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Recent Advances in Kaempferia Phytochemistry and Biological Activity: A Comprehensive Review

Background: Plants belonging to the genus Kaempferia (family: Zingiberaceae) are distributed in Asia, especially in the southeast region, and Thailand. They have been widely used in traditional medicines to cure metabolic disorders, inflammation, urinary tract infections, fevers, coughs, hypertension, erectile dysfunction, abdominal and gastrointestinal ailments, asthma, wounds, rheumatism, epilepsy, and skin diseases. Objective: Herein, we reported a comprehensive review, including the traditional applications, biological and pharmacological advances, and phytochemical constituents of Kaempheria species from 1972 up to early 2019. Materials and methods: All the information and reported studies concerning Kaempheria plants were summarized from library and digital databases (e.g., Google Scholar, Sci-finder, PubMed, Springer, Elsevier, MDPI, Web of Science, etc.). The correlation between the Kaempheria species was evaluated via principal component analysis (PCA) and agglomerative hierarchical clustering (AHC), based on the main chemical classes of compounds. Results: Approximately 141 chemical constituents have been isolated and reported from Kaempferia species, such as isopimarane, abietane, labdane and clerodane diterpenoids, flavonoids, phenolic acids, phenyl-heptanoids, curcuminoids, tetrahydropyrano-phenolic, and steroids. A probable biosynthesis pathway for the isopimaradiene skeleton is illustrated. In addition, 15 main documented components of volatile oils of Kaempheria were summarized. Biological activities including anticancer, anti-inflammatory, antimicrobial, anticholinesterase, antioxidant, anti-obesity-induced dermatopathy, wound healing, neuroprotective, anti-allergenic, and anti-nociceptive were demonstrated. Conclusions: Up to date, significant advances in phytochemical and pharmacological studies of different Kaempheria species have been witnessed. So, the traditional uses of these plants have been clarified via modern in vitro and in vivo biological studies. In addition, these traditional uses and reported biological results could be correlated via the chemical characterization of these plants. All these data will support the biologists in the elucidation of the biological mechanisms of these plants.


Introduction
From the first known civilization, medicinal plants have met primary care and health needs around the world [1][2][3]. Natural products, derived from plants, have enriched the pharmaceutical industry since time immemorial. So far, people of the developing countries depend upon the traditional medicines to cure daily aliments [4]. The medicinal plants are characterized by a diversity of chemical and pharmacological constituents, owing to their complicity and the abundance of secondary metabolites. There are several factors that caused the variations of the secondary metabolites such as ecological zones, weather, climates, and other natural factors via the effects on the biosynthetic pathways [1][2][3].

Introduction
From the first known civilization, medicinal plants have met primary care and health needs around the world [1][2][3]. Natural products, derived from plants, have enriched the pharmaceutical industry since time immemorial. So far, people of the developing countries depend upon the traditional medicines to cure daily aliments [4]. The medicinal plants are characterized by a diversity of chemical and pharmacological constituents, owing to their complicity and the abundance of secondary metabolites. There are several factors that caused the variations of the secondary metabolites such as ecological zones, weather, climates, and other natural factors via the effects on the biosynthetic pathways [1][2][3].
Zingiberaceae (the ginger family) is distributed worldwide comprising 52 genera and more than 1300 plant species [5,6]. Kaempferia is a diverse family with members distributed widely throughout Southeast Asia and Thailand, including some 60 species [5]. Several Kaempferia species are used widely in folk medicine, including K. parviflora, K. pulchra, and K. galanga, (Figure 1). In Laos and Thai, traditional medicines derived from K. parviflora rhizomes are reported for the treatment of inflammation, hypertension, erectile dysfunction, abdominal ailments [6,7], and improvement of the vitality and blood flow [8]. Japanese use the extract of K. parviflora as a food supplement and for the treatment of metabolic disorders [9]. K. pulchra is used extensively as a carminative, diuretic, deodorant, and euglycemic, as well as for the treatment of urinary tract infections, fevers, and coughs [4]. The rhizomes of K. galanga are used as an anti-tussive, expectorant, anti-pyretic, diuretic, anabolic, and carminative, as well as for the curing of gastrointestinal ailments, asthma, wounds, rheumatism, epilepsy, and skin diseases [10]. Extracts and purified compounds from select Kaempferia species are used for the treatment of knee osteoarthritis and the inhibition of a breast cancer resistance protein (BCRP), anti-inflammatory, anti-acne, anticholinesterase, anti-obesity-induced dermatopathy, wound healing, anti-drug resistant strains of Mycobacterium tuberculosis, neuroprotective, anti-nociceptive, human immunodeficiency

