Doi: 10. 1016/j chroma
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jiang2009
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3.1.2. Analysis of PhGsAs the major active components in Cistanche plants, PhGs are usually used as markers for quality evaluation of crude drugs or their corresponding formulations. 3.1.2.1. Analysis of PhGs by thin layer chromatography (TLC). Zhang first established a TLC identification method for acteoside (2), echinacoside (11) and cistanoside A (3) in Herba Cistanches by silica gel, using ethyl acetate–methanol–9% acetic acid (20:3:2) as developing solvent, and FeCl3 solution as coloring reagents [65]. This method was also used in Chinese Pharmacopoeia (2000) for the identification of acteoside (2) [8]. However, in Chinese Pharmacopoeia (2005), the adsorbent of thin layer chromatography has been changed from silica gel to polyamide in order to obtain much better separation effect, and methanol–acetic acid–water (2:1:7) has been used as developing solvent. Two major PhGs in Herba Cistanches, Echinacoside (11) and acteoside (2) could be simultaneously identified [9]. 3.1.2.2. Analysis of PhGs by Ultraviolet Spectrophotometry (UV). As mentioned above, cinnamoyl group often exists in the structures of PhGs, thus, PhGs could be determined directly by UV or by the colorimetric method after reacting with some coloring reagents [66–72]. However, as we known that the colorimetric method is not a reasonable approach for the accurate determination of chemical constituents in TCMs, due to its instability, poor reproducibility and huge testing error. Moreover, it is more tedious to pre-processing the samples detected by UV than by the other methods. Thus, now, UV method is not used for the quantitation of the pure com-
3.1.2.3. Analysis of PhGs by high-performance liquid chromatography (HPLC). Among all of the analysis methods, HPLC is the most frequently used one for the qualitation and quantitation of PhGs, and Table 4 summarizes the methods published on the analyses of PhGs by HPLC in literatures, which will be detailed as follows. 3.1.2.3.1. Analysis of PhGs in crude drugs, biological samples and complex products by HPLC. In 1995, Xu performed a comparison of the chemical constituents of C. deserticola with its substitute C. salsa. The HPLC analysis showed that the chemical composition of PhGs between these two species was similar, but their quantity was different [73]. In the same year, Moriya made a comparison among the chemical components of C. deserticola (Cd), C. tubulosa (Ct) and C. salsa (Cs), using seven PhGs as markers. The results showed that cistanosides A (3), C (5), and a trace amount of tubuloside A (17) existed in Cd and Cs, whereas only tubuloside A (17) existed in Ct, without cistanosides A (3) and C (5). Also, the total amounts of PhGs in Ct and Cs plants had been found to be more than that in Cd plants [28]. It is valuable that this paper has compared the differenceamongCistanchegenusthroughmulti-componentsanalysis; however, it is regretful that the quantitation of these seven PhGs was performed only by one point external standard method, without a systematic methodology validation. Tu made a qualitative analysis of six PhGs and a quantitative analysis of two PhGs in Cistanche genus by Rp-HPLC. The results showed that the chemical constituents of C. deserticola, C. salsa, C. salsa var. albiflora and C. tubulosa were similar, while those of C. sinensis were different from the others [10]. Wang established a method for the analysis of seven PhGs in C. deserticola, C. tubulosa and C. salsa with HPLC/MS/MS, and the results showed that the chemical ingredient distribution of the seven reference PhGs is different in each species [74]. The fragmentation pathways of the glycosidic linkages, the ester bond, and some interesting neural losses of PhGs were discussed in this paper [74] and in another one [75]. However, the advantage of HPLC–MS technique has not been demonstrated thoroughly in these analyses, because only the fragmentation mechanism of PhG standards was studied. Adopting these mechanisms to elucidate unknown PhG compounds to give more chemical information, and the fragmentation mechanism study of other skeleton compounds have been neglected.
