Computational Insights into the Interaction of Pinostrobin with Bcl-2 Family Proteins: A Molecular Docking Analysis.

BACKGROUND: Cancer research has emphasized the Bcl-2 family of proteins because of their interaction in apoptosis process, a critical mechanism that regulates cellular survival and death. Recently small molecules from diverse sources have gained much attention in anticancer research due to their promising inhibitory action against Bcl-2 and Bcl-XL that are pointedly known as the members of anti-apoptotic Bcl-2 family of proteins. Pinostrobin (PN) is a natural flavonoid with diverse pharmacological potential emerged as a molecule of interest as anticancer agent. The present study aims to screen the interaction of PN with anti-apoptotic protagonists Bcl-2 and Bcl- XL at the molecular level through docking studies. METHOD: The molecular docking was performed using the Schrodinger software. The docking score of PN with the Bcl-2 (4IEH) and Bcl-XL (3ZK6) and their molecular interactions was examined and analysed. RESULTS: The result of the molecular docking analysis showed that PN and the anti-apoptotic proteins 4IEH and 3ZK6 had significant interactions and docking energy scores (ΔG) were found to be -5.112 kcal/mol and -7.822 kcal/mol respectively. The small molecule PN illustrated effective interaction with the active site amino acids of the Bcl-2 and Bcl-XL proteins and has been associated through traditional hydrogen bond with 4IEH. Further, it was observed that PN and anti-apoptotic Bcl-2 proteins interaction was stabilized by other non-covalent interactions, such as π-alkyl or π-π interactions and van der Waals forces. CONCLUSIONS: This was the first study to reveal the inhibitory action of PN against anti-apoptotic Bcl-2 and Bcl-XL proteins at the molecular level. The findings of this study concludes that PN ability to inhibit anti-apoptotic proteins, Bcl-2 and Bcl-XL could be useful to induce intracellular apoptosis in tumorous cells.

Pinostrobin attenuates azoxymethane-induced colorectal cytotoxicity in rats through augmentation of apoptotic Bax/Bcl-2 proteins and antioxidants.

Objectives: Pinostrobin (5-hydroxy-7-methoxyflavanone; PN) is a natural active ingredient with numerous biological activities extensively utilized in tumour chemotherapy. The present study investigates the chemo-preventive potentials of PN on azoxymethane-mediated colonic aberrant crypt foci in rats. Methods: Sprague Dawley rats clustered into five groups, normal control (A) and cancer controls were subcutaneously injected with normal saline and 15 mg/kg azoxymethane, respectively, and nourished on 10% tween 20 and fed on 10% tween 20; reference control (C), injected with 15 mg/kg azoxymethane and injected (intraperitoneal) with 35 mg/kg 5-fluorouracil (5-FU); D and E rat groups received a subcutaneous injection of 15 mg/kg azoxymethane and nourished on 30 and 60 mg/kg of PN, respectively. Results: The acute toxicity trial showed a lack of any abnormal signs or mortality in rats ingested with 250 and 500 mg/kg of PN. The gross morphology of colon tissues revealed significantly lower total colonic aberrant crypt foci incidence in PN-treated rats than that of cancer controls. Histological examination of colon tissues showed increased aberrant crypt foci availability with bizarrely elongated nuclei, stratified cells and higher depletion of the submucosal glands in cancer controls. PN treatment caused positive modulation of apoptotic (Bax and Bcl-2) proteins and inflammatory cytokines (TNF-α, IL-6 and IL-10). Moreover, rats fed on PN had significantly higher antioxidants (superoxide dismutase) and lower malondialdehyde concentrations in their colon tissue homogenates. Conclusion: The chemoprotective efficiency of PN against azoxymethane-induced aberrant crypt foci is shown by lower aberrant crypt foci values and higher aberrant crypt foci inhibition percentage, possibly through augmentation of genes responsible for apoptotic cascade and inflammations originating from azoxymethane oxidative stress insults.

Pinostrobin, a dietary bioflavonoid exerts antioxidant, anti-inflammatory, and anti-apoptotic protective effects against methotrexate-induced ovarian toxicity in rats.

