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BioFactors 34 (2008) 147–157 147IOS Press

In search of antioxidants andanti-atherosclerotic agents from herbal

medicines

Ming-Shi Shiaoa,b,∗, Jing-Jing Chiua, Bao-Wen Changb, Jane Wangb, Wei-Ping Jena,b,Ye-Jer Wuc and Yuh-Lien Chend

a Department of Life Science, Chang Gung University, Taoyuan, Taiwan

b Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwanc Internal Medicine, Mackay Memorial Hospital, Taipei, Taiwand Institute of Anatomy and Cell Biology, National Taiwan University, Taipei, Taiwan

Received 12 December 2008

Accepted 14 December 2008

Abstract. Many recent studies have suggested that low-density lipoprotein (LDL) oxidation, endothelial dysfunction, andinflammation are involved in the pathogenesis of atherosclerosis. Herbal regimens in the treatment of blood stasis, a counterpartof atherosclerosis, commonly use medicinal plants of leguminosae and labiatae. We have developed disease-oriented screeningmethods to search for bioactive components, particularly isoflavones in leguminosae and polyphenols in labiatae from Chineseherbal medicines. Many bioactive components and active fractions capable of inhibiting a. Cu(II)-induced LDL oxidation,b. oxidized LDL-induced endothelial damage, c. uptake of oxidized LDL by macrophages (J774A.1), and d. expression of 

cell adhesion molecules (CAMs) have been identified. A polyphenol, namely salvianolic acid B from Salvia miltiorrhiza wasidentified to be a potent antioxidant, endothelial-protecting agent, and an inhibitor to suppress the expression of ICAM andVCAM. This review also briefly describes the strategy for developing herbal medicines as anti-atherosclerotic agents.

Keywords: Atherosclerosis, low-density lipoprotein, antioxidants, leguminosae, labiatae, Salvia miltiorrhiza

1. Introduction

Atherosclerosis is the major cause of coronary heart disease (CHD) in humans [6,21]. Many recent

studies have indicated that oxidative modification of low-density lipoprotein (LDL), endothelial dysfunc-tion, and inflammation are involved in the pathogenesis of atherosclerosis [10,14]. Mildly oxidized LDL(oxLDL) may appear in the plasma whereas extensively oxidized LDL only occurs in the intimal area of 

vessel wall. Functionally disturbed endothelial cells express MCP-1, cell adhesion molecules (CAMs),and selectins to recruit the circulating monocytes. Uptake of oxLDL by the macrophage scavengerreceptors (MSRs) of monocyte-derived macrophages leads to the formation of foam cells, fatty streaks,and fibrous plaques [4].

∗Address for correspondence: Ming-Shi Shiao, Department of Life Science, Chang Gung University, 259 Wen-Hua 1st Road,Queishan, Taoyuan, Taiwan 333. Tel.: +886 3 2118800 ext. 3495; Fax: +886 3 2118258; E-mail: msshiao@mail.cgu.edu.tw.

0951-6433/08/$17.00 2008 – IUBMB/IOS Press and the authors. All rights reserved

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Fig. 1. Antioxidants known to inhibit LDL oxidation or reduce atherosclerosis. The lipophilic antioxidant α-tocopherol is the

major antioxidant within the LDL to resist oxidation. Trolox and probucol were used as positive controls in this study.

Many potential targets have been identified for therapeutic intervention [16]. Among them, plasma

cholesterol-lowering, particularly LDL-cholesterol (LDL-C), remains a major goal of anti-atherosclerotic

treatment. As cholesterol-lowering drugs, statins reduce LDL-C by inhibiting HMG-CoA reductase.

However, recent clinical studies also have indicated that the beneficial effect of very extensive LDL-

cholesterol lowering by combination therapy of lipid-lowering drugs is doubtful [17]. Epidemiological

and animal studies have demonstrated that antioxidantsthat inhibit LDL oxidation may reduce atheroscle-

rosis [3]. However, clinical studies have not been able to demonstrate that antioxidant vitamins, such

as vitamin E and C, reduce cardiovascular disease. Lipophilic antioxidants, such as DPPD [22], probu-

col [12,25], and BO-653 [2], which co-migrate with circulating LDL particles, may protect LDL from

oxidative modification in the intimal area (Fig. 1). Hydrophilic antioxidants also reduce atherosclerosis

in animal models [5,8,26].

