Complete pathway elucidation and heterologous reconstitution of (+)-nootkatone biosynthesis from Alpinia oxyphylla.

(+)-Nootkatone is a natural sesquiterpene ketone widely used in food, cosmetics, pharmaceuticals, and agriculture. It is also regarded as one of the most valuable terpenes used commercially. However, plants contain trace amounts of (+)-nootkatone, and extraction from plants is insufficient to meet market demand. Alpinia oxyphylla is a well-known medicinal plant in China, and (+)-nootkatone is one of the main components within the fruits. By transcriptome mining and functional screening using a precursor-providing yeast chassis, the complete (+)-nootkatone biosynthetic pathway in Alpinia oxyphylla was identified. A (+)-valencene synthase (AoVS) was identified as a novel monocot-derived valencene synthase; three (+)-valencene oxidases AoCYP6 (CYP71BB2), AoCYP9 (CYP71CX8), and AoCYP18 (CYP701A170) were identified by constructing a valencene-providing yeast strain. With further characterisation of a cytochrome P450 reductase (AoCPR1) and three dehydrogenases (AoSDR1/2/3), we successfully reconstructed the (+)-nootkatone biosynthetic pathway in Saccharomyces cerevisiae, representing a basis for its biotechnological production. Identifying the biosynthetic pathway of (+)-nootkatone in A. oxyphylla unravelled the molecular mechanism underlying its formation in planta and also supported the bioengineering production of (+)-nootkatone. The highly efficient yeast chassis screening method could be used to elucidate the complete biosynthetic pathway of other valuable plant natural products in future.

Hepatoprotective effects of Alpinia officinarum rhizome extract on cisplatin-induced hepatotoxicity in rats: A biochemical, histopathological and immunohistochemical study.

BACKGROUND: Alpinia officinarum is a member of the ginger family (Zingiberaceae), which is widely cultivated in Asia and traditionally used for its anti-inflammatory, antimicrobial, and antihyperlipidemic qualities. This study aimed to evaluate the effect of Alpinia officinarum rhizome extract (AORE) on cisplatin (CP)-induced hepatotoxicity in rats. METHODS: Forty-four male rats were divided into six groups including the control group, AORE control group, CP control group, and three groups of CP (7 mg/kg dose, on the 10th day) with AORE (at concentrations of 100, 200 and 400 mg/kg, daily for 14 days). After 14 days, the rats' livers were removed and their liver function was assessed using biochemical marker enzymes including serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH) activities and albumin, total protein, and total bilirubin (T. bilirubin). Oxidative stress was assessed by evaluating malondialdehyde concentration and hepatic superoxide dismutase activity, histopathological and immunohistochemical tests were also conducted. RESULTS: Results demonstrated that treatment with AORE reduced the toxicity in levels of the hepatic biomarkers in cp-induced groups. AORE treatment decreased oxidative stress and improved histopathological indexes. Furthermore, immunohistochemical (IHC) investigation showed the B-cell lymphoma 2 (Bcl-2) upsurging and p53 downregulating expression exhibiting the recovery following AORE administration. CONCLUSION: The founding suggested that AORE administration has positive biochemical, histopathological, and immunohistochemical impacts on the ameliorating of hepatotoxicity in CP-induced rats.

Alpinia officinarum mediated copper oxide nanoparticles: synthesis and its antifungal activity against Colletotrichum gloeosporioides.

Green synthesis offers an environmentally friendly and cost-effective alternative for the synthesis of copper oxide nanoparticles (CuO NPs). In this study, the synthesis of CuO NPs was optimized by using copper sulfate (CuSO4) and the aqueous extract of Alpinia officinarum and its antifungal activity were investigated. The synthesized CuO NPs were characterized by UV-visible spectroscopy (UV-vis), X-ray diffraction (XRD), Fourier-transform infrared radiation spectroscopy (FT-IR), scanning electron microscope (SEM), energy dispersive spectroscopy (EDS), dynamic light scattering (DLS), and transmission electron microscopy (TEM). The results showed that the optimized conditions for the synthesis of CuO NPs were 1:2 ratio of extract and CuSO4 solution, pH 7, and 30 °C. The characteristic UV-vis peak of A. officinarum synthesized CuO NPs was at 264 nm. The synthesized CuO NPs had high crystallinity and purity and were spherical in morphology with the mean size of 46.40 nm. The synthesized CuO NPs reduced the fungal growth of Colletotrichum gloeosporioides in a dose-dependent manner. The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of the CuO NPs were 125 µg·mL-1 and 500 µg·mL-1, respectively. The antifungal activity of CuO NPs may be attributed to its ability to deform the structure of fungal hyphae, induce excessive reactive oxygen species accumulation and lipid peroxidation in fungi, disrupt the mycelium cell membrane, and result cellular leakage.

Alpinia officinarum Rhizome ameliorates the UVB induced photoaging through attenuating the phosphorylation of AKT and ERK.

BACKGROUND: Chronic ultraviolet (UV) exposure is one of the major external factors in skin aging, and repetitive UVB exposure induces extracellular matrix (ECM) damage as well as metabolic disease. Alpinia officinarum Rhizome (AOR) is a medicinal plant that has been traditionally used for treating rheumatism and whooping cough. However, the antiphotoaging effects of AOR remain unclear. We investigated the protective effects of water extracts of AOR (WEAOR) in terms of UVB-mediated ECM damage, wrinkle formation, inflammatory responses, and intracellular signaling on hairless mice and NIH-3T3 skin fibroblast cells. METHODS: WEAOR was administered to UVB-irradiated hairless mice. Wrinkle formation was assessed using the replica assay, epidermal changes through H&E staining, and collagen contents in mice skin through Masson's trichrome staining. The expression of procollagen type-1 (COL1A1), metalloproteinase-1a (MMP-1a), and inflammatory cytokines (IL-6, IL-8, and MCP-3) in hairless mice skin and NIH-3T3 cells was investigated through qRT-PCR. The effects of WEAOR or signaling inhibitors on UVB-induced expression of intracellular mitogen-activated protein kinases (MAPKs) were estimated by Western blotting and qRT-PCR, respectively. RESULTS: Topical WEAOR significantly attenuated the UVB-induced wrinkle formation and epidermal thickening in the skin of hairless mice. WEAOR treatment also attenuated the UVB-induced expression of MMP-1a and COL1A1 and recovered the reduction of collagen content in mouse skin. These effects were confirmed in NIH-3T3 skin fibroblast cells. WEAOR treatment restored the UVB-induced COL1A1 and MMP-1a gene expression and attenuated the UVB-induced expression of IL-6, IL-8, and MCP-3 in NIH-3T3 cells. Notably, WEAOR attenuated UVB-induced phosphorylation of AKT and ERK, but not that of p38 and JNK in NIH-3T3 cells. In addition, the administration of AKT and ERK inhibitors restored the UVB-induced expression of MMP-1a and COL1A1 to an equal extent as WEAOR in NIH-3T3 cells. CONCLUSIONS: The antiphotoaging properties of WEAOR were first evaluated in this study. Our results suggest that WEAOR may be a potential antiphotoaging agent that ameliorates UVB-induced photoaging processes via the AKT and ERK signaling pathways.

Synthesis of silver nanoparticles using Alpinia officinarum rhizome extract induces apoptosis through down-regulating Bcl-2 in human cancer cells.

In this study, silver nanoparticles were synthesized using Alpinia officinarum rhizome extract via an eco-friendly green synthesis method. The silver nanoparticles (AO-AgNPs) were characterized by UV-Vis spectrometry, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and dynamic light scattering. Further, the cytotoxic and apoptotic effects of AO-AgNPs were investigated in human cancer cells with different tissue origins via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and flow cytometric analyses, respectively. The expression levels of anti-apoptotic Bcl-2 protein were evaluated via a real-time polymerase chain reaction. The synthesized AO-AgNPs induced a significant cytotoxic effect in all tested cancer cells but not in normal cells. AO-AgNPs induced the percentage of apoptotic cells and reduced the levels of anti-apoptotic Bcl-2 mRNA levels in cancer cells. These results demonstrate the potential application of AO-AgNPs in cancer treatment.

Alpinia katsumadai Hayata induces growth inhibition and autophagy­related apoptosis by regulating the AMPK and Akt/mTOR/p70S6K signaling pathways in cancer cells.

Alpinia katsumadai Hayata (AKH), a widely used traditional Chinese medicine, exerts various biological functions, including anti­inflammatory, antioxidant, anti­microbial and anti­asthmatic effects. However, studies on its anticancer activity and associated mechanisms are limited. The present study investigated the effects of ethanol extract from AKH on the viability of various human cancer and normal liver LX­2 cells using Cell Counting Kit­8 assay. Apoptosis was detected by Hoechst 33342/PI staining and Annexin­V­FITC/PI double staining. Autophagy was examined by Ad­GFP­LC3B transfection. The association between AKH­induced autophagy and apoptosis was investigated by pre­treatment of the cells with the autophagy inhibitors, 3­methyladenine (3MA) and bafilomycin A1 (Baf­A1), followed by treatment with AKH. The expression levels of cleaved poly(ADP­ribose) polymerase (PARP), caspase­8, caspase­3, caspase­9, phosphorylated (p­)AMP­activated protein kinase (AMPK), Akt, mTOR and p70S6K were examined using western blot analysis. The in vivo antitumor activity of AKH was investigated in nude mice bearing A549 lung cancer xenografts. The components of AKH were detected by liquid chromatography mass spectrometry­ion trap­time­of­flight mass spectrometry. The results revealed that AKH significantly inhibited the proliferation of various cancer cells with the half maximal inhibitory concentration (IC50) values of 203­284 µg/ml; however, its inhibitory effect was much less prominent against normal liver LX­2 cells with an IC50 value of 395 µg/ml. AKH markedly induced apoptosis and autophagy, and upregulated the protein expression of cleaved­caspase­3, caspase­8, caspase­9 and cleaved PARP in a concentration­dependent manner. Of note, the autophagy inhibitors (3MA and Baf­A1) significantly attenuated its pro­apoptotic effects on human pancreatic cancer Panc­28 and lung cancer A549 cells. Furthermore, AKH significantly increased the levels of p­AMPK, and decreased those of p­Akt, p­mTOR and p­p70S6K in Panc­28 and A549 cells. AKH markedly inhibited the growth of A549 tumor xenografts in vivo. In addition, a total of nine compounds were detected from AKH. The present study demonstrates that AKH markedly inhibits the growth and induces autophagy­related apoptosis in cancer cells by regulating the AMPK and Akt/mTOR/p70S6K signaling pathways. AKH and/or its active fractions may thus have potential to be developed as novel anticancer agents for clinical use.

Alpinia nigra fruits mediated synthesis of silver nanoparticles and their antimicrobial and photocatalytic activities.

In the present systematic study, silver nanoparticles have been synthesized using the fruits of Alpinia nigra. Apart from the presence of saponins, glycosides, alkaloids, steroids, the extract of A. nigra fruits are rich in polyphenols. The Total Flavonoid and Phenol Content of A. nigra fruits extract is 718 mgRE/g extract and 74.9 mgGAE/g extract respectively. The formation of the nanoparticles was validated through characterization techniques like UV-Vis spectroscopy, X- ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and Energy dispersive X-ray spectroscopy (EDX). The spherical shape of silver nanoparticles is observed in Transmission Electron Microscopy (TEM) images. The average particle size of the silver nanoparticles is 6 nm. The biomolecules of the fruit extract played the dual role of reducing and capping agents which is evident from Fourier Transform Infrared (FTIR) spectrometer and Scanning Electron Microscopy (SEM) image analysis. The A. nigra capped silver nanoparticles exhibited promising antimicrobial activity against gram negative bacteria Klebsiella pneumoniae, gram positive bacteria Staphylococcus aureus and the pathogenic fungus, Candida albicans. Amongst the three pathogens, Klebsiella pneumoniae is the most susceptible to silver nanoparticles. Furthermore, the nanoparticles efficiently catalysed the degradation of the anthropogenic dyes Methyl orange, Rhodamine B and Orange G in the presence of sunlight. The photocatalytic degradation process follows the pseudo-first order kinetics. These results confirm that the silver nanoparticles can be efficiently synthesized via a green route using A. nigra fruits with applications as antimicrobial and catalytic agents.

