快速制备液相色谱分离库拉索芦荟中10-羟基芦荟大黄素苷

丁雯静，吴小芳，钟佳胜，董银卯，王巧娥，万金志*

1中山大学药学院药物分析实验室，广州 510006;2 中国热带农业科学院分析测试中心;3海南省热带果蔬产品质量安全重点实验室，海口 571101;4北京工商大学理学院北京市植物资源研究开发重点实验室，北京 100048

Introduction

Aloe vera(Aloe barbadensis Mill)is a member of Asphodelaceae (Liliaceae)family which is widely distributed in Europe，Asia and southern parts of North America［1］.It has long been used as ingredients of food products，beverages，cosmetics and pharmaceuticals due to various biological properties and these biological activities lead to the studies of its composition［2］.Among its previously investigated chemical components，anthroneis the main secondary metabolites detected in A.vera［3］.Aloin，a common anthrone，is found in various aloe genuses and occurs as a mixture of the two diastereoisomeric aloin A and aloin B.As oxidation products of aloins，10-hydroxyaloins were also reported as constituents of aloe species which presented in small amounts，naturally occurring as a pair of diastereoisomers as well as aloins A and B［4］(Fig.1).The popularity of A.vera owes to its multiple pharmacological properties including antiproliferative and antitumor activities，wound healing，anti-inflammatory effects，and purgative action.Aloin and other anthrones in A.vera have been shown to possess most of these properties［5］.To date，however，few research on the pharmacological activities of 10-hydroxyaloins A and Bwas reported.There is an urgent need to provide large quantities of pure 10-hydroxyaloins for in depth studies.The isolation of 10-hydroxyaloins was traditionally achieved by multiple column chromatographic procedures［6］，which is time-consuming with low efficiency and yield.Therefore，it would be of great importance to develop an efficient method for the separation and purification of this diastereoisomers.Flash chromatography is basically an air pressure driven hybrid of medium pressure and short column chromatography which has been optimized for particularly rapid separation［7］.It can endure relatively high flow rate with low pressure，offering good separation in a short time at a proper chromatographic condition.Flash chromatography can be applied for normal phase separation and reversed phase separation［8］.

In this paper，a method for preparative isolation of 10-hydroxyaloinsA and B using reversed-phase flash chromatography was developed.The method is simple，fast and efficient，and the compounds obtained are with high purity (over 98.0%)，providing a basis for studies on the analysis and biological properties of 10-hydroxyaloins.

Fig.1 Chemical structures of 10-hydroxyaloins A (a)and B (b)

Experimental

Apparatus

Preparative separation was carried outby an EYELA chromatography system (Kyoto，Japan)，equipped with a low pressure gradientor，intellegent pumps，a UV-9000 UV-Vis detector，a PG-12 gastorr.HPLC analysis was carried out on a liquid chromatography system(Shimadzu，Kyoto，Japan)equipped with two LC-20AD pumps，a CTO-20A column oven and a SPD-M20A DAD detector.HRMS spectra were obtained with aLCMS-IT-TOF mass spectrometer (Shimadzu，Kyoto，Japan)，1H NMR and13C NMR spectra were acquired on a Bruker Avance III 400 Nuclear Magnetic Resonance Spectrometer and optical rotations were measured with a Perkin Eimer 341polarimeter.A SB25-12 DTD ultrasound machine (Scientz Biotechnical.Ltd，Ningbo，China)，an electronic balance (KERN ABT 220-5DM，0.1 mg，Germany)and RE-300 rotational vacuum concentrator (Shanghai YaRong biochemistry instrument factory，China)were utilized for sample preparation.

Reagents

A.vera powder (Batch No:20110601)was purchased from Yunnan Yuanjiang Evergreen Biological Co.，Ltd.(Yuxi，China)and authenticated by Associate Professor Jinzhi Wan (School of Pharmaceutical Sciences，SunYat-Sen University).Preparative C18reversed phase silica gel was purchased from TianjinBonna-Agela Technologies Co.，Ltd (Lot:BL0001L2201，size:20-45 μm，Qty:100 g，Tianjin，China).Methanol used for HPLC was of HPLC grade manufactured by SK Chemicals (Korea).Ultrapure water obtained from Milli-QRG purification unit (Millipore，Bedford，MA，USA)was used for all solutions，dilutions and HPLC analysis.Other chemicals were of analytical grade and purchased from Tianjin Damao Chemical Reagent Factory (Tianjin，China).

Sample preparation

200.0mg of dried A.vera powder was extracted under sonication with 500 mL of methanol at room temperature for 30 min.The extraction procedure was repeated for five times.The extracts were combined and evaporated to dry by rotary evaporator at 40 ℃ under reduced pressure.The extract was yielded after rotary evaporator and dissolved in 50% methanol，affording a sample solution.

Analytical HPLC method

An Agilent TC-C18column (250 mm × 4.6 mm，5 μm)preceded by a Phenomenex C18guard column was used;The binary mobile phase was composed of ultrapure water (A)and methanol (B).The elution was run with a gradient program at 1.0 mL/min:0-25 min，45%B→50%B.Detection wavelength was set at 356 nm.The sample injection volume was 10 μL.The column temperature was ther mostated at 30 ℃.

