The In Vivo Evaluation on Total Components from Extract of Aconitum Based on a New Analytic Method of Area under Absorbance-Wavelength Curve

Objective. To study oral pharmacokinetics of Aconitum extract in randomized Sprague-Dawley (SD) rats and observe the profile change in vivo between plasma concentration and time with an aim to disclose relevant delivery rule for total components from Aconitum extract and finally to evaluate the research of Aconitum sustained-release preparation.Methods. Randomized rats were administered orally with the single and multiple dose of Aconitum extract based on the clinical use of Aconitum injection. Blood samples were collected at predetermined interval time from an eye of rats.-e concentration of total components in those samples was determined by a new analytic method of area under absorbance-wavelength curve, the data were analyzed by Drug and Statistics software, and then the pharmacokinetic parameters and compartment model were acquired and compared, respectively. Results. -e pharmacokinetic parameters of single andmultiple doses were similar, and both of them showed characteristics of the one-compartment model. Cav, DF, and AUCss were (2.075± 0.282) μg/ml, (2.405± 0.175), and (24.901± 0.422) μg/ml/h, respectively. However, the concentrations of total components in the fifth and sixth day were 0.32 and 0.44 μg/ml, which were lower than the value of Cav. Conclusion. -e method of AUAWC is feasible for the pharmacokinetics study in vivo analysis on total components of Aconitum with good sensitivity, specificity, and repeatability. -e results demonstrated that both absorption and elimination of total components from Aconitum extract in rats were rapid and pharmacokinetic behavior was consistent at the level of single and multiple doses, which confirms that Aconitum injection is available to transform the oral sustained-release preparation. -is will bring a good instruction to the research of sustained-release preparation of traditional Chinese medicine (TCM), which still lacks of an evaluation method available for total components in vivo analysis.


Introduction
Aconitum is a famous traditional Chinese medicine that had been used extensively for centuries to treat pain resulted from arthritis and cancer and to strengthen the function of the heart [1][2][3][4].e main bioactive components of Aconitum are Aconitum alkaloids with a narrow therapeutic index, and the activity is closely related to the structure which differs from the number, type, and position of the substituents [5].Aconitum injection composed of raw Chuanwu and Caowu, a kind of traditional Chinese medicine injection, is used for treatment of serious pain in the terminal gastric and liver cancer and has significant analgesic effect without addiction [6][7][8][9].Although it has such promising therapeutic effects, its potential neurotoxicity and cardiotoxicity are frequently observed in clinics [10][11][12][13].Aconitum injection is intramuscular in clinic with once or twice a day at 1∼2 ml every time according to the standard of the ministry of traditional Chinese medicines [14].But, it has no good compliance for the patient and is only injected through the doctors in hospital.
erefore, it is reasonable for us to study oral Aconitum sustained-release preparation transformed from Aconitum injection due to its digestive tract treatment for serious pain from terminal gastric and liver cancer.
Pharmacokinetic study is very important for the development of sustained-release preparation.However, there is no good effectively analytic evaluation method to be used for the pharmacokinetics of total components of TCM. is is the major reason why the sustained-release preparations of TCM were variously reported in references but little marketed in clinic.Up to now, despite many related theories and hypotheses about pharmacokinetics of TCM have been reported to explain its pharmacokinetic behavior, the substantial progresses for total components have not been broken through in nature [15][16][17][18][19][20].
ere were many pharmacokinetic studies on Aconitum from references [21][22][23][24] but still lacking of related information for total components of Aconitum.It was reported that Aconitum alkaloids were absorbed and metabolized in vivo rapidly, which were all based on some certain individual alkaloids with clear chemical structure such as aconitine, meaconitine, and hyaconitine by high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (HPLC-MS) [25][26][27].Nevertheless, the pharmacokinetic results of various individual components are difficult to reasonably clarify the total pharmacokinetic characteristics of TCM with complicated components.Hence, it is very necessary and urgent to establish an analytical method for pharmacokinetic study on total components of Aconitum.
Area under absorbance-wavelength curve (AUAWC) is an analytical method based on UV-visible (UV-Vis) spectrophotometry, which can be used for simultaneous determination of total components of TCM with a combination of mathematics and computer technology, and has being used successfully in our group.At present, despite the methods of HPLC and HPLC-MS are being used in more and more analytical fields, UV-Vis spectrophotometry is also applied for its special merits, especially analysis such as total flavonoids, total alkaloids, and total saponins dealing with total components.In vivo, though part of total components may possibly be metabolized as metabolites, the mother nuclear structure of metabolite does not always change and the absorption behavior in UV-Vis spectrophotometry is generally the same.erefore, AUAWC can accurately illustrate the delivery tendency of total components concentration including metabolites in plasma with time change.It is proportional to the concentration of total components, which was proposed in 2012 and confirmed from the theory based on Lambert-Beer's equation with EL constant [28,29], as follows: And, a series of trials related to AUAWC or its combination with HPLC were well performed and reported in the Journal of Spectroscopy in 2013 [30] and some good Chinese core journals, such as the content determination of total components in TCMs [31][32][33] and the studies on in vitro release tests [34][35][36] and in vivo pharmacokinetics [37][38][39] of total components in compounds preparations of TCMs.As for the operation procedure, firstly, AUAWC can be calculated through Origin software.en, the AUAWC of total components can be obtained after a subtraction of the AUAWC of the drug's plasma sample to the AUAWC of the blank plasma sample, as shown in Figure 1.
irdly, the concentration of total components is calculated through a linear equation in which the concentration of total components is the AUAWC at certain time.Subsequently, the profile between concentration and time can be drawn, and the pharmacokinetic parameters be obtained through DAS software, illustrating absorption, distribution, metabolism, and excretion for total components from TCM and their final delivery rule in vivo.Nowadays, cancer increasingly affects people's health and even becomes the leading cause of death with no presently good treatment criteria.us, in this study, the total components in rats from Aconitum extract, which was prepared according to the technique of Aconitum injection in the standard of the ministry of traditional Chinese medicines, were determined by the method of AUAWC.After an oral administration with the single and multiple doses of Aconitum extract, the pharmacokinetic parameters of total components will be analyzed to search for Aconitum's delivery rule in vivo, which will be helpful to bring a good research base for the Aconitum sustained-release preparation and a convenient oral administration for the cancer patients.

