due to their different structures. Although the contents of yuanhuafine and yuanhuapine were only 1.97 and 1.31 mg/g in the extract, the blood concentrations were extremely high. In contrary, the contents of genkwanine F and Wikstroemia factor M1 in the extract were much higher (11.90 and 23.31 mg/g), but they could not be detected in the blood.

Diterpenoids toxicity and time to death is correlated with their blood concentration In order to explain the mortality and toxic symptom difference at different dosage, the blood concentration of diterpenoids in each rat was further analyzed. The tolerability to toxin and absorption extent changed rat to rat due to individual differences which may lead to different blood concentration and time of death. When the dose is 0.25 g/kg, the blood concentration of yuanhuacine, yuanhuafine, yuanhuatine, yuanhuapine and genkwanin N at the lethal time is approximately 20, 150, 12, 120 and 10 ng/ml. As the dose increased to 0.5 and 1 g/kg, the time to reach the maximum blood concentration decreased, while the maximum blood concentration of the five diterpenoids is similar with that after given 0.25 g/kg. And due to increased dosage, the median time of rat death is shorter. When the dose decreased to 0.1 g/kg, no rats dead and the maximum concentration of yuanhuafine and yuanhuapine is

Fig. 5. The blood concentration of diterpenoids at the death time.

Please cite this article in press as: Chen, Y.-Y., et al., Toxicity of daphnane-type diterpenoids from Genkwa Flos and their pharmacokinetic profile in rat. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.06.012

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6 Table 2 Molecular properties of diterpenoids. Compounds

logP

TPSA

natoms

MW

Yuanhuacine Yuanhuadine Yuanhuafine Yuanhuatine Yuanhuapine Genkwanine N Genkwanine F Wikstroemia factor M1

6.186 4.445 2.266 3.989 2.315 5.09 5.771 6.881

144.29 144.29 144.29 144.29 144.29 127.219 155.147 127.219

47.0 42.0 39.0 44.0 39.0 43.0 47.0 46.0

648 586 542 604 540 590 654 636

Diterpenoids blood concentration is correlated with their structure The final blood concentration of the oral dosing drug depend on its bioavailability, and molecular properties including octanol–water partition coefficient (logP), polar surface area (PSA), number of nonhydrogen atoms (natoms), molecular weight (MW), number of hydrogen-bond acceptors (expressed as the sum of nitrogen and oxygen atoms in the molecule, nON), number of hydrogen-bond donors (expressed as the sum of hydroxyl and amino groups present in a molecule, nOHNH), number of violations (nviolations), number of rotatable bonds (nrotb) and molecular volume always influence the oral bioavailability. The Lipinski “Rule of 5” states that poor absorption or permeation is more likely when logP is over 5, MW is over 500, there are more than 10 hydrogen bond acceptors, and there are more than 5 hydrogen bond donors. If two parameters are out of the range then a poor absorption or permeability is possible (Lipinski et al., 1997, 2012). Here the molecular properties of the diterpenoids were calculated with Molinspiration property engine v2011.04, the details were listed in Table 2. As calculated based on their structures, yuanhuafine and yuanhuapine may have better oral bioavailability and higher blood concentrations than other diterpenoids, which was consistent with our findings. It suggested that the pharmacokinetic profile of the diterpenoids were quite different due to their structure difference. Compounds with both a chain of fatty acid and benzene group were hardly absorbed while compounds without a chain of fatty acid such as yuanhuafine and yuanhuapine were absorbed easily which confirm that suitable polarity is essential in the absorption process of diterpenoids (Table 3). Diterpenoids toxicity is correlated with their structure Compounds with poor bioavailability are inefficient because a major portion of a dose never reaches the plasma to exert a pharmacological effect. Among the analyzed diterpenoids, the oral bioavailability order seemed as: yuanhuafine > yuanhuapine > yuanhuacine > yuanhuatine > genkwanine N > yuanhuadine, genkwanine F, Wikstroemia factor M1 . And since the extent of bioavailability influences the toxic effects resulting from oral administration, yuanhuafine and yuanhuapine might be the major toxic components of TDG due to their high blood concentration.

