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GC–MS method for determination and pharmacokinetic study of seven volatile constituents in rat plasma after oral administration of the essential oil of Rhizoma Curcumae
Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 577–585
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GC–MS method for determination and pharmacokinetic study of seven volatile constituents in rat plasma after oral administration of the essential oil of Rhizoma Curcumae Wenjing Li a,b , Bo Hong b , Zuojing Li a , Qing Li a , Kaishun Bi a,∗ a b
College of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China College of Pharmacy, Qiqihar Medical University, 333 Buikui Street, Qiqihar, 161006, PR China
a r t i c l e
i n f o
Article history: Received 11 July 2017 Received in revised form 25 November 2017 Accepted 25 November 2017 Available online 28 November 2017 Keywords: GC–MS Rhizoma Curcumae Essential oil Pharmacokinetics Rat plasma
a b s t r a c t Rhizoma Curcumae (RC) is perennial herbaceous plant mainly present in China, India and Malaysiabelong, which is belong to the family Zingiberaceae. The rhizomes of RC have been used as a famous traditional Chinese medicine for the treatment of syndrome of blood stasis. A selective, sensitive and accurate gas chromatography–mass spectroscopy (GC–MS) method was developed and validated in this paper for the simultaneous determination and pharmacokinetic study of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione in rat plasma. The GC–MS system was operated under selected ion monitoring (SIM) mode using a DB-5 (30 m × 0.25 mm (ID) × 0.25 m (film thickness)) column. Linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability were used to validate the current GC/MS assay. The lowest limit of quantifications (LLOQ) of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, Curdione were 2.71 ng/mL, 7.76 ng/mL, 3.37 ng/mL, 21.68 ng/mL, 40.21 ng/mL, 24.84 ng/mL and 47.78 ng/mL respectively. After oral administration 1.0 g/kg of RC rhizomes to the rats, the maximum plasma concentration (Cmax ) was 34.72 ± 9.97 ng/mL for ␣-Pinene, 99.86 ± 5.54 ng/mL for 1,8-Cineole, 16.10 ± 3.37 ng/mL for Borneol, 248.98 ± 86.19 ng/mL for -Elemene, 673.75 ± 104.15 ng/mL for Curcumol, 2353.64 ± 637.83 ng/mL for Germacrone and 2420.04 ± 708.51 ng/mL for Curdione. The time to reach the maximum plasma concentration (Tmax ) was 2.33 ± 0.29 h for ␣-Pinene, 0.67 ± 0.29 h for 1,8-Cineole, 1.33 ± 0.58 h for Borneol, 1.83 ± 0.76 h for -Elemene, 0.83 ± 0.29 h for Curcumol, 0.89 ± 0.98 h for Germacrone and 1.17 ± 0.76 h for Curdione. In this study, a validated GC–MS method for simultaneous determination of seven volatile oil compounds in rat plasma after oral administration of the extract of RC rhizomes and research on their pharmacokinetics was validated. The recovery and stability results were satisfactory in this study. © 2017 Published by Elsevier B.V.
1. Introduction Rhizoma Curcumae (RC) is a perennial herbaceous plant, which is belonged to the family Zingiberaceae and mainly presented in China, India, Malaysia. In China, RC is mainly produced in Guangxi, Yunnan, Guizhou, Zhejiang and other south parts provinces. The dried rhizomes of RC, which is known as “E Zhu” in Chinese, is acknowledged in the Chinese Pharmacopoeia [1] and is widely used for treatment of the blood stasis. The essential oil of RC can change the specific viscosity, erythrocyte deposited, erythrocyte
∗ Corresponding author. E-mail address: [email protected] (K. Bi). https://doi.org/10.1016/j.jpba.2017.11.058 0731-7085/© 2017 Published by Elsevier B.V.
sedimentation rate, and other hemorheology parameters of whole blood. Because of these above function, it was used to prevent the platelet aggregation and inhibit the formation of thrombosis in clinical practice [2,3]. The Modern pharmacological studies have shown that the RC rhizomes possessed a lot of other pharmacological and biological activities, such as antitumor [4–6], anti-inflammatory [7], anti-epileptic seizure [8], antibacterial [9], and others [10]. In this paper, we emphasize the pharmacokinetic behavior of the main compounds showed antithrombotic effect in RC. Essential oil of RC as the most important components in promoting blood circulation and removing blood stasis, the content is the only index needed to be qualified for RC rhizomes in Chinese Pharmacopoeia. So it is very important to clarify the pharmacokinetics of the main compounds from essential oil in blood. Among these
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volatile constituents, ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione are key active compounds used for treatment of blood stasis. In the pharmacology study of ␣Pinene [11], removing blood stasis and antitumor effect is the main research direction. Studies have shown that ␣-Pinene can significantly reduce the concentration of TXA2 in the body and further inhibit the activity of TXA2. TXA2 (thromboxaneA2) has the pharmacology effect of activating the platelet, leading to aggregation, and shrinking the blood vessel. In the effect study of ␣-Pinene on the non-small cell lung cancer(NSCLC), Z. Zhang [12] found that ␣-Pinene combined with Paclitaxel can increase the therapeutic effect on lung cancer A549 cells. Besides these, ␣-Pinene also has antifungal and antiinflammatory effect [13,14]. 1,8-Cineole was studied about the anti-inflammatory, expectorant and hypotension effect [15,16]. To the blood system disease, 1,8-Cineole has therapeutic effect and health care function. Borneol was widely used in traditional Chinese medical preparation to treat the cardiovascular and cerebrovascular diseases. Large dose of borneol intravenously administrated to the rabbits can inhibit the platelet aggregation induced by ADP, to the mice can reduce the whole blood clot weight [17]. Borneol can significantly increase the fibrinolysis effect through activating the fibrinlysis system. -Elemene was widely used in the treatment of blood stasis, it also was the second class non cytotoxic antitumor drug. The antithrombotic effect mechanism is affecting the arachidonic acid metabolic pathways and disturbing the synthesis of platelet [18]. Curcumol has been verified that it has strong antiplatelet aggregation(clinically was used to treat blood stasis), antiinflammatory, analgesic, inhibition of cell proliferation, antifibrosis and so on. In addition, Curcumol also has anti-tumor effect through effectively inhibiting the proliferation and inducing the apoptosis of a variety of malignant tumor cells(A549 cells, CEN-2 cells, MDA-MB-231 cells, Hep G2 cells, SPCA-1 cells) [19,20]. Germacrone is a major bioactive component found in volatiles oil product extracted from the RC. It shows an extensive range of biological activities, such as anticoagulation, fibrinolysis, antiinflammatory, antiulcer, antibacterial, antitumor, antitussive, vasodilator and hepatoprotector effects [21,22]. Curdione was found that it has strong antiplatelet aggregation effect and it was mainly used to treat blood stasis [23]. W.H. Qiao [24] reported that Curdione may inhibit thrombin-induced rat platelet activation and aggregation by inhibiting the PLC-PKC-MAPKs pathway, at least in part. So far, we have known that the seven compounds from RC are the key components of activating blood circulation and removing blood stasis, but there is still no method established for the simultaneous determination of these seven constituents in plasma samples after oral administration of essential oil from RC. So, in this study, we studied the pharmacokinetics procedure of RC through detecting the plasma concentration of the 7 compounds in rats. Several analytical methods have been established for determination of sesquiterpenoids from Zedoary turmeric oil in plasma samples, including High Performance Liquid Chromatogrphy (HPLC) for determination of curcumol and germacrone in rat plasma [25], High Performance Liquid Chromatograhpy tandem mass (HPLC–ESI–MS) for determination of individual Curcumol in rats blood [26], GC for curcumol and curdione in rat plasma [27]. GC–MS in selected ion-monitoring mode (SIM) method was developed in this study to quantify ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone and Curdione in rat plasma for the first time. It was fully validated and applied to a pharmacokinetic study in rats after oral administration of the essential oil of RC. GC–MS method is more suitable for volatile constituents determination in selectivity, specificity and sensitivity than LC/MS and HPLC-UV method. The total run time of every sample was 33 min. We expect that the results of this study could not only facilitate to
Fig. 1. The picture of RC and chemical structures of the seven analytes: ␣-Pinene (A), 1,8-Cineole(B), Borneol (C), -Elemene(D), Curcumol(E), Germacrone (F), Curdione(G).
clarify the mechanism of RC, but also to guide the clinical rational use. 2. Experimental 2.1. Materials REFERENCES standards of ␣-Pinene (C10 H16 , 98% purity, Batch No: 101561026), 1,8-Cineole (C10 H18 O, 99% purity, Batch No: 12-2006), Borneol (C10 H18 O, 98% purity, Batch No: 03-2008), Germacrone (C15 H22 O, 99.8% purity, Batch No: 111665-201204), and Curdione (C15 H24 O2 , 99.2% putiry, Batch No: 111800-201001) were purchased from Shanghai Bodun Biochemical Co., Ltd. (Shanghai, PR China); -Elemene (C15 H24 , 99% purity, Batch No:100268-200401), Curcumol (C15 H24 O2 , 99% purity, Batch No: 100185-200506) were purchased from National Institutes of Food and Drug Control(Beijing, PR China). The picture of RC and structures of the seven analytes are shown in Fig. 1. Methanol and acetonitrile were HPLC grade (filtered by 0.22 m filter) and purchased from Oceanpak company (Sweden). Formic acid of HPLC grade was purchased from Dikma (Richmond Hill, NY, USA). Other reagents were analytical grade. Ultra-pure water was prepared by using a Mili-Q water purification system (Millipore, Molsheim, France), 13 mm Luer syringe filter (1513022A) were purchased from USA. The RC rhizomes were collected from Guangxi Province of China in December 2015 and were identified by Prof. Lina Guo in Qiqihar Medical University. The specimen (20151001) was deposited in School of Pharmacy, Qiqihar Medical University, China. 2.2. GC–MS conditions The GC–MS system consisted of a model 7890 B series gas chromatograph coupled with a 7693 autosampler and a model 5977A MSD with Triple-Axis Detector (Agilent, IL, USA). Separation was performed on a DB-5 column(30 m × 0.25 mm, 0.25 m film thickness) supplied by Agilent Technologies (IL, USA). The carrier gas was 99.99% high purity Helium with a flow rate of 1.2 mL/min. The sample volume was 1 L. The injection port and the detector temperatures were 270 ◦ C and 280 ◦ C, respectively. Oven temper-
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Fig. 2. Mass spectrums of ␣-Pinene (A), 1,8-Cineole(B), Borneol (C), -Elemene(D), Curcumol(E), Germacrone (F), Curdione(G).
