Printer Friendly

The sulfur-fumigation reduces chemical composition and biological properties of Angelicae sinensis radix.

ABSTRACT

Angelica Sinensis Radix (roots of Angelica sinensis', ASR) is a popular herbal supplement in China for promoting blood circulation. Today, sulfur-fumigation is commonly used to treat ASR as a means of pest control; however, the studies of sulfur-fumigation on the safety and efficacy of ASR are very limited. Here, we elucidated the destructive roles of sulfur-fumigation on ASR by chemical and biological assessments. After sulfur-fumigation, the chemicals in ASR were significantly lost. The biological activities of antiplatelet aggregation, induction of NO production and estrogenic properties were compared between the water extracts of non-fumigated and sulfur-fumigated ASR. In all cases, the sulfur-fumigation significantly reduced the biological properties of ASR. In addition, application of water extract deriving from sulfur-fumigated ASR showed toxicity to cultured MCF-7 cells. In order to ensure the safety and to achieve the best therapeutic effect, it is recommended that sulfur-fumigation is an unacceptable approach for processing herbal materials.

Keywords:

Angelica Sinensis Radix

Sulfur-fumigation

Chemical composition

Biological function

Introduction

Angelica Sinensis Radix (roots of Angelica sinensis; ASR), well known as "female ginseng", is one of the commonly used traditional Chinese medicines (TCM). ASR contains significant amounts of organic acids, volatile oils and polysaccharides, which are considered to be the biologically active components (Zhao et al., 2003; Yi et al., 2009). Clinically, ASR is good in replenishing and invigorating blood, stopping pain and moistening the intestines (Yi et al., 2009). Very often, ASR is being used to promote blood circulation in treating menstrual disorders, e.g. amenorrhea and dysmenorrheal (Wilasrusmee et al., 2002; Gao et al., 2006). Meanwhile, ASR is also used as a health food supplement for women's care in Europe and America.

Traditionally, the drying of post-harvested ASR under the sun is the standard commodity of herb preparation. In recent years, sulfur-fumigation is commonly used to replace the natural drying process. In fact, sulfur-fumigation has been employed in handling of numerous medicinal herbs as to shorten the drying duration, to control pests and to maintain a better appearance. However, this chemical processing was recently reported to alter bioactive components within the herbs, and consequently the bioactivities and pharmacokinetics of herbs were changed (Wang et al., 2009; Liu et al., 2010). According to this notion, the amount of ferulic acid in ASR was decreased significantly after sulfur-fumigation (Zhao et al., 2003). The chemical comparison of ASR and sulfur-fumigated ASR (S-ASR) is not fully revealed. More important, the role of sulfur-fumigation in the bioactivities of ASR has not been addressed, and the herbal industries do not have a full picture regarding this chemical processing of ASR. Here, the chemical and biological properties of ASR and S-ASR were fully compared for the first time. The results could provide information for a better usage of ASR in clinical practice.

Materials and methods

Plant materials and reagents

Roots of Angelica sinensis (Oliv.) Diels (ASR) were obtained from Minxian of Gansu in China in October of 2009. The authentication of the herbs was confirmed morphologically by Dr. Tina Dong at Hong Kong University of Science and Technology (HKUST). The voucher specimens (voucher # 02-9-1) were deposited in the Centre for Chinese Medicine R&D at HKUST. Butylphthalide, senkyunolide A, Z-butylidenephthalide, senkyunolide I and senkyunolide H were purchased from Weiqike Biotechnology Co. (Sichuan, China). Ferulic acid was obtained from Sigma (St. Louis, MO). Z-Ligustilide was purchased from TLCM (Hong Kong, China). All chemical standards were confirmed to show >98% purity based on theirGC and MS data. Analytical- and HPLC-grade reagents were from Merck (Darmstadt, Germany).

Preparation of sulfur-fumigated ASR

The sulfur-fumigated ASR was prepared following modified procedures similar to those performed by herbal farmers. Five hundred grams of ASR samples were soaked with 50 ml water for 0.5 h, and 50 g of sulfur powder was heated until self-ignition. Then, the burning sulfur and the wetted ASR were carefully put into the lower and upper layer of a desiccator, respectively. The desiccator was then kept closed for 12 h (Jiang et al. 2013). Afterward, the sulfur-fumigated ASR were taken out and dried at room temperature for 12 h.

