Boesenbergia rotunda: From Ethnomedicine to Drug Discovery

Boesenbergia rotunda is a herb from the Boesenbergia genera under the Zingiberaceae family. B. rotunda is widely found in Asian countries where it is commonly used as a food ingredient and in ethnomedicinal preparations. The popularity of its ethnomedicinal usage has drawn the attention of scientists worldwide to further investigate its medicinal properties. Advancement in drug design and discovery research has led to the development of synthetic drugs from B. rotunda metabolites via bioinformatics and medicinal chemistry studies. Furthermore, with the advent of genomics, transcriptomics, proteomics, and metabolomics, new insights on the biosynthetic pathways of B. rotunda metabolites can be elucidated, enabling researchers to predict the potential bioactive compounds responsible for the medicinal properties of the plant. The vast biological activities exhibited by the compounds obtained from B. rotunda warrant further investigation through studies such as drug discovery, polypharmacology, and drug delivery using nanotechnology.


Boesenbergia rotunda and its Morphology.
Boesenbergia rotunda is a ginger species that grows in Southeast Asia, India, Sri Lanka, and Southern China. This species belongs to the family of Zingiberaceae. It was previously categorised under the Kaempferia genus by Baker. However, it is now classified under the Boesenbergia genus [1]. There are many local synonyms to its name, such as Chinese keys or Fingerroot in English, "Temu Kunci" in Malay and Krachai or Krachai-Dang in Thailand. This plant has 8 different botanical names which are Boesenbergia cochinchinensis length polymorphism (AFLP), and single strand conformation polymorphism (SSCP) have made the classification possible [3][4][5][6][7]. Eksomtramage and Boontum [8] have distinguished between B. rotunda and B. aff. rotunda which have similar morphology through chromosome analysis. The chromosome count (2n) for B. rotunda and B. aff. rotunda was found to be 36 and 20, respectively. On the other hand, the chromosome count for B. longipes, B. plicata, and B. xiphostachya was 22. These results were reconfirmed in 2002 [8,9]. The morphology of this ginger species has been well characterized. It is a small perennial plant of about 15-40 cm in height. Its leaves are broad and light green while the leaf sheath is red. Each shoot consists of 3-5 elliptic-oblong-red sheathed leaves of about 7-9 cm in width and 10-20 cm in length. The underground portion of the plant consists of a small globular shaped central subterraneous rhizome (1.5-2.0 cm in diameter) from which several slender and long tubers sprout all in the same direction like the fingers of a hand, thus the common name fingerroot. The tubers are about 1.0-1.5 cm thick in diameter and 5-10 cm long. The tissue of the tuber is looser, softer, and more watery than the central rhizome. Both the colour of the central rhizome and the tubers are dependent on the variety of B. rotunda. The yellow variety produces bright yellow rhizomes, while other varieties produce red and black rhizomes. They are strongly aromatic although different from each other. The flowers are scarlet and bloom throughout the year in tropical countries. These beautiful flowers are usually hidden at the base of the foliage, making them unnoticeable. The morphology of B. rotunda is shown in Figure 1 [10,11].

Ethnomedicinal
Functions of B. rotunda. B. rotunda is a common edible ingredient in many Asian countries such as Malaysia, Thailand, Indonesia, India, and China. It is normally cultivated at small home ranches and used as a condiment in food such as curry and soup due to its aromatic flavour, which promotes appetite. This herbal plant is also used as a traditional medicine to treat illnesses such as rheumatism, muscle pain, febrifuge, gout, gastrointestinal disorders, flatulence, carminative, stomach ache, dyspepsia, and peptic ulcer. In Indonesia, B. rotunda is typically used to prepare "jamu," a popular traditional tonic for women after childbirth as well as a beauty aid for teenage girls and to prevent leukorrhea [12]. The fresh rhizomes are used to treat inflammatory diseases, such as dental caries, dermatitis, dry cough and cold, tooth and gum diseases, swelling, wounds, diarrhoea, and dysentery, and as a diuretic [13,14]. Besides, it is also used as an antifungal and antiparasitic agent to heal fungal infections and eradicate helminth or round worms in human intestine, respectively, as well as an antiscabies agent to relieve skin itchiness from mite bites [15]. Referred to as "Thai ginseng" in Thailand, this plant is used as an aphrodisiac among Thai folk. In addition, consumption of its leaves has been shown to alleviate food allergies and poisoning. Moreover, it has been used as self-medication by AIDS patients in Thailand. Despite the lack of scientific evidence to prove the ethnomedicinal uses of this ginger, the success of current biological researches could potentially explain the significance of its traditional usage.

Pharmaceutical and Medicinal Functions of B. rotunda.
Over the years, using various approaches and technologies, researchers have successfully isolated an array of bioactive compounds from B. rotunda Table 1. Nearly a hundred of compounds were isolated and elucidated, ranging from the flavonoid derivatives, chalcone derivatives, esters, kawains, terpenes and terpenoids (see supplementary data 1 available online at doi:10.1155/2012/473637 for more details about the chemical structures of these isolated compounds). These compounds have shown to exhibit great medicinal potential (Table 2), which will be described in more details on the following subtopics

Toxicity Tests
Given the high consumption of B. rotunda and the fact that its safety has not been scientifically established, recent studies have been focused on investigating the acute toxicity of several extracts in vivo to determine the safety of this plant for consumption. Saraithong et al. [40] demonstrated that the ethanolic extract of B. rotunda was safe for consumption as in vivo studies showed no significant changes in the body weight of B. rotunda fed rats. Furthermore, all haematological and histopathological parameters used to evaluate the toxicity effect did not show any adverse changes [40]. Meanwhile, Charoensin et al. [41] reported that two bioactive compounds from B. rotunda, pinostrobin and pinocembrin, exhibited no mutagenic effect or toxicity towards Wister rats, further confirming the safety of its consumption [41].