ature program was initially set at 50 ◦ C for 1 min, then ramped at 5 ◦ C/min to 120 ◦ C and held for 1.0 min, then ramped at 5 ◦ C/min to 150 ◦ C and held for 5 min, ramped to 200 ◦ C at 3 ◦ C/min and held
for 1 min. The total run time was 33 min with a solvent delay of 10 min. Ionization was performed in electron impact ionization (EI) mode at 70 eV. Selective ion monitoring (SIM) was set for quantifi-
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Table 1 Characteristic ions (m/z) and retention times of the seven analytes. Compound
Characteristic Ions (m/z)*
␣-Pinene 1,8-Cineole Borneol -Elemene Curcumol Germacrone Curdione
137 155 155 205 237 219 237
122 140 140 148 136 176 181
Retention Time (min) 106 109 122 108 122 136 168
94 82 111 94 108 108 110
78 44 96 82 94 92 70
6.64 9.23 13.05 19.70 26.18 30.01 30.90
*The molecular ions which are marked in bold were used for quantification of each analyte.
cation with dwell time 100 ms/ion. Retention times of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone and Curdione were 6.64 min, 9.23 min, 13.05 min, 19.70 min, 26.18 min, 30.01 min and 30.90 min, respectively. The characteristic ions (m/z) and retention times of the seven analytes are listed Table 1. 2.3. Preparation of the essential oil solution of RC rhizomes for administration The dried pieces of the RC rhizomes (12.5 g) were extracted by volatile oil extractor under reflux with 500 mL water for 4 h. The extract solution was concentrated to 100 mL, the oil was collected and combined with tween 80 (1 mL: 0.5 mL) to 100 mL flask, adding the extract solution to 100 mL. The contents of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone and Curdione in essential oil determined by the developed GC–MS method were 0.09%, 3.22%, 1.04%, 0.79%, 3.67%, 4.77% and 9.67%, respectively. 2.4. Preparation of standard samples The stock solution of ␣-Pinene (17.77 mg/mL), 1,8 Cineole (3.18 mg/mL), Borneol (22.09 mg/mL), -Elemene (2.22 mg/mL), Curcumol (16.47 mg/mL), Germacrone (20.35 mg/mL) and Curdione (19.57 mg/mL) were prepared in methanol. Then, we mixed these stock solutions and serially diluted with methanol to provide desired concentration. Calibration solutions were prepared by spiking 20 L of the appropriate standard working solutions into 200 L blank plasma. The ranging value was shown in Table 2. Seven standard curves containing of six-point concentration levels were as followed: ␣-Pinene (2.71, 5.42, 10.84, 21.69, 43.38, 86.77 ng/mL), 1,8 Cineole (7.76, 15.53, 31.05, 62.11, 124.22, 248.44 ng/mL), Borneol (3.37, 6.74, 13.48, 26.96, 53.93, 107.86 ng/mL), -Elemene (21.68, 43.36, 86.72, 173.44, 346.88, 693.75 ng/mL) and Curcumol (40.21, 80.42, 160.84, 321.68, 643.36, 1286.72 ng/mL), sevenpoint concentration levels for Germacrone (24.84, 49.68, 99.36, 198.73, 397.46, 794.92, 1589.84 ng/mL), and Curdione (47.78, 95.56, 191.11, 382.23, 764.45, 1528.91, 3057.81 ng/mL). Quality control samples concentration were 5.42, 21.69, 86.77 ng/mL for ␣-Pinene, 15.53, 62.11, 248.44 ng/mL for 1,8 Cineole, 6.74, 26.96, 107.86 ng/mL for Borneol, 43.36, 173.44, 693.75 ng/mL for Elemene, 80.42, 321.68, 1286.72 ng/mL for Curcumol, 49.68, 198.73, 794.92 ng/mL for Germacrone and 95.56, 382.23, 1528.91 ng/mL for Curdione. All the solutions were stored at 4 ◦ C before use.
auto sample tube. 1 L of the supernatant was injected into GC–MS system for analysis. 2.6. Method validation Selectivity, linearity, intra-day and inter-day precision, accuracy, recovery and stability in accordance to the requirements for the analysis of biological samples were used to validate the current GC–MS assay [28]. 2.6.1. Selectivity After the conditions of chromatography and mass spectrometry were determined, selectivity was assessed by analyzing the chromatograms of six different batches of blank rat plasma. In order to investigate the separation of endogenous substances from analytes, the chromatograms of corresponding spiked with the seven analytes and plasma samples obtained after oral administration of the essential oil of 10 mg/kg (equals to RC rhizomes at a dose of 1.0 g/kg body weight) were supplied. 2.6.2. Linearity and LLOQ Standard plasma samples at series concentrations by least squares linear regression of the peak area of each analyte versus the corresponding concentrations with a weighted (1/X2 ) factor was analyzed to assess the linearity. The lower limit of quantification (LLOQ) was defined as the lowest concentration on the calibration curve. 2.6.3. Precision and accuracy The precision was determined by RSD and the accuracy by RE value of QCL, QCM and QCH samples. Intra- and inter-day precision and accuracy each day and three consecutive days were evaluated by measuring six replicates of QC samples of the seven analytes at three concentration levels respectively. The acceptable criteria for the intra-day and inter-day precision and accuracy are within 15% for three levels. 2.6.4. Extraction recovery QCL, QCM and QCH samples in six replicates were used to analyze the extraction recovery. The peak areas were determined together with the linear samples. The recovery was calculated through comparing the peak areas of QC samples with the analytes spiked into the post extraction supernatant samples. The coefficient of variation should be within 15%. 2.7. Pharmacokinetic study
2.5. Plasma sample preparation The plasma sample (100 L) was put into a 1.5-mL clean eppendorf tube, after that, 100 L hexane and ethyl acetate (1:1) mixture was pipetted into the sample. The sample was shaken for 1 min to extract active compounds in a vortex mixer. Then the sample was centrifuged in high speed cryogenic centrifuge at 12000 rpm for 10 min and the supernatant was transferred to a 1.5 mL clean
Male Wistar rats weighing 250 ± 20 g were supplied by the Laboratory Animal Center of Qiqihar Medical University (Qiqihar, China) and were maintained in temperature of 23 ± 2 ◦ C, relative humidity of 57 ± 2% with free access to food and water for 6 days acclimation. Before the administration of the essential oil of RC rhizomes, the rats were fasted for 12 h with free access to water. The experiment was approved by the Animal Ethics Committee
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Fig. 3. SIM chromatogram of (A) blank plasma, (B) blank plasma spiked with the seven analytes, (C) plasma sample obtained at 2 h after oral administration of the essential oil of RC rhizomes: ␣-Pinene (1), 1,8-Cineole(2), Borneol (3), -Elemene(4), Curcumol(5), Germacrone (6), Curdione(7). Table 2 The regression equations, linear ranges and LLOQs of the analytes in plasma. Compounds
Y = aX + b
R
Linear range (ng/mL)
LLOQ (ng/mL)
␣-Pinene 1,8-Cineole Borneol -Elemene Curcumol Germacrone Curdione
Y = 7 × 108 + 531 Y = 1 × 109 − 7477 Y = 8 × 108 − 852 Y = 1 × 109 + 496 Y = 5 × 108 − 681 Y = 5 × 108 + 7502 Y = 2 × 108 + 403
0.999 4 0.999 3 0.999 9 0.998 0 0.999 6 0.997 1 0.999 7
2.71–86.77 7.76–248.44 3.37–107.86 21.68–693.75 40.21–1286.72 24.84–1589.84 47.78–3057.81
2.71 7.76 3.37 21.68 40.21 24.84 47.78
of Qiqihar Medical University. The single administration was prepared by constituting the essential oil with tween 80 and extract solvent to get a concentration equivalent to 1.0 g/kg RC rhizomes (equivalent to 0.11 mg/kg of ␣-Pinene, 4.02 mg/kg of 1,8-Cineole, 1.29 mg/kg of Borneol, 0.99 mg/kg of -Elemene, 4.58 mg/kg of Curcumol, 5.96 mg/kg of Germacrone, and 12.08 mg/kg of Curdione). The blood samples (400 L) were obtained from the suborbital vein
of rats at 0, 0.17, 0.5, 1, 2, 2.5, 3, 4, 6, 8, 10, 12 and 24 h after dosing. The samples were collected into EP tube pre-coated with heparin and immediately centrifuged at 5000 rpm for 10 min, then the separated plasma samples were stored at −80 ◦ C until analysis. The maximal concentration (Tmax ), maximal concentration (Cmax ) and the elimination rate constant (Ke) were determined directly and calculated by linear regression of the terminal points in a semi-
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Fig. 4. Mean ± S.D. (n = 6) plasma concentration-time curves of ␣-Pinene (A), 1,8-Cineole (B), Borneol (C), -Elemene (D), Curcumol (E), Germacrone (F), Curdione (G) in rats after oral administration of the essential oil of RC rhizomes.
log plot of the plasma concentration against time respectively. The elimination half-life (t1/2 ) was calculated as 0.693/Ke. The area under plasma concentration-time curve (AUC0-t ) was estimated by the linear trapezoidal rule from 0 to 24 h. The area under the plasma concentration-time curve to time infinity (AUC0-∞ ) was calculated as AUC0-∞ = AUC0-t + Ct /Ke, where Ct was the last measurable concentration.
of the seven analytes are presented in Fig. 2. Injector temperature (240, 270 and 300 ◦ C), detector temperature (260, 280 and 300 ◦ C), and column temperature program as the main chromatographic conditions were optimized, in order to obtain better area response, symmetrical peaks and reduced run time.
