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Benzie IFF, Wachtel-Galor S, editors. Herbal Medicine: Biomolecular and Clinical Aspects. 2nd edition. Boca Raton (FL): CRC Press; 2011.

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Herbal Medicine: Biomolecular and Clinical Aspects. 2nd edition.

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Chapter 20Bioactive Components in Herbal Medicine Experimental Approaches

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Modern medicine is a deductive science, whereas traditional Chinese medicine (TCM) is inductive. Deductive medicine has a specific focus, but as a consequence the general need of the individual might be neglected. TCM does not relate to very specific targets or problems, but aims at improving the general well-being of the individual by maintaining an effective balance between various physiological functions. This holistic approach, by which the individual is kept in a biologically balanced state, allows the mobilization of biological reserves to take care of physiological problems (Campion 1993). In contrast, the deductive approach relies on accurate targets, mandates specialization, and is largely disease focused rather than patient focused. Neglect of holistic care by modern medicine is one of the important reasons behind the increasing support for alternative care and over-the-counter health preparations (Eisenberg et al. 1993). If the two divergent systems of medical science can be harmonized, more clinical problems can be solved. Holistic care to promote physiological balance to allow spontaneous adjustment and the building up of better bodily defenses could supplement aggressive single-target modern medicine to remove a specific problem. Such harmonization of the two systems could lead to better and more holistic treatment of individuals within a modern medicine setting that is science based and evidence based. This requires research into efficacy that goes beyond specific targets. However, it also requires research to ensure the quality of herbal medicines, which are often misidentified, contaminated, or adulterated.


Research on herbal medicine in the past century has focused on many aspects, including pharmacognosy, quality control, and laboratory and clinical tests for efficacy. Many resources have focused on the identification of active components in herbs for drug development. One remarkably successful example in China was the discovery of the derivatives of artemisinin (qinghao). This was used traditionally as an herbal treatment for fever and chills, and has been found to be effective against malarial and other parasites and to have cytotoxic effects against some cancers (Valecha and Tripath 1997). Two other examples, from France, are the cytotoxic drugs vincristine, from the periwinkle flower, and taxol, extracted from the bark of the yew tree (National Centre, 1999). However, tremendous resources and facilities are required for such major successes, and there have been many failures. Botanicals are complex and varied mixtures. Extraction and identification of putative active components in the whole herb or herbal formulation require innovative approaches as well as much laboratory investment. Evidence of clinical efficacy is needed, and the overall investment is very costly, although success is not guaranteed. A more cost-effective approach to the search for bioactive components in herbs is needed.

For an approach to be generally accepted in the development and evaluation of herbal medicines, it must address several aspects of an herbal medicine, including efficacy, safety, quality and consistency of composition, and mode of action. A particular research need in this field is to understand the quality of the herbs, which can vary widely. In addition, herbs can be wrongly identified and contamination or adulteration of herbs is common. Herbs need to be thoroughly authenticated. A basic chemical record of quality control can be established through chromatographic studies, and the species details related to the origin of production are established with DNA fingerprinting. Quality in relation to the absence of pesticides, heavy metals, fungi, and microbes is also important. Every batch of an herb should be subjected to screening and counterchecking against records of standard extracts provided by the relevant academic institution in China (Zhan and Lin 2002). However, existing standards for labeling samples of medicinal herbs are far from satisfactory. New technologies can create chemometric profiles of a known herb for quality assessment, as well as helping with the identification and quantitation of biologically active chemical components. This practice takes quality control of herbal medicine to a more effective level and will support its use in modern medicine (Mok and Chau 2006; Zeng et al. 2008).

Ensuring the quality and consistency of herbal medicine is very important, but questions of efficacy and modes of action also must be addressed. An investigative approach that combines biochemical, biological, and chemometric testing in a “layered” system offers a useful platform for the overall evaluation of herbal medicines. Parallel testing of characteristics or biological effects with chemometric screening is performed on crude herbal extracts, before moving to parallel testing of further fractionations and their combinations (Figure 20.1). The aim of the chemometric analysis is to identify biologically active chemical groups, not the compound, and offer a chemical fingerprinting technique that leads to the identification of more target compounds with known and partially known chemical properties. The chemometric fingerprint analysis technique makes possible the comparison of chemical compositions of different samples using all the detected components through their entire chromatograms obtained from liquid chromatography, gas chromatography, mass spectrometry (MS), and others. These techniques also help to discover new biomarkers and active ingredients as well as estimate bioactivity levels more efficiently and effectively (Idborg-Bojorkman et al. 2003; Wang, Wang, and Cheng 2006; Dumarey et al. 2008; Chau et al. 2009).

FIGURE 20.1. The efficacy-driven approach to evaluation of herbal medicine.


The efficacy-driven approach to evaluation of herbal medicine.

In summary, the development and evaluation of herbal medicines requires a comprehensive research approach that encompasses chemometric testing of herbs for quality, consistency, and identification of bioactive components, as well as experimental procedures and clinical trials to confirm the efficacy and mode of action. An example of this comprehensive testing as applied to a formulation for the promotion of cardiovascular well-being is given in Section 20.3.