This study investigated the protective activities of pinostrobin (PIN) against methotrexate (MTX)-induced ovarian toxicity. Female rats were administered with PIN (50 mg/kg) for 4 weeks, while MTX was administered from weeks 2-4 of PIN treatment. Serum hormonal profiles, ovarian oxidative stress, inflammatory and apoptotic biomarkers as well as ovarian histomorphometry were evaluated. MTX administration elicited profound deficit in serum progesterone and estrogen (E2) levels, while luteinizing hormone (LH) and follicle stimulating hormone (FSH) were significantly increased. Additionally, MTX administration was associated with significant increases in ovarian malondialdehyde, nitric oxide, NF-кB, TNF-α, IL-6, IL-1ß, iNOS and caspase-3 activity, as well as notable reduction in the activities of glutathione peroxidase, catalase and superoxide dismutase as well as the level of glutathione. Whereas, treatment with PIN significantly decreased serum levels of FSH and LH, as well as ovarian levels of NO, MDA, caspase 3, NF-κB, IL-1ß, IL-6, TNF-α and iNOS. PIN also significantly upregulated GSH, GPx, CAT and SOD in the ovarian tissues as well as increased serum E2 and progesterone levels compared to the MTX group. Furthermore, PIN significantly restored altered ovarian histoarchitecture in the treated group. These findings suggests that PIN exerts protective effects against MTX-triggered ovarian damages.

Pinostrobin modulates FOXO3 expression, nuclear localization, and exerts antileukemic effects in AML cells and zebrafish xenografts.

Acute myeloid leukemia (AML) is a disease characterized by abnormal cell proliferation in the bone marrow and is the most common quickly progressive leukemia in adults. Pinostrobin, a flavonoid phytochemical, has been reported to exhibit antioxidant, anti-inflammatory, and anticancer properties. In this study, we aimed to investigate the antileukemic effects of pinostrobin and its molecular mechanisms in human AML cells. Our study found that pinostrobin (0-80 µM) significantly reduced the viability of human AML cells, with the pronounced cytotoxic effects observed in MV4-11 > MOLM-13 > HL-60 > U-937 > THP-1 cells. Pinostrobin was found to suppress leukemia cell proliferation, modulate cell cycle progression, promote cell apoptosis, and induce monocytic differentiation in MV4-11 cells. In animal studies, pinostrobin significantly suppressed the growth of leukemia cells in a zebrafish xenograft model. Microarray-based transcriptome analysis showed that the differentially expressed genes (DEGs) in pinostrobin-treated cells were strongly associated with enriched Gene Ontology (GO) terms related to apoptotic process, cell death, cell differentiation, cell cycle progression, and cell division. Combining DisGeNET and STRING database analysis revealed that pinostrobin upregulates forkhead box 3 (FOXO3), a tumor suppressor in cancer development, and plays an essential role in controlling AML cell viability. Our study demonstrated that pinostrobin increases FOXO3 gene expression and promotes its nuclear translocation, leading to the inhibition of cell growth. Finally, the study found that pinostrobin, when combined with cytarabine, synergistically reduces the viability of AML cells. Our current findings shed light on pinostrobin's mechanisms in inhibiting leukemia cell growth, highlighting its potential as a chemotherapeutic agent or nutraceutical supplement for AML prevention or treatment.

In vitro study of pinostrobin propionate and pinostrobin butyrate: Cytotoxic activity against breast cancer cell T47D and its selectivity index.

Backgrounds: Pinostrobin has the potential activity as an anticancer. However, its activity is still lower than the anticancer drugs on the market. To increase its activity, pinostrobin derivatives have been synthesized, namely pinostrobin propionate and pinostrobin butyrate, which are predicted to have better activity and lower toxicity than pinostrobin after being tested by in silico approach. So the compound deserves to be tested for its anticancer activity and selectivity on normal cells. Objective: This study aims to determine the anticancer activity of pinostrobin propionate and pinostrobin butyrate against the T47D breast cancer cell line and its selectivity against the Vero cell line. Methods: The cytotoxicity test which is anticancer activity test and its selectivity on normal cell were carried out using the MTT(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The cells used were breast cancer cell line T47D and normal Vero cells. The test results were analyzed using a microplate reader with a wavelength of 570 nm. Results: From the analysis of anticancer activity on T47D cells, the IC50 values of pinostrobin, pinostrobin propionate, and pinostrobin butyrate were 2.93, 0.57, and 0.40 mM, respectively. While the results of the cytotoxicity test on Vero cells obtained the CC50 value of pinostrobin, pinostrobin propionate, pinostrobin butyrate was 1.27, 0.94, and 0.89 mM, respectively. So the SI value of pinostrobin (SI=0.4) is smaller than its derivatives (SI=1.7 and 2.2). Meanwhile, pinostrobin butyrate is more selective than pinostrobin propionate. Conclusions: It can be concluded that pinostrobin propionate and pinostrobin butyrate compounds have greater activity and selectivity than pinostrobin so these compounds are promising to be further developed as anticancer candidates.

