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J Tradit Chin Med 2016 April 15; 36(2): 238-242 ISSN 0255-2922 © 2016 JTCM. All rights reserved.
EXPERIMENTAL STUDY TOPIC
Correlation between the transdermal characteristics of pseudoephedrine and amygdalin in majiepingchuan in vitro
Kong Hui, Qu Huihua, Qu Baoping, Zeng Wenhao, Zhao Yan, Wang Xueqian, Wang Qingguo aa Kong Hui, Qu Baoping, Wang Xueqian, Wang Qingguo, School of Preclinical Medicine , Beijing University of Chinese Medicine, Beijing 100029, China Qu Huihua, Center of Scientific Experiment, Beijing University of Chinese Medicine, Beijing 100029, China Zeng Wenhao, School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100029, China Supported by the Science and Technology Major Projects for Major New Drugs (Research on innovation of Majiepingchuan effective components cataplasm of transdermal drug delivery system), No. 2012ZX09103201-007. Correspondence to: Prof. Zhao Yan and Wang Xueqian, School of Preclinical Medicine, Beijing University of Chinese Medicine, Beijing 100029, China.
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[email protected] Telephone: +86-10-64286705 Accepted: October 10, 2015
ratios of Qtot, Ttot and Kp between pseudoephedrine and amygdalin, and a correlation between them. © 2016 JTCM. All rights reserved. Key words: Pseudoephedrine; Amygdalin; Majiepingchuan; Transdermal flux; Correlation
INTRODUCTION Majiepingchuan comprises Mahuang (Herba Ephedra Sinica), Kuxingren (Semen Armeniacae Amarum), Baijiezi (Semen Sinapis), Yanhusuo (Rhizoma Corydalis Yanhusuo), and Shengjiang (Rhizoma Zingiberis Recens). It is a topical transdermal formulation employed to treat acute and chronic asthma, and has been used in China for more than 300 years. Effects of majiepingchuan have been noted in clinical studies for asthma treatment.1 It has been reported that amygdalin2 and ephedrine3 can penetrate the skin in vitro. Pseudoephedrine (an isomer of ephedrine) can also be absorbed through the skin. Traditional Chinese Medicine (TCM) compounds have been used as single4 or combined components5 in skin-penetration studies in vitro to show transdermal behavior.6 The relationship among multiple components (small-molecule compounds) has not been reported. However, this relationship could help to reveal the pharmacodynamics of multiple components of TCM compounds. Transdermal experiments using extracts from TCM compounds have revealed the main parameters of the principal components of such extracts: cumulative transdermal flux (Qtot),7 cumulative transmission (Ttot),8 and mean penetration rate (Kp).9 Linear regression analyses10,11 and different analytical methods12 have been adopted for correlation studies. Positive correlations between transdermal behaviors of the TCM ingredients have been documented.
Abstract OBJECTIVE: To analyze the transdermal profile of pseudoephedrine and amygdalin in the Traditional Chinese Medicine majiepingchuan in rat skin and to reveal their interaction. METHODS: A Franz diffusion cell was used in vitro to evaluate the transdermal parameters of cumulative transdermal flux (Qtot), cumulative transmission (Ttot), and mean penetration rate (Kp) of pseudoephedrine and amygdalin in majiepingchuan. Linear regression analyses of Qtot over time of pseudoephedrine vs amygdalin and their ratios was adopted for correlation evaluation. RESULTS: At 1, 2, 4, 6, and 8 h, the Qtot,Ttot and Kp of pseudoephedrine showed a good correlation with that of amygdalin. CONCLUSION: There was a small difference in the JTCM | www. journaltcm. com
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We investigated the transdermal behavior of two ingredients of majiepingchuan, pseudoephedrine and amygdalin, to study their interaction.
Skin preparations Rats were killed and their back skin excised immediately. Skin was fixed along the edge of a smooth foam board, with the hair facing outwards, and dipped in 8% sodium sulfide solution.18 Once the hair color became yellow, the hair was removed with gauze along the same direction. The skin surface was washed with water and subcutaneous fat removed with a scalpel. Samples were stored in physiologic (0.9% ) saline at 4 ℃ but used < 7 days of preparation.
MATERIALS AND METHODS Instruments A 1260 series liquid chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with a quaternary pump, diode array detector, autosampler, and thermostatically controlled column apartment, BT 125D electronic balance (Sartorius Scientific Instruments, Goettingen, Germany), and a modified Franz diffusion cell (Shanghai Kai Kai Technology, Shanghai, China) were employed.