20.3.1. Introduction

Atherosclerosis-induced heart attacks and strokes are leading causes of morbidity and mortality (Muhlestin 2000; Dalal, Evans, and Campbell 2004). Current primary and secondary prevention strategies emphasize control of various atherosclerotic risk factors, including smoking, hypertension, hypercholesterolemia, diabetes mellitus, obesity, inflammation, and homocysteine (Graham et al. 1997; Ridker et al. 1998; Schnyder et al. 2002). Radix Salviae miltiorrhizae (danshen) and Radix puerariae (gegen) are two herbal medicines used in controlling angina and other cardiac symptoms in the Chinese materia medica (Fan, O’Keefe, and Powell 1984; Ji, Tan, and Zhu 2000). Modern pharmacological studies suggest therapeutic values of these herbal preparations, including lowering of blood pressure and lipids (Zheng et al. 2002), antioxidation (Fan, O'Keefe, and Powell 1984; Zhang and Fang 1997; Jiang, Lau, and Lamm 2005), and the promotion of microcirculation (Lei and Chiou 1986; Fung et al. 1993). Therefore, danshen and gegen are worthy of study as cardioprotective agents, and clinical, experimental, and chemometric testing was performed on a dansen–gegen (DG) formulation for efficacy and chemometric fingerprinting.

20.3.2. Efficacy Testing in Experimental Study (Animal and In Vitro Testing)

Antioxidant effects, protection from ischemia–reperfusion injury, effects on blood pressure, and vasodilatory effects have been investigated on the DG formulation using a variety of in vitro and ex vivo models.

The antioxidant effects are discussed first. The compound 2-2′-azo-bis (2-amidinopropane) dihydrochloride (AAPH) is a water-soluble radical-chain initiator that can induce lipid oxidation (Yamamoto et al. 1984; Niki et al. 1986; Miki et al. 1987). The AAPH-induced red blood cell hemolysis assay and AAPH induced cardiomyocyte cell death test are convenient in vitro experiments for the study of antioxidant activity. In the presence of AAPH, the membrane lipids of red blood cells and cardiomyocytes are readily oxidized, resulting in hemolysis and cell death, respectively. The DG formulation inhibited AAPH-induced red blood cell hemolysis and cardiomyocyte cell death in a dose-dependent manner (Leung 2003; Lam et al. 2005; Lam 2006).

The ischemia–reperfusion injury effects are discussed next. In ischemia, oxygen supply is decreased to an extent that is insufficient for maintaining normal metabolism (Hearse 1994). Reperfusion restores the oxygen content but causes injury to the organ at the same time because of the large amount of superoxide produced from the action of xanthine oxidase on the accumulated hypoxanthine (McCord, Roy, and Schaffer 1985; Manning et al. 1988). This ischemia–reperfusion injury occurs in patients undergoing percutaneous transluminal coronary angioplasty (PTCA). To mimic this in ex vivo studies, isolated rat hearts are cannulated on a Langendorff apparatus and subjected to controlled ischemia and reperfusion. The protective effect of DG formulation on isolated rat hearts against ischemia–reperfusion injury was reflected from contractile force recovery, coronary flow rate recovery, and the lower release of enzymes (e.g., creatine kinase and lactate dehydrogenase) from cardiac tissue (Leung 2003).

The antihypertensive effects of DG formulation are as follows: The endothelium plays an important role in regulating vascular tone and blood pressure. It continually releases endothelium-derived relaxing factors (EDRFs) and endothelium-derived contracting factors (EDCFs) to modulate underlying vascular smooth muscle cell (vSMC) function. The EDRFs are comprised of nitric oxide (NO), prostacyclin (PGI2), and endothelium-derived hyperpolarizing factor (EDHF), whereas EDCFs include superoxide anion (O2•-), endothelin, thromboxane A2 (TXA2), and prostaglandin H2 (PGH2). A balance between EDRF and EDCF is maintained in normal physiological situations (Palmer, Ferrige, and Moncada 1987; Akpaffiong and Taylor 1998). To investigate the vasodilative effect of DG formulation, one ex vivo and one in vivo model were employed. In the ex vivo study, rat aortas were isolated and cut into pieces of 2 mm in length. The rings were then mounted on organ baths, containing 37°C Krebs solution aerated with 95% O2 and 5% CO2, and connected to a force transducer to record isometric force. After 1 hr. of equilibration and pretesting with phenylephrine (an α-adrenergic agonist for vSMC contraction testing) and then with acetylcholine (an endothelial muscarinic receptor agonist for endothelium-induced relaxation testing), the aortic rings were rinsed with Krebs solution and allowed to equilibrate for 1. Using this model, DG formulation dose-dependently induced vasodilation of isolated rat aorta (Yam 2005). For in vivo testing, spontaneous hypertensive rats (SHRs) were used, a system in which results consistently correlate efficacy to humans (Lund-Johansen 1990; Doggrell and Brown 1998). Generally, SHRs are prehypertensive at the first 6–8 weeks and hypertension develops over the next 12–14 weeks (i.e., at an age of 6–22 weeks; McGuire and Twietmeyer 1985). Blood pressure of SHRs can be measured by the rat tail-cuff noninvasive blood pressure system.

In testing the DG formulation, conscious rats were put into a transparent container with temperature control and allowed to calm down for at least 15 min. before measurement. An occlusion cuff and a sensor cuff were put onto the rats’ tails. Blood pressure was recorded by the inflation and deflation of the occlusion cuff. Any agitation of the rat could be compensated by another 15-minute calming period before the next measurement. Herbal samples were intragastrically administered to the SHRs once daily for 3 months and blood pressure was measured at 4-week intervals. The DG formulation was found to attenuate hypertension progression in SHRs either at the prehypertensive stage or at the stage of sustained hypertension (Lam et al. 2005; Lam 2006).

The DG formulation shows significant anti-atherosclerosis effects. Atherosclerosis is a continuous pathological process that takes years to develop and progress. It is now possible to assess such vascular abnormalities objectively and noninvasively with ultrasound assessment. For these, measurement of carotid intima-media thickening (IMT) is highly reproducible, correlates well with the severity and extent of coronary artery disease, and is predictive of stroke and coronary events (O'Leary et al. 1999; Simon et al. 2002). Similarly, vascular reactivity (flow-mediated dilation) of the brachial artery as an indicator of overall endothelial function is an emerging sensitive marker of early subclinical atherosclerosis and a reliable index of cardiovascular health (Behrendt and Ganz 2002).

