The Genus Gnaphalium L. (Compositae): Phytochemical and Pharmacological Characteristics

The genus Gnaphalium, a herb distributed worldwide, comprises approximately 200 species of the Compositae (Asteraceae) family that belongs to the tribe Gnaphalieae. Some species are traditionally used as wild vegetables and in folk medicine. This review focuses on the phytochemical investigations and biological studies of plants from the genus Gnaphalium over the past few decades. More than 125 chemical constituents have been isolated from the genus Gnaphalium, including flavonoids, sesquiterpenes, diterpenes, triterpenes, phytosterols, anthraquinones, caffeoylquinic acid derivatives, and other compounds. The extracts of this genus, as well as compounds isolated from it, have been demonstrated to possess multiple pharmacological activities such as antioxidant, antibacterial and antifungal, anti-complement, antitussive and expectorant, insect antifeedant, cytotoxic, anti-inflammatory, antidiabetic and antihypouricemic properties. The present review compiles the information available on this genus because of its relevance to food and ethnopharmacology and the potential therapeutic uses of these species.

knowledge, there have not been any in-depth reviews on this genus from the phytochemical and biological viewpoints. Here, we compile the phytochemical and biological researches on the genus Gnaphalium during the past few decades.

Chemical Constituents
During the past decades, more than 125 secondary metabolites were isolated and identified from species of Gnaphalium. Here, the structures of 68 flavonoids, two sesquiterpenes, 28 diterpenes, five triterpenes, four phytosterols, two anthraquinones, five caffeoylquinic acid derivatives, and 10 other compounds are shown below, of which the names, corresponding plant sources, and references are collected in Table 1. Table 1. Flavonoids, sesquiterpenes, diterpenes, triterpenes, phytosterols, anthraquinones, caffeoylquinic acid derivatives, and other compounds from the genus Gnaphalium. Hispidulin G. antennarioides [24]

Anthraquinones
During the studies on chemical constituents of the petroleum ether fraction from G. affine, two anthraquinones (108 and 109) were obtained [51] (Figure 6).

Antioxidant Activity
The essential oil from G. affine was observed to possess strong radical scavenging activity against 2,2'-azinobis-3-ethylbenzthiazoline-6-sulfonate with the IC 50 being 0.32 ± 0.89 μg/mL (IC 50 of ascorbic acid = 24.06 ± 0.73 μg/mL). A significant inhibitory effect of the essential oil on lipid peroxidation in egg yolk homogenates was shown with an IC 50 value of 0.09 ± 0.75 μg/mL (IC 50 of ascorbic acid = 6.73 ± 0.87 μg/mL). The essential oil had a stronger reducing power than Vc in the reducing of Fe 3+ to Fe 2+ by donating an electron [52]. The scavenging effect elicited by the ethanolic extract of leaves of G. uniflorum was concentration-dependent in 2,2-diphenyl-1-picrylhydrazyl test, the amount of antioxidant necessary to decrease the initial concentration by 50% (SC 50 ) was calculated as 15.96 μg of extract. The extract showed a concentration-dependent antioxidant activity also in the LP-LUV test (IC 50 = 14.86 μg of extract), determination of the accumulation of products of peroxidation in mixed dipalmitoylphosphatidylcholine/linoleic acid unilamellar vesicles induced by the water-soluble peroxyl radical generator 2,2'-azobis(2-amidinopropane)hydrochloride [32].

Anti-Complementary Activity
Various chromatographic procedures on the ethyl acetate fraction of G. affine using silica gel, Sephadex LH-20, ODS, and MCI gel led to the isolation of 27 flavonoids. All compounds were evaluated for their anti-complementary activity on the classical pathway of the complement system in vitro, and some isolated It appears that the hydroxy group at the 4'-position in the flavonoid is essential for anti-complementary activity, but the activity is lost when it is substituted by methoxy group or sugar. However, the anti-complementary activity was also related to the number of hydroxylated groups [17].

