Research Article

Received: 26 April 2010; Revised: 9 June 2010; Accepted: 12 June 2010 Published online in Wiley Online Library: 26 August 2010

(wileyonlinelibrary.com) DOI 10.1002/pca.1244

Simultaneous Analysis of Diosgenin and Sarsasapogenin in Asparagus ocinalis Byproduct by Thin-layer Chromatography
Liwei Wang,a,b Xiaodong Wang,a Xiaofan Yuana and Bing Zhaoa*
ABSTRACT: Introduction Asparagus ocinalis L. has several biological activities including antifungal, antiviral and antitumoral activities due to the steroidal saponins. Normally diosgenin and sarsasapogenin are analysed separately by thin-layer chromatography or high-performance liquid chromatography (HPLC-UV or HPLC-ELSD), which is time-consuming and expensive, so we need to nd a rapid solution to this problem. Objective To develop a sensitive, rapid and validated TLC method for simultaneous detection and quantication of diosgenin and sarsasapogenin. Methodology Samples were prepared by extraction of A. ocinalis with 70% aqueous ethanol to get steroidal saponins, and then hydrolysed using 36 mL 2 M hydrochloric acid for 3 h. The hydrolysis product was extracted with chloroform, and then analysed by TLC, the results of which were veried by HPLC and HPLC-MS. Results The retention factor (Rf) of diosgenin and sarsasapogenin on TLC plate were 0.49 and 0.6, respectively. After calculation from the regression equation of the standard curve, the contents of diosgenin and sarsasapogenin in the A. ocinalis extract were 0.270.46 and 0.110.32%, respectively. Conclusion The study showed that thin-layer chromatography can be applied for the determination of diosgenin and sarsasapogenin in the oldest tissue of A. ocinalis, and also can be conducted for screening of sapogenin in other plant or extracts. Copyright 2010 John Wiley & Sons, Ltd. Keywords: diosgenin; sarsasapogenin; A. ocinalis byproduct; thin-layer chromatography

Introduction
Asparagus ocinalis L. is a well-known healthy vegetable that is mainly consumed for its edible shoots, which have been called the king of vegetables. In traditional Chinese medicine, it is used as a tonic, antifebrile, antitussive, hair growth stimulator and diuretic agent. Recent pharmacological studies showed that A. ocinalis extracts have several biological activities including antifungal (Shimoyamada et al., 1990), antiviral (Aquino et al., 1991), antitumoral (Shao et al., 1996), antioxidant (Sun et al., 2007), cytotoxic (Kim et al., 2005) and molluscicide activities (Sati et al., 1984). A considerable number of chemical constituents, such as avonoids and steroidal saponins, have been isolated from its roots (Zhang et al., 2004). Among all the bioactive compounds present in A. ocinalis, steroidal saponins are the major components (Jang et al., 2004). The sapogenins in A. ocinalis are mainly diosgenin and sarsasapogenin. Diosgenin is a principal starting material for the industrial production of steroidal hormones (corticosteroids and sex hormones, including oral contraceptives). Clinical research showed that the solubility and transport of biliary cholesterol are substantially inuenced by diosgenin-induced increases in biliary cholesterol output (Thewles et al., 1993). Sarsasapogenin has the eects of being antidiabetes and improving memory (Hu et al., 2005). Studies show that sarsasapogenin can induce HepG2 cell apoptosis, leading inhibition of tumour cell growth, through cell cycle arrest on G2/M (Ni et al., 2008).

