Quantitative analysis of silybin A and B in dry extract of milk thistle (Silybum marianum) by UPLC/UV-Vis method

Objectives: To establish an UPLC/UV-Vis method that is suited with current conditions of the laboratory to quantify silybin A and B in dry extract of milk thistle. Materials and methods: Parameters of the method are validated.

QUANTITATIVE ANALYSIS OF SILYBIN A AND B IN DRY

EXTRACT OF MILK THISTLE (Silybum marianum)

BY UPLC/UV-VIS METHOD

Dang Truong Giang*; Vu Tuan Anh*

Hoang Kim Vuong Nam**; Chu Van Men*

SUMMARY

Objectives: To establish an UPLC/UV-Vis method that is suited with current conditions of the laboratory to quantify silybin A and B in dry extract of milk thistle Materials and methods: Parameters of the method are validated including: linear range, precision, accuracy Results: Chromatographic conditions of the method: using Cortecs UPLC BEH column (C 18 ; 1.6 µm, 2.1

x 50 mm), acetonitrile and 0.1% aqueous formic acid as mobile phase following a multistep gradient program, monitored at 320 nm The established method has good precision (RSD = 2.37%) and accuracy (recovery ratio = 97.08 - 103.76%, average = 100.38%, RSD = 2.84%) Conclusions: UPLC/UV-Vis method for quantifying silybin A and B in dry extract of milk thistle has been developed and validated

* Keywords: Milk thistle; Silybin A, B; HPLC

INTRODUCTION

Extract from the milk thistle seeds

(Silybum marianum) is being used as a

herbal therapy for hepatotoxicity and

acute and chronic liver diseases [1, 2]

The pharmacological active ingredient is

the flavonoid complex called silymarin,

the main constituent accounts for about

80% of the extract [3] Silymarin is a

complex of at least seven flavonolignans

that are the most common class of

compounds present in milk thistle extract

and one flavonoid, taxifolin The relative

abundance of each compound may vary

depending on the sources of botanical

materials, suppliers and extraction

processes Silybin represents about 50%

to 70% of the silymarin extract Silybin

(figure 1), a stabilizer of liver cell membrane,

has the effect of protecting the membrane

of liver cells and enhancing its function and also protect the liver function from deterioration resulting from the invasion

by deleterious substances [4] Thus, silybin

is considered as active compound of milk thistle extract Silybin can be resolved into two 1:1 diastereoisomers, silybin A and silybin B The concentration of silybin in the main pharmaceutical products contain silymarin present in the US and other countries ranging from 20% to 40% [5]

Up to now, Vietnamese Pharmacopoeia hasn’t contained any monograph for quality control of dry extract of milk thistle

In this study, we have developed and validated a short UPLC/UV-Vis method for the analysis of silybin A and B in dry extract of milk thistle

* Vietnam Military Medical University

** College of Pharmacy, Lac Hong University

Corresponding author: Chu Van Men (chuvanmen@gmail.com)

Date received: 30/06/2017 Date accepted: 10/08/2017

Figure 1: Chemical structure of silybin A (1a) and B (1b)

MATERIALS AND METHODS

1 Materials and equipments

- Materials: Dry extract of milk thistle

was purchased from Dongtai Kangning

Vegetable Extraction Co., LTD (China), in

December 2016

- Solvents: acetonitrile (ACN) (Merck,

HPLC grade), 0.1% aqueous formic acid

(Merck, analytical grade); methanol

(MeOH) (Merck, HPLC grade);

double-distilled water was prepared using

Hamilton water still system (Hamilton

Laboratory Glass Ltd, UK)

- Chemical: Silybin standard (racemic

mixture of silybin A and B with 93.5%

purity) was purchased from National Drug

Quality Control

- Equipment: Acquity UPLC H-Class system including Acquity Autosampler, Quaternary solvent Manager, column oven and Empower sofware), the Acquity UPLC BEH column (C18; 1.7 µm, 2.1 x 100 mm) was used for the separation; Elmasonic Sultrasonic (Elma), Mettler Toledo MS205DU

2 Methods

- Chromatographic conditions:

+ Detector PDA: detection wavelength was set at 280 nm

+ Flow rate: 0.3 mL/minute

+ Injection volume: 5 µL

+ Column temperature: 25oC

+ A multistep gradient program of mobile phase as shown in table 1

Table 1: Gradient condition for analysis of silybin in dry extract of milk thistle

- Sample preparation: 10 mg of dry extract of milk thistle was ultrasonically extracted with about 25 mL of methanol in a volumetric flask of 25 mL for 15 minutes Methanol was added to volumetric mark After that, this solution was filtered through a 0.45 µm filter membrane and injected into the UPLC system for analysis

- Standard solution preparation: Dissolving and diluting an accurate amount of silybin standard in methanol by using volumetric flasks and Eppendorf micropipets to obtain a suitable range of working standard solutions

- Method validation: The analysis method was fully validated in accordance to ICH guideline [6]

RESULTS AND DISCUSSIONS

1 System suitability

For evaluating system suitability, a standard solution was injected 6 times repeatedly into the UPLC system [6] Results are shown in table 2 and chromatograms

of standard and sample solutions were shown in figure 2

Table 2: Results of system suitability for analysis of silybin

RSD = 0.92%

S = 386,267 RSD = 0.90%

RSD = 0.10 %

t R = 12.99 RSD = 0.09%

Figure 2: Chromagrams of standard silybin (a) and dry extract of milk thistle sample (b);

1: silybin A; 2: silybin B

The results in table 2 show that the

repeatability of peak areas and retention

times are good (both RSD < 2%) Peaks

of silybin A and B are sharp and

proportional (tailing factor for sylibin A and

B were 0.98 and 1.06, respectively)

Theoretic plate number for sylibin A and B

are 11,952 and 10,923, respectively

Therefore, the chromatographic system is

suitable for analyzing samples

2 Specificity

Retention times of the peak in the

chromatogram of sample solution and in

the chromatogram of standard solution are coincident At retention times of each peak of silybin A and B in standard and sample and correlation coefficient of silybin A and B UV spectra in standard and sample were more than 0.999 These results prove that the established method has good specificity

3 Linear range and calibration curves

For evaluating the linear range of the established method, 6 concentrations of silybin A and B were analyzed [6] Results are showed in table 3 and figure 3

Table 3: Correlation between peak areas and concentrations of silybin A and B

Peak area (µV*s)

The results of figure 3 show linear correlation between peak areas and concentrations of silybin A and B following the regression equation Y = 15042.7 X + 1248.35 and Y = 16786.1X - 449.78, respectively with correlation coefficient of 0.9999 for both compounds

4 Precision

Precision of the established method is evaluated based on the repeatability of 6 individual experiments quantifying silybin A and B in the raw materials [6] Results are showed in table 4

Table 4: Precision of the analytical method for silybin A and B

The results in table 4 show the established method has good repeatability with all RSDs less than 2.19% The content of silybin A and B in dry extract of milk thistle are 32.14 mg/g and 62.55 mg/g, respectively

5 Accuracy

Accuracy of the established method is evaluated by spiking an exact amount of silybin A and B into the dry extract sample of milk thistle Then, determine recovery ratio by comparing and recovery amount to the spiked amount of silybin A and B [6] Samples were injected 6 times repeatedly into the UPLC system Results are showed

in table 5

Table 5: Accuracy of the analytical method for silybin A and B

No

The results in table 5 reveal that the established method has good accuracy with recovery ratio = 96.09 - 99.89%, RSDs less than 1.60%

6 LOD and LOQ

By diluting standard solutions of silybin

A and B and analyzing with the UPLC

system, LOD and LOQ of the established

method were determined based on signal

to noise [6] LOD was about 0.31 µg/mL

and LOQ was about 0.93 µg/mL

CONCLUSION

A suitable method had been developed

and validated for quantifying silybin A and

B in dry extract of milk thistle The

established method has good precision

and accuracy This study also provided

valuable information for the content of

silybin A and B in our dry extract of milk

thistle being about 32.14 mg/g and 62.55

mg/g, respectively

ACKNOWLEDGEMENT

This research was supported by

“National Program KC.10/16-20” under

grant number KHCN-KC.10.12/16-20

REFERENCES

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Milk thistle (Silybum marianum) for the

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Silybin dihemisuccinate protects rat erythrocytes

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1996