Preparation of herbal extracts

For GC-QQQ-MS/MS analysis, the ethyl acetate extraction of ASR and S-ASR was performed previously (Zhan et al., 2013a). In water extraction of ASR and S-ASR, about 15 g of root was weighed, boiled in 120 ml of water for 2 h, and extracted twice. For the second extraction, the same extracting conditions were applied. The extracts were dried under vacuum and stored at -80[degrees]C, which was used for HPLC analysis, total chemical parts analysis and biological determination.

Chemical analysis

The water extracts of ASR were quantitative analyzed by HPLC. A Waters HPLC system consisting of a 600 pump, a 717 auto-sampler, and a UV/VIS photodiode array 2996 detector was used for the analysis. The chromatographic condition was as described previously (Zhan et al., 2011). The signals were detected at 325 nm for ferulic acid and Z-ligustilide, and at 280 nm for senkyunolide I and senkyunolide H with a photodiode array detector. The determination of total organic acids (Lu et al., 2010), total volatile oils (Shao, 2010), total polysaccharides and total flavonoids (Dong et al., 2006) were performed, as described previously.

Agilent 7000 GC/MS/MS series system (Agilent, Waldbronn, Germany) was also applied, which was equipped with an Agilent 7890A gas chromatography and GC-QQQ MassHunter workstation software. The extract was separated on an Agilent HP-5MS capillary column (250 [micro]m x 30 m x 0.25 [micro]m). The chromatographic condition was described previously (Zhan et al., 2013a). The volatile compounds were authenticated by comparing the mass spectra with the Kovats retention indices and NIST standard reference database (NIST 08). For the MS/MS analysis, the suitable precursor ion and two product ions were chosen for acquisition in MRM mode for ferulic acid, butylphthalide, Z-butylidenephthalide, senkyunolide A, Z-ligustilide, senkyunolide H, senkyunolide 1 and paeonol (internal standard). Agilent MassHunter software was used for data acquisition and processing.

Sulfur dioxide residue analysis

The distillation method was used for the determination of sulfur dioxide residue, as described in Chinese Pharmacopeia Appendix IX (Shao, 2010). 10 g of the fine powder of crude drug or processed pieces was weighed into an 1000 ml round bottom flask, added with 300-400 ml of water, 10 ml of 6mol/l hydrochloric acid and a few glass beads, shook and mixed well. Heated the flask gently in an electric heating jacket until boiling begins and continued boiling for 3 min. The iodine titrant was used to titrate until the blue color was not changed within 20 s. Sulfur dioxide residue in samples was calculated according to the following equation: P = (A - B) x C x 0.032 x 1000/W.Pwastheamountofsulfurdioxide residue in samples (mg/g); A was the volume of consumed iodine titrant by samples (ml); B was the volume of consumed iodine titrant by blank (ml); C was the concentration of iodine titrant (0.01 mol/1); W was the weight of samples (g); and 0.032 was the weight of sulfur dioxide which was equivalent to 1 ml of iodine titrant (1 M).

Anti-platelet aggregation assay

Blood was collected from adult New Zealand white rabbits. The blood plasma was collected as described previously (Dong et al., 2006). The ASR water extracts were added 5 min before adenosine 5'-diphosphate (ADP, inducer; 10 [micro]M final).The aggregations were recorded using a Sanda-196 platelet aggregator (Shanghai, China). The inhibition activity of platelet aggregation was calculated by the formula: (ADP-induced [A.sub.max] - sample-induced [A.sub.max])/(ADP-induced [A.sub.max]) x 100%.

