Bioactivities of major constituents isolated from Angelica sinensis (Danggui)

Danggui, also known as Angelica sinensis (Oliv.) Diels (Apiaceae), has been used in Chinese medicine to treat menstrual disorders. Over 70 compounds have been isolated and identified from Danggui. The main chemical constituents of Angelica roots include ferulic acid, Z-ligustilide, butylidenephthalide and various polysaccharides. Among these compounds, ferulic acid exhibits many bioactivities especially anti-inflammatory and immunostimulatory effects; Z-ligustilide exerts anti-inflammatory, anti-cancer, neuroprotective and anti-hepatotoxic effects; n-butylidenephthalide exerts anti-inflammatory, anti-cancer and anti-cardiovascular effects.


Background
Angelica sinensis (Oliv.) Diels (Apiaceae) (AS), the root of which is known in Chinese as Danggui (Figure 1), was first documented in Shennong Bencao Jing (Shennong's Materia Medica; 200-300AD) and has been used as a blood tonic to treat menstrual disorders [1]. Danggui is marketed in various forms worldwide [2,3]. Over 70 compounds have been identified from Danggui, including essential oils such as ligustilide, butylphthalide and senkyunolide A, phthalide dimers, organic acids and their esters such as ferulic acid, coniferyl ferulate, polyacetylenes, vitamins and amino acids. Z-ligustilide (water insoluble and heat stable), among which Z-butylidenephthalide and ferulic acid are thought to be the most biologically active components in AS [4] and are often used in quality control and pharmacokinetic studies of Danggui [3][4][5][6].
Z-ligustilide is the main lipophilic component of the essential oil constituents and a characteristic phthalide component of a number of Umbelliferae plants. Z-ligustilide is considered to be the main active ingredient of many medicinal plants, such as Danggui [7] and Ligusticum chuangxiong [8].
Phthalides Phthalides ( Figure 2) consist of monomeric phthalides such as Z-ligustilide and phthalide dimers. In 1990 Danggui was reported in the literature when the Z-ligustilide dimer E-232 was isolated [9]. The majority of the phthalides identified is relatively non-polar, the fraction of which can be extracted with solvents such as hexanes, pentane, petroleum ether, methanol, 70% ethanol and dichloromethane. The amount of Z-ligustilide in Danggui varies between 1.26 and 37.7 mg/g dry weight [6,10,11]. Z-ligustilide facilitates blood circulation, penetrates the blood brain barrier to limit ischemic brain damage in rats and attenuates pain behaviour in mice [12][13][14]. Preclinical studies have indicated that AS and Z-ligustilide may also relax smooth muscle in the circulatory, respiratory and gastrointestinal systems [15].

Organic acids
Danggui contains many organic acids. For example, ferulic acid ( Figure 3) isolated from Danggui is widely used as the marker compound for assessing the quality of Danggui and its products. Methanol, methanol-formic acid (95:5), 70% methanol, 70% ethanol, 50% ethanol or diethyl ether-methanol (20:1) is used as the initial extraction solvent. The amount of ferulic acid in Danggui varies between 0.21 and 1.75 mg/g dry weight [6,16]. We recently extracted ferulic acid from AS using ethyl acetate and obtained 3.75 mg/g dry weight of the whole plant [11]. Abundant in rice bran, wheat, barley, tomato, sweet corn and toasted coffee, ferulic acid is an antioxidant, anti-inflammatory and anti-cancer agent and apart from its effects against Alzheimer's disease, it possesses anti-hyperlipidemic, antimicrobial and anti-carcinogenic properties [17][18][19][20][21].

Polysaccharides
Biochemical and medical researchers have recently been interested in the anti-tumor and immunomodulatory effects of polysaccharides [22]. The efficacy of Danggui is associated with its various polysaccharides [22] which are extracted with water as the initial extraction solvent. Polysaccharides from Danggui consist of fucose, galactose, glucose, arabinose, rhamnose and xylose [23]. Danggui contains a neutral polysaccharide and two kinds of acidic polysaccharides [24].

