Acanthopanax henryi: Review of Botany, Phytochemistry and Pharmacology

Acanthopanax henryi (Oliv.) Harms (Araliaceae), also known as Eleutherococcus henryi and Caoyewujia (Hengliwujia) in Chinese, is a widely used traditional Chinese herb with the effects of expelling wind and removing dampness, relaxing the muscles and stimulating the blood circulation, and regulating the flow of qi to alleviate pain in the theory of Traditional Chinese Medicine. Acanthopanax henryi (AH, thereafter) possesses ginseng-like activities and is known as ginseng-like herb. In the past decade, a great number of phytochemical and pharmacological studies on AH have been carried out. Several kinds of chemical compositions have been reported, including terpenoids (monoterpenoids, diterpenoids, and triterpenoid saponins), phenylpropanoids, caffeoyl quinic acid derivatives, flavonoids, lignans, sterols, fatty acids, etc., among which, triterpenoid saponins were considered to be the most active components. Considerable pharmacological experiments in vitro have demonstrated that AH possessed anti-neuroinflammatory, anti-adipogenic, anti-inflammatory, antibacterial, anti-cancer, anti-oxidation, anti-AChE, anti-BuChE, and antihyaluronidase activities. The present review is an up-to-date and comprehensive analysis of the botany, phytochemistry, and pharmacology of AH.


Introduction
Acanthopanax spp. plays an important role in traditional oriental medicine in China, Korea, Japan, and far-east Russia. Its dried roots and stem barks are famous traditional folk medicine for treating rheumatism, arthritis, paralysis, sinew, and bone pain [1]. Acanthopanax Miq. as an endemic Asian genus with over 100 species (including 54 species and 49 varieties) are mainly distributed in North-eastern Asia including China, Korea, and Japan [2]. In other regions such as Blutan, India, Mongol, Malaysia, Nepal, Philippines, Russia, Thailand, and Vietnam, Acanthopanax spp. are also found [3]. These medicinal plants belonging to Acanthopanax spp. (Araliaceae) possess ginseng-like activities and are known as ginseng-like herb [4], and they are widely used as traditional oriental medicine having tonic, anti-rheumatic, longitudinal bone growth, adaptogenic activity, anti-fatigue, anti-stress, and anti-ischemic heart disease benefits [5,6]. The root barks of the plants

Botany
Acanthopanax henryi (Oliv.) Harms. (AH) is a hermaphroditic deciduous shrub or tree to 1-3 m tall ( Figure 2), with sparse branches and anfractuous rough thorns; branchlets: densely covered with soft, short hair which then gradually falls off; leaves: palmately compound five foliate, rarely three, petioles 4-7 cm long, with bristled hair on both surfaces along veins; leaves like paper, close together on the short branch, are in leaflets of five, elliptic or narrowly oblong, rarely obovate, apex acute or acuminate, base narrow wedgeshaped, 8-12 cm long, 3-5 cm wide, dark green above, rough, gray-green below, leaf veins covered with soft, short hair, leaf margins and above middle part with small serrate, lateral veins in 6-8 pairs, both sides of leaves are raised and obvious but the leaf veins are not obvious; small petioles are 3-6 mm long, with thick, short hair, sometimes with no small petiole; flowers: smaller, umbel inflorescence, 1.5-2.5 cm in diameter, the total pedicel robust and with thick, short hair which then gradually falls off, five petals, five stamens, five ovaries at the base, the style all together generate columnar, blooming from July to September; fruits: drupe, globular, black-ripen from September to October; seeds: two in each fruit, small and flat. AH is grown widely in the provinces of Jiangxi, Shanxi, Shaanxi, Sichuan, Hubei, Henan, Anhui, and Zhejiang. It grows in the forest edge or bushes, at an altitude of 1000-3200 m [1]. AH has the effects of dispelling wind and dampness, promoting blood circulation and relaxing tendons, regulating qi, and relieving pain in the theory of Traditional Chinese Medicine. In folk medicine, it is mainly used in the treatment of rheumatic arthralgia, contracture and numbness, weakness of muscles and bones, edema, traumatic injury, rheumatoid arthritis, and hernia abdominal pain as well as related conditions [9,10].