Traditional Chinese medicine (TCM) takes a holistic approach to maintain body homeostasis. In

which, diseases are considered as the disturbances in body homeostasis. Atherosclerosis has no pre-

cise counterpart in TCM and vise versa. However, blood stasis has been described as dyslipidemia,

poor circulation, hypertension, enhanced platelet aggregation and thrombosis, hyperglycemia, and high

oxidative stress [15]. Since these disturbances are related to atherosclerosis, it strongly suggests that

herbal medicines used in the treatment of blood stasis are good resources to search for anti-atherosclerotic

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Fig. 2. Determination of potential antioxidants using Cu2+-induced LDL oxidation as an in vitro system. Conjugated dieneformation causing by oxidation of polyunsaturated fatty acids (PUFA) in LDL was monitored by the increase of UV absorptionat 234 nm. An herbal extract (sample W1) containing lipophilic antioxidants prolonged the lag phase and reduce the rate of oxidation in the time-course of LDL oxidation. Trolox was used as a positive control in this assay. The five curves fromleft to right indicated the prolongation of lag phases by trolox at 0, 0.5, 1.0, 2.0 and 5.0 µM, respectively. Increased troloxconcentrations prolonged the lag phase without affecting the rate of oxidation.

agents. Extensive literature search further suggested that herbal regimens in blood stasis are composed of medicinal plants taxonomically belonging to leguminosae and labiatae. Their potentials as the resourcesof anti-atherosclerotic agents deserve the greatest attention.

We have developed several disease-oriented, mechanism-based bioassays to search for bioactive com-ponents from the medicinal plants in leguminosae and labiatae. The in vitro assays include a. inhibitionof LDL oxidation [5], b. inhibition of oxLDL-induced endothelial injury, c. inhibition of foam cellformation and d. inhibition of endothelial expression of ICAM and VCAM [6]. The observations aresummarized in this review.

2. Antioxidants to inhibit LDL oxidation

Inhibition of Cu2+-induced LDL oxidation was used as the assay system to search for potential

antioxidants. It is based on the observation that antibodies against Cu

2+

-induced oxLDL can react withoxLDL from atherosclerotic plaques in human and experimental animals and vice versa.Screening for antioxidants was carried out in a 96-well microtiter plate (quartz) and the time course of 

LDL oxidation was followed [24]. Conjugated diene formation causing by oxidation of polyunsaturatedfatty acids (PUFA) in LDL was continuously monitored by the increase of absorption at 234 nm using amicrotiter plate reader. The lag phase (Tlag), a commonly used marker of LDL susceptibility to oxidativedamage, was defined as intercept of the tangent of the slope of UV absorption curve in the propagationphase with the baseline. The maximum rate of oxidation (tangent) in the propagation phase could also

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Fig. 3. 1H-NMR spectra of total lipids extracted from LDL and oxLDL to demonstrate the oxidation of PUFA in oxLDL. Lipidextracts were dissolved in CDCl3 for measurement. A, LDL alone; B. LDL treated with 5.0 µM Cu2+ (before complete PUFAoxidation). The corresponding bis-allylic (-CH=CH–CH2-CH=CH-) proton signals were decreased; C. extensively oxLDLexhibiting no detectable bis-allylic proton signals.

be determined. The half life (T1/2) in LDL oxidation was defined as the time required to cause LDL

oxidation to the stage that the increase of conjugated diene formation reached half of its maximum value

(∆A234). The antioxidant potential of a plant extract or a pure natural product can be evaluated by its

capability to prolong the lag phase (∆Tlag), as compared with that of a control. Trolox (a water-soluble

vitamin E analog) and probucol (a lipophilic antioxidant) were used as the positive controls (Fig. 1).

A representative time course of Cu2+-induced LDL oxidation is illustrated in Fig. 2. The dose-

response effect of trolox in prolongation of LDL lag phase is also shown. To further demonstrate that

PUFA was oxidatively damaged in LDL oxidation, 1H-NMR was used to determine the decrease of 

signal intensity due to the bis-allylic protons (-CH=CH-CH2-CH=CH-) (2.85 ppm in chemical shift)

of PUFA in oxLDL [11] (Fig. 3). In the time course of LDL oxidation, arachidonic acid was oxidized

ahead of linoleic acid. The reduction in α-tocopherol and arachidonic acid can serve as markers for LDL

oxidation in the lag phase and propagation phase, respectively.