Pathological processes of atherosclerosis include accumulation of modified lipids, mainly oxidized low-density lipoprotein (oxLDL), foam cell formation, endothelial cell dysfunction and activation, increase in expression of adhesion molecules, activation and recruitment of inflammatory cells, and induction of proliferation and migration of vSMCs (Gerrity 1981; Pesonen 1989; Steinberg et al. 1989; Spiteller 2005). The LDL oxidation is a lipid-peroxidation process, and antioxidants that can prevent its initiation and cause chain breaking should have a major impact not only on the oxidation resistance of LDL but also on the development of atherosclerosis. The most frequently used assay to assess in vitro oxidation resistance of LDL is the measurement of conjugated dienes formation in isolated LDL, with copper ions as the pro-oxidant (Chomard et al. 1998; Anderson et al. 2001). The DG formulation prevented LDL from being oxidized in a dose-dependent manner, and salvianolic acid B (SAB; Koon 2006), one of the bioactive components from danshen, was at least two times more potent than vitamin C in preventing LDL oxidation. To test its effects on monocyte adhesion, expression of adhesion molecules on the surface of cultured human umbilical vein endothelial cells (HUVECs) is stimulated by tumor necrosis factor (TNF)-α or interleukin (IL)-1β. The next step is the pulsing of human monocytes, THP-1, by tritiated thymidine. After coincubation of the two cell types for 1 hour, the cultured cells are washed thrice with phosphate buffered saline (PBS) to wash away nonadhered monocytes and the cell lysate is collected for scintillation counting. In this assay system, DG formulation was found to decrease the adhesion of THP-1 to HUVECs in a dose-dependent manner (Koon 2006).

The inhibitory effect of DG formulation in foam cell formation was examined using lipid loading of human monocyte–derived macrophages (HMDMs). White cells concentrates were obtained from the peripheral blood of healthy donors, and monocytes were isolated via counterflow centrifugation elutriation. After maturation of the monocytes, lipid loading was achieved by incubation of the HMDMs with acetylated LDL (AcLDL) in culture medium. During lipid loading, the cells were treated with DG formulation. Afterward, cell extracts were prepared and free cholesterol and cholesteryl esters were separated and quantified via reverse-phase high-performance liquid chromatography (HPLC). It was found that DG treatment has significant effects on foam cell formation in vitro by decreasing the accumulation of cholesterol and its esters in a dose-dependent manner (Sieveking et al. 2005).

Vascular proliferation contributes to diffuse intimal thickening in large and medium-sized arteries where the development of atherosclerosis has occurred. Intimal thickening begins after birth and it is composed of vSMCs completely surrounded by an extracellular matrix (ECM) and covered by a monolayer of endothelial cells (Pesonen 1989). Sometimes, macrophages can be found beneath the endothelium layer. And it was found that the ECM in intimal thickening is synthesized by vSMCs (Campbell et al. 1991). Therefore, vSMC proliferation is an important process for plaque formation in primary atherosclerosis (Ross 1995). The DG formulation was found to inhibit platelet-derived growth factor (PDGF)–induced vSMC proliferation by mediating G1/S cell-cycle arrest. The main component that governs the transition from G1 phase to S phase, cyclin D, was found to be downregulated by DG formulation in both protein and messenger ribonucleic acid (mRNA) levels (Koon 2006). DG formulation also showed antimigratory effects against PDGF-induced vSMCs migration (Koon 2006).

Although the atherogenic effect of dietary cholesterol was first demonstrated in rabbits, it has also been observed in several other animals, including pigs and guinea pigs. The New Zealand white rabbit was employed as the animal model for testing whether supplementation of DG formulation could stop the cholesterol-induced progression of atherosclerosis. Fresh aortas were excised, cut longitudinally, and immersed in a solution of Sudan III (saturated solution in 70% alcohol; Hara et al. 1999) for 45 minutes to identify atheromatous lesions on the surface of the aorta. It was reported that DG supplementation significantly decreased the area of atheroma in a dose-dependent manner (Koon 2006).

20.3.3. Efficacy Testing in Clinical Study

One hundred men (n = 87) and women (n = 13) aged 40–70 years (mean [standard deviation or SD] age of 58[8] years) were recruited. They had angiographically documented coronary artery disease (>50% reduction in luminal diameter) in at least one vessel and stable angina status. Patients with recent myocardial infarction (within 3 months) and unstable angina (within 6 months), stroke (within 1 year), significant renal insufficiency (plasma creatinine >140 μmol/L), or history of significant drug hypersensitivity were excluded. Eligible coronary patients were screened in the cardiac catheterization laboratory and cardiac clinic in the Prince of Wales Hospital, Shatin, Hong Kong, People's Republic of China. After signing written informed consent, the patients were randomized to take either 6 capsules of DG formulation (3 g) per day or 6 capsules of appearance-identical placebo capsules daily, in double-blind and parallel fashion for 24 weeks. Other medications were not changed. Clinical visits for progress and tolerability monitoring were arranged at 4-week intervals. Routine hematological, biochemical, and other blood tests (folate, homocysteine, vitamin B12, and proinflammatory biomarkers) and ultrasound vascular imaging were performed at baseline and on completion of the trial.