3. Results and discussion
Extraction methods of liquid–liquid extraction (LLE), solidphase extraction (SPE), and protein precipitation using methanol or acetonitrile were compared for bio-sample preparation. For LLE, organic extracting solvent including hexane, ethyl acetate, and their mixture were investigated. We found that LLE method using hexane and ethyl acetate (1:1) could obtain clean, reproducible extraction and good recovery for the seven analytes.
3.1. Optimization of GC–MS parameters In order to increase the sensitivity of the seven analytes, selective ion monitoring (SIM) mode was chosen to determine. In this paper, the representative spectrogram scanned from 50 to 500 amu
3.2. Selection of extraction method
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Table 3 Summary of accuracy, precision and recovery for the seven analytes in rat plasma (n = 6). Analytes
concentration (ng/mL)
Intra-day RSD(%)
Inter-day RSD(%)
Accuracy RE(%)
Recovery(%, mean ± SD)
␣-Pinene
5.42 21.69 86.77 15.53 62.11 248.44 6.74 26.96 107.86 43.36 173.44 693.75 80.42 321.68 1286.72 49.68 198.73 794.92 95.56 382.23 1528.91
7.75 9.08 8.34 5.25 4.41 4.67 8.92 7.02 2.05 5.05 5.83 5.41 5.17 8.31 5.65 6.54 7.01 1.20 7.82 2.97 1.97
6.80 6.93 5.68 8.56 4.84 9.04 6.55 9.02 8.77 9.62 2.27 2.53 5.90 4.50 2.46 7.22 5.83 4.28 2.95 4.06 3.22
7.46 −2.57 −6.59 −9.75 −6.36 −2.16 −6.36 −5.30 −5.20 6.68 −2.49 −4.77 −4.40 −3.77 1.30 −8.71 −1.96 −4.03 −4.30 −5.16 −4.08
68.3 ± 5.3 79.7 ± 4.9 80.7 ± 1.5 69.5 ± 5.1 77.4 ± 2.6 79.4 ± 3.5 69.0 ± 2.5 76.0 ± 4.4 78.8 ± 1.9 67.7 ± 4.3 76.8 ± 3.3 80.1 ± 8.9 64.8 ± 3.4 74.8 ± 4.3 80.5 ± 8.0 72.5 ± 6.1 75.8 ± 3.9 79.6 ± 4.0 69.0 ± 1.9 78.1 ± 3.1 79.6 ± 4.2
1,8-Cineole
Borneol
-Elemene
Curcumol
Germacrone
Curdione
Table 4 Pharmacokinetic parameters of the seven compounds in rats after oral administration the essential oil of CR rhizomes (mean ± SD, n = 6). compound
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUC0-t (ng h/mL)
AUC0-∞ (ng h/mL)
␣-Pinene 1,8-Cineole Borneol -Elemene Curcumol Germacrone Curdione
34.72 ± 9.97 99.86 ± 5.54 16.10 ± 3.37 248.98 ± 86.19 673.75 ± 104.15 2353.64 ± 637.83 2420.04 ± 708.51
2.33 ± 0.29 0.67 ± 0.29 1.33 ± 0.58 1.83 ± 0.76 0.83 ± 0.29 0.89 ± 0.18 1.17 ± 0.76
8.64 ± 1.46 8.98 ± 1.63 12.43 ± 2.88 19.86 ± 4.05 15.63 ± 5.50 14.17 ± 4.13 7.14 ± 0.67
189.78 ± 89.10 454.74 ± 82.43 100.55 ± 8.27 1067.37 ± 216.55 3154.16 ± 405.94 16501.24 ± 663.88 12524.92 ± 3222.10
229.57 ± 93.50 524.32 ± 81.67 146.28 ± 10.74 2092.70 ± 416.18 5388.65 ± 661.86 28198.873 ± 4102.62 14139.35 ± 3109.19
3.3. Method validation The chromatograms of blank plasma, plasma sample at HQC spiked with the analytes, and the plasma sample from a rat at 2 h after oral administration of the essential oil of RC rhizomes were compared to evaluate the selectivity of the method. From the typical chromatograms with a good separation as well as excellent peak shapes shown in Fig. 3, we saw that there is no endogenous interference observed at the retention times of the seven analytes. According to the Tmax value of seven compounds, we know that the plasma sample obtained at 2 h had the relative higher response to the seven compounds. So in Fig. 3-C, we choose the 2 h after oral administration as the representative plasma sample chromatogram to compare with the other two chromatorams (Fig. 3-A and Fig. 3-B). The retention times of the seven analytes were as followed: ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione were 6.63, 9.22, 13.05, 19.7, 26.17, 30.01 and 30.09 min, respectively. The calibration curves, the linearity ranges and LLOQs value for the seven analytes are summarized in Table 2. A good linearity within the described ranges for seven analytes was indicated from the high correlation coefficient (all higher than 0.9971). All standards met the criteria of less than 15% deviation. For pharmacokinetic studies, the LLOQs were suitable for quantitative detection of the seven analytes. The intra- and inter- day precision and accuracy for seven target analytes are shown in Table 3. The method was reliable and reproducible demonstrated by results for the samples tested within the acceptable criteria of ±15%. The recovery of the assay, which illustrated that the majority of each analyte was successfully extracted are summarized in Table 3. The extraction efficiencies for ␣-Pinene, 1,8-Cineole, Borneol, Elemene, Curcumol, Germacrone, and Curdione were 68.3–80.7%,
69.5–79.4%, 69.0–78.8%, 67.7–80.1%, 64.8–80.5%, 72.5–79.6% and 69–79.6%. The stability of seven analytes in rat plasma under different conditions is summarized in the supplementary. 3.4. Pharmacokinetics study A validated method in this paper was successfully applied to the pharmacokinetics study of the seven bioactive constituents in rat plasma after oral administration of the essential oil from RC rhizomes. We chose an oral dosage for the rats corresponding to the human dosage in Chinese Pharmacopoeia (China Pharma copoeia Committee, partI, 2015). The human dosage of the TCM in Chinese Pharmacopoeia is 8–15 g/day for blood stasis treatment, 1.0 g/kg in rats based on the Meeh-Rubner method [29] was picked as the final oral dosage. Meeh-Rubner method was used to calculate the variation of effective dosage between different species of animals and human. Meeh-Rubner formula: A = K(W2/3 )/10000 is more appropriate to predict body surface area according to the weight, “A” represent body surface area (m2 ), “W” represent weight (g), “K” represent constant (vary with the species of animals, the constant of rat is 9.1). Then we go on calculating the dosage using the conversion factor (CF) from the dosage unit of mg/kg to mg/m2 . In this paper, rat dosage (mg/kg) = human dosage(mg/kg) × (human CF)/(rat CF), in this formula, human dosage is 10 g/60 kg, human CF is 36, rat CF is 6, so the result of rat dosage is 1.0 g/kg. The mean plasma concentration-time profiles of the seven analytes are presented in Fig. 4. Table 4 shows the main pharmacokinetic parameters including half-time (t1/2 ), time to reach the maximum concentrations (Tmax ), maximum plasma concentration (Cmax ), and an under concentration–time curve (AUC0-t and AUC0-∞ ). As seen from Fig. 4, 1,8-Cineole, Borneol, Curcumol, Germacrone, and Curdione were rapidly absorbed following oral administration of the
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essential oil, and they achieved maximum plasma concentration (Cmax ) between 0.67 and 1.33 h, ␣-Pinene and -Elemene were absorbed slowly with the Tmax 2.33 and 1.83 h separately after oral administration. T1/2 of the seven compounds were all more than 7.14 h, the slow elimination may due to the enterohepatic circulation process because of the intragastric administration or the high affinity binded to blood protein. It needed to be proved by the hepatoenteral pharmacokinetic and equilibrium dialysis experiment further. In our previous research, we have evaluated the differences of pharmacokinetic behavior between normal and blood-stasis pregnant rats after intravenous administration using HPLC method for the determination of Curcumol in rat plasma, the LLOQ of Curcumol was 450 ng/mL, while in this paper, the LLOQ of Curcumol was 40.21 ng/mL, the sensitivity increased 10 times than before. D. Ji [30] had studied the pharmacokinetics of Germacrone and curdione in rat plasma using the HPLC method. According to their results, the LLOQ of Germacrone and curdione were 197.2 ng/mL and 1073 ng/mL, while the LLOQ of the method developed in this paper are 24.84 and 47.78 ng/mL for Germacrone and curdione, respectively. In this paper, the AUC and Cmax of these seven analytes showed consistent tendency in plasma concentration–time profiles. It is illustrated that the analytes had the similar oral absorption and distribution after given the essential oil of RC rhizomes, which may contribute to their similar characteristic. To our knowledge, there is no method about simultaneous determination of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione in plasma is available in the literatures. But there are some reports about the determination of some of the seven compounds mentioned above in plasma samples after oral administration of RC prescriptions [31,32]. The results about the pharmacokinetic profiles of the seven analytes in RC in this paper were slightly different from that observed for their pure forms or other prescriptions. We analyzed the reasons might be due to the complexity of TCM for their interactions among so many compounds in vivo. This work can provide some experimental basis for the clinical use of RC and its prescriptions.
4. Conclusion A multi-component pharmacokinetic study of RC was conducted for the simultaneous determination of ␣-Pinene, 1,8Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione in rat plasma after oral administration of RC by GC–MS. The analytical method in this paper was perfect because of the relative short chromatographic running time and simple pretreatment using liquid–liquid extraction sample. The established GC–MS method was suitable for pharmacokinetic study of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione in rat plasma because of the excellent sensitivity, selectivity, precision, accuracy and extraction recovery. The relative rapid absorption and long half-time of bioactive constituents gave a reasonable explanation for the rapid effects and long interval between dosing of RC in the treatment of blood stasis. The results of this study illustrated that the co-existing compounds in RC extract could change the pharmacokinetic behaviors of these seven constituents compared with other prescriptions of TCM. It will be helpful for the mechanism research of action of RC. This is the first report on a GC–MS method for the simultaneous determination of total ␣Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione accurately with a purpose of the pharmacokinetics study of the above mentioned compounds in rats plasma after oral administration of the essential oil of RC rhizomes.