In the past years, over 2000 plant extracts, enriched fractions, and purified natural products have been

screened for potential antioxidants in this laboratory. Since medicines and foods are considered isogenic

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Table 1Prolongation of lag phase by salvianolic acid B (Sal B) andpotency of known antioxidants to inhibit Cu2+-induced LDLoxidation

Tlag (min)∆

Tlag (min) Relative potencyControl 151 − −

Probucol 302 151 1.00Trolox 251 100 0.66Quercetin 422 271 1.79Genistein 216 65 0.43Sal B 1,286 1,135 7.52

1. LDL(50µg/mLLDL-C)oxidationwas induced by 5.0µMCu2+. The potency of probucol (as a positive control) wasset as 1.00. Results are mean values of three determinations.

in TCM, foods and vegetables of leguminosae and labiatae origin were also screened. Results supportedthe view that medicinal plants and edible vegetables taxonomically belonging to leguminosae (rich inisoflavones) and labiatae (rich in polyphenols) were rich in antioxidants to inhibit LDL oxidation.

Leguminosae in herbal medicines, foods and vegetables rich in antioxidants are shown as it follows. A.Herbal medicines: Glycyrrhiza uralensis Fisch.; Astragalus membranaceus Bunge; Dalbergia odorifera

T. Chen.; Cassia obtusifolia L.; Psoralea corylifolia L.; Pueraria lobata (Willd.) Ohwi; Sophora

 flavescens Ait.; Sophora japonica L.; Dolichos lablab L. B. Foods and vegetables:Glycine max (L.)Merr.; Glycine tomentella; Arachis hypogaea L.; Medicago hispida Gaertn.; Vigna radiata (L.) R.Wilczak;Ormosis hosiei Hemsl. et Wils.; Pisum sativum L.; Phaseolus vulgaris L.

Labiatae in herbal medicines, foods and vegetables rich in antioxidants are listed as it follows. A.Herbal medicines: Salvia miltiorrhiza Bunge.; Agastache rugosa (Fisch. et Mey.) O. Kuntze.; Leonurus

 japonicus Houtt.; Scutellaria baicalensis Georgi.; Prunella vulgaris L.; Schizonepeta tenuifolia (Benth.)Briq. B. Foods, vegetables, and others: Rosmarinus officinalis L.; Perilla frutescens (L.) Britt.; Ocimum

basilicum L.; Rabdosia longituba (Miq.) Hara; Scutellaria barbata D. Don (S. rivularis Wall.). Among

them, S. miltiorrhiza was found particularly rich in antioxidants.Bioassay-guided purification leads to the identification of bioactive antioxidants. Salvianolic acid B(Sal B), a water-soluble phenolic natural product, was identified from S. miltiorrhiza as a very potentantioxidant to inhibit Cu2+-induced LDL (Table 1). S. miltiorrhiza is a commonly used medicinal plantin the herbal regimens for the treatment of blood stasis [15,19]. It is particularly rich in water-solublepolyphenols (Fig. 4). Sal B prolonged the lag phase (T lag) in the time course of LDL oxidation withoutaffecting the rate of LDL oxidation. This trend of Sal B to inhibit LDL oxidation was similar to thatof trolox, a water-soluble analogue of α-tocopherol. Sal B was 11.4 times more potent than trolox.Lipophilic antioxidants, such as probucol, reduced the rate of oxidation and prolonged the lag phase.Judged by prolongation of lag phase per se, Sal B was also more potent than probucol (Table 1).

3. Agents protecting oxLDL-induced endothelial damage

Endothelial dysfunction and injuries are the key events in the pathogenesis of atherosclerosis. Agentscapable of reducing endothelial injuries, causing by oxLDL particularly, may have the potential to reduceatherosclerosis. In this study, cultured human aortic endothelial cells (HAECs) were used as the cellularmodel. Treatment with extensively oxLDL (at the range of 20–80 µg oxLDL-cholesterol/mL) inducedapoptosis of HAECs in a dose-dependent manner. At the same concentration range and higher, nativeLDL was not harmful to HAECs.

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Fig. 4. Chemical structures of salvianolic acid B (Sal B) and phenolic compounds in Salvia miltiorrhiza. Sal B is the major andmost potent antioxidant identified in S. miltiorrhiza (labiatae), a medicinal plant very commonly used in the treatment of bloodstasis.

Results showed that many antioxidants capable of inhibiting LDL oxidation also inhibited oxLDL-induced endothelial injuries. Most of them are water-soluble flavones, isoflavones, and polyphenols. Atypical microscopic observation is shown to illustrate that Sal B (5–40 µM) rescued HAECs damagedby extensively oxidized LDL (Fig. 5). Sal B alone, at 100 µM, was not cytotoxic to HAECs.