Antimicrobial Activities
3.1. Anti-Helicobacter pylori Activity. Helicobacter pylori is a prominent Gram-negative bacteria that causes gastritis, dyspepsia, and peptic ulcer and has been linked to the development of gastric and colon cancer. Widespread claims of the antimicrobial activities of B. rotunda prompted scientists to further evaluate the potential of this plant in preventing the infection of H. pylori. Pinostrobin and red oil from the roots of B. rotunda were found to exhibit anti-H. pylori activities against several different isolates of H. pylori [42]. The minimum inhibitory concentration (MIC) for pinostrobin and the red oil were 125 μg/mL and 150 μg/mL, respectively, which were comparable to the positive control, clarithromycin (120 μg/mL); the minimum bactericidal concentration (MBC) was determined to be 150 μg/mL and 175 μg/mL, respectively. Interestingly, while both extracts inhibited H. pylori growth after 3 days, the growth of other bacteria was inhibited by pinostrobin, but not red oil [42]. The ethanolic extract of B. rotunda was also reported to significantly reduce H. pylori infection in Mongolian gerbils. Treated gerbils showed reduced acute and chronic inflammation when fed with B. rotunda 3 weeks before being challenged with H. pylori, and 6 weeks after. Therefore, flavonoid components of B. rotunda could potentially serve as potential drug candidate for inhibition of H. pylori infection [43].

Pathogenic and Spoilage Bacteria Inhibition Activities.
Pathogenic bacteria are a group of bacteria that induce diseases in humans and plants. Spoilage bacteria are another group of bacteria that cause food spoilage through fermentation and decomposition of food products. There has been a rising concern pertaining to food safety and diseases caused by these pathogenic microorganisms, and hence, a renewed interest in finding new antimicrobial agents to combat these pathogens.

Antiparasitic Activity
The  [47]. Although the effect of these plant extracts paled in comparison to metronidazole, a commercial antibiotic with an IC 50 value of 0.48 μg/mL, they are still considered as potential bioactive compounds that could prevent giardiasis.

Inhibition of Biofilm Formation by Oral Pathogens.
Biofilm formation on teeth surfaces is caused by multiple species of oral bacteria, the primary colonisers being mutant Streptococci [72]. Biofilm formation is associated with several acute and chronic infections such as dental caries, gingivitis, and periodontitis and potentially contributes to antibiotic treatment failure against Streptococcus pyogenes [48].  [48].
The following year, Yanti et al. [49] reported the antibiofilm property of B. rotunda extracted panduratin A, which was found to prevent and reduce the spread of multispecies oral bacteria in human mouth. The MIC of panduratin A was determined using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution assay. Mucin-mixed panduratin A at concentrations between 0.5 and 40 μg/mL was coated on 96-well plates, followed by inoculation of three multispecies bacteria, Streptococcus mutans, Streptococcus sanguis, and Actinomyces viscosus, and incubated overnight at 37 • C to allow biofilm formation. Biofilm reduction effect was determined by further treating the bacteria with different concentrations of panduratin A (0.2-10 μg/mL) for up to 60 mins. Panduratin A exhibited bacteria reduction effect at MIC of 1 μg/mL and bactericidal effect against multispecies planktonic cells at 2X MIC, 8 hours after treatment. Reduction of biofilm formation was >50% at 8X MIC, whereas mass reduction of biofilm was observed within 15 mins at a concentration of 10 μg/mL [49]. These results suggested that panduratin A can potentially be used to prevent colonisation of multispecies bacteria, under a dose-dependent manner, and that its effect is equal to commercially available synthetic drugs such as chlorhexidine gluconate [49].

Antiperiodontitis Activity of B. rotunda Extract.
A study conducted by Yanti et al. [49] on the ethanolic extract of B. rotunda revealed the suppressive effect of this extract on the expression of matrix metalloproteinases (MMPs) 2 and 9, both of which are overexpressed by gingival fibroblasts that are activated by Porphyromonas gingivalis during chronic periodontitis. They first described the inhibition of RNA and protein expression of MMP-9 by the B. rotunda ethanolic extract, which were 45% and 52%, respectively. Inhibition of MMP-9 was found to occur through downregulation of mitogen activated protein kinases (MAPK) phosphorylation (ERK1/2, p38, and JNK phosphorylation), thereby reducing the expression of Elk1, c-Jun, and c-Fos transcriptional Evidence-Based Complementary and Alternative Medicine 11 factors. MMP-9 gene-regulating factors, AP-1 and NF-κB, were also blocked [49].
In 2010, Yanti et al. further demonstrated the decrease in RNA and protein expression of MMP-2 in P. gingivalis supernatant-treated human gingival fibroblast-1 (HGF-1), and MMP-9 in P. gingivalis supernatant-treated oral epidermoid cells (KB cells) in a dose-dependent manner, upon treatment with the ethanolic extract of B. rotunda at 2 μg/mL, 5 μg/mL, and 10 μg/mL. Suppression of MMP-2 was found to be mediated by downregulation of c-Jun N-terminal kinase (JNK) and cyclic adenosine monophosphate (cAMP) response element-binding (CREB) signalling pathways [50].

