Chinese Journal of Natural Medicines  2015, Vol. 13 Issue (6): 0410-0408  
0

Cite this article as: 

WANG Lin-Yan, TANG Yu-Ping, LIU Xin, ZHU Min, TAO Wei-Wei, LI Wei-Xia, DUAN Jin-Ao. Effects of ferulic acid on antioxidant activity in Angelicae Sinensis Radix, Chuanxiong Rhizoma, and their combination [J]. Chinese Journal of Natural Medicines, 2015, 13(6): 0410-0408.
[Copy]

Research funding

This work was supported by National Key Technology R&D Program (No. 2008BAI51B01), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20113237110010), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Corresponding author

Tel/Fax: 86-25-85811695, E-mail: yupingtang@njucm.edu.cn

Article history

Received on: 16-June-2014
Effects of ferulic acid on antioxidant activity in Angelicae Sinensis Radix, Chuanxiong Rhizoma, and their combination
WANG Lin-Yan1, 2, TANG Yu-Ping1, 2, LIU Xin1 , ZHU Min1, TAO Wei-Wei1, LI Wei-Xia1, DUAN Jin-Ao1    
1Jingjiang Hospital of Traditional Chinese Medicine, Jingjiang 214500, China;
2Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, and National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China
[Abstract] The present study aimed at exploring different roles of the same compound in different environment, using preparative HPLC, and the significance to investigating bio-active constituents in traditional Chinese medicine (TCM) on the basis of holism. In this study, the depletion of target component ferulic acid (FA) by using preparative HPLC followed by antioxidant activity testing was applied to investigate the roles of FA in Angelicae Sinensis Radix (DG), Chuanxiong Rhizoma (CX) and their combination (GX). The antioxidant activity was performed by 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity testing. FA was successfully and exclusively depleted from DG, CX, and GX, respectively. By comparing the effects of the samples, it was found that FA was one of the main antioxidant constituents in DG, CX and GX, and the roles of FA were DG > CX > GX. Furthermore, the effects of FA varied at different doses in these herbs. This study provided a reliable and effective approach to clarifying the contribution of same compound in different TCMs to their bio-activities. The role of a constituent in different TCMs might be different, and a component with the same content might have different effects in different chemical environments. Furthermore, this study also suggested the potential utilization of preparative HPLC in the characterization of the roles of multi-ingredients in TCM.
[Key words] HPLC    Ferulic acid    Angelica sinensis    Ligusticum chuanxiong    Herb pair    Antioxidant activity    
Introduction

As one of the oldest continuously practiced systems of traditional medicine in the world,herbal medicine has a history of several thousand years and their worldwide utilization has recently increased in both developing and developed countries. The World Health Organization estimated that 65%-80% of the world population used herbal medicines as the primary form of healthcare [1]. Traditional Chinese medicines (TCMs) are natural therapeutic remedies used under the guidance of traditional Chinese medical philosophy and have been prescribed by TCM practitioners in China and the Chinese community worldwide for thousands of years. Most TCMs are multi-ingredient formulae,and it is widely accepted that multiple constituents are responsible for their biological activities [2].

Angelicae Sinensis Radix (Chinese Danggui,DG) is the processed root of Angelica sinensis (Oliv.) Diels (Umbelliferae),which is widely used as one of the TCM materials in prescriptions and composite formulae to nourish blood,activate blood circulation,regulate menstruation,relieve pain,relax bowels,and so forth [3, 4]. Chuanxiong (CX),the rhizome of Ligusticum chuanxiong Hort. (Umbelliferae),is one of the major clinically used cardiovascular-protective traditional Chinese herbs. Having a reputation for facilitating blood circulation and dispersing blood stasis,this herb is commonly prescribed for the treatment of angina pectoris,cardiac arrhythmias,hypertension,and stroke [5, 6]. When two herbs are used in combination,they may produce synergistic,additive,or antagonistic effects,based onTCM theory. The combination of DG and CX,called as Gui-Xiong (GX),is a well-known herbal pair recorded in many monographs [7, 8]. DG,soft and moist in nature,can nourish blood,regulate menstruation,activate blood,relieve pain,resolve stasis,reduce swelling,and moisten the dryness to loosen the intestine. CX,acrid,fragrant and warm in nature,can promote the circulation of Qi and blood,dispel wind,and relieve pain. It is therefore believed that,when the two herbs are combined,they can activate,nourish,and promote blood at the same time. Moreover,DG can reduce the dryness of CX,while CX can prevent DG from greasy,thus resolving stasis without hurting healthy Qi and nourishing the blood without blood obstruction and Qi depression.