MCF-7 cell viability and estrogenic assay

Human mammary epithelial carcinoma MCF-7 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). The cells were grown in modified Eagles medium, supplemented with 10% FBS, L-glutamine, pyruvate and penicillin=streptomycinin in a humidified C[O.sub.2] (5%) incubator at 37[degrees]C. The cell viability was measured by 3-(4,5-dimethylthioazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay (Zhan et al., 2011). Cultured MCF-7 cells were transfected with pERE-Luc to generate stable MCF-7-ERE-Luc cells according to a previous report (Zhan et al., 2011). The pERE-Luc expressed cells were treated with herbal extracts for 48 h. Afterward, the cells were collected by the lysis buffer containing 0.2% Triton X-100, 1 mM dithiothreitol, and 100 mM potassium phosphate (pH 7.8). The lysates were then subjected to luciferase assay (Tropix, Inc.), and the activity was expressed as per milligram of cell protein.

Induction of NO production in HUVECs

Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (San Diego, CA). Cultured HUVECs were maintained at 4.5 x [10.sup.5] cells/ml in EGM Bulletkit medium in a humidified incubator with 95% air, 5% C[O.sub.2]. The medium was replaced by 0.1 ml of serum and growth factor free medium containing ASR or S-ASR extracts every day. The concentrations of NO in the culture medium were measured with the NO detection kit (Biovision, Mountain View, CA) according to the manufacturer's instructions (Leung et al., 2006).

Fluorimetric measurements of NO production were also performed on cultured HUVEC cells using an Olympus Fluoview FV1000 laser scanning confocal system mounted on an inverted 1x81 Olympus microscope, equipped withal 10x objective (NA 0.5). Cultured HUVEC cells, seeded on glass cover slips, were incubated for 30 min at room temperature in normal physiological solution containing 1 M DAF-FM DA. The amounts of NO were evaluated by measuring the fluorescence intensity excited at 495 nm and emitted at 515 nm (Bi et al., 2010).

Statistical analysis and other assays

Protein concentrations were measured routinely by Bradford's method (Hercules, CA). Statistical tests were done by using one-way analysis of variance. The significant differences between treatments were analyzed by independent t-test of SPSS. Statistically significant changes were classed as [*] where p<0.05; [**] where p<0.01 and; [***] wherep<0.001.

Results

Chemical composition of extracts from ASR and S-ASR

The major types of components in ASR, including total organic acids, total volatile oils, total polysaccharides and total flavonoids, were compared between ASR and S-ASR extracts. Having different extracting methods, the extraction efficiencies of these components in water were over 95% (Table 1). By comparing to ASR, S-ASR had similar amount of total polysaccharides, but lower amounts of total organic acids, volatile oils and flavonoids by around 15%, 10% and 25%, respectively (Table 1). In addition, ASR did not contain any sulfur oxide residue, but S-ASR was found to have 138 mg/kg of sulfur oxide residue.

The amounts of ferulic acid, butylphthalide, Z-butylidenephthalide, senkyunolide A, Z-ligustilide, senkyunolide H and senkyunolide 1 in the ethyl acetate extract of ASR were determined. These chemicals are known to be abundant and possessing biological properties (Zhao et al. 2003; Zhan et al. 2013b). The representative GC-QQQ-MS/MS chromatograms of the mixed standards and a typical extract were shown (Fig. 1A). The results of quantitative analysis were summarized in Fig. IB. The results showed that these chemicals in S-ASR were significantly lower than that of ASR. In particular, sulfur-fumigation significantly decreased the amounts of ferulic acid, senkyunolide A and senkyunolide I in S-ASR by ~32%, ~54% and ~29%, respectively.

The major chemical changes of ASR after sulfur-fumigation should occur in the volatile components, and therefore their amounts were compared between the ASR and S-ASR extracts. The relative amounts were expressed in terms of the relative percentages to the total peak areas in original herb, as generated from the GC analyses. Forty-six chemicals of the volatile fractions in ASR, extracted by ethyl acetate, were selected for comparison (Table 2). The volatile fractions were characterized by high percentages of aldehydes, alkalies, acids, ketones, alcohols, terpenes and esters: these components contributed mainly to the fragrance of ASR volatile oils. Having ASR as a reference, the major portion of volatile components in S-ASR was much lower than that in ASR. In comparison with ASR, 10 components were not found in S-ASR samples, and others in that were decreased significantly from 15% to 65% (Table 2).