Inhibition of Candida albicans.
C. albicans is a diploid fungus responsible for oral thrush or oral candidiasis and is a common infection observed in HIV patients. Several studies have demonstrated the potential antifungal role of B. rotunda in inhibiting C. albicans growth. A study conducted by Cheeptham and Towers [51] revealed the antifungal activity of B. rotunda ethanolic extract against C. albicans as well as A. fumigatus. The activation of the antimicrobial activity was found to be light mediated, as treatment performed in the dark showed no fungal inhibition [51]. In 2010, Taweechaisupapong et al. studied the antioral pathogen activities of the oil and 95% ethanolic extracts of B. rotunda and Piper sarmentosum. The extracts were screened against 4 oral pathogens, namely, Streptococcus mutans, Lactobacillus sp., Aggregatibacter actinomycetemcomitans, and C. albican. Results from their study showed that B. rotunda oil extract is a potent inhibitor against all these pathogens, as compared to B. rotunda ethanolic extract, and P. sarmentosum oil and ethanolic extracts. The MIC for S. mutans, Lactobacillus sp., A. actinomycetemcomitans, and C. albicans was 2.0, 1.0, 0.5, and 0.5 mg/mL, respectively, for B. rotunda oil extract, while P. sarmentosum oil did not show any inhibition. In fact, B. rotunda oil extract showed faster killing activity towards C. albicans than the commercial drug, nystatin, in a timekill curve study. Fungistatic activity was observed at concentrations of 1 and 1.5 times the MIC, whereas fungicidal effect was found at concentrations of 2 and 2.5 times the MIC, with a reduction of more than 3 log 10 CFU/mL after 60 and 44 mins incubation, respectively [52]. These studies show that B. rotunda could be a potentially good source of antimicrobial agents in inhibiting oral microbes and fungal infections.

Anticariogenic.
Dental caries or tooth decay (cavity) is a common disease caused by the oral bacteria Streptococcus mutans and Lactobacillus. These acid-producing bacteria cause damage to the tooth in the presence of fermentable carbohydrates such as sucrose and fructose, which wash away the mineral fluoride from the tooth, often resulting in tooth ache, or to a severe extent, death. The anticariogenic activity of B. rotunda was first described in 2004 by Hwang and colleagues. Antibacterial and bactericidal activities of B. rotunda methanolic extract were determined by well diffusion and viable cell count methods, respectively. Antibacterial study revealed a dose-dependent increase in the diameter of inhibition zones, with concentrations of 1 mg/mL, 10 mg/mL, and 20 mg/mL producing inhibition zones of 11 mm, 13 mm,and 14 mm in diameter, respectively. B. rotunda extract also showed rapid bactericidal activity against S. mutans in 2 mins at a concentration of 50 μg/mL, rendering it practically important, given that application in toothpaste and mouthwash should be fast and effective within minutes [53].
Further isolation and purification of B. rotunda rhizomes yielded isopanduratin A, which was identified through 1 HNMR. This compound conferred inhibitory properties against S. mutans, having an MIC value of 4 mg/L, which was lower than some natural anticariogenic agents such as green tea extract (125 mg/L) and eucalyptol (500 mg/L). At 20 mg/L, the bacteria were completely inactivated within 1 min. This compound also showed similar inhibitory activity towards S. sobrinus, S. sanguinis, and S. salivarius at an MIC of 4 mg/L. Microscopic observation through transmission of electron microscope revealed the destruction of the bacterial cell wall and cytoplasmic membrane detachment after treatment with 10 mg/L isopanduratin A, suggesting the potential application of isopanduratin A as a natural anticariogenic agent to prevent cariogenic effect on teeth [54].

Candidal Adhesion Inhibitor.
Candidal adhesion is an essential mechanism for Candida species to adhere to the oral surfaces to colonise the mouth and cause oral diseases. Sroisiri and Boonyanit [55] recently reported that the rhizome extract of B. rotunda could inhibit the adhesion of Candida species on the denture acrylic surfaces in a dosedependent manner. Pretreatment of the dentures with B. rotunda extract at concentrations of 25, 50 and 100 mg/mL significantly inhibited candidal adhesion by approximately 47%, 66%, and 74%, respectively. Ergo, it is theorised that candidal adhesion can possibly be reduced by soaking acrylic dentures in B. rotunda rhizome extract for 30 mins [55].

Antihalitosis. Halistosis (or bad breath) is a condition
where the mouth produces an unpleasant odor when exhaling. This situation is typically associated with oral conditions such as gum diseases and oral hygiene. It can also be caused by the odour from esophagus, tonsil, nose, and stomach. Hwang et al. [56] have patented the optimum oral wash formulation of antihalitosis, containing panduratin derivatives from B. rotunda, that could reduce the effect of halitosis by 70-90% [56].

Inhibition of Biofilm Formation by Intestinal Pathogens
Given the positive findings regarding the inhibitory activity of B. rotunda against biofilm formation of several oral pathogens, Rukayadi

Inhibition of Oxidative Damages by tert-Butylhydroperoxide (t-BHP).
Sohn et al. [58] reported the protective effects of panduratin A against t-BHP, an organic hydroperoxidant that initiates lipid peroxidation through its metabolism to free-radical intermediates, causing oxidative damage to cells. MTT cell viability assay showed a decrement in HepG2 cell growth inhibition by t-BHP, whereas fluorometric measurement revealed a dose-dependent reduction in malondialdehyde (MDA) formation and glutathione (GSH) depletion, upon treatment with panduratin A. Intracellular reactive oxygen species (ROS) production was also reduced from 665±11.79 (mean ± standard deviation) to 170±30.62 when treated with 15 μM of the compound, further implying the potential application of this compound as a natural antioxidant [58].

