Bioassay-guided isolation of antioxidants from Astragalus altaicus by combination of chromatographic...

J. Sep. Sci. 2012, 35, 977–983 977

Chunyan Yang1,2

Yi Yang1

Haji Akber Aisa1

Xuelei Xin1

Hairong Ma1

Abulimiti Yili1Yongxin Zhao1

1Key Laboratory of XinjiangIndigenous Medicinal PlantsResource Utilization, XinjiangTechnical Institute of Physicsand Chemistry, ChineseAcademy of Sciences, Urumqi,China

2Medical College, China ThreeGorges University, Yichang,China

Received December 21, 2011Revised January 15, 2012Accepted January 16, 2012

Research Article

Bioassay-guided isolation of antioxidantsfrom Astragalus altaicus by combinationof chromatographic techniques

An efficient method for bioassay-guided preparative isolation of antioxidants from the n-butanol extract of Astragalus altaicus Bunge was ingeniously developed by combinationof silica gel column chromatography and high-speed counter-current chromatography. Un-der the bioassay-guidance of antioxidant activities, the antioxidants were gradually separatedfrom the crude sample of Astragalus altaicus Bunge by silica gel column chromatography andhigh-speed counter-current chromatography. Silica gel column chromatography separationwas performed with chloroform, chloroform–methanol (100:1∼5:1, v/v) and chloroform–methanol–water (5:1:0.1∼2:1:0.1, v/v/v). High-speed counter-current chromatography sepa-ration was performed with a two-phase solvent system composed of ethyl acetate–n-butanol–water (2:1:6, v/v/v), which was successfully selected by thin layer chromatography analysis,at a flow rate of 1.5 mL/min. As a result, isorhamnetin-3-gentiobioside (20.8 mg), rutin(82.0 mg), and narcissin (12.8 mg) were obtained for the first time from 200 mg of thecrude sample, ABS-5 of Astragalus altaicus Bunge. The purities were all at over 95% by high-performance liquid chromatography analysis, and their structures were unambiguouslyidentified by mass spectroscopy, 1H, and 13C nuclear magnetic resonance spectroscopy.Antioxidant activities of the three compounds were also assayed by in vitro ABTS [2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diamonium salt] radical cation scavenging ac-tivity. Among them, rutin possessed the highest antioxidant capacity with SC50 value of22.15 �g/mL.

Keywords: Antioxidants / Astragalus altaicus Bunge / Bioassay-guided isolation/ Combination of chromatographic techniques / Flavonoids / High-speed counter-current chromatographyDOI 10.1002/jssc.201101104

1 Introduction

Astragalus altaicus Bunge (AA), belonging to the Leguminosaefamily, is an endemic species of Xinjiang, China, which growsat Altaicus mountain about 1700 m high. As a traditionalUighur herb, the roots of AA are used as a tonic, antiper-spirant, and diuretic drug and for the treatment of anemia,chronic nephritis, and chronic ulcer [1]. However, up to date,only few phytochemical investigations of AA have been re-ported [2, 3], although flavonoids and saponins were foundfrom the genus Astragalus in the past several years [4–8]. Asa widely used traditional medicine, scientific data of AA con-cerning the basic compositions are lacking.

Correspondence: Professor Dr. Haji Akber Aisa, Key Laboratoryof Xinjiang Indigenous Medicinal Plants Resource Utilization,Xinjiang Technical Institute of Physics and Chemistry, ChineseAcademy of Sciences, Urumqi 830011, ChinaE-mail: [email protected]: +86-991-3835679

Abbreviations: AA, Astragalus altaicus Bunge; AB, n-butanol extract; ABTS, 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diamonium salt; AC, chloroform extract;AW, water soluble part; HSCCC, high-speed counter-currentchromatography; SGCC, silica gel column chromatography

Flavonoids, as a kind of the major compositions in thegenus Astragalus, are well known for their various biologicalactivities, such as antioxidant activities, metal-ion-chelatingactivities, anti-tumor or antimitotic activities, and inhibi-tion of a variety of enzymes [9]. Due to these particularpharmacological and clinical effects, it is necessary to es-tablish an efficient method for the preparative separationand purification of actives from this plant. As we all know,high-speed counter-current chromatography (HSCCC), aliquid-liquid partition chromatography, eliminates the irre-versible adsorptive loss of samples onto solid support ma-trix columns and has excellent sample recovery comparedwith certain conventional methods [10–12]. Consequently,HSCCC has been successfully applied to the isolation of var-ious natural products [13–17], especially for the flavonoids[18–21].