Agents that stimulate endothelial cell proliferation could also be identified in this assay system.The aqueous ethanolic extract of  S. miltiorrhiza (SME) was found to stimulate the proliferation of HAECs. However, Sal B was not the major active component in SME to stimulate HAECs proliferation.Identification of the major endothelial proliferating agents in SME is currently underway.

4. Inhibition of oxLDL uptake by macrophage scavenger receptors

Since foam cell formation and fatty streaks are the key events in early atherosclerosis, inhibition of theuptake of oxLDL by MSRs may reduce the formation of foam cells and fatty streaks in the subendothelialspace [4].

Murine macrophages (J774A.1), after 24-h culture in a medium supplemented with lipoprotein-deprived serum (LPDS), were used as the cellular model to search for MSR inhibitors [7]. J774A.1macrophages cultured in a LPDS-containing medium expressed LDL-receptor as well as scavengerreceptors. Without fetal calf serum in the medium, macrophages could only uptake extensively oxLDL

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1. Control

Fig. 5. Microscopic observations of Sal B to protect extensively oxidized LDL-induced cell death in cultured human aorticendothelial cells (HAECs). Cell viability was determined by MTT assay. 1. Control; 2. HAECs treated with extensivelyoxidized LDL (40 µg/mL); 3. HAECs treated with oxLDL (40 µg/mL) and Sal B (20 µM); 4. HAECs treated with oxLDL(40 µg/mL) and Sal B (50 µM). Extensively oxidized LDL was used in this study.

via MSRs. The accumulation of oxLDL in macrophage-derived foam cells was readily determined byOil Red O staining. Helvolic acid (HVA) inhibited oxLDL uptake was used as a positive control [20](Fig. 6). In this assay system, inhibitors to MSRs and inhibitors to ACATs can not be distinguished.Since this assay was performed in a kinetic manner and the findings that addition of excess LDL did notcompete against oxLDL uptake, inhibition of ACAT was less likely.

As illustrated in Fig. 7, an active fraction reduced the contents of lipid droplets of macrophage-derivedfoam cells significantly. We have identified several organic anionic compounds, which contained arigid ring skeleton and at least one anionic carboxyl group, as mild to potent MSR inhibitors (SRIs).Ganodermic acid S (GAS), a major oxygenated triterpene identified from the medicinal fungus Gano-

derma lucidum [18], fulfilled the structural requirement as a MSR inhibitor [20]. GAS was more potentthan HVA. The common structural character (a rigid ring skeleton and a negatively-charged carboxylfunctional group) is evident in HVA and GAS (Fig. 7).

Macrophages play a key role in the immunological system in the defense against infection and surveil-lance of neoplasm. The ligands capable of binding to MSRs (namely, SRA, SRB and CD36) includeoxLDL. However, oxLDL-specific MSR inhibition, without interference to the binding of bacterial en-dotoxins, is desirable. Further study is required to establish the potential of MSR inhibitors in reducing

atherosclerosis.

5. Inhibition of the expression of ICAM and VCAM

Adhesion and infiltration of monocytes into the vessel wall can be observed in early atherosclerosis.In which, expression of cell adhesion molecules (CAMs) by the arterial endothelium plays a key role.Agents inhibiting the expression of CAMs may slow down the progression of early atherosclerosis.

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A B

C D

Control OxLDL

OxLDL + HVA OxLDL + VGH-801

Fig. 6. Screening for macrophage scavenger receptor inhibitors. Uptake of extensively oxLDL by J774A.1 macrophages wasdetermined by Oil Red staining. A. Control (cells without treatment); B. Cells treated with extensively oxLDL (40 µg/mL);C. Cells treated with oxLDL (40 µg/mL) and helvolic acid (HVA) (50 µM); D. Cells treated with oxLDL (40 µg/mL) and anactive herbal extract (VGH-801-W1). VGH-801-W1 inhibited the uptake of oxLDL by macrophages more effectively thanHVA without affecting cell viability.

Fig. 7. Helvolic acid (HVA) and ganodermic acid S (GAS) as scavenger receptor inhibitors.

The aqueous ethanolic extract of  S. miltiorrhiza (SME) and Sal B also inhibited the expression of 

CAMs in tumor necrosis factor-α (TNF-α)-treated HAECs. SME or Sal B treatment inhibited TNF-α-induced expression of vascular cell adhesion molecule-1 (VCAM-1). Dose-dependent lowering of 

expression of intercellular cell adhesion molecule-1 (ICAM-1) was also observed with SME or Sal B [1].