Water is a common solvent in TCM preparation (Bo, 2008). Therefore, the water extract of ASR and S-ASR were also quantified by measuring the amount of ferulic acid, senkyunolide I, senkyunolide H and Z-ligustilide. Their amounts were similar as that in the extract of ethyl acetate (Table 3). However, the amount of Z-ligustilide was dramatically decreased after boiling, as compared to ethyl acetate, which could be a possible reason of the loss of volatilized Z-ligustilide during the heating (Zhan et al., 2013b). In addition, the yield of ferulic acid in S-ASR was ~30% lower than that in ASR, and senkyunolide I in S-ASR was ~l-fold different to that of ASR (Table 3).

Biological properties of ASR and S-ASR

The biological properties of water extracts of ASR and S-ASR were compared here. The water extracts were chemically standardized as shown in Table 3, which was to ensure the repeatability of herbal extracts for all biochemical tests. The stimulation of blood circulation is considered as one of the major functions of ASR in treating women's aliments (Dong et al., 2004). Thus, the activity of ASR and S-ASR extracts in preventing platelet aggregation was determined. The blood platelets could be induced by ADP for aggregation. For the inhibition assay, the blood platelets were pre-treated with ticlopidine (TIC, 0.2 mM), which served as a positive control (Fig. 2A). As shown in Fig. 2A, both ASR and S-ASR had the inhibitory effect on the platelet aggregation dose-dependently. Moreover, the effect of S-ASR extract at 1 mg/ml was reduced about 20%, as compared to ASR extract.

We determined cell viability after application of water extracts of ASR and S-ASR in cultured MCF-7 cells. As shown in Fig. 2B, the treatment with S-ASR water extracts caused MCF-7 cell death, in particular which decreased the cell number by ~30% at 3 mg/ml. Meanwhile, a high concentration of ASR extract showed no obvious cell toxicity to the cell growth (Fig. 2B).

To investigate the estrogenic activity, MCF-7 cells stably transfected with pERE-Luc were employed. The water extracts of ASR and S-ASR were applied onto the cultures for 48 h. The promoter (pERE-Luc) driven luciferase activity was subsequently determined. Results showed that both ASR extracts, with and without sulfur-fumigation, were able to stimulate the transcriptional activity of pERE-Luc in a dose-dependent manner. The application of 0.3 and 1 mg/ml ASR extracts led to higher activation of the promoter at ~50% than that of S-ASR (Fig. 2C).

Endothelial cell produces and releases a variety of vasoactive substances, such as nitric oxide (NO). NO is an important regulator of vasodilation in blood vessels (Zhang et al., 2006). Therefore, we would like to test the induction of endothelium-derived NO production by ASR and S-ASR in cultured HUVEC cells. The production of NO was determined in HUVECs after treating ASR water extracts. The data showed that ASR extracts could induce the NO production in HUVECs dose-dependently, and sulfur-fumigation significantly reduced the ASR-induced NO production by ~40% at 1 mg/ml (Fig. 3A). These results were confirmed by another method of NO detection, using laser confocal fluorescent microscopy by labeling with a specific dye (Fig. 3B). Fig. 3C showed that ASR extracts triggered a progressive rise in intracellular NO production, as reflected by the increase of fluorescence intensity within a 15-min recording period. The increased trend, caused by S-ASR extracts, was not as obvious as that by ASR extracts: the difference could be about 10% of fluorescence intensity after 10 min of treatment.

Discussion

Most of the traditional herbal medicines need to undergo a post-harvested processing mainly based on traditional methods, as to convert raw material into a form readily used for prescription. In recent years, sulfur-fumigation is becoming a fast and cheap method being employed in post-harvested handling of many medicinal herbs (Jiang et al., 2013). According to a market survey, eight TCMs exhibited the most serious challenges with sulfur-fumigation, namely, ASR, Fritillaria Hupehensis Bulbus, Gastrodiae Rhizome, Paeoniae Radix Alba, Dioscoreae Rhizoma, Chrysanthemi Flos, Codonopssis Pilosulae Radix and Lycii Fructus. Although the usage of sulfur-fumigation in these herbs is very common, there are very limited data available with regard to the influence of this processing on herbal safety and efficacy. By using ASR as an example, we aimed to provide a comprehensive picture on the changes of chemical profiles, bioactivities and toxicity of ASR after sulfur-fumigation.

