In Silico Study of the Potential of Brazilein Sappan Wood as a Beta-Lactamase Inhibitor against Extended-Spectrum Beta-Lactamase-Encoding Genes.

Background: Infectious illnesses are a serious health concern in Indonesia. Widespread use of self-medication by the community increases the risk of developing multi-drug resistant (MDR) bacteria. This study assessed the potential of sappan wood as an inhibitor of extended-spectrum beta-lactamase (ESBL) encoded by blaSHV, blaTEM and blaCTX-M genes. Method: In silico testing was conducted to develop an effective and economical starting strategy. Thereby, this study significantly advances the development of novel treatments to combat antibiotic resistance. Using clavulanic acid as the benchmark medicine, the potency of the beta-lactamase inhibitor brazilein was predicted. Using the Molegro Virtual Docker computer tool, docking was performed to estimate the chemical and physical properties of the compounds, as well as the biological activity of brazilein toward the required receptor. The receptors used were SHV-1 beta-lactamase, PDB code: 2H0T; TEM-1 beta-lactamase, PDB code: 4OQG and CTX-M-14 beta-lactamase, PDB code: 6VHS. Data analysis was performed by comparing the binding energies of the docking results between the ligands and the target receptor. The more stable the bond that formed between the ligand and the target receptor, the lower the bond energy. Results: The in silico test results on the blaSHV gene were as follows: binding energy of ligand MA4_400[A] = -100.699, brazilein = -82.206, clavulanic acid = -79.3704; in the blaTEM gene: ligand bond energy 2UL_301[B] = -107.681, brazilein = -82.0296, clavulanic acid = -103.3; in the blaCTX-M gene: X57_301[A] ligand bond energy = -86.6197, and brazilein = -88.1586, clavulanic acid = -101.933. Conclusion: The findings of this study demonstrate the significant potential of brazilein sappan wood to block the beta-lactamase activity of blaCTX-M.

Designed complexes combining brazilein and brazilin with betanidin for dye-sensitized solar cell application: DFT and TD-DFT study.

Dye-sensitized solar cells (DSSCs) are promising third-generation photovoltaic cell technology owing to their easy fabrication, flexibility and better performance under diffuse light conditions. Natural pigment sensitizers are abundantly available and environmentally friendliness. However, narrow absorption spectra of natural pigments result in low efficiencies of the DSSCs. Therefore, combining two or more pigments with complementary absorption spectra is considered an appropriate method to broaden the absorption band and boost efficiency. This study reports three complex molecules: brazilin-betanidin-oxane (Braz-Bd-oxane), brazilin-betanidin-ether (Braz-Bd-ether) and brazilein-betanidin-ether (Braze-Bd-ether), obtained from the etherification and bi-etherification reactions of brazilin dye and brazilein dye with betanidin dye. The equilibrium geometrical structure properties, frontier molecular orbital, electrostatic surface potential, reorganization energy, chemical reactivities, and non-linear optical properties of the studied dyes were investigated using density functional theory (DFT)/B3LYP methods, with 6-31+G(d,p) basis sets and LANL2DZ for light atom and heavy atoms respectively. The optical-electronic properties were calculated using TD-DFT/B3LYP/6-31+G(d,p) for isolated dye and TD-DFT/CAM-B3LYP/6-31G(d,p)/LANL2DZ for dyes@(TiO2)9H4. The results reveal that spectra for Braz-Bd-oxane and Braze-Bd-ether complexes red-shifted compared to the individually selected dyes. The simulated absorption spectra of the adsorbed dyes on (TiO2)9H4 are red-shifted compared to the free dye. Moreover, Braz-Bd-oxane and Braz-Bd-ether exhibit better charge transfer and photovoltaic properties than the selected natural dyes forming these complexes. Based on the dyes' optoelectronic properties and photovoltaic properties, the designed molecules Braz-Bd-oxane and Braze-Bd-ether are considered better candidates to be used as photosensitizers in dye solar cells.