nON 10 10 10 10 10 9 10 9

nOHNH

nviolations

nrotb

Volume

3 3 3 3 3 3 5 3

2 1 1 1 1 2 2 2

11 10 5 6 5 6 11 11

586.483 531.636 470.655 525.503 464.442 523.321 608.407 590.514

Discussion To date, there has been some research on GF-induced toxicity. Recently, A urinary metabonomic approach was developed to study the metabolic disturbances caused by the administration of GF to rats. Analysis of serum biochemistry showed remarkably elevated ALT and AST, and histopathological alteration of livers were also observed, which confirmed the toxicty induced by GF. The results showed that eight metabolites (KA, xanthurenic acid, phenylacetylglycine, N2-succinyl-l-ornithine, leucylproline, l-phenylalanyl-l-hydroxyproline, C18 phytosphingosine, cholic acid) were interpreted as potential biomarkers and amino acid metabolism, gut microflora, bile biosynthesis were associated with GF-induced hepatotoxicity (Geng et al., 2013). However, little is known about the toxicological characteristics of the diterpenoids from GF, this could possibly be due to the low content of the diterpenoids in herb and low dose applied in the treatments, which is 1.5–3 g in Chinese Pharmacopeia. However, herbs generally considered as toxic can be used to fight diseases without triggering any undesired effects which made the study about the toxic effect of GF and their main diterpenoids essential. In order to evaluate the toxicity, TDG was administered by gastric intubation into rats. Then, blood samples were collected and blood concentrations of diterpenoids were analyzed by UPLC–TQ-MS technique. High content of toxic ingredients were often chosen to control the quality and safety of toxic herb in previous study. However, this study showed that the toxicological effects were not only related with the contents in herb, but also closely with the blood concentrations, which indicated that it would be necessary to control herb quality by evaluating ingredients of good absorption. Yuanhuafine and yuanhuapine were supposed to be the key compounds of TDG which should be responsible for the toxicity. Previous structure–activity studies showed that esterification of the 20-hydroxyl with fatty acids led to a substantial retention of activity with an overall decrease of toxicity, while formation of an acetonide between the 5- and 20-hydroxyl was detrimental for the activity, as also noticed in the cytotoxicity studies (He et al., 2002a). Genkwanine F, N and Wikstroemia factor M1 were compounds with esterification of the 20-hydroxyl which made the toxicity decreased and yuanhuacine, yuanhuadine and yuanhuatine were hardly to be absorbed. Therefore, yuanhuafine and

Table 3 Information of diterpenoids. Compounds

Dosage (mg/kg)

Cmax (ng/ml)

Absorption coefficient

Absorption extent

Structure difference

Factors affect absorption

Yuanhuacine Yuanhuadine Yuanhuafine Yuanhuatine Yuanhuapine Genkwanine N Genkwanine F Wikstroemia factor M1

7.24 10.70 1.97 1.04 1.31 4.03 11.90 23.31

102.70 ND 318.42 13.12 186.41 17.51 ND ND

102.70/7.24 – 318.42/1.97 13.12/1.04 186.41/1.31 17.51/4.03 – –

+ − +++ + +++ + − −

(CH CH)2 (CH2 )4 CH3 , OCOPh (CH CH)2 (CH2 )4 CH3 , OCOCH3 OCOCH3 , Ph OCOPh, Ph OCOCH3 , Ph OCOPh, Ph (CH CH)2 (CH2 )4 CH3 , Ph (CH CH)2 (CH2 )4 CH3 , Ph

Long chain affect absorption Long chain affect absorption Small MW, good absorption Two benzene rings affect absorption Small MW, good absorption Two benzene rings affect absorption Long chain affect absorption Long chain affect absorption

+++, good absorption; +, poor absorption; −, not absorption.

Please cite this article in press as: Chen, Y.-Y., et al., Toxicity of daphnane-type diterpenoids from Genkwa Flos and their pharmacokinetic profile in rat. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.06.012

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yuanhuapine are likely to be more toxic than other diterpenoids in vivo. Another important issue addressed in this study is that the acute toxicity of TDG correlates with the concentration of deterpenoids in the blood after administration. In fact, toxicology is the presentation of drug actions, while pharmacokinetics is a indispensable approach that realizes the drug actions which means pharmacokinetics is the way while toxicology is the end (Zhang et al., 2012). Although yuanhuafine and yuanhuapine were very low in crude drug, high blood concentrations were observed, and the rats died after a certain concentration, which also suggested a quantity-timetoxic relationship. This result also suggests that the absorption of diterpenoids with a long fatty acid chain such as Genkwanine F and Wikstroemia factor M1 by the animal’s intestine system has its own limit, and no matter how much the orally administered dosage has increased, the absorption rate would not increase at this internal limit. In our future research, we will use Caco-2 human cell line as a model of the intestinal epithelium to demonstrate whether the diterpenoids could be absorbed in the intestine since Caco-2 cells are transporters typical of the small intestine (Maubon et al., 2007), which is considered an excellent model to predict the intestinal absorption rate of different molecules obtaining better approximations to actual intestinal absorption rates, and is a good model for the evaluation of intestinal transport mechanisms. Compounds with lower bioavailability would either be poorly absorbed from the gastrointestinal tract or substantially metabolized prior to becoming systemically available (Turner et al., 2003), so the metabolism of different structure diterpenoids will also be further studied to explain the bioavailability difference. In summary, this paper shows the toxicity of diterpenoids from GF and its correlation with the blood concentration in rats. Based on these findings, it can be inferred that some potential toxicity of the plant was correlated with the blood concentration of diterpenoids. Furthermore, it verified that the blood concentration of diterpenoids were not proportional to the contents in the extract due to their different bioavailability. This study showed that the diterpenoids with high bioavailability may lead to obvious toxicological effects, even if their contents in the herb were low. And compounds of low bioavailability may be less toxic even if their contents in the herb were high. Therefore, it is highly recommended that determination of diterpenoids in herb as well as their concentration in vivo after administration (therapeutic drug monitoring for herbal medicine) were both important for safe usage of this herb in clinic. This would provide a safe application of herbal medicine to patients in clinics.

Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgements This research was financially supported by National Basic Research Program of China (973 Program) (2011CB505300, 2011CB505303). We are pleased to thank Waters China Ltd. for technical support.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phymed.2013. 06.012.

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Please cite this article in press as: Chen, Y.-Y., et al., Toxicity of daphnane-type diterpenoids from Genkwa Flos and their pharmacokinetic profile in rat. Phytomedicine (2013), http://dx.doi.org/10.1016/j.phymed.2013.06.012