Antiulcer Effect
B. rotunda is also used as a traditional medicine to treat ulcer by local communities in Thailand and Indonesia. The antiulcer effect of B. rotunda methanolic extract and its pure compound, pinostrobin, was recently explored by Abdelwahab and coworkers [59]. B. rotunda extract and pinostrobin exhibited cytoprotective effects on ulcer-induced rats, as evidenced by the reduction in ulcer area and mucosal content. In addition, submucosal edema and leukocytes infiltration were significantly reduced or prevented. The antioxidant activity of pinostrobin was proven through its ability to reduce the level of thiobarbituric acid reactive substances (TBARS) and through ferric reducing antioxidant power (FRAP) assay which gave a value of 116.11 ± 0.004 (mean ± standard deviation) [59].

Obesity Treatment
Obesity is a metabolic disorder that poses a global threat to humans. Caused by fat accumulation due to improper energy balance and lipid metabolism, obesity can cause liver and cardiovascular diseases. Panduratin A, previously determined to be a novel natural AMP-activated protein kinase (AMPK) activator, was studied in attempts to decipher the regulatory mechanisms involved in AMPK-PPARα/δ signalling. AMPK is an enzyme that regulates cellular energy through activation of LBK1 and Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ). The activation of AMPK will increase the fatty acid oxidation by activating fatty acid oxidation-related genes. This process will prevent lipid synthesis via reduction of sterol regulatory element-binding protein-1c (SREBP-1c) and PPARγ phosphorylation. When 50 mg/kg/day of panduratin A was applied, AMPK signalling was found to be stimulated, nuclear translocation of AMPKα2 induced, followed by activation of PPARα/δ, with LKB1 being the key mediator of these effects. Activation of PPARα/δ increased fatty acid oxidation, resulting in weight loss, and reduced fat pad mass as observed in the in vivo obese mouse model. Moreover, these mice showed reduction in fatty liver and an improvement in the serum lipid profiles. Myofibre proportion and mitochondria content in muscles were significantly increased, enhancing running endurance [60]. Taken together, these results exemplify the usefulness of panduratin A in treating obesity and associated metabolic disorders.

Antitumour Necrosis Factor Alpha (Anti-TNF-α)
Tumour necrosis factor-α is a pleiotropic inflammatory cytokine that plays an imperative role in immune response to bacterial, fungal, and viral infections, as well as in the necrosis of specific tumours. Extensive production of TNF-α gives rise to health problems such as autoimmune or chronic inflammatory disorders, for instance, rheumatic arthritis. Morikawa et al. [35] recently reported that prenylchalcones and prenylflavanones, extracted from B. rotunda, could inhibit TNF-α. They   [39]. Cell cycle analysis on treated MCF-7 cells revealed B. rotunda to effectively arrest cells at sub-G1 phase, whereas B. pulchella var attenuate arrested the cell cycle at G2/M phase [73].

Inhibition of Prostate Cancers. In 2006, Yun et al. demonstrated that treatment with panduratin A could
inhibit the growth of prostate cancer cell lines (PC3 and DU145) in a time and dose-dependent manner, with IC 50 values of 13.5 and 14 μM for PC3 and DU145 cells, respectively. Immunoflourescence assay showed that panduratin A triggered the induction of apoptosis in both cell lines, through the inhibition of apoptotic-related procaspases 3, 6, 8, and 9. Apoptosis was suggested to occur via the mitochondrial-dependent pathway, as evidenced by the increase of Bax : Bcl-2 ratio by 6-and 15-fold for PC3 and DU145 cells, respectively, and the upregulation of Fas death receptor and TNF-related apoptosis-inducing ligand (TRAIL). Cell cycle analysis revealed cell cycle arrest at G2/M phase in a dose-dependent manner. Moreover, immunoblot analysis showed induction of p21 WAF/Cip1 and p27 Kip1 , and downregulation of cdks 2, 4, and 6, and cyclins D1 and E [64]. These findings suggest that panduratin A could be a potential therapeutic agent against prostate cancer.

Antilung Cancer.
Aside from the anticancer properties towards breast, colon, and prostate cancer, panduratin A also exhibited inhibitory activities against A549 human nonsmall cell lung cancer cells. The IC 50 value of this compound was 4.4 μg/mL, as determined by MTT assay, whereas cell cycle analysis via flow cytometer showed cell cycle arrest at mitotic/M phase. Panduratin A also acted as nuclear factor kappa beta (NF-κB) inhibitor, as treatment at apoptosisinducing concentration was found to inhibit translocation of NF-κB from cytoplasm to nuclei by the activation of tumour necrosis factor alpha (TNF-α) [65].

Antileukemia. B. rotunda rhizome has been suggested
to possess antileukemic property, as demonstrated by Sukari and colleagues [34]. Cytotoxicity assay on B. rotunda extracts and five flavonoid derivatives, pinostrobin, pinocembrin, alpinetin, cardamonin, and boesenbergin A, revealed that most extracts and pure compounds were able to inhibit the growth of HL-60 cancer cell line, particularly the chloroform extract and boesenbergin A [34].