Many published reports have described the determination of saponins using high-performance liquid chromatography (Pant et al., 1988). However, based on a literature search, no studies have been conducted on the simultaneous detection of diosgenin and sarsasapogenin by thin-layer chromatography (TLC). TLC, a common analytical tool for various compounds, has several advantages over HPLC in terms of cost and speed. For example, diosgenin is normally analysed on HPLC using a UV detector at 204210 nm, while sarsasapogenin has no UV absorption and is detected by evaporative light-scattering detector, which is timeconsuming and expensive. The reagents used in HPLC are of chromatography grade, and cost more money than those of analytical grade used in TLC. Therefore, this research aimed to develop a sensitive, rapid and validated TLC method for detecting these steroidal sapogenins in the oldest tissue of A. ocinalis or some

* Correspondence to: Bing Zhao, National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 100190, Beijing, Peoples Republic of China. E-mail: bzhao@home.ipe.ac.cn
a

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, 100190, Beijing, Peoples Republic of China Graduate University of Chinese Academy of Sciences, 100049, Beijing, Peoples Republic of China

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Phytochem. Anal. 2011, 22, 1417

Simultaneous Analysis of Diosgenin and Sarsasapogenin in Asparagus ocinalis extracts, which is benecial for the use of discarded product for producing sapogenin.

Table 1. Validation parameters of TLC and HPLC methods used for diosgenin quantication Parameter LOD (mg/mL) LOQ (mg/mL) Precision (RSD, %) Reproducibility(RSD, %) Stability (RSD, %) Average recovery (%) Analysis time (min) Analysis cost (RMB) TLC 4 10 0.35 1.07 0.58 98.6 10 10 HPLC 1.5 3 0.27 0.81 0.35 99.1 30 120

Experimental
Plant material and reagents
The fresh shoots of green A. ocinalis byproduct were kindly provided by Qinhuangdao Changsheng Agricultural Technology Development Co. Ltd, Hebei Province, China. The fresh samples were immediately cut into small pieces, and ground in a grinding machine to ensure that all samples were uniform, and stored below -80C. The HPLC-grade acetonitrile, deionised water and other reagents of analytical grade were purchased from Thermo Fisher Scientic Inc. Chloroform and hydrochloric acid (HCl) were of analytical reagent grade. The standard compounds of diosgenin and sarsasapogenin were purchased from the National Institute for Control of Pharmaceutical and Biological Products (NICPBP, Beijing China).

HPLC-MS analysis of steroidal saponins

The HPLC-MS was performed on an Agilent 1100 system equipped with an autosampler, a quaternary pump system, a photodiode array and multiple wavelength detector, a thermostated column compartment and a degasser, with an Agilent Zobarx SB C18 column (150 2.1 mm, 5 mm). Solvent A was 0.3% acetic acid in water, and solvent B was 0.3% acetic acid in acetonitrile. The gradient was 010 min, 95100% B; 1020 min, 100% B at a ow rate of 0.2 mL/min. The identication of diosgenin and sarsasapogenin was performed on electrospray ionisation mass spectrometry (ESI-MS) (LCQ DaceXP, Thermo electron, San Jose, CA, USA). The spray voltage was 4.5 kV and the heat capillary was kept at 300C. The scan range was set from m/z 400 to 500. The zoomscan and tandem MS (MS/MS) functions were performed in data-dependent mode. The collision-energy value was 35%. LC-MS and LC-MS/MS in positive ion mode were applied.

Saponin extraction
The shoots of A. ocinalis were extracted by percolation once with a 2.5 L volume of 70% aqueous ethanol at room temperature for 24 h, then sonicated three times at pH 5.0 (3 2.5 L) and the combined extraction solution was transferred to the centrifuge tube and centrifuged at 5000 rpm for 10 min, so that the saponin extract was obtained (Huang and Kong, 2006; Mandal et al., 2006). This extract was then concentrated to dryness by removing the solvent in the rotary evaporator (Shanghai Ya Rong Biochemistry Instrument Factory, Shanghai, China) under reduced pressure.

Acid hydrolysis of steroidal saponins

Saponins (0.8 g) were hydrolysed by 36 mL 2 M hydrochloric acid for 3 h. After cooling, the acid hydrolysis solution was neutralised with 40% sodium hydroxide, and then the hydrolysis product was extracted with chloroform (3 20 mL; Huang and Kong, 2006). The chloroform extracts were combined and removed by evaporation, and concentrated to dryness at 60C. The residue was dissolved in methanol and then chromatographed on silica gel for TLC analysis in comparison with the standards.

Method validation of quantitative analysis

The method was validated in terms of linearity, limits of detection (LOD) and quantication (LOQ), precision, repeatability, stability and recovery test. The results are listed in Table 1.

Calibration curves, LOD and LOQ

The calibration curves were constructed by plotting the peak areas vs the concentration of each analyte. The working solutions of the analytes were further diluted with methanol to yield a series of appropriate concentrations. LOD and LOQ were evaluated on the basis of signal-to-noise ratios of 3 and 10, respectively.

TLC analysis of steroidal saponins

Thin-layer chromatography was carried out on plates precoated with silica gel GF254 (50 100 mm, Yantai Institute of Chemical Industry). The samples were developed using benzeneacetone (9: 1) as the eluent system, dried for complete removal of solvents and detection was achieved with a mixture of ethanol (8% vanillin) and sulfuric acid solution (70%) with the ratio of 0.5:5 (National Pharmacopoeia Committee, 2005). The areas of the spots on the plate were integrated using a KH-3000 TLC-densitometer (Shanghai Kezhe Biochemistry Technological Inc., China) after a full wavelength scanning from 370 to 650 nm. For every sample the procedure was repeated three times. Chromatograms were evaluated by a TLC Scanner at 464 and 452 nm to detect the presence of diosgenin and sarsasapogenin against a blank of 620 and 550 nm, respectively. Mobility of compounds is expressed as retention factor (Rf) values (Rf = distance moved by compound/distance moved by solvent front) (Lapenna and Dinan, 2009).

Precision
Precision of the method was assessed by six replicate analysis of the same concentration of standard solution, and expressed as relative standard deviation (RSD).

Reproducibility and stability

To test the reproducibility of the assay, six independently prepared samples of the hydrolysate of A. ocinalis extract in parallel were prepared and analysed. The stability of the samples was analysed every 2 h within 24 h at room temperature. Variations were expressed as RSD.

Recovery test HPLC analysis of steroidal saponins

HPLC analysis of diosgenin was performed on a Shimadzu 20A system equipped with a C18 column (4.6 250 mm, 5 mm). The solvent system consisted of (A) methanol and (B) water, using a ratio of 95:5 in 20 min at 30C, with a ow rate of 0.8 mL/min. Diosgenin was detected on a UV detector at 204 nm. Recovery was assessed by the method of standard additions. An accurately known amount of the standard solution was added to the known sample and then extraction and analysis were done as described above. Recovery was counted according to the following formula: recovery (%) = (amount found - original amount)/amount spiked 100% (Wei et al., 2010).

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L. Wang et al.

Table 2. The contents of diosgenin and sarsasapogenin in samples from three different places of China Sample no. 1 2 3 Producing area Herbei-A Herbei-B Yunnan Diosgenin content (%) 0.27 0.31 0.46 Sarsasapogenin content (%) 0.19 0.11 0.32

Figure 2. The densitogram of diosgenin (A), sarsasapogenin (B) and the A. ocinalis samples (C). Figure 1. The hydrolysate of saponin in A. ocinalis byproduct: 1, nonhydrolysate of A. ocinalis byproduct extract; 2, standard of sarsasapogenin; 3, hydrolysate of A. ocinalis byproduct extract; 4, standard of diosgenin.

Sample analysis
The method was subsequently applied to simultaneous determination of the two compounds in samples collected from three dierent places of China. All samples were analysed using the optimised extraction and hydrolysis method under optimised TLC and HPLC conditions. The results are shown in Table 2.

Results and Discussion

The TLC results showed the presence of free diosgenin and sarsasapogenin in the chloroform extracts obtained from acid hydrolysis of A. ocinalis byproduct extract, in comparison with authentic samples (Fig. 1). There have been no previous reports of the simultaneous analysis of diosgenin and sarsasapogenin in A. ocinalis byproduct using the same TLC method. According to chromatography in a thin-layer of silica gel, two spots in the hydrolysate of A. ocinalis have a similar colour to the standards of diosgenin and sarsasapogenin, and the retention factors (Rf) of the two spots are 0.49 and 0.6, respectively, which are also the same as the standards. The densitograms of the samples and the standards are shown in Fig. 2. Linearity was observed between concentrations and areas integrated by TLC densitometry (Kivak and Mert, 2001). The TLCdensitometric calibration curve, used to calculate the diosgenin content in the extracts, was expressed by the following linear equation: y = 0.7931x + 2.382 10-13; R2 = 0.9979, where y is the integration unit and x is the diosgenin concentration (mg/mL).

Similarly, the TLC-densitometric calibration curve, used to calculate the sarsasapogenin content in the extracts, was expressed by the following linear equation: y = 0.98665x + 2.382 10-13; R2 = 0.9952, where y is the integration unit and x is the sarsasapogenin concentration (mg/mL). The diosgenin and sarsasapogenin contents in the extracts were calculated from the regression equations of the standard curve. For testing the quantitative accuracy of the TLCdensitometric method, the analyses of the reference substance and of the extracts were repeated on three dierent plates. Finally, the contents of diosgenin and sarsasapogenin in the A. ocinalis extract were 0.270.46 and 0.110.32%, respectively. In order to further identify the structures of the two similar spots above, we use HPLC-MS to analyse the hydrolysate of A. ocinalis extract (Mathias and Halkar, 2004). The results are shown in Fig. 3, and are the same as those for diosgenin and sarsasapogenin, respectivey. The structures of diosgenin and sarsasapogenin are given in Fig. 4, and we can see that there is only a small dierence of one bond, and the molecular weights are 414 and 416, respectively. By comparing the dierence between A and B in Fig. 3, we could conclude that the hydrolysate of A. ocinalis extract contains some trace of diosgenin and sarsasapogenin. The results in Table 1 indicate that the TLC method is precise, accurate and sensitive enough for simultaneous quantitative evaluation of the diosgenin and sarsasapogenin compounds in the hydrolysate of A. ocinalis. During the preparation for acid hydrolysis of steroidal saponins, it is necessary to control the quality of the nished and intermediate products, which suggests that we should optimise the hydrolysis conditions. The nal acid hydrolysis of steroidal saponins was 0.8 g saponins hydrolysed by 36 mL 2 M HCl for 3 h.

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Simultaneous Analysis of Diosgenin and Sarsasapogenin in Asparagus ocinalis

Figure 3.

HPLC analysis of diosgenin (A) and sarsasapogenin (B) in the hydrolysate of A. ocinalis extract.

Figure 4.

Structure of diosgenin and sarsasapogenin (B).

The colour gradually faded as the plate cooled, and after 12 h, spots initially yellow appeared yellow-brown, and then dark brown. Although colour reactions on TLC are not in themselves adequate for compound identication, they do, in conjunction with Rf values, provide good initial indicators of compound identication. Thousands of tons of high-quality A. ocinalis parts are usually rejected as trimmings during canning and fresh packing operations. Studies have shown that the oldest tissue is a rich source of protodiscin (Wang et al., 2003). Here, we also found that this part of the plant is rich in diosgenin and sarsasapogenin, which are usually found in fresh stems (Huang and Kong, 2006; Sun et al., 2010). The diosgenin and sarsasapogenin in the extract of oldest part of the A. ocinalis were analysed by the same TLC methods, and were characterised by HPLC-MS, which suggested that the lower portions of the A. ocinalis shoots that are discarded should instead be considered a promising source of a new valueadded nutraceutical product. This work demonstrates that the proposed TLC densitometry methods are satisfactory for quantication of sapogenin. Acknowledgements We thank Professor Yuchun Wang, Institute of Process Engineering, Chinese Academy of Sciences, for the valuable advice. We also thank Qinhuangdao Changsheng Agricultural Technology Development Co. Ltd for kindly providing the A. ocinalis materials.

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