Pinostrobin and Tectochrysin Conquer Multidrug-Resistant Cancer Cells via Inhibiting P-Glycoprotein ATPase.

Enhanced drug efflux through ATP-binding cassette transporters, particularly P-glycoprotein (P-gp), is a key mechanism underlying multidrug resistance (MDR). In the present study, we investigated the inhibitory effects of pinostrobin and tectochrysin on P-gp in MDR cancer cells and the underlying mechanisms. Fluorescence substrate efflux assays, multidrug resistance 1 (MDR1) shift assays, P-gp ATPase activity assays, Western blotting, and docking simulation were performed. The potential of the test compounds for MDR reversal and the associated molecular mechanisms were investigated through cell viability assay, cell cycle analysis, apoptosis assay, and further determining the combination index. Results demonstrated that pinostrobin and tectochrysin were not the substrates of P-gp, nor did they affect the expression of this transporter. Both compounds noncompetitively inhibited the efflux of rhodamine 123 and doxorubicin through P-gp. Furthermore, they resensitized MDR cancer cells to chemotherapeutic drugs, such as vincristine, paclitaxel, and docetaxel; thus, they exhibited strong MDR reversal effects. Our findings indicate that pinostrobin and tectochrysin are effective P-gp inhibitors and promising candidates for resensitizing MDR cancer cells.

Alleviative effects of pinostrobin against cadmium-induced renal toxicity in rats by reducing oxidative stress, apoptosis, inflammation, and mitochondrial dysfunction.

Introduction: Cadmium (Cd) is a highly toxic heavy metal that can be found everywhere in the environment and can have harmful effects on both human and animal health. Pinostrobin (PSB) is a bioactive natural flavonoid isolated from Boesenbergia rotunda with several pharmacological properties, such as antiinflammatory, anticancer, antioxidant, and antiviral. This investigation was intended to assess the therapeutic potential of PSB against Cd-induced kidney damage in rats. Methods: In total, 48 Sprague Dawley rats were divided into four groups: a control, a Cd (5 mg/kg), a Cd + PSB group (5 mg/kg Cd and 10 mg/kg PSB), and a PSB group (10 mg/kg) that received supplementation for 30 days. Results: Exposure to Cd led to a decrease in the activities of catalase (CAT), glutathione reductase (GSR), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX), whereas levels of reactive oxygen species (ROS) and malondialdehyde (MDA) increased. Cd exposure also caused a substantial increase in urea, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and creatinine levels. Moreover, a noticeable decline was noticed in creatinine clearance. Moreover, Cd exposure considerably increased the levels of inflammatory indices, including interleukin-1b (IL-1b), tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), nuclear factor kappa-B (NF-kB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) activity. Cd treatment decreased the expression of the antiapoptotic markers (Bcl-2) while increasing the expression of apoptotic markers (Bax and Caspase-3). Furthermore, Cd treatment substantially reduced the TCA cycle enzyme activity, such as alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, and isocitrate dehydrogenase. Moreover, mitochondrial electron transport chain enzymes, succinatedehydrogenase, NADH dehydrogenase, cytochrome c-oxidase, and coenzyme Q-cytochrome reductase activities were also decreased following Cd exposure. PSB administration substantially reduced the mitochondrial membrane potential while inducing significant histological damage. However, PSB treatment significantly reduced Cd-mediated renal damage in rats. Conclusion: Thus, the present investigation discovered that PSB has ameliorative potential against Cd-induced renal dysfunction in rats.

Pinostrobin induces acute leukemia cell apoptosis via the regulation of miR-410-5p and SFRP5.

AIMS: This study attempted to explore the mechanisms involved in pinostrobin (PN)-mediated acute leukemia cell apoptosis regulated by miR-410-5p. MATERIAL AND METHODS: NB4 and MOLT-4 cells were cultured and treated with PN at the IC50 concentration. Apoptosis was examined by Annexin V-FITC/PI staining. RT-qPCR was used to measure the expression of caspase-3, BAK, BCL-W, and MCL-1. The target protein of PN was identified using LC-MS/MS followed by bioinformatic analysis. TargetScan, DIANA, and miRDB were used for the prediction of miRNAs involved in the PN-induced apoptosis mechanism. miRNA mimic transfection, RT-qPCR, and western blot analysis were performed to evaluate the regulatory effect of miRNA on its target and the involvement of miRNA in apoptosis induction by PN. In addition, the synergistic effect of PN and daunorubicin (DNR) were investigated by using the MTT assay. KEY FINDINGS: The results showed that PN reduced cell viability and induced apoptosis in both leukemia cell lines. From the LC-MS/MS and bioinformatics analysis, SFRP5 and miR-410-5p were selected as a potential PN target protein and miRNA, respectively. After miRNA mimic transfection, miR-410-5p, which is an onco-miRNA, was decreased and led to increased apoptosis in both cell lines, indicating that this miRNA is involved in PN-mediated apoptosis mechanisms. Moreover, PN demonstrated a synergistic effect with DNR, suggesting that PN may be used in combination with conventional chemotherapy drugs. SIGNIFICANCE: PN regulates the expression of miR-410-5p and SFRP5 to promote apoptosis in acute leukemia cells. It could be developed as an alternative treatment for leukemia in the future.

Pinostrobin alleviates testicular and spermatological damage induced by polystyrene microplastics in adult albino rats.

BACKGROUND: Polystyrene microplastics (PS-MPs) have become major environmental pollutants that adversely effects multiple organs specifically testicles. Pinostrobin (PN) is an important flavonoid which, shows several pharmacological potentials. PURPOSE: The current study was designed to elucidate the mitigative effects of PN against PS-MPs induced testicular toxicities in rats. METHODS: 48 male albino rats were randomly distributed into 4 groups, control, PS-MPs group (0.01 mg/kg), PS-MPs + PN group (0.01 mg/kg of PS-MPs; 40 mg/kg of PN) and PN group (40 mg/kg). RESULTS: PS-MPs intoxication substantially lessened the activities of glutathione peroxidase (GPx), glutathione reductase (GSR), superoxide dismutase (SOD) along with catalase (CAT) while, raised the level of malondialdehyde (MDA) as well as reactive oxygen species (ROS). Additionally, PS-MPs reduced luteinizing hormone (LH), plasma testosterone, follicle-stimulating hormone (FSH) concentration, sperm motility, sperm count, expression of steroidogenic enzymes and Bcl-2 (anti-apoptotic protein) along with the count of spermatogenic cells. While, dead sperm count, sperm abnormalities (tail, neck and head), Bax and caspase-3 (apoptotic proteins) expression along with histopathological anomalies were elevated. Moreover, PS-MPs exposure increased the level of inflammatory markers. However, PN treatment considerably decreased oxidative stress (OS) by reducing ROS as well as increased sperm motility and alleviated all the damages induced by the PS-MPs. CONCLUSION: Therefore, it is concluded that PN may prove a potential therapeutic candidate to restore all the PS-MPs-induced testicular toxicities.

Pinostrobin alleviates chronic restraint stress­induced cognitive impairment by modulating oxidative stress and the function of astrocytes in the hippocampus of rats.

Chronic stress has been recognized to induce the alterations of neuronal and glial cells in the hippocampus, and is thus implicated in cognitive dysfunction. There is increasing evidence to indicate that natural compounds capable of exerting neuroprotective and antioxidant activities, may function as potential therapeutic agents for cognitive impairment. The present study examined the neuroprotective effects of pinostrobin from Boesenbergia rotunda (L.) against chronic restraint stress (CRS)-induced cognitive impairment associated with the alterations of oxidative stress, neuronal density and glial fibrillary acidic protein (GFAP) of astrocytes in the hippocampus. For this purpose, male Wistar rats were administered once daily with pinostrobin (20 and 40 mg/kg, per os) prior to exposure to CRS (6 h/day) for 21 days. The cognitive behaviors, the concentration of malondialdehyde, and the activities of superoxide dismutase and catalase were determined. Histologically, the alterations in astrocytic GFAP and excitatory amino acid transporter 2 (EAAT2) in the hippocampus were examined. The results revealed that pinostrobin potentially attenuated cognitive impairment in the Y-maze and in novel object recognition tests, with a reduction in oxidative stress. Furthermore, pinostrobin effectively increased neuronal density, as well as the immunoreactivities of GFAP and EAAT2 in the hippocampus. Taken together, these findings indicate that treatment with pinostrobin alleviates chronic stress-induced cognitive impairment by exerting antioxidant effects, reducing neuronal cell damage, and improving the function of astrocytic GFAP and EAAT2. Thus, pinostrobin may have potential for use as a neuroprotective agent to protect against chronic stress-induced brain dysfunction and cognitive deficits.

Pinostrobin Suppresses the α-Melanocyte-Stimulating Hormone-Induced Melanogenic Signaling Pathway.

Pinostrobin is a dietary flavonoid found in several plants that possesses pharmacological properties, such as anti-cancer, anti-virus, antioxidant, anti-ulcer, and anti-aromatase effects. However, it is unclear if pinostrobin exerts anti-melanogenic properties and, if so, what the underlying molecular mechanisms comprise. Therefore, we, in this study, investigated whether pinostrobin inhibits melanin biosynthesis in vitro and in vivo, as well as the potential associated mechanism. Pinostrobin reduced mushroom tyrosinase activity in vitro in a concentration-dependent manner, with an IC50 of 700 µM. Molecular docking simulations further revealed that pinostrobin forms a hydrogen bond, as well as other non-covalent interactions, between the C-type lectin-like fold and polyphenol oxidase chain, rather than the previously known copper-containing catalytic center. Additionally, pinostrobin significantly decreased α-melanocyte-stimulating hormone (α-MSH)-induced extracellular and intracellular melanin production, as well as tyrosinase activity, in B16F10 melanoma cells. More specifically, pinostrobin inhibited the α-MSH-induced melanin biosynthesis signaling pathway by suppressing the cAMP-CREB-MITF axis. In fact, pinostrobin also attenuated pigmentation in α-MSH-stimulated zebrafish larvae without causing cardiotoxicity. The findings suggest that pinostrobin effectively inhibits melanogenesis in vitro and in vivo via regulation of the cAMP-CREB-MITF axis.

Hepatoprotective effect of pinostrobin against thioacetamide-induced liver cirrhosis in rats.

The study in vivo assessed the effect of pinostrobin on the histology, immunohistochemistry, and biochemical parameters of thioacetamide (TAA) induced liver cirrhosis in Sprague Dawley rats. The rats were noticeably gavaged with two doses of pinostrobin (30 mg/kg and 60 mg/kg) with TAA and exhibited a substantial decrease in the liver index and hepatocyte propagation with much minor cell injury. These groups meaningfully down-regulated the proliferation of cellular nucleus antigen (PCNA) and alpha-smooth muscle actin (α-SMA). The liver homogenate displayed augmented antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT) activities escorted with reducing in malondialdehyde (MDA) level. The serum level of bilirubin, total protein, albumin, and liver enzymes (ALP, ALT, and AST) returned to normal and was similar to that of normal control and silymarin with TAA-treated groups. pinostrobin-fed groups also decreased the level of Tumor necrosis factor-alpha (TNF-α), Interleukin-6 (IL-6), and increased the level of Interleukin-10 (IL-10). Acute toxicity with a higher dose of 500 mg/kg of pinostrobin did not manifest any toxicological signs in rats. The hepatoprotective effect of pinostrobin could be due to potentially inhibited the progression of liver cirrhosis, down-regulation of PCNA and α-SMA proliferation, prevented oxidation of hepatocytes, improved SOD and CAT enzymes, condensed MDA, repairs of liver biomarkers, reduced cellular inflammation and modulation of inflammatory cytokines.