Acknowledgments The work was financially supported by National Natural Science Foundation of China (81403173) and China Postdoctoral Science Foundation (2014M551291), and in part by “Young Excellent and Innovative Talent Training Program” of Heilongjiang Ordinary Undergraduate University (7UNPYSCT-201611).
References [1] China Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of China, China Chemical Industry Press, Beijing, China, 2015, pp. 274–275. [2] L. Wan, J.M. Zhang, S. Fu, J.S. Wang, C.M. Fu, Chromatography activity relation of stagnation of vital energy and blood stasis syndrome influencing hemorheology by stirbaked Curcumae Rhizoma with vinegar, Chin. Tradi. Patent Medi. 35(2013) 330-335. [3] X. Wang, Q. Xia, D.J. Xu, Research on the anticoagulant and antithrombotic effects of Rhizoma Curcumae, Chin. Tradit. Pate. Medi. 34(2012) 550-553. [4] J.J. Lu, Y.Y. Dang, M. Huang, W.S. Xu, X.P. Chen, Y.T. Wang, Anti-cancer properties of terpenoids isolated fromRhizomaCurcumae-a review, J. Ethnopharmacol. 143 (2012) 406–411. [5] D.C. Tang, W.H. Zang, H.H. Feng, Influences of medicated serum of different cultivars of Ezhu (Rhizoma Zedoariae) on proliferation, apoptosis and nucleo-cytoplasmic ratio in human gastric carcinoma cells BGC82, J. Beijing Univ. Tradit. Chin. Med. 36 (2013) 254–257. [6] L.Z. Huang, J. Wang, F.T. Lu, et al., Mechanism study on anti-proliferative effects of curcumol in human hepatocarcinoma Hep G2 cells, Chin. J. Chin. Mater. Medi. 38 (2013) 1812–1815. [7] X. Chen, C.J. Zong, Y. Gao, R.L. Cai, L. Fang, J. Lu, F. Liu, Y. Qi, Curcumol exhibits anti-inflammatory properties by interfering with the JNK-mediated AP-1 pathway in lipopolysaccharide-activated RAW264. 7cells, Eur. J. Pharmacol. 723 (2014) 339–345. [8] J. Ding, J.J. Wang, C. Huang, L. Wang, S.N. Deng, T.L. Xu, W.H. Ge, W.G. Li, F. Li, Curcumol from Rhizoma Curcumae suppresses epileptic seizure by facilitation of GABA(A) receptors, Neuropharmacology 81 (2014) 244–255. [9] H.F. Huang, C.J. Zheng, G.Y. Chen, W.Q. Yin, M.O. Zheng, Comparison of chemical composition and antibacterial activity between zedoary turmeric oil and dregs residual oil, Chin. J. Exper. Tradit. Medi. Form 21 (2015) 67–70. [10] Z. Liu, W.Y. Gao, S.L. Man, Y. Zhang, H.F. Li, S.S. Wu, J.Z. Zhang, C.X. Liu, Synergistic effects ofRhizomaParidisandRhizomaCurcuma longaon different animal tumor models, Environ. Toxico. Pharmacol 38 (2014) 31–40. [11] H. Türkez, E. Aydin, In vitro assessment of cytogenetic and oxidative effects of ␣-pinene, Toxi. Ind Heal. 32 (2016) 168–176. [12] Z. Zhang, S. Guo, X. Liu, X. Gao, Synergistic antitumor effect of ␣-pinene and -pinene with paclitaxel against non-small-cell lung carcinoma (NSCLC), Drug Res. 55 (2014) 113–119. [13] P. Dhar, P. Chan, D.T. Cohen, F. Khawam, S. Gibbonset, T.S. Leiby, E. Dickstein, P.K. Rai, G. Watal, Synthesis, antimicrobial evaluation, and structure-activity relationship of ␣-pinene derivatives, J. Agric. Food Chem. 62 (2014) 3548–3552. [14] S.Y. Nam, C.K. Chung, J.H. Seo, S.Y. Rah, H.M. Kim, H.J. Jeong, The therapeutic efficacy of ␣-pinene in an experimental mouse model of allergic rhinitis, Int. Immunopharmacol. 23 (2014) 273–282. [15] C. Morcia, M. Malnati, V. Terzi, In vitro antifungal activity of terpinen-4-ol, eugenol, carvone, 1,8- cineole (eucalyptol) and thymol against mycotoxigenic plant pathogens, Food Addit. Contami. Part A. 29 (2012) 415–422. [16] M. Takaishi, F. Fujita, K. Uchida, S. Yamamoto, S.M. Sawada, U.C. Hatai, M. Shimizu, M. Tominaga, 1,8-cineole, a TRPM8 agonist, is a novel natural antagonist of human TRPA1, Mol. Pain. 8 (2012) 86–98. [17] Q. Zhang, D. Wu, J. Wu, Y. Ou, C.L. Mu, B. Han, Q.L. Zhang, Improved blood-brain barrier distribution: effect of borneol on the brain pharmacokinetics of kaempferol in rats by in vivo microdialysis sampling, J. Ethnopharmacol. 162 (2015) 270–277. [18] Y. Zhao, R.G. Yang, M. Luo, The pharmacology effect and clinical research development of Zedoary turmeric oil, J. Pract. Tradit. Chin. Inter. Medi. 20 (2006) 1250–1261. [19] H. Wang, Y. Fang, Y. Wang, Z. Wang, Q. Zou, Y. Shi, J. Chen, D. Peng, Inhibitory effect of curcumol on jak2-STAT signal pathway molecules of fibroblast-like synoviocytes in patients with rheumatoid arthritis, Evid. Based Complement Alternat. Med. 746 (2012) 4–26. [20] Q.L. Tang, J.Q. Guo, Q.Y. Wang, H.S. Lin, Z.P. Yang, T. Peng, X.D. Pan, B. Liu, S.J. Wang, L.Q. Zang, Curcumol induces apoptosis in SPC-A-1 human lung adenocarcinoma cells and displays anti-neoplastic effects in tumor bearing mice, Asian Pac. J. Cancer Prev. 16 (2015) 2307–2312. [21] W. Cho, J.W. Nam, H.J. Kang, T. Windono, E.K. Seo, K.T. Lee, Zedoarondiol isolated from the rhizoma of Curcuma heyneana is involved in the inhibition of Inos, COX-2 and proinflammatory cytokines via the downregulation ofNFB pathway in LPS-stimulated murine macrophages, Inter. immunopharmacol. 9 (2009) 1049–1057. [22] X. Xiao, Y. Zhao, H. Yuan, W. Xia, J. Zhao, X. Wang, Study on the effect of Rhizoma Curcuma Longa on gastrin receptor. Zhong yao cai, J. Chin. Medi. Mater. 25 (2002) 184–185.
W. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 577–585 [23] Q. Xia, X. Wang, D.J. Xu, X.H. Chen, F.H. Chen, Inhibition of platelet aggregation by curdione from Curcuma wenyujin essential oil, Thromb. Res. 130 (2012) 409–414. [24] W.H. Qiao, D.L. Zhang, Y.L. Zhao, Y. Huang, J.M. Sun, D.J. Xu, Q. Xia, Inhibition by curdione of thrombin-induced platelet activation and aggregation, Acta Univer. Medi. Anhui. 52 (2017) 376–382. [25] Y. Pan, Y. Zhang, Z. Xiang, P.C. Yan, K.X. Huang, Determination of curcumenol and germacrone in plasma and its pharmacokinetics in rats, Chin. Tradit. Pat. Medi. 35(2013) 252-255. [26] T.J. Xu, S.Q. Tang, H. Rong, Y.C. Niu, Metabolic differences of curcumol between normal and blood-stasis rats using liquid chromatography-mass spectrometry, Lishizhen Medi. Mater. Medi. Res. 27 (2016) 3053–3056. [27] Y.T. Sun, Z.Q. Zhang, X. Li, S.C. Liu, H.J. Bian, J. Sun, Analysis of curcumol and curdione in plasma of rats by CGC. Chin. Tradi. Pat. Medi. 32(2010) 991-995. [28] Z.B. Wang, Q.H. Wang, B.Y. Yang, et al., GC–MS method for determination and pharmacokinetic study of four phenylpropanoids in rat plasma after oral
[29] [30]
[31]
[32]
585
administration of the essential oil of Acorus tatarinowii Schott rhizomes, J. Ethnopharmacol. 155 (2014) 1134–1140. Y.P. Zhang, «Pharmacology experiment» (the second edition), People’s Medical Publishing House (Appendix V), 238-239. D. Ji, C.Q. Mao, J.C. Li, T.L. Lu, H. Xie, G.F. Jiang, Y.Q. Xiao, Pharmacokinetics of curdione and germacrone in zedoary turmeric oil in rats in vivo, Chin. Tradi. Pate. Medi. 35(2013) 2622-2626. D. Lv, Y. Cao, X. Dong, X. Chen, Z. Lou, Y. Chai, Analysis and pharmacokinetic study of curdione in Rhizoma Curcumae by UPLC/QTOF/MS, Biomed. Chromatogr. 28 (2014) 782–787. Y.X. Song, Y.J. Liu, D.P. Zou, G. Wang, Z.C. Li, H.Y. Zou, Pharmacokinetic study of rhizoma Curcumae oil and rhizoma Curcumae oil--cyclodextrin inclusion complex in pigs after oral administration, J. Vet. Pharmacol. Ther. 35 (2012) 47–51.
