Therapeutic components possess direct therapeutic effects of a herbal medicine. They may be used as chemical markers for both qualitative and quantitative assessments.

Originated from the bulbs of Fritillaria species (family Liliaceae), Bulbus Fritillariae (Beimu) is commonly prescribed as an antitussive and expectorant in Chinese medicine practice. Five different Bulbus Fritillariae derived from nine Fritillaria species are documented in the Chinese Pharmacopoeia 3. Isosteroidal alkaloids of Bulbus Fritillariae, including verticine, verticinone and imperialine, were identified as the major therapeutic components that account for the antitussive effect 789. Therefore, isosteroidal alkaloids were selected as the chemical markers for the quality assessment of Bulbus Fritillariae using a series of chromatographic techniques such as pre-column derivatizing gas chromatography – flame ionization detection (GC-FID), direct GC-FID, gas chromatography – mass spectrometry (GC-MS), pre-column derivatizing high-performance liquid chromatography – ultraviolet detection (HPLC-UV), high-performance liquid chromatography – evaporative light scattering detection (HPLC-ELSD) and high-performance liquid chromatography – mass spectrometry (HPLC-MS) methods 10.

Artemisinin from Herba Artemisiae Annuae (Qinghao) is another example of therapeutic component. Herba Artemisiae Annuae is well known for its potent anti-malarial activity 11. Artemisinin inhibits Plasmodium falciparum and Plasmodium vivax, two pathogens that cause malaria 1213. Artemisinin is now used as a chemical marker in HPLC-ELSD 14, GC-FID 15 and GC-MS 1516 for assessing the quality of the plant (parts and whole) at various stages 15, including the green and dead leaves of the plant 16.

Bioactive components are structurally different chemicals within a herbal medicine; while individual components may not have direct therapeutic effects, the combination of their bioactivities does contribute to the therapeutic effects. Bioactive components may be used as chemical markers for qualitative and quantitative assessment.

According to Chinese medicine theories, Radix Astragali (Huangqi), derived from the roots of Astragalus membranaceus (Fish.) Bge. or A. membranaceus var. mongholicus (Bge.) Hsiao, is used to reinforce qi. Isoflavonoids, saponins and polysaccharides of Radix Astragali showed pharmacological actions in immune and circulatory systems, which were consistent with the Chinese medicine indications 17. These bioactive components, including isoflavonoids and saponins, were used simultaneously in the evaluation of the quality of Radix Astragali 181920.

Synergistic components do not contribute to the therapeutic effects or related bioactivities directly. However, they act synergistically to reinforce the bioactivities of other components, thereby modulating the therapeutic effects of the herbal medicine. Synergistic components may be used as chemical markers for qualitative and quantitative assessment.

The products of St John's wort (Hypericum perforatum L.) are popular for treating mild depression 21. Butterweck et al. reviewed the research progress on the phytochemistry and pharmacology of St John's wort 2223. Naphthodianthrone, hypericin, and hyperforin (a phloroglucinol derivative) were identified as the major components that contribute to the pharmacological activities of St John's wort. Rutin, a ubiquitous flavonoid of natural products, demonstrated synergistic antidepressant actions in St John's wort 24. In a forced swimming test on rats, extracts of St John's wort with various chemical profiles were tested, among which the extract containing about 3% of rutin showed positive effects, whereas the extracts containing less than 3% of rutin were inactive. The extracts became active when the level of rutin was increased to about 3%. However, rutin alone did not show any effects under the same conditions 24. These results suggest that chemicals in St John's wort work synergistically to achieve the antidepressant effects. Therefore, naphthodianthrones, phloroglucinols and flavonoids may be used as chemical markers for the quality control of St John's wort 2526272829.

While characteristic components may contribute to the therapeutic effects, they must be specific and/or unique ingredients of a herbal medicine.

Terpene lactones in the leaves of Ginkgo biloba L. (Yinxing) exemplify characteristic components. EGb 761, a standardized leave extract of Ginkgo biloba is a well defined product for the treatment of cardiovascular diseases, memory loss and cognitive disorders associated with age-related dementia 30. Flavonoids and terpene lactones are responsible for the medicinal effects of EGb 761 31. Flavonoids, terpene lactones including ginkgolides A, B and C, and bilobalide are chemical markers for the quality control of Ginkgo biloba leave extracts 31323334. EGb 761 contains 6% of terpene lactones (2.8–3.4% of ginkgolides A, B and C, and 2.6–3.2% of bilobalide) and 24% of flavone glycosides. Aglycons are primarily quercetin, kaempferol and isorhamnetin.