Effects of Encapsulation of Caesalpinia sappan L. with Cyclodextrins for Bovine Mastitis.

The main components of Caesalpinia sappan L. (CS) are brazilin and brazilein, which show high potential in pharmacologic applications. However, these have been drastically limited by the poor water solubility and stability. The present study investigates the formation of inclusion complexes F1, F2, and F3 between CS and ß-cyclodextrin (ßCD), hydroxypropyl-ß-cyclodextrin (HPßCD), and methyl-ß-cyclodextrin (MßCD), respectively. These complexes were characterized by Fourier transform infrared spectroscopy (FT-IR). The results showed that the highest encapsulation efficiency and loading capacity of CS extract were 44.24% and 9.67%, respectively. The solubility and stability of CS extract were significantly increased through complexation in phase solubility and stability studies. The complexes F1-F3 showed mainly significant antibacterial activities on gram-positive bacteria pathogens causing mastitis. Moreover, the expression levels of COX-2 and iNOS were significantly decreased in LPS-induced inflammatory cells at concentrations of 50 and 100 µg/mL. In addition, treatment of complex F3 (CS/MßCD) in bovine endothelial cells remarkably increased the chemokine gene expression of CXCL3 and CXCL8, which were responsible for immune cell recruitment (9.92 to 11.17 and 8.23 to 9.51-fold relative to that of the LPS-treated group, respectively). This study provides a complete characterization of inclusion complexes between CS extract and ßCD, HPßCD, and MßCD for the first time, highlighting the impact of complex formation on the pharmacologic activities of bovine mastitis.

Brazilein inhibits epithelial-mesenchymal transition (EMT) and programmed death ligand 1 (PD-L1) expression in breast cancer cells.

Triple-negative breast cancer (TNBC) exhibits high levels of Epithelial-mesenchymal transition (EMT) and Programmed death ligand 1 (PD-L1) expression, which promotes immune escape and metastasis. Brazilein is a natural compound extracted from Caesalpinia sappan L., and has been demonstrated to be an anti-inflammatory anti- proliferative and apoptosis-inducer in various cancer cells. Here, we investigated the effect of brazilein on EMT and PD-L1 expression in breast cancer cells and its related molecular mechanisms using MCF-7 and MDA-MB-231 cells as a model. Since the AKT, NF-κB, and GSK3ß/ß-catenin signaling were reported to be important mechanisms in immune escape and metastasis, the effect of brazilein on these signaling pathways were also found out in our study. Firstly, brazilein was treated on breast cancer cells at various concentrations to study cell viability, apoptosis, and apoptosis proteins. Then, breast cancer cells were treated with non-toxic concentrations of brazilein to study its influence on EMT and expression of PD-L1 protein using MTT, flow cytometry, western blot, and wound healing analysis, respectively. We found that brazilein exerts an anti-cancer effect by reducing cell viability via induction of apoptosis, while it also downregulated EMT and PD-L1 through suppression of phosphorylation of AKT, NF-κB, and GSK3ß/ß-catenin. Moreover, the migration ability was diminished by inhibiting the activation of MMP-9 and MMP-2. Taken together, brazilein might delay cancer progression through inhibition of EMT, PD-L1, and metastasis suggesting it might be a potential therapeutic option in breast cancer patients having a high level of EMT and PD-L1.

Protective Effects of Caesalpinia sappan Linn. and Its Bioactive Compounds on Cardiovascular Organs.