Chemical Constituents
To date, the chemical components (Table 1) of AH mainly contain monoterpenoids and their glycosides, diterpenoids, triterpenoid saponins, phenylpropanoids and their glycosides, caffeoyl quinic acids, flavonoids, lignans and their glycosides, steroids, fatty acids, and other compounds. Of these compounds, monoterpenoids and their glycosides, triterpenoid saponins, caffeoyl quinic acids, and lignans and their glycosides as the major active components.

Monoterpenoids
AH contains abundant secondary metabolites; among them, the monoterpenes are the major ones, as listed in Table 1. To date, 14 of these compounds have been isolated from fruits of AH [11][12][13]. Of them, four are monoterpene aglycones, and ten are monoterpenoid glycosides ( Figure 3). The above mentioned compounds include the three new monoterpenoid glycosides, (2E,6R)-6-hydroxy-2,6-dimethyl-2,7-octadien-1-yl- , which were isolated from the methanol extract of fruits of AH, based on our previous work [11]. The basic skeleton of these compounds can be divided into two types: linalool and menthene. The information of the monoterpenoids is shown in Table 1.

Lignans
Lignans have been suggested to be evolutionarily derived by elaboration of the phenylpropanoid pathway for a plant's own benefits, as its immunoprotection and protection, as it were, from harmful free radicals [27]. According to our previous work, 14 lignans and their glycosides were isolated and purified from the fruit [11,12], root [15,19,24,25], stem [20], and flower [21] (Figure 8). Of them, (−)-sesamin is one of the main active ingredients [12,19,20]. Several studies revealed that (−)-sesamin possesses potent anti-cancer properties. The anti-cancer effects of sesamin have been mainly attributed to its anti-proliferative, pro-apoptotic, anti-inflammatory, anti-metastatic, antiand pro-angiogenic, and pro-autophagocytic activities. Also, the previous researches indicate that NF-κB, STAT3, JNK, ERK1/2, p38 MAPK, PI3K/AKT, caspase-3, and p53 signaling pathways are critically involved in mediating the anti-cancer effects of (−)-sesamin [28]. In addition, oral sesamin administration (50 mg/kg·bodyweight/day) significantly attenuated depressive, aversive, repetitive, and anxiety-like behaviors in a long-term multiple nonsocial stress-treated CD-1 mice model. Sesamin inhibited stress-induced gut barrier integrity damage, reduced circulating lipopolysaccharide (LPS) levels, and suppressed neuroinflammatory responses. Moreover, sesamin treatment also restructured the gut microbiome by enhancing the relative abundances of Bacteroidales and S24-7. The correlation analysis indicated that the microbiota composition changes were strongly correlated with behavioral disorders, serotonin, norepinephrine, and LPS levels. In conclusion, sesamin has preventive effects on stress-induced behavioral and psychological disorders, which might be highly related to the reshaped microbiota composition [29].

Pharmacology Research
The traditional functions of AH are for paralysis, arthritis, rheumatism, lameness, edema, injuries from fall, hernia, and abdominal pain applications. According to its traditional efficacies, researchers have conducted a series of studies and found that the plant also possesses anti-neuroinflammatory, anti-adipogenic, anti-inflammatory, antibacterial, anticancer, anti-oxidation, anti-AChE, anti-BuChE, and antihyaluronidase functions ( Table 2). The active ingredients related to these functions are mainly lignans, pentacyclic triterpenoid saponins, flavonoids, and caffeoyl quinic acid derivatives. Table 2. Biological activities of the Acanthopanax henryi shown by in vitro studies.

Biological Activity
In Vitro Studies Ref.

