Curculigo orchioides polysaccharide COP70-1 stimulates osteogenic differentiation of MC3T3-E1 cells by activating the BMP and Wnt signaling pathways.

The crude polysaccharide CO70 isolated from Curculigo orchioides could alleviate ovariectomy-induced osteoporosis in rats. To clarify the bioactive components, a new heteropolysaccharide (COP70-1) was purified from CO70 in this study, which was consisted of ß-D-Manp-(1→, →4)-α-D-Glcp-(1→, →4)-ß-D-Manp-(1→, →3,4)-ß-D-Manp-(1→, →4,6)-ß-D-Manp-(1→, and →4,6)-α-D-Galp-(1→. COP70-1 significantly promoted the osteoblastic differentiation of MC3T3-E1 cells through improving alkaline phosphatase activity, the deposition of calcium as well as up-regulating the expression of osteogenic markers (RUNX2, OSX, BSP, OCN, and OPN). Furthermore, COP70-1 stimulated the expression of critical transcription factors of the BMP and Wnt pathways, including BMP2, p-SMAD1, active-ß-catenin, p-GSK-3ß, and LEF-1. In addition, LDN (BMP pathway inhibitor) and DKK-1 (Wnt pathway inhibitor) suppressed the COP70-1-induced osteogenic differentiation of MC3T3-E1 cells. Therefore, COP70-1 was one of the bioactive constituents of C. orchioides for targeting osteoblasts to treat osteoporosis by triggering BMP/Smad and Wnt/ß-catenin pathways.

Toxicity Assessment of Curculigo orchioides Leaf Extract Using Drosophila melanogaster: A Preliminary Study.

Curculigo orchioides is used in Indian and Chinese traditional medicinal systems for various health benefits. However, its toxicological effects are mostly unknown. This study assesses the potential toxicity of aqueous leaf (A.L.) extract of C. orchioides using Drosophila melanogaster as an experimental model. Preliminary phytochemical tests were followed by the Fourier transform infrared (FTIR) tests to identify the functional group in the A.L. extract of C. orchioides. Drosophila larvae/adults were exposed to varying concentrations of C. orchioides A.L. extract through diet, and developmental, lifespan, reproduction, and locomotory behaviour assays were carried out to assess the C. orchioides toxicity at organismal levels. The cellular toxicity of A.L. extract was examined by analysing the expression of heat shock protein (hsps), reactive oxygen species (ROS) levels, and cell death. The FTIR analysis showed the presence of functional groups indicating the presence of secondary metabolites like saponins, phenolics, and alkaloids. Exposure to A.L. extract during development resulted in reduced emergence and wing malformations in the emerged fly. Furthermore, a significant reduction in reproductive performance and the organism's lifespan was observed when adult flies were exposed to A.L. extract. This study indicates the adverse effect of C. orchioides A.L. extract on Drosophila and raises concerns about the practice of indiscriminate therapeutic use of plant extracts.