Transdermal experiments The modified Franz diffusion cell19 was fixed with a volume in the accepting pool of 6.5 mL and an effective contact area in the receiving pool of 2.8 cm2. Then, 30% ethanolic saline20 was added to the accepting pool. The dermal side of isolated rat skin faced the accepting pool (thereby ensuring the absence of bubbles) while excess skin was excised. Paste samples were applied to the cutting side of isolated rat skin, and were clamped. The receptor compartment was filled with receiver solution, which was stirred by an electromagnetic stirring bar (200 rpm) and maintained at 32 ± 1 ℃ 21 by a circulating water bath. Aliquots of the receiving phase (50 mL) were withdrawn at 1, 2, 4, 6, and 8 h, and were replaced immediately with an equal volume of fresh solution to the pool. The sample was filtered with a microporous membrane (size, 0.45 μm) and was injected into the high-performance liquid chromatography (HPLC) system. The peak area, reference drug concentration, and cumulative transdermal flux at each time point (Qn) were calculated using the following formula:22
Medicines and reagents Mahuang (Herba Ephedra Sinica), Kuxingren (Semen Armeniacae Amarum), Baijiezi (Semen Sinapis), and Yanhusuo (Rhizoma Corydalis Yanhusuo) were purchased from Beijing Tongrentang Herbal Medicines (Beijing, China). Standards of pseudoephedrine (batch No. 171237-200304) and amygdalin (110820201004) were purchased from the National Institutes for Food and Drug Control (Beijing, China). All other reagents were available commercially, including reagents for chromatography. Animals Animal studies were undertaken according to the Guidelines for the Care and Use of the Laboratory Animals (National Institutes of Health, Bethesda, MD, USA). The study protocol was approved by the Animal Experimentation Ethics Committee of the Beijing University of Chinese Medicine (Beijing, China). Male Sprague-Dawley rats (6-8 weeks; 18-22 g) were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China).
where Cn is the measured concentration at n sampling points, Ci is the measured concentration at the first sampling point, V0 is the accepted pool volume, V is the sampling volume at each time, A is the skin diffusion area, and Qn (μg·cm−2) is the cumulative transdermal flux at each time point. Linear regression of Qn vs t1/2 was the Higuchi equation and the slope of the line was Kp (μg·cm−2·h−1/2).
Sample preparation Weighed quantities of ephedra, mustard seed, bitter almond, and corydalis tuber were mixed according to prescription proportions, followed by addition of 6 × volumes of ethanol at different concentrations.13–16 The mixture was extracted in different times under nine conditions,17 and nine extractions were obtained. Ethanol was recovered through a vacuum at 60 ℃ and the remainder was plaster. The plaster was dried at 60 ℃ by dry-box decompression (－0.08 MPa). The dry plaster was smashed to a fine powder and named "majiepingchuan extractions" before being stored in a dryer. Ginger was squeezed to obtain its juice, which was then centrifuged at 8000 × g. for 5 min at room temperature. The supernatant was stored at 4 ℃. This powder was mixed with the corresponding amount of ginger juice and stirred to obtain paste samples, which were the transdermal test preparations. JTCM | www. journaltcm. com
RESULTS Donor paste Quantity of pseudoephedrine and amygdalin from nine majiepingchuan extractions were analyzed by HPLC (Table 1). Ratios of the quantities of pseudoephedrine and amygdalin in different samples were equal to those of the donor (Table 2). Receiver solution For transdermal experiments, each paste sample was tested in replicate. The receiver solution was withdrawn according to preset time points, and injected into the HPLC system. Characteristics of the penetration 239
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of pseudoephedrine and amygdalin for the nine conditions was a constant value of 1.19±0.20. Ratios of Qtot (μg·cm−2), Ttot (%) in 8 h, and Kp (μg·cm−2·h−1/2) were constant values of 2.55 ± 0.49, 1.92 ± 0.23, and 2.03 ± 0.47, respectively. From the results, we concluded that the other components in the herbs could not have affected the characteristics of the extraction and penetration of pseudoephedrine and amygdalin. Hence, the other components could be considered to be "inert". Quantities of the nine extractions were different, but the ratio of pseudoephedrine and amygdalin was constant. This finding demonstrated that the extraction conditions affected only the quantity (and not the ratio) of pseudoephedrine and amygdalin. The same scenario was observed with Qtot, Ttot in 8 h, and Kp, which showed that transdermal conditions (i.e., skin and receiver solution) affected only the absolute values and not the ratios. Ttot in the receiver solution was about two-fold that of the extractions. Hence, for each amygdalin molecule that penetrated the skin, approximately two molecules of pseudoephedrine penetrated the skin (Table 3). There was a very good linear correlation (r2 > 0.90) (Table 4, Figure 1), suggesting that pseudoephedrine and amygdalin had analogous transdermal behaviors with a good interrelation between them. In conclusion, during extraction of majiepingchuan under different conditions, a small difference in the extraction ratio of pseudoephedrine and amygdalin was noted even though they are different types of compound (pseudoephedrine is an alkaloid and amygdalin is a glycoside). There was a small difference in the ratios of Qtot, Ttot and Kp between pseudoephedrine and amygdalin, and a correlation between them.
