Ethanol-assisted kneading of apigenin with arginine for enhanced dissolution rate of apigenin: development of rapidly disintegrating tablets.

Apigenin is a natural flavonoid which is claimed to have many pharmacological activities ranging from simple anti-inflammatory to anticancer action. However, poor dissolution slowed the advancement of this drug through the development pipelines. The objective of this work was to probe ethanol-aided kneading of apigenin with arginine as a new strategy for enhanced dissolution rate. The work was extended to develop rapidly disintegrating tablets of apigenin. Apigenin was mixed with increasing molar ratios of arginine before ethanol-aided kneading. The resulting products were examined using Fourier transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction in addition to probing the dissolution characteristics of apigenin. The analytical techniques highlighted the existence of new crystalline species with a possibility of salt formation. The recorded alterations in the crystalline properties were associated with a significant enhancement in the dissolution rate of apigenin. The presence of arginine did not have any negative effect of the cytotoxic power of apigenin. Optimum formulation was successfully prepared as rapidly disintegrating tablets which showed fast liberation of apigenin. The study introduced arginine as a potential excipient for enhanced dissolution of apigenin after ethanol-assisted kneading.

Synthesis, pharmacological evaluation and mechanistic study of scutellarin methyl ester -4'-dipeptide conjugates for the treatment of hypoxic-ischemic encephalopathy (HIE) in rat pups.

A series of novel scutellarin methyl ester-4'-dipeptide conjugates exhibiting active transport characteristics and protection against pathological damage caused by hypoxic-ischemic encephalopathy (HIE) were successfully designed and synthesized. The physiochemical properties of the obtained compounds, as well as the Caco-2 cell-based permeability and uptake into hPepT1-MDCK cells were evaluated using various analytical methods. Scutellarin methyl ester-4'-Val-homo-Leu dipeptide (5k) was determined as the optimal candidate with a high apparent permeability coefficient (Papp A to B) of 1.95 ± 0.24 × 10-6 cm/s, low ER (Papp BL to AP/Papp AP to BL) of 0.52 in Caco-2 cells, and high uptake of 25.47 µmol/mg/min in hPepT1-MDCK cells. Comprehensive mechanistic studies demonstrated that pre-treatment of PC12 cells with 5k resulted in more potent anti-oxidative activity, which was manifested by a significant decrease in the malondialdehyde (MDA) and reactive oxygen species (ROS) levels, attenuation of the H2O2-induced apoptotic cell accumulation in the sub-G1 peak, and improvement in the expression of the relevant apoptotic proteins (Bcl-2, Bax, and cleave-caspase-3). Moreover, evaluation of in vivo neuroprotective characteristics in hypoxic-ischemic rat pups revealed that 5k significantly reduced infarction and alleviated the related pathomorphological damage. The compound was also shown to ameliorate the neurological deficit at 48 h as well as to decrease the brain tissue loss at 4 weeks. Conjugate 5k was demonstrated to reduce the amyloid precursor protein (APP) and ß-site APP-converting enzyme-1 (BACE-1) expression. Pharmacokinetic characterization of 5k indicated favorable druggability and pharmacokinetic properties. The conducted docking studies revealed optimal binding of 5k to PepT1. Hydrogen bonding as well as cation-π interactions with the corresponding amino acid residues in the target active site were clearly observed. The obtained results suggest 5k as a potential candidate for anti-HIE therapy, which merits further investigation.

Apigenin-mediated Alterations in Viability and Senescence of SW480 Colorectal Cancer Cells Persist in The Presence of L-thyroxine.

INTRODUCTION: Deregulation of Thyroid Hormones (THs) system in Colorectal Cancer (CRC) suggests that these hormones may play roles in CRC pathogenesis. Flavonoids are polyphenolic compounds, which possess potent antitumor activities and interfere, albeit some of them, with all aspects of THs physiology. Whether the antitumor actions of flavonoids are affected by THs is unknown. Therefore, we investigated the effects of apigenin (Api), a well-known flavone, on some tumorigenic properties of SW480 CRC cells in the presence and absence of L-thyroxine (T4). METHODS: Cell viability was assessed by MTT assay. Flow cytometry and DNA electrophoresis were used to evaluate cell death. Cell senescence was examined by in situ detection of ß-galactosidase activity. Protein expression was assessed by antibody array technique. RESULTS: While T4 had minimal effects, Api reduced cell growth and senescence by induction of apoptosis. Expression of anti-apoptotic and pro-apoptotic proteins were differentially affected by Api and T4. Survivin, HSP60 and HTRA were the most expressed proteins by the cells. Almost all Api-induced effects persisted in the presence of T4. CONCLUSION: These data suggest that Api may inhibit CRC cell growth and progression through induction of apoptosis rather than cell necrosis or senescence. In addition, they suggest that T4 has minimal effects on CRC cell growth, and is not able to antagonize the anti-growth effects of Api. Regardless of the treatments, cells expressed high levels of survivin, HSP60 and HTRA, indicating that these proteins may play central roles in SW480 CRC cell immortality.