Reagents. Aconitine was purchased from the National
Institute for Control of Pharmaceutical and Biological Products (Beijing, China).e raw material of Chuanwu and Caowu was purchased from medicine market (Bozhou) and were authenticated by Professor Zehao Huang from Fujian University of Traditional Chinese Medicine.Extract of Aconitum was obtained from the Department of Pharmaceutics of Fujian University of Traditional Chinese Medicine.All other reagents were of analytical grade, and the water used in the experiment was double distilled.

Animals.
Healthy Sprague-Dawley rats, weighing 0.20-0.22kg, were purchased from SLAC Laboratory Animal Company (Shanghai, China, SCXK (HU) 2018-0005).All the rats were allowed to acclimate one week before experiment.All studies were in compliance with the Guidelines for the Care and Use of Laboratory Animals and approved by Institutional Animal Care and Use Committee in XBL-China.

Animal Administration and
Sampling.SD rats were randomly divided into nine groups (eight rats each group) based on different interval times, one group of which was regarded as the blank group and the others were administered orally the extract of Aconitum.e rats were fasted overnight and administered Aconitum extract at a single dose of 0.6 mg/kg in the morning, which is equivalent to the one-time dose of Aconitum injection.Blood samples (about 2.5 ml) were collected from intraocular angular veins of each rat and placed into tubes with heparin sodium anticoagulant at predetermined interval times (0, 0.25, 0.5, 1, 2, 4, 6, 8, and 12 h).en, blood samples were centrifuged immediately at 3800 r/min for 10 min, and the separated plasma samples were stored at −20 °C until analysis.e rats were fed to acclimate for one week after taking blood and then administered the Aconitum extract again at a dose of 0.6 mg/kg for 7 d continuously.e blood samples were collected and processed as the same as single dose except the blood samples for stable drug concentration determination, which were taken in the morning of the fth and sixth day before the rats were administered.

Method Validation 2.5.1. Preparation of Calibration Standards.
e reference substance of aconitine was prepared in methanol before use with a total concentration of 213 μg/ml.Working solutions were prepared by diluting stock solution with methanol for further concentration series of 42.6, 17.04, 8.52, 4.26, 1.06, and 0.53 μg/ml.2.5.2.Speci city.Speci city of the method was investigated by comparing chromatograms of processed blank plasma samples, the standard of aconitine, the blank plasma spiked with the aconitine, and plasma after oral administration of Aconitum extract.e AUAWC of plasma samples should be larger than that of blank plasma samples, so the interference of blank plasma can be eliminated by the difference value.

