H2O-Induced Hydrophobic Interactions in MS-Guided Counter-Current Chromatography Separation of Anti-Cancer Mollugin from Rubia cordifolia.

Counter-current chromatography (CCC) is a unique liquid-liquid partition chromatography and largely relies on the partition interactions of solutes and solvents in two-phase solvents. Usually, the two-phase solvents used in CCC include a lipophilic organic phase and a hydrophilic aqueous phase. Although a large number of partition interactions have been found and used in the CCC separations, there are few studies that address the role of water on solvents and solutes in the two-phase partition. In this study, we presented a new insight that H2O (water) might be an efficient and sensible hydrophobic agent in the n-hexane-methanol-based two-phase partition and CCC separation of lipophilic compounds, i.e., anti-cancer component mollugin from Rubia cordifolia. Although the n-hexane-methanol-based four components solvent systems of n-hexane-ethyl acetate-methanol-water (HEMWat) is one of the most popular CCC solvent systems and widely used for natural products isolation, this is an interesting trial to investigate the water roles in the two-phase solutions. In addition, as an example, the bioactive component mollugin was targeted, separated, and purified by MS-guided CCC with hexane-methanol and minor water as a hydrophobic agent. It might be useful for isolation and purification of lipophilic mollugin and other bioactive compounds complex natural products and traditional Chinese medicines.

A new pyrano coumarin from Clausena excavata roots displaying dual inhibition against α-glucosidase and free radical.

A new pyrano coumarin, identified as excavatin A (1) together with two known compounds nordentatin (2) and binorpocitrin (3) was isolated from the 95% EtOH extract of Clausena excavata. All structures were elucidated by using spectroscopy methods such as extensive NMR and HR-FAB-MS spectrometry. All the isolated compounds were tested on antidiabetes activity by using α-glucosidase inhibition assay and the antioxidant activity by DPPH assay. Compounds 1-3 showed antioxidant activity with IC50 values 0.286, 0.02, 0.278 mM. Among them, 2 exhibited inhibition activity against maltase (IC50 5.45 µM) and sucrase (IC50 43.57 µM). However, compounds (1) and (3) displayed inhibition on yeast α-glucosidase with IC50 values 1.92 and 5.58 mM.[Figure: see text].

Isolation of bioactive components with soluble epoxide hydrolase inhibitory activity from Stachys sieboldii MiQ. by ultrasonic-assisted extraction optimized using response surface methodology.

Stachys sieboldii MiQ (SSM) is an important food and medicinal herb in Korea, used to improve memory of patients with senile dementia and cardiovascular diseases. However, little information on bioactive components from SSM or standardized extraction methods for these components is available. This study isolated and purified major components from SSM for the first time, and assessed their ability to inhibit soluble epoxide hydrolase (sEH). The results showed that acteoside is the most potent inhibitor of sEH, with an IC50 of 33.5 ± 0.5 µM. Additional active components, including harpagide, tryptophan, and 8-acetate-harpagide, along with acteoside, were tentatively identified using high-performance liquid chromatography photodiode array tandem mass spectrometry (HPLC-PDA-MS/MS) and quantified using an ultraviolet detector at 210 nm. Further, an ultrasonic-assisted extraction technique for extraction of four bioactive compounds in SSM was developed and optimized using response surface methodology (RSM). The optimal extraction conditions were: extraction time, 30.46 minutes; extraction temperature, 67.95 °C, and methanol concentration 53.85%. The prediction model of RSM was validated with laboratory experiments. The similarity between predicted and actual values was 97.84%. The extraction method is thus a rapid, environment-friendly, energy-saving method can be applied to extract bioactive components from SSM in large quantities.

