Unveiling the Antiviral Properties of Panduratin A through SARS-CoV-2 Infection Modeling in Cardiomyocytes.

Establishing a drug-screening platform is critical for the discovery of potential antiviral agents against SARS-CoV-2. In this study, we developed a platform based on human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to investigate SARS-CoV-2 infectivity, with the aim of evaluating potential antiviral agents for anti-SARS-CoV-2 activity and cardiotoxicity. Cultured myocytes of iPSC-CMs and immortalized human cardiomyocyte cell line (AC-16) were primarily characterized for the expression of cardiac markers and host receptors of SARS-CoV-2. An infectivity model for the wild-type SARS-CoV-2 strain was then established. Infection modeling involved inoculating cells with SARS-CoV-2 at varying multiplicities of infection (MOIs) and then quantifying infection using immunofluorescence and plaque assays. Only iPSC-CMs, not AC16 cells, expressed angiotensin-converting enzyme 2 (ACE-2), and quantitative assays confirmed the dose-dependent infection of iPSC-CMs by SARS-CoV-2, unlike the uninfectable AC16 cells lacking the expression of ACE2. Cytotoxicity was evaluated using MTT assays across a concentration range. An assessment of the plant-derived compound panduratin A (panA) showed cytotoxicity at higher doses (50% cytotoxic concentration (CC50) 10.09 µM) but promising antiviral activity against SARS-CoV-2 (50% inhibition concentration (IC50) 0.8-1.6 µM), suppressing infection at concentrations 10 times lower than its CC50. Plaque assays also showed decreased viral production following panA treatment. Overall, by modeling cardiac-specific infectivity, this iPSC-cardiomyocyte platform enables the reliable quantitative screening of compound cytotoxicity alongside antiviral efficacy. By combining disease pathogenesis and pharmacology, this system can facilitate the evaluation of potential novel therapeutics, such as panA, for drug discovery applications.

Potential Lipolytic Effect of Panduratin A Loaded Microspicule Serum as a Transdermal Delivery Approach for Subcutaneous Fat Reduction.

Boesenbergia rotunda (L.) Mansf contained a potent anti-obesity agent. The objectives of this study were to investigate the anti-adipogenesis and lipolysis effects of panduratin A from B. rotunda extract and develop extract-loaded lipolytic body microspicule (MS) serum. Panduratin A that was separated from the ethanolic extract of B. rotunda in fraction 3 (BP-3) were studied the bioactivity of 3T3-L1 preadipocyte cells. The extract-loaded MS serum was formulated and evaluated for safety and efficacy. The BP-3 extract containing panduratin A at 0.29 g per g of the extract was not toxic to the cells at concentrations lower than 10 µg/mL, and the antiadipogenesis and lipolysis effects of the BP-3 extract were strong at 10 µg/mL. To deliver bioactive panduratin A into and through the skin, MS serum was successfully formulated. Application of BP-3 extract-loaded MS serum to the human thigh for 14 d reduced the thigh circumference and increased skin hydration and firmness. Although the skin erythema was increased, no severe redness or pain was found. In conclusion, BP-3 extract acts as a potent bioactive compound to inhibit adipocyte cells, and the antiadipogenesis and lipolysis effects of BP-3 extract in MS serum might play an important role as a potential lipolytic body product for reducing human subcutaneous fat mass.

Absolute oral bioavailability and possible metabolic pathway of panduratin A from Boesenbergia rotunda extract in beagle dogs.

CONTEXT: Attempts are ongoing to develop medications to fight against the COVID-19 pandemic. Our previous study revealed the in vitro anti-SARS-CoV-2 activity of fingerroot [Boesenbergia rotunda (L.) Mansf. (Zingiberaceae)] and its phytochemical, panduratin A. OBJECTIVE: To investigate the pharmacokinetic profiles of panduratin A as a pure compound and in a fingerroot extract formulation in beagle dogs. MATERIALS AND METHODS: A total of 12 healthy dogs were randomly divided into three groups, a single dose of 1 mg/kg panduratin A by intravenous and multiple doses of 5 and 10 mg/kg panduratin A fingerroot extract formulation by oral administration for seven consecutive days. The plasma concentration of panduratin A was determined by LCMS. RESULTS: The peak concentrations of a single dose of 5 and 10 mg/kg panduratin A fingerroot extract formulation were 12,416 ± 2,326 and 26,319 ± 8,221 µg/L, respectively. Increasing the oral dose of fingerroot extract formulation, equivalent to panduratin A 5-10 mg/kg, showed dose proportionality, with an approximately 2-fold increase in Cmax and AUC. The absolute oral bioavailability of panduratin A in the fingerroot extract formulation was approximately 7-9%. The majority of panduratin A was biotransformed into several products via oxidation and glucuronidation, and predominantly excreted via the faecal route. CONCLUSION: The oral formulation of fingerroot extract was safe in beagle dogs, and increasing dose showed dose proportionality in terms of the systemic exposure of panduratin A. This information will support the phytopharmaceutical product development of fingerroot extract against the COVID-19 pandemic.

Genome Assembly and Analysis of the Flavonoid and Phenylpropanoid Biosynthetic Pathways in Fingerroot Ginger (Boesenbergia rotunda).

Boesenbergia rotunda (Zingiberaceae), is a high-value culinary and ethno-medicinal plant of Southeast Asia. The rhizomes of this herb have a high flavanone and chalcone content. Here we report the genome analysis of B. rotunda together with a complete genome sequence as a hybrid assembly. B. rotunda has an estimated genome size of 2.4 Gb which is assembled as 27,491 contigs with an N50 size of 12.386 Mb. The highly heterozygous genome encodes 71,072 protein-coding genes and has a 72% repeat content, with class I TEs occupying ~67% of the assembled genome. Fluorescence in situ hybridization of the 18 chromosome pairs at the metaphase showed six sites of 45S rDNA and two sites of 5S rDNA. An SSR analysis identified 238,441 gSSRs and 4604 EST-SSRs with 49 SSR markers common among related species. Genome-wide methylation percentages ranged from 73% CpG, 36% CHG and 34% CHH in the leaf to 53% CpG, 18% CHG and 25% CHH in the embryogenic callus. Panduratin A biosynthetic unigenes were most highly expressed in the watery callus. B rotunda has a relatively large genome with a high heterozygosity and TE content. This assembly and data (PRJNA71294) comprise a source for further research on the functional genomics of B. rotunda, the evolution of the ginger plant family and the potential genetic selection or improvement of gingers.

Protective Effect of Panduratin A on Cisplatin-Induced Apoptosis of Human Renal Proximal Tubular Cells and Acute Kidney Injury in Mice.

BACKGROUND: Cisplatin is an effective chemotherapy but its main side effect, acute kidney injury, limits its use. Panduratin A, a bioactive compound extracted from Boesenbergia rotunda, shows several biological activities such as anti-oxidative effects. The present study investigated the nephroprotective effect of panduratin A on cisplatin-induced renal injury. METHODS: We investigated the effect of panduratin A on the toxicity of cisplatin in both mice and human renal cell cultures using RPTEC/TERT1 cells. RESULTS: The results demonstrated that panduratin A ameliorates cisplatin-induced renal toxicity in both mice and RPTEC/TERT1 cells by reducing apoptosis. Mice treated with a single intraperitoneal (i.p.) injection of cisplatin (20 mg/kg body weight (BW)) exhibited renal tubule injury and impaired kidney function as shown by histological examination and increased serum creatinine. Co-administration of panduratin A (50 mg/kg BW) orally improved kidney function and ameliorated renal tubule injury of cisplatin by inhibiting activation of extracellular signal-regulated kinase (ERK)1/2 and caspase 3. In human renal proximal tubular cells, cisplatin induced cell apoptosis by activating pro-apoptotic proteins (ERK1/2 and caspase 3), and reducing the anti-apoptotic protein (Bcl-2). These effects were significantly ameliorated by co-treatment with panduratin A. Interestingly, panduratin A did not alter intracellular accumulation of cisplatin. It did not alter the anti-cancer efficacy of cisplatin in either human colon or non-small cell lung cancer cell lines. CONCLUSIONS: The present study highlights panduratin A has a potential protective effect on cisplatin's nephrotoxicity.

Cytotoxic mechanisms of panduratin A on A375 melanoma cells: A quantitative and temporal proteomics analysis.

Melanoma is a lethal form of skin cancer with rising global incidence. However, limited treatment options are available for advanced melanoma and this is further compounded by the development of resistance toward existing drugs. Panduratin A (PA), a cyclohexanyl chalcone found in Boesenbergia rotunda, was investigated for its cytotoxic potentials against human malignant melanoma A375 cells. Our initial findings revealed that mitochondrion is the primary acting site of PA on A375 cancer cells and the cytotoxic mechanisms of PA were further investigated using a temporal quantitative proteomics approach by iTRAQ 2D-LC-MS/MS. Comprehensive proteomics analysis identified 296 proteins that were significantly deregulated in PA-treated A375 cells and revealed the involvement of mitochondrial oxidative phosphorylation, secretory and ER stress pathway, and apoptosis. We further confirmed that the PA-induced apoptosis was mediated by prolonged ER stress at least in part via the PERK/eIF2α/ATF4/CHOP pathway. Pretreatment with cycloheximide, an ER stress inhibitor rescued PA-induced cell death, which was accompanied by the suppression of ER-stress-related HSPA5 and CHOP proteins. The present study provides comprehensive mechanistic insights into the cytotoxic mechanisms of PA.

Enhancing effect of natural adjuvant, panduratin A, on antibacterial activity of colistin against multidrug-resistant Acinetobacter baumannii.

Colistin- and carbapenem-resistant Acinetobacter baumannii is a serious multidrug resistant (MDR) bacterium in clinical settings. Discovery of new antibacterial drugs against MDR is facing multiple challenges in drug development. Combination of known antibiotics with a robust adjuvant might be an alternative effective strategy for MDR treatment. In the study herein, we report an antibiotic adjuvant activity of a natural compound panduratin A from fingerroot (Boesenbergia rotunda) as a potent adjuvant to colistin. The present study investigated the antibiotic adjuvant effect of panduratin A against 10 colistin- and carbapenem-resistant A. baumannii. Antibacterial activities were tested by broth microdilution method. Biofilm assay was used to determine the efficacy of panduratin A in biofilm formation inhibition on two representative strains Aci46 and Aci44. Genomic and transcriptomic analyses of colistin- and carbapenem-resistant A. baumannii strains were used to identify potential resistance and tolerance mechanism in the bacteria. Panduratin A-colistin combination showed an increased effect on antibacterial in the A. baumannii. However, panduratin A did not improve the antibacterial activity of imipenem. In addition, panduratin A improves anti-biofilm activity of colistin against Aci44 and Aci46, the colistin- and carbapenem-resistant A. baumannii. Panduratin A markedly enhances bactericidal and anti-biofilm activity of colistin against colistin- resistant A. baumannii. Based on genome comparisons, single nucleotide polymorphism (SNP) patterns in six genes encoding biofilm and lipid A biosynthesis were shared in Aci44 and Aci46. In Aci44, we identified a partial sequence of pmrB encoding a polymyxin resistant component PmrB, whereas a full length of pmrB was observed in Aci46. RNA-seq analyses of Aci44 revealed that panduratin A-colistin combination induced expression of ribosomal proteins and oxidative stress response proteins, whereas iron transporter and MFS-type transporter systems were suppressed. Panduratin A-colistin combination could promote intracellular reactive oxygen species (ROS) accumulation could lead to the cidal effect on colistin-resistant A. baumannii. Combination of panduratin A and colistin showed a significant increase in colistin efficacy against colistin- resistant A. baumannii in comparison of colistin alone. Genomic comparison between Aci44 and Aci46 showed mutations and SNPs that might affect different phenotypes. Additionally, based on RNA-Seq, panduratin A-colistin combination could lead to ROS production and accumulation. These findings confirmed the potency of panduratin as colistin adjuvant against multidrug resistant A. baumannii.

Oral Bioavailability, Tissue Distribution, Metabolism, and Excretion of Panduratin A from Boesenbergia rotunda Extract in Healthy Rats.

Background: Our previous studies in vitro and in vivo have shown anti-severe acute respiratory syndrome coronavirus 2 activity of fingerroot extract (Boesenbergia rotunda) and its phytochemical panduratin A. Aim of Study: Therefore, the objective of this study was to determine the pharmacokinetic profiles of panduratin A, as a pure compound and in fingerroot extract, in rats. Materials and Methods: Male rats were randomly divided into four groups. Rats underwent intravenous administration of 4.5 mg/kg panduratin A, a single oral administration of 45 mg/kg panduratin A, or a multiple oral administration of 45 mg/kg panduratin A-consisted fingerroot extract for 7 consecutive days. The concentrations of panduratin A in plasma, tissues, and excreta were measured by using LCMS with a validated method. Results: The rats showed no change in health status after receiving all test preparations. The absolute oral bioavailability of panduratin A administered as pure panduratin A and fingerroot extract were approximately 9% and 6%, respectively. The peak concentrations for the single oral doses of 45 mg/kg panduratin A and fingerroot extract, were 4833 ± 659 and 3269 ± 819 µg/L, respectively. Panduratin A was mostly distributed in gastrointestinal organs, with the highest tissue-to-plasma ratio in the stomach. Approximately 20-30% of unchanged panduratin A from the administered dose was detected in feces while a negligible amount was found in urine. The major metabolites of administered panduratin A were identified in feces as oxidation and dioxidation products. Conclusion: Panduratin A from fingerroot extract showed low oral bioavailability, good tissue distribution, and partially biotransformed before excretion via feces. These findings will assist in developing fingerroot extract as a phytopharmaceutical product for COVID-19 treatment.

A computational study on the molecular mechanisms of panduratin A as a potential inhibitor on SARS-CoV-2 protein targets.

Panduratin A from Boesebergia rotunda was recently reported as a potent anti-SARS-CoV-2 compound. However, the molecular mechanisms underlying the inhibition by Panduratin A and its target remained unclear. Molecular docking calculations were performed between panduratin A and five important proteins, i.e., main protease (Mpro), papain-like protease (PLpro), receptor binding domain (RBD) of spike proteins, RNA-dependent-RNA-polymerase (RdRp), and 2'-O-methyltransferase (MTase). The estimated binding free energy and the interaction networks extracted from the best docking mode for each complex suggested that MTase was the most probable target for panduratin A inhibition. To further validate the ability of panduratin A to inhibit MTase, molecular dynamics (MD) simulations and binding free energy calculations were performed for panduratin A-MTase complex, in comparison with another MTase complex with sinefungin as a positive control. Chemical features of panduratin A and sinefungin were compared for their contribution in MTase binding. It was found that both molecules could bind to the S-Adenosyl methionine (SAM) binding pocket and prevent the SAM entrance co-substrate, which could eventually halt the function of MTase. Despite a slightly weaker binding free energy, the equilibrated positional binding of panduratin A was found at a closer distance to the active sites. Therefore, this study proposed MTase as a possible target of panduratin A, along with the mechanisms of inhibition, prompting another future in vitro study as a verification.

Pharmacological Activities of Fingerroot Extract and Its Phytoconstituents Against SARS-CoV-2 Infection in Golden Syrian Hamsters.

Background: The outbreak of COVID-19 has led to the suffering of people around the world, with an inaccessibility of specific and effective medication. Fingerroot extract, which showed in vitro anti-SARS-CoV-2 activity, could alleviate the deficiency of antivirals and reduce the burden of health systems. Aim of Study: In this study, we conducted an experiment in SARS-CoV-2-infected hamsters to determine the efficacy of fingerroot extract in vivo. Materials and Methods: The infected hamsters were orally administered with vehicle control, fingerroot extract 300 or 1000 mg/kg, or favipiravir 1000 mg/kg at 48 h post-infection for 7 consecutive days. The hamsters (n = 12 each group) were sacrificed at day 2, 4 and 8 post-infection to collect the plasma and lung tissues for analyses of viral output, lung histology and lung concentration of panduratin A. Results: All animals in treatment groups reported no death, while one hamster in the control group died on day 3 post-infection. All treatments significantly reduced lung pathophysiology and inflammatory mediators, PGE2 and IL-6, compared to the control group. High levels of panduratin A were found in both the plasma and lung of infected animals. Conclusion: Fingerroot extract was shown to be a potential of reducing lung inflammation and cytokines in hamsters. Further studies of the full pharmacokinetics and toxicity are required before entering into clinical development.

Interaction of panduratin A and derivatives with the SARS-CoV-2 main protease (mpro): a molecular docking study.

Panduratin A (Pa-A) is a prenylated cyclohexenyl chalcone isolated from the rhizomes of the medicinal and culinary plant Boesenbergia rotunda (L.) Mansf., commonly called fingerroots. Both an ethanolic plant extract and Pa-A have shown a marked antiviral activity against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), responsible for the COVID-19 pandemic disease. Pa-A functions as a protease inhibitor inhibiting infection of human cells by the virus. We have modeled the interaction of Pa-A, and 26 panduratin analogues with the main protease (Mpro) of SARS-CoV-2 using molecular docking. The natural product 4-hydroxypanduratin showed a higher Mpro binding capacity than Pa-A and isopanduratin A. The interaction with MPro of all known panduratin derivatives (Pa-A to Pa-Y) have been compared, together with more than 60 reference products. Three compounds emerged as potential robust MPro binders: Pa-R, Pa-V, Pa-S, with a binding capacity significantly higher than 4-OH-Pa-A and Pa-A. The empirical energy of interaction (ΔE) calculated with the best compound in the panduratin series, Pa-R bound to Mpro, surpassed that measured with the top reference protease inhibitors such a ruprintrivir, lufotrelvir, and glecaprevir. Structure-binding relationships are discussed. Compounds with a flavanone moiety (PA-R/S) are the best binders, better than those with a chromene unit (Pa-F/G). The extended molecules (such as Pa-V) exhibit good Mpro binding, but the dimeric compound Pa-Y is too long and protrudes outside the binding cavity. The work provides novel ideas to guide the design of new molecules interacting with Mpro.Communicated by Ramaswamy H. Sarma.

Panduratin A is the main bioactive molecule in extracts of the medicinal plant Boesenbergia rotunda.Extracts of B. rotunda and Pa-A have shown activity against the virus SARS-CoV-2.We modeled the interaction of 27 panduratin derivatives with the main protease (M^pro) of the virus.Three molecules (Pa-R/V/S) revealed high M^pro binding capacity compared to reference compounds.Structure­binding relationships are discussed, to guide the design of compounds to treat COVID-19.

Anti-Inflammatory Activity of Panduratin A against LPS-Induced Microglial Activation.

Uncontrolled and excessive microglial activation is known to contribute to inflammation-mediated neurodegeneration. Therefore, reducing neurotoxic microglial activation may serve as a new approach to preventing neurodegeneration. Here, we investigated the anti-inflammatory effects of panduratin A against microglial activation induced by lipopolysaccharides (LPS) in the SIMA9 microglial cell line. We initially examined the anti-inflammatory properties of panduratin A by measuring LPS-induced nitric oxide (NO) production and the levels of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6). Panduratin A significantly reduced NO levels and pro-inflammatory cytokines' production and secretion. In addition, panduratin A enhanced the production of anti-inflammatory cytokines IL-4 and IL-10. The anti-inflammatory effects of panduratin A are related to the suppression of the NF-κB signaling pathway. Together, these results demonstrate the anti-inflammatory properties of panduratin A against LPS-induced microglial activation, suggesting panduratin A has the potential to be further developed as a new agent for the prevention of neuroinflammation-associated neurodegenerative diseases.

Toward the use of Boesenbergia rotunda extracts and the chalcone panduratin A to treat periodontitis.

Novel affordable medications are needed to treat chronic periodontitis, which is one of the most common dental pathologies worldwide. Extracts prepared from the rhizome of the medicinal plant Boesenbergia rotunda (L.) Mansf., commonly known as fingerroot, are used to treat a variety of human pathologies. These extracts contain potent anti-inflammatory compounds, including the chalcone derivative panduratin A (Pa-A), which is the lead compound of a series of analogues, designated panduratins A to Y. The anti-inflammatory properties of the extracts of B. rotunda and the most abundant bioactive products found in these extracts (including Pa-A, 4-hydroxyoanduratin, isopanduratin, and others) have been reviewed. A standardized extract of the plant has promising utility in the treatment of gingival inflammation. The effects are characterized by three actions: (i) a direct antimicrobial effect against fungi and oral pathogens such as Porphyromonas gingivalis, (ii) a marked anti-inflammatory effect via a reduced production of mediators, like prostaglandin E2 and different interleukins, and (iii) a dual bone-preserving effect, with a reduction in bone resorption and an increase in bone formation. Acting as a protease inhibitor, Pa-A is one of the main active ingredients of the extract, implicated in these actions. A Pa-A-standardized extract of B. rotunda has been used in humans for treating dyspepsia. The product is safe and well-tolerated. The development of panduratin-containing dental products, for the prevention and treatment of periodontitis, has been proposed. The structural analogues, Pa-A to-Y, should also be investigated for the treatment of dental inflammation.

Nephroprotective potential of Panduratin A against colistin-induced renal injury via attenuating mitochondrial dysfunction and cell apoptosis.

Colistin is a last-resort polypeptide antibiotic widely used to treat against multidrug-resistant Gram-negative bacterial infections. However, this treatment is associated with nephrotoxicity. The aim of this study was to examine the potential protective effect of panduratin A, a bioactive compound of Boesenbergia rotunda, on colistin-induced nephrotoxicity in both in vivo and in vitro models. Intraperitoneal injection of 15 mg/kg colistin for 7 days markedly promoted renal tubular degeneration, increased blood urea nitrogen (BUN) levels, and upregulated the expression of renal injury biomarker and apoptosis proteins. In addition, treatment with colistin increased oxidative stress and apoptosis in mice kidney tissues. Interestingly, these defects were attenuated when co-administered of colistin with panduratin A (2.5 or 25 mg/kg). The underlying mechanisms of panduratin A attenuating colistin toxicity was investigated in human renal proximal tubular cells (RPTEC/TERT1). The mechanisms by which colistin-triggered cytotoxicity was determined by analysis of cell death, reactive oxygen species (ROS) levels, mitochondria function as well as the expression of proteins related to apoptosis pathway. Colistin treatment (200 µg/ml) significantly increased cell apoptosis, elevated ROS production, reduced mitochondrial membrane potential, and decreased anti-apoptotic protein (Bcl-2) expression. These effects were notably suppressed by co-treatment with panduratin A (5 µM). Collectively, panduratin A exerts as a novel nephroprotective agent to protect against colistin-induced renal injury by attenuating mitochondrial damage and renal cell apoptosis.

Estimation of an Appropriate Human Dose of Boesenbergia pandurata Extracts Based on Allometric Scaling Data of Panduratin A in Mice, Rats, and Dogs.

It can be difficult to identify health/functional foods that exert therapeutic benefits for alleviating gingivitis and periodontitis. Recently, extracts of Boesenbergia pandurata (Roxb.), which is a tropical plant, have shown promising inhibitory activity against lipopolysaccharide-induced periodontitis. As a result, a clinical trial is being planned to assess utility of B. pandurata (Roxb.) extracts for promoting oral health; this study was designed to determine an appropriate human dose of the extracts for the trial. Pharmacokinetic studies of panduratin A, which is an active substance in fingerroot, were carried out in mice, rats, and dogs after oral administration of the extracts. The clearance data for each species were used to estimate clearance in humans through allometric scaling based on the maximum lifespan potential, and a daily dose providing sufficient anti-periodontitis activity was estimated for use in the clinical trial. The findings indicated that allometric scaling is a reasonable approach that is relatively free of safety issues and can be used to determine doses of substances for incorporation into health/functional foods appropriate for humans.

Panduratin A induces protective autophagy in melanoma via the AMPK and mTOR pathway.

BACKGROUND: Targeting autophagy is emerging as a promising strategy in cancer therapeutics in recent years. Autophagy can be modulated to drive cancer cell deaths that are notoriously resistant to apoptotic-inducing drugs. In addition, autophagy has been implicated as a prosurvival mechanism in mediating cancer chemoresistance. Our previous study has demonstrated that Panduratin A (PA), a plant-derived active compound exploits ER-stress-mediated apoptosis as its cytotoxic mechanism on melanoma. PURPOSE: Our previous proteomics analysis revealed that treatment with PA resulted in the upregulation of an autophagy marker, LC3B in melanoma cells. Therefore, the present study sought to investigate the role of PA-induced autophagy in melanoma cells. METHODS: Transmission electron microscopy was performed for examination of autophagic ultra-structures in PA-treated A375 cells. Cytoplasmic LC3B and p62/SQSMT1 punctate structures were detected using immunofluorescene staining. Expression levels of LC3B II, p62/SQSMT1, ATG 12, Beclin 1, phospho S6 (ser235/236), phospho AMPK (Thr172) and cleaved PARP were evaluated by western blotting. RESULTS: Autophagosomes, autolysosomes and punctuates of LC3 proteins could be observed in PA-treated A375 cells. PA-induced autophagy in A375 melanoma cells was found to be mediated through the inhibition of mTOR signaling and activation of AMPK pathway. Furthermore, we showed that PA-induced apoptosis was increased in the presence of an autophagy inhibitor, signifying the cytoprotective effect of PA-induced autophagy in melanoma cells. CONCLUSION: Taken together, results from the present study suggest that the inhibition of autophagy by targeting mTOR and AMPK could potentiate the cytotoxicity effects of PA on melanoma cells.

Hepatoprotectivity of Panduratin A against liver damage: In vivo demonstration with a rat model of cirrhosis induced by thioacetamide.

This experiment evaluated Panduratin A (PA), a chalcone isolated from Boesenbergia rotunda rhizomes, for its hepatoprotectivity. Rats were subjected to liver damage induced by intra-peritoneal injection of thioacetamide (TAA). PA was tested first for its acute toxicity and then administered by oral gavage at doses 5, 10, and 50 mg/kg to rats. At the end of the 8th week, livers from all rats were excised and evaluated ex vivo. Measurements included alkaline phosphatase (AP), alanine transaminase (ALT), aspartate transaminase (AST) and gamma-glutamyl transferase (GGT), serum platelet-derived growth factor (PDGF) and transforming growth factor (TGF-ß1), and hepatic metalloproteinase enzyme (MMP-2) and its inhibitor extracellular matrix protein (TIMP-1). Oxidative stress was measured by liver malondialdehyde (MDA) and nitrotyrosine levels, urinary 8-hydroxy 2- deoxyguanosine (8-OH-dG), and hepatic antioxidant enzyme activities. The immunohistochemistry of TGF-ß1 was additionally performed. PA revealed safe dose of 250 mg/kg on experimental rats and positive effect on the liver. The results suggested reduced hepatic stellate cells (HSCs) activity as verified from the attenuation of serum PDGF and TGF-ß1, hepatic MMP-2 and TIMP-1, and oxidative stress. The extensive data altogether conclude that PA treatment could protect the liver from the progression of cirrhosis through a possible mechanism inhibiting HSCs activity.

Inhibitory Effects of Panduratin A on Periodontitis-Induced Inflammation and Osteoclastogenesis through Inhibition of MAPK Pathways In Vitro.

Periodontitis is an inflammatory disease caused by microbial lipopolysaccharide (LPS), destroying gingival tissues and alveolar bone in the periodontium. In the present study, we evaluated the anti-inflammatory and anti-osteoclastic effects of panduratin A, a chalcone compound isolated from Boesenbergia pandurata, in human gingival fibroblast-1 (HGF-1) and RAW 264.7 cells. Treatment of panduratin A to LPS-stimulated HGF-1 significantly reduced the expression of interleukin-1ß and nuclear factor-kappa B (NF-κB), subsequently leading to the inhibition of matrix metalloproteinase-2 (MMP-2) and MMP-8 compared with that in the LPS control (**p < 0.01). These anti-inflammatory responses were mediated by suppressing the mitogen-activated protein kinase (MAPK) signaling and activator protein-1 complex formation pathways. Moreover, receptor activator of NF-κB ligand (RANKL)-stimulated RAW 264.7 cells treated with panduratin A showed significant inhibition of osteoclastic transcription factors such as nuclear factor of activated T-cells c1 and c-Fos as well as osteoclastic enzymes such as tartrate-resistant acid phosphatase and cathepsin K compared with those in the RANKL control (**p < 0.01). Similar to HGF-1, panduratin A suppressed osteoclastogenesis by controlling MAPK signaling pathways. Taken together, these results suggest that panduratin A could be a potential candidate for development as a natural anti-periodontitis agent.

Inhibitory Effects of Standardized Boesenbergia pandurata Extract and Its Active Compound Panduratin A on Lipopolysaccharide-Induced Periodontal Inflammation and Alveolar Bone Loss in Rats.

Periodontitis, an inflammatory disease of the gingival tissue, triggered by microbial-derived elements, such as lipopolysaccharide (LPS), collapses the periodontal tissues and resorbs the alveolar bone. This study evaluated the inhibitory effects of standardized Boesenbergia pandurata extract (BPE) and panduratin A (PAN) on periodontitis-induced inflammation and alveolar bone loss. Sprague-Dawley rats with LPS-induced periodontitis were orally administered BPE (50 and 200 mg/kg/day) and PAN (20 mg/kg/day) for 8 days. Histological analysis revealed that BPE- and PAN-administered groups showed decreased cell infiltration and alveolar bone resorption. Furthermore, the BPE and PAN significantly alleviated the mRNA and protein expression levels of nuclear factor kappa B (NF-κB), interleukin-1ß, matrix metalloproteinase (MMP)-2, and MMP-8. BPE and PAN also inhibited the expression of nuclear factor of activated T cells, cytoplasmic 1, c-Fos, and ostoclastogenesis-related enzymes, including cathepsin K and tartrate-resistant acid phosphatase (ALP). BPE and PAN not only upregulated the osteoblastogenesis-associated markers, such as collagen type I (COL1A1) and ALP, but also increased the ratio of osteoprotegerin to receptor activator of NF-κB ligand. Collectively, BPE and PAN efficiently prevent destruction of periodontal tissues and stimulating the loss of alveolar bone tissues, strongly indicative of their potential as natural antiperiodontitis agents.

Panduratin A Inhibits Cell Proliferation by Inducing G0/G1 Phase Cell Cycle Arrest and Induces Apoptosis in Breast Cancer Cells.

Because of the unsatisfactory treatment options for breast cancer (BC), there is a need to develop novel therapeutic approaches for this malignancy. One such strategy is chemotherapy using non-toxic dietary substances and botanical products. Studies have shown that Panduratin A (PA) possesses many health benefits, including anti-inflammatory, anti-bacterial, anti-oxidant and anti-cancer activities. In the present study, we provide evidence that PA treatment of MCF-7 BC cells resulted in a time- and dose-dependent inhibition of cell growth with an IC50 of 15 µM and no to little effect on normal human MCF-10A breast cells. To define the mechanism of these anti-proliferative effects of PA, we determined its effect critical molecular events known to regulate the cell cycle and apoptotic machinery. Immunofluorescence and flow cytometric analysis of Annexin V-FITC staining provided evidence for the induction of apoptosis. PA treatment of BC cells resulted in increased activity/expression of mitochondrial cytochrome C, caspases 7, 8 and 9 with a significant increase in the Bax:Bcl-2 ratio, suggesting the involvement of a mitochondrial-dependent apoptotic pathway. Furthermore, cell cycle analysis using flow cytometry showed that PA treatment of cells resulted in G0/G1 arrest in a dose-dependent manner. Immunoblot analysis data revealed that, in MCF-7 cell lines, PA treatment resulted in the dose-dependent (i) induction of p21WAF1/Cip1 and p27Kip1, (ii) downregulation of Cyclin dependent kinase (CDK) 4 and (iii) decrease in cyclin D1. These findings suggest that PA may be an effective therapeutic agent against BC.