Having the standard method here, the S-ASR contained up to 138mg/kg of sulfur dioxide residue. This amount of sulfur dioxide was comparable to the previous works (Wang et al., 2009). Although sulfur dioxide is known to kill several microbes and insects, as well as sulfurous acid is serving as a bleaching and anticorrosion agent (Jiang et al., 2013), the residue in herbal medicine, nevertheless, is harmful to human health. The concentration of sulfur dioxide greater than 0.05% in herbal medicine was reported to cause an unpleasant taste sensation (Branen, 1985). Moreover, a long-term contact with sulfur dioxide, even at low dosage, induced cough, chest tightness, throat irritation and other respiratory symptoms (Liu et al., 2010; Wu et al., 2012). Therefore, the determination of sulfur dioxide residue has been included in Chinese Pharmacopeia 2010. In addition, the State Administration of Food and Drugs of China has formulated a set of standards for Chinese herbal medicines, stipulating that sulfur dioxide residue should not exceed 150 mg/kg for most of the herbs.

Although the sulfur dioxide residue of S-ASR prepared in this study was lower than the regulated limit of China at 150 mg/kg, the residue significantly decreased the chemical components and the biological properties of ASR. In view of our chemical analysis on ASR extracts, the data showed that sulfur-fumigation decreased the amounts of total organic acids, volatile oils and flavonoids. In the S-ASR extract, the amount of ferulic acid, a chemical marker for quality control of the herb, was remarkably reduced; meanwhile most volatile components were decreased, even disappeared. Similarly, it has been reported that the contents of major furocoumarins in Angelicae Dahuricae Radix were significantly reduced, and at least 60% of imperatorin was lost due to the sulfur-fumigation (Wang et al., 2009). Moreover, S-ASR had strong anti-cell viability effects against MCF-7 cell lines, but ASR did not have such effect. The biological properties of ASR were shown to have superior effects to those of S-ASR in many aspects, including anti-platelet aggregation, induction of NO production in cultured HUVEC cells and the estrogenic activity in MCF-7 breast cells. ASR showed better bioactivities and lower cytotoxic than S-ASR, which might be the result of two reasons. First, sulfur-fumigation changed the physical state of the herbs, such that which suppressed the chemical solubility of those active ingredients. The negative effects on the bioactivities could be a result of decreased chemical composition. Second, sulfur dioxide was harmful to human body, especially in respiratory system (Liu et al., 2010; Wu et al., 2012).

In the present study, the efficacy and safety of sulfur-fumigated ASR were systematically investigated. The results suggested that sulfur-fumigation was an unacceptable approach for processing herbal materials. Alternatives to sulfur-fumigation for the preservation of ASR should also be developed.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phymed. 2014.07.002.

Conflict of interest

We declare that no one of us have financial/commercial conflicts of interest.

Acknowledgements

This research was supported by the grants of the project of Hong Kong Chinese Materia Medica Standards by Hong Kong Government; Research Grants Council Theme-based Research Scheme (T13-607/12R), GRF (661110, 662911, 660411, 663012, 662713), The Hong Kong Jockey Club Charities Trust and Foundation of The Awareness of Nature (TAON12SC01) and 1TF (GHP/059/12SZ) to Karl Tsim. Janis Zhan was supported by Scholarship from Hong Kong Chiu Chow Chamber of Commerce and China Postdoctoral Science Foundation (2014M552188).

References

Bi, C.W., Xie, Q.H., Xu, L., Li, J., Cheung, A.W.H., Zhu, J.T.T., Zheng, K.Y.Z., Chen, V.P., Law, B.W., Choi, R.C., Wang, T.J., Dong, T.T.X., Tsim, K.W.K., 2010. Fo Shou San, an ancient herbal decoction prepared from Chuanxiong Rhizoma and Angelica Sinensis Radix, stimulates the blood functions. Planta Med. 76, 1525-1529.

Bo, K., 2008. Study on scientific decoction of traditional Chinese medicine from the view of pharmacy. Chin. Tradit. Pat. Med. 37,363-364.

Branen, A.F., 1985. Antimicrobials in Foods. Marcel Dekker Inc., New York, pp. 191-199.