Both DG and CX contain many bio-active aromatic acids,especially ferulic acid (FA) [3, 4, 5, 6, 7, 8, 9, 10, 11]. FA is usually used to assess the quality of DG and CX [12] and has been clinically used to treat angina pectoris and hypertension in China [13]. Previous investigations have suggested that it could significantly improve blood fluidity,inhibit platelet aggregation,decrease serum lipids,prevent thrombus formation,protect neurons,and exhibit anticancer and antioxidant activities [14, 15, 16, 17, 18, 19, 20]. FA also has anti-inflammatory action [21],prevents ethanol-induced liver injury [22],inhibits viral infections including AIDS [23],suppresses the production of interleukin-8 (IL-8) which was the main cause of the local accumulation of neutrophils,and modulates various inflammatory reactions [21]. The content of FA in DG,CX and GX has been comparatively analyzed,and the results show that combination of DG and CX could demote the dissolution of FA [6]. It has been reported that FA is one of the main absorbed components in rat plasma [24, 25].

It is unknown whether the role of FA is same in DG and CXand whether its role will change when the two herbs are used in combination. In the present study,depletion of target component FA by preparative HPLC was applied to investigate the role of FA in DG,CX and GX (DG : CX = 1 : 1),Based on antioxidant activity testing. This study provided an effective approach to clarifying the contribution of same compound in different TCM preparations in relation to their bio-activities. Furthermore,this study demonstrated the utility of preparative HPLC in characterization of multi-ingredients in TCMs.

Materials and Methods Chemicals, reagents and materials

Ferulic acid was purchased from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing,China),with a purity of more than 98%. DPPH (1,1-diphenyl-2-picrylhydrazyl) was purchased from Sigma- Aldrich (Shanghai,China). Methanol was HPLC-grade and obtained from Jiangsu Hanbon Science & Technology (HuaiAn,China) and deionized water was purified by an EPED Ultra-purification system (Eped,Nanjing,China). Other reagents and solutions such as ethanol and acetic acid were of analytical grade (Sinopharm Chemical Reagent Co.,Ltd.,Shanghai,China).

Angelicae Sinensis Radix was collected at Min County,Gansu Province,China,in 2011. Chuanxiong Rhizoma was collected at Pengzhou,Sichuan Province,China,also in 2011. They were authenticated by Dr. YAN Hui (Department of Pharmacognosy,Nanjing University of Chinese Medicine,Nanjing,China). The voucher specimens (Nos. NJUTCM-2011081 and NJUTCM-2011082) were deposited in the Herbarium of Nanjing University of Chinese Medicine.

Apparatus and chromatographic conditions Preparative HPLC

The depletion of FA from DG,CX or GX was performed on the Waters AutoPurificationTM system (Waters Corp.,Milford,MA,USA),including 2545 Binary Gradient Module,2767 Sample Manager,column fluidics Organizer,2489 UV/Visible Detector,Fractionlynx TM Software and XBridgeTM prep C18 OBDTM column (30 mm × 150 mm,5 μm). The mobile phase was composed of A (deionized water) and B (methanol) at the flow rate of 30 mL·min-1; the detection wavelength was set at 286 and 311 nm (λmax of FA). The details are shown in Table1; the two conditions differed in the gradient elution pattern.

Table 1 Gradient elution method for preparative HPLC
Analytical HPLC-PDA analysis

The analyses of the samples were performed on a Waters 2695 Alliance HPLC system (Waters Co.,Milford,MA,USA),equipped with a quaternary pump solvent management system,an auto-sampler,and a non-line degasser. The separation was carried out on a Hypersil ODS2 column (4.6 mm × 250 mm,5 μm) at a column temperature of 30 ℃. The mobile phase was composed of A (Methanol) and B (0.1% aqueous acetic acid,V/V) with a gradient elution at the flow rate of 0.8 mL·min-1: 0-10 min,10%-20% A; 10-25 min,20%-40% A; 25-33 min,40%-80% A; 33-34 min,80%-10% A. Re-equilibration duration was 5 min between individual runs. A Waters 2998 photo diode array (PDA) was connected to the liquid chromatography for detection of elutes,with the full scan spectrum from 210 to 400 nm.