Total synthesis of scutellarin and apigenin 7-O-ß-d-glucuronide.

A general protocol for direct glucuronic linkages formation featuring Au(I)-catalyzed appropriately protected glucuronyl o-alkynylbenzoate-involved glycosylation reaction, as well as a concise approach for easy access of scutellarein prominent for the mild and efficient hydroxyl group installation via borylation-oxidation sequence from flavanone derivative, has been established, based on which a novel route for scutellarin derivatives preparation has been devised. The developed strategies, among which the stepwise deprotection process was also included, guarantee the high whole synthetic efficiency, and definitely will find broad application in diversity-oriented synthesis of bioactive flavonoid glycosides.

Synthesis and Biological Evaluation of Scutellarein Alkyl Derivatives as Preventing Neurodegenerative Agents with Improved Lipid Soluble Properties.

BACKGROUND: Exogenous antioxidants are considered as a promising therapeutic approach to treat neurodegenerative diseases since they could prevent and/or minimize the neuronal damage by oxidation. OBJECTIVE: Three series of lipophilic compounds structurally based on scutellarein (2), which is one metabolite of scutellarin (1) in vivo, have been designed and synthesized. METHODS: Their antioxidant activity was evaluated by detecting the 2-thiobarbituric acid reactive substance (TBARS) produced in the ferrous salt/ascorbate-induced autoxidation of lipids, which were present in microsomal membranes of rat hepatocytes. The lipophilicity of these compounds indicated as partition coefficient between n-octanol and buffer was investigated by ultraviolet (UV) spectrophotometer. RESULTS: This study indicated that compound 5e which had a benzyl group substituted at the C4'- OH position showed a potent antioxidant activity and good lipophilicity. CONCLUSION: 5e could be an effective candidate for preventing or reducing the oxidative status associated with the neurodegenerative processes.

Synthesis of scutellarein derivatives with antiproliferative activity and selectivity through the intrinsic pathway.

To explore antitumor agents with potent efficacy and low toxicity, scutellarein derivatives with benzoic acid mustard (10a-c, 11a-c and 13a-c) were designed and synthesized. The antiproliferative activities of the target derivatives against A549, MCF-7 and Bel-7402 cancer cell lines were tested. Compound 10a showed the strongest potency with an IC50 value of 1.50 µM against MCF-7 cell line, and displayed low toxicity against human liver L-O2 normal cells (IC50 > 50 µM), showing specificity between normal and malignant cells. The mechanism studies revealed that 10a could induce apoptosis in MCF-7 cells, arrest MCF-7 cell cycle at the G1 phase and cause mitochondrial dysfunction in a concentration-dependant manner. Furthermore, the enhanced expression of the pro-apoptotic proteins caspase-9, caspase-3, Bax and cytochrome c, and the reduced expression of the anti-apoptotic protein Bcl-2 confirmed that 10a induced the intrinsic apoptosis pathway in MCF-7 cells. The potent antiproliferative activity and good selectivity guaranteed 10a a lead compound for the further development into anticancer therapeutics, especially for breast cancer.

Synthesis of Scutellarein Derivatives with a Long Aliphatic Chain and Their Biological Evaluation against Human Cancer Cells.

Scutellarin is the major active flavonoid extracted from the traditional Chinese herbal medicine Erigeron breviscapus (Vant.) Hand-Mazz., which is widely used in China. Recently, accumulating evidence has highlighted the potential role of scutellarin and its main metabolite scutellarein in the treatment of cancer. To explore novel anticancer agents with high efficiency, a series of new scutellarein derivatives with a long aliphatic chain were synthesized, and the antiproliferative activities against Jurkat, HCT-116 and MDA-MB-231 cancer cell lines were assessed. Among them, compound 6a exhibited the strongest antiproliferative effects on Jurkat (IC50 = 1.80 µM), HCT-116 (IC50 = 11.50 µM) and MDA-MB-231 (IC50 = 53.91 µM). In particular, 6a even showed stronger antiproliferative effects than the positive control NaAsO2 on Jurkat and HCT-116 cell lines. The results showed that a proper long aliphatic chain enhanced the antiproliferative activity of scutellarein.