Norlignans and phenolics from genus Curculigo protect corticosterone-injured neuroblastoma cells SH-SY5Y by inhibiting endoplasmic reticulum stress-mitochondria pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The plants of genus Curculigo are divided into the Section Curculigo and the Section Capitulata, which are mainly distributed in southeastern and southwestern China. Various ancient chinese books record that these plants were used as an important herb for tonifying kidney yang. Traditional Chinese medicine often draws on this property to treat depression syndrome. Thus genus Curculigo has potential for the treatment of neurodegenerative diseases (ND). The study showed that phenolics were the main characteristic components of plants in the Section Curculigo, represented by orcinol glucoside and curculigoside; the norlignans, with Ph-C5-Ph as the basic backbone, were the main characteristic components of the Section Capitulata. However, there is a lack of sufficient scientific evidence as to whether these two types of ingredients have neuroprotective effects. AIM OF THE STUDY: To determine the neuroprotective effects of phenolics and norlignans in genus Curculigo on human neuroblastoma cells SH-SY5Y. To discuss their structure-activity relationship and screen for compounds with high activity and neuroprotective effects. To reveal that the amelioration of endoplasmic reticulum (ER) stress by two classes of compounds is mediated by the PERK/eIF2α/ATF4 pathway. MATERIALS AND METHODS: The cytotoxicity of 17 compounds was assayed by MTT. SH-SY5Y cells were damaged by corticosterone (Cort) (200 µM) for 24 h and then co-administered with 17 compounds (0.1-100 µM) and Cort (200 µM) for 24 h. Cell survival was determined by MTT assay. Apoptosis rate, mitochondrial membrane potential (MMP) and intracellular reactive oxygen species (ROS) levels were detected using flow cytometry. Intracellular Ca2+ levels were detected using a fluorescent probe. Cellular mitochondrial and ER damage was observed using transmission electron microscopy (TEM). ER stress and apoptotic pathway-related proteins (BiP, CHOP, cleaved caspase-3, cleaved caspase-9, Bax/Bcl-2), and the expression level of PERK/eIF2α/ATF4 pathway was measured via western blot (WB). RESULTS: The experimental data showed that Cort treatment of SH-SY5Y cells resulted in decreased cell survival and increased apoptosis, mitochondrial depolarization, ROS, and intracellular Ca2+ levels. The co-action of 17 compounds and Cort for a period of time significantly increased cell survival. Compounds 3, 7, 12, 13 also reduced apoptosis rate, mitochondrial depolarization, ROS and intracellular Ca2+ levels in the subsequent experiments. In addition, TEM observed that Cort caused mitochondrial and ER damage, and the damage was improved after treatment. WB analysis obtained that Cort increased the expression of apoptotic and ER stress-related proteins and activated pathway expression. However, in the presence of compounds 3, 7, 12, 13, the expression of BiP, CHOP, cleaved caspase-3, cleaved caspase-9, and Bax/Bcl-2 was significantly reduced, and the phosphorylation of PERK and eIF2α and the expression of ATF4 were inhibited. CONCLUSION: This study found that one phenolic (3) and three norlignans (7, 12, 13) from genus Curculigo have significant neuroprotective effects. The results of the structure-activity relationship indicated that the glucosyl polymeric norlignans and the phenolics with benzoic acid as the parent nucleus were more active. The neuroprotective effect of three norlignans is the latest discovery. This finding has important research value in the field of prevention and treatment of neurodegenerative diseases.

De novo transcriptome analysis and comparative expression profiling of genes associated with the taste-modifying protein neoculin in Curculigo latifolia and Curculigo capitulata fruits.

BACKGROUND: Curculigo latifolia is a perennial plant endogenous to Southeast Asia whose fruits contain the taste-modifying protein neoculin, which binds to sweet receptors and makes sour fruits taste sweet. Although similar to snowdrop (Galanthus nivalis) agglutinin (GNA), which contains mannose-binding sites in its sequence and 3D structure, neoculin lacks such sites and has no lectin activity. Whether the fruits of C. latifolia and other Curculigo plants contain neoculin and/or GNA family members was unclear. RESULTS: Through de novo RNA-seq assembly of the fruits of C. latifolia and the related C. capitulata and detailed analysis of the expression patterns of neoculin and neoculin-like genes in both species, we assembled 85,697 transcripts from C. latifolia and 76,775 from C. capitulata using Trinity and annotated them using public databases. We identified 70,371 unigenes in C. latifolia and 63,704 in C. capitulata. In total, 38.6% of unigenes from C. latifolia and 42.6% from C. capitulata shared high similarity between the two species. We identified ten neoculin-related transcripts in C. latifolia and 15 in C. capitulata, encoding both the basic and acidic subunits of neoculin in both plants. We aligned these 25 transcripts and generated a phylogenetic tree. Many orthologs in the two species shared high similarity, despite the low number of common genes, suggesting that these genes likely existed before the two species diverged. The relative expression levels of these genes differed considerably between the two species: the transcripts per million (TPM) values of neoculin genes were 60 times higher in C. latifolia than in C. capitulata, whereas those of GNA family members were 15,000 times lower in C. latifolia than in C. capitulata. CONCLUSIONS: The genetic diversity of neoculin-related genes strongly suggests that neoculin genes underwent duplication during evolution. The marked differences in their expression profiles between C. latifolia and C. capitulata may be due to mutations in regions involved in transcriptional regulation. Comprehensive analysis of the genes expressed in the fruits of these two Curculigo species helped elucidate the origin of neoculin at the molecular level.

Crassifoside H improve the depressive-like behavior of rats under chronic unpredictable mild stress: Possible involved mechanisms.