Preparation of sample solutions for depletion of FA

DG and CX were crushed into small pieces. DG,CX and GX (each 200 g) were weighed accurately and then extracted twice in 2.0 and 1.8 L of 50% aqueous ethanol (V/V),with refluxing times of 2 and 1.5 h,respectively. The decoction was combined and the solvent was removed below 60 ℃ till certain volume at the ratio of 1 : 1 (W/W,weight of all herbs and the extracted filtrates) under vacuum,and the samples of DG,CX and GX were obtained (1 mL was equivalent to 1.00 g of crude herbs). The extracts were diluted with 20% aqueous methanol in ultrasonic machine. After centrifugation at 13 000 r·min-1 for 10 min,the supernatants were prepared for the depletion of FA as the original solutions.

The sample solutions were injected onto the preparative HPLC system,and the injection volume was 500 μL each time. The condition I was used for the depletion of FA from DG and CX,and the condition II for the depletion of FA from GX. All fractions were collected according to the retention time of FA with 10 mL per fraction. The remnant solutions were collected,and then the solvent was removed at 60 ℃ under vacuum. Three new samples were obtained from the depletion solutions,i.e.,DG without FA (DG - FA),CX without FA (CX - FA) and GX without FA (GX - FA).

Fig.1 The preparative and analytical chromatograms of the depletion of FA from DG: (A1) preparative chromatogram at 286 nm; (A2) preparative chromatogram at 311 nm; (B1) analytical chromatogram of DG at 270 nm; (B2) analytical chromatogram of DG-FA at 270 nm; and (B3) analytical chromatogram of FA depleted from DG at 270 nm
Preparation of the DPPH solution for antioxidant activity test

DPPH was weighed accurately and was dissolved in absolute ethanol to acquire the DPPH solution ( 0.1 mg·mL-1). The solution was stored at 4 ℃ away from light.

Preparation of sample solutions for antioxidant activity test

FA was dissolved in DMSO (dimethyl sulfoxide,control) to obtain a stock solution (0.396 mg·mL-1 ). In order to eliminate the effect of the solvent on the antioxidant assay,the final concentration of DMSO was fixed at 1% (V/V). The sample solutions including the original and depletion solutions were prepared by diluting the solution with 1% DMSO to a series of concentrations (every mL was equivalent to 10,5,2.5,1.25 and 0.625 mg crude herbs,respectively). FA was added into the depletion solutions to acquire a series of concentrations as the recovery solutions,and the concentrations of FA were the same as the extracts before the depletion. After all the sample solutions were centrifuged at 13 000 r·min-1 for 10 min,the supernatants were collected and stored at 4 ℃ until analysis.

The depletion extracts were dissolved in DMSO to obtain three stock solutions of each crude herb (20 mg·mL-1). The depletion solutions were prepared by diluting the stock solutions with 1% DMSO to two concentrations: 5 and 1.25 mg·mL-1. FA was weighed accurately and was dissolved in 1% DMSO as standard stock solution. Then it was prepared by diluting the stock solutions with 1% DMSO to a series of concentrations (3.125,12.5,50,100,and 200 μg·mL-1). Deionized water or FA was added to the depletion solutions with a ratio of 1 : 1 (V/V) to gain a series of working solutions. After centrifugation at 13 000 r·min-1 for 10 min,the supernatants were collected and stored at 4 ℃ until analysis.

DPPH radical-scavenging activity assay

The radical-scavenging activity was evaluated by a standard spectrophotometric assay using the DPPH radical in a 96-well microplate [26, 27]. A 100 μL of working solutions and 100 μL of 0.1 mg·mL-1 ethanolic DPPH solution were added to each well [28]. After 30 min incubation,the absorbance was determined at 517 nm (λmax). Inhibition of free radical in percent was calculated by using the following equation: I (%) = (ADPPH - Asample/ADPPH) × 100,where ADPPH is the absorption of the DPPH control solution against the blank of solvent,and Asample is the absorption of the extract against the blank of sample solution [29]. Student’s t-test was used to determine the significance of differences between the different samples.