Antitussive and Expectorant Activity
For thousands of years, the herb G. affine was decocted for treating respiratory diseases. The herbs of G. affine were extracted twice by the water decoction method for 1 h each time, and evaporation of the solvent gave a viscous material. The water extract (be equal to 18 g/kg, 12 g/kg, and 6 g/kg of plant material) was orally administrated to coughing mice induced by ammonium hydroxide and coughing guinea pigs induced by acitric acid, and mice injected with phenol red, respectively, to evaluate its potential expectorant and antitussive activity. The extract significantly prolonged the tussive delitescence and decreased the cough frequency caused by ammonium hydroxide and acitric acid, as well as the mucus secretion from mouse tracheas obviously increased by measuring the tracheal output of phenol red [55]. Campos-Bedolla et al. investigated the effect of methanol extract from G. conoideum on the responses to contractile agonists in guinea pig tracheas and the possible role of L-type Ca 2+ channels in tracheal guinea pig isolated myocytes. Cumulative concentration-response curves to carbachol or histamine, as well as contractile responses to KCl were evaluated with or without 30 min preincubation with 20 or 100 μg/mL methanol extract, and intracellular Ca 2+ concentrations were measured by microfluorometric method in isolated tracheal myocytes with or without preincubation with 0.1 μg/mL, 0.31 μg/mL, and 1 μg/mL methanol extract. The results showed that the extract significantly diminished the contractile responses to histamine, but not to carbachol or KCl, and significantly reduced the intracellular Ca 2+ rise induced by 60 mM KCl in isolated myocytes. Because histamine contractile responses are largely dependent on extracellular Ca 2+ and KCl responses are mainly mediated through L-type Ca 2+ channels, the results suggested that methanol extract from G. conoideum might be acting as a partial blocker of these Ca 2+ channels [10]. Hexane extract of G. liebmannii was the most active relaxant extract (IC 30 = 54.23 ± 19.47 μg/mL with 99.5 ± 3.2% of relaxation) than dichloromethane extract (IC 30 = 120.22 ± 5.27 μg/mL with 76.44 ± 2.3% of relaxation) and methanol extract (IC 30 = 190.25 ± 30.02 μg/mL with 45.94 ± 10.3 % of relaxation) on guinea pig trachea smooth muscle. Hexane extract produced a parallel rightward shift of the concentration-response curve of carbachol (IC 50 = 0.04 ± 0.0013 μM) in a competitive manner at concentrations of 177 μg/mL (IC 50 = 0.20 ± 0.0089 μM) and 316 μg/mL (IC 50 = 0.19 ± 0.001 μM), but did not modify the concentration-response curves for histamine (IC 50 = 4.4 ± 0.36 μM) at concentrations of 100 μg/mL, 177 μg/mL, and 316 μg/mL. The relaxant effect of hexane extract (100μg/mL, 133μg/mL, 177 μg/mL, 237 μg/mL, and 316 μg/mL for block of ATP-sensitive potassium channel or 31 μg/mL, 100 μg/mL, 177 μg/mL, and 316 μg/mL for β-adrenergic receptors) of G. liebmannii was unaffected by the presence of propranolol or glibenclamide. However, hexane extract (87 μg/mL, 130 μg/mL, and 316 μg/mL) produced a leftward shift of the concentration-response curves of forskolin, nitroprusside, isoproterenol, and aminophylline, suggesting that G. liebmannii induced relaxation of the tracheal muscle, probably via phosphodiesterase inhibition [56]. By employing a bioassay-guided fractionation of the active hexane extract of G. liebmannii, using the model of isolated trachea from guinea pig, gnaphaliin A (53) and gnaphaliin B (46) were identified as the active relaxant compounds. Gnaphaliin A (EC 50 = 195.97 ± 36.07 μM) and gnaphaliin B (EC 50 = 134.04 ± 6.41 μM) showed more potent relaxant properties than aminophylline (EC 50 = 534.50 ± 27.88 μM), a well-known relaxant drug can be used to treat bronchial asthma, chronic asthmatic bronchitis and chronic obstructive pulmonary disease [37].