Hepatoprotective species from the Chilean medicinal flora: Junellia spathulata (Verbenaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Chilean population relies on medicinal plants for treating a wide range of illnesses, especially those of the gastrointestinal system. Junellia spathulata (Gillies & Hook.) Moldenke var. spathulata (Verbenaceae), called as "verbena-azul-de-cordilleira", is a medicinal plant native to Argentina and Chile traditionally used for treating digestive disorders. Although the species of the genus are important as therapeutic resources for the Andean population, the plants are very scarcely studied. AIMS OF THE STUDY: The purpose of the present study was to find out the main constituents and investigate the protective effect of J. spathulata against oxidative stress induced by the potent oxidant 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) in human hepatoblastoma cells. MATERIALS AND METHODS: The crude methanol extract of J. spathulata and an iridoid obtained by chromatographic processes were tested to access the hepatoprotective effect and cytotoxicity in HepG2 cell. In addition, the reducing power of the samples and their ability to scavenge free radicals were evaluated using FRAP and ORAC assay systems. RESULTS: The iridoid asperuloside, the main compound of the crude methanol extract of J. spathulata, was isolated and identified by means of NMR analysis. The crude methanol extract of J. spathulata and asperuloside protected HepG2 cells against oxidative damage triggered by AAPH-derived free radicals. This effect can be credited to the ability of the extract and asperuloside to protect the liver cells from chemical-induced injury, which might be correlated to their free radical scavenging potential. CONCLUSIONS: This study experimentally evidenced the ethnopharmacological usefulness of J. spathulata as a treatment of digestive disorders. Our result could stimulate further investigations of hepatoprotective agents in other Chilean Junellia species.

Harpagide: Occurrence in plants and biological activities - A review.

In this review article, the occurrence of harpagide in the plant kingdom and its associated biological activities are presented and detailed for the first time. The presence of harpagide has been reported in several botanical families within Asteridae, and harpagide has been observed to exert a wide number of biological activities such as cytotoxic, anti-inflammatory, and neuroprotective. These results show how harpagide can be recovered from several natural sources for several pharmacological purposes even if there is a lot to still be studied. Nowadays, the interest is related to its presence in phytomedicines. Threfore, these studies are useful to support and validate the large use of several plants in the folklore medicine.

Methoxytetrahydro-2H-pyran-2-yl)methyl benzoate inhibits spinal cord injury in the rat model via PPAR-γ/PI3K/p-Akt activation.

Spinal cord injury (SCI) is the most commonly seen trauma leading to disability in people worldwide. The purpose of current study was to determine the protective effect of methoxytetrahydro-2H-pyran-2-yl)methyl benzoate (HMPB) on SCI in rat model. TUNEL staining was used to examine apoptotic changes in spinal cord of SCI rats. The ELISA kits were employed to assess inflammatory processes and oxidative factors in the spinal cord tissues. Behavioral changes in SCI rats were assessed using Basso, Beattie, and Bresnahan (BBB) scoring system. Western blotting was used for assessment of proteins. The HMPB treatment of SCI rats reduced apoptotic cell number based on the concentration of dose administered. Treatment of SCI rats with HMPB enhanced BBB score and decreased accumulation of water content in SCI rats significantly. On treatment with HMPB the TNF-α and interleukin-6/1ß/18 levels were suppressed in SCI rats. Treatment with HMPB induced excessive release of SOD, CAT, and GSH molecules and decreased overproduction of MDA. The SCI induced upregulation of caspase-3/9 activity was completely alleviated by HMPB at 2 mg/kg dose. The HMPB treatment of SCI rats promoted peroxisome proliferator-activated receptor γ (PPAR-γ) expression, reduced cyclooxygenase (COX)-2 production and increased expression of p-Akt and phosphoinositide 3-kinase (p-PI3K). The study demonstrated that HMPB suppressed apoptosis, raised BBB score and inhibited inflammation in SCI rats. Moreover, activation of PI3K/Akt in the spinal cord tissues of SCI rats was promoted by HMPB. Therefore, HMPB has protective effect on SCI in the rat model.

Harpagide from Scrophularia protects rat cortical neurons from oxygen-glucose deprivation and reoxygenation-induced injury by decreasing endoplasmic reticulum stress.