Fig.2 The preparative and analytical chromatograms of the depletion of FA in CX: (A1) preparative chromatogram at 286 nm; (A2) preparative chromatogram at 311 nm; (B1) analytical chromatogram of CX at 270 nm; (B2) analytical chromatogram of CX-FA at 270 nm; and (B3) analytical chromatogram of FA depleted from CX at 270 nm
Results and Discussion The depletion results of preparative HPLC

The preparation chromatograms for FA depletion from DG,CX and GX are showed in Figs. 1A,2A and 3A,respectively. Analytical HPLC was used to analyze the depletion results and confirm its validity. Based on the comparison with standard substances and related literatures [30, 31, 32, 33],the peak to be depleted was identified as the target component FA (tR = 25.966 min),and their purity was validated with wavelength scanning by analytical HPLC (Figs. 1B,2B,and 3B). Their purity was over 90%,and FA was depleted from each sample. Using this method,three samples DG - FA,CX - FA and GX - FA were obtained. Thereafter,the antioxidant activities of DG,DG - FA,(DG - FA) + FA' (sample DG - FA adding standard FA at same content as the original sample DG),CX,CX - FA,(CX - FA) + FA' (sample CX - FA adding standard FA at same content as the original sample CX),GX,GX - FA and (GX - FA) + FA' (sample GX - FA adding standard FA at same content as the original sample GX) were evaluated.

Fig.3 The preparative and analytical chromatograms of the depletion of FA in GX: (A1) preparative chromatogram at 286 nm; (A2) preparative chromatogram at 311 nm; (B1) analytical chromatogram of GX at 270 nm; (B2) analytical chromatogram of GX-FA at 270 nm; (B3) analytical chromatogram of FA depleted from GX at 270 nm
Antioxidant activity

As shown in Fig.4,the inhibitory activities of the tested samples were concentration-dependent,and the trend of antioxidant activity was as follows: DG ≈ (DG - FA) + FA' > DG - FA,CX ≈ (CX - FA) + FA' > CX - FA,GX ≈ (GX - FA) + FA' > GX - FA. The inhibitory effects of the recovery samples ((DG - FA) + FA',(CX - FA) + FA' and (GX - FA) + FA'),in which the content of FA was the same as the original sample (DG,CX and GX),showed no obvious difference from their original samples. The results revealed that the depletion of FA reached the demand in the antioxidant activity evaluation,so the depletion samples could be used to evaluate the roles of FA in DG,CX and GX in their antioxidant activity.

Fig.4 Comparing DPPH radical-scavenging activity of the original,depletion,and recovery samples (mean ± SD,n = 5). *P < 0.05,**P < 0.001,***P < 0.001 vs the respective original solutions

The effects of all the depletion samples (DG - FA,CX - FA and GX - FA) were obviously lower than that of the corresponding original samples (DG,CX,and GX),which demonstrated that FA was one of the main antioxidant constituents in DG,CX,and GX. In addition,the effects of all the depletion samples still had certain antioxidant activity,indicating that there were other constituents contributing to the antioxidant activity in these herbs.

As shown in Fig.5,the trends of relative inhibition ratio of FA on DPPH scavenging-activity were same in DG,CX,and GX. The effect increased as the concentration increased till 2.5 mg·mL-1,but decreased above this concentration. The results might be related to other herbal constituents that might not show certain effect or showed weak effect at lower s,especially below 2.5 mg·mL-1,but might show certain effect or potent effect at higher concentration. These results supported the well-known complexity of bio-active constituents in TCMs.

