Pharmacological effects of Radix Angelica Sinensis (Danggui) on cerebral infarction

Radix Angelica Sinensis, the dried root of Angelica sinensis (Danggui), is a herb used in Chinese medicine to enrich blood, promote blood circulation and modulate the immune system. It is also used to treat chronic constipation of the elderly and debilitated as well as menstrual disorders. Research has demonstrated that Danggui and its active ingredients, as anti-arthrosclerotic, anti-hypertensive, antioxidant anti-inflammatory agents which would limit platelet aggregation, are effective in reducing the size of cerebral infarction and improving neurological deficit scores.


Background
Danggui, the dried root of Angelica Sinensis (Radix Angelica Sinensis), is a commonly used Chinese medicinal herb to enrich blood, promote blood circulation and treat blood deficiency pattern and menstrual disorders such as dysmenorrhea and irregular menstrual cycle [1]. Wilasrusmee et al. [2] reported that Danggui (105 μg/ml) plays an immunostimulatory role in mitogen-stimulated murine lymphocytes in vitro. Angelan, a purified polysaccharide component of Angelica nakai thought to improve immune function, increases the expression of cytokines in splenocytes as Angelan enhances and the production of interleukin-6 (IL-6) and interferon-γ(IFN-γ) of activated macrophages, helper T cells and natural killer cells [3]. The chemical constituents of the Danggui extract are classified into essential oil and water soluble parts including lipid compounds, phenolic compounds, carbohydrates, organic acids and other constituents [4]. The most active ingredients are polysaccharides, Z-Ligustilide (3-butylidene-4,5-dihydrophthalide) and ferulic acid (4hydroxy-3-methoxycinnamicacid) [1].
This article aims to provide an overview of the pharmacological effects of Danggui in reducing the size of cerebral infarction and improving neurological deficit scores.

Vasodilation and improving microcirculation
Nitric oxide (NO) is synthesized with nitric oxide synthase (NOS) which includes three different isoforms, namely endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) [5]. While nNOS and eNOS are induced under different conditions, their activation relies on intracellular Ca 2+ for binding calmodulin [5,6]. Due to its vasodilative effects, eNOS is considered neuro-protective [6]. Hypertension and a lack of endothelium-derived relaxing factor activity are found in eNOS knockout mice [7]. Moreover, the cerebral infarction size is larger in a model of eNOS mutant mice with middle cerebral artery occlusion (MCAo) [8]. Therefore, eNOS has a vasodilation effect and is neuro-protective by increasing the blood flow [9]. It is possible that Danggui increases NO formation and relaxes the endothelium [10], thereby limiting infarction size. In rabbits on a high-lipid diet, treatment with ferulic acid, an active component of Danggui, increases the generation of NO, thereby inhibiting platelet aggregation on endothelium and proliferation of smooth muscles and preventing leucocytes adhering to the endothelium [11].
Z-Ligustilide (3-butylidene-4,5-dihydrophthalide), a component of Danggui, inhibits (4-8 μg/ml) the spontaneous contraction of isolated rat uterus in a dose-dependent manner [12]. Moreover, Z-Ligustilide may also inhibit prostaglandin F-2α, oxytocin, acetylcholine chloride and potassium depolarization-induced uterine contraction, suggesting that Ligustilide modulates the function of uterine tissue and has a non-specific anti-spasmodic effect [12]. Z-Ligustilide enhances the recovery of conjunctival capillary and venue diameter after dextran T500 administration to rabbits and increases the number of opened capillaries as well as blood flow, suggesting that Z-Ligustilide improves microcirculation [13].
Ferulic acid is the main organic acid component of Danggui. Ferulic acid (10 -3 mol/L) relaxes the phenylephrine-induced contraction of aorta ring in spontaneous on rat (SHR) whereas the effects of ferulic acid may be partially blocked by pretreatment of the aorta with N Gnitro-L-arginine methyl ester (L-NAME, 10 -4 mol/L) which inhibits the production of NO from L-arginine [14]. Ferulic acid (10 -3 mol/L) reduces the production of thromboxane B 2 in the aorta ring of SHR [14]. Ferulic acid (10 -4 mol/L) also significantly reduces the generation of NADPH-dependent production of the superoxide anion [14] and enhances the acetylcholine-induced vasodilation whereas hydroxyhydroquinone (HHQ) inhibits this effect [14]. Taken together, ferulic acid reduces blood pressure in SHR via effects on (1) eNOS; (2) the inhibition of thromboxane B 2 to relax aorta ring; (3) reactive oxygen species (ROS) scavenging activity to increase the availability of NO in endothelial cell of aorta [14].