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Short communication
GC–MS method for determination and pharmacokinetic study of seven volatile constituents in rat plasma after oral administration of the essential oil of Rhizoma Curcumae Wenjing Li a,b , Bo Hong b , Zuojing Li a , Qing Li a , Kaishun Bi a,∗ a b
College of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, PR China College of Pharmacy, Qiqihar Medical University, 333 Buikui Street, Qiqihar, 161006, PR China
a r t i c l e
i n f o
Article history: Received 11 July 2017 Received in revised form 25 November 2017 Accepted 25 November 2017 Available online 28 November 2017 Keywords: GC–MS Rhizoma Curcumae Essential oil Pharmacokinetics Rat plasma
a b s t r a c t Rhizoma Curcumae (RC) is perennial herbaceous plant mainly present in China, India and Malaysiabelong, which is belong to the family Zingiberaceae. The rhizomes of RC have been used as a famous traditional Chinese medicine for the treatment of syndrome of blood stasis. A selective, sensitive and accurate gas chromatography–mass spectroscopy (GC–MS) method was developed and validated in this paper for the simultaneous determination and pharmacokinetic study of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione in rat plasma. The GC–MS system was operated under selected ion monitoring (SIM) mode using a DB-5 (30 m × 0.25 mm (ID) × 0.25 m (film thickness)) column. Linearity, intra-day and inter-day precisions, accuracy, extraction recovery and stability were used to validate the current GC/MS assay. The lowest limit of quantifications (LLOQ) of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, Curdione were 2.71 ng/mL, 7.76 ng/mL, 3.37 ng/mL, 21.68 ng/mL, 40.21 ng/mL, 24.84 ng/mL and 47.78 ng/mL respectively. After oral administration 1.0 g/kg of RC rhizomes to the rats, the maximum plasma concentration (Cmax ) was 34.72 ± 9.97 ng/mL for ␣-Pinene, 99.86 ± 5.54 ng/mL for 1,8-Cineole, 16.10 ± 3.37 ng/mL for Borneol, 248.98 ± 86.19 ng/mL for -Elemene, 673.75 ± 104.15 ng/mL for Curcumol, 2353.64 ± 637.83 ng/mL for Germacrone and 2420.04 ± 708.51 ng/mL for Curdione. The time to reach the maximum plasma concentration (Tmax ) was 2.33 ± 0.29 h for ␣-Pinene, 0.67 ± 0.29 h for 1,8-Cineole, 1.33 ± 0.58 h for Borneol, 1.83 ± 0.76 h for -Elemene, 0.83 ± 0.29 h for Curcumol, 0.89 ± 0.98 h for Germacrone and 1.17 ± 0.76 h for Curdione. In this study, a validated GC–MS method for simultaneous determination of seven volatile oil compounds in rat plasma after oral administration of the extract of RC rhizomes and research on their pharmacokinetics was validated. The recovery and stability results were satisfactory in this study. © 2017 Published by Elsevier B.V.
1. Introduction Rhizoma Curcumae (RC) is a perennial herbaceous plant, which is belonged to the family Zingiberaceae and mainly presented in China, India, Malaysia. In China, RC is mainly produced in Guangxi, Yunnan, Guizhou, Zhejiang and other south parts provinces. The dried rhizomes of RC, which is known as “E Zhu” in Chinese, is acknowledged in the Chinese Pharmacopoeia [1] and is widely used for treatment of the blood stasis. The essential oil of RC can change the specific viscosity, erythrocyte deposited, erythrocyte
∗ Corresponding author. E-mail address: [email protected] (K. Bi). https://doi.org/10.1016/j.jpba.2017.11.058 0731-7085/© 2017 Published by Elsevier B.V.
sedimentation rate, and other hemorheology parameters of whole blood. Because of these above function, it was used to prevent the platelet aggregation and inhibit the formation of thrombosis in clinical practice [2,3]. The Modern pharmacological studies have shown that the RC rhizomes possessed a lot of other pharmacological and biological activities, such as antitumor [4–6], anti-inflammatory [7], anti-epileptic seizure [8], antibacterial [9], and others [10]. In this paper, we emphasize the pharmacokinetic behavior of the main compounds showed antithrombotic effect in RC. Essential oil of RC as the most important components in promoting blood circulation and removing blood stasis, the content is the only index needed to be qualified for RC rhizomes in Chinese Pharmacopoeia. So it is very important to clarify the pharmacokinetics of the main compounds from essential oil in blood. Among these
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volatile constituents, ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione are key active compounds used for treatment of blood stasis. In the pharmacology study of ␣Pinene [11], removing blood stasis and antitumor effect is the main research direction. Studies have shown that ␣-Pinene can significantly reduce the concentration of TXA2 in the body and further inhibit the activity of TXA2. TXA2 (thromboxaneA2) has the pharmacology effect of activating the platelet, leading to aggregation, and shrinking the blood vessel. In the effect study of ␣-Pinene on the non-small cell lung cancer(NSCLC), Z. Zhang [12] found that ␣-Pinene combined with Paclitaxel can increase the therapeutic effect on lung cancer A549 cells. Besides these, ␣-Pinene also has antifungal and antiinflammatory effect [13,14]. 1,8-Cineole was studied about the anti-inflammatory, expectorant and hypotension effect [15,16]. To the blood system disease, 1,8-Cineole has therapeutic effect and health care function. Borneol was widely used in traditional Chinese medical preparation to treat the cardiovascular and cerebrovascular diseases. Large dose of borneol intravenously administrated to the rabbits can inhibit the platelet aggregation induced by ADP, to the mice can reduce the whole blood clot weight [17]. Borneol can significantly increase the fibrinolysis effect through activating the fibrinlysis system. -Elemene was widely used in the treatment of blood stasis, it also was the second class non cytotoxic antitumor drug. The antithrombotic effect mechanism is affecting the arachidonic acid metabolic pathways and disturbing the synthesis of platelet [18]. Curcumol has been verified that it has strong antiplatelet aggregation(clinically was used to treat blood stasis), antiinflammatory, analgesic, inhibition of cell proliferation, antifibrosis and so on. In addition, Curcumol also has anti-tumor effect through effectively inhibiting the proliferation and inducing the apoptosis of a variety of malignant tumor cells(A549 cells, CEN-2 cells, MDA-MB-231 cells, Hep G2 cells, SPCA-1 cells) [19,20]. Germacrone is a major bioactive component found in volatiles oil product extracted from the RC. It shows an extensive range of biological activities, such as anticoagulation, fibrinolysis, antiinflammatory, antiulcer, antibacterial, antitumor, antitussive, vasodilator and hepatoprotector effects [21,22]. Curdione was found that it has strong antiplatelet aggregation effect and it was mainly used to treat blood stasis [23]. W.H. Qiao [24] reported that Curdione may inhibit thrombin-induced rat platelet activation and aggregation by inhibiting the PLC-PKC-MAPKs pathway, at least in part. So far, we have known that the seven compounds from RC are the key components of activating blood circulation and removing blood stasis, but there is still no method established for the simultaneous determination of these seven constituents in plasma samples after oral administration of essential oil from RC. So, in this study, we studied the pharmacokinetics procedure of RC through detecting the plasma concentration of the 7 compounds in rats. Several analytical methods have been established for determination of sesquiterpenoids from Zedoary turmeric oil in plasma samples, including High Performance Liquid Chromatogrphy (HPLC) for determination of curcumol and germacrone in rat plasma [25], High Performance Liquid Chromatograhpy tandem mass (HPLC–ESI–MS) for determination of individual Curcumol in rats blood [26], GC for curcumol and curdione in rat plasma [27]. GC–MS in selected ion-monitoring mode (SIM) method was developed in this study to quantify ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone and Curdione in rat plasma for the first time. It was fully validated and applied to a pharmacokinetic study in rats after oral administration of the essential oil of RC. GC–MS method is more suitable for volatile constituents determination in selectivity, specificity and sensitivity than LC/MS and HPLC-UV method. The total run time of every sample was 33 min. We expect that the results of this study could not only facilitate to
Fig. 1. The picture of RC and chemical structures of the seven analytes: ␣-Pinene (A), 1,8-Cineole(B), Borneol (C), -Elemene(D), Curcumol(E), Germacrone (F), Curdione(G).
clarify the mechanism of RC, but also to guide the clinical rational use. 2. Experimental 2.1. Materials REFERENCES standards of ␣-Pinene (C10 H16 , 98% purity, Batch No: 101561026), 1,8-Cineole (C10 H18 O, 99% purity, Batch No: 12-2006), Borneol (C10 H18 O, 98% purity, Batch No: 03-2008), Germacrone (C15 H22 O, 99.8% purity, Batch No: 111665-201204), and Curdione (C15 H24 O2 , 99.2% putiry, Batch No: 111800-201001) were purchased from Shanghai Bodun Biochemical Co., Ltd. (Shanghai, PR China); -Elemene (C15 H24 , 99% purity, Batch No:100268-200401), Curcumol (C15 H24 O2 , 99% purity, Batch No: 100185-200506) were purchased from National Institutes of Food and Drug Control(Beijing, PR China). The picture of RC and structures of the seven analytes are shown in Fig. 1. Methanol and acetonitrile were HPLC grade (filtered by 0.22 m filter) and purchased from Oceanpak company (Sweden). Formic acid of HPLC grade was purchased from Dikma (Richmond Hill, NY, USA). Other reagents were analytical grade. Ultra-pure water was prepared by using a Mili-Q water purification system (Millipore, Molsheim, France), 13 mm Luer syringe filter (1513022A) were purchased from USA. The RC rhizomes were collected from Guangxi Province of China in December 2015 and were identified by Prof. Lina Guo in Qiqihar Medical University. The specimen (20151001) was deposited in School of Pharmacy, Qiqihar Medical University, China. 2.2. GC–MS conditions The GC–MS system consisted of a model 7890 B series gas chromatograph coupled with a 7693 autosampler and a model 5977A MSD with Triple-Axis Detector (Agilent, IL, USA). Separation was performed on a DB-5 column(30 m × 0.25 mm, 0.25 m film thickness) supplied by Agilent Technologies (IL, USA). The carrier gas was 99.99% high purity Helium with a flow rate of 1.2 mL/min. The sample volume was 1 L. The injection port and the detector temperatures were 270 ◦ C and 280 ◦ C, respectively. Oven temper-
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Fig. 2. Mass spectrums of ␣-Pinene (A), 1,8-Cineole(B), Borneol (C), -Elemene(D), Curcumol(E), Germacrone (F), Curdione(G).