Crassifoside (CH) is a novel chlorine-containing compound isolated from rhizomes of Curculigo glabrescens. This study aimed to explore the antidepressant-like effect of CH and involved mechanisms. A rat depression model was established using chronic unpredictable mild stress (CUMS) paradigm. Behavioral tests including sucrose preference test (SPT), open field test (OFT) and forced swimming test (FST) were used to evaluate the antidepressant-like effect of CH. The levels of plasma corticosterone (CORT) and corticotrophin-releasing factor (CRF) in hypothalamus were measured to determine the activity of hypothalamic pituitary-adrenal (HPA) axis. Protein expression of 5-hydroxytryptamine 1A (5-HT1A) receptor, brain-derived neurotrophic factor (BDNF), as well as the total and phosphorylated extracellular signal-regulated kinase (ERK)1/2 in hippocampus were also analyzed by Western blotting. The CH administration effectively ameliorated the depressive-like behaviors of CUMS rats, as indicated by the increased sucrose intake in SPT, reduced immobility time in FST, and the increased rearing and grooming numbers, spent more time in inner zone and less time in outer zone in OFT. CH improved CUMS-induced HPA axis hyperactivity by reduced plasma CORT and CRH expression in hypothalamus. Moreover, CH reversed CUMS-induced decrease of 5-HT1A receptor expression, and up-regulated BDNF and phosphorylated-ERK1/2 levels in hippocampus. These findings suggest that CH improved depressive behaviors of CUMS rats by modulating of HPA axis dysfunction, increasing 5-HT1A receptor expression, and activating BDNF-ERK signaling pathway.

Curculigoside improves osteogenesis of human amniotic fluid-derived stem cells.

Curculigoside, a phenolic glycoside, is the main active compound of Curculigo orchioides (Amaryllidaceae, rhizome). C. orchioides is a traditional Chinese herbal medicine and has been commonly used to treat orthopedic disorders and bone healing in Asia. This study evaluated the effect of curculigoside on osteogenic differentiation of human amniotic fluid-derived stem cells (hAFSCs). The results showed that curculigoside stimulated alkaline phosphatase activity and calcium deposition of hAFSCs during osteogenic differentiation in a dose-dependent manner (1-100 µg/mL), while the effects were reduced at the higher concentration of 200 µg/mL. From reverse transcriptase-polymerase chain reaction analysis, the osteogenic genes osteopontin (OPN) and Collagen I were upregulated with curculigoside treatment (1-100 µg/mL). Concurrently, the ratio of osteoprotegerin (OPG) to receptor activator of nuclear factor kappa-B ligand (RANKL) was increased, indicating the inhibition of osteoclastogenesis by curculigoside. Moreover, the role of Wnt/ß-catenin signaling during curculigoside treatment was revealed by the upregulation of ß-catenin and Cyclin D1. In summary, curculigoside improved osteogenesis and inhibited osteoclastogenesis of hAFSCs, suggesting its potential use to regulate hAFSC osteogenic differentiation for treating bone disorders.

Non-acidic compounds induce the intense sweet taste of neoculin, a taste-modifying protein.

Neoculin, a sweet protein found in the fruit of Curculigo latifolia, has the ability to change sourness into sweetness. Neoculin turns drinking water sweet, indicating that non-acidic compounds may induce the sweetness. We report that ammonium chloride and certain amino acids elicit the intense sweetness of neoculin. Neoculin can thus sweeten amino acid-enriched foods.

Neoculin, a taste-modifying sweet protein, accumulates in ripening fruits of cultivated Curculigo latifolia.

Neoculin is a sweet protein with a taste-modifying activity of converting sourness to sweetness. It occurs in the fruits of Curculigo latifolia, a wild plant found in tropical Asia. We successfully cultivated the plant and evaluated the production of neoculin. The neoculin content of the fruit was high for 10 weeks after flowering, following which the yield decreased gradually. The optimal period for harvesting the fruits with sensory activity coincided with this 10-week peak period during which the amount of neoculin was 1-3mg in the whole fruit and 1.3mg/g of pulp. Immunohistochemical staining showed that neoculin occurred in the whole fruit, especially at the basal portion. Although it is known that neoculin comprises an acidic subunit (NAS) with an N-glycosylated moiety and a basic subunit (NBS), protein gel blot analysis revealed the presence of a non-glycosylated NAS species. This suggests the presence of multiple NAS-NBS heterodimers in our cultivar.

Curculin exhibits sweet-tasting and taste-modifying activities through its distinct molecular surfaces.