[76]. Considering that it is a little tedious to determine two constituents in the same plant using two different solvents, a simpler Rp-HPLC method was established for simultaneous detection of echinacoside (11) and acteoside (2) [77]. Chen then adopted this method to determine the contents of echinacoside (11) and acteoside (2) in the wild and cultivated C. tubulosa [78]. The results showed that the content of echinacoside (11) was markedly higher than that of acteoside (2) in all the samples, and the content of these two active components in wild was higher than that in cultivated [78]. It deserves to mention that the sample preparation in this paper adopted the method of mixing multi-batches together, which means that the author has considered the obvious individual quality differences in Herba Cistanches. SincethewildCistanchespeciesareontheedgeofextinction,the planting of C. deserticola and C. tubulosa is being carried out in Xinjiang and Inner Mongolia, China. In order to give some theoretical guidance for the cultivation, different cultivated samples have been analyzed.CaodeterminedthecontentofPhGsinspringandautumn Fig. 2. Structures of benzanoid glycosides from Cistanche species. with LC–MS, in order to compare C. deserticola samples collected in different seasons. Four PhGs, i.e. echinacoside (11), acteoside (2), cistanoside A (3) and 2-acetylacteoside (1) were selected as markers, and their total content was observed to be higher in spring than in autumn [79]. Wang made a study on the chemical constituents variation at different growing times and different parts of the cultivated C. tubulosa, using the fingerprint and the content of echinacoside (11) as index. He found that the chemical distribution of PhGs was similar among different samples, but the quantity of each PhG, especially of echinacoside (11), was significantly different among various samples. After comparison, he concluded that the cultivating time of C. tubulosa should be over three years, and its harvest time must be controlled strictly before blooming. In this paper,thequalityofcultivatedplantswasfoundtobenobetterthan wild ones, so the author suggested that the cultivating technology should be promoted to improve the content of active compounds [80]. Yang studied the dry matter accumulation and echinacoside (11) content of C. tubulosa in Huabei plain. The result showed that the dry matter accumulation of C. tubulosa was in “S”-shaped variant, and the content of echinacoside (11) was the highest when C. tubulosa grew up for 5 months [81]. Other than these, fingerprint, a new technique focusing on the systemic and comprehensive characteristics of the analyzed samples, has also been used for the quality evaluation of Cis- Fig. 3. Structures of saccharide derivatives and alkaloids from Cistanche species. tanche plants. As mentioned above, Wang analyzed the chemical constituents and its variation in cultivated C. tubulosa by using fingerprint and other indicators [80]. Xie established a chromatographic fingerprint of C. deserticola by HPLC (Fig. 1) and used it to evaluate the difference of inherent qualities of samples from different habitats. Finally, notable differences were found in the quality of the samples from different habitats [82]. The same phenomenon was even observed during our research on the quality evaluation of C. deserticola, so later more serious concern should be paid to the quality consistency of Cistanche species. Except planting, cultivation by cells or callus is another attractive alternative to solve the resource shortage of Herba Cistanche. During the cultivation process, many analytical methods have been established to analyze the cultivated result and to optimize the cultivated conditions [83–88]. The corresponding analytical conditions have also been summarized in Table 4. Table 5Iridoids from Cistanche species As for the analysis of PhGs in complex products, Zhang reported a quantitation method of isoacteoside (12) in the total Cistanche glycosides capsules by HPLC [89]. Wu established a method for the analysis of acteoside (2) in Shenqiyinao capsules by HPLC [90], and Lei made a determination of echinacoside (11) in Congrong spirit by HPLC [91]. The key point of analyzing PhGs in the complex products is to avoid the disturbance of other components, so, pretreatment of the sample and optimization of the chromatographic conditions are usually necessary. 3.1.2.3.2. Metabolic analysis of PhGs by HPLC. In 2001, Lei first reported the metabolism process of PhGs in the gastro intestine of beagle dogs, and four metabolites, echinacoside (11), acteoside (2), isoacteoside (12) and 2-acetylacteoside (1) were isolated from feces by preparative HPLC [96]. In 2006, the metabolism of acteoside(2)andechinacoside(11),themainactiveconstituentsofHerba Cistanches were studied respectively, and their pharmacokinetics
a Cd: C. deserticola; Ct: C. tubulosa; Cp: C. phelypaea; Csa: C. salsa; Csi: C. Sinensis. b “a” means axial bond. c “e” means equatorial bond. and bioavailabilities in rats were analyzed [97,98,134]. Wu established a sensitive LC–MS/MS method with a simple solid-phase extraction for the detection of acteoside (2) in rat plasma and tissue homogenates for the investigation of bioavailability and brain distribution in freely moving rats [97,134]. Jia developed a rapid and simple HPLC coupling with UV method to determine the content of echinacoside(11)inratserum[98].However,thebioavailabilitiesof echinacoside and acteoside were found to be only 0.83% and 0.12%, respectively, which was contradictory to the significant biological effects of PhGs in animals [58–64]. In the future, further researches have to be made to elucidate the metabolic process and absorption mechanism of PhGs. 3.1.2.4. Analysis of PhGs by high-speed counter-current chromatography (HSCCC). HSCCC, a support-free liquid–liquid partition chromatography, eliminates irreversible adsorption of sample onto the stationary phase, and has been widely used in preparative separation of natural products. Compared with the traditional solid–liquid column chromatography, it yields higher recovery and efficiency. Lei applied HSCCC to the separation and purification of acteoside (2) and 2-acetylacteoside (1) from C. salsa with a quaternary two-phase solvent system composed of ethyl acetate–n-butanol–ethanol– water (4:0.6:0.6:5, v/v). HPLC analysis oftheCCCfractionsrevealedthatthetwoPhGswereover98%purity [99]. Later, Li established a method using two solvent systems, one consisting of ethyl acetate–ethanol–water (5:0.5:4.5, v/v/v), and the other of ethyl acetate–n-butanol–ethanol–water (0.5:0.5:0.1:1, v/v/v/v) to isolate PhGs from C. deserticola. Five PhGs, echinacoside (11), cistanoside A (3), acteoside (2), isoacteoside (12) and 2-acetylacteoside (1) were isolated and purified, and the purities of these isolated compounds were all above 92.5% as determined by HPLC [100]. These methods supplied good references for the future isolation and purification of PhGs’ standards. Download 1.52 Mb. Do'stlaringiz bilan baham: 
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