In the present study, a simple and feasible methodfor the separation of three antioxidants, isorhamnetin-3-gentiobioside, rutin, and narcissin (Fig. 1), was established forthe first time from AA by combination of chromatographictechniques, silica gel column chromatography (SGCC) andHSCCC, under the bioassay-guidance of antioxidant activi-ties. Furthermore, the solvent system of HSCCC was suc-cessfully selected by TLC analysis.

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978 C. Yang et al. J. Sep. Sci. 2012, 35, 977–983

Figure 1. The structures of isolated com-pounds from Astragalus altaicus Bunge.

2 Experimental

2.1 Apparatus

The HSCCC instrument employed in this study was amodel TBE-300A high-speed counter-current chromatogra-phy (Shanghai, Tauto Biotech, China) with three polytetra-fluoroethylene (PTFE) coils (diameter of tube 2.6 mm, totalvolume 290 mL). The revolution radius or the distance be-tween the holder axis and the central axis of the centrifuge (R)was 5 cm, and the β-value varied from 0.5 at the internal termi-nal to 0.8 at the external terminal (β = r/R, where r is the dis-tance from the coil to the holder shaft). An optimum speed of800 rpm was used in this study.

The solvent was pumped into the column with a modelTBP-50A constant-flow pump (Shanghai, Tauto Biotech,China). Continuous monitoring of the effluent was achievedwith a model 8823A-UV monitor at 254 nm and a man-ual sample injection valve with a 20 mL loop for HSCCCwas used to introduce the sample into the column. A modelN2000 workstation (Zhejiang University, Hangzhou, China)was used to draw the chromatogram.

The HPLC (DIONEX, USA) equipment used was aDIONEX system including a P680 pump, an ASI-100 Au-tomated sample injector, a TCC-100 Thermostatted columncompartment, a UVD170U detector. The analysis was car-ried out with a Gemini 110A-C18 column (5 �m, 4.6 mm× 250 mm) from Phenomenex Inc., Guangzhou, China.Evaluation and quantification were made on a Chromeleonworkstation.

2.2 Reagents

All organic solvents used for HSCCC were of analyticalgrade and purchased from Tianjin chemical Factory (Tianjin,China). Methanol used for HPLC was of HPLC-grade andpurchased from Fisher Scientific Company (Fair Lawn, NJ,USA). The following reagents were used in the antioxidantactivity experiments: 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diamonium salt (ABTS) (Sigma, St.Louis, USA), potassium persulfate (Sigma, St. Louis,USA).

Raw plants of AA were collected in May 2009 from Al-taicus, Xinjiang, China. A voucher specimen of the plant(NO.050052) was deposited in the Herbarium of the DrugInspection Institute of Altaicus, Xinjiang, China.

Figure 2. Separation procedure of target compounds from Astra-galus altaicus Bunge.

2.3 Preparation of the crude samples

As shown in Fig. 2, raw material (3.70 kg) of AA was powderedto a homogeneous size by a mill and extracted with methanolexhaustively in a Soxhlet apparatus. After filtration, the ex-tract was combined and evaporated under reduced pressure,which yielded 663.33 g of the extractum (A). Subsequently,the extractum obtained from the combined extract was sus-pended with water. Then, the aqueous solution was extractedwith chloroform and n-butanol (water-saturated) successively,which yielded 143.00 g chloroform extract (AC), 274.00 g n-butanol extract (AB), and 150.48 g water soluble part (AW).Thereafter, 40 g AB was further subjected to silica gel col-umn (1 kg silica gel G 200–300, Qingdao Haiyang Chemical,Qingdao, China) by eluting stepwise with CHCl3, CHCl3–MeOH (100:1–5:1, v/v) and CHCl3–MeOH–H2O (5:1:0.1–2:1:0.1, v/v/v) to obtain seven fractions (ABS-1–7). Amongthose, ABS-5 yielded 571.9 mg of dried material, which wasused for subsequent HSCCC separation (Fig. 2).