However, the expression of endothelial cell selectin (E-selectin) was not affected. Sal B also significantly

reduced the binding of the human monocytic cell line, U937, to TNF-α-stimulated HAECs. Both SME

and Sal B inhibited TNF-α-induced activation of nuclear factor kappa B (NF-kB) in HAECs [1]. These

findings suggest that SME and Sal B have anti-inflammatory activities.

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6. Discussion

In this study, extensive search for antioxidants, endothelium-protecting agents, macrophage scavengerreceptor inhibitors, and CAMs expression inhibitors have been carried out. The screening methods are

based on the following bioassays in vitro. a. Cu(II)-induced LDL oxidation, b. oxLDL-induced endothe-lial damage, c. uptake of oxLDL by macrophages (J774A.1), and d. expression of CAMs in HAECs.The above-mentioned events are considered as the key steps in the pathogenesis of atherosclerosis.Semi-automation has been designed for these assays, especially in the screening for antioxidants. Forantioxidant assay, over 2000 herbal extracts, enriched fractions, and pure natural products have beenscreened. Many herbal regimens in the treatment of blood stasis use medicinal plants of leguminosaeand labiatae. Our results support the speculation and further provide scientific basis that herbal regimensin the treatment of blood stasis contain antioxidants and anti-atherosclerotic agents. Although differentspecies may be used, Glycyrrhiza (licorice) contains potential antioxidant and anti-atherosclerotic agents.It has been reported that licorice extract and its major polyphenol glabridin protect LDL against oxidationin vitro and ex vivo [5].

Atherosclerosis is the key underlying mechanism in CHD. Beyond hypercholesterolemia, multiple risk factors are involved in this degenerative disease [6]. Besides, the disease pathogenesis may take twentyyears or longer until clinical symptoms become obvious. It can be anticipated that a single chemicalentity alone is less likely to be effective to interrupt the progression of atherosclerosis. It may alsopartly explain the observations that antioxidant vitamins are not effective for CHD patients in clinicalstudies. Combination therapy is an emerging trend and should be a better strategy for the management of atherosclerosis. A current study has predicted that a polypill containing a statin, three antihypertensives,folic acid, and aspirin may reduce cardiovascular disease by more than 80% [23]. Since the outcome of intensive reduction of a single risk factor, such as cholesterol-lowering by a combination therapy, remainscontroversial and risky [17], it may not be the most appropriate strategy for combination therapy.

It is worthwhile to mention that TCM uses combination therapy. A regimen usually contains four ormore herbal materials and their soluble components in hot water or water-ethanol are orally consumed.

Our results also showed that most antioxidant activities, MSR inhibitors, and endothelial-protectingagents in these herbal regimens could be extracted readily by an aqueous ethanolic solution. It isanticipated that herbal medicines, if effective, may exhibit therapeutic potential in the early, reversiblestage of atherosclerosis. Development of combined, enriched active herbal preparations to inhibit themultiple key events in early atherosclerosis is the best strategy. Further pharmacological evaluationand clinical studies are yet required to prove the concept and demonstrate the potential for treatment of atherosclerosis.

In this study, Sal B was identified as a potent antioxidant and an effective endothelium-protecting agentfrom Salvia miltiorrhiza, one of the most popular medicinal herbs in TCM [19]. Therapeutic potentialsof Sal B alone or a Sal B-enriched fraction of  S. miltiorriza deserves great attention. New Zealand white(NZW) rabbits fed with a high-cholesterol diet and apolipoprotein E-deficient mice are the two animalmodels commonly used for the evaluation of pharmacological function of ant-atherosclerotic agents [9,

13,27]. The anti-atherosclerotic potential of Sal B-enrich fraction using cholesterol-fed NZW rabbits asthe model has been studied [26]. Treatment of Sal B-enriched fraction reduced atherosclerotic lesionarea, inhibited LDL oxidation ex vivo, and preserved vitamin E in LDL. The Sal B-enriched fraction alsoinduced neointimal cell apoptosis in a rabbit angioplasty model [8].

In conclusion, this study demonstrated the fruitfulness to search for potential antioxidants and anti-atherosclerotic agents from herbal medicines traditionally used in the treatment of blood stasis. Basedon recent advances in atherosclerotic research, this study also provided platform technology for theevaluation of herbal medicines which are originated from ancient wisdom.

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Acknowledgements

This work was supported by the National Science Council, ROC (NSC95-2320B-075-009 to M.-S.

Shiao), the Program for Promoting University Academic Excellence (91-B-FA09-2-4 to M.-S. Shiao andY.-L. Chen), and Chang Gung University (EMRPD170201).

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