Novel vitexin-inspired scaffold against leukemia.

Acute lymphoblastic leukemia (ALL) is the most common type of leukemia in children. Up to a quarter of ALL patients relapse and face poor prognosis. To identify new compound leads, we conducted a phenotypic screen using terrestrial natural product (NP) fractions against immortalized ALL cellular models. We identified vitexin, a flavonoid, as a promising hit with biological activity (EC50 = 30 µM) in pre-B cell ALL models with no toxicity against normal human tissue (BJ cells) at the tested concentrations. To develop more potent compounds against ALL and elucidate its potential mode of action, a vitexin-inspired compound library was synthesized. Thus, we developed an improved and scalable protocol for the direct synthesis of 4-quinolone core heterocycles containing an N-sulfonamide using a one-pot condensation reaction protocol. The newly generated compounds represent a novel molecular scaffold against ALL as exemplified by compounds 13 and 15, which demonstrated EC50 values in the low micromolar range (0.3-10 µM) with little to no toxicity in normal cellular models. Computational studies support the hypothesis that these compounds are potential CDK inhibitors. The compounds induced apoptosis, caused cell arrest at G0/G1 and G2/M, and induced ROS in cancer cells.

Scaffold Hopping Strategy for the Design, Synthesis and Biological Activity Evaluation of Novel Hexacyclic Scutellarein Derivatives with a 1,3-Oxazine Ring Fused at A-ring.

BACKGROUND: Discovery of novel agents with anticoagulant and antioxidant activity is very important to treat cerebrovascular disease. Lead compound LR3d discovered in our laboratory exhibited stronger anticoagulant ability and good antioxidant activity, compared with scutellarein (2), which is the major in vivo active metabolite of the natural product scutellarin (1). OBJECTIVE: Design and synthesis novel scutellarein derivatives with improved anticoagulant and antioxidant activity. METHODS: By utilizing a scaffold hopping strategy on LR3d, we describe the design and synthesis of a series of novel hexacyclic scutellarein derivatives 4 with a 1,3-oxazine ring fused at positions 7 and 8 in A ring. The thrombin inhibitory activities of all these new compounds were studied by the analysis of thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen (FIB). The antioxidant abilities of these analogs were evaluated by using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method through 1,1- diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay. RESULTS: Nine new hexacyclic scutellarein derivatives with a 1,3-oxazine ring fused at A-ring were synthesized, the results of the biological activity evaluation showed that compound 4e exhibited stronger anticoagulant and antioxidant ability compared to LR3d. CONCLUSION: 4e could be used for further development to treat ischemic cerebrovascular disease.

Synthesis and Bioactivity Characterization of Scutellarein Sulfonated Derivative.

Scutellarin (1) has been widely used to treat acute cerebral infarction in clinic, but poor aqueous solubility decreases its bioavailability. Interestingly, scutellarin (1) could be metabolized into scutellarein (2) in vivo. In this study, a sulfonic group was introduced at position C-8 of scutellarein (2) to enhance the aqueous solubility of the obtained derivative (3). DPPH (1,1-diphenyl-2-picrylhydrazyl)-radical scavenging ability and antithrombic activity were also conducted to determine its bioactivity. The result showed that scutellarein derivate (3) could be a better agent for ischemic cerebrovascular disease treatment.

Scutellarin derivatives as apoptosis inducers: Design, synthesis and biological evaluation.

To explore novel antitumor agents with high efficiency and low toxicity, a series of NO-donating scutellarin derivatives (14-17) were synthesized and the antiproliferative activities against MCF-7, HCT-116, PC-3 and HepG2 cancer cell lines were assessed. Among them, compound 14b was the strongest with IC50 values of 2.96 µM, 7.25 µM, 0.09 µM and 0.50 µM, respectively, and displayed low toxicity against normal human liver L-O2 cells with an IC50 of 47.96 µM, showing good selectivity between normal and malignant liver cells. Moreover, NO releasing ability of the derivatives has been studied. Mechanism studies of the most promising compounds 14b and 15a were carried out. The results indicated that 14b and 15a could induce apoptosis, cell cycle arrest at the S phase and led to mitochondrial dysfunction in the HepG2 and PC-3 cell lines, respectively. Furthermore, Human Apoptosis Protein Array kit assay demonstrated that 14b could induce apoptosis through down-regulating the levels of procaspase-3 and inhibiting the expression of survivin, c-IAP1, HSP27, HSP60, HSP70, HO-1/HMOX1/HSP32 and HO-2/HMOX2 in HepG2 cell line. These results guaranteed compound 14b to be a drug candidate against liver cancer for further investigation.