Fig.5 Comparing the relative roles of FA on DPPH radical-scavenging activity in the original herbs: relative role (%) = (Esample - Esample - FA) / Esample × 100,Esample (EDG,ECX and EGX) DPPH radical-scavenging activity of sample (DG,CX and GX); Esample - FA (EDG - FA,EDG - FA and EDG - FA) DPPH radical-scavenging activity of the deletion samples (DG - FA,CX - FA and GX - FA)

The main antioxidant constituents in DG are phenolic acids (such as FA and caffeic acid) and phthalides (such as Z-butylidenephthalide,Z-ligustilide,senkyunolide I and senkyunolide H); the main antioxidant constituents in CX are alkaloids (such as ligustrazine and perlolyrine) besides phenolic acids and phthalides [34, 35, 36, 37, 38, 39, 40, 41, 42]. Therefore,the bio-active components in GX are the combination of DG and CX; the role of a compound (such as FA) in a single herb (such as DG or CX) may differ from that in combined herb formulae (such as GX).The antioxidant constituents in DG were fewer than CX [5],and thus the role of FA in DG might be greater than that in CX. These results are also shown in Fig.5.

For further comparing the roles of FA in antioxidant activity in DG,CX,and GX,the effects of FA at different doses in DG,CX,and GX were also investigated on the basis of the depleted samples in the present study. As shown in Fig.6,the roles of FA in DG,CX,and GX in their antioxidant activity showed in an apparent dose-dependent manner,and their dose-effect relationship curves had the same trend at the tested concentrations,which demonstrated that FA could produce a significant effect on antioxidant activity in DG,CX and GX. The results also showed that the trends of FA action were DG > CX ≈ GX (Fig.6),further demonstrating that the role of FA in DG was greater than that in CX and GX. Our results revealed that the role of a constituent in different TCM preparations might be different,and a component with the same content level might have different effects in different chemical environments. Thus,the reasonable applications of TCMs need to consider their physicochemical and biological environments.

Fig.6 Comparing the roles of FA at different doses on DPPH radical-scavenging activity in DG,CX and GX (mean ± SD,n = 5). *P < 0.05,**P < 0.001,***P < 0.001 vs GX: (A) each sample concentration was 10 mg·mL-1; (B) each sample concentration was 2.5 mg·mL-1; (C) each sample concentration was 0.625 mg·mL-1; Relative inhibition ratio (%) = E(sample - FA) + FA' - E(sample - FA),Esample - FA (EDG - FA,EDG - FA and EDG - FA) DPPH radical-scavenging activity of the deletion samples (DG - FA,CX - FA and GX - FA),E(sample - FA) + FA' (E(DG - FA) + FA',E(CX - FA) + FA' and E(GX - FA) + FA'). DPPH radical-scavenging activity of the deletion samples including FA at different doses
Conclusion

In the present study,a convenient and effective approach was established for studying complicated bio-active constituents of TCM with preparative HPLC followed by bioactivity evaluation. The role of the target component in different TCMs can be demonstrated by using this method. In the future,a constituent of interest could be depleted in more TCMs and more bioactivity tests could be designed for specific evaluation,providing more and stronger evidences for clarifying the roles of complicated bioactive constituents in TCM and the underlying mechanisms for the variability resulted from their compatibility.

