Chromatographic determination of siphonodin content: A rapid and simple strategy for discriminating between Hemsleya omeiensis and other sources of Xuedan

Purpose: To develop a rapid and simple siphonodin content-based high performance liquid chromatography (HPLC) method to distinguish Hemsleya omeiensis from other sources of xuedan. Methods: Siphonodin was isolated from Hemsleya omeiensis and identified by x-ray crystallographic analysis. An optimized HPLC method was applied for the determination of siphonodin contents of H. omeiensis, H. dolichocarpa and H. gigantha. Results: Siphonodin was successfully separated by the optimized HPLC method in < 10 min, and the results of validation showed that the HPLC method was stable and very accurate for the quantification of siphonodin. The mean content of siphonodin in 10 batches of H. omeiensis was 3.78 mg/g, but the compound was not detectable in H. dolichocarpa and H. gigantha using the developed HPLC method. Conclusion: These results indicate that the developed HPLC method is suitable for distinguishing H. omeiensis from other sources of xuedan.


INTRODUCTION
It is known that herbal medicines are beneficial for protecting humans from diseases, and they are important sources of new drugs for the cure and prevention of diseases [1,2]. In Chinese folk medicine, xuedan is a traditional herbal medicine used for clearing heat and removing toxins. It has for long been widely used in clinics as a folk and conventional herb for the treatment of ulcers, bacillary dysentery and all kinds of inflammations [3,4]. Xuedan is derived mainly from three varieties of Hemsleya: Hemsleya omeiensis, Hemsleya gigantha and Hemsleya dolichocarpa. Compared with other varieties, Hemsleya omeiensis is not bitter, and it is associated with better clinical compliance and high price. Several studies have reported appreciable differences in compositions among the three sources of xuedan, especially in their bitter taste [5][6][7][8]. However, it is usually difficult to distinguish them in morphology when these medicinal herbs are dried. Previous studies revealed that xuedan contains chikusetsu saponin IVa, hemslosides Ma 1 , hemslosides G 1 , hemslosides H 1 , dihydrocucurbitacin F-25-O-acetate, dihydrocucurbitacin F and oleanolic acid-3-O-a-L-arabinopyranosyl glucurropyranoside E [7]. Siphonodin [4-hydroxymethyl-2(5H)-furanone] is a natural compound found in a variety of Euonymus leaves and small nest moths, and it has also been isolated from Rambutan seeds and H. Ellipsoidea [9][10][11]. It is one of the 2(5)furanone compounds structurally similar to the bacterial quorum sensing system signaling molecule N-acyl homoserine lactone (AHL), and it is widely studied as bacterial quorum sensing inhibitor [12][13][14]. Previous studies reported that siphonodin exerted bacterial biofilm inhibitory activity against Pseudomonas aeruginosa, and that its structure can be modified [15]. Consequently, the aim of this study was to develop a rapid and simple siphonodin contentbased HPLC method for distinguishing H. omeiensis from other sources of xuedan. Fresh, whole tubers were cut into slices and oven-dried at 55 o C. The dried tubers were ground with a universal high-speed milling machine (Bingdu Electric Co. Ltd, Shanghai, China), and then sieved through a 65 mesh prior to use in subsequent experiments. The water used was purified with a UPH-1-10T model water purifier system (Chengdu ultrapure Technology Co., Ltd, China). All other chemicals were of HPLC or reagent grade. Silica gel (200 -300 mesh, and 60 -80 mesh) were supplied by Qingdao Marine Chemical Corporation, China.

Sample purification
For thin layer chromatography (TLC) analysis, powdered tuber (2 g) was dissolved with 20 mL of methanol in a tightly sealed conical flask. It was then subjected to ultrasonic processing for 40 min, and filtered. Thereafter, 10 mL of the filtrate was concentrated to 4 mL. For HPLC analysis, powdered tuber (0.2 g) was weighed precisely and placed in a weighed 50 mL conical flask to which HPLC-grade methanol (25 mL) was added, and the flask and contents were subjected to ultrasonic treatment for 30 min. On cooling and weighing, some methanol (MeOH) was added, with shaking, and the solution was through 0.45 m polytetrafluoroethylene filter before injection.

TLC analysis
The TLC separations were performed on G plates (Qingdao Marine Chemical Corporation, China). The sample solutions (5 L) were applied at the origin on the TLC plate, and developed with petroleum ether/ethyl acetate/methanol at a volume ratio of 5:1:0.5. The developed plates were air-dried and the spots were made visible by spraying with 2 % vanillic aldehyde in 10 % ethanol sulfate solution, followed by heating at 105 °C for 2 min. The spots were visualized at 365 nm in a UV trans-illuminator (Baoshan Gucun Electro Optic Instrument Factor, Shanghai, China).

Instrument and chromatographic conditions
The HPLC separations were carried out on a Shimadzu LC-2030C3 D model instrument (Shimadzu Corp., Kyoto Japan) using a reversephase Shim-pack GIST C 18 (250 × 4.6 mm, 5 m) column (Shimadzu Corp., Kyoto Japan) at a column temperature of 30 °C. The samples were eluted that using a gradient program of the mixture of acetonitrile and 0.1 % phosphoric acid (Table 1). The flow rate was 0.8 mL/min, and the sample injection volume was 5 L. The UV detection wavelength was set at 210 nm.

