Investigation of the unstable components of Achyranthes root using liquid chromatography-nuclear magnetic resonance/mass spectrometry (LC-NMR/MS) [3]

The crude drug, Achyranthes root (牛膝), originates from the roots of Achyranthes fauriei, and although there are many reports on the constituents of Achyranthes root, in addition to the long-known ecdysteroids such as inokosterone [4], specific dicarboxylic acids bound to the sugar moiety, such as Triterpene saponins, have been reported. The latter was first isolated from A. fauriei by Ida et al. in the form of methyl esters and named achyranthoside [5], whereas Yoshikawa et al. isolated the same compound from Beta vulgaris in the form of a free carboxylic acid and named it betavulgaroside [6]. These saponins have a structure in which a specific dicarboxylic acid is attached via an acetal to the sugar moiety attached to the hydroxyl group at C-3 of the oleanane skeleton and are considered to be relatively unstable. Thus, many unstable saponins and achyranthosides are present immediately after being harvested from the field; however, structural analysis of unstable components requires separation, purification, and immediate spectral analysis.

LC-NMR, in which NMR is directly connected to the HPLC, allows the NMR spectrum of a compound separated by HPLC to be measured directly, enabling the structural analysis of even unstable compounds immediately after separation (Fig. 1). The NMR spectrum is measured after the peaks of the target compounds separated in the HPLC unit are incorporated into the loop; however, for compounds without UV absorption, such as sugars and some terpenes, detecting the peaks of the target compounds is an issue. LC-NMR/MS, which combines LC-NMR with MS, uses a mass spectrometer to detect peaks eluted from HPLC, enabling the detection of compounds with no UV absorption, and the addition of molecular weight information facilitates compound estimation. The addition of NMR also made it possible to determine the structures of isomers that could not be determined by LC–MS. Thus, LC-NMR/MS complements the advantages of mass spectrometry and NMR spectroscopy to enable the structural analysis of samples that are difficult to analyze using conventional LC–MS or LC-NMR alone. Structural changes in the saponin components of Achyranthes fauriei roots after harvesting and drying under various temperature conditions were investigated using HPLC and LC-NMR/MS. As a result, it was deduced that significant changes in achyranthosides occur around 70 °C, and that the final degradation process takes place around the drying temperature of 70 °C, as shown in the Fig. 2. LC-NMR has the disadvantage of using either deuterium or a solvent as the mobile phase for the LC when it was first developed, which is costly; however, this disadvantage has been overcome in recent years by coupling it with SPE.

Fig. 1

Schematic overview of liquid chromatography-nuclear magnetic resonance (LC-NMR)

Fig. 2

Estimated achyranthoside degradation processes in Achyranthes roots [3]

Determination of unstable components of Leonurus herb using thin-layer chromatography/mass spectrometry (TLC/MS) [7]

The crude drug, Leonurus herb (益母草), is the above-ground part of Leonurus japonicus (Labiatae) during the flowering season, and TLC confirmed that significant differences in the leaf constituents occurred depending on the drying temperature. First, it was assumed that essential oil components, such as monoterpenes, were lost during drying, as this is a constituent of the leaves of Labiatae plants. A clear difference was observed in the GC–MS and TLC comparisons of the extracts immediately after extraction and after approximately six months (refrigerated sealed storage). The lack of a clear correlation between the GC–MS and TLC spots for the target compounds in this case led to a trial of 2D-TLC-MS. The target compound was estimated to be a diterpene based on the high-resolution MS results of the target spot in the 2D-TLC (Fig. 3). The assumption of loss due to drying temperatures caused by volatile components was disproven as diterpenes do not exhibit volatility. The compound was successfully isolated by conducting the entire preparative process at a low temperature, and its structure was eventually revealed by NMR to be a labdane-type diterpene, which decomposed almost completely to a ring-fused compound in approximately three days when heated at 40 °C (Fig. 4). Diterpene compounds in the starting materials are rarely found in marketable crude drugs.