Anti-inflammatory effects
Ferulic acid and isoferulic acid inhibit macrophage inflammatory protein-2 (MIP-2) production by murine macropharge RAW 264.7 cells, suggesting that these compounds contribute to the anti-inflammatory activity of AS [25,26]. Z-ligustilide also shows anti-inflammatory effects, probably related to inhibition of the TNF-α and NF-B activities [27]. Using an NF-B-dependent trans-activation assay as a pre-screening tool, our study demonstrated the anti-inflammatory effect of the ethyl acetate fraction of AS or Danggui [28]. AS suppresses NF-B luciferase activity and decreases NO and PGE 2 production in lipopolysaccharide (LPS)/IFN-γ-stimulated murine primary peritoneal macrophages. Ferulic acid and Z-ligustilide, two major compounds in AS, decrease NF-B luciferase activity, which may contribute to the anti-inflammatory activity of AS [11]. Our in vivo study further confirmed that the ethyl acetate extract of AS inhibits the production of inflammatory mediators thereby alleviating acute inflammatory

Immunomodulatory effect
Treatment of BALB/c mice spleen cells with AS polysaccharide (100 μg/ml) increases the production of IL-2 and IFN-γ and decreases the production of IL Excess adipose tissue can lead to insulin resistance and increases the risk of type II diabetes and cardiovascular diseases. Water and 95% ethanol extracts of AS effectively decrease fat accumulation in 3T3-L1 adipocytes and reduce triglyceride content [49]. Yeh et al. demonstrated that n-butylidenephthalide is anti-angiogenic and is associated with the activation of the p38 and ERK1/2 signaling pathways [50].

Neuroprotective effects
Z-ligustilide treatment decreases the level of malondialdehyde (MDA) and increases the activities of the antioxidant enzymes glutathione peroxidise (GSH-Px) and superoxide dismutase (SOD) in the ischemic brain tissues in ICR mice; meanwhile there is a decrease in Bax and caspase-3 protein expression [51]. Z-ligustilide increases the choline acetyltransferase activity and inhibits the acetylcholine esterase activity in ischemic brain tissue from Wistar rats [52]. Huang et al. reported that AS extract protects Neuro 2A cell viability against β-amyloid (Aβ) peptide induced oxidative damage by ROS, MDA and glutathione (GSH) and rescues mitochondrial transmembrane potential levels [53]. Z-ligustilide inhibits the TNF-α-activated NF-B signaling pathway, which may contribute to Z-ligustilide's protective effect against Aβ peptide-induced neurotoxicity in rats [54].

Anti-oxidative activities
Water AS extract can be further purified into various AS polysaccharide fractions, such as a highly acidic polysaccharide fraction consisting of galacturonic acid. BALB/c murine peritoneal macrophages pretreated with various AS polysaccharide fractions alleviate the decrease in cell survival caused by tert-butylhydroperoxide, with an increased intracellular GSH content [58]. Furthermore, acidic polysaccharide fraction is also the most active fraction in terms of inhibiting the decrease in cell viability caused by H 2 O 2 . Acidic polysaccharide fraction also decreases the MDA formation, reduces the decline in SOD activity and inhibits the depletion of GSH in murine peritoneal macrophages caused by H 2 O 2 [59]. Ethanol extract of AS combined with eight other Chinese herbs significantly increase the radical scavenging ability of 1,1diphenyl-2-picryl hydrazine (DPPH) [60].