2.4 Selection of two-phase solvent system

The two-phase solvent systems were selected according to Rfvalues of the target components in the TLC analysis. Different


J. Sep. Sci. 2012, 35, 977–983 Liquid Chromatography 979

ratios of ethyl acetate–n-butanol–water were prepared andequilibrated in a separation funnel at room temperature. Theupper organic phase of solvent systems as the developingreagents and a suitable amount of samples (1 mg/mL) wereapplied to TLC analysis. When the Rf value is less than 0.5,the lower aqueous phase can be used as the mobile phasefor HSCCC separation [22]. And the separation factor (�) wascalculated using the following equation:

� = Rf Large/Rf small (1)

2.5 HSCCC separation

The HSCCC was performed with a model TBE-300A HSCCCinstrument as follows: the multiplayer coiled column wasfirst entirely filled with the upper phase as stationary phase.After rotation at 800 rpm, the sample solution (200 mgof the ABS-5 in 20 mL of a mixture of upper and lowerphases) was injected through the sample port. Subsequently,the lower aqueous phase was pumped into the head endof the HSCCC coil column, and the effluent from the out-let of the column was monitored with a UV detector at 254nm. Peak fractions were manually collected according to thechromatogram.

2.6 HPLC analysis and identification of the crude

samples and the peak fractions from HSCCC

The crude samples and the peak fractions from HSCCC wereanalyzed by HPLC. The analysis was performed with a Gem-ini 110A-C18 column (5 �m, 4.6 mm × 250 mm) at a columntemperature of 30�C. The mobile phase was eluted with alinear gradient of methanol (A) and 0.2% formic acid (B) asfollows: A–B (15:85, v/v) to A–B (75:25, v/v) in 60 min. Theflow-rate was 1.0 mL/min and the effluent was monitored at254 nm by a UV detector.

Identification of the HSCCC peak fractions was carriedout by mass spectroscopy (MS), 1H-NMR, and 13C-NMR.

2.7 Evaluation of antioxidant activity by ABTS

radical cation decolorization assay

The spectrophotometric analysis of ABTS radical scavengingactivity was determined according to the traditional method[23] with some modifications. Briefly, ABTS•+ radical cationwas prepared by reacting 10 mL of 2 mM ABTS water so-lution with 100 �L of 70 mM potassium persulfate, and themixture was allowed to stand in the dark at room temper-ature for 12 h before use. Prior to the assay, the solutionwas diluted in ethanol to give an absorbance at 734 nm of0.70 ± 0.02 in a 96-well flat-bottom microtiter plates and wasequilibrated at 25�C. The ABTS solution (110 �L) was addedto the samples solution (total volume: 200 �L), stirred for 30 s,and the absorbance of AA fractions (Ai) were read at 734 nm

after 10 min. The blank Absorbance (A0) was measured us-ing ethanol. Antioxidant activity was expressed as percentageinhibition of the ABTS•+ radical cation and was determinedby the following equation:

AA(%) = [1 − (Ai/A0)] × 100% (2)

Sample concentration providing 50% scavenging capabil-ity (SC50) was calculated from the graph plotting inhibitionpercentage. All the tests were run in triplicates, and the aver-age value was calculated.

3 Results and discussion

3.1 HPLC analysis of the crude samples and the

isolated compounds

HPLC method was developed for the analysis of active sam-ples at first. Several elution systems were tested in HPLCfor the separation of active samples, such as gradient elutionof methanol-water, acetonitrile-water, methanol-acetonitrile-water, acetonitrile-aqueous acid, methanol-aqueous acid, etc.At last, formic acid was introduced into the mobile phaseof HPLC to alleviate the peak tailing of compositions in thecrude samples, and the appropriate concentration of formicacid was 0.2%. When methanol–0.2% formic acid was used asthe mobile phase in gradient mode, good resolution of targetcompound could be obtained. The active samples and peakfractions separated by HSCCC were analyzed by HPLC underthe optimum analytical conditions. The result of the samplesAB and ABS-5 contained isorhamnetin-3-gentiobioside (re-tention time 33.94 min), rutin (retention time 35.90 min),and narcissin (retention time 38.91 min) with some impuri-ties is shown in Fig. 3A and B.

3.2 Separation procedures

The selection of two-phase solvent system for the target com-pound is the most important step in HSCCC where searchingfor a suitable two-phase solvent system may be estimated as90% of the entire work in HSCCC [24]. Usually, two-phasesolvent system is selected according to the partition coeffi-cient (K) of the target components which can be determinedby HPLC [25]. The partition coefficient (K) is the ratio ofsolute distributed between the mutually equilibrated two sol-vent phases. The suitable K values for HSCCC are 0.4 ≤ K≤ 2.5 [26]. In this report, however, a simple and convenientmethod for two-phase solvent system selection was used ac-cording to Rf values of target compounds in the TLC plate[22], although there were very few reports about using TLC toselect solvent systems in HSCCC. When the Rf value is lessthan 0.5, the lower aqueous phase can be used as the mobilephase for HSCCC separation [22]. Table 1 shows Rf valuesand separation factors (�) of three target compounds in TLCplates developed by different mobile phase. All Rf values were



