A Review of Recent Research Progress on the Astragalus Genus

Astragalus L., is one of the largest genuses of flowering plants in the Leguminosae family. Roots of A. membranaceus Bge. var. mongholicus (Bge.) Hsiao, A. membranaceus (Fisch.) Bge. and its processed products are listed in the China Pharmacopeia for “qi deficiency” syndrome treatment. However, more and more researches on other species of Astragalus have been conducted recently. We summarize the recent researches of Astragalus species in phytochemistry and pharmacology. More than 200 constituents, including saponins and flavonoids, obtained from 46 species of Astragalus genus were collected for this article. In pharmacological studies, crude extracts of Astragalus, as well as isolated constituents showed anti-inflammatory, immunostimulant, antioxidative, anti-cancer, antidiabetic, cardioprotective, hepatoprotective, and antiviral activities. The goal of this article is to provide an overview of chemical and pharmacological studies on the Astragalus species over the last 10 years, which could be of value to new drug or food supplement research and development.


Saponins
Saponin is the major chemical constituent type in the Astragalus genus. Cycloartane-and oleanane-type saponins from it showed interesting biological properties.
Some uncommon cycloartane triterpene glycosides such as 108-142 were also isolated from the Astragalus genus. For example, the eremophilosides E-I (118-121) [24] are 16β,23-epoxycycloartanes. Among them, eremophilosides E (118) and F (119) are characterized as having an unusual loss of a four carbon side chain from C-24 to C-27 and a six-membered lactone ring between C-23 and C-16, while eremophilosides G-I (117, 120 and 121) show an unusual modification of the cyclic side chain.
In the study of isoprenoid plants of the Astragalus genus like A. orbiculatus, researchers found several derivatives of 16β,23,16α,24-diepoxycycloartane (127-133) from its aerial parts [38], which have been found only in this species.

Oleanane-Type Saponins
Apart from the cycloartane triterpene glycosides, many oleanane-type saponins shown in Table 2 were also isolated and identified from the Astragalus genus. Structure characterizations of this kind of saponin indicated they were substituted with a β-hydroxymethyl, instead of methyl in the 23-position.     160 Azukisaponin V A. cruciatus aerial parts and roots [2] A. hareftae whole plant [10] 161 Astragaloside VIII

Biological Activities of the Astragalus Genus
A. membranaceus, A. mongholicus and A. complanatus have been mainly used in folk medicine for their anti-inflammatory, immunostimulant, antioxidative, anti-cancer, antidiabetic, cardioprotective, hepatoprotective, and antiviral properties in recent years. The active constituents for the above-mentioned effects were proved to be polysaccharides, saponins, and flavonoids.

Anti-Inflammatory Activity
Astragalus extract, along with its polysaccharides, and saponins showed anti-inflammatory activity both in vitro and in vivo. Kim et al., found that the extract of A. membranaceus not only improved the atopic dermatitis skin lesions in 1-chloro-2,4-dinitrobenzene-induced mice as well as restoring nuclear factor-κB expression markedly, but also suppressed the expression of Th2 cytokines and significantly decreased the TNF-α level. They then figured out that A. membranaceus was effective for treating atopic dermatitis by regulating cytokines [80]. Ryu et al., verified that Astragali Radix had an anti-inflammatory effect mediated by the MKP-1-dependent inactivation of p38 and Erk1/2 and the inhibition of NFkappaB-mediated transcription [81]. As the main composition of Astragalus, Astragalus polysaccharides can effectively ameliorate the palmitate-induced pro-inflammatory responses in RAW264.7 cells through AMPK activity [82]. They also showed anti-inflammatory activity, along with structure protective properties for lipopolysaccharide-infected Caco2 cells [83]. On the other hand, the anti-inflammatory activity of saponins was also studied. The results, of agroastragalosides I, II, isoastragaloside II, and astragaloside IV showed the ability to inhibit lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophages [84]. Meanwhile, astragaloside IV was shown to be a promising natural product with both healing and anti-scar effects for wound treatment [85], could be used as a novel anti-inflammatory agent, and attenuated diabetic nephropathy in rats by inhibiting NF-κB mediated inflammatory gene expression [86].