Anti-hepatotoxic effects
The liver contains a series of microsome hemoproteins called cytochrome P450s (CYPs). CYPs play an important role in the metabolic oxygenation of a variety of lipophilic chemicals including drugs, pesticides, food additives and environmental pollutants. The most important isoenzyme forms of cytochrome are CYP1A2 (13%), CYP2C (20%), CYP2D6 (2%), CYP2E1 (7%) and CYP3A (29%) [61]. Tang et al. reported that water and ethanol extracts of AS strongly increase the CYP2D6 and CYP3A activity in the microsome fraction of male Wistar rat livers [62]. Gao et al. demonstrated that treatment with Danggui Buxue Tang (DBT), which contains the roots of Astragali and AS, induces erythropoietin mRNA expression in a dosedependent manner in human hepatocellular carcinoma cell line Hep3B [63]. Dietz et al. reported that Z-ligustilide targets cysteine residues in human Keap1 protein thereby activating Nrf2 and the transcription of antioxidant response element (ARE) regulated genes and inducing NADPH:quinine oxidoreductase 1 (NQO1) [64].

Kidney protective effects
There has been a 60% increase in the number of people needing treatment for chronic kidney disease between 2001 and 2010. Characterized by an increase in interstitial fibrosis and tubular epithelial cell atrophy, renal tubulointerstitial fibrosis is the common pathogenetic process of chronic kidney disease [65][66][67]. Angiotensin II appears to play a key role in several mechanisms involved in tubulointerstitial fibrosis. Angiotensin II up-regulates the expression of TGFβ1, a profibrotic cytokine involved in many of the events leading to renal fibrosis. Angiotensin converting enzyme inhibitors (ACEi) can reduce renal tubulointerstitial fibrosis [68].
Oral administration of Astragalus membranaceus var. mongholicus and AS (14 g/kg/day) to Wistar rats increases the constitutive ROS activity in the kidneys. The treatment also enhances NO production via eNOS activation and the scavenging of ROS in the obstructed kidney in Wistar rats after unilateral ureteral obstruction [69]. In analysis with genechips, gene expression is induced, including transient receptor protein 3 (TRP3), bone marrow stromal cell antigen 1 (BST-1), peroxisomal biogenesis factor 6 (PEX6), xanthine dehydrogenase (XDH), CYP1A1, serine/cysteine proteinase inhibitor clade E member 1 (PAI-1) and fibroblast growth factor 23 (FGF23). These genes may be involved in the increased degeneration of the extracellular matrix (ECM), decreasing ROS and regulating calcium phosphate metabolism [70]. Administration of Astragalus membranaceus var. mongholicus and AS into Sprague Dawley rats, as a unilateral ureteral obstruction model, decreases TGFβ1 levels, fibroblast activation, macrophage accumulation and tubular cell apoptosis [71]. Song et al. reported that oral administration of Astragalus membranaceus var. mongholicus and AS (12 g/kg/ day) shows renoprotective effects, possibly associated with a reduction of proteinuria and up regulation of VEGF. These changes may have reduced the loss of capillaries and improve microstructure dysfunction in nephrectomized rats [72].
A study on 47 herbs of potential interest in the context of renal or urinary tract pathologies demonstrated that AS, Centella asiatica, Glycyrrhiza glabra, Scutellaria lateriflora and Olea europaea have strong antioxidant effects in tubular epithelial cells or apoptotic effects on renal mammalian fibroblasts or both [73].

Other effects
Ethanol extract of AS (100, 300 mg/kg) prolongs estrus in rats and the estrogenic activity of AS extract is likely due to the presence of Z-ligustilide [74]. A dichloromethane extract of AS exhibits a potent inhibitory effect on melanin production [75]. AS polysaccharide (ASP, 2.5 mg/day) enhances recovery in platelet, red blood cells and white blood cells counts in BALB/c mice after irradiation (4 Gy) [76]. ASP also reduces hepcidin expression by inhibiting signal transducer and activator of transcription (STAT) 3/5 and decapentaplegic protein (SMAD) 4 expression in the liver. Thus, ASP is suggested to be used for treating hepcidin-induced diseases [77].

Conclusion
Bioactive components extracted from AS roots include Z-ligustilide, ferulic acid and AS polysaccharides. Major pharmacological effects of Danggui extract or its components include anti-inflammatory, anti-cancer, immunomodulatory, anti-cardiovascular, neuroprotective, anti-oxidative, anti-hepatotoxic and renoprotective activities.