ETHNOPHARMACOLOGICAL RELEVANCE: Harpagide is the main ingredient in Scrophularia ningpoensis Hemsl which is used for the therapeutic purpose of treating encephalopathy. Harpagide has shown promise in the treatment of oxygen-glucose deprivation and reoxygenation (OGD/R)-induced brain injury. However, the underlying mechanisms remain unclear. AIM OF STUDY: In this study, we aimed to determine the neuroprotective effect of harpagide on rat cortical neurons under OGD/R conditions that induce the development of ischaemia-reperfusion (I/R). MATERIALS AND METHODS: To explore the biological function of harpagide in cerebral ischaemia-reperfusion injury (CIRI), The CIRI model was established by oxygen-glucose deprivation and reoxygenation (OGD/R) on rat cortical neurons. It tested cell survival rate by 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, apoptosis by flow cytometry, intracellular Ca2+ concentration [Ca2+] i by cofocal laser, and expressions related to endoplasmic reticulum stress (ERS) by RT-PCR and Western blot. RESULTS: We found that pretreatment with harpagide (50 µM) prevented OGD/R-induced apoptotic cell death. Harpagide also significantly decreased the gene expression levels and protein production of ERS-related proteins. We found that harpagide also exerted a neuroprotective effect on TG-induced apoptosis in rat cortical neurons and decreased the gene expression levels and protein production of GRP78, caspase-12 and CHOP. We also measured the intracellular calcium ion concentration ([Ca2+]i) in neurons and found that harpagide significantly decreased the [Ca2+]i induced by OGD/R and TG. CONCLUSION: These results suggest that harpagide protects against OGD/R-induced cell apoptosis, likely by decreasing ERS. Collectively, harpagide was demonstrated to be a prominent suppressor of ERS and prevented the apoptosis of rat cortical neurons. Based on the results, harpagide could potentially serve as a therapeutic agent of ischaemia-like injury associated with excessive ERS and apoptosis.

A new phenanthropyran and a new biphenanthrene from the rhizomes of Dioscorea septemloba and their antioxidant activities.

A new phenanthropyran, dioscorone B (1), and a new phenanthrene (2), together with seven known compounds (3-9), were isolated from the 75% ethanol extract of Dioscorea septemloba rhizomes. The chemical structures of these compounds were elucidated by comprehensive spectroscopic methods including NMR, HRESIMS, IR, and UV spectra. Compounds 1-5 were first isolated from genus Dioscorea. The proton and carbon chemical shifts of compounds 1-9 were unambiguously assigned based on the 1D-NMR and 2D-NMR data. Compounds 1-5 and 8-9 were first tested for their antioxidant activities. Compounds 1 and 2 showed excellent activities with IC50 values of 0.07 ± 0.10 µM and 0.13 ± 0.09 µM, respectively.

A novel Ursane-type triterpene saponin from Ajuga multiflora.

A phytochemical study was carried out on Ajuga multiflora, and obtained a novel ursane-type triterpene saponin, i.e., 3-epi-ursolic acid 3-O-palmitoyl 28-O-α-L-arabinofuranosyl ester (1), named ajugoside. Its structure and relative configuration were identified mainly by spectroscopic methods including 1D- and 2D-NMR techniques as well as HR-MS analysis. This paper described the isolation and structural elucidation of ajugoside (1) for the first time.

Evaluation of Oxasqualenoids from the Red Alga Laurencia viridis against Acanthamoeba.

Acanthamoeba genus is a widely distributed and opportunistic parasite with increasing importance worldwide as an emerging pathogen in the past decades. This protozoan has an active trophozoite stage, a cyst stage, and is dormant and very resistant. It can cause Acanthamoeba keratitis, an ocular sight-threatening disease, and granulomatous amoebic encephalitis, a chronic, very fatal brain pathology. In this study, the amoebicidal activity of sixteen Laurencia oxasqualenoid metabolites and semisynthetic derivatives were tested against Acanthamoeba castellanii Neff. The results obtained point out that iubol (3) and dehydrothyrsiferol (1) possess potent activities, with IC50 values of 5.30 and 12.83 µM, respectively. The hydroxylated congeners thyrsiferol (2) and 22-hydroxydehydrothyrsiferol (4), active in the same value range at IC50 13.97 and 17.00 µM, are not toxic against murine macrophages; thus, they are solid candidates for the development of new amoebicidal therapies.

Smoke-Isolated Karrikins Stimulated Tanshinones Biosynthesis in Salvia miltiorrhiza through Endogenous Nitric Oxide and Jasmonic Acid.