Linearity.
In accordance with the UV-Vis spectrophotometry, methanol was used as the blank control.Each 0.2 ml blank plasma was precisely added above series of standard solutions to 4 ml, respectively, vortexed for 2 min, and centrifuged at 3800 r/min for 10 min.en supernatant was used for a full UV-Vis spectrum scanning from 200 nm to 500 nm wavelength.An eight-point calibration curve was constructed by plotting the AUAWC against the concentration.e linearity of the calibration curve was evaluated by linear regression analysis.

Recovery.
e extraction recovery and relative recovery of aconitine were determined by analyzing six replicates of QC samples at three concentration levels.e extraction recovery was calculated by comparing the AUAWC values (a) of extracted QC samples with those (a) in which the compounds were spiked directly to methanol.
e relative recovery was evaluated by comparing the concentration calculated by substituting the AUAWC values (a) into the regression equation with the actual concentration.

Precision.
e precision of the method was determined by analyzing QC samples at three concentration levels in six replicates.Intraday precision was assessed from replicate analyses (n 6) of QC samples at each concentration level on the same day.e precision was expressed as relative standard deviation (RSD) for each QC concentration.e precisions were required to be within ±15% RSD.

Di erence.
e di erence of the blank plasma samples from randomized SD rats in a group was determined.e di erence could be ignored if the RSD < ±15%.

Stability.
e stability of drug plasma samples from rats was determined at di erent time periods, including their stability after three freeze-thaw cycles with a frozen temperature of −20 °C and the stability of processed samples kept at room temperature for 8 h.Samples were considered to be stable if the values were within requirement of precision (<±15% RSD).

Sample Preparation and Test.
A plasma sample of 0.2 ml was precisely diluted with methanol to 4 ml, vortexed for 2 min, and centrifuged at 3800 r/min for 10 min.en, supernatant was used for a full UV-Vis spectrum scanning from 200 nm to 500 nm wavelength with methanol as a blank control.(2) AUAWC of blank plasma: a full spectrum scanning for the plasma sample from the rat without drug.

Pharmacokinetics Study.
e values of AUAWC from the blank plasma group and drug groups at di erent interval time periods were calculated using Origin software.When the mean AUAWC values of drug group were subtracted from that of the blank group, respectively, the increased AUAWC caused by total components was obtained correspondingly and then substituted into the linear equation to get the total components concentration in vivo.e pharmacokinetic models (one-versus two-compartment) were compared according to the AIC rule, with minimum AIC values being regarded as the best representation of the blood concentration-time dynamic data.A compartmental model of total components in rats was proposed by the DAS software.
e pharmacokinetic parameters of total components in rats were expressed as mean ± RSD and were compared by the unpaired Student's t-test using the Statistical Package for the Social Science (SPSS, version 18.0).
e result was obtained according to the process as showed in Figure 2.

Validation of the Method
3.1.1.Linearity.From Figure 3, there were no interference from endogenous or exogenous materials and linear in the range from 1.02 to 65.00 μg/ml.e typical calibration curve of aconitine in rat plasma was ΔAUAWC 1.2355C + 3.2935 with a coe cient of correlation r 0.9993.

Recovery
. Six replicates at low, medium, high QC concentrations for aconitine were prepared for recovery, as shown in Table 1.e recoveries of aconitine were satisfactory and reproducible.
e mean absolute recovery of aconitine was from 80.45 to 99.52% at three QC levels.e mean relative recovery was from 100.64 to 107.34% for aconitine at three QC levels.

Precision.
e intraprecision values were within ±15% RSD at all QC levels of aconitine in rats plasma as shown in Table 1.
e data were considered to be acceptable for subsequent analysis of all the samples.

Di erence.
e UV-Vis scanning spectrum of blank plasma from randomized eight SD rats in single-dose and multidose trials was shown, respectively, in Figures 4 and 5. RSD of the AUAWC value was within ±15% as shown in Table 2, which indicated that there is little di erence for the determination of the total components in di erent plasma from di erent randomized rats.