Dong, T.T.X., Zhao, K.J., Gao, Q.T., Ji, Z.N., Zhu, T.T., Li, J., Duan, R., Cheung, W.H., Tsim, K.W.K., 2006. Chemical and biological assessment of a Chinese herbal decoction containing Astragali Radix and Angeiicae Sinensis Radix: determination of drug ratio in having optimized properties. J. Agr. Food Chem. 54, 2767-2774.

Dong, W.G., Liu, S.P., Zhu, H.H., Luo, H.S., Yu, J.P., 2004. Abnormal function of platelets and role of Angelica sinensis in patients with ulcerative colitis. World J. Gastroenterol. 10, 606-609.

Gao, Q.T., Cheung, J.K.H., Li, J., Chu, G.K.Y., Duan, R., Cheung, A.W.H., Zhao, K.J., Dong, T.T.X., Tsim, K.W.K., 2006. A Chinese herbal decoction Danggui Buxe Tang, prepared from Radix Astragali and Radix Angeiicae Sinensis stimulates the immune responses. Planta Med. 72, 1227-1231.

Jiang, X., Huang, L.F., Zheng, S.H., Chen, S.L., 2013. Sulfur fumigation, a better or worse choice in preservation of traditional Chinese medicine? Phytomedicine 20, 97-105.

Leung, K.W., Cheng, Y.K., Mak, N.K., Chan, K.K.C., Fan, T.P.D., Wong, R.N.S., 2006. Signaling pathway of ginsenoside-Rgi leading to nitric oxide production in endothelial cells. FEBS Lett. 580,3211 -3216.

Liu.J.J., Liu, X., Li, S.L., Cai, B.C., Cai, H., 2010. Current situation in studies on traditional medicinal materials and Yinpian by sulfur-fumigated process. Chin. Herbal Med. 8,1403-1406.

Lu, J.Q., He, D.L, Ruan, X.Y., Wang, Q., Xu. J.Q., Hu, X.Y., Cao, R.B., 2010. Study on the quality standards of total organic acids in Angelica roots. J. Hubei Univ. Chin. Med. 12,33-35.

Shao, M.L, 2010. Pharmacopoeia of the People's Republic of China (Part I). Chemical Industry Press, Beijing, China.

Wang, X.H., Xie, P.S., Urn, C.W., Yan, Y.Z., Yu, Q.X., 2009. Study of the destructive effect to inherent quality of Angeiicae dahuricae radix (Baizhi) by sulfur-fumigated process using chromatographic fingerprinting analysis, j. Pharm. Biomed. Anal. 49,1221-1225.

Wilasrusmee, C., Kittur, S., Siddiqui, J., Bruch. D., Wilasrusmee, S., Kittur, D.S., 2002. in vitro immunomodulatory effects of ten commonly used herbs on murine lymphocytes. J. Altern. Complement. Med. 8.467-475.

Wu, P.A., Li, Y., Li, Y.F., Liu, F.L., Jin, H., Du, S., 2012. The effect of sulfur-smoking on the total polysaccharides of Angelica Sinensis Radix. J. Gansu Coll. Tradit. Chinese Med. 1,364-367.

Yi, L.Z., Liang, Y.Z., Wu, H., Yuan, D.L, 2009. The analysis of Angelicae Sinensis Radix (Danggui). J. Chromatogr. A 1216,1991-2001.

Zhan, J.Y.X., Zhang, W.L., Zheng, K.Y.Z., Zhu, K.Y., Chen, J.P., Chan, P.H., Dong, T.T.X., Choi, R.C.Y., Tsim, K.W.K., Lau, D.T.W., 2013a. The wine treatment on Angelicae Sinensis Radix and Chuanxiong Rhizoma: chemical distinction using gas chromatography coupled with triple quadrupole mass spectrometry. Chin. Med. 8, 12-19.

Zhan, J.Y.X., Zheng, K.Y.Z., Zhu, K.Y., Zhang, W.L, Bi, C.W.C., Chen, J.P., Du. C.Y.Q., Dong, T.T.X., Lau, D.T.W., Tsim, K.W.K., 2013b. Importance of wine-treated Angelica Sinensis Radix in a herbal formula Si Wu Tang, in treating woman aliments. Planta Med. 79, 533-537.

