1.1 History of traditional Chine medicine (TCM)
Herbal medicines are substances originated from plant and they occurred naturally with minimal or without industrial processing for use in medicinal purposes. Herbal medicines are now obtaining considerable attention in worldwide healthcare because of their great medicinal and economic importance (Li et al., 2009).
In far Eastern countries, especially for the Chinese people, there is a long history of the usage of herbal medicine where they have utilized herbs and plants for the treatment of various diseases for more than 8000 years (Drasar and Moravcova, 2004, p.4). The natural products made from these herbs are now developed for various medicinal uses, such as immunosuppressive agents, hypocholesterolemic agents, enzyme inhibitors, antimigrane agents, antiparasitic agents and ruminant growth promoters (Chen et al., 2009).
According to the Rodale's Illustrated Encyclopedia of Herbs (1998, p. 316), the medicine systems in China, India (Ayurveda), and ancient Egypt are the oldest known systems of medicine in the world. The Chinese medicine system is widely accepted as one of the most complete and effective herbal traditions which has survived into modern times. The term Ben Cao (Herbal) in Chinese medicine means material medica, a book that including all aspects of natural medicaments. In ancient China, the Emperor Shen-nong, who is a great cultural hero and believed to live from 3737 to 2697 B.C., has great contribution in the work of the oldest Chinese pharmacopoeias.
Shen-nong is said to have compounded and tested hundreds of herbal preparation by himself. His work of Shen-nong Ben Cao Jing or Shen Nong's Materia Medica has formally published in the Han dynasty (100B.C.). This piece of work is the first official Ben Cao in Chinese history and it consists of 365 different natural medicaments. These medicaments are divided according to their sources, which including 237 medicaments from botanical, 65 from animal, 43 from mineral and 20 from unknown origin (Hou and Jin, 2004, p. 37).
In China, herbal medicines are still a main part of their medical system, such as the traditional Chinese medicine (TCM) in their country (Li et al., 2009). TCM is a healthcare system based on human experiences which are accumulated for several thousand years. There are more than 500 examples of crude drugs from plants and 400 TCMs which are worldwide used and are recorded in the Chinese Pharmacopoeia (Drasar and Moravcova, 2004, p.4).
Chromatographic analysis of traditional Chinese medicine
Based on the research by Li et al. (2009), they conclude that the effects of Chinese medicinal herbs are probably given by their chemical compounds and these compounds are called effective compounds. Since the effects of these herbs is brought about by their chemical constituents, thus the chemical analysis of these herbs is important for determining chemical components present in them and to determine which ingredients give the therapeutic effect.
To ensure the efficacy of these herbs, the reproduction and the stability of the effective compounds are important. The efficacy evaluation of Chinese medicinal herbs is mainly done according to its pharmacological effects and chemical research. The purpose of doing this is to determine the effective compounds of the crude medicinal materials and thus the qualitative and quantitative analytical methods can be established. As the result, the quality of Chinese medicinal herbs can be assessed by this way (Li et al., 2008).
However, most of the crude plant extracts and their derivative products are a complex mixture of different phytochemical constituents. Chen et al. (2009) reported that these herbs might contained more than hundreds of active components which are chemically different. The presences of these compounds make the quality control of crude drugs and medical preparation become very difficult. Besides that, Drasar et al. (2004) stated that there are only one or a few compounds are responsible for the beneficial or hazardous effects of these drugs.
Therefore, the methods and extraction techniques which involved in identification and quantitative analysis of the active compounds have to be very efficient for the purpose of drug standardization. The main techniques applied in this field are chromatography. This is because they have powerful separation efficiency together with sensitive detection (Drasar et al., 2004). Li et al. (2008) reported that there are different analytical techniques which have been developed for the analysis of complex constituents in these herbs or its products, which including gas chromatography (GC), thin layer chromatography (TLC), liquid chromatography-mass spectrometry (LC-MS), high speed counter-current chromatography (HSCCC) and high-performance liquid chromatography (HPLC).
According to Fong et al. (2006, p. 18), TLC procedures posses advantage of providing simple, rapid and useful characteristic profile patterns and it is inexpensive to use. However, its disadvantage is limited resolving power and quantitative data for minor constituents are hard to be obtained. GC is able to provide high resolution of the more volatile polar complex mixtures. Unfortunately, it provides limited value in the case of non-volatile polar components such as the polar polyhydroxylated and glycosidic compounds. Compared to TLC and GC, HPLC is able to resolve complex mixtures of polar and non-polar components and it has become the choice of chromatographic method for qualitative and quantitative analysis of botanical extracts and products.