Bibliometric Analysis of Research on Boesenbergia rotunda, Pinostrobin, and Their Derivatives: Dominance of Southeast Asian Researchers

Pinostrobin, marker compounds from Boesenbergia rotunda with various pharmacological activities, have been studied extensively, including synthesizing its derivatives, which have potent pharmacological activities. This study aims to describe research related to B. rotunda, pinostrobin, and their derivatives. Metadata information was collected from Scopus in August 2022, with three keywords searched for article titles, abstracts, and keywords. Analysis and research mapping were carried out with VOSviewer. The most widely used synonym for the plant name was "Boesenbergia rotunda", in which Norzulaani Khalid from the University of Malaya, Malaysia, mostly reported research with the keywords "Boesenbergia rotunda", "pinostrobin", and "derivative". The majority of researchers come from institutions in Southeast Asia, such as Malaysia, Thailand, and Indonesia. Interestingly, no Chinese researchers have reported studies on this topic. The journals and publishers that publish the most articles with these three keywords are Bioorganic and Medicinal Chemistry Letters and Elsevier, respectively. This information will make it easier for researchers on this topic to find partners for collaboration and determine journals to publish their research results.

La pinostrobina, compuesto de marcadores de Boesenbergia rotunda con diversas actividades farmacológicas, se ha estudiado ampliamente, incluida la síntesis de sus derivados que tienen potentes actividades farmacológicas. Este estudio tuvo como objetivo describir investigaciones relacionadas con B. rotunda, pinostrobina y sus derivados. La información de metadatos se recopiló de Scopus en agosto de 2022, con tres palabras clave buscadas para títulos de artículos, resúmenes y palabras clave. El análisis y el mapeo de la investigación se realizaron con VOSviewer. El sinónimo más utilizado para el nombre de la planta fue "Boesenbergia rotunda", en el que Norzulaani Khalid de la Universidad de Malaya, Malasia, informó principalmente sobre investigaciones con las palabras clave "Boesenbergia rotunda", "pinostrobina" y "derivado". La mayoría de los investigadores provienen de instituciones del sudeste asiático como Malasia, Tailandia e Indonesia. Curiosamente, ningún investigador chino ha informado de estudios sobre este tema. Las revistas y editoriales que más artículos publican con estas tres palabras clave son Bioorganic and Medicinal Chemistry Letters y Elsevier. Esta información facilitará a los investigadores sobre este tema encontrar colaboraciones y determinar las revistas para publicar los resultados de sus investigaciones.

Pinostrobin mitigates neurodegeneration through an up-regulation of antioxidants and GDNF in a rat model of Parkinson's disease.

Background: One of the most common neurodegenerative diseases is Parkinson's disease (PD); PD is characterized by a reduction of neurons containing dopamine in the substantia nigra (SN), which leads to a lack of dopamine (DA) in nigrostriatal pathways, resulting in motor function disorders. Oxidative stress is considered as one of the etiologies involved in dopaminergic neuronal loss. Thus, we aimed to investigate the neuroprotective effects of pinostrobin (PB), a bioflavonoid extracted from Boesenbergia rotunda with antioxidative activity in PD. Methods: Rats were treated with 40 mg/kg of PB for seven consecutive days before and after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. After completing the experiment, the brains including SN and striatum were used for histological studies and biochemical assays. Results: PB treatment demonstrated a reduction of free radicals in the SN as indicated by significantly decreased MDA levels, whereas the antioxidative enzymes (SOD and GSH) were significantly increased. Furthermore, PB treatment significantly increased glial cell line-derived neurotrophic factor (GDNF) immunolabelling which has neurotrophic and neuroprotective effects on the survival of dopaminergic neurons. Furthermore, PB treatment was shown to protect CA1 and CA3 neurons in the hippocampus and dopaminergic neurons in the SN. DA levels in the SN were increased after PB treatment, leading to the improvement of motor function of PD rats. Conclusions: These results imply that PB prevents MPTP-induced neurotoxicity via its antioxidant activities and increases GDNF levels, which may contribute to the therapeutic strategy for PD.