Although smoke-isolated karrikins (KAR1) could regulate secondary metabolism in medicinal plants, the signal transduction mechanism has not been reported. This study highlights the influence of KAR1 on tanshinone I (T-I) production in Salvia miltiorrhiza and the involved signal molecules. Results showed KAR1-induced generation of nitric oxide (NO), jasmonic acid (JA) and T-I in S. miltiorrhiza hairy root. KAR1-induced increase of T-I was suppressed by NO-specific scavenger (cPTIO) and NOS inhibitors (PBITU); JA synthesis inhibitor (SHAM) and JA synthesis inhibitor (PrGall), which indicated that NO and JA play essential roles in KAR1-induced T-I. NO inhibitors inhibited KAR1-induced generation of NO and JA, suggesting NO was located upstream of JA signal pathway. NO-induced T-I production was inhibited by SHAM and PrGall, implying JA participated in transmitting signal NO to T-I accumulation. In other words, NO mediated the KAR1-induced T-I production through a JA-dependent signaling pathway. The results helped us understand the signal transduction mechanism involved in KAR1-induced T-I production and provided helpful information for the production of S. miltiorrhiza hairy root.

Enhanced accumulation of harpagide and 8-O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures analyzed by UPLC-MS/MS.

Harpagide and its derivatives have valuable medicinal properties, such as anti-inflammatory, analgesic and potential antirheumatic effects. There is the demand for searching plant species containing these iridoids or developing biotechnological methods to obtain the compounds. The present study investigated the effects of methyl jasmonate (MeJa, 50 µM), ethephon (Eth, 50 µM) and L-phenylalanine (L-Phe, 2.4 g/L of medium), added to previously selected variant of Murashige and Skoog medium (supplemented with plant growth regulators: 6-benzylaminopurine 1.0 mg/L, α-naphthaleneacetic acid 0.5 mg/L, gibberellic acid 0.25 mg/L) on the accumulation of harpagide and 8-O-acetyl-harpagide in Melittis melissophyllum L. agitated shoot cultures. Plant material was harvested 2 and 8 days after the supplementation. Iridoids were quantitatively analyzed by the UPLC-MS/MS method in extracts from the biomass and the culture medium. It was found that all of the variants caused an increase in the accumulation of harpagide. In the biomass harvested after 2 days, the highest harpagide content of 247.3 mg/100 g DW was found for variant F (L-Phe and Eth), and the highest 8-O-acetyl-harpagide content of 138 mg/100 g DW for variant E (L-Phe and MeJa). After 8 days, in some variants, a portion of the metabolites was released into the culture medium. Considering the total amount of the compounds (in the biomass and medium), the highest accumulation of harpagide, amounting to 619 mg/100 g DW, was found in variant F, and the highest amount of 8-O-acetyl-harpagide, of 255.4 mg/100 g DW, was found in variant H (L-Phe, MeJa, Eth) when harvested on the 8th day. These amounts were, respectively, 24.7 and 4.8 times higher than in the control culture, and were, respectively, 15 and 6.7 times higher than in the leaves of the soil-grown plant. The total amount of the two iridoids was highest for variant F (0.78% DW) and variant H (0.68% DW) when harvested on the 8th day. The results indicate that the agitated shoot cultures of M. melissophyllum can be a rich source of harpagide and 8-O-acetyl-harpagide, having a potential practical application. To the best of our knowledge we present for the first time the results of the quantitative UPLC-MS/MS analysis of harpagide and 8-O-acetyl-harpagide in M. melissophyllum shoot cultures and the enhancement of their accumulation by means of medium supplementation with elicitors and precursor.

Plagiochiline A Inhibits Cytokinetic Abscission and Induces Cell Death.

We previously reported on the isolation and biological activities of plagiochiline A (1), a 2,3-secoaromadendrane-type sesquiterpenoid from the Peruvian medicinal plant, Plagiochila disticha. This compound was found to have antiproliferative effects on a variety of solid tumor cell lines, as well as several leukemia cell lines. Other researchers have also noted the cytotoxicity of plagiochiline A (isolated from different plant species), but there are no prior reports regarding the mechanism for this bioactivity. Here, we have evaluated the effects of plagiochiline A on cell cycle progression in DU145 prostate cancer cells. A cell cycle analysis indicated that plagiochiline A caused a significant increase in the percentage of cells in the G2/M phase when compared with control cells. When cells were stained and observed by fluorescence microscopy to examine progress through the mitotic phase, we found a significant increase in the proportion of cells with features of late cytokinesis (cells connected by intercellular bridges) in the plagiochiline A-treated samples. These results suggest that plagiochiline A inhibits cell division by preventing completion of cytokinesis, particularly at the final abscission stage. We also determined that plagiochiline A reduces DU145 cell survival in clonogenic assays and that it induces substantial cell death in these cells.

New alkenylated tetrahydropyran derivatives from the marine sediment-derived fungus Westerdykella dispersa and their bioactivities.

