서울特別市 保健環境硏究院報 第40號	(2004)

Report of S.I.H.E., 40:175~186	(2004)

HPLC를 이용한 복방카르두스마리아누스엑스 연질캅셀 중 실리빈의 분리 정량

약품화학팀

황인숙․박주성․한은정․김진곤․김명희

Analysis of Silybin in Compound

Cardus Marianus Extract Soft Capsules

Pharmaceutical Chemistry Team

In-sook Hwang, Ju-sung Bak, Eun-jung Han,

Jin-gon Kim and Myung-hee Kim

Abstract

카르두스마리아누스엑스는 간세포에 작용하여 세포막을 안정시켜 간독성물질로부터 간세포를 보호하고 손상된 조식의 재생부활을 촉진시키는데 이 때 약효발현의 주성분은 실리마린이라는 보고가 되어 있다. 실리마린은 보다 정밀한 분석기기의 활용으로 실리빈, 이소실리빈, 실리디아닌, 실리크리스틴 등 4가지 주요 플라보노리그난체로으로 분리결과가 보고되었는데 이들 플라보노리그난류 중 실리빈은 주요 약효성분으로 알려져 있다. 식품의약품안전청고시 제98-127호에 의하면 “복방카르두스마리아누스엑스ㆍ니코틴산아미드ㆍ리보플라빈제제”는 만성간질환, 지방간, 간경변, 중독성간질환에 치료보조제로 응용하는데 이 제제의 정량법에서는 카르두스마리아누스엑스중 실리빈을 함량기준으로 하고 있다. 본 연구에서는 복방카르두스마리아누스엑스ㆍ니코틴산아미드ㆍ리보플라빈제제 중 실리빈을 분리할 수 있는 조건을 찾아 실리빈의 확인 및 정량을 시도하고자 하였다. 국내에서 유통중인 9개의 제품을 임의로 구매하여 이 분석법을 적용하였다. 역상컬럼(Symmetry C18), 메탄올, 1% 초산, 50 mM 헵탄설폰산나트륨을 시간에 따른 농도구배조건으로 하여 HPLC- PDA 분석기기를 사용하였다. 검출한계와 정량성 있는 농도조건은 각각 0.2 μg/ml과 1 μg/ml이었다. 실리빈 표준액을 1～500  μg/ml의 4단계 농도로 검량선을 작성한 결과 농도에 따른 직선성을 보였으며 r값은 1.0000으로 나타났고 회수율도 양호하였다.

Key words : silybin, HPLC, nicotinamide, riboflavin, pyridoxine, thiamine

Introduction

In recent years, antioxidants have been subjected to many epidemiological studies that have related their consumption with a reduction in the incidence of cardiovascular diseases and cancers. Among the recognised antioxidants(carotenoids, tocopherols, vitamin C and E, etc) there is an extensive family of diverse components. Silymarin is an antihepatotoxic polyphenolic substance isola- ted from the milk thistle plant, Silybum marianum(1). They are currently enjoying re-emergence for therapy of liver diseases, as other natural remedies have become increasingly popular in the USA. Their use has been widespread throughout Europe since preparations became officially available for clinical use there in 1969(2). Silymarin was considered as a pure compound with the structure of 7-chromanol-3-methyl-taxifolin, but after the introduction of more accurate methods of analysis and separation it was shown that silymarin consists of a large number of flavonolignans, including silybin diastereomers, isosilybin diastereomers, silydianin, and silychristin(3). Possibly due to their antioxidants and membrane stabilizing properties, the compounds have been shown to protect different organs and cells against a number of insults. Currently the most important medicinal applications of milk thistle is its use as a hepatoprotectant and as supportive treatment of chronic inflammatory liver disorders such as cirrhosis, hepatitis, and fatty infiltration due to alcohol and toxic chemicals(4). Most liver toxins produce their damaging effects by free radical mechanisms. Silymarin protects red blood cell membranes against lipid peroxidation and haemolysis caused by certain red blood cell poisons.

Silybin(silibinin) is the principal component and main active substance of silymarin(5). Only two diastereomers of silybin are produced. The absolute assignment was achieved by X-ray analysis of the natural diastereomeric mixture showing that isome- rism occurs only in the benzodioxane part of the molecule. Up to now the individual diastereomers cannot be separated on a preparative scale.

In this paper, it has been studied identification and determination of silybin diastereomers which are described as the major component of compound Cardus marianus extract, nicotinamide and riboflavin soft capsules preparations in Korea by HPLC-PDA.

Experimental

1. Samples

Nine commercial products were purchased randomly in Korea. See the composition of each compound Cardus marianus extract, nicotinamide and riboflavin commercial products(Table 1).

