Journal of Chromatography B, 974 (2015) 75–82
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Structural characterization of steroidal saponins from Smilax trinervula using ultra high-performance liquid chromatography coupled with LTQ-Orbitrap mass spectrometry Yonghong Liang a , Shun Yao b , Fang Liang a , Jian Liang a , Guangyu Huang a , Min Liu a , Huilian Huang a,∗ a b
Key Laboratory of Modern Preparation of TCM, Jiangxi University of Traditional Chinese Medicine, Ministry of Education, Nanchang 330004, China Department of Pharmaceutical and Biological Engineering, Sichuan University, Chengdu 610065, China
a r t i c l e
i n f o
Article history: Received 7 May 2014 Accepted 27 September 2014 Available online 24 October 2014 Keywords: Steroidal saponins Smilax trinervula UHPLC/HRMS LTQ-Orbitrap mass
a b s t r a c t Steroid saponins are a class of naturally existing substances widely distributed in the plants of Smilacaceae. Their biological activities have been attracting the interest of scientists in the chemical field for the past few years. To our best knowledge, there has been no study on structural characterization of steroidal saponins from Smilax trinervula (S. trinervula) using LTQ-Orbitrap mass spectrometry, which could provide an excellent approach for rapid screening of steroidal saponins in other plants of Smilacaceae. An ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC/HRMS) method was therefore developed to characterize steroidal saponins in S. trinervula. This method was operated in both negative and positive ion modes with HRMS, as a result, a total of twenty-two steroidal saponins with three aglycone skeletons were elucidated in the crude extract from the root of S. trinervula. The characteristic-fragment ions could well identify and differentiate the three types of aglycone skeletons (diosgenyl saponins, furostanol saponins, C27 -hydroxy diosgenyl saponins). © 2014 Elsevier B.V. All rights reserved.
1. Introduction The saponins, classified as steroids or triterpenoid saponins depending upon the nature of the aglycone, are widely distributed in the plant kingdom. The name ‘saponin’ is derived from the saponin molecules that form soap-like foams when shaken with water. Steroid saponins exist in Dioscoreaceae, Smilacaceae, Liliaceae and Scrophulariaceae in general and can be subcategorized into spirostanol, isospirostanol, furostanol, pseudospirostanol and cholestanol saponins on skeletal basis [1]. Steroid saponins were reported to possess a variety of bioactivities, such as cytotoxic, antitumor, cardiac, antifungal, hemolytic and immunoregulatory activities. Sapogenins, such as diosgenin, are the starting material of industrial interest in the synthesis of many steroids which are on the market as anti-inflammatory, androgenic, estrogenic, and contraceptive drugs [2]. It is a well-known fact the Smilacaceae family is rich in steroidal saponins. This family comprises about 370 species that are mainly
∗ Corresponding author. Tel.: +86 79187118658; fax: +86 79187118658. E-mail address: [email protected] (H. Huang). http://dx.doi.org/10.1016/j.jchromb.2014.09.040 1570-0232/© 2014 Elsevier B.V. All rights reserved.
distributed in the tropical and temperate zones throughout the world, especially in East Asia and North America [3]. Steroidal saponins are widely recognized as the ‘marker compounds’ in the plants of Smilacaceae, because they have been isolated and identified from the family [4–9]. Smilax trinervula (S. trinervula), a perennial plant widely distributed in South China, such as Jiangxi, Guizhou and Anhui Province, has been being used to eliminate dampness and treat pelvic inflammation and chronic pelvic inflammation as in traditional Chinese medicine (TCM). In previous papers, isolation and structural determination of some new steroidal saponins, phenylpropanoid substituted catechins and epicatechins from Smilax china have been reported [10,11]. To our best knowledge, there is no report on the constituents of S. trinervula. It is known to all that nuclear magnetic resonance is the most accurate method for complete elucidation of chemical compounds; nevertheless, isolation and purification is a timeenergy-and-labor-consuming job, especially for trace compounds. The availability of effective methods for the structural characterization and identification of steroidal saponins in S. trinervula is therefore of significant importance. UHPLC–MS, combining the efficient separation capabilities of UHPLC with the great power
76
Y. Liang et al. / J. Chromatogr. B 974 (2015) 75–82
in structural characterization of MS, is ultimately used to identify target compounds by comparing retention time, UV shift, molecular weight and fragmentation data. The UHPLC–MS method provided a new powerful approach to identify the most abundant constituents in plant extracts, rapidly and accurately. Recently, UHPLC-PDA-ESI/HRMS/MSn method has been successfully applied to the identification and characterization of target compounds in various fields [12,13]. The combination of Orbitrap technology with a linear ion trap has achieved fast, sensitive and reliable detection and identification for small molecules regardless of relative ion abundance [14]. This hybrid configuration offers greatly strengthened selectivity and suitability of different scan types according to different data requirements. Furthermore, with the external calibration, high mass accuracy of all measured spectra could be ensured, which results in a simplified experimental protocol. Orbitrap consists of an outer barrel-like electrode and a coaxial inner spindle-like electrode that forms an electrostatic field with quadro-logarithmic potential distribution [15,16]. It provides a high mass accuracy (