Pinostrobin from Boesenbergia rotunda attenuates oxidative stress and promotes functional recovery in rat model of sciatic nerve crush injury

Oxidative stress plays a role in the delay of peripheral nerve regeneration after injury. The accumulation of free radicals results in nerve tissue damage and dorsal root ganglion (DRG) neuronal death. Pinostrobin (PB) is one of the bioflavonoids from Boesenbergia rotunda and has been reported to possess antioxidant capacity and numerous pharmacological activities. Therefore, this study aimed to investigate the effects of PB on peripheral nerve regeneration after injury. Male Wistar rats were randomly divided into 5 groups including control, sham, sciatic nerve crush injury (SNC), SNC + 20 mg/kg PB, and SNC + 40 mg/kg PB. Nerve functional recovery was observed every 7 days. At the end of the study, the sciatic nerve and the DRG were collected for histological and biochemical analyses. PB treatment at doses of 20 and 40 mg/kg reduced oxidative stress by up-regulating endogenous glutathione. The reduced oxidative stress in PB-treated rats resulted in increased axon diameters, greater number of DRG neurons, and p-ERK1/2 expression in addition to faster functional recovery within 4 weeks compared to untreated SNC rats. The results indicated that PB diminished the oxidative stress-induced nerve injury. These effects should be considered in the treatment of peripheral nerve injury.

Pinostrobin ameliorates lipopolysaccharide (LPS)-induced inflammation and endotoxemia by inhibiting LPS binding to the TLR4/MD2 complex.

Pinostrobin is a natural flavonoid with valuable pharmacological properties, including anti-cancer, anti-viral, and anti-oxidant activities. However, the anti-inflammatory effects of pinostrobin have not been well studied. In this study, we investigated whether pinostrobin attenuates lipopolysaccharide (LPS)-induced inflammation and endotoxemia. Additionally, the target molecule of pinostrobin was identified through molecular docking simulation. Pinostrobin decreased LPS-induced nitric oxide (NO) and prostaglandin E2 production, and reduced the expression of inducible NO synthase and cyclooxygenase-2. Furthermore, pinostrobin inhibited the production of proinflammatory cytokines, including interleukin-12 and tumor necrosis factor-α in LPS-stimulated RAW 264.7 macrophages accompanied by inhibiting nuclear translocation of nuclear factor-κB. The anti-inflammatory effect of pinostrobin was further confirmed in LPS-microinjected zebrafish larvae by diminishing the recruitment of macrophages and neutrophils, and proinflammatory gene expression. Moreover, LPS-microinjected zebrafish larvae showed a decrease in heart rate and an increase in mortality and abnormalities. However, pinostrobin significantly attenuated these adverse effects. Molecular docking showed that pinostrobin fits into myeloid differentiation factor (MD2) and Toll-like receptor 4 (TLR4) with no traditional hydrogen bonds (pose 1). The 2D ligand interaction diagram showed that pinostrobin forms a carbon hydrogen bond with LYS89 in MD2 and many non-covalent interactions, including π-alkyl or alkyl and van der Waals interactions, indicating that pinostrobin hinders LPS binding between MD2 and TLR4 and consequently inhibits TLR4/MD2-mediated inflammatory responses. These data suggest that pinostrobin attenuates LPS-induced inflammation and endotoxemia by binding to the TLR4/MD2 complex.

Discovery of Pinostrobin as a Melanogenic Agent in cAMP/PKA and p38 MAPK Signaling Pathway.

Melanogenesis is the process of melanin synthesis to protect the skin against ultraviolet radiation and other external stresses. The loss of skin pigmentation is closely related to depigmented skin disorders. The melanogenic effects of pinostrobin, an active flavanone found in honey, were evaluated. B16F10 cells were used for melanin content, tyrosinase activity, and the expression of melanogenesis-related markers. Moreover, computational simulations were performed to predict docking and pharmacokinetics. Pinostrobin increased melanin levels and tyrosinase activity by stimulating the expression of melanogenic regulatory factors including tyrosinase, tyrosinase-related protein (TRP) 1 and microphthalmia transcription factor (MITF). Specifically, the phosphorylation of cAMP response element binding (CREB) involved in the MITF activation was augmented by pinostrobin. Moreover, the compound upregulated the ß-catenin by cAMP/PKA-mediated GSK-3ß inactivation. Co-treatment with a PKA inhibitor, inhibited melanin production, tyrosinase activity, and expression of MITF, p-CREB, p-GSK-3ß and p-ß-catenin, demonstrating that pinostrobin-stimulated melanogenesis was closely related to cAMP/PKA signaling pathway. Furthermore, the combination of pinostrobin and a specific p38 inhibitor, showed that MITF upregulation by pinostrobin was partly associated with the p38 signaling pathway. Docking simulation exhibited that the oxygen group at C-4 and the hydroxyl group at C-5 of pinostrobin may play an essential role in melanogenesis. In silico analysis revealed that pinostrobin had the optimal pharmacokinetic profiles including gastrointestinal absorption, skin permeability, and inhibition of cytochrome (CYP) enzymes. From the present results, it might be suggested that pinostrobin could be useful as a potent and safe melanogenic agent in the depigmentation disorder, vitiligo.