References
[1] Hao H, Cui N, Wang G, et al. Global detection and identification of nontarget components from herbal preparations by liquid chromatography hybrid ion trap time-of-flight mass spectrometry and a strategy [J]. Anal Chem, 2008, 80(21): 8187-8194.
[2] Li T, Roy R. Studying traditional Chinese medicine [J]. Science, 2003, 300(5620): 740-741.
[3] Lü JL, Zhao J, Duan JA, et al. Quality evaluation of Angelica sinensis by simultaneous determination of ten compounds using LC-PDA [J]. Chromatographia, 2009, 70(3-4): 455-465.
[4] Li SJ, Lin H, Tang YP, et al. Comparative metabolomics analysis on invigorating blood circulation for herb pair Gui-Hong by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and pattern recognition approach [J]. J Pharmaceut Biomed, 2015, 107: 456-463.
[5] Li WX, Tang YP, Chen YY, et al. Advances in the chemical analysis and biological activities of chuanxiong [J]. Molecules, 2012, 17(9): 10614-10651.
[6] Li WX, Tang YP, Guo JM, et al. Comparative metabolomics analysis on hematopoietic functions of herb pair Gui-Xiong by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry and pattern recognition approach [J]. J Chromatogr A, 2014, 1346: 49-56.
[7] Li WX, Wang H, Tang YP, et al. The quantitative comparative analysis for main bio-active components in Angelica sinensis, Ligusticum chuanxiong, and the herb pair Gui-Xiong [J]. J Liq Chromatogr Relat Technol, 2012, 35(17): 2439-2453.
[8] Tang YP, Zhang YH, Li WX, et al. Comparative characterization of ten aromatic acids in Siwu series decoctions and their constituting herbs by HPLC-DAD method [J]. J Liq Chromatogr Relat Technol, 2012, 35(17): 2425-2438.
[9] Huang WY, Sheu SJ. Separation and identification of the organic acids in Angelicae Radix and Ligustici Rhizoma by HPLC and CE [J]. J Sep Sci, 2006, 29(17): 2616-2624.
[10] Li WX, Tang YP, Qian YF, et al. Comparative analysis of main aromatic acids and phthalides in Angelicae Sinensis Radix, Chuanxiong Rhizoma, and Fo-Shou-San by avalidated UHPLC–TQ-MS/MS [J]. J Pharmaceut Biomed, 2014, 99: 45-50.
[11] Li WX, Tang YP, Shang EX, et al. Identification on the metabolites of main aromatic acids from Danggui, Chuanxiong and the Gui-Xiong herb pair in blood deficiency rats [J]. China J Tradit Chin Med Pharm, 2013, 28(5): 1212-1218.
[12] Chinese Pharmacopoeia Commission. Pharmacopeia of the People’s Republic of China [M]. Beijing: People’s Medicine Publishing House, 2010.
[13] Hou YZ, Yang J, Zhao GR, et al. Ferulic acid inhibits vascular smooth muscle cell proliferation induced by angiotensin II [J]. Eur J Pharmacol, 2004, 499(1-2): 85-90.
[14] Kobayashi S, Mimura Y, Naitoh T, et al. Chemical structure-activity of Cnidium rhizome-derived phthalides for the competence inhibition of proliferation in primary cultures of mouse aorta smooth muscle cells [J]. Jpn J Pharmacol, 1993, 63(3): 353-359.
[15] Hou YZ, Zhao GR, Yang J, et al. Protective effect of Ligusticum chuanxiong and Angelica sinensis on endothelial cell damage induced by hydrogen peroxide [J]. Life Sci, 2004, 75(14): 1775-1786.
[16] Mathew S, Abraham TE. Ferulic acid: An antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and their applications [J]. Crit Rev Biotechnol, 2004, 24(2-3): 59-83.
[17] Lin Z, Zhu D, Yan Y, et al. Neuroprotection by herbal formula FBD and its active compounds [J]. Pharm Boil, 2009, 47(7): 608-614.
[18] Barone E, Calabrese V, Mancuso C. Ferulic acid and its therapeutic potential as a hormetin for age-related diseases [J]. Biogerontology, 2009, 10(2): 97-108.
[19] Madhujith T, Shahidi F. Antioxidant and antiproliferative potential of pearled barley (Hordeum vulgarae) [J]. Pharm Biol, 2008, 46(1-2): 88-95.
[20] Li WX, Li NG, Tang YP, et al. Biological activity evaluation and structure-activity relationships analysis of ferulic acid and caffeic acid derivatives for anticancer [J]. Bioorg Med Chem Lett, 2012, 22(19): 6085-6088.