In this review, the chemical analysis of three selected TCMs (Danggui, Danshen and Gan-Cao) is mainly focused on the use of HPLC due to their advantages in the chemical analysis of herbal medicines. According to Ye and Zhang (2009), analysis with HPLC gives high sensitivity, reproducibility, good resolution and linearity. Besides that, HPLC is able to analyze multiple constituents present in medicinal herbs. Up to date, HPLC has been widely in qualitative and quantitative analyses of many TCMs. A schematic diagram of HPLC equipment.
Cheng, Leung and Xue (2003, p. 117) stated that HPLC is a general term of liquid chromatography and its separation modes including size exclusion, ion exchange, hydrophobic interaction and reversed phase. Different separation modes exhibit different separation mechanism for different applications in separating biomolecules. Among these modes, reverse phase chromatography (RPC) poses the excellent resolving and separation power. The availability of volatile mobile phase has made RPC a favorable method for both the analytical and preparative separations of herbal active components (including peptide, protein, polysaccharide and metabolites). HPLC is originated from classical LC but it uses a high pressure and super-packing gel matrix system to provide a high performance (rapid) and high-resolution (good separation) separation process.
HPLC fingerprinting has been employed as a fast and reliable method for authentication of herb and herbal product. This method is dependent on the chemical components (chemical markers) in the herb and herbal product to generate a fingerprint (chromatographic map) (Cheng et al., 2003). HPLC is able to detect most of the chemical compounds in the herb because it is equipped with different mobile phases and detectors. Among these chemical markers, some of them are herb-specific and it is effective to identify the herb and herbal product. Therefore, HLPC became a powerful tool for the quick characterization of natural product extracts when it is couples with a variety of detections (Li et al., 2008).
For an example, Cheng et al. (2003) has mentioned that the combination of HPLC and ultra-violet (UV) photo-diode array detector can be use to check the purity of resolved chromatographic peak by comparing the spectrophotometric spectrum with the identified marker. This method is based on the internal property of the single chemical marker that has an unique and comparable spectral characteristic.
Fong et al. (2006) stated that a range of analytical detection techniques including ultra-violet (UV) spectroscopy and mass spectrometry (MS) can be coupled with HPLC to generate a fingerprint of the botanical product and/or to quantify the concentration of one or more of the active chemical markers. UV detection is normally available in most laboratories and it is carried out with a single or dual wavelength, or a full spectrum (e.g. photo-diode array) detector. This is the most suitable technique for routine analysis of components that consists of a UV-active chromophore.
Besides that, HPLC can also be coupled with a mass spectrometer (MS) to calculate the molecular weight and identify the structure of the chemical marker (Cheng et al., 2003). The combination of HPLC with MS has been increasingly used for herbs analysis. The advantage of this combination is that the compound's mass can be determined by the mass spectrometer using exact mass measurement as the compound elutes from the system. Then, structurally significant fragmentation can be detected and this provides the specific identification of the eluting peak (Fong et al., 2006).
Ye et al. (2009) in their published research reported that recently a powerful technique called HPLC-diode array detection (DAD) has been extensively used in analyzing plant extracts because it is able to systematically profile the composition of samples and focuses on the identification and consistency assessment of the components found in the samples. However, a reliable and convincing chromatographic fingerprint should have most of its peaks assigned, especially those corresponding to the active constituents and toxic ingredients.
Unfortunately, this technique could only provide very limited structural information such as UV spectrum and the standard compounds which are necessary for identification of individual constituents are unavailable for most of the cases. As the consequence, isolation and purification from crude plant extracts of pure compounds (more than 90% purity) for nuclear magnetic resonance (NMR) identification was needed before it can serve as reference compounds. The whole procedure could be tedious and this appears to be the disadvantages of using this technique (Ye et al., 2009).
Since TCMs has gained increasing attention and it has been extensively used for therapeutic purposes, thus the comprehensive quality control is crucial to ensure the efficacy and safety in clinical use. In this review, the focus is on the qualitative analysis of three selected TCMs including Danggui, Danshen and licorice by using chromatographic methods.