2.1 Analysis of Radix Angelicae Sinensis (Danggui)
2.1.1 Bioactivities and Chemistry
Radix Angelicae Sinensis, or named as Danggui in Chinese, appeared to be one of the most important and widely used traditional Chinese medicines (TCM). According to the research done by Liang et al. (2009), it is stated that Danggui is well known in treating various gynecological conditions that are generally difficult to be treated by conventional therapies. As the result, Danggui has been called the “female ginseng”. Danggui is useful for tonifying blood and treating irregular menstruation, improving blood circulation, relieving pain and alleviating constipation by lubricating the bowels (Cao et al., 2008). Besides that, the research done by Liang et al. (2009) claim that Danggui is also widely used to treat anaemia, cardiovascular disease and hepatic fibrosis.
Up to now, there are more than 70 chemical compounds has been extracted and identified from Danggui. These compounds can be categorized into a few gropus, which including essential oils (primarily those monomeric phthalides), phthalide dimers, coumarins, organic acids and their esters, polysaccharides, polyacetylenes, vitamins, amino acids, and others (Liang et al., 2009). However, Cao et al. (2008) concluded that the main pharmacological activities of Danggui are brought by the presence of essential oils and organic acids within it. Other than these low molecular weighted compounds, the polysaccharides present in Danggui also grab the attention of scientist because of its widespread pharmacological activities (Liang et al., 2009).
Phthalides are commonly found in plants, fungi and liverworts. Most of the plants that contained phthalides have been widely used as herbal remedies in traditional and folk medicines, dietary supplements and food flavorings. Study also shown that biological activities of these phthalides including actions on the central nervous system, anti-platelet aggregation and anti-thrombosis, cardiac function modulation and anti-angina, inhibition of smooth muscle cell proliferation, protection against cerebral ischemia and smooth muscle relaxation (Chan et al., 2007).
Based on the published research done by Liang et al. (2009), phthalides are one of the active compounds found in Danggui, with parent nucleus of phthalide. In all the Angelica species which are being examined by now, phthalides can be found in them and the differences among the species are given by the structures and relative amount of phthalides present in them.
Liang et al. (2009) stated that the polysaccharides extracted from Danggui have raised the attention of researchers because of their significant biological activities. Most of the drug effects of Danggui are related with its polysaccharides. The study reported that polysaccharide from Danggui consists of fucose, galactose, glucose, arabinose, rhamnaose, and xylose. According to Huang and Williams (1998, p. 316), polysaccharide of Danggui can improve hemopoiesis by directly or indirectly stimulating macrophages, fibroblasts, erythrocytes, granulocytes, and lymphocytes, and can lead to an increased secretion of some human growth factors from myscle tissues. Besides that, the polysaccharide also shows an immune stimulating activity and blood-tonifying effect.
188.8.131.52 Organic acids and their esters
Research done by Liang et al. (2009) stated that there were a series of organic acids can be found in Danggui, which have various bioactive effects. They reported that ferulic acid was isolated from Danggui for a long time ago and this compound can also be found in other plants. Ferulic acid has a simple chemical structure but it has extensive bioactivities that are related to the medicinal functions of Danggui. Thus, ferulic acid is widely used as one of the marker compounds to evaluate the quality of Danggui and its products.
The ester of ferulic acid, which is the coniferyl ferulate is also found in Danggui sample. Since coniferyl ferulate is likely to hydrolyze into ferulic acid and coniferyl alcohol, two new terms, ‘free ferulic acid' and ‘total ferulic acid' was introduced. Free ferulic acid indicates the natural content of ferulic acid in herb whereas total ferulic acid refers to the amount of ferulic acid in medicinal function. Total ferulic acid is recommended as one of the markers to evaluate the quality of Danggui (Liang et al., 2009).