Anti-arthrosclerosis effects
Stroke is divided into two major groups according to the cerebral damage, cerebral infarction and cerebral hemorrhage. Eighty percent (80%) of stroke patient suffer from cerebral infarction [15]. Cerebral infarction is mainly caused by thrombosis, embolism or systemic hemodynamic hypertension. Atherosclerosis in large and small arteries is a major contributor to cerebral thrombosis. The etiology of atherosclerosis and stroke is related to inflammation and genetic factors. Ischemic cerebral infarction may be prevented by anti-inflammatory agents or the treatment of vascular diseases, heart diseases and hypertension [16][17][18].
A principal contributor to cerebral infarction and atherosclerosis is believed to be initiated by an excessive inflammatory-fibro-proliferative response [19]. Atherosclerosis involves growth factors, cytokines and vaso-regulatory factors such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGH), transforming growth factor-β (TGF-β), interleukin-1 (IL-1) and tumor necrosis factor -α (TNF-α) [19,20]. Cytokines are both pro-and anti-atherogenic; for example, IL-1 and TNF-α mediate the production of monocyte chemoattractant protein-1 (MCP-1) to induce monocyte migration directly into the intima. By contrast, cytokines can induce NO production which regulates the vasomotor tone of artery, thereby influencing the initiation and progression of the atherosclerosis process [20]. A previous study [21] found that nicotine can mediate the development and progression of atherosclerosis via the inhibition of TGF-β1 and basic fibroblast growth factor (bFGF). The alteration of TGB-β activity leads to the atherosclerotic change of vessel wall and increased TGB-β signaling plays a protective role of atherosclerosis [22]. A study [23] reported that bFGF enhances smooth muscle migration and proliferation via the regulation of interstitial collagenase expression in the early stages of atherosclerosis. Wang et al. [24] found that the levels of TGB-β reduced and those of bFGF increased in human umbilical vein endothelial cells damaged by hyperlipidemic serum. Moreover, under electromicroscopy the morphology of endothelial cell was also damaged which was reversed by Danggui (20 mg/ml) and its component of sodium ferulate (0.3 mg/ml). These results indicate that both Danggui and sodium ferulate have anti-atherogenic effects [24]. Yu et al. [25] found that the levels of total cholesterol (TC, 0.95 mmol/L vs. 11.79 mmol/L), triglyceride (TG) and high density lipoprotein cholesterol (HDLC) and low density lipoprotein cholesterol (LDLC) increased in rabbits on a high-lipid diet compared to the control group which was on a normal diet. After 25% Danggui was administered (i.v.) for four weeks, the levels of TG decreased from 3.52 mmol/L to 1.68 mmol/L. The plaque area of thoracic aorta was also reduced after Danggui treatment, from 63.31% to 35.58% [25]. Moreover, Danggui reduced the increase of the serum malonyldialdehyde (MDA) levels caused by the high-lipid diet [25]. In a similar study [11], after treatment with sodium ferulate, the plaque area of thoracic aorta was reduced while the TG level was reduced to 1.75 mmol/L in rabbits on a high-lipid diet [11]. Moreover, the sodium ferulate-treated group increased the production of NO from epithelium cells. Both Danggui and sodium ferulate inhibit the formation of atherosclerosis because Danggui reduces the TG and lipid peroxidation levels or increases NO production, or both.

Anti-platelet aggregation effects
Anti-platelet aggregation agents such as aspirin, ticlopidine and clopidogrel are widely used to prevent secondary ischemic stroke [16,26]. A clinical trial [26] reported that administration of aspirin within six hours of the onset of ischemic stroke reduces the patients' mortality rate.