The formulation used was as follows: Danshen and gegen raw herbs (in a ratio of 7:3 by weight) supplied from Sichuan farms were prepared, authenticated (Sun, Shaw, and Fung 2007), and extracted in one batch following good manufacturing practices at the Hong Kong Institute of Biotechnology, Science Park, Hong Kong, People's Republic of China. The prepared herbs were subjected to aqueous extraction (with an herb:water ratio of 1:10) at 100°C twice for 60 minutes and once for 30 minutes, spray dried at –660 mmHg and 50–60°C, and the dried powder encapsulated (500 mg/capsule) after an accelerated stability test. A dosage of 3 g/day of the DG formulation was chosen for the clinical study, based on the recommendation from Chinese materia medica (Fan, O'Keefe, and Powell 1984; Ji, Tan, and Zhu 2000) and our previous in vitro experiment (Fung et al. 1993).

The endothelial function of the brachial artery was studied at baseline using high-resolution ultrasound, as described in Section 20.3.2 (Woo et al. 1993, 2004). In brief, the diameter of the brachial artery was measured on B-mode ultrasound images using a linear array transducer (L10-5 median frequency: 7.5 MHz) and a standard Advanced Technology Laboratories 3000 ultrasound system (Bothell, Washington, USA) and forearm tourniquet cuff placement to induce hyperemia after deflation (Celermajer et al. 1992; Woo et al. 1993; Corretti et al. 2002; Woo et al. 2004; Deanfield et al. 2005). Scans were acquired at rest, during post-tourniquet-reactive hyperemia (to induce flow-mediated endothelium-dependent dilation [FMD]; Celermajer et al. 1992), and 4 minutes after administration of 400 μg sublingual glyceryltrinitrate (GTN; for endothelium-independent dilation). The FMD is predominantly due to endothelial NO release (Jannides et al. 1995), and the endothelial responses of the brachial artery measured by this method correlate significantly with coronary endothelial function in the same subjects (Anderson et al. 1995) and with the severity of coronary atherosclerosis (Schroeder et al. 1999). The accuracy, reproducibility, and low interobserver error for this measurement of arterial physiology have been demonstrated previously in the literature (Celermajer et al. 1992; Woo et al. 1993; Sorensen et al. 1995; Woo et al. 2004). All carotid scans were performed by a single and blinded operator after a predetermined and standardized scanning protocol for the right and left carotid arteries, as described previously (Salonen and Salonen 1991; Bots et al. 1997). All scans were recorded on super-VHS (video hyperspace) videotape for subsequent offline measurement of intima-media thickness (IMT) by a blinded investigator, using a verified automatic edge-detecting and measurement software package as described previously (Woo et al. 1999). The intraobserver variability for the mean IMT was 0.03 ± 0.01 mm (coefficient of variation [CV]: 1.0%).

After the double-blind trial phase, all patients were offered an option of continuing open-label DG (1.5 g/day) for an additional 6 months for lower dose-titration and consolidation of the therapeutic effects. Ten placebo patients who had not opted for open-label DG after the double-blind phase but had consented to be followed up were restudied at 1 year for comparison (negative control). The study protocol was approved by the institutional ethics committee on human research of the Chinese University of Hong Kong, People's Republic of China, in compliance with the Declaration of Helsinki. Data were processed to give group mean values and standard deviations where appropriate. In case of normal distribution, possible intergroup differences were identified with a Student's t-test. Otherwise, the possible intergroup differences were assessed by a Kruskal–Wallis's test. In case of detecting any significant intergroup differences, the subsequent identification of the groups was carried out with a Wilcoxon Rank–Sum test. The primary study end points were FMD, GTN, and IMT, whereas other, nonprimary outcome variables were compared with Bonferroni adjustment for multiple comparisons. Multivariate linear regression analysis was carried out to assess the major determinants of FMD changes, including age, changes in total and LDL cholesterols, and treatment groups. A p value less than 0.05 is considered statistically significant. The statistical analyses were made with SPSS for Microsoft Windows 10.0. On our pilot finding of impaired FMD in the range of 5.0 ± 1.2% and of a mean interobserver relative difference of 3% in FMD over time, and assuming a 10% dropout rate in 24 weeks, enrollment of 100 coronary artery disease (CAD) patients can detect a 12% relative improvement from baseline in brachial FMD after DG treatment and in FMD difference between DG- and placebo-treated groups with 80% power and α = 0.05.

In total, 92 patients finished the 24-week, double-blind phase study. Eight patients dropped out (two withdrew consent, two had adverse events while on placebo, one had poor compliance, and three defaulted on nonmedical grounds). Baseline characteristics, clinical features, cardiac medication, and vascular function in the two groups were found to be similar (Table 20.1).

TABLE 20.1. Characteristics of Danshen–Gegen and Placebo Groups.

TABLE 20.1

Characteristics of Danshen–Gegen and Placebo Groups.

After 24 weeks, when compared with the baseline, there were no significant changes in blood pressure, blood folate, homocysteine, C-reactive protein, and other proinflammatory markers levels in either group. A small decrease in total and LDL cholesterols (p < .05) in the DG group and a mild decrease in LDL cholesterol in the placebo group were observed (p < .05; Table 20.2).

TABLE 20.2. Results of Placebo-Controlled Trial.

TABLE 20.2

Results of Placebo-Controlled Trial.

The FMD and GTN of the brachial artery improved significantly with DG (p < .01), although FMD also was significantly improved after placebo treatment also (p < .05). However, FMD was significantly higher after DG treatment than after placebo treatment (p < .001) (Figure 20.2a and b). A slight decrease in carotid IMT (p < .05) was seen after 24 weeks of DG but not after placebo treatment (Table 20.3, Figure 20.3a and b). On multivariate analysis, improvement in FMD was related to DG treatment (β = 0.32, R = 0.3, p = .03), but not to age or changes in total or LDL cholesterols.

FIGURE 20.2. Changes of (a) brachial flow-mediated dilation and (b) intima-media thickness after 6 months of placebo and subsequent 6 months of open-label herbal medicine (DG) treatment.