Contents lists available at ScienceDirect
Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Structural characterization of steroidal saponins from Smilax trinervula using ultra high-performance liquid chromatography coupled with LTQ-Orbitrap mass spectrometry Yonghong Liang a , Shun Yao b , Fang Liang a , Jian Liang a , Guangyu Huang a , Min Liu a , Huilian Huang a,∗ a b
Key Laboratory of Modern Preparation of TCM, Jiangxi University of Traditional Chinese Medicine, Ministry of Education, Nanchang 330004, China Department of Pharmaceutical and Biological Engineering, Sichuan University, Chengdu 610065, China
a r t i c l e
i n f o
Article history: Received 7 May 2014 Accepted 27 September 2014 Available online 24 October 2014 Keywords: Steroidal saponins Smilax trinervula UHPLC/HRMS LTQ-Orbitrap mass
a b s t r a c t Steroid saponins are a class of naturally existing substances widely distributed in the plants of Smilacaceae. Their biological activities have been attracting the interest of scientists in the chemical field for the past few years. To our best knowledge, there has been no study on structural characterization of steroidal saponins from Smilax trinervula (S. trinervula) using LTQ-Orbitrap mass spectrometry, which could provide an excellent approach for rapid screening of steroidal saponins in other plants of Smilacaceae. An ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC/HRMS) method was therefore developed to characterize steroidal saponins in S. trinervula. This method was operated in both negative and positive ion modes with HRMS, as a result, a total of twenty-two steroidal saponins with three aglycone skeletons were elucidated in the crude extract from the root of S. trinervula. The characteristic-fragment ions could well identify and differentiate the three types of aglycone skeletons (diosgenyl saponins, furostanol saponins, C27 -hydroxy diosgenyl saponins). © 2014 Elsevier B.V. All rights reserved.
1. Introduction The saponins, classified as steroids or triterpenoid saponins depending upon the nature of the aglycone, are widely distributed in the plant kingdom. The name ‘saponin’ is derived from the saponin molecules that form soap-like foams when shaken with water. Steroid saponins exist in Dioscoreaceae, Smilacaceae, Liliaceae and Scrophulariaceae in general and can be subcategorized into spirostanol, isospirostanol, furostanol, pseudospirostanol and cholestanol saponins on skeletal basis [1]. Steroid saponins were reported to possess a variety of bioactivities, such as cytotoxic, antitumor, cardiac, antifungal, hemolytic and immunoregulatory activities. Sapogenins, such as diosgenin, are the starting material of industrial interest in the synthesis of many steroids which are on the market as anti-inflammatory, androgenic, estrogenic, and contraceptive drugs [2]. It is a well-known fact the Smilacaceae family is rich in steroidal saponins. This family comprises about 370 species that are mainly
∗ Corresponding author. Tel.: +86 79187118658; fax: +86 79187118658. E-mail address: [email protected] (H. Huang). http://dx.doi.org/10.1016/j.jchromb.2014.09.040 1570-0232/© 2014 Elsevier B.V. All rights reserved.
distributed in the tropical and temperate zones throughout the world, especially in East Asia and North America [3]. Steroidal saponins are widely recognized as the ‘marker compounds’ in the plants of Smilacaceae, because they have been isolated and identified from the family [4–9]. Smilax trinervula (S. trinervula), a perennial plant widely distributed in South China, such as Jiangxi, Guizhou and Anhui Province, has been being used to eliminate dampness and treat pelvic inflammation and chronic pelvic inflammation as in traditional Chinese medicine (TCM). In previous papers, isolation and structural determination of some new steroidal saponins, phenylpropanoid substituted catechins and epicatechins from Smilax china have been reported [10,11]. To our best knowledge, there is no report on the constituents of S. trinervula. It is known to all that nuclear magnetic resonance is the most accurate method for complete elucidation of chemical compounds; nevertheless, isolation and purification is a timeenergy-and-labor-consuming job, especially for trace compounds. The availability of effective methods for the structural characterization and identification of steroidal saponins in S. trinervula is therefore of significant importance. UHPLC–MS, combining the efficient separation capabilities of UHPLC with the great power
76
Y. Liang et al. / J. Chromatogr. B 974 (2015) 75–82
in structural characterization of MS, is ultimately used to identify target compounds by comparing retention time, UV shift, molecular weight and fragmentation data. The UHPLC–MS method provided a new powerful approach to identify the most abundant constituents in plant extracts, rapidly and accurately. Recently, UHPLC-PDA-ESI/HRMS/MSn method has been successfully applied to the identification and characterization of target compounds in various fields [12,13]. The combination of Orbitrap technology with a linear ion trap has achieved fast, sensitive and reliable detection and identification for small molecules regardless of relative ion abundance [14]. This hybrid configuration offers greatly strengthened selectivity and suitability of different scan types according to different data requirements. Furthermore, with the external calibration, high mass accuracy of all measured spectra could be ensured, which results in a simplified experimental protocol. Orbitrap consists of an outer barrel-like electrode and a coaxial inner spindle-like electrode that forms an electrostatic field with quadro-logarithmic potential distribution [15,16]. It provides a high mass accuracy (