2.1.2 Analysis of chemical components in Danggui
The chromatograms for Danggui sample was obtained by using an Agilent/HP 1100 series HPLC-diode array detection (DAD) system consists of a vacuum degasser, binary pump, autosampler, thermostated column compartment and DAD (Agilent, Palo Alto, CA, USA). Besides that, mass spectrometric measurements were obtained by using an Applied Biosystems/PE-SCIEX API 365 LC-MS-MS system with atmospheric pressure chemical ionization source (Applied Biosystems, Foster City, CA, USA). Branson 5120E-MTH ultrasonic bath (Branso Ultrasonic Corporation, CT, USA) was used for sample extraction (Chan et al., 2004).
Based on the experiment done by Chan et al. (2004), an Alltima C18 column (5 µm, 250 mm X 4.6 mm) with a suitable guide column (C18, 5 µm, 7.5 mm X 4.6 mm) was used for chromatographic analysis. The mobile phase used is composed of 1.0 % acetic acid in water (A) and acetonitrile (B) using a gradient program of 19 % (B) in 0-18 minutes, 19-100% (B) in 18-60 minutes and 100 % (B) in 60-75 minutes. The flow rate was 1.0 mL/min and column temperature was maintained at 30°C. DAD detector was set at 280 nm for obtaining chromatograms.
184.108.40.206 Solvent and chemicals
For sample preparation, analytical grade methanol (Labscan, Bangkok, Thailand) and formic acid (Unichem, Warsaw, Poland) were used in this experiment. Besides that, HPLC grade acetonitrile (Labscan, Bangkok, Thailand), deionized water obtained from a Milli-Q water system (Millipore, Bedford, MA, USA) and analytical grade acetic acid (Unichem, Warsaw, Poland) were used for preparation of mobile phase (Chan et al., 2004).
220.127.116.11 Reference compounds
Ferulic acid used was purchased from the Institute for the Control of Pharmaceutical and Biological Products of China (Beijing, China). For Z-ligustilide, it was extracted, isolated and purified from fresh roots of Danggui. Then, purified Z-ligustilide was identified by electron impact ionization (EI) MS, -NMR and -NMR spectrometric techniques. According to the percentage of total peak are by HPLC analysis, the purity of Z-ligustilide was found to be more than 98 % (Chan et al., 2004).
18.104.22.168 Sample preparation
Below is the general procedure done by Chan et al. (2004) to prepare sample solution of Danggui for HLPC analysis.
2.1.3 Results and discussion
In this experiment, 40 Danggui samples were analyzed. Based on the results, it shows that these samples exhibit consistent chromatographic patterns although the absorption intensity of some peaks was different. The Computer Aided Similarity Evaluation System was used to calculate the correlation coefficients of entire chromatographic patterns among the samples and simulative mean chromatogram was generated. The correlation coefficient of each chromatogram to their simulative mean chromatogram was 0.9810.017 (mean S.D., n = 40). From the observation, it was clearly seen that these chromatograms are associated with similar chemical properties or components in Danggui (Chan et al., 2004).
Table 2.1 Chemical compounds identified in HPLC fingerprint (Chan et al., 2004).
Liang et al. (2009) reported that the distinctive fragrance in Danggui is used to evaluate Danggui quality and grade. In their work, it was stated that modern researches shows that its fragrance is related to one of the major monomeric phthalides, which is the Z-ligustilide (compound 9). Thus, Z-ligustilide is commonly used as an important marker for evaluating Danggui's quality due to its bioactivity and it was found in relatively higher content.
Besides that, ferulic acid (compound 1), which was comparatively lower in Danggui, is also chosen as an additional marker in evaluating the quality of Danggui and its products. Therefore, both compounds 1 and 9 which fall in different chemical groups of compounds were chosen as characteristic compounds for detection in this study (Chan et al., 2004). Liang et al. (2009) reported that in herbs materials and products of their volatile oils, ligustilide (including E-ligustilide and Z-ligustilide) are the volatile components that is often analyzed by gas chromatography-flame ionization detector (GC-FID) or GC-MS.
However, ligustilides and some other phthalides may be easily isomerizes under high temperature because these compounds are thermally labile. There are several publications reported that ligustilides identified in fingerprints using HPLC-DAD or LC-MS is actually interfered by some other compounds. For example, determination of Z-ligustilide is affected by E-butylidenephthalide, Z-butylidenephthalide and other possible impurities in the published chromatographic conditions (Liang et al., 2009).