Changes of (a) brachial flow-mediated dilation and (b) intima-media thickness after 6 months of placebo and subsequent 6 months of open-label herbal medicine (DG) treatment. In each box plot, the bottom and top of the box represent the twenty-fifth percentile (more...)

TABLE 20.3. Changes in Vascular Profiles as Primary Efficacy End Points in a Danshen–Gegen (DG) Trial.

TABLE 20.3

Changes in Vascular Profiles as Primary Efficacy End Points in a Danshen–Gegen (DG) Trial.

FIGURE 20.3. Changes of (a) brachial flow-mediated dilation and (b) intima-media thickness after 6 and 12 months of herbal medicine (DG) treatment.


Changes of (a) brachial flow-mediated dilation and (b) intima-media thickness after 6 and 12 months of herbal medicine (DG) treatment. Changes in carotid IMT at 24 weeks after placebo and herbal medicine (DG) treatment are shown. In each box plot, the (more...)

The study drugs were well tolerated in both groups, with no significant symptomatic complaints and no derangement in hematological or routine biochemical profiles. Eight severe adverse events were reported, mostly in the placebo group, due to the occurrence of chest pain, left heart failure, sciatica, gastrointestinal bleeding, and a road traffic accident. The 45 patients who opted to take 6-month open-label DG treatment were not significantly different in their baseline characteristics from the remaining patients. Sustained improvement of their brachial FMD (5.2 ± 1.3 to 5.8 ± 1.0%; p < .0001) and carotid IMT (0.92 ± 0.23 to 0.89 ± 0.25 mm; p < .0001) at 1 year from baseline was observed. In the 10 placebo patients who had not opted for open-label DG, a nonsignificant trend of mean carotid IMT deterioration (1.17 ± 0.57 to 1.20 mm ± 0.57; p > .05) was seen; they were restudied after 6 months.

20.3.4. Summary of the Cardioprotective Effects of the Danshen–Gegen Formulation

Based on the results of a variety of in vitro, ex vivo, and in vivo biological assays, the beneficial effects of DG formulation have been demonstrated to resemble those of antioxidants, vasodilators, antihypertensives, and antiatherosclerotics. In the in vitro systems, DG formulation could directly scavenge free radicals and prevent oxidative stress exerted on red blood cells, cardiomyocytes, and LDL. Moreover, DG formulation exhibited ex vivo vasodilatory effects on precontracted rat aortas and suppressed further progression of severe hypertension in primary hypertensive rats. Also, DG formulation inhibited the pathological processes of atherosclerosis, including vSMC proliferation and migration as well as monocyte–endothelial cell adhesions. Furthermore, the in vivo antiatherosclerotic effects of DG formulation were demonstrated in the rabbit model. The clinical trial showed the DG formulation was well tolerated and treatment of patients with CAD was associated with sustained improvements in FMD and IMT, although no marked changes were seen in the biochemical markers of CAD risk such as lipids or homocysteine. In total, these findings provide credible and reliable experimental evidence in support of DG formulation for cardiovascular well-being. However, for clinical use, herbs and herbal formulations of consistently high quality are needed, and this requires effective quality control.


20.4.1. Background

In general, one to a few representative chemical constituents with known chemical structures or “markers” are used for authentication of herbal medicines (Zschocke, ClaBen-Houben, and Bauer 2001b; Upton 2003a; State Pharmacopoeia Commission of People’s Republic of China 2005; United States Pharmacopoeial Conversion 2009). Unknown herbs are identified and the identity of a known herb is confirmed by the markers found within it. The same approach is applied very often to identify the component herbs within a combination preparation, such as the DG formulation. Furthermore, to ensure the quality of a formulation, it is essential to maintain the correct amounts of each herb. In the DG formulation, the major classes of the chemical constituents of danshen are phenolic acid and diterpene (Zhu et al. 2007), while that of gegen is isoflavonoid (Chen et al. 2007; Jiang, Lau, and Lamm 2005). According to the Chinese Pharmacopoeia (State Pharmacopoeia Commission of People’s Republic of China 2005), the recommended markers of danshen are tanshinone IIA, a diterpene, and SAB, a phenolic acid. These components are reported to promote blood circulation (Gu et al. 2004; Gao et al. 2009). The isoflavonoid, puerarin, is the marker for gegen, and is an antioxidant (Chung et al. 2008).

20.4.2. The Pattern-Oriented Approach in Chemometrics

The use of a few markers for the authentication of herbal medicine has been widely accepted by authorized agencies and by the pharmaceutical industry (CDER 2004; Zeng et al. 2008). However, some markers are not unique and may be found in other herbs also. For instance, Z-ligustilide, the widely used marker of Radix Angelicae sinensis, danggui (Zschocke, Classen-Houben, and Bauer 2001a), can also be found in Radix Ligustici chuanxiong (Zschocke, ClaBen-Houben, and Bauer 2001c). Moreover, only one marker, that is, salvianic acid A, is recommended for the Chinese medicine combination preparation fufang danshen diwan, with three component herbs (State Pharmacopoeia Commission of P. R. China 2005). Ginseng (Qian, Guo and Li 2009) and Ginkgo biloba (Upton 2003b) are a few examples of herbs with more than 10 chemical standards available. Furthermore, for many herbs, there are no reference materials and markers are not established. Therefore, from the quality control point of view, the previously accepted marker approach is barely satisfactory, and a multicompound approach is now adopted for authentication of herbal medicines (Mok and Chau 2006; Zeng et al. 2008). This approach makes use of chemical constituents with known chemical structures and also those on whom only partial chemical information is available, for example, retention times, mass spectra, and ultraviolet (UV) spectra. This approach provides a more accurate and reliable means for quality control. The marker and multicompound approaches are grouped together and referred to as a “compound-oriented approach” (Zeng et al. 2008; Figure 20.4).

