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Determination of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in rat plasma and tissues by HPLC-UV method and their application to a pharmacokinetic and tissue distribution study.

1. Introduction

In traditional Chinese medicine, Atractylodis Rhizoma is the dried root and stems from Atractylodes lancea (Thunb.) DC. or Atractylodes chinensis (DC.) Koidz. The medicinal herb is widely known as Cangzhu in China. And it is widely used for the treatment of rheumatic diseases, digestive disorders, mild diarrhea, and influenza [1]. In clinic, Atractylodis Rhizoma is often processed by stir-frying with wheat bran with the aim of reducing its dryness and increasing the function of tonifying spleen [2, 3].

Atractylodis Rhizoma is rich of essential oil including sesquiterpenes and polyethylene alkynes, which are the main active components in this medicine [4]. Recent researches have shown that polyethylene alkynes exhibit various desirable pharmacological effects including anti-inflammatory, antibacterial, and antiarrhythmic activity [5]. (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate is one of polyethylene alkyne components. Some literature reported HPLC method for determination its content in Atractylodis Rhiomzoma [6-8].

However, there have been few methods available for its quantification in biosamples and few reports on its pharmacokinetic study and tissues distribution until now. The in vivo study of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate, an active component of Atractylodis Rhizoma, would be necessary and helpful for further clinical application and explanation of the processing mechanism. The present paper developed a new and simple RP-HP LC method for quantification of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in rat plasma and tissues after oral administration of raw and processed Atractylodis Rhizoma, respectively. This fully validated method was successfully applied to a pharmacokinetic and tissue distribution study of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in rats for the first time.

2. Experimental

2.1. Chemicals and Reagents. The (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate (purity, 98%) was supplied by Traditional Chinese Medicine Standardization Research Center (Shanghai, China). The IS called Emodin (purity, 98%) was supplied by the National Institute for Food and Drug Control (Beijing, China). The chemical structures of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate and IS are shown in Figure 1.

HPLC grade acetonitrile was purchased from Fisher Scientific Company (New Jersey, USA) and Pure water was supplied by Wahaha Company (Hangzhou, China). Analytical grade ethanol and chloroform were from Baierdi Company (Beijing, China). Atractylodis Rhizoma was identified by Professor Li Feng (Liaoning University ofTCM) according to the standards of Chinese Pharmacopoeia 2010. The processed Atractylodis Rhizoma comes from the same batch Atractylodis Rhizoma. The herb was stored in a cool and dry place.

2.2. Preparation of Atractylodis Rhizoma Solution. Atractylodis Rhizoma (50 g) was crushed into powder and soaked into 600 mL of 80% ethanol for 24 h and then percolated at 2 mL x [min.sup.-1], and Ethanol was evaporated to near dryness under reduced pressure to get the residue. Distilled water was added into the residue and then vortexed for 10 min. The final concentration of Atractylodis Rhizoma solution was 2 g x [mL.sup.-1] [9]. The sample was stored in dry and dark place before use.

2.3. Apparatus and HPLC Conditions. The liquid chromatographic system consisted of an LC-10 AD pump (Shimadzu, Kyoto, Japan) with a 20 [micro]L loop (Cotata, CA, USA) and an SPD-10A ultraviolet-visile detector (Shimadzu, Kyoto, Japan). A LC-10 AD workstation was used for data acquisition. A Diamonsil [C.sub.18] analytical column (250 x 4.6 mm; 5 [micro]m) from Dikma Technologies (Dalian, China) was used. The mobile phase consisted of acetonitrile and 0.1% phosphoric acid water (60: 40, v/v) at a flow rate of 1 mL x [min.sup.-1]. The detection wavelength was set at 336 nm. All the measurements were performed at room temperature, and the injection volume was 20 [micro]L.

2.4. Preparation of Standard Solution and Quality Control Samples. Stock solutions of (4E,6E,12E)-tetradecatriene-8.10-diyne-1,3-diyl diacetate and IS with concentrations of 0.143 mg x [mL.sup.-1] and 0.0504 mg x [mL.sup.-1], respectively, were prepared in methanol and stored at -20[degrees]C and dark place until use. When we used the standard solution, the working concentration of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate and IS were 286 [micro]g x [L.sup.-1] and 5.04 [micro]g x [L.sup.-1],respectively. Calibration standards of (4E,6E,12E)-tetradecatriene-8.10-diyne-1,3-diyl diacetate were prepared by spiking the appropriate amount of the working solutions into 200 [micro]L blank rat plasma or tissue homogenates. To plasma samples, the final concentrations of calibration standard samples were 0.003575, 0.00715, 0.0143, 0.03575, 0.0715, 0.143, and 0.2145 [micro]g x [mL.sup.-1]. Quality control (QC) samples were prepared at low, medium, and high concentrations of 0.00375, 0.03575, and 0.0715 [micro]g x [mL.sup.-1]. To tissue homogenates, the final concentrations of calibration standard samples were 0.00143, 0.003575, 0.00715, 0.0143, 0.0286, 0.03575, and 0.0715 [micro]g x [mL.sup.-1]. Quality control (QC) samples were prepared at low, medium, and high concentrations of 0.00375, 0.03575, and 0.0715 [micro]g x [mL.sup.-1] for tissue homogenates.

2.5. Sample Preparations. For plasma samples, the 200 [micro]L of rat plasma was mixed with 20 [micro]L IS (0.00504 mg x [mL.sup.-1]). After protein was precipitated with 1,000 [micro]L of acetonitrile in 1.5 mL polypropylene tube by vortexing for 2 min, the sample was centrifuged at 10,000 rpm-[min.sup.-1] for 5 min. The supernatant was transferred into a 5.0 mL tube and added with 1,000 [micro]L of chloroform, extract and the under organic phase was transferred to another tube and evaporated to dryness at 40[degrees] C with nitrogen. The resulting extract was dissolved in 50 [micro]L of methanol, and vortex mixed for 2 min. After centrifugation at 10,000 rpm-[min.sup.-1] for 5 min, 20 [micro]L supernatant was injected for analysis [10-12]. For tissue homogenate, each weighed tissue sample was thawed and the homogenized in ice-cold physiological saline (2 mL). Then a 200 [micro]L of tissue homogenate was taken and processed further like the plasma samples.

2.6. Method Validation

2.6.1. Specificity. The selectivity of the method was demonstrated by comparing chromatograms of blank plasma samples and tissue homogenate (without IS) obtained from rats and plasma samples and tissue homogenate spiked with the analytes and IS and plasma samples and tissue homogenate after an oral dose. All blank plasma samples and tissue homogenates were prepared and analyzed to ensure the absence of interfering peaks.

2.6.2. Calibration Procedure. The linearity of the method was assessed by plotting calibration curves in plasma at seven concentration levels in triplicate on three consecutive days. The lower limit of quantification (LLOQ) was defined as the lowest concentration of the calibration curve that was measured with accuracy and precision by analyzing samples in six replicates at the concentration of 0.00143 [micro]g-mL 1 for (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate.

2.6.3. Accuracy and Precision. Intraday precision and accuracy were evaluated by analysis of the three QC samples with six determinations per concentration at the same day, whilst the interday precision and accuracy were measured over three consecutive days. The precision was defined as the relative standard deviation (RSD%), while accuracy was determined by calculating the percentage deviation observed in the analysis of QC samples and expressed by relative error (RE%). The accepted criteria for the data were that the precision and accuracy should not exceed 15%, except at the LLOQ, where it should not exceed 20%.

2.6.4. Extraction Recovery and Stability. The extraction recoveries of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate were determined at low, medium, and high level of QC samples. Recoveries were calculated by comparing the observed peak area ratios in biosamples to those non-processed standard solutions at the same concentrations. The recovery of IS was determined in the same way at the concentration of 0.00504 mg x [mL.sup.-1].