Biological Activity
Extracts and purified compounds of Kaempferia species are used for the treatment of knee osteoarthritis and the inhibition of a breast cancer resistance protein (BCRP), anti-inflammatory, antiacne, anticholinesterase, anti-obesity-induced dermatopathy, wound healing, anti-drug resistant strains of Mycobacterium tuberculosis, neuroprotective, anti-nociceptive, human immunodeficiency virus type-1 (HIV-1) inhibitory activity, in vitro anti-allergenic, and larvicidal activity against Aedes aegypti [11]. Kaempheria plant extracts and isolated compounds demonstrate numerous and promising biological and pharmaceutical activities, which are summarized in Figure 2.

Anticancer Activity
Rhizome ethanolic extracts of K. galanga and the purified component ethyl trans p-methoxycinnamate (105) demonstrate moderate cytotoxic activity against human cholangiocarcinoma (CL-6) cells with IC 50 of 64.2 and 49.4 µg mL −1 , respectively. Significant cholangiocarcinoma (CCA) efficacy as indicated by suppressing tumor growth and lung metastasis in CL6-xenografed mice [15] is also observed. Swapana et al. [16] documented that K. galanga isopimarene diterpenoids, sandaracopimaradiene-9α-ol (2), kaempulchraol I (14), and kaempulchraol L (17) exhibit promising activity against human lung cancer with IC 50 of 75 µM, 74 µM, and 76 µM, respectively, and mouth squamous cell carcinoma (HSC-2) inhibition with IC 50 of 70 µM, 53 µM, and 58 µM, respectively [16]. The latter compound, isolated from K. pulchra, is reported to have weak anti-proliferative activity against human pancreatic and cervix cancers [17]. Chawengrum et al. [18] stated that K. pulchra labdene diterpenoids, (−)-kolavelool (81), and (−)-2β-hydroxykolavelool (82) exhibit cytotoxic activity against human leukemia cells (HL-60) with IC 50 values of 9.0 ± 0.66 and 9.6 ± 0.88 µg mL −1 , respectively [18]. Acetone, petroleum ether, chloroform, and MeOH extracts of K. galanga rhizomes show moderate cytotoxicity in a brine shrimp lethality bioassay compared with vincristine sulfate as the reference compound [19]. Moreover, a methanolic extract of K. galanga rhizomes induces Ehrlich ascites carcinoma (EAC) cell death in a dose-dependent manner [20]. 5,7-Dimethoxyflavone (86) isolated from K. galanga was found to reduce cancer resistance to tyrosine kinase inhibitors (TKI) by inhibiting breast cancer resistance protein (BCRP), one of the efflux transporters that increased efflux of TKI out of cancer cells. This was observed both in vitro with a dose-dependent increase in the intracellular concentration of sorafenib in MDCK/BCRP1 breast cancer resistance cells, with an EC 50 of 8.78 µM as well as in vivo by increasing sorafenib AUC in mice tissues when co-administered with compound 88, as reported by kinetic results [21]. The isolated methyl-β-D-galactopyranoside specific lectin from the rhizome of K. rotunda exhibited in vitro antitumor activity against Ehrlich ascites carcinoma cells at a pH between 6-9 and a temperature range between 30-80 • C. Tumor inhibition was also observed in vivo in EAC-bearing mice [22]. The cytotoxicity of MeOH, petroleum ether, and EtOAc extracts against C33A cancer cells via MTT and scratch assays compared with essential oils of K. galanga rhizomes showed activity for the EtOAc and MeOH fractions at 1000 µg mL −1 with 11% and 14% cell viability and weak efficacy with petroleum ether extracted essential oils in a MTT assay. Cell growth inhibition was observed with all extracts in the scratch assay [23]. Compound (140) isolated from K. angustifolia was described to have strong activity with an IC 50 of 1.4 µg mL −1 , which was comparable to 5-fluorouracil as a reference drug. Compound (138) also showed moderate inhibition against human lung cancer. 2 -Hydroxy-4,4 ,6 -trimethoxychalcone (flavokawain A; 119) exhibited potent activity against HL-60 and MCF-7 cell lines. The results of Tang et al. [24] revealed that flavokawain A (119) exhibited cytotoxic activity against MCF-7 and HT-29 cell lines with GI 50 values of 17.5 µM (5.5 µg mL −1 ) and 45.3 µM (14.2 µg mL −1 ), respectively. Kaempfolienol (65) and zeylenol (133) were also found to have moderate activity against HL-60 and MCF-7 cells with IC 50 values <30 µg mL −1 and against HL-60 only with an IC 50 value of 11.6 µg mL −1 respectively [24].

Anti-Obesity Activity
An ethanolic extract, a polymethoxyflavonoid-rich fraction (PMF) and a polymethoxyflavonoid-poor fraction from K. parviflora were screened against an obesity-induced dermatopathy system using Tsumura Suzuki obese diabetes (TSOD) mice as an obesity model (Hidaka, Horikawa, Akase, Makihara, Ogami, Tomozawa, Tsubata, Ibuki, and Matsumoto) [11]. The ethanolic extract reduced mouse body weight and the thickness of the subcutaneous fat layer more than the PMF fraction that is used as a dietary supplement in controlling skin disorders caused by obesity [11].

Anti-HIV Activity
Viral protein R (Vpr) is one of the HIV accessory proteins that can be targeted for controlling viral replication and pathogenesis. A CHCl 3 fraction of K. pulchra exhibits Vpr-inhibitory activity at 25l g mL −1 . In addition, isopimarene type diterpenoids isolated from the rhizomes of the

Antioxidant Activity
The CHCl 3 and MeOH extracts of the rhizomes of K. angustifolia showed strong antioxidant activity against DPPH expressed with 615.92 mg trolox equivalent (TE)/g of extract. In an azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) assay, MeOH extracts showed good antioxidant properties with a value of 38.87 mg TE/g. However, n-hexane extract exhibited significant antioxidant activity with 901.76 mg TE/g in a cupric-reducing antioxidant capacity assay, while EtOAc extract exhibited significant reduction ability against ferric reducing antioxidant power (FRAP) with a value of 342.23 mg TE/g. Also, kaempfolienol (65) showed potent free radical scavenging activity in a DPPH assay, as well as, 2 -hydroxy-4,4 ,6 -trimethoxychalcone (119) in ABTS, CUPRAC, and FRAP assays [33,34]. A methanol extract of rhizomes of K. galanga exhibited a concentration-dependent antioxidant activity in DPPH, ABTS, and nitric oxide (NO) radical scavenging assays [20]. Moreover, the essential oil extracts of conventionally propagated and in vitro propagated K. galanga had significant DPPH radical scavenging activity [35]. As well, the ethanol extract of K. rotunda exhibited antioxidant activity in a DPPH assay with IC 50 (67.95 µg mL −1 ) [30].

Anti-Inflammatory Activity
The cyclohexane, chloroform, and ethyl acetate extracts with diarylheptanoids isolated from K. galanga showed a pronounced inhibition of Lipopolysaccharides (LPS)-induced nitric oxide in macrophage RAW 264.7 cells compared with indomethacin [13]. The EtOH extract and compounds (1, 52, 53, 119, 120) isolated from K. marginata had promising anti-inflammatory activity based on the suppression of NO production and inducible nitric oxide synthase (iNOS) mRNA and cyclooxygenase-2 (COX-2) genes expression [36,37]. Diterpenoids (9-10) isolated from K. pulchra had topical anti-inflammatory activity in 12-O-tetradecanoylphorbol-13-acetate-induced ear edema in rats with ID 50 330 and 50 µg/ear, respectively. Biological activity may be due to the activation of Maxi-K channels in neurons and smooth muscles [38]. The ethanol extract of K. parviflora exhibited potent inhibition of PGE2. The plant extract and 3 ,4 ,5,7-tetramethoxyflavone (86) were also reported to exhibit a dose-dependent inhibition of iNOS-mRNA expression. Additionally, H 2 O, EtOH, EtOAC, CHCl 3 , and n-hexane soluble sub-fractions exhibited good in vivo anti-inflammatory activity by decreasing rat paw edema [39]. An 80% EtOH extract reduced UV-induced COX-2 expression in mice skin that was attributed to the anti-oxidative activity of polyphenolics against the oxidizing properties of UV radiation [40]. A 60% EtOH and EtOAc-soluble fraction of 100% methanol extracts of K. parviflora decreased knee osteoarthritis, which was likely due to methoxylated flavones [41]. Ethyl p-methoxycinnamate (105) isolated from K. galana inhibited cytokines as IL-1 and TNFα and endothelial function in rats [42].

Anticholinesterase Activity
According to Sawasdee et al. [46], a MeOH extract as well as compounds (86-87) isolated from K. parviflora rhizomes inhibited acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with greater cholinesterase inhibitory toward AChE and BChE for (86), which was an observation of significance in the treatment of Alzheimer's disease [46].

Effect on Cytochromes CYP 450
The results listed by Ochiai et al. [48] stated that the continued ingestion of (88) isolated from K. parviflora decreases liver CYP3A expression, which in turn increased levels of compounds metabolized by CYP3As such as midazolam [48].

Vascular Activity
The oral administration of CH 2 Cl 2 extract of K. parviflora in middle-aged rats was found to decrease vascular responses to phenylephrine, increase acetylcholine-induced vasorelaxation and the production of nitric oxide (NO) from blood vessels, and decrease visceral, subcutaneous fat, fasting serum glucose, triglyceride, and liver lipid accumulation [49]. The effect of intravenous administration of a CH 2 Cl 2 extract of K. galanga to rats reduced the mean arterial blood pressure [50]. This anti-hypertensive effect was attributed to ethyl cinnamate, which is a major compound in the extract [50]. The ethanol extract of rhizomes of K. parviflora caused dose-dependent relaxation on aortic rings as well as ileum pre-contracted with phenylephrine and acethylcholine [51].

Adaptogenic Activity
Hexane, chloroform, methanol, and ethanol extracts of K. parviflora exhibited adaptogenic activity compared with a crude ginseng root powder used as a reference [52]. A single oral dose of K. parviflora rhizome (60% EtOH extract) increased the whole-body potential expenditure in humans [53]. K. parviflora was also found to improvement physical fitness and health by decreasing oxidative stress [54].

Neurological Activity
A methanolic extract (95% MeOH) of K. parviflora exhibited neuroprotective activity by increasing rat hippocampus serotonin, norepinephrine, and dopamine levels in comparison with a vehicle-treated group [56]. An acetone extract of K. galanga rhizomes and leaves also exhibited central nervous system depressant activity [57].

Nociceptive Activity.
A K. galanga rhizome extract exhibited anti-nociceptive activity in rats that was stronger than aspirin but weaker than morphine. The efficacy was abolished by naloxone, suggesting that the analgesic effect may be centrally and peripherally mediated [58].

Wound-Healing Activity
The co-administration of a K. galanga rhizomes extract (95% EtOH) with dexamethazone was found to have wound-healing activity in mice comparable to dexamethazone only [59].
Recently, K. parviflora alcoholic extract at 3-30 µg mL −1 was evaluated regarding the molecular mechanisms associated with rheumatoid arthritis for up to 72 h compared with the dexamethasone as positive control [67]. They documented that the EtOH extract significantly decreased the gene expression levels of pro-inflammatory cytokines, inflammatory mediators, and matrix-degraded enzymes, but neither induced apoptosis nor altered the cell cycle. They also reported that the alcoholic extract inhibits cell migration, reduces the mRNA expression of cadherin-11, and selectively reduces the phosphorylation of mitogen-activated protein kinases (P38, MAPKs), signal transducers, and activators of transcription 1 (STAT1) and 3 (STAT3) signaling molecules, without interfering with the NF-κB pathway [67].
A K. galanga extract (acetone, petroleum ether, chloroform, or methanolic) exhibited dose-dependent anthelmintic activity with strong paralytic activity within one hour and death within 80 min at a 25 mg mL −1 concentration [68].

Chemical Metabolites of Kaempferia Species
Chemical profiles of Kaempferia exhibited the presence of different types of secondary metabolites such as terpenoids, especially isopimarane phenolic compounds, diarylheptanoids [13], flavonoids [69][70][71], and essential oils [72,73]. This review summarized the reported variety of compound types, including isopimarane, abietane, labdan, and clerodane diterpenoids, flavonoids, phenolic acids, phenyl-heptanoids, curcuminoids, tetrahydropyrano-phenolic, and steroids. Diterpenoids, especially isopimarane types, were the most reported compounds from the plants of this genus, in addition to phenolics, flavonoids, and essential oils. Each class will be described and listed in the following items, and the structures will be summarized in Tables 1-3.