Valerenic acids, the characteristic components of valerian derived from the roots of Valeriana officinalis L., have sedative effects and improve sleep quality 3536. Valerenic acids are used as chemical markers to evaluate the quality of valerian preparations although their sedative effects have not been fully elucidated 37. These chemical markers are also used for studying in vitro release of coated and uncoated tablets 38 and stability test for valerian ground materials and extracts 39.

Main components are the most abundant in a herbal medicine (or significantly more abundant than other components). They are not characteristic components and their bioactivities may not be known. Main components may be used for both qualitative and quantitative analysis of herbal medicines especially for differentiation and stability evaluation.

Four well-known Chinese herbal medicines derived from the genus Panax, namely (1) Radix et Rhizoma Ginseng (Renshen), (2) Radix et Rhizoma Ginseng Rubra (Hongshen), (3) Radix Panacis Quinquefolii (Xiyangshen) and (4) Radix et Rhizoma Notoginseng (Sanqi) 39, contain triterpenoid saponins including ginsenoside Rg1, Re, Rb1 and notoginsenoside R1 as their main components 404142. Through qualitative and quantitative comparison of the saponin profiles 434445, these four herbs can be differentiated from one another 4246.

Herba Epimedii (Yinyanghuo), derived from the aerial parts of Epimedium brevicornum Maxim., E. sagittatum (Sieb. et Zucc.) Maxim., E. pubescens Maxim., E. wushanense T. S. Ying or E. koreanum Nakai, has been traditionally used to reinforce kidney-yang (shenyang), strengthen tendons and bones, and relieve rheumatic conditions 47. Flavonoids including epimedin A, B, C and icariin are the main components of Herba Epimedii 48. A 24-month randomised, double-blinded and placebo-controlled clinical study showed that flavonoids from Herba Epimedii exerted beneficial effects on preventing osteopenia in late post-menopausal women 49. According to the Chinese Pharmacopoeia (2005 edition), total flavonoids and icariin are used as chemical markers for Herba Epimedii 47. In a recent study, Chen et al. developed an HPLC method to simultaneously quantify up to 15 flavonoids, of which epimedin A, B, C and icariin were selected as chemical markers for the quality assessment of the Epimedium species documented in the Chinese Pharmacopoeia (2005 edition) 50.

Correlative components in herbal medicines have close relationship with one another. For example, these components may be the precursors, products or metabolites of a chemical or enzymatic reaction. Correlative components can be used as chemical markers to evaluate the quality of herbal medicines originated from different geographical regions and stored for different periods of time.

According to the Chinese Pharmacopoeia (2005 edition), only psoralen and isopsoralen (Figure 1) are used as chemical markers for assessing the quality of Fructus Psoraleae (Buguzhi) 51. Recently, our group identified glycosides, psoralenoside and isopsoralenoside (Figure 1) found them useful as the chemical markers for Fructus Psoraleae 52. The levels of psoralen and isopsoralen are inversely correlated to the level of glycosides psoralenoside and isopsoralenoside 53. When extracted with 50% methanol, samples had high levels of psoralen and isopsoralen and a minute amount of psoralenoside and isopsoralenoside. When moistened with 100% methanol, dried and extracted with 50% methanol, samples contained all four components. Psoralen and isopsoralen may be the enzymatic reaction products of psoralenoside and isopsoralenoside respectively. After incubation of a psoralenoside and isopsoralenoside solution with β-glucosidase at 36°C for 24 hours, the amount of psoralen and isopsoralen became detected. Psoralenoside and isopsoralenoside, therefore, may be used as chemical markers for the quality control of Fructus Psoraleae 53.

Traditional Chinese medicine literature and modern toxicological studies documented some toxic components of medicinal herbs. For instance, aristolochic acids (AAs) and pyrrolizidine alkaloids (PAs) may cause nephrotoxicity and heptotoxicity respectively 5455.

The use of three herbal medicines that contain AAs, namely Radix Aristolochiae Fangchi (Guangfangji), Caulis Aristolochiae Manshuriensis (Guanmutong) and Radix Aristolochiae (Qingmuxiang), have been prohibited in China since 2004 56. These three herbs were traditionally used to relieve pain and treat arthritis. Radix et Rhizoma Asari (Xixin) was traditionally sourced from the whole plants of Asarum heterotropoides Fr. Schmidt var. mandshurcum (maxim.) Kitag., A. sieboldii Miq. var. seoulense Nakai or A. seiboldii Miq. Its medicinal use has now been officially limited to the roots and rhizomes because the roots and rhizomes contain a much lower level of AAs than the aerial parts 57. AAs are now used as markers to control nephrotoxic herbs and proprietary herbal products 58596061.