Preparation of inclusion complex of apigenin-hydroxypropyl-ß-cyclodextrin by using supercritical antisolvent process for dissolution and bioavailability enhancement.

In this study, an inclusion complex of apigenin (AP)-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was prepared via supercritical antisolvent (SAS) method using N, N-dimethylformamide (DMF) as solvent and carbon dioxide as antisolvent. The mole ratio of AP and HP-ß-CD (1:1) was established by phase solubility equilibrium experiment. The optimal conditions were determined through single-factor experiments; these conditions included precipitation pressure of 22.5MPa, precipitation temperature of 50°C, and AP concentration of 20mg/ml. The load efficiency and encapsulation efficiency of the AP-HP-ß-CD inclusion complex, with a mean particle size of 392.13±7.56nm, were 13.97%±0.17% and 93.22%±1.17%, respectively, under the optimal conditions. FTIR, (1)H NMR, SEM, XRD, DSC, and TG analyses were also conducted. Results showed that the inclusion complex was formed because of the interaction between AP and HP-ß-CD. DMF residue in the inclusion complex was 0.033% lower than the ICH limit for class II solvents. The solubility of the inclusion complex was approximately 152.43 times higher than that of the raw AP. In the in vitro study, the dissolution rate of the AP-HP-ß-CD inclusion complex was about 7.60 times higher than that of the raw AP. In the in vivo study, the bioavailability of the inclusion complex increased by 6.45 times compared with that of the raw AP. Hence, the prepared AP-HP-ß-CD inclusion complex exhibits potential as a new oral therapeutic agent formulation for clinical applications.

Total synthesis of 8-(6″-umbelliferyl)-apigenin and its analogs as anti-diabetic reagents.

The naturally occurring flavone 8-(6″-umbelliferyl)apigenin, a hybrid structure of apigenin and coumarin, as well as seven of its analogues were synthesized for the first time by using iodination and Suzuki coupling reactions as key steps. The synthesis of 8-(6″-umbelliferyl)-apigenin was achieved in seven linear steps from the commercially available 1-(2,4,6-trihydroxyphenyl)ethan-1-one and 7-hydroxyl coumarine with 31% overall yield. Effects of these compounds on glucose disposal were investigated in adipocytes. All of the flavonoid and coumarin hydrids were found to have better bioactivities than their corresponding flavonoid cores. The most potent compound 15 (10 µΜ) could promote glucose consumption by 57% which exhibited similar effect as the positive control metformin at 1 mM. Moreover, fluorescence microscopy showed that four 8-(6″-umbelliferyl)apigenin analogues 2, 15, 30 and 31 could promote the 2-NBDG uptake into 3T3-L1 cells, which consist with those observed in the regulation of glucose.

An Efficient Chemical Synthesis of Scutellarein: An in Vivo Metabolite of Scutellarin.

Scutellarein (2), which is an important in vivo metabolite of scutellarin (1), was synthesized from 3,4,5-trimethoxyphenol (3) in high yield in four steps. This strategy relies on acetylation, aldolization, cyclization and hydrolysis reactions, respectively.

Design, Synthesis, and Biological Evaluation of Scutellarein Derivatives Based on Scutellarin Metabolic Mechanism In Vivo.

Three series of scutellarein derivatives have been designed and synthesized based on metabolic mechanism of scutellarin (1) in vivo. Their thrombin inhibition activities were tested through the analyzation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB). The antioxidant activities of these target products were assessed by 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay and the ability to protect PC12 cells against H2 O2 -induced cytotoxicity, and their solubilities were evaluated by ultraviolet (UV) spectrophotometer. The results showed that the two isopropyl groups substituted derivative (18c) demonstrated stronger anticoagulant activity, better water solubility, and good antioxidant activity compared with scutellarein (2), which warrants further development of 18c as a promising agent for ischemic cerebrovascular disease treatment.

Synthesis of scutellarein derivatives to increase biological activity and water solubility.

In order to improve the biological activity and water solubility of scutellarin (1), some derivatives of its main metabolite (scutellarein) were designed and synthesized. All the compounds were tested for their thrombin inhibition activity through the analyzation of thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen (FIB). Their antioxidant activities were assessed by measuring their scavenging capacities toward 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and the ability to protect PC12 cells against H2O2-induced cytotoxicity, their water solubility were also assessed by ultraviolet (UV) spectrophotometer. The results showed that compound 8b demonstrated stronger anticoagulant and antioxidant activity, better water solubility compared with scutellarein (2), which warrants it as a promising agent for the treatment of ischemic cerebrovascular disease.