Curculin isolated from Curculigo latifolia, a plant grown in Malaysia, has an intriguing property of modifying sour taste into sweet taste. In addition to this taste-modifying activity, curculin itself elicits a sweet taste. Although these activities have been attributed to the heterodimeric isoform and not homodimers of curculin, the underlying mechanisms for the dual action of this protein have been largely unknown. To identify critical sites for these activities, we performed a mutational and structural study of recombinant curculin. Based on the comparison of crystal structures of curculin homo- and heterodimers, a series of mutants was designed and subjected to tasting assays. Mapping of amino acid residues on the three-dimensional structure according to their mutational effects revealed that the curculin heterodimer exhibits sweet-tasting and taste-modifying activities through its partially overlapping but distinct molecular surfaces. These findings suggest that the two activities of the curculin heterodimer are expressed through its two different modes of interactions with the T1R2-T1R3 heterodimeric sweet taste receptor.

Extracellular production of neoculin, a sweet-tasting heterodimeric protein with taste-modifying activity, by Aspergillus oryzae.

Neoculin (NCL), a protein with sweetness approximately 500-fold that of sugar, can be utilized as a nonglycemic sweetener. It also has taste-modifying activity to convert sourness to sweetness. NCL is a heterodimer composed of an N-glycosylated acidic subunit (NAS) and a basic subunit (NBS), which are conjugated by disulfide bonds. For the production of recombinant NCL (rNCL) by Aspergillus oryzae, alpha-amylase with a KEX2 cleavage site, -K-R-, was fused upstream of each of NAS and NBS and the resulting fusion proteins were simultaneously expressed. For accurate and efficient cleavage of the fusion construct by KEX2-like protease, a triglycine motif was inserted after the KEX2 cleavage site. As NBS showed lower production efficiency than did NAS, a larger amount of the NBS expression plasmid than of NAS expression plasmid was introduced during cotransformation, resulting in successful production of rNCL in the culture medium. Moreover, to obtain a higher production yield of rNCL, the active form of hacA cDNA encoding a transcription factor that induces an unfolded protein response was cloned and expressed constitutively. This resulted in a 1.5-fold increase in the level of rNCL production (2.0 mg/liter). rNCL was purified by chromatography, and its NAS was found to be N-glycosylated as expected. The original sweetness and taste-modifying activity of rNCL were comparable to those of native NCL when confirmed by calcium imaging with human embryonic kidney cells expressing the human sweet taste receptor and by sensory tests.

Actividad antioxidante in vitro del extracto de metanol de los rizomas de Curculigo orchioides Gaertn / In vitro antioxidant activity of methanol extract of rhizomes of Curculigo orchioides Gaertn

Se analizó la actividad antioxidante in vitro del extracto de metanol de Curculigo orchioides mediante ensayo DPPH, barrido de superóxido, barrido de óxido nítrico, determinación del poder reductor y peroxidación lipídica in vitro vitro. El extracto de metanol resultó ser extremadamente eficaz en el barrido del radical superóxido (IC 50 29.28 ìg/ml), mientras que la actividad fue moderada en el barrido del radical DPPH (IC 50 105.94 µg/ml), el radical de óxido nítrico (IC 50 90.96 µg/ml) y la inhibición de la peroxidación lipídica (IC 50 94.78 µg/ml). El extracto de metanol presentó distintos niveles de actividad antioxidante en los modelos anali- analizados. También apoyó la actividad antioxidante al presentar un poder reductor significativo. Para facilitar litar su identificación y caracterización, se estableció un perfil de huella de cromatografía de capa fina de alto rendimiento (HPTLC) adicional del extracto de metanol. El presente estudio ha demostrado la actividad antioxidante in vitro del extracto de metanol de Curculigo orchioides

Methanol extract of Curculigo orchioides was screened for in-vitro antioxidant activity using DPPH assay, superoxide scavenging, nitric oxide scavenging, determination of reducing power and in-vitro lipid peroxidation. Methanol extract was found to be extremely effective in scavenging superoxide radical (IC 50 29.28 µg/ml) whereas activity was moderate in scavenging DPPH radical (IC 50 105.94 µg/ml), nitric oxide radical(IC 50 90.96 µg/ml) and in inhibition of lipid peroxidation (IC 50 94.78 ìg/ml). Methanol extract showed different levels of antioxidant activities in tested models. It also supported anti oxidant activity by showing significant reducing power. Further HPTLC finger print profile of the methanol extract was established to facilitate its identification and characterization. Present study demonstrated antioxidant activity of methanol extract of Curculigo orchioides in-vitro