Six new alkenylated tetrahydropyran derivatives belonged to polyketides, designated as (12R,13R)-dihydroxylanomycinol (1), (12S,13S)-dihydroxylanomycinol (2), (12R,13S)-dihydroxylanomycinol (3) and (12S,13R)-dihydroxylanomycinol (4), (12S,13R)-N-acetyl-dihydroxylanomycin (5) and (12S,13S)-N-acetyl-dihydroxylanomycin (6), together with one related known compound lanomycinol (7) were isolated from the liquid cultures of Westerdykella dispersa obtained from the marine sediments. Their structures and absolute configurations were determined on the basis of extensive analysis of NMR spectra combined with comparison of NMR data to those of known compound, and computational method via calculation of the electronic circular dichroism (ECD). The anti-agricultural pathogenic fungal and antibacterial activities of all isolated compounds were evaluated. Compounds 1-7 exhibited moderate antifungal activities selectively against tested fungal strains with minimum inhibitory concentration (MIC) values ranging from 12.5 to 50µg/mL, and weak antibacterial activities selectively against tested antibacterial strains with MIC value at 100µg/mL.

Potential anti-cholinesterase and ß-site amyloid precursor protein cleaving enzyme 1 inhibitory activities of cornuside and gallotannins from Cornus officinalis fruits.

Cholinesterase (ChE) and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors are promising agents for the treatment of Alzheimer's disease (AD). In the present study, we examined the inhibitory activity of seven compounds isolated from the fruits of Cornus officinalis, cornuside, polymeric proanthocyanidins, 1,2,3-tri-O-galloyl-ß-D-glucose, 1,2,3,6-tetra-O-galloyl-ß-D-glucose, tellimagrandin I, tellimagrandin II, and isoterchebin, against acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and BACE1. All of the compounds displayed concentration-dependent in vitro inhibitory activity toward the ChEs and BACE1. Among them, tellimagrandin II exhibited the best inhibitory activity toward ChEs, whereas the best BACE1 inhibitor was 1,2,3,6-tetra-O-galloyl-ß-D-glucose. Isoterchebin and polymeric proanthocyanidins were also significant ChE inhibitors. The kinetic and docking studies demonstrated that all compounds interacted with both the catalytic active sites and the peripheral anionic sites of the ChEs and BACE1. Tellimagrandin II, isoterchebin, and the polymeric proanthocyanidins exhibited concentration-dependent inhibition of peroxynitrite-mediated protein tyrosine nitration. In conclusion, we identified significant ChE and BACE1 inhibitors from Corni Fructus that could have value as new multi-targeted compounds for anti-AD agents.

A new pyranoxanthone from Garcinia nervosa.

Phytochemical studies on the stem bark of Garcinia nervosa has resulted in the discovery of one new pyranoxanthone derivative, garner xanthone (1) and five other compounds, 1,5-dihydroxyxanthone (2), 6-deoxyisojacareubin (3), 12b-hydroxy-des-D-garcigerrin A (4) stigmasterol (5), and ß-sitosterol (6). The structures of these compounds were elucidated with the aid of spectroscopic techniques, such as NMR and MS. The crude extracts of the plant were assessed for their antimicrobial activity.

Antibacterial activity of 14, 15-dihydroajugapitin and 8-o-acetylharpagide isolated from Ajuga bracteosa Wall ex. Benth against human pathogenic bacteria.

Ajuga bracteosa Wall ex. Benth. (Lamiaceae) commonly known as Bungle Weed has been in use since ancient times and is mentioned Ayurvedic literature. The upper ground parts of the plant are used for treatment of various diseases. The weed is credited with astringent, febrifugal, stimulant, aperient, tonic, diuretic and depurative properties and is used for the treatment of gout and rheumatism, palsy and amenorrhoea. Two compounds 1) 14, 15-dihydroajugapitin and 2) 8-o-acetylharpagide were isolated from the aerial parts of the plant and tested for antibacterial activity against various pathogenic bacteria by agar well diffusion method. Compound 1 and 2 showed maximum antibacterial activity against Escherichia coli with zone of inhibitions of 25. 0 ± 1.4 mm and 22.6 ± 0.9 mm respectively. The MIC value of compound 1 and 2 ranged between 500 and 1000 µg/ml. It could be concluded that both compounds isolated from the aerial parts of Ajuga bracteosa possess antibacterial activity against pathogens.

