Bioactive Compounds from Zingiber montanum and Their Pharmacological Activities with Focus on Zerumbone

The genus Zingiber consists of about 85 species and many of these species are used as food, spices, and medicines. One of the species, Zingiber montanum (J. Koenig) Link ex A. Dietr. is native to Southeast Asia and has been extensively used as traditional medicines and food. The aim of this review was to collect and critically analyze the scientific information about the bioactive compounds and pharmacological activities of Z. montanum with focus on one of the main components, zerumbone (ZER). Various studies have reported the analysis of volatile constituents of the essential oils from Z. montanum. Similarly, many phenylbutanoids, flavonoids and terpenes were also isolated from rhizomes. These essential oils, extracts and compounds showed potent antimicrobial, anti-inflammatory and antioxidant activities among others. Zerumbone has been studied widely for its anticancer, anti-inflammatory, and other pharmacological activities. Future studies should focus on the exploration of various pharmacological activities of other compounds including phenylbutanoids and flavonoids. Bioassay guided isolation may result in the separation of other active components from the extracts. Z. montanum could be a promising source for the development of pharmaceutical products and functional foods.


Introduction
The Zingiberaceae family consists of about 50 genera and more than 1500 species which are distributed all over the world and most of them are found in Asia, Central Zerumbone (ZER) (Figure 1c), a sesquiterpenoid, is one of the major compounds in the essential oils and rhizomes of Z. montanum [4]. In recent years, it has received much attention among researchers as a potent antitumor and anti-inflammatory compound [8][9][10][11][12][13][14][15]. Z. montanum being extensively used in traditional medicine but very few investigations were found for their bioactive constituents and mechanism based pharmacological actions. Thus, the main aim of this review is to scientifically analyze the available scientific Zerumbone (ZER) (Figure 1c), a sesquiterpenoid, is one of the major compounds in the essential oils and rhizomes of Z. montanum [4]. In recent years, it has received much attention among researchers as a potent antitumor and anti-inflammatory compound [8][9][10][11][12][13][14][15]. Z. montanum being extensively used in traditional medicine but very few investigations were found for their bioactive constituents and mechanism based pharmacological actions. Thus, the main aim of this review is to scientifically analyze the available scientific information about the chemical constituents and pharmacological activities of extracts and compounds isolated form Zingiber montanum along with the various activities of ZER.

Pharmacological Activities of Z. montanum Extracts and Compounds
Various pharmacological activities such as antimicrobial, anti-inflammatory, antioxidant, antihistaminic, smooth muscle relaxant, insecticidal activities are reported for the essential oils, extracts and some isolated compounds of Z. montanum. Some of these activities are discussed in detail in following sections.

Anti-Inflammatory Activity
The hexane extract of Z. montanum showed remarkable inhibitory effect on carrageenan-induced rat paw edema, acetic acid-inducing writhing reaction in mice and yeasttriggered hyperthermia in rats [56]. Moreover, phenylbutanoids have been reported as active constituents for anti-inflammatory activities [45]. Sabinene and terpinene-4-ol from essential oil of Z. montanum significantly reduced nuclear factor-kappa B (NF-κB) protein expression in human leukemic monocyte lymphoma cells and interleukin-6 (IL-6) secretion in lipopolysaccharide (LPS) stimulated mice macrophage (RAW264.7) [57]. Methyl tbutyl ether (MTBE) and methanol extracts of Zingiber were effective to inhibit LPS induced in vitro production of prostaglandin E2 (PGE2) and TNF-α in human promonocytic U937 cells [58]. The methanol extract and phenylbutanoids of Z. montanum rhizome showed inhibitory effects on the production of NO from LPS induced peritoneal macrophages from mouse [52]. Methanol extract and its fractions (petroleum ether, hexane and aqueous) of Z. montanum showed anti-inflammatory activity in carrageenan-induced edema in rats, and acetic acid-induced vascular permeability and writhing test in mice [45].

Antifungal Activity
Jantan et al. reported that the Z. montanum rhizome oil at a dose of 0.75 mg/disc showed significant fungicidal activity against five dermatophytes fungi (Trichophyton mentagrophytes, T. rubrum, Microsporum canis, M. nanum and Epidermophyton floccosum) and

Extraction Solvent Compounds References
Chloroform extract Methanol extract Acetone extract Acetone extract Hexane extract

Extraction Solvent Compounds References
Ethanol extract Methanol extract Methanol extract Methanol extract Chloroform extract Methanol extract

Pharmacological Activities of Z. montanum Extracts and Compounds
Various pharmacological activities such as antimicrobial, anti-inflammatory, antioxidant, antihistaminic, smooth muscle relaxant, insecticidal activities are reported for the essential oils, extracts and some isolated compounds of Z. montanum. Some of these activities are discussed in detail in following sections.

Anti-Inflammatory Activity
The hexane extract of Z. montanum showed remarkable inhibitory effect on carrageenaninduced rat paw edema, acetic acid-inducing writhing reaction in mice and yeast-triggered hyperthermia in rats [56]. Moreover, phenylbutanoids have been reported as active constituents for anti-inflammatory activities [45]. Sabinene and terpinene-4-ol from essential oil of Z. montanum significantly reduced nuclear factor-kappa B (NF-κB) protein expression in human leukemic monocyte lymphoma cells and interleukin-6 (IL-6) secretion in lipopolysaccharide (LPS) stimulated mice macrophage (RAW264.7) [57]. Methyl t-butyl ether (MTBE) and methanol extracts of Zingiber were effective to inhibit LPS induced in vitro production of prostaglandin E 2 (PGE 2 ) and TNF-α in human promonocytic U937 cells [58]. The methanol extract and phenylbutanoids of Z. montanum rhizome showed inhibitory effects on the production of NO from LPS induced peritoneal macrophages from mouse [52]. Methanol extract and its fractions (petroleum ether, hexane and aqueous) of Z. montanum showed anti-inflammatory activity in carrageenan-induced edema in rats, and acetic acid-induced vascular permeability and writhing test in mice [45].

Antifungal Activity
Jantan et al. reported that the Z. montanum rhizome oil at a dose of 0.75 mg/disc showed significant fungicidal activity against five dermatophytes fungi (Trichophyton mentagrophytes, T. rubrum, Microsporum canis, M. nanum and Epidermophyton floccosum) and three filamentous fungi (Aspergillus niger, A. fumigatus and Mucor sp.) [59]. Another study reported that the essential oil of the rhizome showed antifungal activity against Thanetophorus cucumeris [60]. Z. montanum exhibited high activity against the yeasts namely Saccharomyces cerevisiae, Cryptococcus neoformans, Candida albicans, C. tropicalis, C. glabrata [59]. Tripathi et al. reported the essential oils of Z. montanum at 500 mg/L showed 100% growth inhibition of fruit fungus Botrytis cinerea [61].

Antioxidant Activity
Many studies have demonstrated the antioxidant properties of Z. montanum. Extract from Z. montanum exhibited potent antioxidant activity hydroxyl radical (OH) scavenging assay [62]. Anastasia et al. reported the antioxidant activities of different fractions of Z. montanum by using 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide (H 2 O 2 ), β-carotene bleaching assays. Among different fractions, chloroform fraction showed highest antioxidant activities in DPPH radical scavenging assay, hexane fraction showed highest activity in H 2 O 2 assays and ethyl acetate fraction in β-carotene bleaching assay [63]. Masuda et al. studied the antioxidant activity of cassumunins A, B and C isolated from Z. montanum rhizomes acetone extract using a thiocyanate method which demonstrated that all cassumunins at a dose of 2.7 µM inhibited accumulation of linoleic acid hyperoxide [64]. Bua-in and Paisooksantivatana reported the antioxidant activity of the extracts obtained from the rhizomes of Z. montanum collected from various localities in Thailand [65].

Antibacterial Activity
Z. montanum essential oil showed potent antibacterial activity against a number of Gram-positive and Gram-negative bacteria. Compared to methanolic extract, chloroform extract showed significant antimicrobial activity against a wide range of pathogens [66]. The rhizomes of Z. montanum are reported to be rich in essential oil effective against a range of pathogenic bacteria including Escherichia coli, Klebsiella pneumonia, Salmonella paratyphi, S. typhi and Shigella flexneri [27]. Z. montanum oil showed potent antimicrobial activity against seventy-four microbial strains with most potent activity against bacteria as such Bacillus subtilis, E. coli, and Salmonella typhi evaluated by disc-diffusion broth dilution method [19]. Boonyanugomol et al. reported significant antimicrobial activity of the essential oil of Z. montanum against Gram-negative Acinetobacter baumannii strains by agar disc-diffusion tests [67]. Sesquiterpenes, monoterpenes and diterpenes from Z. montanum showed various degrees of antimicrobial action against B. cereus, Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa [68].

Analgesic and Antipyretic Activity
Plai cream, a water in oil emulsion prepared from the essential oil of rhizomes of Z. montanum, was reported to reduce the delayed onset of muscle soreness in healthy volunteers [30,69]. Strong antipyretic action of Z. montanum hexane extract was observed in yeast induced hyperthermia rats and analgesic activity was observed on acetic acidinduced writhing response in mice [56]. In another study, strong analgesic activity was observed in hot plate method compared to the standard pentazocine in case of chloroform and dichloromethane extract of Z. montanum [70].

Antiulcer Activity
Al-Amin et al. evaluated the antiulcer activity of methanol extract of Z. montanum in mice and it showed 62.0% and 83.1% inhibition of stomach lesions induced by 1N hydrochloric acid (HCl) at doses of 200 mg/kg and 400 mg/kg, respectively. The major compound isolated from the extract i.e., zerumbone also showed potent antiulcer activity in ethanol and indomethacin induced gastric lesions in mice [16]. Another study reported that different concentration of rhizome extracts of Z. montanum showed significant antiulcer activity in comparison with control group in aspirin-induced rat model [71].

Anti-Allergic Activity
Ethanolic and aqueous extracts of Z. montanum exhibited the most potent anti-allergic activity in antigen induced beta hexosaminidase release in RBL-2H3 cell lines [72]. Capsules prepared from Z. montanum inhibited wheal and flare responses (Type 1 allergic reaction) induced by the mite skin prick test in allergic rhinitis patients [73].

Cytotoxicity Activity
Zulkhairi et al. reported the cytotoxicity activity of different extracts and compounds from rhizomes in human T-acute lymphoblastic leukemia cancer cells (CEMss) and human cervical cancer cells (HeLa) [74]. Crude methanolic extract of Z. montanum rhizomes showed significant cytotoxic activity in NIH 3T3 fibroblast cell line [65].

Other Activities
Dulpinijthmma et al. reported that Z. montanum capsule had remarkable role in the treatment of asthma by reducing the bronchial hyperresponsiveness [75]. Crude ethanolic Z. montanum extracts showed potent inhibitory effect on phorbol 12-myristate 13-acetate (PMA) induced mucous producing gene (MUC2, MUC5AC) as well as its protein expression in epithelial cell via inhibition of extracellular signal-regulated kinase pathway [76]. Dichloromethane extract from the rhizome of Z. montanum showed significant mosquito larvicidal activity [77]. Kato et al. reported the neutrophilic activity of phenylbutanoid constituents [17]. Okonogi et al. reported that essential oil showed moderate butyrylcholinesterase inhibitory [78].

Anticancer Activity
Cancer is one of the leading causes of death worldwide [91]. Various studies have evaluated the anticancer potential of zerumbone. It was assessed against HeLa cell line and interestingly it showed a selective inhibition of HeLa cells proliferation (IC 50 of 14.2 ± 0.5 µmol/L) via enhancement of cellular uptake compared to the normal cell line L929 [92]. Moreover, Rosa and co-workers revealed the anticancer mechanism of ZER on three cell lines including HeLa, B16F10 and undifferentiated Caco-2 cell lines. It was shown that ZER altered the total lipid and fatty acid profile in cancer cells, inducing marked changes in the phospholipid/cholesterol ratio [93]. In addition, the anticancer activity was assessed on Jurkat cells, human T cell leukemia, and it was found that ZERpendant derivatives showed antiproliferative effects (IC 50 values as low as 1-10 µM for most derivatives) [94]. A recent study reported that ZER inhibited cell migration of human esophageal squamous cancer by suppressing Rac1 expression, which is achieved through promoting Rac1 ubiquitination and degradation [95]. Wide number of studies had reported the in vivo, in vitro and in silico anticancer activities of ZER. Herein, an in vivo study showed that ZER significantly controls the growth of tumor and metastasis in BALB/c female mice injected with 4T1 (6-thioguanine resistant cell line) to spontaneously produce highly metastatic tumor [96]. Sithara et al. reported the anticancer activity of ZER against colorectal cancer cells, where they showed that ZER activates caspase 3, caspase 8, and caspase 9. ZER resulted in cell cycle arrest at the G2/M phase [97]. Similarly, other study reported induction of apoptosis in hepatoma HepG2 cells by ZER [98]. Eid and co-workers attempted to explore the underlying mechanism of ZER against breast cancer using in silico study. Since estrogen mediates several pathophysiological signaling pathways associated with cancer progression, the author had selected estrogen as a target for breast cancer and found that the promising molecular interaction, binding interaction, and stability of ZER and estrogen receptor-β (ERβ) suggests ZER as lead compound for breast cancer [99]. More details of these activities are given in Table 2. Table 2. Anticancer activities of zerumbone (ZER).

Experimental Models Results and Possible Mechanisms Reference
HeLa cells ZER selectively inhibited the proliferation of HeLa cells and also enhanced the anti-proliferative activity of anticancer agents vinblastine and paclitaxel. [92] HeLa cells ZER stimulated the apoptosis.

Experimental Models Results and Possible Mechanisms Reference
BALB/c female mice ZER controled the growth of tumor and metastasis via delayed progression of cancer cell cycle and apoptosis. [96] Male ICR mice ZER effectively suppressed mouse colon and lung carcinogenesis through multiple modulatory mechanisms of growth, apoptosis, inflammation and expression of NFκB and HO-1. [118] Female Balb/c mice ZER induced apoptosis in cervical tissues from female Balb/c mice treated prenatally with diethylstilboestrol. [119] Caov-3 and HeLa cells ZER inhibited cancer cell growth through the induction of apoptosis and arrested cell cycle at G2/M phase. [120] Female BALB/c Mice Combination of ZER and cisplatin modulated the serum level of interleukin 6 in mice with cervical intraepithelial neoplasia. [121] HeLa cells ZER caused prominent growth retardation of HeLa cells. [122,123] HepG2 cells ZER increased apoptosis in HepG2 cells by up-regulating pro-apoptotic Bax protein and suppressing anti-apoptotic Bcl-2 protein expression. [124] MCF-7 and MDAMB-231cells ZER inhibited the viability of MCF-7 and MDA-MB-231 cells [125] HepG2 cells Highly soluble inclusion complex of ZER-hydroxypropyl-β-cyclodextrin induced apoptosis of HepG2 via Caspase8/BH3 interacting-domain death agonist cleavage switch and modulating Bcl2/Bax ratio. [12] HepG2, human umbilical vein endothelial cells (HUVECs) ZER inhited prolieration and migration of HepG2 cells and inhibited angiogenesis, and expression of matrix metalloproteinase-9, vascular endothelial growth factor (VEGF) and VEGF receptor proteins in HUVECs cell line. Human PaCa cell lines BxPC-3 and MIA PaCa-2 ZER blocked the PaCa-associated angiogenesis through the inhibition of NF-κB and NF-κB dependent proangiogenic gene products. [128] Human renal cell carcinoma (RCC) cell line 786-O and Caki-1 ZER acted as a novel blocker of STAT3 signaling cascade. [129] Oral squamous cell carcinoma (OSCC) lines ZER inhibited the activation of CXCR4-RhoA and PI3K-mTOR signaling pathways resulting into reduced cell viability of OSCC cells. Murine leukemia induced with WEHI-3B cells ZER-loaded nanostructured lipid carrier (ZER-NLC) induced mitochondrial-dependent apoptotic pathway in murine leukemia. [132] Human gastric cancer cell line SGC-7901 ZER induced human gastric cancer cells apoptosis.

Anti-Inflammatory Activity
The anti-inflammatory property of ZER is also reported by many studies in vitro and in vivo studies using different models. Various cellular mechanisms of anti-inflammatory activities are also reported. The details of these activities are given in Table 3. Table 3. Anti-inflammatory activity of ZER.

Antimicrobial Activity
Various studies have reported the potent antibacterial activity of zerumbone [87,149]. A recent study reported the inhibitory effect of ZER extract and its compounds against multi-drug resistant and methicillin resistant Staphylococcus aureus [20]. In addition, ZER has an anti-biofilm potential; where it is reported to significantly suppress the expression level of BmeB12 along with antibacterial activity against Bacteroides fragilis [150]. Moreover, a study reported the bactericidal action of ZER against the carcinogenic bacterium Streptococcus mutans (ATCC35668) [151]. Synthetic derivatives of zerumbone are also reported as potent antimicrobial compounds [152].

Other Pharmacological Activities
Various other pharmacological activities are also reported for ZER such as immunomodulatory activity, neuroprotective effect, antinociceptive, anti-platelet and anti-melanogenic activities ( Table 4). Different studies reported the immunomodulatory properties of ZER. Keong et al. revealed ZER activates mice thymocytes, splenocytes and peripheral blood mononuclear cells (PBMC) at dose dependent pattern [153]. A similar study assessed a commercially obtained ZER on human peripheral blood, where it showed that ZER activates human lymphocytes and upregulates interleukin-12p70 cytokine [154]. For neuroprotective effect of ZER, Hamdi et al. reported that ZER oxide protects NG108-15 cells from H 2 O 2 induced oxidative stress [155]. Apart from that, ZER has a gastroprotective effect, where ZER reduces submucosal edema and leukocyte infiltration. On the other hand, a recent in vivo study reported the antinociceptive activity of ZER on mouse, where ZER suppresses inflammatory mediators without any signs of sedation [156]. An in vivo assessment reported the anti-platelet action of ZER investigated from human blood [157]. For the anti-melanogenic activity, a recent study reported that ZER attenuates melanin accumulation in α-melanoma cells [158]. Table 4. Other pharmacological activities of ZER.

Hepatoprotective activity
In vitro C57BL/6 mice In a chronic liver injury model induced by CCl 4 , ZER treatment alleviated the hepatocellular toxicity and inhibitd activation of primary hepatic stellate cells. [159] In vivo Mice ZER restored the activities of antioxidant enzymes such as superoxide dismutase and glutathione peroxidase. It also reduces the release of pro-inflammatory cytokines such as IL-6 and TNF-α, and inactivated the TLR4/NF-κB/COX-2 pathway in acute liver injury induced by CCl 4 in mice.
[ 168] In vivo C57BL/6 mice ZER decreased the levels of plasma triglycerides well as plasma insulin and leptin. [169]

Anti-hyperglycemia and related activities
In vitro MDCK cells ZER increased AMPK phosphorylation at Thr172 under normal/high glucose without affecting mitochondrial function. [170] In vivo STZ-diabetic rats ZER ameliorated diabetic nephropathy by inhibiting hyperglycemia-induced inflammation. [171] In vivo STZ-diabetic rats ZER protected from hyperglycemia-induced retinal damage. [172] In vitro INS-1 rat pancreatic β cells ZER protected against high glucose-induced apoptosis of INS-1 pancreatic β cells. [173] Wound healing activity In vivo ZER treated wound sections showed greater tissue regeneration and more fibroblasts possibly through the inhanced expression of VEGF, TGF-β1 and collagen IV. [174]

Antiallergic activity
In vivo Female BALB/c and C57BL/6 mice ZER showed antiallergic effect via modulation of Th1/Th2 cytokines in an asthmatic mouse model [164] Although zerumbone shows promising biological activities, its low water solubility and poor bioavailability are the limiting factors for wider applications of various formulations containing zerumbone. Few studies have been reported aimed at improving the solubility and bioavailability of zerumbone such as formulation inclusion complexes with cyclodextrin [8,175], nanostructured lipid careers [9], etc.

Conclusions and Future Prospects
This review highlighted the traditional food and medicinal uses, bioactive chemical constituents, and pharmacological activities of Z. montanum. Various bioactive compounds have been isolated and identified form the different plant parts. The most widely used and studied part was rhizome. Studies have reported both volatile and non-volatile compounds from the rhizomes. Sesquiterpene lactone, ZER was one of the main components in the rhizomes. ZER has been studied widely for its anticancer, anti-inflammatory, and other pharmacological activities. Future studies should focus on the exploration of various pharmacological activities of other compounds including flavonoids and phenylbutanoids. Bioassay guided isolation may result in the isolation of other active components from the extracts. Future studies should also focus on in vivo studies dealing with pharmacological and pharmacokinetic evaluations. Moreover, clinical studies should be conducted to validate the promising biological activities of ZER. Based on these data, Z. montanum can be a potential source for the development of functional and health beneficial food products.