FIGURE 20.4. Different approaches for the quality control of herbal medicine.


Different approaches for the quality control of herbal medicine.

20.4.3. Separation Techniques Used in the Multicompound Chemometric Approach

The use of advanced hyphenated separation techniques is one of the key factors that makes the multicompound approach feasible (Chau et al. 2004; Zeng et al. 2008). An example is the coupling of an HPLC or its faster version and a rapid-resolution liquid chromatography (RRLC) with a multichannel diode array detector (DAD), which can acquire an absorption spectrum over a wide wavelength range for every retention time, greatly increasing the amount of data obtained. Figure 20.5a shows the three-dimensional (3D) chromatogram of the DG formulation obtained within 60 minutes by HPLC–DAD (the 1100 instrument of Agilent Technology, Inc., California) in the spectral range of 245–400 nm. The total number of spectrochromatographic data points obtained is 1.27 million. Figure 20.5b gives the commonly used two-dimensional (2D) chromatograms of the same herbal medicine measured at 268 and 328 nm, with each peak representing a chemical. Each of these 2D chromatograms consists of 12,000 data points, which is hundredfold less than that of the 3D chromatogram. As the chemical constituents are sensitive to the wavelength selected, the two 2D chromatograms do not have the same profile structure, and care has to be taken in determining the chemical composition of herbal medicine by selecting the right wavelength. The 3D chromatogram provides information across the spectrum, providing more information for identification. In quantitative analysis, the wavelength of the highest absorptivity is usually selected for the component concerned as it produces the most accurate results. However, DAD has its limitation: it cannot detect those ingredients that have no or very low absorption. In this situation, MS and other techniques are used, for example, gas chromatography (GC)-MS, liquid chromatography (LC)-MS, and liquid chromatography-tandem mass spectrometry (LC-MS/MS). There are different classes of chemical compounds present in herbal medicine. In chemical fingerprinting, HPLC is commonly utilized to analyze more polar compounds, whereas GC is good for volatile compounds. This leads to the use of more than one kind of fingerprint in a multipattern approach instead of just one in the single-pattern approach for assessment (Mok and Chau 2006; Zeng et al. 2008).

FIGURE 20.5. (See color insert.


(See color insert.) Chromatograms of DG formulation: (a) the three-dimensional chromatogram of DG formulation from 245 to 400 nm, and (b) the two-dimensional chromatograms of DG formulation measured at wavelengths of 268 and 328 nm.

The huge amount of data obtained from hyphenated instruments embeds valuable information. How to dig or mine it out is a great challenge to scientists. In recent years, chemometrics has been found to be very useful in this aspect. The development of new chemometrics data-processing methods boosts the application of multicompound and other approaches in the study of herbal medicine. Very often, a chromatogram of an herbal medicine has overlapping peaks. It is not easy to separate them well by adjusting the experimental conditions alone. Here, chemometric resolution methods (CRMs) and other mathematical tools, such as heuristic evolving latent projection (HELP), multicomponent spectral correlative chromatography (MSCC), window factor analysis (WFA), subwindow factor analysis (SFA), augmented evolving window orthogonal projection (AEWOP), principal component analysis (PCA), and partial least squares (PLS), can help (Otto 1999; Malinowski 2002; Chau et al. 2004; Mok and Chau 2006; Zeng et al. 2008). For instance, the chemical compositions of the head, body, and tail of danggui were studied in detail by GC-MS and chemometrics (Wei et al. 2008). The tools SFA and AEWOP were applied to resolve the GC-MS data sets of these parts to get the pure mass spectra of many chemical ingredients. Up to 42 common components were found, and 35 were identified by matching their resolved spectra with those available in the National Institute of Standards and Technology (NIST) MS database and the Wiley Registry of Mass Spectral data (McLafferty 1989). This approach provides a better understanding on the variation in different parts of danggui. Through LC-DAD-atmospheric pressure chemical ionization mass spectrometry (LC-DAD-APCI-MS) and data analysis using such specialized tools, Wang et al. (2005) investigated the absorption and metabolite components in plasma samples from a rabbit that was given an oral solution of danggui. More than 32 chemical components were discovered in both danggui and biofluid samples, but over 10 components were detected only in plasma. The different chemical patterns provide information about what danggui ingredients were absorbed and what metabolites were generated.

In the year 2000, the State Food and Drug Administration (SFDA) of the People’s Republic of China announced that all the commercial injectable Chinese medicines were required to have their chemical fingerprints submitted for approval (SFDA 2000). Here, “fingerprint” refers to patterns like the chromatogram, spectrum, and others. The use of this fingerprint incorporates the concept of photoequivalence (Tyler 1999). From an authentication point of view, a chromatogram and spectrum contain information of all the chemical components of a sample that can be detected by the utilized instrument or method. Of course, the appearance of a pattern depends very much on the tools used, and the spectral pattern of an herb or herbal mixture may look very complicated with overlapping profile structures. This can make data analysis and interpretation difficult without the use of chemometric tools to resolve the pattern and reveal interesting chemical features.