2. Chemicals and Reagents

Standards of silymarin and silybin, and heptane sulfonic acid sodium salt were purchased from Sigma-Aldrich. Methanol, acetic acid used in this work were HPLC grade and other reagents were analytical grade. Ultra pure water system(Barnstead, USA) treated water(with resistivity more than 18 MΩ cm) was used throughout the experiment.

Table 1. Composition of samples
Samples	Composition
1	Cardus Marianus extract 200 mg (Silymarin 140 mg, Silybin 60 mg) Thiamine HCl 8 mg	Pyridoxine HCl 8 mg Nicotinamide 24 mg Calcium pantothenate 1 6 mg Cyanocobalamine 2.4 ㎍ Riboflavin 8 mg
2	Cardus Marianus extract 200 mg (Silymarin 140 mg, Silybin 60 mg) Thiamine nitrate 4 mg	Pyridoxine HCl 4 mg Nicotinamide 12 mg Calcium pantothenate 8 mg Cyanocobalamine 1.2 ㎍
3	Cardus Marianus extract 200 mg (Silymarin 140 mg, Silybin 60 mg) Thiamine HCl 8 mg	Pyridoxine HCl 8 mg Nicotinamide 24 mg Calcium pantothenate 16 mg Cyanocobalamine 2.4 ㎍ Riboflavin 8 mg
4	Cardus Marianus extract 200 mg (Silymarin 140 mg, Silybin 60 mg) Thiamine HCl 8 mg	Pyridoxine HCl 8 mg Nicotinamide 24 mg Calcium pantothenate 16 mg Cyanocobalamine 2.4 ㎍ Riboflavin 8 mg
5	Cardus Marianus extract 200 mg (Silymarin 140 mg, Silybin 60 mg) Thiamine nitrate 8 mg	Pyridoxine HCl 8 mg Nicotinamide 24 mg Calcium pantothenate 16 mg Cyanocobalamine 2.4 ㎍ Riboflavin 8 mg
6	Cardus Marianus extract 200 mg (Silymarin 140 mg, Silybin 60 mg) Thiamine HCl 8 mg	Pyridoxine HCl 8 mg Nicotinamide 24 mg Calcium pantothenate 16 mg Cyanocobalamine 2.4 ㎍ Riboflavin 8 mg
7	Cardus Marianus extract 200 mg (Silymarin 140 mg, Silybin 60 mg)	Thiamine nitrate 4 mg Pyridoxine HCl 4 mg Nicotinamide 12 mg Calcium pantothenate 8 mg Cyanocobalamine 1.2 ㎍
8	Cardus Marianus extract 200 mg (Silymarin 140 mg, Silybin 60 mg) Thiamine nitrate 4 mg	Pyridoxine HCl 4 mg Nicotinamide 12 mg Calcium pantothenate 8 mg Cyanocobalamine 1.2 ㎍
9	Cardus Marianus extract 200 mg (Silymarin 140 mg, Silybin 60 mg)	Thiamine nitrate 4 mg Pyridoxine HCl 4 mg Nicotinamide 12 mg Calcium pantothenate 8 mg Cyanocobalamine 1.2 ㎍

3. Instrument

HPLC analyses were performed on Waters Alliance System instrument consisting of Separation Modules(Waters 2695, USA) and Photodiode Array Detector(Waters 2996, USA) controlled by Empower.

4. Condition of analyses

1) Working Standard Solution

The silybin was used as standard for quantitative analysis and external standard method(four concentration levels  1, 10, 100, and 500 μg/ml) was used. Standard and  each sample were dissolved in methanol.

2) Sample Solution

Take 20 capsules of each sample prepara- tion and weigh accurately mass of each capsule. Open the capsule, remove the contents by washing with volatile solvent, such as ether, remove the remaining solvent of the empty capsule and leave the capsules in a room temperature to remove the remaining solvent. Weigh the mass of the empty capsule. Calculate the mass of the content by subtracting the mass of the empty capsule from the mass of the corresponding capsule.  Calculate the mean mass of the contents of 20 capsules.

Weigh accurately 20 capsules(compound Cardus marianus extract, nicotinamide and riboflavin soft capsules) respectively, cut open, transfer the contents and mix well. Weigh portion of the suspension oil, equivalent to about 6 mg of silybin, to a 100 ml volumetric flask. Add about 70 ml of methanol and shake with stirring bar for 30minutes. Add methanol to make 100 ml and filter through a membrane filter with pore size of 0.45 ㎛. Discard the first 10 ml of the filtrate and perform the test with 10 ㎕ each of the test solution and the standard solution as directed under the HPLC assay.