Antifungal Activities against AIDS-Related Fungal Infections. AIDS is one of the major infectious diseases in
Thailand and is most commonly transmitted sexually. HIV patients are susceptible to fungal infections such as candidiasis by Candida species, cryptococcosis by Cryptococcus species, and histoplasmosis by Histoplasma capsulatum, and traditional herbs are typically sought after as natural treatment. Phongpaichit et al. [66] reported that the chloroform extract from rhizome of B. rotunda could inhibit the propagation of Candida neoformans and Microsporum gypseum but showed low effect against C. albicans by using antifungal assay. From the disc diffusion assay, the plant extract of B. rotunda showed the smallest diameter range as compared to other plant extracts, with inhibition zones of 8.0 ± 0.1-0.6 mm and ±9.0 mm in diameter for C. albicans and Cryptococcus neoformans, respectively. The MIC for the chloroform extract of B. rotunda was 64 μg/mL against C. neoformans and M. gypseum, which was determined to be the lowest and most active among the plant extracts in inhibiting fungal growth. The methanolic extract, on the other hand, had an MIC of 128 μg/mL against C. neoformans. Conversely, both extracts had little inhibition against C. albicans (MIC > 512 μg/mL) [66].

Inhibition of Spoilage and Aflatoxin Producing Fungi.
Food spoilage due to spoilage fungi, such as aflatoxin fungi, is a major concern among consumers. Pattaratanawadee et al. [44] showed that the ethanolic extract of B. rotunda could inhibit spoilage fungi activities (Aspergillus flavus, Aspergillus niger, Aspergillus parasiticus, and Fusarium oxysporum) with MICs of >10% (v/v), 8% (v/v), 10% (v/v), and <8% (v/v), respectively [44]. This shows that B. rotunda is a good choice to inhibit the growth of certain spoilage fungi.

Inhibition of Ca 2+ Signal in Yeast Model
Ca 2+ signalling is one of the crucial physiological pathways in most living organisms, playing imperative roles in regulating diverse cellular processes such as T-cell activation and apoptosis. Extensively studied in the yeast Saccharomyces cerevisiae, Ca 2+ signalling is implicated in the regulation of G2/M cell cycle progression, and inappropriate activation of this signalling pathway can cause physiological and developmental defects. Wangkangwan et al. [67] reported that the bioactive compounds from the crude rhizome extract of B. rotunda could inhibit Ca 2+ signalling in S. cerevisiae mutant zds1Δ strain. Further purification of the crude extract led to isolation of three compounds, namely, pinostrobin, alpinetin, and pinocembrin chalcone. Yeast proliferation assay showed significant inhibition by pinostrobin (low cytotoxicity), alpinetin (high cytotoxicity), and pinocembrin chalcone (high cytotoxicity) with MICs of <0.5, 1, and 0.5, respectively. Given the low toxicity of pinostrobin at 1 mM, it was further subjected to biochemical studies and was found to relieve hyperactivation of Ca 2+ signals in yeast, which is responsible for abnormal morphology and growth arrest at G2 phase. Flow cytometry analysis revealed that treatment with 1 mM pinostrobin prevented G2 arrest of yeast cells, and normal morphology characterised by equal nuclei distribution and the absence of abnormal budding was observed [67].

Anti-HIV-1 Protease Activity.
The HIV-1 protease (aspartyl protease class), a highly conserved protein component for viral maturation, propagation, and infectivity in the human body, is a promising drug target currently under extensive research for the development of drugs and therapeutics to combat HIV/AIDS. The anti-HIV protease activity of B. rotunda rhizomes was previously characterised by Cheenpracha et al. [30]. Purification of methanolic extract of B. rotunda rhizomes yielded cyclohexenyl chalcones panduratin A, panduratin C, hydroxypanduratin A, and chalcone derivatives, helichrysetin, 2 ,4 ,6trihydroxyhydrochalcone, and uvangoletin. Their results showed that hydroxypanduratin A and panduratin A exhibited high inhibition, with IC 50 values of 5.6 μM and 18.7 μM, respectively, as compared to other bioactive compounds which showed weaker inhibition. Structure activity relationship (SAR) study revealed that the effectiveness of the HIV-1 protease inhibition is related to the hydroxylation and prenylation of chalcones [30]. In a separate study, Tewtrakul and colleagues [26] investigated the anti-HIV protease activity of chloroform, methanol and water extracts of several traditional herbs used by Thai locals as selfmedication for AIDS. The chloroform extract of B. rotunda exhibited the most potent inhibition against HIV-1 protease (64.92±4.75%), followed by methanolic extract with 51.92± 0.22% inhibition as compared to other plant species [26]. In another study, four flavonoids, namely, pinostrobin, pinocembrin, cardamonin, and alpinetin, were isolated from the ethanolic extract of B. rotunda rhizomes. Antiviral assay showed that cardamonin exhibited the highest inhibition of 75.11 ± 1.44% with an IC 50 value of 31.0 μg/mL [27]. Therefore, cardamonin, panduratin A, and hydroxypanduratin A are potential drug targets to inhibit HIV-1 protease activity.

Inhibition of Dengue NS2B/NS3 Protease.
Dengue virus serotype-2 (Flaviviridae family) is one of the four dengue serotypes responsible for causing dengue fever, dengue haemorrhagic fever, and dengue shock syndrome worldwide. To date, there is no commercial vaccine available to circumvent the spread of the virus. Research is still underway to develop an effective vaccine or drug and natural compounds are currently among the important antiviral resources.