By nature, the application of the pattern or fingerprint approach for quality control is very different from that of the conventional compound-oriented approach, which uses known markers and target compounds. Fingerprinting makes use of all the components at the same time, even though their chemical properties may not be fully known. Therefore, all the bioactive and inactive ingredients are recorded within the fingerprint. This is very important because the biological activity of a Chinese medicine is usually induced by more than one constituent, and there may be important interactions among constituents (Xie 1998; Xie 2000; Mok and Chau 2006). In the fingerprint approach for qualitative analysis, the retention times of components from different samples as found in the entire or selected regions of their chromatograms are utilized for comparison. Using the “true” retention time to identify an unknown compound is a common practice of analytical chemists. Chromatograms under study have to be run under identical experimental conditions, and data preprocessing is used to minimize the noise level and to do the peak alignment so as to get the true chromatogram of a sample, after which quantitative analysis can be carried out. The content of a constituent in a certain sample is usually determined by its chromatographic peak height or area. As for the pattern approach, a common assessment scheme is to evaluate how similar the components of different samples are to one another. The similarity index (SI) parameter provides a quantitative measure of this kind. The index is usually represented by the correlation coefficients between the fingerprints under study (Yi et al. 2009). In this way, every data point corresponding to a retention time and related height is considered in the comparison. If the SI value is close to 1.0, then the chemical compositions of the two samples concerned are almost identical to each other. If one of them is an unknown herb and the other one is a known or reference herb, then the unknown herb is identified or authenticated. Furthermore, the use of SI incorporates a fuzzy logic concept in assessing the quality of the sample because it allows tolerance of the natural variations and differences between different samples of the same Chinese medicine.

Using the whole fingerprint approach emphasizes the integral chemical feature of a system with the help of a high-throughput measurement technique. The demand of SFDA has triggered other organizations, including the World Health Organization (WHO; Department of Technical Cooperation for Essential Drugs and Medicines Policy 2005), Food and Drug Administration (FDA) of the United States (CDER 2004), and European Agency for the Evaluation of Medicinal Products (EMEA; CDER 2004), to consider chemical fingerprinting as a means of screening botanical and related products (Mok and Chau 2006; Zeng et al. 2008).

To improve quality monitoring, an effort has been made to put both chemical and bioactivity information together (SFDA 2000; Upton 2003a; Cheng, Wang, and Wang 2006; Dong et al. 2006; Lu et al. 2006; Dumarey et al. 2008; Zeng et al. 2008). This is another kind of multipattern approach. Together with the single-pattern approach mentioned earlier in this section, they are grouped as a pattern-oriented approach (Figure 20.4), in contrast to the compound-oriented approach mentioned earlier in this section (Zeng et al. 2008).

20.4.4. Application of Chemometric Approach in the Quality Control of Herbs

Two different batches of DG (as described and used in the clinical study in Section 20.3.3) were prepared in 2002 and 2005, but they were based on the same prescription. The authentication procedures as recommended by the Chinese Pharmacopoeia 2000 (State Pharmacopoeia Commission of People’s Republic of China 2000) were applied to these Chinese medicine samples. They are briefly described here. They cover the markers used, chemical analysis procedures and techniques, as well as related experimental conditions. Thin-layer chromatography (TLC) and HPLC were the basic analysis techniques used. Some of these procedures were modified by us or taken from the literature so as to acquire more information and more accurate results with a minimal number of experiments. In addition, the HPLC-DAD and LC-DAD-MS hyphenated instruments were used to acquire 3D chromatograms of the samples in order to get more information for chemometric data analysis. Other target compounds of sodium danshensu, daidzin, daidzein, and SAB were also included. For those compounds with reference standards available, different concentrations of the standards were prepared and calibration curves were set up by plotting chromatographic peak area against concentration. Through these, the concentrations of these compounds in unknown samples were determined.

The compound-oriented and pattern approaches were applied to the HPLC-DAD and LC-DAD-MS data of the samples. We also looked into the UV or MS spectral data at those retention times of interest to further confirm the identification. In cases of profiles with overlapping or “embedded” peaks, CRMs and other chemometric methods were applied to resolve them, as described previously in a study on the Chinese medicine formulation ping-wei powder (Gong et al. 2001). It is worthwhile to look into another useful application of CRM here. In our experience, CRM can help to obtain more chemical information from a single experiment, thereby shortening the analysis time. As more chemical information, like retention times and UV or MS spectra of the components of an herbal medicine, can be obtained through the combination of experimental study and chemometric data analysis, quality control is carried out more efficiently, more effectively, and more reliably. Even components for whom only partial information is available can be included for the purpose. This is very helpful in dealing with a Chinese medicine formulation with more than one component herb. We adopted this way throughout our project and some of the results obtained will be presented in Section 20.4.5.

The HPLC-DAD and LC-MS fingerprints of the DG samples were also utilized for quality control in the pattern-oriented approach in this work. In applying the whole fingerprint to do the job, the chromatograms themselves have to be preprocessed to get the true ones as mentioned in Section 20.4.3. Here, data preprocessing includes noise removal, peak alignment, background shift, and other activities. Afterward, the similarity of these true chromatographic patterns was obtained quantitatively through correlation analysis to give the related SIs for assessment. Moreover, the mean chromatogram based on those from different samples of the same Chinese medicine was also obtained. This served as the representative or the “standard” fingerprint of the medicine, provided the different individual chromatograms were not too diverse. All chemometrics data analyses were carried out by the computer-aided similarity evaluation (CASE) system method, which was coded and run under the MATLAB® environment (Wang et al. 2008). A CRM was then applied to the preprocessed fingerprints to resolve overlapping peak clusters to get more accurate pure spectra and retention times of the components involved.

20.4.5. Example with Danshen, Gegen, and Danshen–Gegen Formulation

The TLC separation technique was applied to investigate the Chinese medicine samples of danshen, gegen, the reference herb of dashen, as well as the two batches of DG. The two markers tanshinone IIA and puerarin were used in the TLC study for quality control. Visible light was utilized to detect these two markers in the developed TLC plates. These markers were found in all the samples concerned.