The stability of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in plasma and tissue was determined under different storage or handling conditions. Short-term stability was assessed by analyzing QC samples kept at room temperature for 8 h. Freeze-thaw stability was evaluated at three consecutive freeze-thaw cycles. Long-term stability was studied by assaying samples following a period of 10 days of storage at -20[degrees] C.

2.7. Applications in Pharmacokinetic Studies. All the studies on animals were in accordance with the Guidelines for the Care and Use of Laboratory Animals. Healthy Sprague-Dawley rats (250 [+ or -] 20 g) were purchased from The Medical University of Dalian (Dalian, China) and acclimated in the laboratory for one week before to the experiments. Rats for oral ingestion were fasted for 12 h with free access to water. Rats were oral administration raw and processed Atractylodis Rhizoma at a single dose of 40 g x [kg.sup.-1], respectively.

For plasma samples, twelve rats were randomly assigned to two groups for pharmacokinetic investigation (n = 6 per group). The blood sample (0.5 mL) was collected at 0, 0.17, 0.33, 0.5, 1, 2, 3, 4, 6, 8, 12, and 24 h. All samples were immediately transferred into heparinized tubes and centrifuged for 5 min at 10,000 rpm-[min.sup.-1]. The supernatant was stored at -20[degrees]C and dark place until use.

2.8. Tissue Distribution Study. For tissue distribution study, forty-eight rats were divided into eight groups (n = 6 per group) randomly.

After oral administration four groups rats raw Atractylodis Rhizoma at a single dose of 40 g x [kg.sup.-1] and the other four groups rats processed Atractylodis Rhizoma at a single dose of 40 g x [kg.sup.-1]. Heart, liver, spleen, lung, kidney, stomach, small intestine, and large intestine were collected at 0.5, 2, 4, and 8h. Tissue samples were weighed 0.1 g rapidly, rinsed with physiological saline to remove the blood or content, blotted on filter paper, and then stored at -20[degrees]C and dark place until use.

2.9. Statistical Analysis. HPLC analysis procedure was applied to analyze plasma concentration-time profiles of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate. Data was processed by noncompartmental method using Drug and Statistics (DAS) 2.0 software package (Chinese Pharmacological Society, Shanghai, China).

3. Results and Discussion

3.1. Method Validation

3.1.1. Specificity. The representative chromatograms for determination of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in plasma and tissues are shown in Figure 2. The retention time of Emodin (IS) was about 16.30 min and (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate was about 30.45 min. It was indicated that analytes and IS were well separated and no interferences were detected from endogenous substances or metabolites.

3.1.2. Linearity of Calibration Curve and Lower Limit of Quantification. The calibration curves were linear over the concentration range of 0.003575-0.2145 [micro]g x [mL.sup.-1] in rat plasma and 0.00143-0.0715 [micro]g x [mL.sup.-1] in tissue homogenates by weighted (1/[chi square]) linear least-squares regression method. The correlation coefficient values of the calibration curves were over 0.9900. The RE of the back-calculated values of the standards from their nominal values were constantly within 15% for all values, including the LLOQ. The LLOQ measurement showed the respective averages 0.003575 [micro]g x [mL.sup.-1] with RSD 7.68% for rat plasma and 0.00143 [micro]g x [mL.sup.-1] with RSD 8.68% for tissue homogenates. Typical linear regression equations, correlation coefficients in plasma and each tissue were listed in Table 1.

3.1.3. Precision and Accuracy. The intraday and interday precision and accuracy were assessed by analyzing six aliquots of low, medium, and high concentration samples. The intraday precision of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in rat plasma and tissues ranged between 1.83 and 6.71% with RE of -14.47--7.81% and the interday precision ranged between 1.57 and 8.68% with RE of-14.38~ -5.13%.