Stability.
e data in Table 3 obtained from determination of stability demonstrated that there was no signi cant degradation under the conditions tested.e plasma samples were stable at room temperature for 8 h, during three freeze-thaw cycles at −20 °C.

e Concentration-Time Curve.
e validated method was applied to the study on pharmacokinetics of Aconitum extract in rats.e full wavelength scan for di erent plasma samples is illustrated in Figure 3. e mean AUAWC values of blank plasma samples and the AUAWC increased by total components are provided in Table 4. e pro le of kinetic curve on concentration versus time in rats after administration is shown in Figures 6 and 7.    Journal of Spectroscopy

Pharmacokinetics.
e main pharmacokinetic parameters of total components in rats after a single and multiple dose administration of Aconitum extract are shown in Table 5.Comparison of the pharmacokinetic parameters of two groups by the unpaired Student's t-test revealed signi cant di erence (P < 0.05) among the important pharmacokinetic parameters, such as C max , AUC 0∼48 , and half-life (t 1/2 ) in Table 6.
Following the above gures and tables, the concentration of total components in the fth and sixth day was lower than the value of C av , but close to that of 0.25 h after administration in the seventh day during the course of multiple oral dose of Aconitum extract.Meanwhile, the concentrationtime curve in rats presented a single-peak phenomenon after a single or multiple oral dose of Aconitum extract.ey indicated the concentration of total components decreased quickly after reaching the C max .In addition, T max of total components stayed the same for 2 h, but t 1/2 was 3.395 h after a multiple dose, and there was a trend of decrease compared with single dose; however, no signi cant di erence was seen.
e results further demonstrated that the absorption and excretion of total components in rats were rather rapid, which were characterized by the one-compartment model in this study, and they were consistent with the results in the studies [22,[40][41][42].From Table 6, there was no signi cant di erence (P > 0.05) in those pharmacokinetic parameters between the single-dose group and multiple-dose group.e cumulative ratio of C max and AUC 0∼t (the value of AUC 0∼12 in seventh day divided by that of the rst day) were 0.658 and

6
Journal of Spectroscopy 0.418, and there were no accumulation of total components in rats after continuous administration for 7 d and once a day.

Discussion.
From the view of biopharmaceutics, the drug concentration in rat plasma will wave greatly if the rats get a fright with blooding repeatedly, so randomized rats were sampled only one time for a rat and eight rats were divided in a group to improve the accuracy of the data.Moreover, the concentration of total components in the fifth and sixth day was lower than the value of C av , which demonstrated that the valley concentration of Aconitum extract is a little small, and suggested the dose of sustainedrelease preparation, which is transformed from Aconitum injection, can be larger than that of injection.UV-Vis scan spectrum can well illustrate absorbance characteristics of total components in TCM, which is better than the method of HPLC in this point.

Conclusion
e method of AUAWC used is feasible for the in vivo evaluation study on total components of Aconitum with a convenience, quickness, and specificity response.In this study, compared with single dose, there were no significant change of the pharmacokinetic behavior and no risk of drug accumulation after continuous administration of Aconitum extract, which is beneficial to the research on sustainedrelease preparation of Aconitum and other TCMs.

Figure 1 :
Figure 1: AUAWC of blank plasma and drug plasma sample.(1) AUAWC of drug plasma: a full spectrum scanning for the plasma sample from the rat with the administered drug.(2) AUAWC of blank plasma: a full spectrum scanning for the plasma sample from the rat without drug.

Figure 2 :Figure 3 :
Figure 2: e in vivo analysis process of total components by the new method of AUAWC.(1) UV-Vis spectrophotometer for a full spectrum scanning; (2) exported .xldata les and preprocessed; (3) the data preprocessed are then exported into Origin software; (4) the value of AUAWC then were calculated; (5) the concentration acquired by the regression equation and kinetic time were analyzed by DAS 2.0 software; (6) the pharmacokinetic parameters and the concentration-time curves were known.

Figure 4 :
Figure 4: e spectrum of blank plasma from randomized rats in single dose (n 8).

Figure 5 :
Figure 5: e spectrum of blank plasma from randomized rat in multidose (n 8).

Figure 6 :
Figure 6: e pro le of concentration-time curve after a single dose (n 8).

Figure 7 :
Figure 7: e pro le of concentration-time curve after a multiple dose (n 8).

Table 1 :
e recoveries and precision for aconitine in rats plasma (n 6).

Table 2 :
e AUAWC values of blank plasma from randomized rats (n 8).

Table 3 :
Stability of plasma samples under di erent storage conditions (n 6).

Table 6 :
Main pharmacokinetic parameters for the statistics and analysis (n � 8).Method P value Conclusion In C max Independent sample t-test 0.424 No significance In AUC 0∼12 Independent sample t-test 0.399 No significance t 1/2 Independent sample t-test 0.300 No significance T max Independent sample t-test 0.106 No significance