Molecular Insight and Mode of Inhibition of α-Glucosidase and α-Amylase by Pahangensin A from Alpinia pahangensis Ridl.

The inhibition of carbohydrate-hydrolyzing enzymes in human digestive organs is crucial in controlling blood sugar levels, which is important in treating type 2 diabetes. In the current study, pahangensin A (1), a bis-labdanic diterpene characterized previously in the rhizomes of Alpinia pahangensis Ridl., was identified as an active dual inhibitor for α-amylase (IC50 =114.80 µm) and α-glucosidase (IC50 =153.87 µm). This is the first report on the dual α-amylase and α-glucosidase inhibitory activities of a bis-labdanic diterpene. The Lineweaver-Burk plots of compound 1 indicate that it is a mixed-type inhibitor with regard to both enzymes. Based on molecular docking studies, compound 1 docked in a non-active site of both enzymes. The dual inhibitory activity of compound 1 makes it a suitable natural alternative in the treatment of type 2 diabetes.

Biogenic synthesis of gold nanoparticles and their application in photocatalytic degradation of toxic dyes.

Plants and their extracts play an important role in the green synthesis of nanoparticles mainly because of their environmental benignity. Based on plant extracts number of metal nanoparticles have been synthesized. In our study, we report a green technique for the synthesis of gold nanoparticles using the aqueous extracts of Alpinia nigra leaves and their photocatalytic activities. The antioxidant, antibacterial and antifungal potential of the synthesized nanoparticles were also evaluated. The aqueous extract of the plant is rich in flavonoids with Total Flavonoid Content of 491mgRE/g extract. The presence of flavonoids was further confirmed through analytical High Performance Liquid Chromatography (HPLC) analysis. The A. nigra mediated syntheses of gold nanoparticles (ANL-AuNPs) were characterized by UV-Vis spectrophotometer, Fourier Transform Infrared (FTIR) Spectroscopy, X-ray Diffraction (XRD) and Transmission Electron Microscopy (TEM). The crystalline nature of the ANL-AuNPs was confirmed by the powder XRD analysis. The TEM micrographs showed that the ANL-AuNPs was predominantly spherical in shape and the average particle size was 21.52 nm. The polyphenolics and other functional groups present in the aqueous extract that acted as reducing and capping agent in the synthesis of the Au-NPs were identified via FTIR spectral analysis. These green synthesized nanoparticles exhibited antioxidant activity with IC50 value of 52.16 µg/ml and showed inhibition in the growth of both gram-positive and gram-negative bacteria. The pathogenic fungus, Candida albicans was also susceptible to these nanoparticles. The ANL-AuNPs in the presence of sunlight catalyzed the degradation of the anthropogenic pollutant dyes, Methyl Orange and Rhodamine B with percent degradation of 83.25% and 87.64% respectively. The photodegradation process followed pseudo first order kinetic model. These results confirm that Alpinia nigra is a potential bioresource for the synthesis of Au-NPs with versatile applications.

Therapeutic Potential of Alpinia officinarum.

The plant Alpinia officinarum of the ginger family originated in China and is used throughout South and South-East Asian countries to flavor food and as a traditional medicine to treat a variety of diseases. This review summarizes the biological, pharmacological and phytochemical properties of extracts and subsequently isolated compounds from A. officinarum. In vitro and in vivo studies of both extracts and pure compounds indicate a wide variety of potent bioactivities including antiinflammatory, antibacterial, antioxidant, antiobesity, anticancer, enzyme inhibitory and remarkable antiviral properties. The latter is particularly promising in the face of emerging, virulent respiratory diseases in Asia and the Middle East.

Essential Oil from Fructus Alpiniae Zerumbet Protects Human Umbilical Vein Endothelial Cells In Vitro from Injury Induced by High Glucose Levels by Suppressing Nuclear Transcription Factor-Kappa B Signaling.