Anti-oxidative effects
Reactive oxygen species (ROS) including the superoxide anion, hydrogen peroxide and hydroxyl radical are generated after cerebral ischemia. The ROS affect mitochondrial function, DNA repair and transcription factors, leading to apoptosis [37,6]. Superoxide dismutase 1 (SOD1), an endogenous antioxidant, blocks the early release of cytochrome c from mitochondria and reduces the development of apoptosis in focal cerebral ischemic mice [38]. Apolipoprotein E, via its anti-oxidative effects against cerebral ischemia, is neuro-protective in transient forebrain ischemia induced by bilateral common carotid artery occlusion (BCCAo) in mice [39]. Anti-oxidant nutrients such as vitamin E and Ginkgo biloba extract reduce cerebral damage in rodent models of ischemia and reperfusion [40]. GABA B receptor agonist baclofen may be neuro-protective via the inhibition of N-methyl-D-asparate (NMDA) receptor-mediated NO production in brain ischemic injury [41]. Ferulic acid (100 mg/kg, i.v.) enhanced the expression of GABA B1 in the reperfusion period (three and 24 hours after ischemia) in rats [34]. Z-ligustilide reduced the size of cerebral infarction from 22.1% to 11.8% (5 mg/kg, i.p.) and 2.60% (20 mg/kg, i.p.) [42]. Z-ligustilide reduced the MDA levels and increased glutathione peroxidase (GSH-Px) and SOD activities in the ischemia-reperfusion brain tissues induced by BCCAo in mice [42]. Danggui or its components or both demonstrate anti-oxidant activity in ischemia-reperfusion injury models.

Effect of Danggui on cerebral infarction
Danggui (5 g/kg, i.p.) increased blood circulation and neuronal metabolism in an MCAo rat model [43]. Danggui reduced the size of cerebral infarction, neurological deficit scores and increased blood flow and SOD activity in the MCAo rat model [44]. Z-ligustilide reduced cerebral infarction size to 10.90% and 3.19% in rats orally dosed with 20 m/kg or 80 mg/kg respectively (21.08% in the control group) in a MCAo model [45]. Moreover, Z-ligustilide (10 mg/kg or 40 mg/kg, p.o.) increased choline acetyltransferase activity and inhibited acetylcholinesterase to improve cognitive function in rats with hypoperfusion [46]. Our previous study [33] found that ferulic acid (80 mg/kg or 100 mg/kg, i.v.) reduced cerebral infarction size and neurological deficit scores in rats. Liu et al. [47] reported that Danggui (25%, i.v.) administered to 1040 patients with acute cerebral infarction improved neuro-function scores and the Barthel index score more than Salvia miltiorrhiza (78.7% vs. 59.3%). Danggui reduces the size of cerebral infraction and improves neurological deficit scores.

Conclusion
The effects of Danggui on cerebral infarction are through multiple pathways, including anti-arthrosclerosis, improving microcirculation, anti-platelet aggregation, anti-inflammatory and anti-oxidative effects (Table 1). Danggui may be useful in treating the cerebral infarction type of stroke.  reverse the reduction of TGB-β/reverse the increase of bFGF [24] Danggui reduce the increase of serum malonyldialdehyde (MDA) levels [25] sodium ferulated decrease the levels of triglyceride [11] Vasodilatation and improving microcirculation effects Danggui increase the formation of NO and mediate the inhibition of calcium influx [10] sodium ferulate increase the generation of NO [11] Ligustilide inhibit prostaglandin F-2α, oxytocin, acetylcholine chloride, and potassium depolarization-induced muscle contraction [12] Ligustilide increase the number of opened capillary and the speed of blood flow [13] Ferulic acid enhance acetylcholine-induced vasodilatation and reduce the production of thromboxane B 2 [14] Anti-platelet aggregation effects Danggui and sodium ferulate inhibit ADP-induced and collagen-induced platelet aggregation [27] Z-Ligustilide inhibit ADP-induced platelet aggregation [28] Anti-inflammatory effects Ferulic acid inhibit ICAM-1 and NF-B expression [33] Ferulic acid enhance gamma-aminobutyric acid type B1 (GABA B1 ) receptor expression [34] Danggui reduce TNF-α and TGF-ß1 mRNA expression [35] Danggui polysaccharides reduce TNF-α levels [36] Anti-oxidative effects Ferulic acid reduce the generation of NADPH-dependent production of superoxide anion [14] Ferulic acid enhances the expression of GABA B1 receptor expression [34] Z-ligustilide reduce MDA levels and increase GSH-PX and SOD activities [42]