[21] Chawla AS, Singh M, Murthy MS, et al. Anti-inflammatory action of ferulic acid and its esters in carrageen induced rat paw edema model [J]. Indian J Exp Biol, 1987, 25(3): 187-189.
[22] Chotimarkorn C, Ushio H. The effect of trans-ferulic acid and gamma-oryzanol on ethanol-induced liver injury in C57BL mouse [J]. Phytomedicine, 2008, 15(11): 951-958.
[23] Nakashima H, Murakami T, Yamamoto N, et al. Lignified materials as medicinal resources V. anti-HIV (human immunodeficiency virus) activity of some synthetic lignins [J]. Chem Pharm Bull, 1992, 40(8): 2102-2105.
[24] Zuo AH, Wang L, Xiao HB, et al. Identification of the absorbed components and metabolites in rat plasma after administration of Rhizoma Chuanxiong decoction by HPLC-ESI-MS/MS [J]. J Pharm Biomed Anal, 2011, 56(5): 1046-1056.
[25] Li WX, Guo JM, Tang YP, et al. Pharmacokinetic comparison of ferulic acid in normal and blood deficiency rats after oral administration of Angelica sinensis [J]. Int J Mol Sci, 2012, 13(3): 3583-3597.
[26] Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity [J]. Lebensm Wiss Technol, 1995, 28(1): 25-30.
[27] Terpinc P, Abramovic H. A kinetic approach for evaluation of the antioxidant activity of selected phenolic acids [J]. Food Chem, 2010, 121(2): 366-371.
[28] Choudhury RP, Kumar A, Garg AN. Analysis of Indian mint (Mentha spicata) for essential, trace and toxic elements and its antioxidant behavior [J]. J Pharm Biomed Anal, 2006, 41(3): 825-832.
[29] Kintzios S, Papageorgiou K, Yiakoumettis I, et al. Evaluation of the antioxidants activities of four Slovene medicinal plant species by traditional and novel biosensory assays [J]. J Pharm Biomed Anal, 2010, 53(3): 773-776.
[30] Lu GH, Chan K, Leung K, et al. Assay of free ferulic acid and total ferulic acid for quality assessment of Angelica sinensis [J]. J Chromatogr A, 2005, 1068(2): 209-219.
[31] Ho CC, Kumaran A, Hwang LS. Bio-assay guided isolation and identification of anti-Alzheimer active compounds from the root of Angelica sinensis [J]. Food Chem, 2009, 114(1): 246-252.
[32] Lu GH, Chan K, Liang YZ, et al. Development of high-performance liquid chromatographic fingerprints for distinguishing Chinese Angelica from related umbelliferae herbs [J]. J Chromatogr A, 2005, 1073(1-2): 383-392.
[33] Yan R, Li SL, Chung HS, et al. Simultaneous quantification of 12 bioactive components of Ligusticum chuanxiong Hort. By high-performance liquid chromatography [J]. J Pharm Biomed Anal, 2005, 37(1): 87-95.
[34] Cheng CY, Ho TY, Lee EJ, et al. Ferulic acid reduces cerebral infarct through its antioxidative and anti-Inflammatory effects following transient focal cerebral ischemia in rats [J]. Am J Chin Med, 2008, 36(6): 1105-1109.
[35] Ou SY, Kwok KC. Ferulic acid: pharmaceutical functions, preparation and applications in foods [J]. J Sci Food Agric, 2004, 84(11): 1261-1269.
[36] Li WD, Wu Y, Liu XD, et al. Isolation, identification and screen of lactone compounds from Angelica sinensis [J]. Chin Tradit Pat Med, 2011, 33(12): 2114-2118.
[37] Yu Y, Du JR, Wang CY, et al. Protection against hydrogen peroxide-induced injury by Z-ligustilide in PC12 cells [J]. Exp Brain Res, 2008, 184(3): 307-312.
[38] Kuang X, Du JR, Liu YX, et al. Postischemic administration of Z-Ligustilide ameliorates cognitive dysfunction and brain damage induced by permanent forebrain ischemia in rats [J]. Pharmacol Biochem Behav, 2008, 88(3): 213-221.
[39] Li WM, Liu HT, Li JJ, et al. Isolation and antioxidative activity of the substrate isolated from Chuanxiong [J]. J Food Sci Biotechnol, 2010, 29(1): 64-70.
[40] Qi HY, Siub SO, Chen Y, et al. Senkyunolides reduce hydrogen peroxide-induced oxidative damage in human liver HepG2 cells via induction of heme oxygenase-1 [J]. Chem Biol Interact, 2010, 183(3): 380-389.
[41] Chen XP, Li Wei, Xiao XF, et al. Phytochemical and pharmacological studies on Radix Angelica sinensis [J]. Chin J Nat Med, 2013, 11(6): 577–587.
[42] Jiang FR, Qian JC, Chen SY, et al. Ligustrazine improves atherosclerosis in rat via attenuation of oxidative stress [J]. Pharm B