3.1.4. Recovery and Stability. The extraction recoveries of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate ranged from 86.6% to 91.6% in plasma and tissue samples and the method recovery ranged from 85.6% to 94.9%, while the recovery of IS was above 80%. These data indicated that the biosample preparation procedure was satisfied and can achieve the acceptable extraction recovery. Stability of analytes showed no significant sample loss over 12 h at room temperature, three freeze-thaw cycles, and 10 days storage condition. The RE of three conditions were within [+ or -] 15%.

3.2. Pharmacokinetics of (4E,6E,12E)-Tetradecatriene-8,10-diyne-1,3-diyl Diacetate in Rats. The assay was applied to a preliminary pharmacokinetic experiment after oral administration of 40 g x [kg.sup.-1] raw and processed Atractylodis Rhizoma to rats, respectively. Mean concentration-time curves were shown in Figures 3 and 4. The pharmacokinetic parameters were shown in Table 2.

A significant result of this study is finding that (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate showed double peaks after oral administration, which demonstrated that a hepatoenteral circulation may exist. For raw Atractylodis Rhizoma the absorption peaks in rat plasma was at 2h and 4h, respectively and the [C.sub.max] was 38 [+ or -] 24 [micro]g x [L.sup.-1]. And for processed Atractylodis Rhizoma the absorption peaks was at1h and 3 h, respectively and the [C.sub.max] is 42 [+ or -] 17 [micro] x g[L.sup.-1]. So the time of absorption peak was 1 hour in advance and the concentration of rat plasma was increased after processing. The value of [T.sub.max] and [T.sub.1/2] indicated that the (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate was rapidly distributed but slowly eliminated. The reason for this result also requires further study.

3.3. Tissue Distribution Study. The tissue concentrations of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate determined at 0.5, 2, 4, and 8 h after oral administration raw and processed Atractylodis Rhizomaat a dose of 40 g x [kg.sup.-1] are shown in Table 3 and Figures 5 and 6, which indicated that (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate could be distributed to all collected tissues, for raw Atractylodis Rhizoma, the concentrations of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in tissue were distributed, followed by spleen, liver, small intestine, heart, stomach, large intestine, kidney, and lungs; for processed Atractylodis Rhizoma, the concentrations of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in tissue were distributed, followed by spleen, heart, kidney, liver, lungs, stomach, small intestine, large intestine. Relatively, the concentrations of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate of raw and processed Atractylodis Rhizoma was higher in the spleen. And the concentrations in the spleen were increased after stir-frying with bran. The results showed that processing can promote the rate of absorption of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate.

4. Conclusion

A simple, specific, and rapid RP-HPLC method with UV detection for quantification of (4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate in rat plasma has been developed for the first time. It has been successfully applied to a preliminary pharmacokinetic and tissue distribution study of (4E,6E,12E)-tetradedecatriene-8,10-diyne-1,3-diyl diacetate after oral administration of 40 g x [kg.sup.-1] raw and processed Atractylodis Rhizoma, respectively. We found that the [T.sub.max] have significant difference (P < 0.05), and other pharmacokinetics have no significant difference after using Student's t-test. The result indicated that processing can promote and accelerate the absorption and the concentration of (4E,6E,12E)-tetradecatrinen-8,10-diyne-1,3-diyl diacetate is the highest in the spleen Which proved that the traditional theory of processing Atractylodis Rhizoma can increase its function of tonifying the spleen.

Conflict of Interests

The authors declare that they have no conflict of interests.


This project was supported by grants from the National Natural Science Foundation of China (no. 81202919) and the Special Scientific Research for Traditional Chinese Medicine of State Administration of Traditional Chinese Medicine of China (no. 20110700712).


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Yan Huo, Yu-Qiang Liu, Zhong-Xu Bai, and Qian Cai

School of Medicine, Liaoning University of Traditional Chinese Medicine, Dalian 116600, China

Correspondence should be addressed to Qian Cai;

Received 31 October 2013; Accepted 21 January 2014; Published 24 February 2014

Academic Editor: Yu-Ming Fan

TABLE 1: The Linear regression analysis of
(4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate
in rat plasma and tissues (n = 7).