ature program was initially set at 50 ◦ C for 1 min, then ramped at 5 ◦ C/min to 120 ◦ C and held for 1.0 min, then ramped at 5 ◦ C/min to 150 ◦ C and held for 5 min, ramped to 200 ◦ C at 3 ◦ C/min and held
for 1 min. The total run time was 33 min with a solvent delay of 10 min. Ionization was performed in electron impact ionization (EI) mode at 70 eV. Selective ion monitoring (SIM) was set for quantifi-
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Table 1 Characteristic ions (m/z) and retention times of the seven analytes. Compound
Characteristic Ions (m/z)*
␣-Pinene 1,8-Cineole Borneol -Elemene Curcumol Germacrone Curdione
137 155 155 205 237 219 237
122 140 140 148 136 176 181
Retention Time (min) 106 109 122 108 122 136 168
94 82 111 94 108 108 110
78 44 96 82 94 92 70
6.64 9.23 13.05 19.70 26.18 30.01 30.90
*The molecular ions which are marked in bold were used for quantification of each analyte.
cation with dwell time 100 ms/ion. Retention times of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone and Curdione were 6.64 min, 9.23 min, 13.05 min, 19.70 min, 26.18 min, 30.01 min and 30.90 min, respectively. The characteristic ions (m/z) and retention times of the seven analytes are listed Table 1. 2.3. Preparation of the essential oil solution of RC rhizomes for administration The dried pieces of the RC rhizomes (12.5 g) were extracted by volatile oil extractor under reflux with 500 mL water for 4 h. The extract solution was concentrated to 100 mL, the oil was collected and combined with tween 80 (1 mL: 0.5 mL) to 100 mL flask, adding the extract solution to 100 mL. The contents of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone and Curdione in essential oil determined by the developed GC–MS method were 0.09%, 3.22%, 1.04%, 0.79%, 3.67%, 4.77% and 9.67%, respectively. 2.4. Preparation of standard samples The stock solution of ␣-Pinene (17.77 mg/mL), 1,8 Cineole (3.18 mg/mL), Borneol (22.09 mg/mL), -Elemene (2.22 mg/mL), Curcumol (16.47 mg/mL), Germacrone (20.35 mg/mL) and Curdione (19.57 mg/mL) were prepared in methanol. Then, we mixed these stock solutions and serially diluted with methanol to provide desired concentration. Calibration solutions were prepared by spiking 20 L of the appropriate standard working solutions into 200 L blank plasma. The ranging value was shown in Table 2. Seven standard curves containing of six-point concentration levels were as followed: ␣-Pinene (2.71, 5.42, 10.84, 21.69, 43.38, 86.77 ng/mL), 1,8 Cineole (7.76, 15.53, 31.05, 62.11, 124.22, 248.44 ng/mL), Borneol (3.37, 6.74, 13.48, 26.96, 53.93, 107.86 ng/mL), -Elemene (21.68, 43.36, 86.72, 173.44, 346.88, 693.75 ng/mL) and Curcumol (40.21, 80.42, 160.84, 321.68, 643.36, 1286.72 ng/mL), sevenpoint concentration levels for Germacrone (24.84, 49.68, 99.36, 198.73, 397.46, 794.92, 1589.84 ng/mL), and Curdione (47.78, 95.56, 191.11, 382.23, 764.45, 1528.91, 3057.81 ng/mL). Quality control samples concentration were 5.42, 21.69, 86.77 ng/mL for ␣-Pinene, 15.53, 62.11, 248.44 ng/mL for 1,8 Cineole, 6.74, 26.96, 107.86 ng/mL for Borneol, 43.36, 173.44, 693.75 ng/mL for Elemene, 80.42, 321.68, 1286.72 ng/mL for Curcumol, 49.68, 198.73, 794.92 ng/mL for Germacrone and 95.56, 382.23, 1528.91 ng/mL for Curdione. All the solutions were stored at 4 ◦ C before use.
auto sample tube. 1 L of the supernatant was injected into GC–MS system for analysis. 2.6. Method validation Selectivity, linearity, intra-day and inter-day precision, accuracy, recovery and stability in accordance to the requirements for the analysis of biological samples were used to validate the current GC–MS assay [28]. 2.6.1. Selectivity After the conditions of chromatography and mass spectrometry were determined, selectivity was assessed by analyzing the chromatograms of six different batches of blank rat plasma. In order to investigate the separation of endogenous substances from analytes, the chromatograms of corresponding spiked with the seven analytes and plasma samples obtained after oral administration of the essential oil of 10 mg/kg (equals to RC rhizomes at a dose of 1.0 g/kg body weight) were supplied. 2.6.2. Linearity and LLOQ Standard plasma samples at series concentrations by least squares linear regression of the peak area of each analyte versus the corresponding concentrations with a weighted (1/X2 ) factor was analyzed to assess the linearity. The lower limit of quantification (LLOQ) was defined as the lowest concentration on the calibration curve. 2.6.3. Precision and accuracy The precision was determined by RSD and the accuracy by RE value of QCL, QCM and QCH samples. Intra- and inter-day precision and accuracy each day and three consecutive days were evaluated by measuring six replicates of QC samples of the seven analytes at three concentration levels respectively. The acceptable criteria for the intra-day and inter-day precision and accuracy are within 15% for three levels. 2.6.4. Extraction recovery QCL, QCM and QCH samples in six replicates were used to analyze the extraction recovery. The peak areas were determined together with the linear samples. The recovery was calculated through comparing the peak areas of QC samples with the analytes spiked into the post extraction supernatant samples. The coefficient of variation should be within 15%. 2.7. Pharmacokinetic study
2.5. Plasma sample preparation The plasma sample (100 L) was put into a 1.5-mL clean eppendorf tube, after that, 100 L hexane and ethyl acetate (1:1) mixture was pipetted into the sample. The sample was shaken for 1 min to extract active compounds in a vortex mixer. Then the sample was centrifuged in high speed cryogenic centrifuge at 12000 rpm for 10 min and the supernatant was transferred to a 1.5 mL clean
Male Wistar rats weighing 250 ± 20 g were supplied by the Laboratory Animal Center of Qiqihar Medical University (Qiqihar, China) and were maintained in temperature of 23 ± 2 ◦ C, relative humidity of 57 ± 2% with free access to food and water for 6 days acclimation. Before the administration of the essential oil of RC rhizomes, the rats were fasted for 12 h with free access to water. The experiment was approved by the Animal Ethics Committee
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Fig. 3. SIM chromatogram of (A) blank plasma, (B) blank plasma spiked with the seven analytes, (C) plasma sample obtained at 2 h after oral administration of the essential oil of RC rhizomes: ␣-Pinene (1), 1,8-Cineole(2), Borneol (3), -Elemene(4), Curcumol(5), Germacrone (6), Curdione(7). Table 2 The regression equations, linear ranges and LLOQs of the analytes in plasma. Compounds
Y = aX + b
R
Linear range (ng/mL)
LLOQ (ng/mL)
␣-Pinene 1,8-Cineole Borneol -Elemene Curcumol Germacrone Curdione
Y = 7 × 108 + 531 Y = 1 × 109 − 7477 Y = 8 × 108 − 852 Y = 1 × 109 + 496 Y = 5 × 108 − 681 Y = 5 × 108 + 7502 Y = 2 × 108 + 403
0.999 4 0.999 3 0.999 9 0.998 0 0.999 6 0.997 1 0.999 7
2.71–86.77 7.76–248.44 3.37–107.86 21.68–693.75 40.21–1286.72 24.84–1589.84 47.78–3057.81
2.71 7.76 3.37 21.68 40.21 24.84 47.78
of Qiqihar Medical University. The single administration was prepared by constituting the essential oil with tween 80 and extract solvent to get a concentration equivalent to 1.0 g/kg RC rhizomes (equivalent to 0.11 mg/kg of ␣-Pinene, 4.02 mg/kg of 1,8-Cineole, 1.29 mg/kg of Borneol, 0.99 mg/kg of -Elemene, 4.58 mg/kg of Curcumol, 5.96 mg/kg of Germacrone, and 12.08 mg/kg of Curdione). The blood samples (400 L) were obtained from the suborbital vein
of rats at 0, 0.17, 0.5, 1, 2, 2.5, 3, 4, 6, 8, 10, 12 and 24 h after dosing. The samples were collected into EP tube pre-coated with heparin and immediately centrifuged at 5000 rpm for 10 min, then the separated plasma samples were stored at −80 ◦ C until analysis. The maximal concentration (Tmax ), maximal concentration (Cmax ) and the elimination rate constant (Ke) were determined directly and calculated by linear regression of the terminal points in a semi-
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Fig. 4. Mean ± S.D. (n = 6) plasma concentration-time curves of ␣-Pinene (A), 1,8-Cineole (B), Borneol (C), -Elemene (D), Curcumol (E), Germacrone (F), Curdione (G) in rats after oral administration of the essential oil of RC rhizomes.
log plot of the plasma concentration against time respectively. The elimination half-life (t1/2 ) was calculated as 0.693/Ke. The area under plasma concentration-time curve (AUC0-t ) was estimated by the linear trapezoidal rule from 0 to 24 h. The area under the plasma concentration-time curve to time infinity (AUC0-∞ ) was calculated as AUC0-∞ = AUC0-t + Ct /Ke, where Ct was the last measurable concentration.
of the seven analytes are presented in Fig. 2. Injector temperature (240, 270 and 300 ◦ C), detector temperature (260, 280 and 300 ◦ C), and column temperature program as the main chromatographic conditions were optimized, in order to obtain better area response, symmetrical peaks and reduced run time.