Anti-Inflammatory
Inflammation is a biological process that is activated in response to extracellular stimulants such as pathogens and chemicals, to mitigate the effects or heal the organism. B. rotunda has been traditionally used in treating several inflammatory-related diseases such as gout, allergy, and peptic ulcer. Scientific research has proven the antiinflammatory properties of this plant, as discussed below. (−)-hydroxypanduratin A inhibited TPA-induced ear oedema by 73% at 2000 μg/ear concentration and 10 hrs treatment, whereas 94% inhibition was obtained with (−)-panduratin A treatment at the same concentration and incubation time point. Ear oedema thickness was also significantly reduced to 48 ± 6 μm from 94 ± 6 μm and 11 ± 4 μm from 81 ± 8 μm for (−)-hydroxy panduratin A and (−)-panduratin A treated rats, respectively [25]. Further investigation is warranted to study the mechanisms of action and targets of both compounds with regards to their anti-inflammatory activities.

Anti-Inflammatory Effect Caused by Opisthorchis viverrini.
Opisthorchis viverrini is a parasite from the Opisthorchiidae family that causes cholangiocarcinoma in humans. It is disseminated through consumption of raw and uncooked fish and can cause inflammation during the infection. Boonjaraspinyo et al. [70] reported that B. rotunda rhizome could inhibit inflammation caused by O. viverrini and induced by N-nitrosodimethylamine administration (NDMA) in rats. Histopathological study showed that the liver tissues of normal and B. rotunda-treated rats exhibited similar morphology (no inflammation observed) compared to NDMA-treated (higher cytotoxicity effect) and O. viverrini-infected rats which showed inflammation around the hepatic bile ducts after one month. Upon treatment with B. rotunda plant extract, O. viverrini-infected and NDMA-treated liver cells showed a reduction in the inflammatory cells surrounding the hepatic bile ducts, which correlated with the decrement in the levels of serum alanine transaminase and direct bilirubin, but not of alkaline phosphatase, which remained the same level as untreated group [70].

Inhibition of Platelet-Activating Factor (PAF) Receptor Binding Effect
PAF is a phospholipid mediator that is involved in many negative physiological functions and pathological conditions such as bronchoconstriction-induced asthma, hyperacute organ-transplant rejection, gastrointestinal ulceration, thrombosis, and allergic reaction. Jantan et al. [71] showed that the PAF receptor binding effect could be inhibited by using local medicinal herb extracts obtained in Malaysia.  [71]. Therefore, these plant extracts can be used to potentially treat PAF-mediated diseases.

Wound Healing Properties
Ethanolic extracts of B. rotunda rhizome have been shown to accelerate wound healing in rats [75]. Visually, it was shown that wounds dressed with rhizomes extract and Intrasite gel significantly healed earlier than those treated with vehicle. Histological analysis of healed wounds dressed with rhizomes extract showed comparatively less scar width at wound closure and healed wound contained less inflammatory cells and more collagen with angiogenesis compared to wounds dressed with vehicle only. From the empirical results mentioned previously, B. rotunda contains potential bioactive compounds with multiple medicinal properties that can prevent, mitigate, or treat various diseases as well as prevent them from spreading. Table 2 summarises the inhibition activities of various B. rotunda extracts towards diseases investigated and reported by scientists worldwide. 19.1. Tissue Culture and Metabolite Engineering. In vitro culture provides an immediate source of compounds in a sustainable approach and as study subjects for molecular biology, crop improvements, and genetics studies. Furthermore, it offers a feasible platform with defined, controllable chemical and physical conditions for metabolite engineering. Rao and Ravishankar [76] described a nonredundant list of plant-derived pharmaceuticals and highlighted the importance of the availability of contaminant-free samples, optimum multiplication, and regeneration protocol for metabolite engineering studies. With that, uniform quality and yield of the compounds could be achieved without problem with variation due to seasonal and geographical reason [76].