Zhan, J.Y.X., Zheng, K.Y.Z., Zhu, K.Y., Bi, C.W.C., Zhang, W.L, Du, C.Y.Q., Fu, Q.. Dong. T.T.X., Choi, R.C.,Tsim, K.W.K., Lau, D.T.W., 2011. Chemical and biological assessment of Angelicae Sinensis Radix after processing with wine: an orthogonal array design to reveal the optimized conditions. J. Agr. Food Chem. 59. 6091-6098.

Zhang, R.Z., Yang, Q., Yim, A.P.C., Huang, Y., He, G.W., 2006. Role of NO and EDHF-mediated endothelial function in the porcine pulmonary circulation: comparison between pulmonary artery and vein. Vase. Pharmacol. 44,183-191.

Zhao, K.J., Dong, T.T.X., Tu, P.F., Song, Z.H., Lo, C.K., Tsim, K.W.K., 2003. Molecular genetic and chemical assessment of Angelica Radix (Danggui) in China. J. Agr. Food Chem. 51, 2576-2583.

Janis Ya-Xian Zhan (a,b), Ping Yao (b), Cathy Wen-Chuan Bi (b), Ken Yu-zhong Zheng (c), Wendy Li Zhang (b), Jian-Ping Chen (b), Tina Ting-Xia Dong (b), Zi-Ren Su (a), Karl Wah-Keung Tsim (b), *

(a) School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, China

(b) Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Clear Water Bay Road, Hong Kong, China

(c) Department of Biology, Hanshan Normal University, Chaozhou, Guangdong, China

Abbreviations: ASR, Angelicae Sinensis Radix; TCM, traditional Chinese medicine.

* Corresponding author. Tel.: +852 2358 7332; fax: +852 2358 1559.

E-mail address: botsim@ust.hk (K.W.-K. Tsim).

http://dx.doi.org/10.1016/j.phymed.2014.07.002

Table 1

The contents of total chemical parts in ASR and S-ASR.

Sample        Organic acids (a) (%)   Volatile oils (b) (mg/g)

ASR           2.15 [+ or -] 0.11      5.73 [+ or -] 0.06
S-ASR         1.83 [+ or -] 0.04 *    5.12 [+ or -] 0.08 *

Sample        Polysaccharides (c) (%)   Flavonoids (d) (%)

ASR           2.12 [+ or -] 0.08        0.72 [+ or -] 0.03
S-ASR         2.31 [+ or -] 0.04        0.52 [+ or -] 0.02 *

Sample        Sulfur oxide residue (e) (mg/kg)

ASR           --
S-ASR         138.3 [+ or -] 11.21 ***

Values are expressed in % or mg/g or mg/kg of dried powder, and
in means [+ or -] SD, where n = 5.

* p < 0.05.

*** p <0.001.

(a) Organic acids were determined according to Lu et al. (2010).

(b) Volatile oils were determined according to Shao (2010).

(c) Polysaccharides were determined according to Dong et al. (2006).

(d) Flavonoids were determined according to Dong et al. (2006).

(e) Sulfur oxide residue was determined according to Shao (2010).

(a-d) The extraction efficiency was >95%.

"--": Not detectable.

Table 2
Components of the volatile chemicals in ethyl acetate extracts of
different ASRs.

No.   RRI    Components (b)                    Amount (%)    No.   RRI
      (a)                                          (c)