Preparation of standard solution
The standard solution was prepared with siphonodin dissolving in methanol to a final concentration of 0.2224 mg/mL.

Method validation
The method validation contains for linearity and range, limit of detection (LOD) and limit of quantification (LOQ), accuracy, precision, and recovery test [16].

Linearity assessment
Linearity of the method was obtained by the determination of the peak area of the same reference solution with sample sizes of 1, 2, 3, 5, 7, 9 and 10 μL. Calibration curves were constructed by plotting the peak areas against the corresponding concentrations of the compound. The regression equation was calculated by using the reference quantity of the control sample as the horizontal axis (x), and the peak area of the chromatogram as the ordinate (y).

Precision assessment
The precision was obtained by injecting the same reference solution of 5μl sample size 6 times into the HPLC column. From the areas obtained, the % relative standard deviation (RSD) value for siphonodin was calculated.

Repeatability
The repeatability was obtained from six copies of determinations of a 5-μL sample solution (SI) from H. Omeiensis. The RSD was calculated based on the mass fraction of siphonodin.

Solution stability
Stability was tested with one sample solution stored at room temperature at several time points (0, 3, 6, 9, 12, 15, 18 and 24 h) after preparation, and the RSD values were calculated. The sample size used was 5 μL.

Recovery test
Six samples (S6, 0.1 g) of H. omeiensis, determined already, were weighed precisely. Then, 1 mL reference solution in which the concentration of the siphonodin was 2.251 mg/mL was added to each sample. Using the above method of sample preparation to make one concentration level (100 %), the peak areas of mixed reference solutions were determined, with sample size of 5 μL, and the recovery was obtained. The average recovery was estimated as percentage of analyte.

Limits of detection and of quantification
The LOD and LOQ values were the corresponding concentrations when the signal-tonoise ratio was 3:1 and 10:1.

Identification of compounds
Siphobodin: C 5 H 6 O 3 , light yellow crystal (petroleum ether). The absolute configurations of this compound were identified by X-ray crystallographic analysis (Figure 2). Comparing the published physical and NMR data, the compound was siphonodin [4-hydroxymethyl-2(5H)-furanone] [17]. This structurally simple butenolide was first isolated from H. omeiensis while it was reported that siphonodin in the

Precision
In the precision test, the RSD value of the peak area of the siphonodin was 0.33 %, which indicated that the instrument had high precision.

Repeatability
In the repeatability test, the RSD values of each component was less than 0.99 %, indicating that the method had good repeatability.

Solution stability
In the stability test, the RSD value of siphonodin was 0.64 %. The solution stability results showed that peak area of siphonodin remained approximately unchanged up to 24 h: no significant degradation was observed within the indicated period.

Recovery
In the recovery test, average recovery was 96.50 % and RSD value was 1.34 %, as shown in Table 2.

Limits of detection and of quantification
The LOD and LOQ for siphonodin were estimated at signal-to-noise ratios of 3:1 and 10:1 as 0.0741 ng and 0.2964 ng, respectively.

Content of siphonodin in three sources of Xuedan
The results of analysis of 30 batches of samples in the three sources of xuedan revealed that siphonodin was detected only in H. omeiensis, while there was no chromatographic evidence of siphonodin in H. gigantha and H. dolichocarpa ( Figure 3). Thus, the new HPLC-UV method for determination of siphonodin would be useful for discriminating between H. omeiensis and other sources of xuedan.
Under the above chromatographic conditions, chromatographic peaks of the sample solution and reference solution had the same retention time. The degree of separation of siphonodin in all samples was greater than 1.5, the theoretical plates were greater than 60000, and the method gave good specificity.  The quantitative results on siphonodin from the 30-batch samples in the HPLC-PDA assays are shown in Table 3. In H. omeiensis, the average content of siphonodin was 3.78 mg/g. However, the contents of siphonodin in H. dolichocarpa and H. gigantha were 2.02 x 10 -3 and 1.56 x 10 -3 mg/g, respectively, and were approximately below the limit of quantitation. Therefore, siphonodin content could be a marker for identification of H. omeiensis.

DISCUSSION
In a previous study, 25-O-acetyl-23,24dihydrocucurbitacin F and 23, 24-dihydrocucurbitacin F were identified as responsible for the bitter taste in xuedan [18]. These cucurbitacins are usually used as markers of quality control, and for distinguishing between various xuedans [19]. However, these compounds are not suitable markers for H. omeiensis. From the results obtained in this study, the light green spot with Rf 0.53 was set as a specific marker for the identification of H. omeiensis. This was the basis of subsequent guided isolation using TLC analysis.

Conflict of interest
No conflict of interest is associated with this study.

Authors' contribution
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors. QWH is corresponding author on the study. YQ was first author and responsible for collecting materials, doing experiment, writing the paper. JLS and JW edited in the article pictures. HLZ and QWH analyzed the article and made recommendations. RCY provided samples. All authors read and approved the final manuscript.

Open Access
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