3. Results and discussion
Extraction methods of liquid–liquid extraction (LLE), solidphase extraction (SPE), and protein precipitation using methanol or acetonitrile were compared for bio-sample preparation. For LLE, organic extracting solvent including hexane, ethyl acetate, and their mixture were investigated. We found that LLE method using hexane and ethyl acetate (1:1) could obtain clean, reproducible extraction and good recovery for the seven analytes.
3.1. Optimization of GC–MS parameters In order to increase the sensitivity of the seven analytes, selective ion monitoring (SIM) mode was chosen to determine. In this paper, the representative spectrogram scanned from 50 to 500 amu
3.2. Selection of extraction method
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Table 3 Summary of accuracy, precision and recovery for the seven analytes in rat plasma (n = 6). Analytes
concentration (ng/mL)
Intra-day RSD(%)
Inter-day RSD(%)
Accuracy RE(%)
Recovery(%, mean ± SD)
␣-Pinene
5.42 21.69 86.77 15.53 62.11 248.44 6.74 26.96 107.86 43.36 173.44 693.75 80.42 321.68 1286.72 49.68 198.73 794.92 95.56 382.23 1528.91
7.75 9.08 8.34 5.25 4.41 4.67 8.92 7.02 2.05 5.05 5.83 5.41 5.17 8.31 5.65 6.54 7.01 1.20 7.82 2.97 1.97
6.80 6.93 5.68 8.56 4.84 9.04 6.55 9.02 8.77 9.62 2.27 2.53 5.90 4.50 2.46 7.22 5.83 4.28 2.95 4.06 3.22
7.46 −2.57 −6.59 −9.75 −6.36 −2.16 −6.36 −5.30 −5.20 6.68 −2.49 −4.77 −4.40 −3.77 1.30 −8.71 −1.96 −4.03 −4.30 −5.16 −4.08
68.3 ± 5.3 79.7 ± 4.9 80.7 ± 1.5 69.5 ± 5.1 77.4 ± 2.6 79.4 ± 3.5 69.0 ± 2.5 76.0 ± 4.4 78.8 ± 1.9 67.7 ± 4.3 76.8 ± 3.3 80.1 ± 8.9 64.8 ± 3.4 74.8 ± 4.3 80.5 ± 8.0 72.5 ± 6.1 75.8 ± 3.9 79.6 ± 4.0 69.0 ± 1.9 78.1 ± 3.1 79.6 ± 4.2
1,8-Cineole
Borneol
-Elemene
Curcumol
Germacrone
Curdione
Table 4 Pharmacokinetic parameters of the seven compounds in rats after oral administration the essential oil of CR rhizomes (mean ± SD, n = 6). compound
Cmax (ng/mL)
Tmax (h)
T1/2 (h)
AUC0-t (ng h/mL)
AUC0-∞ (ng h/mL)
␣-Pinene 1,8-Cineole Borneol -Elemene Curcumol Germacrone Curdione
34.72 ± 9.97 99.86 ± 5.54 16.10 ± 3.37 248.98 ± 86.19 673.75 ± 104.15 2353.64 ± 637.83 2420.04 ± 708.51
2.33 ± 0.29 0.67 ± 0.29 1.33 ± 0.58 1.83 ± 0.76 0.83 ± 0.29 0.89 ± 0.18 1.17 ± 0.76
8.64 ± 1.46 8.98 ± 1.63 12.43 ± 2.88 19.86 ± 4.05 15.63 ± 5.50 14.17 ± 4.13 7.14 ± 0.67
189.78 ± 89.10 454.74 ± 82.43 100.55 ± 8.27 1067.37 ± 216.55 3154.16 ± 405.94 16501.24 ± 663.88 12524.92 ± 3222.10
229.57 ± 93.50 524.32 ± 81.67 146.28 ± 10.74 2092.70 ± 416.18 5388.65 ± 661.86 28198.873 ± 4102.62 14139.35 ± 3109.19
3.3. Method validation The chromatograms of blank plasma, plasma sample at HQC spiked with the analytes, and the plasma sample from a rat at 2 h after oral administration of the essential oil of RC rhizomes were compared to evaluate the selectivity of the method. From the typical chromatograms with a good separation as well as excellent peak shapes shown in Fig. 3, we saw that there is no endogenous interference observed at the retention times of the seven analytes. According to the Tmax value of seven compounds, we know that the plasma sample obtained at 2 h had the relative higher response to the seven compounds. So in Fig. 3-C, we choose the 2 h after oral administration as the representative plasma sample chromatogram to compare with the other two chromatorams (Fig. 3-A and Fig. 3-B). The retention times of the seven analytes were as followed: ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione were 6.63, 9.22, 13.05, 19.7, 26.17, 30.01 and 30.09 min, respectively. The calibration curves, the linearity ranges and LLOQs value for the seven analytes are summarized in Table 2. A good linearity within the described ranges for seven analytes was indicated from the high correlation coefficient (all higher than 0.9971). All standards met the criteria of less than 15% deviation. For pharmacokinetic studies, the LLOQs were suitable for quantitative detection of the seven analytes. The intra- and inter- day precision and accuracy for seven target analytes are shown in Table 3. The method was reliable and reproducible demonstrated by results for the samples tested within the acceptable criteria of ±15%. The recovery of the assay, which illustrated that the majority of each analyte was successfully extracted are summarized in Table 3. The extraction efficiencies for ␣-Pinene, 1,8-Cineole, Borneol, Elemene, Curcumol, Germacrone, and Curdione were 68.3–80.7%,
69.5–79.4%, 69.0–78.8%, 67.7–80.1%, 64.8–80.5%, 72.5–79.6% and 69–79.6%. The stability of seven analytes in rat plasma under different conditions is summarized in the supplementary. 3.4. Pharmacokinetics study A validated method in this paper was successfully applied to the pharmacokinetics study of the seven bioactive constituents in rat plasma after oral administration of the essential oil from RC rhizomes. We chose an oral dosage for the rats corresponding to the human dosage in Chinese Pharmacopoeia (China Pharma copoeia Committee, partI, 2015). The human dosage of the TCM in Chinese Pharmacopoeia is 8–15 g/day for blood stasis treatment, 1.0 g/kg in rats based on the Meeh-Rubner method [29] was picked as the final oral dosage. Meeh-Rubner method was used to calculate the variation of effective dosage between different species of animals and human. Meeh-Rubner formula: A = K(W2/3 )/10000 is more appropriate to predict body surface area according to the weight, “A” represent body surface area (m2 ), “W” represent weight (g), “K” represent constant (vary with the species of animals, the constant of rat is 9.1). Then we go on calculating the dosage using the conversion factor (CF) from the dosage unit of mg/kg to mg/m2 . In this paper, rat dosage (mg/kg) = human dosage(mg/kg) × (human CF)/(rat CF), in this formula, human dosage is 10 g/60 kg, human CF is 36, rat CF is 6, so the result of rat dosage is 1.0 g/kg. The mean plasma concentration-time profiles of the seven analytes are presented in Fig. 4. Table 4 shows the main pharmacokinetic parameters including half-time (t1/2 ), time to reach the maximum concentrations (Tmax ), maximum plasma concentration (Cmax ), and an under concentration–time curve (AUC0-t and AUC0-∞ ). As seen from Fig. 4, 1,8-Cineole, Borneol, Curcumol, Germacrone, and Curdione were rapidly absorbed following oral administration of the
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essential oil, and they achieved maximum plasma concentration (Cmax ) between 0.67 and 1.33 h, ␣-Pinene and -Elemene were absorbed slowly with the Tmax 2.33 and 1.83 h separately after oral administration. T1/2 of the seven compounds were all more than 7.14 h, the slow elimination may due to the enterohepatic circulation process because of the intragastric administration or the high affinity binded to blood protein. It needed to be proved by the hepatoenteral pharmacokinetic and equilibrium dialysis experiment further. In our previous research, we have evaluated the differences of pharmacokinetic behavior between normal and blood-stasis pregnant rats after intravenous administration using HPLC method for the determination of Curcumol in rat plasma, the LLOQ of Curcumol was 450 ng/mL, while in this paper, the LLOQ of Curcumol was 40.21 ng/mL, the sensitivity increased 10 times than before. D. Ji [30] had studied the pharmacokinetics of Germacrone and curdione in rat plasma using the HPLC method. According to their results, the LLOQ of Germacrone and curdione were 197.2 ng/mL and 1073 ng/mL, while the LLOQ of the method developed in this paper are 24.84 and 47.78 ng/mL for Germacrone and curdione, respectively. In this paper, the AUC and Cmax of these seven analytes showed consistent tendency in plasma concentration–time profiles. It is illustrated that the analytes had the similar oral absorption and distribution after given the essential oil of RC rhizomes, which may contribute to their similar characteristic. To our knowledge, there is no method about simultaneous determination of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione in plasma is available in the literatures. But there are some reports about the determination of some of the seven compounds mentioned above in plasma samples after oral administration of RC prescriptions [31,32]. The results about the pharmacokinetic profiles of the seven analytes in RC in this paper were slightly different from that observed for their pure forms or other prescriptions. We analyzed the reasons might be due to the complexity of TCM for their interactions among so many compounds in vivo. This work can provide some experimental basis for the clinical use of RC and its prescriptions.
4. Conclusion A multi-component pharmacokinetic study of RC was conducted for the simultaneous determination of ␣-Pinene, 1,8Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione in rat plasma after oral administration of RC by GC–MS. The analytical method in this paper was perfect because of the relative short chromatographic running time and simple pretreatment using liquid–liquid extraction sample. The established GC–MS method was suitable for pharmacokinetic study of ␣-Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione in rat plasma because of the excellent sensitivity, selectivity, precision, accuracy and extraction recovery. The relative rapid absorption and long half-time of bioactive constituents gave a reasonable explanation for the rapid effects and long interval between dosing of RC in the treatment of blood stasis. The results of this study illustrated that the co-existing compounds in RC extract could change the pharmacokinetic behaviors of these seven constituents compared with other prescriptions of TCM. It will be helpful for the mechanism research of action of RC. This is the first report on a GC–MS method for the simultaneous determination of total ␣Pinene, 1,8-Cineole, Borneol, -Elemene, Curcumol, Germacrone, and Curdione accurately with a purpose of the pharmacokinetics study of the above mentioned compounds in rats plasma after oral administration of the essential oil of RC rhizomes.