                                                      S-
                                               ASR    ASR

 1    1218   3-Ethyl-3-methylheptane           0.02   --     24    1640
 2    1220   2-Methyl nonane                   0.22   0.10   25    1645
 3    1224   o-Cymene                          0.69   0.42   26    1651
 4    1228   Limonene                          0.63   0.21   27    1655
 5    1233   b-Pinene                          3.63   2.55   28    1661
 6    1246   4-Octanone                        0.09   --     29    1675
 7    1256   b-Myrcene                         1.06   0.43   30    1678
 8    1260   Heneicosane                       1.65   1.38   31    1683
 9    1266   Ferulic acid                      6.38   4.25   32    1687
10    1400   Tetradecane                       0.13   0.05   33    1692
11    1423   Caryophyllene                     0.24   --     34    1697
12    1429   Pentyl benzene                    0.57   0.35   35    1804
13    1434   Eicosane                          0.71   0.53   36    1809
14    1438   Di-tert-dodecyl disulfide         0.51   0.22   37    1816
15    1443   b-Linalool                        0.79   0.43   38    1849
16    1448   3-Butylidene-4-hydroxyphthalide   0.33   --     39    1852
17    1452   a,p-Dimethylstyrene               0.48   0.30   40    1866
18    1461   2,5-Di-tert-Butylaniline          0.93   0.41   41    1869
19    1466   Lignocerol                        0.12   0.12   42    1872
20    1470   4,8-Epoxyterpinolene              0.55   0.39   43    1925
21    1474   7-Hexyltridecan-1-ol              0.49   0.22   44    2014
22    1479   b-Funebrene                       0.62   0.31   45    2052
23    1603   b-Humulene                        0.12   --     46    2465

No.   Components                       Amount (%)

                                              S-
                                       ASR    ASR

 1    Butylphthalide                   4.35   2.47
 2    Pentadecane, 8-heptyl-           0.29   --
 3    Z-Butylidenephthalide            8.79   4.61
 4    b-Eudesmol                       0.13   --
 5    E-Butylidenephthalide            0.15   0.11
 6    Senkyunolide A                   3.14   2.43
 7    Muurola-4,11-diene               0.14   0.08
 8    Z-Ligustilide                    49.8   41.5
 9    1-Nonadecene                     0.56   0.36
10    Cryptone                         0.58   0.31
11    3,9-Diethyl-6-tridecanol         0.21   0.14
12    5-(2-Thienyl)-4-pyrimidinamine   0.19   0.13
13    g-Eudesmol                       0.27   0.10
14    E-Ligustilide                    8.30   4.55
15    6,7-Dihydroxyligustilide         0.12   0.09
16    Senkyunolide F                   0.18   0.10
17    Hexadecanoic acid                0.01   --
18    Senkyunolide H                   0.12   0.04
19    Senkyunolide I                   0.38   0.13
20    1-Octadecanol                    0.20   --
21    Methoxsalen                      0.22   0.12
22    Marmesin                         0.21   --
23    Lomatin                          0.70   0.53

(a) RRI: Related retention indices calculated against n-alkanes.

(b) Volatile components were obtained from ethyl acetate extraction
of ASR, as described in the Materials and methods, and the
extraction efficiency was >98%.

(c) The amount is referring to relative percentages of the total
peak areas in the original crude herb. A total of 46 chemicals were
identified, and the unknown peaks (not shown) covered 4% in ASR(n =
5).

"--": Not detectable.

Table 3
Quantitative assessment of four marker chemicals in water extracts
of ASR and S-ASR.

Chemical         Amount (a)

                 ASR                            S-ASR

Ferulic acid     305.24 [+ or -] 23.43 *, (b)   203.55 [+ or -] 15.37
Z-Ligustilide    266.28 [+ or -] 31.16          237.31 [+ or -] 21.08
Senkyunolide H    55.83 [+ or -] 7.28            42.35 [+ or -] 5.22
Senkyunolide I   243.95 [+ or -] 26.92 **        95.66 [+ or -] 10.56

(a) The water extracts of ASR and S-ASR were analyzed by HPLC. Values
are expressed in [micro]g/g dried materials and in mean [+ or -] SD,
where n = 3, each with triplicate samples.

(b) Statistical comparison was made with S-ASR.

* p < 0.05.

** p < 0.01.
COPYRIGHT 2014 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2014 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Zhan, Janis Ya-Xian; Yao, Ping; Bi, Cathy Wen-Chuan; Zheng, Ken Yu-zhong; Zhang, Wendy Li; Chen, Jia
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Sep 25, 2014
Words:4967
Previous Article:Silibinin, a novel chemokine receptor type 4 antagonist, inhibits chemokine ligand 12-induced migration in breast cancer cells.
Next Article:Mechanism of action of Rhodiola, salidroside, tyrosol and triandrin in isolated neuroglial cells: an interactive pathway analysis of the downstream...
Topics:

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters