The Genus Alnus, A Comprehensive Outline of Its Chemical Constituents and Biological Activities

The genus Alnus (Betulaceae) is comprised of more than 40 species. Many species of this genus have a long history of use in folk medicines. Phytochemical investigations have revealed the presence of diarylheptanoids, polyphenols, flavonoids, terpenoids, steroids and other compounds. Diarylheptanoids, natural products with a 1,7-diphenylheptane structural skeleton, are the dominant constituents in the genus, whose anticancer effect has been brought into focus. Pure compounds and crude extracts from the genus exhibit a wide spectrum of pharmacological activities both in vitro and in vivo. This paper compiles 273 naturally occurring compounds from the genus Alnus along with their structures and pharmacological activities, as reported in 138 references.


Introduction
Alnus is a genus in the family Betulaceae, which comprises more than 40 species mainly distributed in Asia, Africa, Europe and North America. A total of seven species and one variant are distributed in the south and north of China [1]. The plants in the genus are commonly used as traditional medicines [2]. Alnus hirsuta Turcz. indigenously distributed in Korea, China, Japan, and Russia, has been used in Oriental medicine as a remedy for fever, haemorrhages, burn injuries, diarrhea, and alcoholism [3]. It was reported that many bioactive natural components including diarylheptanoids, polyphenols, flavonoids, terpenoids, and steroids, were isolated from the genus [4][5][6][7][8]. Diarylheptanoids comprise a class of natural products formed from 1,7-diphenylheptane, which appears in linear and cyclic forms [9]. They have been regarded as the primary bioactive compounds of Alnus and have drawn attention due to their physiological activities, especially their anticancer and antioxidative activities [10,11].
Alnus japonica Steudel., Alnus hirsuta Turcz. and Alnus glutinosa (L.) Gaertn. have a long-standing medical history and extensive research on their irreplaceable functions has been reported. A. japonica, a famous medical herb in China, Japan and Korea as well as a functional food consumed in health drinks, contains abundant diarylheptanoid derivatives, in addition to methylated and acylated diarylheptanoids and diarylheptanoid glycosides [12][13][14]. It has been known to exert antioxidative, anti-inflammatory, anticancer and hepatoprotective effects and its antioxidative properties are presumed to contribute to its hepatoprotective activity in certain situations [15]. Diarylheptanoids with strong antioxidative activity isolated from A. japonica and A. hirsuta showed significant hepatoprotective effects on t-butyl hydroperoxide (t-BHP)-induced toxicity in primary rat hepatocytes and a human hepatoma cell line (HepG2), respectively [3,16]. Alnus glutinosa (L.) Gaertn., used on the market as a food supplement to help reduce the risk of different chronic dermatological conditions, has increasingly become a research hot-spot for its potent chemo-protective, antioxidant and antimicrobial effects [17][18][19]. Diarylheptanoids from the bark of A. glutinosa may serve as protectors of normal cells during chemotherapy without significantly diminishing the effect of the applied chemotherapeutic agents [4,20]. Sati et al. have summarized 192 chemical constituents and biological activities of genus Alnus [2]. Further studies on the Alnus genus were carried out in recent years, and many chemical components from this genus have been isolated. Therefore, a new comprehensive and systematic review of Alnus genus is much needed. Most of the papers not only covered new chemical components, but also refered to their pharmacology activities, structure-activity relationships and functional mechanisms, especially the anticancer meshanism of hirsutenone [11,[21][22][23]. Hirsutenone can sensitize resistant ovarian cancer cells to cisplatin, so that co-treatment with it may have the potential to overcome chemoresistance [24]. Futhermore, an induction of oxidative stress and topo II-mediated DNA damage may play a role in hirsutanone-induced cancer cell death [21]. In this review, we mainly summarized 273 chemical constituents and biological activities of the genus Alnus, based on 138 cited references. It is hoped that the information presented in this paper will be useful for further research and the application of this genus.  Table 17. The occurrence of diarylheptanoids appears to be a characteristic feature of this genus.
Compounds 1-89, the linear-diarylheptanoids shown in Tables 1-3 and Figure 1, can be further divided according to the features of the aliphatic carbons. The heptane chains of compounds 1-28 (Table 1) are saturated, and the C-3 or C-5 position is always linked to hydroxyls. Diarylheptanoid derivatives 9-28 possess a monosaccharide or a disaccharide at the C-3 or C-5 position of the heptane chain to form O-glycosides. Moreover, the C-6 position in the glucosyl unit of 26-28 is attached to a cinnamoyl moiety. Compounds 29-71 (Table 2) are classified as 1,7-bis-(p-hydroxyphenyl)-3-heptanones. Compounds 30-37 always contain an oxygen substituent at the C-5 position. The hydroxyl at C-5 of 38-44 is replaced by an aliphatic hydrocarbon. Diarylheptanoids 45-50, 62-69, as well as 7, 9, 14, 18, 33, 38 all could be isolated from the bark of A. glutinosa. Structure-activity analysis revealed a high dependence of their cytotoxic action on the presence of a carbonyl group at C-3, substitution of the heptane chain on C-5 and the number of hydroxyl groups in the aromatic rings [11]. What s more, in 53-69, the sugar groups attached at the C-5 position in the heptane group are connected with aromatic acyl radical moieties. These compounds are a class of natural product called cinnamic acid sugar ester derivatives (CASEDs), with one or several phenylacrylic moieties such as -coumaroyl, -cinnamoyl, -benzoyl and -vanilloyl, linked with the non-anomeric carbon of a glycosyl skeleton part through ester bonds. Several CASEDs are reported in traditional medicine as compounds to calm the nerves and display anti-depression and neuroprotective activity [25][26][27]. Compound 52 has a disaccharide unit with β-D-apiofuranosyl bonded to β-D-glucopyranose. Compound 69 is an O-(2-methyl)-butanoyl derivative of oregonin (48).

Polyphenols
Tannins are the major components of antioxidant polyphenols in genus Alnus, mainly including four groups: gallotannins (compounds 100-104), ellagitannins (compounds 105-120), dimeric ellagitannins (compounds 121-124) and C-glycosidic tannins (compounds 125-128). They are always composed of a galloyl group, a hexahydroxydiphenoyl (HHDP) group and a valoneoyl group with glucose core(s) in which the mode of linkage is different. In addition, some polyphenols and their glycosides are also found in this genus. Table 4 lists compounds 100-111. 100-104 from A. japonica, A. hirsuta and Alnus sieboldiana Matsum. that belong to the gallotannins, in which the galloyl group is directly attached to the hydroxyl of the glucose moiety through an ester bond [5,36,37]. Compound 104 contains a methylated galloyl group, which is linked to the C-1 of the glucose core. Ellagitannins 105-111 are esterified by one or two HHDP group(s) at C-1, C-2 and/or C-3, C-4 in the glucosyl moiety to form sugar aryl ester linkages, meanwhile, galloyl groups are often present, except in 108 and 109. Glutinoin (111), a novel antioxidative ellagitannin with a glutinoic acid dilactone moiety, was isolated from A. glutinosa cones [38]. The C-1 hydroxyl group of the sugar is coupled with a galloyl group, which is connected to the ellagoyl group via the C-4′ (para) hydroxyl group. The compound with a p-COC-type junction between two units, corresponding to the ellagoyl-galloyl motif in glutinoin, was named glutinoic acid dilactone and the corresponding group was named glutinoyl [38]. To our knowledge, no similar structure has been annotated so far.
Compounds 112-115 in Figure 4 isolated from the cones of A. glutinosa, the leaves of A. japonica and Alnus hirsuta var. microphylla based have structures with a valoneoyl group and its depsidone form at C-4/C-6 of the glucose core [5,36,38].

Polyphenols
Tannins are the major components of antioxidant polyphenols in genus Alnus, mainly including four groups: gallotannins (compounds 100-104), ellagitannins (compounds 105-120), dimeric ellagitannins (compounds 121-124) and C-glycosidic tannins (compounds 125-128). They are always composed of a galloyl group, a hexahydroxydiphenoyl (HHDP) group and a valoneoyl group with glucose core(s) in which the mode of linkage is different. In addition, some polyphenols and their glycosides are also found in this genus. Table 4 lists compounds 100-111. 100-104 from A. japonica, A. hirsuta and Alnus sieboldiana Matsum. that belong to the gallotannins, in which the galloyl group is directly attached to the hydroxyl of the glucose moiety through an ester bond [5,36,37]. Compound 104 contains a methylated galloyl group, which is linked to the C-1 of the glucose core. Ellagitannins 105-111 are esterified by one or two HHDP group(s) at C-1, C-2 and/or C-3, C-4 in the glucosyl moiety to form sugar aryl ester linkages, meanwhile, galloyl groups are often present, except in 108 and 109. Glutinoin (111), a novel antioxidative ellagitannin with a glutinoic acid dilactone moiety, was isolated from A. glutinosa cones [38]. The C-1 hydroxyl group of the sugar is coupled with a galloyl group, which is connected to the ellagoyl group via the C-4′ (para) hydroxyl group. The compound with a p-COC-type junction between two units, corresponding to the ellagoyl-galloyl motif in glutinoin, was named glutinoic acid dilactone and the corresponding group was named glutinoyl [38]. To our knowledge, no similar structure has been annotated so far.

Polyphenols
Tannins are the major components of antioxidant polyphenols in genus Alnus, mainly including four groups: gallotannins (compounds 100-104), ellagitannins (compounds 105-120), dimeric ellagitannins (compounds 121-124) and C-glycosidic tannins (compounds 125-128). They are always composed of a galloyl group, a hexahydroxydiphenoyl (HHDP) group and a valoneoyl group with glucose core(s) in which the mode of linkage is different. In addition, some polyphenols and their glycosides are also found in this genus. Table 4 lists compounds 100-111. 100-104 from A. japonica, A. hirsuta and Alnus sieboldiana Matsum. that belong to the gallotannins, in which the galloyl group is directly attached to the hydroxyl of the glucose moiety through an ester bond [5,36,37]. Compound 104 contains a methylated galloyl group, which is linked to the C-1 of the glucose core. Ellagitannins 105-111 are esterified by one or two HHDP group(s) at C-1, C-2 and/or C-3, C-4 in the glucosyl moiety to form sugar aryl ester linkages, meanwhile, galloyl groups are often present, except in 108 and 109. Glutinoin (111), a novel antioxidative ellagitannin with a glutinoic acid dilactone moiety, was isolated from A. glutinosa cones [38]. The C-1 hydroxyl group of the sugar is coupled with a galloyl group, which is connected to the ellagoyl group via the C-4 (para) hydroxyl group. The compound with a p-COC-type junction between two units, corresponding to the ellagoyl-galloyl motif in glutinoin, was named glutinoic acid dilactone and the corresponding group was named glutinoyl [38]. To our knowledge, no similar structure has been annotated so far.
Compounds 112-115 in Figure 4 isolated from the cones of A. glutinosa, the leaves of A. japonica and Alnus hirsuta var. microphylla based have structures with a valoneoyl group and its depsidone form at C-4/C-6 of the glucose core [5,36,38].  Figure 4 contain a tergalloyl group at C-4/C-6 and a HHDP group at C-2/C-3 of the glucose core. Compounds 118 and 119 ( Figure 5) from the extracts of fruits and leaves of A. sieboldiana both have a monolactonized tergalloyl group [37,39]. The A, C benzene rings of the tergalloyl group and the HHDP group in alnusiin (118) are linked with C-4/C-6 and C-2/C-3 in glucosyl moiety through ester bonds, respectively, while the A and B rings in tergallin (119) are attached to the C-2/C-3 positions. Additionally, tergallin (119) has a characteristic ellagic acid moiety symmetrically coupled with two galloyl groups at C-4/C-6 by C-C bonds. Hirsunin (120, Figure 6), represents a rare example of a hydrolysable tannin, and consists of diarylheptanoid glycoside (oregonin) and ellagitannin (praecoxin A) moieties. It was isolated from Alnus hirsuta var. microphylla in 1992 and remains the sole example of an ellagitannin with a diarylheptanoid moiety up to now [36,40].
Triterpenic acids 201 and 202 were both isolated from the leaves of A. nepalensis [8,31]. Particularly, the cycloartane type mangiferonic acid (202), which was not detected in any other Alnus species, has been considered as a specific chemical marker of A. nepalensis from a chemotaxonomical point of view [8]. The tetracyclic triterpenes 203-208 and 216-223, isolated from flowers and leaves of A. sieboldiana, Alnus serrulatoides Call. and A. pendula, are characterized by their C 31 -dammarane-type and C 31 -3, 4-seco-dammarane-type skeletons [55]. All the tetracyclic triterpenes 210-215 occurring in A. japonica male flowers were of the C 30 -3, 4-seco-dammarane-type [55]. In particular, alnuselide (208), structurally similar to alnuseric acid (207), has a lactone ring formed by the 3-carboxyl and the 11-hydroxyl groups [58]. Triterpenoid saponins 218-223 are connected with a sugar moiety at C-12 through an O-glycosidic bond. In addition, positions C-2 in the sugar unit of compounds 221-223 were always linked with an acetyl group.