Cardiovascular diseases are the leading cause of death worldwide. The long-term aim of cardiovascular disease therapy is to reduce the mortality rate and decelerate the progression of cardiovascular organ damage. Current therapies focus on recovering heart function and reducing risk factors such as hyperglycemia and dyslipidemia. However, oxidative stress and inflammation are important causes of further damage to cardiovascular organs. Caesalpinia sappan Linn. (Fabaceae), a flowering tree native to tropical Asia, has antioxidant and anti-inflammatory properties. It is used as a natural dye to color food and beverages and as a traditional treatment for diarrhea, diabetes, and blood stasis. The phytochemical compounds in C. sappan, mainly the homoisoflavonoids brazilin, sappanone A, protosappanin, and hematoxylin, can potentially be used to protect cardiovascular organs. This review aims to provide updates on recent developments in research on C. sappan in relation to treatment of cardiovascular diseases. Many studies have reported protective effects of the plant's bioactive compounds that reduce cardiac damage and enhance vasorelaxation. For example, brazilin and sappanone A have an impact on molecular and cellular changes in cardiovascular disease pathogenesis, mainly by modulating oxidative, inflammatory, and apoptotic signaling pathways. Therefore, bioactive compounds of C. sappan have the potential to be developed as therapeutic agents to combat cardiovascular diseases like myocardial infarction and vascular disease. This review could help further the understanding of the possible modulatory role of the compounds in cardiovascular diseases, thereby facilitating future studies.

Working goal of Brazilein sappan wood as a candidate for SARS-coV-2 antivirus drug against spike (S) glycoprotein, papain-like proteinase, and main protease: In silico study.

Brazilein sappan wood, played by Spike (S) glycoprotein, Papain-Like proteinase (PLpro), and Main protease (Mpro), is expected to be a candidate for the antiviral drug SARS-CoV-2, which can inhibit viral attachment to the human body, replication, and transcription processes. The aim of this study was to predict in silico, using the comparative drug hydroxychloroquine, the working goal of brazilein sappan wood as a candidate for the antiviral drug SARS-CoV-2 against protein S, PLpro, and Mpro. The approach used is the in silico docking test using the computer program Molegro Virtual Docker. Receptor used by protein S, Protein Data Bank (PDB) code: 6M0J, NAG_601[E] ligand; PLpro, PDB code: 7JIT, Y95_501[A] ligand; and Mpro, PDB code: 1WOF, I12_1145[A] ligand. Data analysis was carried out by comparing the docking bond energies between the ligands at the target receptor. Silico test results for protein S: ligand bond energy NAG_601 [E] = -59.4555, brazilein = -71.5537, hydroxychloroquine = -79.3704; PLpro protein: Ligand bond energy Y95_501 [A] = -129.561, brazilein = -94.9761, hydroxychloroquine = -100.984; Mpro protein: Ligand bond energy I12 1145 [A] = -141.135, brazilein = -96.6169, hydroxychloroquine = -104.88. The above test results indicate that brazilein sappan wood has potential as a SARS-CoV-2 drug candidate, has a stable bond, and that the biological activity of the compound is stronger against S protein than the proteins of PLpro and Mpro.

Effects of brazilein on PSD-95 protein expression and neurological recovery in mice after sciatic nerve injury.

PURPOSE: To evaluate the local nerve myelin recovery and the expression of PSD-95 protein and mRNA in the L4-L6 segment of the spinal cord after applying Brazilein to sciatic nerve injury BALB/c mice model and investigate the regulatory effects of Brazilein on myelin recovery after peripheral nerve injury. METHODS: A total of 160 BALB/c mice were selected to establish the unilateral sciatic nerve injury model and randomly divided into four groups: saline blank control, Brazilein high-dose, medium-dose, and low-dose. Mice were assessed at different time points (1 w, 2 w, 4 w, 8 w) after sciatic nerve injury for the sciatic functional index (SFI) and sciatic nerve function recovery of the injured side by myelin Luxol Fast Blue (LFB) staining of the sciatic nerve. In addition, immunohistochemistry, real time-PCR, and Western blot were used to detect the PSD-95 expression in the spinal cord L4-L6 segments of the injured sciatic nerve at each time point. RESULTS: The results of SFI and sciatic nerve function recovery, as well as, myelin LFB staining of the injured side indicated that all indexes of the Brazilein middle- and high-dose groups were significantly better than the low-dose and blank control groups at each time point. The PSD-95 expression in the L4-L6 segment of the spinal cord was statistically lower in the high- and medium-dose groups than in the low-dose and blank control groups at 1 w, 2 w, and 4 w, while the differences between the groups were not significant at 8 w. CONCLUSION: Brazilein inhibits PSD-95 activation in the corresponding segment of sciatic nerve spinal cord in BALB/c mice after sciatic nerve injury, thereby inhibiting the excessive expression of free radicals and promoting myelin regeneration.

[Preparation of brazilein from Caesalpinia sappan by high performance countercurrent chromatography].

Brazilein is among the main chemical constituents of Caesalpinia sappan. It has diverse pharmacological activities. Modern pharmacological studies have shown that the compound has antitumor, anti-inflammatory, antibacterial, antioxidant, immunomodulatory, and other pharmacological activities. Brazilein is often used as a stain in various industries. The separation of brazilein by traditional column chromatography will not only result in contamination of the chromatographic column materials, but also lead to loss of the active ingredient. Countercurrent chromatography is an advanced liquid-liquid chromatographic separation technique. It has been widely used for natural product separation and isolation as it offers several advantages, such as low solvent consumption, a highly selective solvent system, and high recoveries. Typical countercurrent chromatography techniques include centrifugal partition chromatography (CPC), high-speed countercurrent chromatography (HSCCC), and high performance countercurrent chromatography (HPCCC). It is well known that choosing a suitable solvent system is vital in countercurrent separation. Therefore, two methods were introduced for choosing a suitable solvent system. One is the generally useful estimation of solvent systems (GUESS) method, which employs thin-layer chromatography (TLC) to identify a suitable solvent system with minimal labor for the rapid purification of target compounds, and another is the Shake-Flash method. The solvent system could be determined by observing the distribution of the sample in the upper and lower phases. Two kinds of solvent systems were screened using the TLC-GUESS and Shake-Flash methods, and tested through the analysis mode of the HPCCC instrument. The results showed that chloroform-methanol-water (4:3:2, v/v/v) was the optimal solvent system for HPCCC separation. A total of 15.2 mg of brazilein and 5.7 mg of caesappanin C were obtained from an ethyl acetate extract with high purities (95.6% and 89.0%, analyzed by HPLC) in one step using the preparation mode of HPCCC, the reversed-phase liquid chromatography mode with the apparatus rotated at 1600 r/min, a flow rate of 10 mL/min, separation temperature of 25℃, and detection wavelength of 285 nm. Their structures were determined by spectroscopic and spectrometric analyses. Brazilein stained the solid packing material in the column and was difficult to elute. The results showed that the use of HPCCC for the separation of brazilein can not only prevent the loss of target active ingredients in Caesalpinia sappan, but also shorten the separation and purification times and improve the operating efficiency. Therefore, HPCCC can be used for the separation and preparation of other pigment compounds in Caesalpinia sappan and other dye plants.

RETRACTED: Brazilein induces apoptosis and G1/G0 phase cell cycle arrest by up-regulation of miR-133a in human vestibular schwannoma cells.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Editor-in-Chief. Given the comments of Dr Elisabeth Bik regarding this article "... the Western blot bands in all 400+ papers are all very regularly spaced and have a smooth appearance in the shape of a dumbbell or tadpole, without any of the usual smudges or stains. All bands are placed on similar looking backgrounds, suggesting they were copy/pasted from other sources, or computer generated", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request and therefore the Editor-in-Chief decided to retract the article.

Two Active Compounds from Caesalpinia sappan L. in Combination with Cisplatin Synergistically Induce Apoptosis and Cell Cycle Arrest on WiDr Cells.

Purpose: The aim of this study is to observe the synergistic effect of two active compounds of secang, brazilin and brazilein, combined with cisplatin on WiDr colon cancer cells. Methods: Cytotoxic activities of brazilin (Bi) and brazilein (Be) in single and in combination with cisplatin (Cisp) were examined by MTT assay. Synergistic effect was analyzed by combination index (CI) parameter. Apoptosis and cell cycle profiles were observed by using flow cytometry. Results: The result of MTT assay showed that IC50 value of brazilin and brazilein on WiDr cancer cells were 41 µM and 52 µM respectively. The combination of ½ IC50 of Bi-Cisp reduced cells viability up to 64% and showed synergistic effect with CI value less than 1 (CI = 0.8). The combinations of ½ IC50 of Be-Cisp also reduced cells viability up to 78% and showed synergistic effect (CI=0.65). Combination of Bi-Cisp and Be-Cisp induced apoptosis higher than the single treatments. Further analysis on the cell cycle progression showed that single treatment of ½ IC50 of Be and Bi induced S-phase and G2/M-phase accumulation, while combination of Be-Cisp and Bi-Cisp enhanced S-phase accumulation. Conclusion: Both combination of Bi-Cisp and Be-Cisp showed synergistic effect on WiDr cells through induction of apoptosis and halted the cell cycle progression, thus, WiDr cells growth were significantly reduced.

Brazilein inhibits neuronal inflammation induced by cerebral ischemia and oxygen-glucose deprivation through targeting NOD2 expression.

Brazilein is reported to have immunosuppressive effect on cardiovascular and cerebral-vascular diseases. The essential roles of innate immunity in cerebral ischemia are increasingly identified, but no studies concerning the influence of brazilein on the innate immunity receptors have been reported. The present study was designed to investigate the regulation of NOD2 (Nucleotide-binding oligomerization domain-containing protein 2) by brazilein for its protection of neuron in cerebral ischemia in vivo and oxygen-glucose deprivation in vitro. The results showed that brazilein could reverse the elevated expression of NOD2 and TNFα (tumor necrosis factor alpha) elicited by cerebral ischemia and reperfusion. This reduction could also be detected in normal mice and C17.2 cells, indicating that this suppressive effect of brazilein was correlated with NOD2. The results from GFP reporter plasmid assay suggested brazilein inhibited NOD2 gene transcription. In conclusion, brazilein could attenuate NOD2 and TNFα expression in cerebral ischemia and NOD2 may be one possible target of brazilein for its immune suppressive effect in neuro-inflammation.

Reproductive toxicity of brazilein in ICR mice.

Brazilein is an active small molecular compound extracted from Caesalpinia sappan L. with favorable pharmacological properties on immune system, cardiovascular system, and nervous system. C. sappan has been used as a traditional medicine in China for hundreds of years for various diseases. However, the general reproductive toxicity of brazilein is still unknown. The purpose of the present study was to thoroughly evaluate the general reproductive toxicity of brazilein in ICR mice to support the future drug development and modernization of this potent traditional Chinese medicine. The results showed that, although no apparent toxicity on the reproducibility of the male was observed, brazilein might cause considerable risks to the fetuses and females as indicated by the ratios of dead fetuses and reabsorptions. In conclusion, our results from the present study provided some useful insights about the safety profile of brazilein, suggesting that brazilein should be used with caution in pregnant women.

Brazilein Suppresses Inflammation through Inactivation of IRAK4-NF-κB Pathway in LPS-Induced Raw264.7 Macrophage Cells.

The medicinal herbal plant has been commonly used for prevention and intervention of disease and health promotions worldwide. Brazilein is a bioactive compound extracted from Caesalpinia sappan Linn. Several studies have showed that brazilein exhibited the immune suppressive effect and anti-oxidative function. However, the molecular targets of brazilein for inflammation prevention have remained elusive. Here, we investigated the mechanism underlying the inhibitory effect of brazilein on LPS-induced inflammatory response in Raw264.7 macrophage cells. We demonstrated that brazilein decreased the expression of IRAK4 protein led to the suppression of MAPK signaling and IKKß, and subsequent inactivation of NF-κB and COX2 thus promoting the expression of the downstream target pro-inflammatory cytokines such as IL-1ß, MCP-1, MIP-2, and IL-6 in LPS-induced Raw264.7 macrophage cells. Moreover, we observed that brazilein reduced the production of nitrite compared to the control in LPS-induced Raw264.7. Thus, we suggest that brazilein might be a useful bioactive compound for the prevention of IRAK-NF-κB pathway associated chronic diseases.