Current Research on B. rotunda
Optimization of the culture conditions for B. rotunda has been carried out by Tan et al. [77]. By manipulating the concentration of the plant growth regulator, 2,4dichlorophenoxyacetic acid (2,4-D), in Murashige and Skoog [78] nutrient medium, Tan et al. [77] have successfully induced somatic embryo from meristematic tissue of the young plant. Somatic embryogenesis is a process whereby a somatic cell undergoes development analogous to the zygotic embryo and regenerates into a clonal plant. With the medium formulation, 23.3 ± 4.3% of embryogenic callus formed, and the plantlet regeneration rate was 6.6 ± 0.1 plantlets from a callus aggregate of 1 cm diameter. This finding has made somatic embryogenesis a model system to mass propagate identical cell/tissue samples of B. rotunda for metabolite engineering.
Besides somatic embryogenesis, another route of plantlets regeneration of B. rotunda has been investigated. Yusuf et al. [79] reported a mass production method from young shoot bud in MS medium with sucrose (30.0 g/L), gelrite (2.0 g/L), different concentrations of 6-benzylaminopurine (BAP), and α-naphthaleneacetic acid (NAA). Combination of 2.0 mg/L BAP and 0.5 mg/L NAA was found to be the best treatment for callus induction while multiple shoots (90%, 5 shoots/explant) were induced from day 10 to 14. In all treatments, roots spontaneously grew after 10-14 days. Acclimatization of the in vitro plantlets in soil was successfully performed.
Cell culture in liquid suspension is also an important alternative source for metabolite engineering, mainly owing to the fast propagating rate and ease of scaling-up [80]. Furthermore, secondary metabolites accumulation in suspension culture can be manipulated and enhanced by external chemical and physical treatments with simplicity. Direct contact between cell and nutrients or treatment agents present in the liquid media permits a quick response of the cell. Chemical treatments such as nutrient level, sucrose level, plant growth regulator (PGR), precursor, and substrate feeding are the common methods facilitating the enhancement of compound yield in suspension cultures. By modifying growth-associated factors such as sucrose level, nutrient level especially phosphate and nitrogen composition, and the addition of different types of PGR, productivity of both biomass and compounds could be increased [81]. On the other hand, relative productivity of compounds per dry weight of cell biomass could be enhanced with nongrowthrelated precursors and elicitors.
Precursors are substrates or intermediates found in the biosynthesis pathways from which the secondary metabolites formed [82], while elicitors are physiological stimuli from various abiotic or biotic sources that trigger secondary metabolites accumulation [83]. Metal salts and inorganic ions are common abiotic elicitors used in treatments of plant cell cultures for compounds enhancement. Biotic elicitors include microbial cells or components of microbial cell and even plant signalling molecules or compounds such as yeast extract (YE), salicylic acid, and methyl jasmonate.
Zhao et al. [84] examined the effects of four classes of biotic and abiotic elicitors, heavy metal ions (cobalt, silver nitrate, and cadmium chloride), polysaccharides from YE and chitosan, salicylic acid, methyl jasmonate, and sorbitol on the production of diterpenoid tanshinones in Salvia miltiorrhiza cell culture. Amongst the treatments, 25 μM Ag + (silver nitrate), 25 μM Cd + (cadmium chloride), and 100 mg/L polysaccharide from YE successfully enhanced tanshinone production by more than ten fold compared to the control. Interestingly, the authors also reported suppressed cell growth with a decrease in biomass yield by about 50% in treated S. miltiorrhiza cell culture. In another example, treatment of Taxus chinensis cells with Ag + following adaptation in chitosan through several subcultures resulted in a 4.6-fold increase in paclitaxel production compared to the unadapted cells [85]. The authors also suggested that the treatment using elicitors needed to be optimized and explored thoroughly for promising results.
Physical treatments such as stress factors, light sources, modification of culture environment and electric current, and Pulsed Electric Field (PEF) methods are also widely used for metabolite engineering in plant cell cultures [86]. Kaimoyo et al. successfully enhanced the production of medicarpin by 168-fold in cell suspension [87].
Cai et al. [88] examined the effects of elicitors and high hydrostatic pressure on secondary metabolism of Vitis vinifera suspension culture. The authors used the concentration of phenolic compounds as a measure of the secondary metabolism level and found that it was significantly higher than in the control when treated with ethephon. When the treatment was carried out with combination of ethephon and high hydrostatic pressure, extracellular phenolic acids and 3-O-glucosyl-resveratrol were increased. The results showed that hydrostatic pressure can be used for compound secretion into the liquid medium. Cai et al. [89] further explained that high hydrostatic pressure might alter the permeability of cell/membrane and cause secretion of the compounds as a defense response of plant cells. In V. vinifera cv. Chasselas 9 and Vitis berlandieri cell suspension cultures in a 2 L stirred bioreactor, 90% of the total resveratrol can be secreted into the liquid medium [90].
In conclusion, with the availability in vitro sources for B. rotunda, the study and enhancement of secondary metabolite production can be robustly performed and the bioreactor technology could be employed for maximising the yield in the near future.  [91]. Although important progress has been made in the syntheses of these compounds, the systematic study of B. rotunda-derived flavonoids, that is, (−)-pinostrobin and (±)-panduratin A is still hampered by the inaccessibility of enantiomeric pure starting materials.

Chemical Syntheses of Bioactive Compounds
The enantioselectivity of (−)-pinostrobin was first reported by Hodgetts [92]. The method was based on an intramolecular Mitsunobu cyclisation of the chiral hydroxyphenol 3, which was prepared from the Weinreb amide 2 and the methoxymethyl (MOM)-protected phenol 1 (Scheme 1). Subsequent cleavage of the protecting groups on 3 allowed the intramolecular Mitsunobu cyclisation to produce methoxylated pinostrobin 4. Finally, regioselective demethylation of 4 with aluminium chloride gave enantiomeric pure (−)-pinostrobin in 60% overall yield from 3.
We have recently reported the synthesis of (±)-panduratin A and its regioisomer (±)-isopanduratin A in four steps from (E)-ocimene via a Diels-Alder cycloaddition reaction (Scheme 3) [94]. However, attempts to use the chiral auxiliaries or catalysts such as the CBS-oxazaborolidines and MacMillan's imidazolidinones for the enantioselective synthesis of panduratin A were not successful.
Amaral and coworkers described a strategy to synthesise yangonin and derivatives based on the Heck crosscoupling reaction of pyrone 7 and aryl iodides 8 ( Figure 5). Reaction of the pyrone 7, aryl iodides, and 10 mol% of tetrakis(triphenylphosphine)palladium (0) in the presence of Hünig's base in DMF under microwave irradiation (300 W) for 5.5 min produced the desired kavalactones in moderate yields [118].