Biosynthesis of Isopimarane-Type Diterpenoids
Isopimarane diterpenoids are the most characteristic compounds for Kaempheria plants.

Diterpenoids
Kaempferia plants were characterized with a predominance of diterpenoids, especially the isopimaranes in addition to abietane, labdane, and clerodane types (Table 1).

Abietane-Type Diterpenoids
Seven abietanes (58-64) ( Table 1) have been isolated and characterized from the rhizomes of K. roscoeana, and one (65) was isolated and characterized from K. angustifolia [26,33,34]. These highly oxygenated metabolites contain one or more double bonds and an absence of exomethylenes, except for roscotane D (61), which contains no double bonds.

Volatile Oils
Kaempheria species were documented as very rich plants with volatile oils such as K. galanga [29,73,82,83], K. angustiflora [29], and K. marginata [29]. The volatile oil of K. galanga has been reported as a potential market product in India and over all the world with market values around 600-700 US$/kg on the international market [83]. Phenylpropanoids and/or cinamates were represented as major constituents of volatile oils derived from Kaempheria species followed by monoterpenes [29,73,82]. The phenylpropanoid compound, trans-ethyl cinnamate, was documented as a principal component of volatile oils of all the studied Kaempheria species up to date with concentrations varied from 16-35% of the total identified [29,73,82,83]. The volatile oils of Kaempheria species were reported to have numerous biological activities such as anti-microbial [83], antioxidant [35], nutraceutical [83], nematicidal toxicity [82], and larvicide activities [29]. Table 4 summarized the main components (142-157) of the reported volatile oils of Kaempheria species.

Principal Components Analysis (PCA) and Agglomerative Hierarchical Clustering (AHC) for Kaempferia Species
To assess the correlation between the various Kaempferia species, chemical classes of different compounds were subjected to PCA and AHC (Figure 3). According to the similarity, the analysis showed that we can group the Kaempferia species under three groups: the first group comprised K. galanga, K. marginata, K. pulchra, and K. roscoeana, and these species are correlated to isopimaranes compounds. The Pearson correlation coefficient (r) between K. marginata and K. pulchra was the highest with r = 0.938, while between K. marginata and K. roscoeana, it was 0.771, between K. roscoeana and K. pulchra, it was 0.766, and between K. marginata and K. galanga, it was 0.615 (Table 5). The second group contained K. angustifolia and K. parviflora (r = 0.833), and this group showed a close correlation to flavonoids and phenolics. However, the K. elegans was separated alone, and showed a close relation to labdane and clerodane compounds. The similarities within each group might be ascribed to the genetic relations, as well as the environmental and microclimatic conditions [1][2][3].
In a study of a genetic variation of Kaempferia species based on chloroplast DNA [5], K. marginata and K. galanga were grouped together, which is agreeable with our results (r = 0.615) according to the PCA data of the present study based on the chemical composition. However, in contrast to the data from the PCA, K. angustifolia and K. parviflora were separated in different groups, but K. elegans and K. parviflora were grouped together. In another recent study, based on the DNA and morphological characteristics [84], K. angustifolia and K. parviflora were grouped together in agreement with the chemical variation of the present study.

Conclusions
Kaempheria species are widely used plants in traditional medicine worldwide. All the biological activity data for these plants and their isolated constituents have resulted in numerous leads for medicinal drugs. Mainly, seven rhizomes of Kaempheria plants afforded a vast array of diterpenoids, especially the isopimarane type, along with significant bioactive methoxylated flavonoids. From all these documented chemical and biological results, these plants have been and continue to be a promising source for medicinal natural products and food industrial products.

Conclusions
Kaempheria species are widely used plants in traditional medicine worldwide. All the biological activity data for these plants and their isolated constituents have resulted in numerous leads for medicinal drugs. Mainly, seven rhizomes of Kaempheria plants afforded a vast array of diterpenoids, especially the isopimarane type, along with significant bioactive methoxylated flavonoids. From all these documented chemical and biological results, these plants have been and continue to be a promising source for medicinal natural products and food industrial products.