Preparative flash chromatography method

A reversed-phase C18column (manually packed，300 mm × 20 mm，particle size:20-45 μm).The mobile phase was composed of water (A)and methanol (B)=65∶35.The flow rate was 10 mL/min and the effluent was monitored at 356 nm.1.0 mL of the sample solution was injected into the reversed-phase C18column and the effluent from the column was collected into test tubes with a fraction collector set at 5 mL for each tube.Fractions within the same peak were combined，concentrated and dried under reduced pressure.Peak 1 and peak 2 were collected separately (Fig.2).

Fig.2 Chromatogram of the sample solution eluted with flash chromatography

Results and Discussion

Selection of the extraction solvent

In this study，10-hydroxyaloins in A.vera powder were extracted by ultrasonic extraction method.According to the solubility of the two targeted compounds，three solvents including methanol，ethyl acetate and acetone were selected to extract 10-hydroxyaloins.After HPLC analysis，both methanol and acetone were found to give higher extraction efficiencies.Due to the lower cost of methanol，it was selected as the extraction solvent(Fig.3).

Fig.3 HPLC chromatogram of the methanol extract of A.vera powder

Optimization of flash chromatography conditions

The effects of particle size and injection volume on chromatographic separation were investigated in this study.As the polarity of 10-hydroxyaloinsA and B were similar，C18reversed-phase silica gel with 20-45 μm of the particle size was selected，and the result gave a satisfactory resolution.Regarding the sample injection volume，it gave rise to low yield and purity when loading 0.5 mL of the sample solutions onto the manually packed column，while bad resolution was found when enlarging the injection volume more than 1.0 mL.Finally the sample injection volume was set at 1.0 mL.The gradient of the mobile phase was optimized as well，and isocratic elution with methanol-water (35 ∶65，v/v)as the mobile phase gave an ideal resolution and a relatively short analysis time.

Compound 1 and compound 2 were isolated by the optimized reversed-phase flash chromatography conditions.After dried under reduced pressure，6.5 mg of 10-hydroxyaloin B and 4.7 mg of 10-hydroxyaloin A were yielded，with their chromatographic purities of 98.9%and 98.2%(Fig.4)，respectively.

Fig.4 HPLC chromatogramof isolated 10-hydroxyaloin B (a)and 10-hydroxyaloin A (b)

Confirmation of the two compounds

The structural data of compound 1 were listed as follows，which were the same as 10-hydroxyaloin B［4，6，9］.Yellow amorphous powder;UV (MeOH)λmaxnm:269，301，364;HRMS (ESI)calcd.forC21H22O10［M+Na］+457.0439，found457.0448;-49.7°(c 0.95，MeOH);1H NMR and13C NMR spectral data were summarized in Table 1.

The structural data of compound 2 were listed as follows，which matched the data of 10-hydroxyaloin A［4，6，10］.Yellow amorphous powder;UV (MeOH)λmaxnm:269，301，364;HRMS(ESI)calcd.for C21H22O10［M+H］+435.1015，found 435.1012;+102.8° (c 1.07，MeOH);1H NMR and13C NMR spectral data were summarized in Table 1.

Table 1 13C and 1H NMR data of 10-hydroxyaloin Band 10-hydroxyaloin A (CD3OD，J in Hz，δin ppm)

Conclusion

10-hydroxyaloin B and 10-hydroxyaloin A were successfully separated by reversed-phase flash chromatography on a manually-packed C18column，with purities of 98.9% and 98.2%，respectively.The established method was proved to be simple，fast and reproductive.It can be applied to the preparation of reference substances of 10-hydroxyaloins，providing a basis for studies on the analysis and biological properties.

AcknowledgementsThis work was financially supported by the Twelfth Five Plan of National Science and Technology Project in Rural Areas of China(2012BAD36B02).

1 Waller GR，Mangiafico S，Ritchey CR.A chemical investigation of Aloe BarbadensisMiller.Proc Okla Acad Sci，1978，58:69-76.

2 Li TD，Luo Y，Han WJ.Development in the research of the pharmacological action of aloe and its application.China J Mod Med，2007，17:2881-2886.

3 Wan JZ，Chen XX，Qiu CM，et al.Isolation and purification of isoaloeresin D and aloin from Aloe vera by high-speed counter-current chromatography.Chin Herb Med，2010，2:148-152.

4 Manitto P，Monti D，Speranza G.Studies on Aloe.Part 6.conformation and absolute configuration of aloins A and B and related 10-C-Glucosyl-9-anthrones.J Chem Soc Perkin Trans，1990，1:1297-1300.

5 Yamamoto M，Masui T，Sugiyama K，et al.Anti-inflammatory active constituents of Aloe arborescens Miller.Agric Biol Chem，1991，55:1627-1629.

6 Okamura N，Hine N，Harada S，et al.Diastereomeric C-glucosylanthrones of Aloe vera leaves.Phytochemistry，1997，45:1519-1522.

7 Still WC，Kahn M，Departm AM.Rapid chromatographictechnique for preparative separations with moderate resolution.J Org Chem，1978，43:2923-2925.

8 Weber P，Hamburger M，Schafroth N，et al.Flash chromatography on cartridges for the separation of plant extracts:rules for the selection of chromatographic conditions and comparison with medium pressure liquid chromatography.Fitoterapia，2011，82:155-161.

9 DagneE，Wyk BV，Stephenson D，et al.Three oxanthrones from Aloe littoralis.Phytochemistry，1996，42:1683-1687.

10 Park MK，Park JH，Shin YG，et al.Chemical constituents in Aloe barbadensis.Arch Pharm Res，1997，20:88-90.