Drug Discovery through
Bioinformatics. Some bioactive compounds of B. rotunda had been studied computationally by Othman et al. [120], Paul et al. [121], and Frimayanti et al. [122]. Three flavanones, pinostrobin, pinocembrin, and alpinetin, and four chalcones, pinostrobin chalcone, pinocembrin chalcone, and cardamonin, had been subjected to automated docking towards dengue virus type 2 NS2B/NS3 protease (Protein Data Bank id: 2FOM) to understand the interactions of these reported inhibitors [123] with the binding sites of the protease [120]. In this study, it was reported that the estimated ΔG (free energy of binding) for the flavanones were lower than those of their chalcone derivatives.  that all the ligands studied did not bind to the active site of the protease, which are consistent with the bioassay results, illustrating the noncompetitive inhibitory activities for most of the ligands [74]. Through SAR analysis, it was also suggested that the higher noncompetitive inhibitory activity shown by pinostrobin compared to the other compounds could be accounted for by H-bonding interaction with the backbone carbonyl of Lys74, which is bonded to Asp75 (one of the catalytic triad residues). As shown in Figure 2, the rigid structure of flavanone, the C5 hydroxyl and C7 methoxy groups on ring A, and the phenyl ring (B) was also suggested to be important features to consider in designing new compounds with potential inhibitory activities against dengue virus type 2 NS2B/NS3 protease.
Another docking study was performed in 2011 by Frimayanti et al. using 2FOM structure, and the homology model of dengue virus type 2 NS2B/NS3 protease with reported competitive inhibitors [74], 4-hydroxypanduratin A and panduratin A from B. rotunda as reference compounds. The derivatives of these compounds were then used as ligands for docking, and subsequently, new competitive inhibitors were designed based on the docking result. Based on Figure 4, substitutions were performed individually on positions 1, 2, 3, 4, and 5 of the benzyl ring A of 4hydroxypanduratin A and panduratin A. It was found that substitutions at positions 4 and 5 gave the lowest and closest energies to the reference compounds from the calculated complexation energies, and new ligands were designed by substituting various functional groups on these positions. This strategy was suggested to be an early stage drug discovery for identifying drug candidates.
In 2010 [121], Paul and Choudhury conducted molecular docking studies on the activity of naturally occurring pyranochalcones on the transcriptional regulator enzyme of Pseudomonas putida, a gram-negative bacteria that is resistant to antibiotics. In his studies, the HTH-type transcriptional regulator TTgR (Protein Data Bank id: 2UXI) in P. putida (bound with phloretin) was taken as the target for docking with pyranochalcones as ligands. One of the pyranochalcones, boesenbergin A (Figure 3), was isolated from B. rotunda rhizomes and reported as a highly potential candidate to be active against the multidrug resistant strain of bacteria. From the SAR analysis, the binding affinity of the pyranochalcones was found to increase with increasing number of methoxy moiety in the aromatic part of the ligands. In contrast, the effect of the methoxy moiety connected to the fused aromatic unit, seemed to be less pronounced, could be due to steric hindrance. The results are hoped to be useful in designing new series of drugs especially against the antibiotic-resistant bacteria.

Protein Profile of B. rotunda
Since B. rotunda has many medicinal uses, it is important to explore the molecular level of the biosynthesis of the targeted plant metabolites, especially panduratin A and 4hydroxypanduratin A, that have high therapeutic values. In 2011, Chong et al. conducted a preliminary study using proteomic approaches to extract the total protein from the callus (normal and treated callus). This is the initial step to obtain the protein profile of the callus that can be used to analyse the protein expression level of the callus after treatment with phenylalanine, a precursor of phenylpropanoid biosynthesis pathway, to produce CCD, through an unknown biosynthesis pathway in B. rotunda [124].

Future Prospects of Plant-Based Drug
The ethnomedicinal usages of B. rotunda, supported by strong scientific evidence of its potential medicinal properties, clearly justify that this plant should indeed be brought to the next level of drug discovery studies, directed towards metabolomics, genomics, transcriptomics, proteomics, and bioinformatics aspects to further characterise the mechanisms and pathways that contribute to its desired properties.
The wide inhibition range of B. rotunda against multiple diseases such as cancers, microbes, viruses, and parasites should be further explored through new drug discovery studies such as plant-derived compounds, polypharmacology, drug-DNA/protein interactions, and, specific drug stability, solubility, and delivery to the targeted organ, by using nanotechnology.

Conclusion
B. rotunda is a native ingredient in many Asian countries and is used as a condiment in food. It is also used as traditional medicine to treat several illnesses, consumed as traditional tonic especially after childbirth, beauty aid for teenage girls, and as a leukorrhea preventive remedy for women. Its fresh rhizomes are also used to treat inflammatory diseases, in addition to being used as an antifungal, antiparasitic, and aphrodisiac among Thai folks. Its leaves are used by locals to alleviate food allergic and poisoning. Moreover, AIDS patients self-medicate themselves with B. rotunda to cure the infection. With the advancement in technology, the ethnomedicinal usages of herbal plants can be explained through in vitro and in vivo studies to prove the activities of the plant extracts. The current state of research on B. rotunda clearly shows that the isolated bioactive compounds have high potential in treating many diseases. With the development of medicinal chemistry and bioinformatics, we are well on our way to successfully develop plant-based drugs. Molecular progressions further encourage scientists to delve deeper into the biosynthetic pathways of B. rotunda bioactive compounds to obtain a bigger picture of the whole process, which in turn could accelerate the development of better and stronger drugs to counter the diseases in the future.