Sample            Calibration curves      r       Linear range

Plasma            Y = 23.628X + 0.044   0.9950   0.00361-0.18814
Heart             Y = 13.259X + 0.031   0.9958   0.00149-0.07322
Liver             Y = 14.351X + 0.064   0.9951   0.00144-0.06925
Spleen            Y = 12.936X + 0.028   0.9951   0.00146-0.07431
Lungs             Y = 14.704X + 0.059   0.9956   0.00143-0.07872
Kidney            Y = 12.936X + 0.028   0.9951   0.00143-0.06716
Stomach           Y = 15.161X + 0.012   0.9964   0.00143-0.06786
Large intestine   Y = 11.271X + 0.019   0.9952   0.00146-0.08179
Small intestine   Y = 12.956X + 0.037   0.9985   0.00144-0.07339

TABLE 2: The pharmacokinetic parameters of
(4E,6E,12E)-tetradecatriene-8,10-diyne-1,3-diyl diacetate of
raw and processed AtractylodisRhi-zoma at a dose of 40 g
x [kg.sup.-1] to rats, respectively (n = 6).

Parameters                          Raw

[AUC.sub.(0-t)]             0.260 [+ or -] 0.137
  (mg x h x [L.sup.-1])
[AUC.sub.(0-[infinity])])   0.317 [+ or -] 0.157
  (mg x h x [L.sup.-1]
[MRT.sub.(0-t)] (h)         7.706 [+ or -] 0.888
[MRT.sub.(0-t)] (h)         14.86 [+ or -] 6.813
[t.sub.1/2z] (h)            9.905 [+ or -] 7.382
[T.sub.max] (h)             2.333 [+ or -] 0.816
[C.sub.max] (mg-L-1)        0.038 [+ or -] 0.024

Parameters                       Processed

[AUC.sub.(0-t)]             0.265 [+ or -] 0.100
  (mg x h x [L.sup.-1])
[AUC.sub.(0-[infinity])])   0.303 [+ or -] 0.111
  (mg x h x [L.sup.-1]
[MRT.sub.(0-t)] (h)         7.384 [+ or -] 0.86
[MRT.sub.(0-t)] (h)         12.22 [+ or -] 3.364
[t.sub.1/2z] (h)            8.047 [+ or -] 3.347
[T.sub.max] (h)             1.000 [+ or -] 0.817
[C.sub.max] (mg-L-1)        0.042 [+ or -] 0.017

TABLE 3: The tissue concentrations of (4E,6E,12E)/
 and processed tetradecatriene-8,10-diyne-1,3-diyl diacetate after
oral administration raw Atractylodis Rhizoma at a dose of
440 g [kg.sup./1] to rats, respectively ([micro]g/g).

                       0.5 h                 2h

Tissues            Raw     Processed    Raw     Processed

Heart             125.31    137.68     128.97    244.30
Liver             80.37     158.37     155.10    208.56
Spleen            170.71    119.41     253.18    292.69
Lung              69.35      70.01     46.54     174.26
Kidney            108.76    105.54     76.15     242.21
Stomach           112.31    107.95     109.77    122.96
Large Intestine   20.33      30.68     79.02      54.78
Small Intestine   91.25     106.29     139.04    115.53

                         4h                  8h

Tissues            Raw     Processed    Raw     Processed

Heart             77.35      61.62     35.94      30.56
Liver             158.07    173.38     115.10    128.98
Spleen            186.45    204.92     17.34      30.66
Lung              84.79     100.26     40.20      60.61
Kidney            81.83      84.78     24.51      21.12
Stomach           65.22      78.53     43.17      59.93
Large Intestine   112.25    143.34     166.77    176.64
Small Intestine   191.22    159.02     44.91      54.78
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Title Annotation:Research Article
Author:Huo, Yan; Liu, Yu-Qiang; Bai, Zhong-Xu; Cai, Qian
Publication:Journal of Analytical Methods in Chemistry
Article Type:Report
Geographic Code:9CHIN
Date:Jan 1, 2014
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