Table 1 Quantities (mg) of PE and Am in nine extractions Extract No.
PE
Am
1
2.59
2.57
2
4.93
5.29
3
6.05
4.82
4
3.99
3.05
5
5.29
5.08
6
3.57
3.31
7
5.16
4.02
8
3.39
2.16
9
5.17
4.29
Notes: PE: pseudoephedrine; Am: amygdalin.
that were analyzed were Qtot (μg·cm −2), Ttot (%) in 8 h, and Kp (μg·cm −2·h −1/2) (Table 3) with their ratios are shown in Table 2. Correlation between quantities before and after penetration of skin To describe transdermal changes in pseudoephedrine and amygdalin, their quantities in donor paste and receiver solution at the final time point were compared. Their ratios were very similar (1.95), along with the relative standard deviation (11.22%) (Table 2). Correlation of Qtot A linear correlation model was developed using the Qtot of pseudoephedrine and amygdalin corresponding to different time points, and the regression equation in each sample was obtained (Table 4).
DISCUSSION Table 2 shows that the ratio of the extraction quantity
Table 2 Ratios of PE and Am in majiepingchuan before and after skin penetration in vitro (n = 3, xˉ ± s) Extract No.
A
B
C
D
B/A
1
1.01
1.56±0.06
1.58±0.08
1.42±0.03
1.57
2
0.93
1.79±0.47
1.93±0.50
1.46±0.80
1.93
3
1.25
2.81±0.80
2.24±0.64
2.48±1.35
2.25
4
1.31
2.54±0.80
1.94±0.61
2.14±1.09
1.94
5
1.04
2.35±0.44
2.25±0.42
2.52±0.50
2.26
6
1.08
1.96±0.27
1.81±0.25
1.68±0.29
1.81
7
1.28
2.60±1.35
2.02±1.06
2.63±1.47
2.03
8
1.57
2.66±0.36
1.70±0.23
2.25±0.54
1.96
9
1.20
2.13±0.20
1.78±0.17
1.71±0.39
1.78
1.19±0.20
2.55±0.49
1.92±0.23
2.03±0.47
1.95±0.22
16.47
19.37
12.04
23.25
11.22
Mean ± SD RSD (%)
Notes: A: the ratio of PE and Am in the donor sample (μg). B: the ratio of PE and Am in Qtot (μg·cm－2); results were calculated based on the mean ± SD of triplicate observations. C: the ratio of PE and Am in Ttot (%); results were calculated based on the mean ± SD of triplicate observations. D: the ratio of PE and Am in Kp (μg·cm－2·h－1/2); results were calculated based on the mean ± SD of triplicate observations. PE: pseudoephedrine; Am: amygdalin; RSD: relative standard deviation. JTCM | www. journaltcm. com
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Hui K et al. / Experimental Study Table 3 Results of Qtot (μg·cm－2), Ttot (%) in 8 h and Kp (μg·cm－2·h－1/2) ( xˉ ±s, n = 3) Extract number
Qtot of PE (μg·cm－2)
Qtot of Am (μg·cm－2)
Ttot of PE (%)
Ttot of Am (%)
Kp of PE (μg·cm−2·h−1/2)
Kp of Am (μg·cm−2·h−1/2)
1
597±191
5352±158
64±20
41±15
244±75
173±57
2
673±459
7792±163
38±26
23±18
274±259
225±182
3
691±548
890±99
32±25
18±19
251±117
172±198
4
769±451
517±65
54±32
33±24
258±179
145±91
5
607±277
1245±117
32±15
15±9
248±85
104±46
6
896±297
4467±146
70±23
40±19
389±174
231±106
7
1036±795
7627±157
59±45
41±34
437±331
239±196
8
561±204
3614±161
46±16
28±14
234±109
106±47
9
1276±297
6801±151
72±18
41±13
495±224
277±78
Notes: PE: pseudoephedrine; Am: amygdalin; Qtot: cumulative transdermal flux; Ttot: cumulative transmission; Kp: mean penetration rate. Table 4 Correlation in transdermal flux (Qn) between pseudoephedrine (PE) and amygdalin (Am) (n = 3) Extract No.
Regression equation
Correlation coefficient (r2)
1
Y=6.3638x–634.02
0.94
2
Y=8.6341x–1510.9
0.96
3
Y=1.3442x–139.98
0.95
4
Y=1.2825x–187.11
1.00
5
Y=1.6975x+32.419
1.00
6
Y=3.641x–238.84
0.99
7
Y=5.2077x–601.91
0.99
8
Y=4.0073x–112.57
0.98
9
Y=3.6975x–299.26
0.99
5
6
7
8
9
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