Figure 3. HPLC chromatogram of crude samples and HSCCC fractions. Column: Gemini 110A-C18 column (5 �m, 4.6 mm × 250 mm);column temperature: 30�C. The mobile phase: a linear gradient of methanol (A) and 0.2% formic acid (B) as follows: A–B (15:85, v/v) toA–B (75:25, v/v) in 60 min; flow rate: 1.0 mL/min; detection wavelength: 254 nm. (A) AB, (B) ABS-5, (C) fraction 1, (D) fraction 2, and (E)fraction 3.

less than 0.5, which means the lower aqueous phases of allsolvent system in Table 1 can be used as the mobile phasefor the separation of target compounds in HSCCC. How-ever, their separation factors (�) were different. As seen from

Table 1, solvent system 1 was the favorite for fraction 1 and2 separation, but solvent system 2 was suitable for fraction 2and 3 separation. However, �1,2 values were much greaterthan �2,3 values in all solvent systems. So if we want to



Table 1. Rf values and separation factors (�) of three target com-pounds in different solvent system

No. Volume ratio (v/v/v) Fractions Rf �1,2 = Rf2/Rf1 �2,3 = Rf3/Rf2

1 2:1:3 1 0.11 1.63 1.052 0.183 0.19

2 2:1:6 1 0.12 1.42 1.182 0.173 0.20

3 1:1:2 1 0.20 1.35 1.042 0.273 0.28

4 2:3:5 1 0.23 1.21 1.112 0.283 0.31

Note: TLC mobile phase: upper phase of different two-phase sol-vent system composed of ethyl acetate–n-butanol–water.

Figure 4. HSCCC chromatogram of antioxidants separation fromAstragalus altaicus Bunge. Sample: ABS-5; sample size: 200 mg;flow rate: 1.5 mL/min; rotational speed: 800 rpm; detection wave-length: 254 nm; Sf: 55%.

separate all of them in a single run using HSCCC, �2,3 had tobe considered first of all. Finally, two-phase solvent system 2composed of ethyl acetate–n-butanol–water (2:1:6, v/v/v) wasselected for the separation of our target compounds usingHSCCC.

Figure 4 shows the HSCCC chromatogram of ABS-5 us-ing ethyl acetate–n-butanol–water (2:1:6, v/v/v). HSCCC wasperformed at a flow rate of 1.5 mL/min with the rotationalspeed of 800 rpm. Isorhamnetin-3-gentiobioside (20.8 mg),rutin (82.0 mg) and narcissin (12.8 mg) were obtained for thefirst time from 200 mg ABS-5 of AA. The purities were allat over 95% by HPLC analysis (Fig. 3C–E). Retention ratio ofthe stationary phase (Sf) was 55%.

The practical K values can be calculated from Fig. 4according to the following equation:

K = (VR − VM)/VS (3)

where VR is the retention volume of the solute, and VM and VS

are the volume of upper phase and lower phase, respectively.The practical K values of fraction 1–3 were approximately 0.71,2.04, and 3.15, respectively. Combined with the Rf values, wecan summarize that when the Rf value of target compound isless than 0.1, K value may be less than 0.5 which results in aloss of peak resolution. When Rf value of target compound isbetween 0.1 and 0.5, the lower aqueous phase can be used asmobile phase for HSCCC separation. Otherwise, the upperorganic phase has to be used as mobile phase.

3.3 Structural identification

Fraction 1, yellowish amorphous powder, negative ESI-MSm/z 639 [M-H]−, m/z 315 [(M-H)-(162+162)]−. 1H-NMR (400MHz, DMSO-d6): 2.80 (1H, m, H-5′ ′ ′), 2.85 (1H, m, H-2′ ′ ′),2.94 (1H, m, H-3′ ′ ′), 2.98 (1H, m, H-4′ ′), 3.24 (1H, overlapped,H-2′ ′), 3.26 (2H, overlapped, H-3′ ′, 4′ ′ ′), 3.33 (2H, overlapped,H-5′ ′, H-6′ ′ ′), 3.51 (1H, m, H-6′ ′ ′), 3.53 (1H, m, H-6′ ′), 3.84(3H, s, 3′-OMe), 3.89 (1H, d, J = 11.8 Hz, H-6′ ′), 4.08 (1H, d,J = 7.2 Hz, H-1′ ′ ′, anomeric), 5.51 (1H, d, J = 7.2 Hz,anomeric), 6.16 (1H, d, J = 2.0 Hz, H-6), 6.38 (1H, d,J = 1.6 Hz, H-8), 6.91 (1H, d, J = 8.8 Hz, H-5′), 7.49 (1H,dd, J = 2.0, 8.4 Hz, H-6′), 7.93 (1H, d, J = 1.6 Hz, H-2′). 13C-NMR (400 MHz, DMSO-d6): 156.1 (C-2), 133.0 (C-3), 171.2(C-4), 161.2 (C-5), 99.0 (C-6), 165.5 (C-7), 93.9 (C-8), 156.5 (C-9), 103.7 (C-10), 121.0 (C-1′), 113.3 (C-2′), 146.9 (C-3′), 149.4(C-4′), 115.3 (C-5′), 122.1 (C-6′), 101.1 (C-1′ ′), 74.2 (C-2′ ′), 76.3(C-3′ ′), 69.7 (C-4′ ′), 76.4 (C-5′ ′), 67.2 (C-6′ ′), 103.2 (C-1′ ′ ′), 73.5(C-2′ ′ ′), 76.7 (C-3′ ′ ′), 69.6 (C-4′ ′ ′), 76.6 (C-5′ ′ ′), 60.7 (C-6′ ′ ′), 55.8(3′-OMe). A detailed comparison of the NMR data of fraction1 showed that it was identical with [27].

Fraction 2, yellowish amorphous powder, negative ESI-MS m/z 609 [M – H]−. 1H-NMR (400 MHz, pyridine-d5): 1.53(3H, d, J = 5.6 Hz), 4.03 (2H, H-4′ ′, H-6′ ′, t-like, J = 8.0Hz), 4.13 (1H, m, H-5′ ′), 4.18 (1H, m, H-4′ ′ ′), 4.20 (1H, m,H-5′ ′ ′), 4.29 (1H, m, H-2′ ′), 4.33 (1H, m, H-3′ ′), 4.43 (1H,overlapped, H-3′ ′ ′), 4.44 (1H, overlapped, H-2′ ′ ′), 4.54 (1H, d,J = 8.0 Hz, H-6′ ′), 5.37 (1H, s, anomeric), 6.02 (1H, d, J = 7.6Hz, anomeric), 6.66 (1H, d, J = 1.6 Hz, H-8), 6.69 (1H, d, J= 2.0 Hz, H-6), 7.37 (1H, d, J = 8.4 Hz, H-5′), 8.14 (1H, dd, J= 8.0, 2.0 Hz, H-6′), 8.38 (1H, d, J = 2.0 Hz, H-2′). 13C-NMR(400 MHz, pyridine-d5): 158.5 (C-2), 135.8 (C-3), 179.0 (C-4),163.0 (C-5), 100.2 (C-6), 166.3 (C-7), 95.0 (C-8), 158.1 (C-9),105.6 (C-10), 122.8 (C-1′), 118.3 (C-2′), 147.1 (C-3′), 151.1 (C-4′), 116.7 (C-5′), 123.3 (C-6′), 105.3 (C-1′ ′), 76.4 (C-2′ ′), 79.1(C-3′ ′), 71.7 (C-4′ ′), 77.9 (C-5′ ′), 68.9 (C-6′ ′), 103.0 (C-1′ ′ ′), 72.5(C-2′ ′ ′), 72.9 (C-3′ ′ ′), 74.4 (C-4′ ′ ′), 70.0 (C-5′ ′ ′), 18.9 (C-6′ ′ ′). Adetailed comparison of the NMR data of fraction 2 showedthat it was identical with [27, 28].