There are over 6,000 plant species containing PAs which can cause hepatic veno-occlusive disease 62. There have been various PA restriction guidelines issued by government bodies and organisations. According to the WHO guidelines issued in 1989, the lowest intake rate of toxic PAs that may cause veno-occlusive disease in human is 15 μg/kg/day 63. In 1993, the American Herbal Products Association (AHPA) alerted its members to restrict the use of comfrey, a herbal medicine that contains PAs for external applications. In 2001, the Food and Drug Administration (FDA) of the United States recalled comfrey from all dietary supplements 64. In 2007, the Medicines and Healthcare Products Regulatory Agency (MHRA) of the United Kingdom advised all herbal interest groups to withdraw all unlicensed proprietary products that may contain hepatotoxic PAs from Senecio species 65. PAs are thus markers for detection of hepatotoxic components in herbs 66676869707172.

General components are common and specific components present in a particular species, genus or family. These components may be used with 'fingerprints' for quality control purposes.

Lobetyolin, a polyacetylene compound, is used as a marker for Radix Codonopsis (Dangshen) in thin-layer chromatography (TLC). Radix Codonopsis is derived from the roots of three Codonopsis species, namely Codonopsis pilosula (Franch.) Nannf., C. pilosula Nannf. var. modesta (Nannf.) L. T. Shen or C. tangshen Oliv. 73. Our study showed that other five Codonopsis species that are common substitutes of Radix Codonopsis also contain lobetyolin. They are C.tubulosa Kom., C.subglobosa W. W. Smith, C. clematidea (Schynek) C. B. Cl., C.canescens Nannf. and C.lanceolata (Sieb. et Zucc.) Trautv. Moreover, the roots of Campanumoea javanica Bl. and Platycodon grandiflorum (Jacq.) A. DC. (familyCampanulaceae), which are easily confused with Radix Codonopsis, also contained lobetyolin. Therefore, lobetyolin may be used as a general chemical marker coupled with HPLC-UV 'fingerprints' to differentiate Radix Codonopsis from its substitutes and adulterants 74.

As a chemical component may have more than one attribute, a component may belong to multiple categories. For example, ginkgolides A, B and C, and bilobalide are not only characteristic components, but also bioactive components of Ginkgo biloba. Ginsenoside Rg1, Re and Rb1 are both main and bioactive components of Panax ginseng. Categories of chemical markers are summarised in Table 1.

Derived from the resin of Garcinia hanburyi Hook f. (family Guttiferae), gamboges (Tenghuang) has been used in China to treat scabies, tinea and malignant boil 75, and in Thailand to treat infected wounds, pain and oedema 76. Characteristic polyprenylated caged xanthones including gambogic acid, gambogenic acid were isolated as the main and bioactive components of gamboges 77. In our previous study, an adulterant of gamboges was differentiated from the authentic sample by an HPLC-UV method using eight caged xanthones as chemical markers. The chromatogram of gamboges had all eight compounds, while the adulterant showed none of them (Figure 2).

Radix Stemonae (Baibu) is a traditional antitussive and insecticidal herbal medicine derived from the roots of three Stemonae species, namely Stemona tuberosa Lour, S. sessilifolia (Mig.) Mig. and S. japonica Mig. 78. The Stemona alkaloids were pharmacologically proven to be responsible for the antitussive and insecticidal effects of Radix Stemonae 7980818283. In our studies, we observed that the chemical profiles of these three Stemona species varied greatly. Croomine-typealkaloids such as croomine were detected in all three species, while protostemonine-type alkloids such as protostemonine and maistemonine were detected in S. japonica and S. sessilifolia. Moreover, stichoneurine-type alkaloids such as stemoninine, neotuberostemonine and tuberostemonine were only found in S. tuberosa. Stemona alkaloids may be used as markers to discriminate the three Stemona species 8485.