Anti-Inflammatory Activity
The air-dried flowers of G. stramineum were successively extracted with n-hexane, methanol, and water. Each extract was tested orally for anti-inflammatory activity using carrageenan-induced edema in rat paws. The methanol extract (140 mg/kg) was the most active, displaying 36.8% inhibition of edema (5 h), while the n-hexane extract (200 mg/kg), inhibiting the edema by 35.7%, was less active. The aqueous extract did not show significant anti-inflammatory activity. Bioassay-guided fractionation of the methanol extract of G. stramineum resulted in the isolation of four caffeoylquinic acid derivatives, 4-O-caffeoylquinic, 3,5-di-O-caffeoylquinic, 4,5-di-O-caffeoylquinic, and 3,4,5-tri-O-caffeoylquinic acids (111 and 113-115, resp.), and quercetin glycosides, isoquercitrin, quercetin 3-O-β-D-galactopyranoside, and rutin (36-38, resp.). Caffeoylquinic acid derivatives were tested in activated human macrophages for their activities on some human leukocyte functions related to inflammatory mechanism such as on monocyte migration and superoxide anion production. Compounds 113 and 114 exhibited an appreciable anti-inflammatory activity, while compound 115 was inactive. Compound 113 inhibited the peak of chemotactic index at a concentration of 1 × 10 −11 M, but revealed a significant activity at a concentration as low as 1 × 10 −13 M; compound 114 blocked the chemotaxis only at a concentration of 1 × 10 −7 M; compound 115 was completely at any of the tested concentration (1 × 10 −7 M-1 × 10 −17 M). Quercetin glycosides (glucoside, galactoside and rutinoside) were able to reduce the edema induced by carrageenan and the exudative response induced by cotton pellet granuloma. The comprehensive evaluation showed that the anti-inflammatory activities of the extracts of G. stramineum may be due to a combination of caffeoylquinic acid derivatives and flavonol glycosides [36].

Hypoglycemic Activity
The decoction of G. uliginosum was documented to reduce experimental epinephrine and diabetic hyperglycemia but not to elevate the decreased blood insulin level in an epinephrine hyperglycemia model and alloxan diabetes in rats and mice [57]. In 1995, Tachibana, et al. found that the ethyl acetate and methanol extracts of G. affine displayed inhibitory effects on aldose reductase (IC 50 = 4.3 μg/mL and 1.4 μg/mL, respectively). Bioassay-guided fractionation resulted in the isolation of luteolin (6), quercetin (31), gnaphalin (68), and scopoletin (121). The aldose reductase inhibitory assay test revealed that compounds 6, 31, and 68 exhibited potent activities with IC 50 values of 0.7 μM, 2.6 μM, and 4.5 μM, respectively, whereas compound 121 was less active [46].

Antihypouricemic Activity
The hypouricemic actions of G. affine was in vivo examined using oxonate-induced hyperuricemic mice. The water extracts of G. affine at 25, 12.5, and 6.5 g/kg injected intraperitoneally were demonstrated to possess potent hypouricemic effects [58].

Other Activities
The water extract of G. affine (2.0 g/kg, 1.0 g/kg, and 0.5 g/kg) exhibited the protective effect for carbon tetrachloride-induced acute liver injury [59]. A gel formulation containing the extract from G. uniflorum proved to afford a significant in vivo protection against UV-B-induced skin erythema in healthy human volunteers [32]. As reported by Kubo et al., the methanol extract of G. cheiranthifolium showed significant (<200 μg/mL) inhibitory activity for the oxidation of L-3,4-dihydroxyphenylalanine mediated by mushroom tyrosinase. The compounds luteolin 4'-O-β-D-glucopyranoside (7) and gnaphalin (68) exhibited significant (almost 100% at 100 μg/mL) inhibition of the oxidation of L-3,4dihydroxyphenylalanine by tyrosinase. Their limited availability prevented further study, so neither ID 50 values nor their mode of inhibition were investigated. [25]. The ethyl acetate extract of G. affine promoted the rabbit platelet aggregation induced by ADP, whereas its methanol extract inhibited the aggregation. Compound 68 isolated from this plant showed an inhibitory effect (IC 50 = 1.6 mM) on the rabbit platelet aggregation induced by PAF of about the same potency as that of quercetin (IC 50 = 1.7 mM) [47].

Conclusions
Plants of the genus Gnaphalium are widely distribute all over the World, and many species are traditionally used as wild vegetables and inh folk medicine. In this review, we summarized the secondary metabolites reported from Gnaphalium species, as well as their biological activities. From our review, it can be concluded that phytochemistry investigations mainly focused on ca. 31 species. With regard to the 200 species of this genus, there are still many species that have received little or no attention. Further studies to exploit phytochemical constituents and biological activities from the plant of this genus are necessary to develop more potentially value-added products used in food and pharmaceutical industry.