Research done by Chan et al. (2004) stated that in previous studies, organic solvents including hexane, aqueous methanol and ethyl acetate-methanol (70:30) were used to generate Danggui fingerprints in optimal extraction. Since methanol allow for effective extraction of a variety of compounds with different polarity, it becomes the first choice of extraction solvent in the present study. These compounds including coniferyl ferulate (compound 1), senkyunolide A (compound 5), ligustilide and so on. Other than this, interference caused by the presence of sugar in raw herbs could be reduced by carry out the extraction using methanol.
However, Chan et al. (2004) in their report stated that extraction using methanol leads to the unstabilization of compound 1 and 4 (Senkyunolide A). They observed that the peak height of compound 4 was found to be decreased while the peak for compound 1 was increasing during the storage period of sample solution. Chan et al. (2004) concluded that this is due to the hydrolyzation of compound 4 into compound 1. Furthermore, coniferyl alcohol might contribute for the change in HPLC chromatographic pattern. Then, they examined the extraction efficiency of methanol-formic acid at ratios of 99:1, 97:3, 95:5, 93:7 and 90:10 by ultrasonic extraction to find out the optimal ratio for extraction.
The stability of compound 1 and 4 extracted with methanol-formic acid at ratios of 95:5 was further evaluated by determining their peak areas after storage for 0-40 hours respectively. The recovery of compound 1 and 4 were found having relative standard deviation (R.S.D) of 1.16 and 2.1% (n = 8) respectively by comparing the chromatographic peak areas. This observation shows that both compound 1 and 4 were relatively stable in the extraction solvent of methanol-formic acid in ratio of 95:5 (Chan et al., 2004). Thus, in the study done by Chan et al. (2004), they have chosen the methanol-formic acid in ratio of 95:5 as the extraction solvent.
In reported literatures, concentration of Z-ligustilide in Danggui determined by various methods falls in the range from 1.26 mg/g to 37.7mg/g. Table 2.2 shows the variation in Z-ligustilide contents for Danggui which was analyzed by variety of methods in literatures (Liang et al., 2009).
Table 2.2 The variation in concentration of Z-ligustilide for Danggui analyzed by the different methods in literatures (Liang et al., 2009).
Among the variety of methods, HPLC and GC are the major instruments used for determination of Z-ligustilide. Based on the result in Table 2.2, content of Z-ligustilide determined by HPLC is much higher than those from GC. This indicates that GC is not very suitable for analysis of Z-ligustilide. The high temperature used in GC could lead to this problem (Liang et al., 2009).
Study done by Liang et al. (2009) reported that for analysis using HPLC, different sample preparations and HPLC analytical conditions will give different quantification of the sample. This is mainly caused by two aspects, which is the instrumental systematic error and artificial operational wrong doing. Thus, to prevent any detectable degradation of the phthalides and unacceptable fluctuation during the analysis, the sample preparation and HPLC analytical conditions should be strictly under controlled.
According to the published research done by Liang et al. (2009), GC instrument is mainly used to analyze the composition of monosaccharides of polysaccharide in Danggui. For GC analysis of sugars, it is crucial to convert them into volatilizable and stable derivatives. Trimethylsilyl derivatives and acetate derivatives are the two kinds of derivatives used in GC analysis of sugars. For trimethylsilyl derivatives, it can be separated perfectly in GC with moderate polar stationary phase. However, strong polar stationary liquids are the first choice for separation of acetate derivatives. In the analysis of composition of monosaccharides of polysaccharide, HPLC and ion chromatography (IC) are widely used for their high separation efficiency and simple operations.
Among the different methods mentioned above, HPLC gives the most accurate outcome compared to others. In a nutshell, hyphenation of different analytical techniques is required for the analysis of polysaccharides, which including pretreatment, extraction, purification, purity measurement, molecular weight measurement, constituent monosaccharides identification and structural analysis. Table 2.3 shows the hyphenation techniques for analysis of polysaccharides in Danggui (Liang et al., 2009).