Other Compounds
About 19 other compounds were isolated from this genus. Five stilbenes 255-259 (Table 16)

Biological Activities
Many species of the genus Alnus have been used as remedies for diarrhea, dysentery, fever and inflammatory diseases in traditional Chinese and Korean medicine [107]. Alnus species have remarkable anticancer activity, which is the most notable and important pharmacological activity [21]. Beyond that, they also display antioxidant, anti-inflammatory, antimicrobial, antiviral and hepatoprotective activities, etc. [16,52,57,105]. Diarylheptanoids are the the main effective components in the genus Alnus for their remarkable biological activities [2]. Phenolic compounds and flavonoids, which are widely found as secondary metabolites in Alnus plants, are important due to their ability to act as antioxidants [18]. Additionally, several triterpenoids have revealed antitumor activity, HIV-1 viral enzyme inhibition and hepatoprotective effects [52,61,104].

Anticancer Activity
Many researchers have focused on the pharmacological effects of hirsutenone (73) isolated from A. japonica, A. hirsuta and A. pendula, etc. [13,69,87]. It has a similar chemical structure and anti-cancer properties as curcumin, the most well-known diarylheptanoid in turmeric (Curcuma longa Linn, Zingiberaceae), which has been ranked as a third generation chemoprevention agent by the U.S. National Cancer Institute [21,108,109]. Hirsutenone (73) inhibited the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced upregulation of cyclooxygenase-2 (COX-2) and matrix metalloproteinases-9 (MMP-9) in human breast epithelial cells, which has been implicated in the pathogenesis of different kinds of cancer [110]. Further experiments confirmed the cytotoxic activity of 73 against HT-29 human colon carcinoma cells via the induction of oxidative stress and topo II-mediated DNA damage [21]. It can enhance the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) on epithelial ovarian carcinoma cell lines by increasing the activation of the caspase-8-and Bid-dependent pathways and mitochondrial pathway, leading to caspase activation as well [111]. Kang et al. found that hirsutenone suppressed human prostate cancer by targeting Akt1 and 2 as a key component to explain for anti-cancer activity [112]. A research in 2014 indicated that hirsutenone (73) could sensitize chemoresistant ovarian cancer cells to cisplatin via modulation of apoptosis-inducing factor and X-linked inhibitor of apoptosis [24].
Compounds 46, 47, 50, 62, 66 and 73 from A. glutinosa bark exhibited strong anticancer activity compared with other diarylheptanoids from the same species and considerably higher than curcumin, which served as a positive control, in human non-small cell lung carcinoma cell lines. Structure-activity analysis revealed a high dependence of the cytotoxic action on the presence of a carbonyl group at C-3, substitution of a heptane chain on C-5 and the number of hydroxyl groups in the aromatic rings [11]. In addition, Choi et al. found that platyphylloside (50), which has a keto-enol moiety and one hydroxyl group in the aromatic ring, showed the most potent cytotoxic activity on B16 mouse melanoma and human stomachic adenocarcinoma cells. This study also suggested that the ketone or keto-enol moiety and hydroxyl groups in the aromatic rings were essential to the higher cytotoxic activity of diarylheptanoids [73].
A rencent comparative study was performed on structurally analogous diarylheptanoids isolated from the bark of A. viridis and A. glutinosa to address their biological effects and determine structure-activity relationship. (5S)-O-methylplatyphyllonol (40) and platyphyllenone (74) do not possess 3 and 3 -OH groups showed significantly higher cytotoxicity compared to that of analogues 5(S)-O-methylhirsutanonol (38) and hirsutenone (73). The C-4/C-5 double bond instead of a methoxy group in compounds 74 and 73 positively influenced cell growth inhibition and pro-apoptotic potential. These results indicated that minor differences in the chemical structure can greatly influence the effect of diarylheptanoids on apoptosis and redox status and determine their selectivity towards cancer cells [76].
Oregonin (48) and hirsutanonol (33) are potential cancer chemopreventive agents. They showed significant inhibitory effects on TPA-induced COX-2 expression in immortalized human breast epithelial MCF10A cells [113]. In addtion, a novel immunomodulator 48 exhibited powerful anticancer activity through augmenting the acttivities of macrophage and natural killer cells [114,115]. Jin et al. isolated six diarylheptanoids from the stem bark of A. hirsuta. Among them, cyclic-type diarylheptanoids 90 and 91 inhibited the HIF-1 activation with IC 50 values of 11.2 µM and 12.3 µM while the other diarylheptanoids showed very weak activity with IC 50 values greater than 100 µM. It suggested that the big cyclic ring contributes to the strong HIF-1 activity [30]. The leaves, barks, and cones extracts of A. incana and A. viridis showed potent cytotoxic inhibition effect on HeLa cells with IC 50 values ranging from 26.02 to 68.5 µg/mL. The most active extract of A. incana bark has been found to contain great amounts of total phenolics (316.2 mg of gallic acid (137)) [116]. Pedunculagin (109), which is an ellagitannin, exhibited dose-dependent cytotoxicity in vitro and a lengthening effect on the lifespan in mice bearing S 180 tumors in vivo [117]. Galangin (154), isolated from A. sieboldiana, significantly inhibited tumor necrosis factor-α (TNF-α) gene expression in A549 cells. It may also be useful in cancer prevention [100].

Antioxidant Activity
Free radical damage is linked to the occurrence of many degenerative diseases, including cancer, cardiovascular disease, cataracts, and aging. Antioxidants can attenuate the damaging effects of reactive oxygen species (ROS) in vitro and have attracted major interest, not only for health care and cosmetics, but also in the food industry [118].
Kim et al. isolated nine known diarylheptanoids from the barks of A. japonica. Among these diarylheptanoids, oregonin (48) and hirsutenone (73) exhibited apparent inhibitory effects on lipopolysaccharide (LPS)-induced NO production and COX-2 production [121]. An analysis of the structure-activity relationship suggested that the presence of a keto-enol group in the heptane moiety or a caffeoyl group in the aromatic ring was important for the inhibitory activity efficacy [121]. Later on, another study also suggested that the carbonyl group is important for the inhibitory activity of diarylheptanoids against LPS-induced NO production [41]. Lee et al. provided new evidence for the anti-inflammatory actions of oregonin (48), which include the inhibition of inducible nitric oxide synthase (iNOS) gene transcription via suppressing transcriptional activity of NF-κB and activator protein-1 (AP-1), as well as the up regulation of anti-inflammatory molecule HO-1 [123]. In addition, oregonin (48) reduces lipid accumulation, inflammation and ROS production in primary human macrophages, indicating its anti-inflammatory bioactivity [124]. Hirsutenone (73) may exert a preventive effect against microbial endotoxin lipopolysaccharide-induced inflammatory skin diseases through inhibition of extracellular signal-regulated kinase (ERK) pathway-mediated NF-κB activation [29,125].
Compounds 12, 33, 90 and 91 in the bark of A. hirsuta, four phenolic glycosides 129-132 and six diarylheptanoids 36, 41, 45, 48, 50, 53 isolated from A. firma showed significant ability to inhibit LPS-induced inflammation in macrophages or BV2 microglial cells [10,33,41]. Aguilar et al. confirmed the traditional uses of A. acuminata in acute inflammatory conditions and its safety for consumption. Several triterpenoids from the hexane extract and diarylheptanoids from the methanol extract of A. acuminata were isolated and characterized [57]. The methanol extract of A. glutinosa leaves and shikimic acid (134) were found to exhibit remarkable anti-inflammatory effect by "inhibition of acetic acid-induced capillary permeability", "carrageenan-induced hind paw edema" and "TPA-induced ear edema" assays [98]. A recent study in 2017 evaluated the methanol extract and derived fractions of A. nitida stem bark for anti-inflammatory activity by using in vitro heat induced albumin denaturation assay and various in vivo assays. It suggested that the presence of polyphenols, sterols, terpenoids and other constituents of A. nitida stem bark might contribute towards the anti-inflammatory and analgesic activities [99].

Antimicrobial and Antiviral Activities
It was reported that the EtOH extract of A. pendula bark had significant antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Oregonin (48) and hirsutenone (73) isolated from the active fractions inhibited MRSA strains with the minimum inhibitory concentrations (MICs) ranged from 31.25 to 250 µg/mL. Moreover, two fold MIC of 73 could completely suppress the growth of MRSA [126]. Later on, the antibacterial evaluation of the fractions by bioautography on Staphylococcus aureus revealed that 48 was the most active, with an antibacterial inhibitory effect comparable to antibiotics [17]. The extracts of cone, leaves, and bark of A. incana and A. viridis showed antimicrobial activities against 15 microorganisms with MIC values ranging from 0.117 to 0.292 mg/mL [116]. Genkwanin (143) isolated from the seeds of A. glutinosa showed high antimicrobial activity against seven strains of Gram-positive and Gram-negative bacteria [47]. Betulin (237), betulone (238) and betulinic acid (240) were identified as the major antimycobacterial constituents in the bark of A. incana. The functionality at C-3 and C-28 of the lupane skeleton was seemed to be important in determining the antimycobacterial activity [105].

Hepatoprotective Activity
The methanolic extract of the bark of A. firma exhibited significant antifibrotic activity. Meawhlie, compounds 73 and 245 isolated from A. firma barks showed potent inhibitory effect on the proliferation of hepatic stellate cell (HSC). The authors determined that the presence of substitution at C-3 and C-5 might be responsible for the inhibitory activity of diarylheptanoids on HSC proliferation [61]. It was reported that A. japonica was used for hepatitis as an endemic species in Korea. There were evidences that the methanol extract of A. japonica stem bark displays hepatoprotective effects against acetaminophen-induced cytotoxicity in cultured rat hepatocytes in vitro [127]. Compounds 12, 14, 22, 33, 37, 38, 45, 46, 48, 49 and 73 isolated from the A. japonica bark and the A. hisuta stem bark showed significant hepatoprotective effects on tert-butyl hydroperoxide (t-BHP) -induced damage to HepG2 cells. According to structure characteristics, the authors considered that the cytoprotective effect was closely related to catechol moiety [3,16,80]. Sajid et al. explored the antioxidant and hepatoprotective properties of A. nitida stem bark crude methanol extract on rats. The study concluded that the hepatoprotective activity of A. nitida bark is likely due to the antioxidant potential [119]. Furthermore, some other studies also suggested that the hepatoprotective effect is closely linked with the antioxidant property [16,127].

DNA Damage Protection Activity
Novaković et al. isolated twenty-one diarylheptanoids and two polyphenols from the barks of A. glutinosa and A. viridis. All isolated compositions were evaluated for their in vitro protective effects on chromosome aberrations in peripheral human lymphocytes using cytokinesis-block micronucleus (CBMN) assay. Many of them exerted a pronounced effect of decreasing DNA damage of human lymphocytes, acting stronger than the known synthetic protector amifostine [4,42]. Both platyphylloside (50) and 62 isolated from the bark of A. glutinosa could protect HaCaT cells against doxorubicin-induced DNA damage. They showed chemo-protective effects of at multiple subcellular levels, and could be considered as protective agents for non-cancerous dividing cells during chemotherapy [19,20].

Anti-Adipogenic Activity
Martineau et al. found that the extracts of the inner bark of A. incana strongly inhibited the formation of triglyceride-laden mature adipocytes from 3T3-L1 pre-adipocytes. A. incana extracts acted early in the differentiation process and acted as partial agonists toward peroxisome proliferator activated receptor gamma (PPAR-γ) activity. The diarylheptanoid glycoside oregonin (48) was isolated and confirmed to be the active principle exerting the anti-adipogenic effect in A. incana [82,128]. On the evaluation of antiadipogenic activities of diarylheptanoids isolated from A. hirsuta, eighteen compounds were obtained and most of them could decrease lipid accumulation in 3T3-L1 pre-adipocytes. In the assay system, the most potent compound 47, platyphyllonol-5-O-β-D-xylopyranoside had anti-adipogenic activity mediated by the regulation of PPAR-γ dependent pathway. Furthermore, the ketone functionality at C-3, the substituent at C-5, the double bond in heptanone and the hydroxyl groups in the benzene rings were related to the activity [69]. The lupane-type triterpenoid 239 isolated from the methanol extract of A. hirsuta showed significant inhibitory activity to diacylglycerol acyltransferase with IC 50 value of 9.6 µM in the rat liver microsomes. In addition, it also inhibited the triglyceride formation in human HepG2 cells. The results indicated that betulinic acid (239) may be a potential lead agent in the treatment of obesity [60].

Anti-Atopic Activity
Atopic dermatitis (AD) is a common inflammatory skin disease. Choi and his co-workers did many studies about the anti-atopic dermatitis effect of oregonin (48) and hirsutenone (73). The AD animal models were treated with them via topical application as well as intraperitoneal injection. The Th2-related cytokines IL-4, IL-5, IL-13 levels, IgE inflammatory factors, eosinophil levels in blood and lymphocytes were all reduced in AD-like skin lesions of rat model. These results revealed that the two diarylheptanoids were effective for the treatment of AD [129,130]. The leaves and barks extract from A. japonica was also proved useful in the treatment of atopic dermatitis and other allergic skin diseases in NC/Nga mice [131]. In addition, hirsutenone is an attractive source for developing a topical drug for T cell-based antiatopic dermatitis by its actions as a calcineurin inhibitor [132]. Furthermore, they developed hirsutenone-loaded and oregonin-loaded Tat peptide-admixed elastic liposomal formulations to treat AD, which aid to increasing the skin permeation of medicine [133,134].

Other Activities
Apart from the summarized functions above, the constituents or extracts from Alnus plants also have some other activities. Oregonin (48) could improve glucose metabolism and insulin signal transduction of HepG2 cells partly by enhancing the expression level of insulin receptor and insulin receptor substrate-1 [85]. The cyclic diarylheptanoids 95, 98 and 99 isolated from the branch wood of A. sieboldiana were assayed for α-glucosidase inhibitory activities with the IC 50 values 2.34, 8.69 and 1.35 µg/mL, respectively. In comparison, they have a stronger inhibitory effect than acarbose (IC 50 = 451 µg/mL), a positive control, which is currently used as an antidiabetic agent [97]. Diarylheptanoids 7, 14, 33 and 48 isolated from the bark of A. hirsuta could reduce melanin level and tyrosinase activity in melanoma cell [136]. Furthermore, it was reported that 5(R)-O-methylhirsutanonol (41) and oregonin (48) might be useful in the prevention and treatment of atherosclerosis through attenuation of adhesion molecule expression by inhibition of NF-κB activation [81]. Hirsutenone (73) was able to protect retinal ganglion cells from oxidative stress-induced death. Therefore, it was considered to be a neuroprotective agent for the treatment of neurodegenerative disease, such as glaucoma [137]. The methanol extract of A. glutinosa subsp. glutinosa leaves increased wound tension, contraction capacity and tissue hydroxyproline levels. Shikimic acid (134) was found to be the major compound responsible for the wound healing effect [88]. The methanolic extract of A. rugosa stems showed anticholinesterase activity and it was very interesting for further isolation of acetylcholinesterase inhibitors, which are widely used in the treatment of Alzheimer s disease [138].

Conclusions
This article summarized a total of 273 compounds that have been reported from the genus Alnus, with 138 cited references. Many species in the genus Alnus have been used as traditional herbal medicines in Korea, Japan and China [2]. So far, phytochemical research on the genus has revealed the extensive presence of diarylheptanoids, flavones, polyphenols, terpenoids, steroids and other compound types. The pharmacological activities of pure compounds and crude extract from this genus were mainly focused on anticancer, antioxidant and anti-inflammatory properties. For their significant anticancer activities, diarylheptanoids are a research hotspot in the genus Alnus.
Some researchers have pointed out the anticancer mechanism of hirsutenone (73), which should be more thoroughly tested as a potential anticancer agent in the future. Some experiments involved the structure-function relationships of antioxidant, anticancer, anti-adipogenic and insecticidal activities, but were not deep enough except structure-antioxidant activity relationship [11,69,75,90]. Hence, researchers may need to pay attention to them in future studies. As a whole, the phytochemical and biological investigations were mainly concentrated on the seven Alnus species (A. japonica, A. hirsuta, A. glutinosa, A. incana, A. nepalensis, A. sieboldiana and A. firma), with little or no attention being paid to other folk medical species. In view of this background, plenty of further studies are necessary in order to examine the other plants of the Alnus genus, extend the use of Alnus species and provide clinical rationale for the development of new therapeutic agents from traditional medicine sources. The authors hope this review will provide valuable data for the exploration and advanced research on Alnus species.