Iridoids and phenylethanoid glycosides from the aerial parts of Ajuga tenorei, an endemic Italian species.

We report the first analysis in absolute, and in particular, concerning the phytochemical pattern, about an endemic Italian species, Ajuga tenorei C. Presl. The analysis, performed by means of techniques such as Column Chromatography and NMR spectroscopy and Mass spectrometry, led to the isolation and the identification of five compounds namely verbascoside (1), echinacoside (2), ajugoside (3), harpagide (4) and 8-O-acetylharpagide (5). The presence of these compounds is important from both chemotaxonomic and ethno-pharmacological point of view. For what concerns the first point is confirmed the correct botanical classification of the species. The isolated compounds are also known to exert peculiar pharmacological activities and their presence may give a rationale to the historical medicinal properties associated to the Ajuga genus in general, since these plants have a long traditional use in many parts of the world. Such fact might suggest the use of also this species in this sense.

HPLC-ELSD Quantification and Centrifugal Partition Chromatography Isolation of 8-O-Acetylharpagide from Oxera coronata (Lamiaceae).

INTRODUCTION: Iridoid glycosides possess highly functionalised monoterpenoid aglycon with several contiguous stereocentres. For the most common, they are often present in quantities reaching several percentage of the fresh plant weight, and thus they may be regarded as starting material for the synthesis of a number of new chiral and bioactive molecules. OBJECTIVE: To quantify and to isolate 8-O-acetylharpagide (AH) from several extracts of Oxera coronata R.P.J. de Kok, a Lamiaceae species endemic to New Caledonia, using HPLC-ELSD (evaporative light scattering detector) and centrifugal partition chromatography (CPC). METHODOLOGY: Oxera coronata produces high amounts of AH in leaves, twigs and fruits. Water and methanol extracts of these plant parts were prepared. The content of AH in each extract was quantified by HPLC-ELSD, using acetonitrile-water (+0.1% formic acid) gradient elution. The HPLC method was validated for precision, linearity, limit of detection (LOD), limit of quantification (LOQ) and accuracy. A ternary solvent system ethyl acetate/n-propanol/water (3:2:5, v/v/v) was selected and applied to recover the target compound using Spot CPC from the leaves aqueous extract. RESULTS: HPLC-ELSD analysis followed by CPC purification led to the efficient isolation of AH from O. coronata leaves aqueous extract. CONCLUSION: HPLC-ELSD has proven to be a well-adapted detection and quantification method for iridoid glycosides, while CPC confirmed to be an efficient technique for the isolation of polar compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Inhibition of Hypoxia-Induced Retinal Angiogenesis by Specnuezhenide, an Effective Constituent of Ligustrum lucidum Ait., through Suppression of the HIF-1α/VEGF Signaling Pathway.

Specnuezhenide (SPN), one of the main ingredients of Chinese medicine "Nü-zhen-zi", has anti-angiogenic and vision improvement effects. However, studies of its effect on retinal neovascularization are limited so far. In the present study, we established a vascular endothelial growth factor A (VEGFA) secretion model of human acute retinal pigment epithelial-19 (ARPE-19) cells by exposure of 150 µM CoCl2 to the cells and determined the VEGFA concentrations, the mRNA expressions of VEGFA, hypoxia inducible factor-1α (HIF-1α) & prolyl hydroxylases 2 (PHD-2), and the protein expressions of HIF-1α and PHD-2 after treatment of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1, 1.0 µg/mL) or SPN (0.2, 1.0 and 5.0 µg/mL). Furthermore, rat pups with retinopathy were treated with SPN (5.0 and 10.0 mg/kg) in an 80% oxygen atmosphere and the retinal avascular areas were assessed through visualization using infusion of ADPase and H&E stains. The results showed that SPN inhibited VEGFA secretion by ARPE-19 cells under hypoxia condition, down-regulated the mRNA expressions of VEGFA and PHD-2 slightly, and the protein expressions of VEGFA, HIF-1α and PHD-2 significantly in vitro. SPN also prevented hypoxia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy in vivo. These results indicate that SPN ameliorates retinal neovascularization through inhibition of HIF-1α/VEGF signaling pathway. Therefore, SPN has the potential to be developed as an agent for the prevention and treatment of diabetic retinopathy.