Rhizoma Chuanxiong (Chuanxiong) is one of the traditional Chinese medicinal herbs frequently used to treat cerebro- and cardio-vascular diseases. Various chemical compounds have been isolated and identified from Rhizoma Chuanxiong, including ferulic acid, senkyunolide I, senkyunolide H, senkyunolide A, coniferyl ferulate, Z-ligustilide, 3-butylidenephthalide, riligustilide and levistolide A 86878889909192. These chemicals have multiple biological activities which may contribute to the therapeutic effects of the herb 93949596979899. Thus, major bioactive components senkyunolide A, coniferyl ferulate, Z-ligustilide, ferulic acid, 3-butylidenephthalide, riligustilide and levistolide A may be used as markers to select the best harvesting time. A previous study using these markers suggested that the best harvesting time for Rhizoma Chuanxiong is from mid April to late May 100.

In our studies on the chemistry and antitussive activities of Radix Stemonae 101102103, four chemical profiles of S. tuberosa of different geographic sources were characterised using croomine, stemoninine, neotuberostemonine or tuberostemonine as markers 102. Moreover, the total alkaloid of S. tuberosa exhibited various levels of antitussive activities in a citric acid-induced guinea pig cough model 82. Croomine, stemoninine, neotuberostemonine and tuberostemonine all possess significant antitussive activities, however, croomine (croomine type) act on the central nervous system pathway, whereas the other three alkaloids (stichoneurine type) acted on the peripheral pathway of cough reflex 82. In terms of safety, those containing stichoneurine-type alkaloids are more suitable Radix Stemonae sources than those containing croomine as the major component. Croomine, stemoninine, neotuberostemonine, and tuberostemonine may be used as markers to confirm the collection sites for S. tuberosa (e.g. Shizhu and Erbian in Sichuan province, Masupo and Baoshan in Yunnan province, Shanglin in Guangxi province or Yudu in Jiangxi province, China) which contains higher levels of stemoninine, neotuberostemonine or tuberostemonine, and a low level of croomine 102.

In general practice, most herbs must be processed to reduce toxicity. For example, Radix Aconiti (Chuanwu) derived from the root of Aconitum carmichaeli Debx 104, is a well known toxic and potent herbal medicine. Cases of intoxication and even death were reported in China and Japan 105106107. The herb is processed by boiling in water for 4–6 hours or steaming for 6–8 hours 108. The toxic components of this herb are diester-diterpene Aconitum alkaloids, such as aconitine, mesaconitine and hypaconitine. When processed, these alkaloids hydrolyse into their respective analogues collectively known as monoester alkaloids 109. Monoester alkaloids are much less toxic than diester alkaloids 110. These six Aconitum alkaloids may be used to evaluate Radix Aconiti 111.

Traditionally, Radix Astragali is graded according to its diameter, length and physical appearance. Isoflavonoids and saponins were recognised as the major bioactive components attributed to the therapeutic effects of Radix Astragali. These two types of components were used to evaluate the quality of Radix Astragali in our study, in which 25 samples of Radix Astragali were collected from four cultivating regions in China 112. The contents of 11 main isoflavonoids and three major astragalosides were analysed. Contrary to the traditional notion, thin roots contained more astragalosides than thick ones. Moreover, the content of astragalosides in the bark were over 74-fold higher than that in the xylem. There was no difference in isoflavonoid content between the thin and thick roots, or the bark and the xylem. These results suggest that the thin root Radix Astragali is of better quality 112.

Qingfu Guanjie Shu (QGS, also known as JCICM-6) capsule is a proprietary product to treat rheumatoid arthritis. QGS has significant suppressive effects on arthritic 113 and acute inflammation in animal models 114. The formula of QGS is composed of five anti-inflammatory and anti-arthritic herbs, namely Caulis Sinomenii, Radix Paeoniae Alba, Cortex Moutan, Rhizoma Curcumae Longae and Radix Aconiti Lateralis Preparata. Sinomenine, paeoniflorin, paeonol, cucurmin and hypaconitine are the major constituents of the five herbs respectively, all of which have significant in vivo and in vitro effects including anti-inflammation, analgesia, anti-arthritis and immunosuppression 115116117118. Thus, HPLC methods were developed with these five chemicals as markers in the manufacturing process of QGS 111119. Immediately after production, three batches of QGS products were examined and no remarkable variations in terms of the five chemicals were found 119.

Stability test is used to evaluate product quality over time and determine recommended shelf life. The five markers mentioned above were used as indicators to evaluate the product stability of QGS. For example, the accelerated conditional stability test was carried out with four time points in a period of three months in chambers at 40 ± 2°C and 75 ± 5% of humidity. The five markers were quite stable during the period; only paeonol showed a slight decrease of 5% immediately after production 119.