The HPLC provides better separation power of chemical components than TLC, and the markers considered in the TLC work were further investigated by HPLC-DAD and LC-MS. Again the respective markers of danshen and gegen (tanshinone IIA and puerarin) were identified in the single herb samples. These component herbs were also found in the DG products based on these two markers and the other target compounds in Section 20.4.1. In doing so, we compared both their retention times obtained and the observed UV spectra at the same times in different chromatograms.

The stability of the DG product using both the marker and the pattern approach was then evaluated. Stability is very important to assure the consistency of the quality of encapsulated herbs such as the DG capsules used in the clinical trial mentioned in Section 20.3.3. Here, we scrutinized the variation in the chemical compositions of the DG samples before and after 3 months of “accelerated aging” treatment, which is commonly used to establish the shelf life of a product. All samples were analyzed by HPLC-DAD with the sample preparation and experimental procedures all exactly the same. Results showed that the marker contents at time 0 and after 3 months were 0.42 and 0.45 mg/g, respectively, for puerarin and 0.07 and 0.03 mg/g, respectively, for tanshinone IIA. This indicates the product was stable.

We also expanded stability testing using all the detected chemical compounds in these samples for comparison via the pattern approach. By visual inspection, the time-0 and 3-month chromatograms were very similar (result not shown), but in order to determine how similar they are quantitatively, their similarity index (SI) was calculated and was found to be 0.99. Here, the profile structure used for comparison included the peak height or intensity measured at every retention time. It should be noted that the HPLC-UV chromatographic height is related directly to the content of a component in the sample concerned, although different peaks indicate different components. Therefore, this approach to stability testing simultaneously investigates loss of individual components (by peak height comparison) as well as their transformation into other components (by SI comparison).

It is worth mentioning another application of SI here, and that is performance monitoring of the separation instrument. Multiple successive injections of the same extract give SIs that should be in high agreement (based on our experience, at least >90% to be acceptable).

In testing the 2002 and 2005 batches of DG and their chemical compositions, an LC-DAD-MS study was carried out. Again, we used both the multicompound and pattern approach to scrutinize the variation of their chemical components based on the 3D chromatograms obtained. The target compounds (markers) used were sodium danshensu, SAB, puerarin, daidzin, and daidzein. The HPLC-UV chromatograms acquired at 280 and 254 nm were chosen, respectively, for the first two compounds and the remaining three in quantitative analysis. Calibration curves were set up based on the chromatographic peak areas of individual compounds. All had values of linear regression coefficient >0.95. The contents (in milligram per gram) of markers in the 2002 and 2005 batches of DG were determined to be, respectively, 5.54 and 6.25 for sodium danshensu, 10.89 and 12.18 for puerarin, 1.47 and 1.72 for daidzin, 1.72 and 2.36 for SAB, and 0.87 and 1.03 for daidzein, indicating acceptable stability.

The chromatograms of the two DG products (not shown here) exhibited complicated profiles in the early elution phase. To look into their chemical composition in more detail, chemometric CRM and alternate moving window factor analysis (AMWFA) methods were applied to their LC-MS chromatograms to resolve the overlapping peaks. In this way, a total of 28 common components were identified based on how close their retention times were. In addition, comparison was made on the resolved mass spectra. The matching factors of common components were all >91% with nine components of the two batches matching at >99%. Therefore, using the multicompound approach, a more detailed comparison of the chemical compositions of the 2002 and 2005 DG batches was revealed.

The whole HPLC-UV fingerprints of the two batches of DG were also used for similarity assessment. First, data preprocessing was carried out on the HPLC-UV chromatograms acquired. The calculated SI values at 254 and 280 nm were 88% and 87%, respectively, indicating a high similarity in the chemical compositions of the two batches.

20.4.6. Summary

Authentication of the samples of DG combination preparation and its component herbs danshen and gegen were studied in detail using the traditional marker approach by using TLC, HPLC-DAD, and LC-DAD-MS. In addition, the multicompound approach, as well as the newly developed pattern or fingerprint approach, coupled with chemometric data processing, was performed. By applying CRM and other mathematical methods to two batches of DG, 28 common components were found by comparing their resolved retention times and pure MS spectra obtained from the respective LC-DAD-MS chromatograms. This improves the quality control of complex systems like Chinese medicine combination preparation via the multicomponent approach. Furthermore, all the components of the two batches as detected by LC-DAD-MS were used in the pattern approach for assessing their chemical compositions and the related contents of components. The HPLC-UV SI value of the two batches (from 2002 and 2005) was high, and the stability of the DG products was assessed using the same approach.


There is a need for more objective and scientific ways to authenticate individual herbs, identify chemical constituents, detect adulteration or contamination of herbs, and monitor the quality of herbs and herbal medicines. There is also a need to check the consistency of different batches of herbs used in clinical studies and to identify bioactive components in herbs reported to have physiological effects. New technologies are now providing chemical fingerprinting of herbs for such purposes. This chemometric approach enables standardized formulations or “fingerprint models” to be produced, and these are described in this chapter along with their application and clinical studies of an herbal medicine containing danshen and gegen.

The chemometric approach is developing rapidly, and lends itself to the study of various herbs and foods. For example, different types of green tea have been studied, and models have been built using the chromatographic fingerprints and antioxidant capacities obtained (Dumarey et al. 2008). Wang, Wang, and Cheng (2006) proposed a quantitative composition–activity relationship to botanical drug design. Chau et al. (2009) developed the quantitative pattern–activity relationship (QPAR) technique and applied it to discover the antioxidative active regions in the fingerprint of the Chinese medicine gegen. Therefore, in addition to standardizing herbal medicines and assessing quality, the chemometrics approach can help identify a drug lead from plants and herbs.


The research was supported by an Area of Excellence grant from the University Grants Committee of the Hong Kong Special Administrative Region, People’s Republic of China (Project number: AoE/B-10/01).


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