Fraction 3, yellowish amorphous powder, negative ESI-MS m/z 623 [M – H]−, m/z 315 [(M-H)-(162+146)]−. 1H-NMR(400 MHz, pyridine-d5): 1.50 (3H, d, J = 5.6 Hz), 3.93 (3H, s,3′-OMe), 4.08 (1H, m, H-6′ ′), 4.10 (1H, m, H-4′ ′), 4.12 (1H,m, H-4′ ′ ′), 4.13 (1H, m H-5′ ′ ′), 4.18 (1H, m, H-5′ ′), 4.31 (1H,m, H-3′ ′), 4.33 (1H, m, H-2′ ′), 4.36 (1H, m, H-2′ ′ ′), 4.40 (1H,



Figure 5. ABTS radical scavenging activities screening of the crude samples and the isolated compounds from Astragalus altaicus Bunge.The concentration of the tested samples was 10 �g/mL.

overlapped, H-3′ ′ ′), 4.55 (1H, d, J = 11.2 Hz, H-6′ ′), 5.38 (1H,s, anomeric), 6.28 (1H, d, J = 8.0 Hz, anomeric), 6.71 (1H,d, 2.0 Hz, H-6), 6.74 (1H, d, J = 1.6 Hz, H-8), 7.31 (1H, d,J = 8.8 Hz, H-5′), 7.93 (1H, dd, J = 8.4, 2.0 Hz, H-6′), 8.42(1H, d, J = 2.0 Hz, H-2′). 13C-NMR (400 MHz, pyridine-d5):158.2 (C-2), 135.4 (C-3), 179.1 (C-4), 163.3 (C-5), 100.3 (C-6),166.5 (C-7), 95.2 (C-8), 158.2 (C-9), 105.8 (C-10), 122.5 (C-1′),114.8 (C-2′), 148.5 (C-3′), 151.8 (C-4′), 116.8 (C-5′), 123.5 (C-6′), 104.5 (C-1′ ′), 76.7 (C-2′ ′), 79.1 (C-3′ ′), 72.1 (C-4′ ′), 78.2 (C-5′ ′), 68.8 (C-6′ ′), 103.1 (C-1′ ′ ′), 73.1 (C-2′ ′ ′), 72.6 (C-3′ ′ ′), 74.4(C-4′ ′ ′), 70.2 (C-5′ ′ ′), 56.6 (3′-OMe), 19.0 (C-6′ ′ ′). A detailedcomparison of the NMR data (1H, 13C) of fraction 3 showedthat it was identical with [28, 29].

3.4 Antioxidant activities of crude samples and

HSCCC fractions

The aim of this experiment was to screen antioxidants fromthe crude extract (A) of AA, which possessed potential antiox-idant activity (inhibition percentage: 8.9% at 10 �g/mL) asshown in Figs. 1 and 5. After extracted with organic solvents,each sample including AB, AC, AW was assayed using in vitroABTS radical cation scavenging activity. The results (as seenfrom Fig. 5) showed that the antioxidant activity of AB withinhibition percentage of 13.5% at 10 �g/mL was much betterthan the others. And then, 40 g AB was subjected to SGCCand eluted by CHCl3, CHCl3–MeOH (100:1–5:1, v/v) andCHCl3–MeOH-H2O (5:1:0.1–2:1:0.1, v/v) respectively, whichgave samples ABS-1–7. Through screening of antioxidant ac-tivity, ABS-5 showed the best antioxidant activity with 23.5%inhibition percentage at 10 �g/mL. Subsequently, ABS-5 wassubjected to HSCCC separation, which produced fractions1–3. Antioxidant activities of the three fractions were still

Table 2. ABTS radical scavenging activities of isolated com-pounds from Astragalus altaicus Bunge.

Compounds MW IC50 values (�g/mL)

Fraction 1 639 33.43Fraction 2 609 22.15Fraction 3 623 40.97Vitamin C 176 17.10

assayed by in vitro ABTS radical cation scavenging activity.The SC50 as shown in Table 2 were 33.43, 22.15, and 40.97�g/mL, respectively, compared with positive control refer-ence, ascorbic acid with 17.10 �g/mL. Among them, fraction2, rutin, possessed the highest antioxidant capacity, whichwas slightly lower than that of the positive control reference(as shown in Fig. 5 and Table 2).

4 Conclusion remarks

In this study, we have developed a combination of chro-matographic techniques for bioassay-guided separation of an-tioxidants from Astragalus altaicus Bunge. Three active com-pounds were received for the first time from AA, which ex-hibited the very good antioxidant activities. In addition, thepresent study also provides a method for selecting of two-phase solvent system of HSCCC according to Rf values ofTLC analysis, which is simple and convenient. As a whole,compared with the traditional methods of separating activecompositions, the present experiment provides a simpler andmore efficient method to obtain active compounds directlyfrom natural resources.



This work was funded by the China National Founds forDistinguished Young Scientists (Grant No. 30925045). Researchwas also supported by the CAS/SAFEA International PartnershipProgram for Creative Research Teams.

The authors have declared no conflict of interest.

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