Immunoregulatory Activity
Qin et al., studied the effect of A. membranaceus extract on the advanced glycation end product-induced inflammatory response in α-1 macrophages. The results suggested that it could inhibit advanced glycation end product-induced inflammatory cytokine production to down-regulate macrophage-mediated inflammation via p38 mitogen-activated protein kinase and nuclear factor (NF)-κB signaling pathways [87]. Du et al., investigated the potential adjuvant effect of Astragalus polysaccharides on humoral and cellular immune responses to hepatitis B subunit vaccine. The result suggested that it was a potent adjuvant for the hepatitis B subunit vaccine and could enhance both humoral and cellular immune responses via activation of the Toll-like receptor 4 signaling pathway and inhibit the expression of transforming growth factor β [88]. Nalbantsoy et al., studied the in vivo effects of two Astragalus saponins on the immune response cytokines by using six to eight week old male Swiss albino mice. The results showed that astragaloside VII and macrophyllosaponin B showed powerful immunoregulatory effects without stimulation of inflammatory cytokines in mice, and had no significant effect on the inflammatory cellular targets in vitro [89]. Huang et al. found that astragaloside IV could rival the suppressing effect of high mobility group box 1 protein on immune function of regulatory T cells with dose-dependent in vitro [90].

Anti-Tumor Activity
Recently, many active screening results have indicated that Astragalus polysaccharides, saponins, and flavonoids have anti-tumor activities. Tian et al., investigated the adjunct anticancer effect of Astragalus polysaccharides on H22 tumor-bearing mice, and found that it exerted a synergistic anti-tumor effect with adriamycin and to alleviate the decrease in the sizes of the spleen and thymus induced by adriamycin in H22 tumor-bearing mice [91]. As a potential anti-tumor saponin, astragaloside IV could down-regulate Vav3.1 expression in a dose-and time-dependent manner [92]. Meanwhile, astragaloside II could down regulate the expression of the P-glycoprotein and mdr1 gene, which suggested it was a potent multidrug resistance reversal agent and could be a potential adjunctive agent for hepatic cancer chemotherapy [93]. On the other hand, the experimental data showed that the total flavonoids of Astragalus and calycosin could inhibit the proliferation of K562 cells [94].

Cardioprotection Activity
Ma et al., studied the cardio protective effect of the extract of Radix Astragali on myocardial ischemia and its underlying mechanisms in ROS-mediated signaling cascade in vivo by using different rat models, and drew the conclusion that the cardio protection was due to a protection of tissue structure and a decrease in serum markers of the ischemic injury [95]. The total flavonoids of A. mongholicus are the active components, which benefit cardiovascular disease attributed to the potent antioxidant activity in improving the atherosclerosis profile [96]. Isoflavones, calycosin and formononetin from the Astragalus root, could promote dimethylarginine dimethylaminohydrolase-2 protein and mRNA expressions in Madin Darby Canine Kidney (MDCK) II cells, and up regulate the neuronal nitric oxide synthase levels [97]. Astragaloside IV could prolong the action potential duration of guinea-pig ventricular myocytes, which might be explained by its inhibition of K + currents [98].

Antidiabetic
The study of Liu et al., indicated that Astragalus polysaccharide could regulate part of the insulin signaling in insulin-resistant skeletal muscle, and could be a potential insulin sensitizer for the treatment of type 2 diabetes [99]. Zhou et al., found Astragalus polysaccharide could up regulate the expression of galectin-1 in muscle of type I diabetes mellitus mice [100]. Saponins and astragaloside IV could exert protective effects against the progression of peripheral neuropathy in streptozotocin-induced diabetes in rats [101]. In addition, astragaloside V was found to inhibit the formation of N-(carboxymethyl)lysine and pentosidine during the incubation of bovine serum albumin with ribose, which suggested that it might be a potentially useful strategy for the prevention of clinical diabetic complication by inhibiting advanced glycation end products [102].