3)  HPLC assay

The separation of silybin diastereomers of compound Cardus marianus extract, nicotinamide and riboflavin commercial products was carried out using stationary phase Symmetry C18(3.9×150 mm, 5 ㎛). Methanol and 50 mM heptane sulfonic acid sodium salt with 1% acetic acid were served as mobile phase at a flow rate 0.5 ml/min. The elution has been made in a gradient mode(Table 2) with photodiode array detector.

Table 2. Time table of gradient mode.
Time	Mobile A(%)	Mobile B(%)	Flow rate(ml/min)
0	85.0	15.0	0.5
3.00	85.0	15.0	0.5
15.00	50.0	50.0	0.5
30.00	50.0	50.0	0.5
35.00	85.0	15.0	0.5
40.00	85.0	15.0	0.5
Mobile phase A - 50 mM heptane sulfonic acid with 1% acetic acid Mobile phase B - 100% methanol Post time 20 minutes

Results and discussion

1. Linearity and recovery of method

Four solutions containing silybin at con- centrations ranging from 1.0 to 500.0 μg/ml were analyzed. The silybin was resolved into two different peaks. Silybin concentra tion is expressed as a sum of both diastereo mers. The calibration functions of silybin standard calculated with peak area(y, mAU) and concentration(x, μg/ml) was y = 53928x + 2499.7(r = 1.0000), over the concentration range 1 to 500 μg/ml.(Fig. 1) For recovery test, known amounts of silybin standards are added to the known concentration of sample solution and analyzed 3 times by HPLC-PDA. The efficiency of recovery by the HPLC-PDA was shown in Table 3. The average recovery of silybin diastereomers was about 99.7% and RSD is 0.25%.

Fig. 1. Calibration curve of silybin standard solutions. (Silybin concentration is expressed as a sum of both diastereomers).

Table 3. Recovery rate of silybin diastereomers from the compound Cardus marianus extract, nicotinamide and riboflavin commercial products by the HPLC method(mean of n=3)

	1	2	3	mean	RSD
Added (μg/ml)	155.594	155.594	155.594	155.594
Found (μg/ml)	154.816	155.127	155.591	155.178	0.25
Recovery rate(%)	99.5	99.7	100.0	99.7	0.25

2. Discussion

We also observed the impurity test by impurity-plot. The results of impurity test of the spiked solutions were good. The purity angle was lower than the purity threshold. We named the silybin diasteromers, silybin diastereomer I, II, in sequence of retention time. Silybin diastereomer I showed 0.154, 0.264 and silybin diastereomer II 0.148, 0.260, respectively(Fig. 2). The results of purity of the standard solution of silybin 100 μg/ml were 0.118, 0.295 of silybin dia- stereomer I and 0.125, 0.302 of diastereomer II, in sequence. Besides those of sample solution indicated 0.219, 0.367 of silybin diastereomer I and 0.311, 0.334 of dia- stereomer II.

We performed the library match of spectrum of spiked solution, sample solution compared to standard solution. The spectrum of spiked solution and sample solution corresponded with the spectrum of silybin standard. Merck Index thirteenth edition reports that UV max(methanol)of silybin is 288 nm(6). We figured out maximum uv absorbency was 287 nm of two silybin diastereomers in this study(Fig. 3).

While the silybin was isolated into two different peaks, DAD scanning showed that

their UV absorbency spectra were the same. As Ding et al.(7) suggested, it seemed that the silybin was a mixture of two different diastereoisomers at C2', C3'. They reported that the mass spectra of both substances showed the molecular peak at 433 and very similar fragmentation. Most fragments were found in each mass spectrum and differ only in intensity.

The observed linearity, recovery, and impurity indicated this method is suitable and applicable for qualitative and quanti- tative evalution of the silybin diastereomers in the compound Cardus mari anus extract, nicotinamide and riboflavin commercial products(Fig. 4).

Fig. 2. Impurity test of spiked solutions.

*SB I : silybin diastereomer I, SB II : silybin diastereomer II

2R, 3R, 2'S, 3'S

Fig. 3. Spectrum of silybin standard solutions(upper), sample solutions(middle) and spiked solutions(lower).

2R, 3R, 2'R, 3'R

Fig. 4. Structure of silybin diastereomers.

The chromatogram of the sample solutions obtained as directed in the HPLC-PDA method(Fig. 5) exhibits a major peak for silybin diastereomers, retention time of which, relative to that of external standard, corresponds to that exhibited in the chromatogram of the standard solution obtained as directed in the HPLC-PDA method. The third chromatogram was obtained by adding standards into the known sample and the retention time of it corresponded to that of standard solution, too.