Anti-Neuroinflammatory Activity
In our previous study, the lignan savinin (73), with anti-neuroinflammatory activity, was isolated from the roots of AH. Savinin showed inhibitory activity against lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E 2 (PGE 2 ) production with IC 50 values of 2.22 ± 0.11 and 2.28 ± 0.23 µM, respectively. The effects of savinin were associated with the suppression of LPS-induced expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein. Furthermore, savinin negatively regulated the production of interleukin (IL)-1β and tumor-necrosis factor (TNF)-α at the transcriptional level in LPS-stimulated BV2 microglial cells. These anti-neuroinflammatory effects of savinin were mediated by p38 mitogen-activated protein kinase (MAPK), but not ERK or JNK MAPKs [19].
In addition, the phytochemical investigation on the fruits of AH resulted in the discovery of three novel monoterpene glycosides: eleuhenryiside A (1), eleuhenryiside B (2), and eleuhenryiside C (3), as well as a known lignan, (−)-kobusin (69). The antineuroinflammatory activities of these compounds were evaluated with LPS-stimulated BV2 microglia. The results showed that new compounds, eleuhenryiside A and eleuhenryiside C, have inhibitory effects on NO production with IC 50 values of 32.50 ± 1.60 and 3.54 ± 0.20 µM in LPS-stimulated BV2 microglia. Also, the lignan (−)-kobusin has ability to inhibit NO production with the IC 50 values of 14.25 ± 2.69 µM in BV2 cells [11].
Our previous work showed that the methanol extracts of flowers and stems of AH as well as their ethyl acetate extraction part could inhibit the production of NO in LPSstimulated BV2 microglia, respectively [20,21].

Anti-Inflammatory Activity
Some studies have reported that triterpenoid saponins isolated from the leaves of AH had a significant anti-inflammatory effect [11,20,21,[31][32][33]. For instance, Ciwujianoside C3 (29) exhibited no cytotoxicity at the measured concentrations in RAW264.7 macrophages. Treatment with Ciwujianoside C3 inhibited NO production, proinflammatory cytokine levels, including IL-6, TNF-α, and PGE 2 , and protein and mRNA expression levels of iNOS and COX-2. Furthermore, Ciwujianoside C3 suppressed phosphorylation of extracellular signal-regulated kinases and c-jun N-terminal kinases. It was also able to suppress activation of NF-κB via inhibition of the TLR4 signaling pathway. In brief, Ciwujianoside C3 exerts inhibitory effects on LPS-induced PGE 2 , NO, IL-6, and TNF-α production. In addition, iNOS and COX-2 expressions were decreased in RAW264.7 murine macrophages. These inhibitory effects may be achieved via suppression of MAPKs and NF-κB phosphorylation following inhibition of the TLR4 signaling pathway [31].
In the cell model mentioned above, Araliasaponin II (32) markedly inhibited the production of NO and PGE 2 and reduced iNOS and COX-2 expression at the transcriptional and translational levels. Also, it downregulated the expression of interleukin-6 and tumor necrosis factor-α at the protein and mRNA levels. Furthermore, pre-treatment with Araliasaponin II significantly suppressed the TLR-4-NF-κB signaling pathway; this effect may be caused by Araliasaponin II competing with LPS for binding to TLR-4 and subsequently inhibiting translocation of the NF-κB/p65 protein to the nucleus [32].
In our previous study, the rare anthraquinone, glyceroyl-1,6,8-trihydroxy-3-methyl-9,10-dioxo-2-anthracene carboxylate (102), which was isolated from leaves of AH, significantly decreased the production of NO and the levels of other inflammatory factors, such as TNF-α and IL-6, in LPS-stimulated RAW264.7 macrophages in a dose-dependent manner. This is the first report of the anti-inflammatory effect of this compound [16]. Additionally, the lignan (−)-kobusin (69) purified from the fruits of AH showed abilities to inhibit NO production and had no influence on cell viability, with the IC 50 value of 36.35 ± 6.27 µM in RAW264.7 cells [11].
Recent research reported that the hot methanol extract of the root bark of AH was subjected to XAD-4 column chromatography eluting with a gradient of methanol in water. The cytotoxicity and anti-inflammatory effects of the MeOH fractions were evaluated on the inhibition on LPS-induced nitric oxide, prostaglandin E 2 , interleukin-1β, and interleukin-6 production in RAW264.7 macrophages. Results showed that the 80% MeOH fraction was a better inhibitor of LPS-induced NO, PGE 2 , IL-1β, and IL-6 production, and expression of iNOS at the protein levels in a concentration-dependent manner [33].
Additionally, our previous studies have shown that the MeOH extracts of flowers and stems of AH, as well as their petroleum ether, ethyl acetate, and n-butanol extraction parts, had a potential anti-inflammatory activity based on the inhibition of NO production in LPS-induced RAW264.7 cells, respectively [20,21].

Antimicrobial Activity
A recent study showed that the pentacyclic triterpenoid saponin, ursolic acid 3-O-α-L-arabinopyranoside (URS, 18), isolated from the leaves of AH, has antibacterial activity in combination with oxacillin (OXA) against methicillin-resistant Staphylococcus aureus (MRSA). The synergistic effects of URS and OXA were determined using a checkerboard dilution test and time-kill curve assay. The minimal inhibitory concentration (MIC) value of URS against MRSA was found to be 6.25 µg/mL and there was a partial synergistic effect between OXA and URS. The time-kill growth curves were suppressed by OXA combined with URS at a sub-inhibitory level. Compared to the optical density at 600 nm (OD600) value of URS alone (0.09 µg/mL), the OD600 values of the suspension in the presence of 0.09 µg/mL URS and 0.00001% Triton X-100 or 250 µg/mL N,N -dicyclohexylcarbodiimide reduced by 56.6 and 85.9%, respectively. The transmission electron microscopy (TEM) images of MRSA indicated damage to the cell wall, broken cell membranes, and cell lysis following treatment with URS and OXA. Moreover, an inhibitory effect on the expression of penicillin-binding protein 2a (PBP2a) protein was observed when cells were treated with URS and OXA compared with untreated controls [34].
Moreover, one latest study reported that another pentacyclic triterpenoid saponin, Eleutheroside K (20), isolated from the leaves of AH, can effectively inhibit the growth of MRSA through down-regulating the expression of PBP2a [35].

Anticancer/Antitumor Activities
A recent study showed that the 100% MeOH fraction (after MeOH extract of AH was chromatographed on XAD-4 column) of the root bark of AH has moderate inhibitory effects on HL-60 human leukemia cells, HT-29 human colon cancer cells, and A549 human lung cancer cells with IC 50 values of 50.41, 102.05, and 147.57 µg/mL, respectively [14]. Besides this, the 75% MeOH extract of roots of E. henryi cultivated in Poland exhibited moderate inhibition effect of HL-60 cell line growth with an IC 50 value of 270 µg/mL [36].

Anti-Oxidant, Anti-AChE, and Anti-BuChE Activities
In recent years, a study was focused on the chemical constituents of the leaves of AH along with their antioxidant and acetyl cholinesterase (AChE) inhibitory activities. Caffeoyl quinic acid derivates and flavonoids were obtained from AH through column chromatography technologies, and the content of major constituents was determined by the HPLC-UV method. Anti-oxidant activity of the isolated metabolites was analyzed by free radical scavenging (DPPH, ABTS radicals) and superoxide anion scavenging. The results showed that antioxidant activity of di-caffeoyl quinic acid derivates, including 1,5-di-Ocaffeoyl quinic acid (46), 3,4-di-O-caffeoyl quinic acid (47), 3,5-di-O-caffeoyl quinic acid (48), and 4,5-di-O-caffeoyl quinic acid (49) was stronger than that of the positive controls (ascorbic acid, trolox, and allopurinol). AChE inhibitory activity of the constituents was estimated. Among which, 4,5-di-O-caffeoyl quinic acid (49), 4-O-caffeoyl quinic acid (52), and quercetin (64) were found to have strong AChE inhibitory activity with IC 50 values ranging from 62.6 to 121.9 µM. This study showed that some of the tested components from the leaves of AH exhibit strong anti-oxidant and anti-AChE activities in vitro [22].
A study showed that the 75% methanol extract of roots of A. henryi cultivated in Poland exhibited certain anti-AChE and anti-DPPH activities, with inhibitory rates at 19.6% (at the concentration of 100 µg/0.19 mL of the reaction mixture) and 14.7% (in 90 min), respectively. The extract reduced DPPH in a time-dependent mode of 90 min, at the concentration of 0.8 mg/mL [36].
Another work reported that, using the HPTLC assay method, the 75% ethanol extract of A. henryi exhibited certain anti-AChE and anti-BuChE activities with IC 50 values of 1.75 ± 0.1 and 0.13 ± 0.02 mg/mL, respectively. Also, the chloroform extract of A. henryi showed weak BuChE inhibitory activity with an IC 50 value of 1.21 ± 0.2 mg/mL. Moreover, the 75% ethanol and chloroform extracts of A. henryi exhibited anti-DPPH antioxidant activities [37].
Załuski and Janeczko reported that the ethanol extracts from fruits of A. henryi, freshly dried and stored for one year, had the effects of anti-DPPH with the EC 50 values of 0.2 ± 0.01 and 0.23 ± 0.02 mg/mL, respectively [38].

Anti-Hyaluronidase Activity
A recent study reported that the 75% methanol extract of roots of A. henryi cultivated in Poland exhibited moderate antihyaluronidase activity, and the inhibitory rate was 40.7% at the concentration of 100 µg/0.16 mL of the reaction mixture [36]. In addition, the ethanol extracts from the fruit of A. henryi, after being freshly dried and stored for one year, had the effects of antihyaluronidase with the IC 50 values of 0.61 ± 0.05 and 0.70 ± 0.04 mg/mL, respectively [38].

Discussion
The research findings indicated that the main chemical constituents of AH are monoterpenoids and their glycosides, triterpenoid saponins, caffeoyl quinic acid, and lignans. As to A. gracilistylus, included in Chinese Pharmacopoeia (Edition 2020), its major chemical components are diterpenoids, triterpenoids and their saponins, and ceramides [39]; A. senticosus, another species of traditional medicinal plant of Acanthopanax, has been widely studied by domestic and foreign scholars. A large number of documents reported that the main chemical constituents of A. senticosus are triterpenoid saponins and lignans [27]. Comparison of the three species of Acanthopanax mentioned above found that although they have similarities in traditional usage, such as they can be used to treat rheumatic arthralgia, contracture and numbness, weakness of muscles and bones, asthenia, and edema, etc., there are some differences in their main chemical components. Whether they can replace each other needs to be confirmed by more in-depth pharmacological and clinical studies. Therefore, they should be distinguished from each other when they are used. So far, there is no report on the essential oil of AH; further studies on the volatile components and their pharmacological activities of AH need to be carried out.

Conclusions
In summary, this review presents 102 chemical components of AH, mainly including monoterpenoids and their glycosides, diterpenoids, triterpenoid saponins, phenylpropanoids, caffeoyl quinic acid derivatives, flavonoids and their glycosides, lignans and their glycosides, sterols, fatty acids, and other compounds isolated from 1993 to 2021. Of them, triterpenoid saponins exhibit very good anti-inflammatory, anti-adipogenic, and antibacterial activities. The lignans showed potential anti-neuroinflammatory activity, and the extracts and fractions enriched in caffeoyl quinic acid derivatives and flavonoids have antioxidation and anti-AChE activities. The methanol and ethanol extracts showed anticancer, anti-BuChE, and antihyaluronidase activities.
However, the studies on pharmacological activities of crude extracts and isolated compounds are mostly focused on the cell level in vitro. A deeper investigation of isolated metabolites in preclinical and clinical studies are necessary to define the active constituents of AH. Therefore, further evaluation of in vivo activities of isolated compounds is urgently needed. Meanwhile, the discovery of new metabolites is important and promising and can provide clues for the development of new drugs. In this paper, the current research status of AH was reviewed in order to provide some scientific theoretical basis and reference for the follow-up research of the Chinese endemic medicinal plant.