Anti-Aging
According to the study of the anti-aging effect of astragalosides, Lei et al., suggested that the mechanism might be related to the improvement of brain function and immunomodulatory effects [105]. Gao et al., concluded that Astragalus polysaccharides could lengthen the living time of mice, improve the activity of superoxide dismutase and decrease the malonaldehyde content in mice blood serum compared with the control group, which suggested that Astragalus polysaccharides have anti-aging effects [106].

Other Biological Activities
Additionally, according to previous research, the plants of Astragalus species also have pharmacological effects such as antiviral, hepatoprotective, neuron protective, and so on.

Analyses
Researchers have conducted numbers of qualitatively and quantitatively analytical experiments on the plants in the Astragalus genus by different methods. Among them, the analyses of flavonoids and saponins from the radix of A. membranaceus and A. mongholicus were well done.
Han et al., studied ultra-performance liquid chromatography for quantification of flavone in A. membranaceus, the outcome of which showed that the linear ranges of calycosin glycoside, formononetin glycoside, calycosin, and formononetin were 0.1313-1.313 g/L (r = 0.9997), 0.1186-1.186 g/L (r = 0.9994), 0.0206-0.206 g/L (r = 0.9995), and 0.0150-0.150 g/L (r = 0.9995), respectively. The average recoveries were 97.07%, 97.26%, 97.45% and 96.97% respectively [107]. The research results of Zhang et al., indicated that the content of flavonoids in Radix Astragali of different growth years increased along with the growth period, and the types of flavonoids remained the same [108]. By comparing the retention time and MS data with those obtained from the authentic compounds and the published data, Ye et al., identified a total of 19 compounds including 11 isoflavonoids and eight saponins [109].
In addition, analyses for other constituents in A. membranaceus and A. mongholicus and composition analysis for other species of Astragalus were also conducted. Huang et al., studied the water extract of Radix Astragali by infrared spectroscopy, and found it contained an abundance of polyose, and the residue from it included some substances such as starch, cellulose, and lignin. The study provided an effective reference for the further analyses of chemical components and the improvement of extraction-separation technologies of TCM [110]. Using solid phase microextraction followed by GC-MS analysis, Movafeghi et al., identified the volatile organic compounds in the leaves, roots and gum of A. compactus. As a result, a range of volatile compounds were recognized in different parts of it under optimized conditions, but tetradecane 1-chloro only existed in roots [111]. Sun et al., determined the chemical components of A. hamiensis with a systematic extract method and TLC, the results showed that it contained alkaloids, polysaccharides, glucosides, amino acids, steroids, terpenoids, oils, tannins, phenols, organic acids, flavonoids-chinones, cardiac glycosides, and coumarins. Futhermore, they found the alkaloids were mainly swainsonine and analogous indolizidine. Meanwhile, A. hamiensis was found to contain small amount of ermopsine and anagyrine, which belong to quinolizidine [112].

Conclusions
As a TCM, the root of the Astragalus plant, Huang Qi, has been used in Chinese medicine for thousands of years. There are over two thousand species of Astragalus, among which only A. membranaceus and A. mongholicus are primarily used for medicinal purposes. In light of the considerable interest generated in the chemistry and pharmacological properties of the Astragalus plant, this review summarizes the retrieved literature published over the last 10 years dealing with several aspects including phytochemistry, bioactivity, and the research of the analysis .
In the field of phytochemistry and analysis, the chemical constituents from 46 kinds of Astragalus species have been studied. Although more than 200 compounds, including cycloartane-type saponins and flavonoids, were obtained from them, the systematic phytochemistry research needs to be improved. In addition, though the researchers conducted a number of qualitatively and quantitatively analytical experiments for flavonoids and saponins from the radix of A. membranaceus and A. mongholicus by different methods, the simultaneous analysis of two kinds of constituents was rarely found. In the field of pharmacology, the bioactivity evaluation of extracts and isolated compounds focused on anti-inflammatory, immunostimulant, antioxidative, anti-cancer, antidiabetic, cardioprotective, hepatoprotective, and antiviral, but anti-inflammation activity research of flavonoids was rare. Though a lot of results of pharmacological studies were carried out using crude extract of Astragalus species, the relationship between chemical constituents and activity is still unclear. Additionally, data on pharmacokinetics and bioavailability, especially related to the target tissue, need to be further supplemented.