Study of the dermal anti-inflammatory, antioxidant, and analgesic activity of pinostrobin.

Pinostrobin is a flavanone isolated from Renealmia alpinia (Rottb.) Maas, which is used to treat painful diseases and ailments; indigenous peoples use it as plasters. Different plant species have been reported as a source of this flavonoid, among which are: Boesenbergia rotunda, Cajanus cajan, Piper ecuadorense, Piper hispidum, Teloxys graveolens, Kaempferia pandurata, among others. Pinostrobin expresses potentially useful biological activities such as antioxidant, analgesic, and dermal anti-inflammatory, at low levels nonetheless due to its low solubility. The formation of inclusion complexes deems a good strategy to improve the pharmacologic effects of many substances. In the present work, we evaluated the dermal toxicity, analgesic and dermal anti-inflammatory activity of pinostrobin included in cyclodextrins, to improve those effects on experimental animals. To include pinostrobin, we used two of beta cyclodextrin (ßCD) and hydroxypropil beta cyclodextrin (HPßCD) complexes using two methods developed by Benesi-Hildebrand and Higuchi-Connors. Dermal anti-inflammatory activity was evaluated in experimental mice by inhibiting the edema generated by 12-O-tetradecanoylforbol-13-acetate (TPA). Analgesic activity was evaluated by inducing chemical pain by means of a Siegmund test. Antioxidant activities were measured with two in vitro tests. Analgesic and dermal anti-inflammatory activities of pinostrobin, as included in control and experimental complexes, showed comparatively better effects than pinostrobin without inclusion complexes. Our results indicate that both beta cyclodextrin (ßCD) and hydroxypropyl beta cyclodextrin (HPßCD) enhance the different effects of pinostrobin, which may indicate greater bioavailability.

In silico analyses of major active constituents of fingerroot (Boesenbergia rotunda) unveils inhibitory activities against SARS-CoV-2 main protease enzyme.

Boesenbergia rotunda (L.) Mansf., commonly known as fingerroot is a perennial herb in the Zingiberaceae family with anticancer, anti-leptospiral, anti-inflammatory, antioxidant, antiulcer, and anti-herpes viral activities. While the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) inhibitory activity of B. rotunda extract has been recently found, the active compounds contributing to this activity are yet unknown. The main protease (Mpro) enzyme is one of the most well established therapeutic targets among coronaviruses which plays a vital role in the maturation and cleavage of polyproteins during viral replication. The current work aims to identify active phytochemical substances from B. rotunda extract that can inhibit the replication of SARS-CoV-2 by using a combined molecular docking and dynamic simulation approaches. The virtual screening experiment revealed that fifteen molecules out of twenty-three major active compounds in the plant extract have acceptable drug-like characteristics. Alpinetin, Pinocembrin, and Pinostrobin have binding energies of -7.51 kcal/mol, -7.21 kcal/mol, and -7.18 kcal/mol, respectively, and can suppress Mpro activity. The stability of the simulated complexes of the lead compounds with the drug-receptor is demonstrated by 100-ns MD simulations. The binding free energies study utilizing molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and molecular mechanics generalized Born surface area (MM-GBSA) show that the compounds and Mpro enzyme have favourable thermodynamic interactions, which are majorly driven by van der Waals forces. Thus, the selected bioactive phytochemicals from B. rotunda might be used as anti-SARS-CoV-2 candidates that target the Mpro enzyme.