3. Resolution of silybin and vitamins

According to Pharmaceutical codex second edition, the identification and assay of silybin of compound Cardus marianus extract, nicotinamide and riboflavin commercial products are based on HPLC method with aminopropylsilyl silica column, acetonitrileㆍacetic  acidㆍwater(95:2:3). Other vitamins like thiamine, nicotinamide, riboflavin, and pyridoxine are analyzed by general vitamin method, separately(8). But the determination of silybin of compound Cardus marianus extract, nicotinamide and riboflavin commer- cial products were not easy because of complicated composition. Most soft capsules have soybean oil, lecithin, beeswax, other ingredients and preservatives which contri- bute to sustain suspension state and preserve.

Up to now, main components of silymarin (silybin diastereomers, isosilybin diastereo mers, silydianin and silychristin) have been separated and determined by HPLC with UV, electrochemical or MS detection and capillary electorphoresis(7, 9-10).

There was a dissolution test for silymarin tablets and capsules(11). Maybe the preparations were single major component, silymarin. Silymarin percentage by HPLC was expressed as a sum of taxifoline, silycristine, silydianine, silybin, and isosilybin. The extraction process used moderate sonication(5 min) to avoid exposing samples to heat generated from prolonged treatment. Instead, we extracted silybin from the sample preparations by using magnetic stirring apparatus(Fisher brand). The solu- bilities of silybin were soluble in methanol, ethanol, acetone, ethyl acetate(6). We adopted methanol as many other references.

We surveyed the simultaneous analysis method of silybin and vitamins. By use of ion-pairing effect of heptane sulfonic acid, we employed gradient mobile phase changing method(Table 2). We found simultaneous identification of silybin diastereomers and

Fig. 5. Chromatograms of silybin standard solutions(above), sample solutions(middle), and spiked solutions(below), UV 287 nm,

*SB I : silybin diastereomer I, SB II : silybin diastereomer II

Fig. 6. Chromatograms of standard mixture of four vitamins and silybin sample solutions. Silymarin standard(UV 270 nm) and spectrum of silymarin.

*SB I : silybin diastereomer I, SB II : silybin diastereomer II

Table 4. Analytical results of silybin diastereomers in each preparation
Sample	Labeled amount	Content of silybin diastereomers(%) No. of experiment			Mean	RSD
1	60 mg/cap	90.1	92.5	90.9	91.2	1.3
2	60 mg/cap	90.4	93.8	92.5	92.2	1.9
3	60 mg/cap	100.7	102.9	103.4	102.3	1.4
4	60 mg/cap	88.9	90.4	92.9	90.7	2.2
5	60 mg/cap	95.1	90.9	91.1	92.4	2.6
6	60 mg/cap	115.4	110.0	114.3	113.2	2.5
7	60 mg/cap	101.8	100.9	101.5	101.4	0.5
8	60 mg/cap	104.9	110.2	109.4	108.2	2.6
9	60 mg/cap	90.7	100.1	98.5	96.4	5.2
* Areas are calculated as a sum of both diastereomers peak area.

vitamins of compound Cardus marianus extract, nicotinamide and riboflavin commercial products. Although the UV max is 287 nm of silybin, we employed 270 nm because of its wide spectrum of absorbance in vitamins. But there was some problem to determine vitamins, separately. The results of resolution of silybin diastereomers were excellent, but those of vitamins were not suitable.(Fig 6) More extraction and clean-up process should be necessary to analyze vitamins of compound Cardus marianus extract, nicotinamide and riboflavin com mercial products, absolutely. We found that the retention time of some vitamins were similar to that of some peaks in silymarin. Silymarin standard solution was analyzed by this method. Refer to Ding et al(7). We guessed that one of two peaks (peak 6 and peak 7) after silybin diastere- omers was isosilybin. Throughout this study, this HPLC-PDA method with a reversed phase Symmetry column is an analytical aid not only for the separation of other vitamins, like nicotinamide, pyridoxne, riboflavin, and thiamine and silybin, but also for determina tion of silybin (two diastereomers). We could catch a glimpse of simultaneous analysis of silybin diastereomers and vitamins of com pound Cardus marianus extract, nicotinamide and riboflavin soft capsules. Each compound Cardus marianus extract, nicotinamide and riboflavin commercial product contained more than 90.0% of the labeled amount of silybin diastereomers(C25H22O10 : 482.44) (Table 4). They all conformed to the Korean Pharma- ceutical codex second edition regulation.

Conculsion

The determination of silybin diastereomers in compound Cardus marianus extract, nicoti namide and riboflavin commercial products developed by HPLC-PDA. The 3D-PDA meth od provides a linear response and is precise. It also may be used for quality assesment and to study isolation and identification. The linearity, recovery, and impurity of silybin diastereomers indicated that this method may be used as pharma cotechnical quality indicator.

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