Fig. 3

Left: thin-layer chromatography (TLC) of flowers, stems, and leaves at various drying temperatures (arrows indicate spots observed under low-temperature drying conditions for leaves). Right: 2D-TLC/MS results are shown (purple spots were presumed to be diterpenes based on high-resolution MS) [7]. (Modification of Figs. 1 and 3 from Reference 7)

Fig. 4

Estimated decomposition processes of the components of Leonurus japonica leaves. (Modification of Fig. 7 from Reference 7)

Change in constituents of Scutellaria root as a consequence of growth years and drying temperature conditions [8]

Scutellaria root (黄芩) is the root of Scutellaria baicalensis (Labiatae) and has medicinal properties such as anti-inflammatory, promotion of bile secretion, and antipyretic effects (Fig. 5). The main constituent is the flavonoid glycoside baicalin, whereas the other major constituents include baicalein, wogonoside, and wogonin. In preparation and processing, the best type of root has a round shaft with a heavy outer bark that is brownish with a yellow-green interior and is not decayed and hollow. All products marketed in Japan are produced in China. In recent years, the shortage of this plant in China has become a problem, and there has been a switch from wild to cultivated products, often using a method known as "free-range cultivation" to produce properties similar to those of wild plants. It has also been reported that the flavonoid content is higher in cultivated varieties. There is also the issue of the suspected causative crude drug of interstitial pneumonia in the recently problematic Kampo medicine shosaikoto (小柴胡湯); thus, the correlation between the constituents of Scutellaria root and wild and cultivated products is important.

Fig. 5

One-year-old root (left) and perennial (right) roots of Scutellaria baicalensis [8]

With the processing and preparation of Scutellaria root, the major flavonoid component baicalin is easily hydrolyzed to the aglycones baicalein and glucuronic acid by the enzyme baicalinase, which is present in plants. Therefore, the quality of Scutellaria roots is expected to change significantly during the processing and preparation stages. As mentioned previously, as the cultivation period of Scutellaria roots increases, the roots become old and hollow, resulting in a decline in quality. Therefore, a comparison was made between the composition of 1st-, 2nd-, and 3rd-year roots of the seedling cultivars and the roots from the divided plants.

The contents of the glycosides baicalin and wogonoside tended to decrease with increasing drying temperature when 1st, 2nd, and 3rd year roots were compared. The contents of their respective aglycone parts, baicalein and wogonin, tended to increase with increasing drying temperature, although there was considerable variation in baicalein. No significant differences were observed in the content of the C-glycoside, chrysin-6-C-arabinosyl-8-C-glucoside, which is presumed to be relatively less susceptible to cleavage by temperature or enzymes at any drying temperature.

Degradation process of perillaldehyde in Perilla herb and discovery of α-asarone (AS) [9]

Perilla herb (蘇葉) is the leaf and branch tip of Perilla frutescens var. crispa, and its odor component is perillaldehyde (PA); however, its structure contains an intramolecular aldehyde, which is assumed to rapidly transform into acetal in the presence of alcohol (Fig. 6). The extraction solution and storage conditions of Perilla herb were studied, and finally the identification test method and quantification method in The Japanese Pharmacopoeia were established; however, it was found that some Chinese samples in the process contained little PA and instead contained AS (Fig. 7). Perilla herbs have a phenylpropanoid-rich type (PP-type), but AS was not observed in comparison with domestic PP-type samples. The AS is the E-form, while the Z-form is a compound that is prohibited as a food additive by the FDA due to its mutagenic properties. This study led to the first detection of this compound in Perilla herbs.

Fig. 6

Degradation processes of perillaldehyde

Fig. 7

Perillaldehyde (PA) and α-asarone (AS) contents (mg) of Perilla herbs distributed in the Japanese market. (No.1–7; imported (China), No.8–10; domestic). Each value is the mean ± SE (n = 3)[9]