Toxic components may be used as chemical markers in screening methods, e.g. rapid diagnosis of acute hidden aconite poisoning in urine samples by HPLC-MS 107. Five pairs of aconite alkaloids (i.e. aconitine and benzoylaconitine, yunaconitine and deacetyl-yunaconitine, mesaconitine and benzoylmesaconitine, hypaconitine and benzoylhypaconitine, and crasscauline A and deacetyl-crasscauline A) were chosen as markers to develop a LC-MS screening method. The screening method was applied to a clinical investigation of 15 cases of suspected herbal poisoning, of which 11 cases were tested by LC-MS 107.

The components responsible for the therapeutic effects may be investigated as lead compounds for new drug discovery. Gambogic acid (Figure 2), one of the major caged xanthones of gamboges, is used as a chemical marker for quality control and safety evaluation of gamboges 120121122123. As its cytotoxicity is attributed to cell apoptosis induction 124125126127128, gambogic acid is a potential lead compound for new anti-cancer drugs 124125126 and has recently been approved by the State Food and Drug Administration of China for clinical trials of cancer treatment 129.

At present, some herbs do not have markers for quality control. According to the Chinese Pharmacopoeia (2005 edition), only 281 out of 551 herbs have one or two chemical markers for quality control. A total of 282 chemical markers have been listed 3 for qualitative or quantitative analysis of herbs. Moreover, many herbal medicines share the same chemical markers for quality control (Table 2).

Inconsistency in quality is a common prolem among the commercially available chemical markers. The overall quality of chemical markers may be influenced by various physical and chemical factors such as described and exemplified below.

Gambogic acid is one of the major characteristic caged xanthones in gamboges and is therefore an ideal chemical marker for quality control. However, during the isolation and purification processes, gambogic acid can be transformed by the nucleophilic addition of methanol to the olefinic bond at C-10 when stored in methanol solution at room temperature (Figure 3) 130.

Bioactive components of Radix Astragali, isoflavonoids have been used as chemical markers in the quality control of the herb 181920. The relative contents of flavonoids varied significantly among samples obtained with different extraction methods (Figure 4). The chemical profiles of samples with microwave assisted extraction, reflux or Soxhlet were compared. Isoflavonoid glycoside malonates were converted into their respective glycosides or flavonoid glycons (Figure 5) during the prolonged conventional extraction procedures with Soxhlet and reflux at higher temperature 18.

Cinnamaldehyde is the chemical marker for the quantitative evaluation of Cortex Cinnamomi 131. This compound is light-sensitive. When exposed to light at room temperature for six hours, 10% of the content of cinnamaldehyde was lost; and 36 hours later, only 25% was left (Figure 6). Later studies indicated that this compound gradually transformed to crystallized cinnamic acid when exposed to light (Figure 7).

Stereoisomers of some phytochemicals often co-exist in nature and are sometimes mistakenly isolated as 'pure' compounds. Most stereoisomers possess very different bioactivities from each other. Gambogic acid, a chemical marker for the gamboges 120121, has two epimers i.e. 2(R)-gambogic acid and 2(S)-gambogic acid in nature 77132. The epimers have different inhibitory effects on CYP2C9 which is an important enzyme in the liver. The two epimers should be used as separate chemical markers 77. The two epimers were eluted as a fine peak on a C₁₈ column; they can be separated on a C₈ column under optimized conditions 122123.

Spectral complexity of a compound sometimes leads to confusion on its purity. Biflavonoids, for instance, always show substantial spectral complexity at the dimeric level due to hindered rotation between the flavanone and the flavanonol moieties around the C-3/C-8" axis. GB1 (3",4',4"',5,5",7',7"-heptahydroxy-3,8"-biflavanone) is a major biflavanoids of Garcinia kola Heckel which is a plant native to Nigeria and Ghana and is often chewed by the locals for tooth cleaning. GB1 is an ideal marker for G. kola. The NMR spectra of GB1 obtained at 21°C exhibited two sets of signals, which appeared to be a mixture of two characteristic conformations. When measured at 70°C and 90°C, GB1 showed a single set of signals, the higher the temperature, the faster the hindered rotation between the flavanone and the flavanonol moieties around C-3/C-8" axis. When the rotation was fast enough, the compound existed at a relatively stable conformation, and the spectral complexity at the dimeric level disappeared. When measured at much lower temperatures such as -30°C and -50°C, GB1 showed the same behaviour 133. We finally confirmed that GB1 was a pure compound 133.