Inhibitory effect of brazilein on tyrosinase and melanin synthesis: Kinetics and in silico approach.

In our present study, the inhibitory effect of brazilein from Caesalpinia sappan on tyrosinase activity was investigated through multi-spectroscopic and molecular docking techniques. The result has shown that brazilein reversibly inhibited tyrosinase in a mixed type manner. The interaction of brazilein with the amino acid residues of tyrosinase has been validated through fluorescence quenching studies. Copper interaction studies suggested that brazilein could bind with copper ions of the enzyme. Circular dichroism analysis confirmed that brazilein induced secondary structural changes in tyrosinase. Docking studies further authenticate that brazilein forms hydrophobic and hydrogen bonding with the active site residues of tyrosinase. Moreover, in vitro studies confirmed that the inhibitory mechanism of cellular tyrosinase and melanin synthesis by brazilein in B16F0 melanoma cells. These results suggested that brazilein act as a potential tyrosinase inhibitor and it would contribute as a of novel tyrosinase inhibitor in food, cosmetic and pharmaceutical industry.

Brazilwood, sappanwood, brazilin and the red dye brazilein: from textile dyeing and folk medicine to biological staining and musical instruments.

Brazilin is a nearly colorless dye precursor obtained from the heartwood of several species of trees including brazilwood from Brazil, sappanwood from Asia and the Pacific islands, and to a minor extent from two other species in Central America, northern South America and the Caribbean islands. Its use as a dyeing agent and medicinal in Asia was recorded in the 2(nd) century BC, but was little known in Europe until the 12(th) century AD. Asian supplies were replaced in the 16(th) century AD after the Portuguese discovered vast quantities of trees in what is now Brazil. Overexploitation decimated the brazilwood population to the extent that it never fully recovered. Extensive environmental efforts currently are underway to re-create a viable, sustainable population. Brazilin is structurally similar to the better known hematoxylin, thus is readily oxidized to a colored dye, brazilein, which behaves like hematein. Attachment of the dye to fabric is by hydrogen bonding or in conjunction with certain metallic mordants by coordinative bonding. For histology, most staining procedures involve aluminum (brazalum) for staining nuclei. In addition to textile dyeing and histological staining, brazilin and brazilein have been and still are used extensively in Asian folk medicine to treat a wide variety of disorders. Recent pharmacological studies for the most part have established a scientific basis for these uses and in many cases have elucidated the biochemical pathways involved. The principal use of brazilwood today is for the manufacture of bows for violins and other stringed musical instruments. The dye and other physical properties of the wood combine to produce bows of unsurpassed tonal quality.

Brazilein, a compound isolated from Caesalpinia sappan Linn., induced growth inhibition in breast cancer cells via involvement of GSK-3ß/ß-Catenin/cyclin D1 pathway.

Caesalpinia sappan Linn. has long been used in traditional medicine in China. Here, the anticancer activity of brazilein, a compound isolated from C. sappan Linn. was investigated. MTT assay showed that the IC50 value of brazilein against human breast cancer MCF-7 cells was 7.23 ± 0.24 µmol/L. PI staining and flow cytometry analysis indicated that brazilein caused cell cycle arrest in G1 phase. Western blot and RT-PCR assay demonstrated that cyclin D1, a key factor of the G1 to S phase progression, was downregulated in a concentration-dependent manner by brazilein treatment. Further Western blot and RNA interference assay showed that brazilein treatment activated GSK-3ß and following reduced ß-Catenin protein, which accounted for the downregulation of cyclin D1 and blockage of cell cycle at G1 phase. Together, all these results illustrated that brazilein induced growth inhibition of breast cancer cells and downregulation of GSK-3ß/ß-Catenin pathway was involved in its action mechanism.