Acknowledgments The work was financially supported by National Natural Science Foundation of China (81403173) and China Postdoctoral Science Foundation (2014M551291), and in part by “Young Excellent and Innovative Talent Training Program” of Heilongjiang Ordinary Undergraduate University (7UNPYSCT-201611).
References [1] China Pharmacopoeia Committee, Pharmacopoeia of the People’s Republic of China, China Chemical Industry Press, Beijing, China, 2015, pp. 274–275. [2] L. Wan, J.M. Zhang, S. Fu, J.S. Wang, C.M. Fu, Chromatography activity relation of stagnation of vital energy and blood stasis syndrome influencing hemorheology by stirbaked Curcumae Rhizoma with vinegar, Chin. Tradi. Patent Medi. 35(2013) 330-335. [3] X. Wang, Q. Xia, D.J. Xu, Research on the anticoagulant and antithrombotic effects of Rhizoma Curcumae, Chin. Tradit. Pate. Medi. 34(2012) 550-553. [4] J.J. Lu, Y.Y. Dang, M. Huang, W.S. Xu, X.P. Chen, Y.T. Wang, Anti-cancer properties of terpenoids isolated fromRhizomaCurcumae-a review, J. Ethnopharmacol. 143 (2012) 406–411. [5] D.C. Tang, W.H. Zang, H.H. Feng, Influences of medicated serum of different cultivars of Ezhu (Rhizoma Zedoariae) on proliferation, apoptosis and nucleo-cytoplasmic ratio in human gastric carcinoma cells BGC82, J. Beijing Univ. Tradit. Chin. Med. 36 (2013) 254–257. [6] L.Z. Huang, J. Wang, F.T. Lu, et al., Mechanism study on anti-proliferative effects of curcumol in human hepatocarcinoma Hep G2 cells, Chin. J. Chin. Mater. Medi. 38 (2013) 1812–1815. [7] X. Chen, C.J. Zong, Y. Gao, R.L. Cai, L. Fang, J. Lu, F. Liu, Y. Qi, Curcumol exhibits anti-inflammatory properties by interfering with the JNK-mediated AP-1 pathway in lipopolysaccharide-activated RAW264. 7cells, Eur. J. Pharmacol. 723 (2014) 339–345. [8] J. Ding, J.J. Wang, C. Huang, L. Wang, S.N. Deng, T.L. Xu, W.H. Ge, W.G. Li, F. Li, Curcumol from Rhizoma Curcumae suppresses epileptic seizure by facilitation of GABA(A) receptors, Neuropharmacology 81 (2014) 244–255. [9] H.F. Huang, C.J. Zheng, G.Y. Chen, W.Q. Yin, M.O. Zheng, Comparison of chemical composition and antibacterial activity between zedoary turmeric oil and dregs residual oil, Chin. J. Exper. Tradit. Medi. Form 21 (2015) 67–70. [10] Z. Liu, W.Y. Gao, S.L. Man, Y. Zhang, H.F. Li, S.S. Wu, J.Z. Zhang, C.X. Liu, Synergistic effects ofRhizomaParidisandRhizomaCurcuma longaon different animal tumor models, Environ. Toxico. Pharmacol 38 (2014) 31–40. [11] H. Türkez, E. Aydin, In vitro assessment of cytogenetic and oxidative effects of ␣-pinene, Toxi. Ind Heal. 32 (2016) 168–176. [12] Z. Zhang, S. Guo, X. Liu, X. Gao, Synergistic antitumor effect of ␣-pinene and -pinene with paclitaxel against non-small-cell lung carcinoma (NSCLC), Drug Res. 55 (2014) 113–119. [13] P. Dhar, P. Chan, D.T. Cohen, F. Khawam, S. Gibbonset, T.S. Leiby, E. Dickstein, P.K. Rai, G. Watal, Synthesis, antimicrobial evaluation, and structure-activity relationship of ␣-pinene derivatives, J. Agric. Food Chem. 62 (2014) 3548–3552. [14] S.Y. Nam, C.K. Chung, J.H. Seo, S.Y. Rah, H.M. Kim, H.J. Jeong, The therapeutic efficacy of ␣-pinene in an experimental mouse model of allergic rhinitis, Int. Immunopharmacol. 23 (2014) 273–282. [15] C. Morcia, M. Malnati, V. Terzi, In vitro antifungal activity of terpinen-4-ol, eugenol, carvone, 1,8- cineole (eucalyptol) and thymol against mycotoxigenic plant pathogens, Food Addit. Contami. Part A. 29 (2012) 415–422. [16] M. Takaishi, F. Fujita, K. Uchida, S. Yamamoto, S.M. Sawada, U.C. Hatai, M. Shimizu, M. Tominaga, 1,8-cineole, a TRPM8 agonist, is a novel natural antagonist of human TRPA1, Mol. Pain. 8 (2012) 86–98. [17] Q. Zhang, D. Wu, J. Wu, Y. Ou, C.L. Mu, B. Han, Q.L. Zhang, Improved blood-brain barrier distribution: effect of borneol on the brain pharmacokinetics of kaempferol in rats by in vivo microdialysis sampling, J. Ethnopharmacol. 162 (2015) 270–277. [18] Y. Zhao, R.G. Yang, M. Luo, The pharmacology effect and clinical research development of Zedoary turmeric oil, J. Pract. Tradit. Chin. Inter. Medi. 20 (2006) 1250–1261. [19] H. Wang, Y. Fang, Y. Wang, Z. Wang, Q. Zou, Y. Shi, J. Chen, D. Peng, Inhibitory effect of curcumol on jak2-STAT signal pathway molecules of fibroblast-like synoviocytes in patients with rheumatoid arthritis, Evid. Based Complement Alternat. Med. 746 (2012) 4–26. [20] Q.L. Tang, J.Q. Guo, Q.Y. Wang, H.S. Lin, Z.P. Yang, T. Peng, X.D. Pan, B. Liu, S.J. Wang, L.Q. Zang, Curcumol induces apoptosis in SPC-A-1 human lung adenocarcinoma cells and displays anti-neoplastic effects in tumor bearing mice, Asian Pac. J. Cancer Prev. 16 (2015) 2307–2312. [21] W. Cho, J.W. Nam, H.J. Kang, T. Windono, E.K. Seo, K.T. Lee, Zedoarondiol isolated from the rhizoma of Curcuma heyneana is involved in the inhibition of Inos, COX-2 and proinflammatory cytokines via the downregulation ofNFB pathway in LPS-stimulated murine macrophages, Inter. immunopharmacol. 9 (2009) 1049–1057. [22] X. Xiao, Y. Zhao, H. Yuan, W. Xia, J. Zhao, X. Wang, Study on the effect of Rhizoma Curcuma Longa on gastrin receptor. Zhong yao cai, J. Chin. Medi. Mater. 25 (2002) 184–185.
W. Li et al. / Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 577–585 [23] Q. Xia, X. Wang, D.J. Xu, X.H. Chen, F.H. Chen, Inhibition of platelet aggregation by curdione from Curcuma wenyujin essential oil, Thromb. Res. 130 (2012) 409–414. [24] W.H. Qiao, D.L. Zhang, Y.L. Zhao, Y. Huang, J.M. Sun, D.J. Xu, Q. Xia, Inhibition by curdione of thrombin-induced platelet activation and aggregation, Acta Univer. Medi. Anhui. 52 (2017) 376–382. [25] Y. Pan, Y. Zhang, Z. Xiang, P.C. Yan, K.X. Huang, Determination of curcumenol and germacrone in plasma and its pharmacokinetics in rats, Chin. Tradit. Pat. Medi. 35(2013) 252-255. [26] T.J. Xu, S.Q. Tang, H. Rong, Y.C. Niu, Metabolic differences of curcumol between normal and blood-stasis rats using liquid chromatography-mass spectrometry, Lishizhen Medi. Mater. Medi. Res. 27 (2016) 3053–3056. [27] Y.T. Sun, Z.Q. Zhang, X. Li, S.C. Liu, H.J. Bian, J. Sun, Analysis of curcumol and curdione in plasma of rats by CGC. Chin. Tradi. Pat. Medi. 32(2010) 991-995. [28] Z.B. Wang, Q.H. Wang, B.Y. Yang, et al., GC–MS method for determination and pharmacokinetic study of four phenylpropanoids in rat plasma after oral
[29] [30]
[31]
[32]
585
administration of the essential oil of Acorus tatarinowii Schott rhizomes, J. Ethnopharmacol. 155 (2014) 1134–1140. Y.P. Zhang, «Pharmacology experiment» (the second edition), People’s Medical Publishing House (Appendix V), 238-239. D. Ji, C.Q. Mao, J.C. Li, T.L. Lu, H. Xie, G.F. Jiang, Y.Q. Xiao, Pharmacokinetics of curdione and germacrone in zedoary turmeric oil in rats in vivo, Chin. Tradi. Pate. Medi. 35(2013) 2622-2626. D. Lv, Y. Cao, X. Dong, X. Chen, Z. Lou, Y. Chai, Analysis and pharmacokinetic study of curdione in Rhizoma Curcumae by UPLC/QTOF/MS, Biomed. Chromatogr. 28 (2014) 782–787. Y.X. Song, Y.J. Liu, D.P. Zou, G. Wang, Z.C. Li, H.Y. Zou, Pharmacokinetic study of rhizoma Curcumae oil and rhizoma Curcumae oil--cyclodextrin inclusion complex in pigs after oral administration, J. Vet. Pharmacol. Ther. 35 (2012) 47–51.