Design, synthesis and evaluation of scutellarein-O-alkylamines as multifunctional agents for the treatment of Alzheimer's disease.

A series of scutellarein-O-alkylamine derivatives were designed, synthesized and tested as multifunctional agents for the treatment of Alzheimer's disease (AD). The results showed that most of these compounds exhibited good multifunctional activities. Among them, compound 16d demonstrated significant metal chelating properties, moderate acetylcholinesterase (AChE) inhibitory and anti-oxidative activity, and excellent inhibitory effects on self-induced Aß(1-42) aggregation, Cu(2+)-induced Aß(1-42) aggregation, human AChE-induced Aß(1-40) aggregation and disassembled Cu(2+)-induced aggregation of the well-structured Aß(1-42) fibrils. Both kinetic analysis of AChE inhibition and molecular modeling study suggested that 16d binds simultaneously to the catalytic active site and peripheral anionic site of AChE. Moreover, compound 16d showed a good protective effect against H2O2-induced PC12 cell injury, with low toxicity in SH-SY5Y cells. Furthermore, the step-down passive avoidance test showed this compound significantly reversed scopolamine-induced memory deficit in mice. Thus, 16d was shown to be an interesting multifunctional lead compound worthy of further study.

Novel pyrano and vinylphenol adducts of deoxyanthocyanidins in sorghum sourdough.

This study determined the fate of deoxyanthocyanidins in sorghum sourdoughs. Sourdoughs prepared from the red sorghum variety Town were fermented with the caffeic acid-decarboxylating strains Lactobacillus plantarum FUA3171 and the decarboxylase negative L. casei FUA3166. Deoxyanthocyanidins were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Apigeninidin and methoxyapigeninidin were the major deoxyanthocyanidins prior to fermentation. During fermentation, novel deoxyanthocyanidins were formed. Purification by preparative LC, followed by NMR analysis and high-resolution MS identified two of the compounds as 6-deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin. To identify pathways for their formation, sorghum was fermented with single strains, L. plantarum or L. casei. 6-Deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin were formed only during fermentation with L. plantarum FUA3171, indicating a role of vinylphenol in their formation. Chemical synthesis confirmed that 6-deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin are formed from apigeninidin with vinylphenol but not with p-coumaric acid as reactants. In conclusion, the products of microbial decarboxylation of hydroxycinnamic acids convert apigeninidin and methoxyapigeninidin to pyrano-3-deoxyanthocyanidins and vinylphenol adducts.

Preparation of two flavonoid glycosides with unique structures from barley seedlings by membrane separation technology and preparative high-performance liquid chromatography.

Barley seedlings are rich in flavones that can have positive effects on people with antihypoxia and antifatigue. Lutonarin and saponarin are two major flavonoid glycosides that have unique structures in barley seedlings. This study presents a new approach for the preparation of lutonarin and saponarin from barely seedlings by membrane separation technology and preparative high-performance liquid chromatography. Preparative conditions of these two flavonoid glycosides by membrane separation technology were studied using response surface methodology. Under the optimized conditions, the total contents of these two flavonoid glycosides amounts to 17.0%.

Novel apigenin based small molecule that targets snake venom metalloproteases.

The classical antivenom therapy has appreciably reduced snakebite mortality rate and thus is the only savior drug available. Unfortunately, it considerably fails to shield the viper bite complications like hemorrhage, local tissue degradation and necrosis responsible for severe morbidity. Moreover, the therapy is also tagged with limitations including anaphylaxis, serum sickness and poor availability. Over the last decade, snake venom metalloproteases (SVMPs) are reported to be the primary component responsible for hemorrhage and tissue degradation at bitten site. Thus, antivenom inability to offset viper venom-induced local toxicity has been a basis for an insistent search for SVMP inhibitors. Here we report the inhibitory effect of compound 5d, an apigenin based molecule against SVMPs both in silico and in vivo. Several apigenin analogues are synthesized using multicomponent Ugi reactions. Among them, compound 5d effectively abrogated Echis carinatus (EC) venom-induced local hemorrhage, tissue necrosis and myotoxicity in a dose dependant fashion. The histopathological study further conferred effective inhibition of basement membrane degradation, and accumulation of inflammatory leucocytes at the site of EC venom inoculation. The compound also protected EC venom-induced fibrin and fibrinogen degradation. The molecular docking of compound 5d and bothropasin demonstrated the direct interaction of hydroxyl group of compound with Glu146 present in hydrophobic pocket of active site and does not chelate Zn2+. Hence, it is concluded that compound 5d could be a potent agent in viper bite management.