BACKGROUND In China, the essential oil of the fruit, Fructus Alpiniae zerumbet (FAZ), is used to treat cardiovascular diseases. Recent in vitro studies have shown that the essential oil of FAZ (EOFAZ) can protect endothelial cells from injury. Because of the prevalence of diabetes mellitus and its effects on the cardiovascular system, the aim of this study was to investigate the mechanism of the effects of EOFAZ on human umbilical vein endothelial cells (HUVECs) treated with high levels of glucose in vitro. MATERIAL AND METHODS The lactate dehydrogenase (LDH) leakage assay was used to detect HUVEC injury. Tumor necrosis factor-alpha (TNF-α), interleukin-8 (IL-8), and nuclear transcription factor-kappa B (NF-κB) p65 subunit DNA-binding activity was detected. The expression of NF-κB pathway-associated proteins, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) was studied by Western blotting. The cellular location of NF-κB in HUVECs was evaluated using immunofluorescence. RESULTS Cell viability and LDH leakage assays showed that high glucose-induced HUVEC injury was reduced by EOFAZ. High glucose-induced secretion of IL-8, TNF-α, ICAM-1, and VCAM-1 was reduced, and translocation of the p65 subunit of NF-κB to the endothelial cell nucleus was inhibited by EOFAZ. Western blotting confirmed that EOFAZ blocked the activation of NF-κB induced by high glucose levels. EOFAZ reduced high glucose-induced p65/DNA binding to inhibit NF-κB activation. CONCLUSIONS The findings of this in vitro study showed that treatment of HUVECs with EOFAZ had a protective role against the effects of high glucose levels via the NF-κB signaling pathway.

The effects of sesquiterpenes-rich extract of Alpinia oxyphylla Miq. on amyloid-ß-induced cognitive impairment and neuronal abnormalities in the cortex and hippocampus of mice.

As a kind of medicine which can also be used as food, Alpinia oxyphylla Miq. has a long clinical history in China. A variety of studies demonstrated the significant neuroprotective activity effects of chloroform (CF) extract from the fruits of Alpinia oxyphylla. In order to further elucidate the possible mechanisms of CF extract which mainly contains sesquiterpenes with neuroprotection on the cognitive ability, mice were injected with Aß(1-42) and later with CF in this study. The results showed that the long-term treatment of CF enhanced the cognitive performances in behavior tests, increased activities of glutathione peroxidase (GSH-px) and decreased the level of malondialdehyde (MDA), acetylcholinesterase (AChE), and amyloid-ß (Aß), and reversed the activation of microglia, degeneration of neuronal acidophilia, and nuclear condensation in the cortex and hippocampus. These results demonstrate that CF ameliorates learning and memory deficits by attenuating oxidative stress and regulating the activation of microglia and degeneration of neuronal acidophilia to reinforce cholinergic functions.

[Photosynthetic characteristics of wildlife tending Alpinia oxyphylla].

OBJECTIVE: To investigate the photosynthetic characteristics of wildlife tending Alpinia oxyphylla, and provide a theoretical basis for choosing wildlife tending environment and cultivation management. METHOD: The response parameters of the net photosynthetic rate to light intensity, CO2 concentration and photosynthetic characteristics were measured by Li-6400 portable photosynthesis in blossom bud forming stages under different treated conditions. RESULT: The maximum net photosynthetic rate (Pmax), daily average photosynthetic rate (Pn), apparent quantum efficiency (AQY), apparent carboxylation efficiency (CE), light using efficiency (LUE), and water use efficiency (WUE) were optimal in the wild tending treatment at the light transmission rate of 17.4%-24.1%, beyond the light transmission rate, the photosynthetic capacity utilization of A. oxyphylla would not have a significant increase or be inhibited. The light compensation point (LCP) and light saturation point (LSP) of A. oxyphylla improved with light intensity enhancing. Wildlife tending could enhance the scope of A. oxyphylla to CO2 adaptation. CONCLUSION: A. oxyphylla as sciophytes, and the optimum light transmission rate for wild tending and cultivating was at 17.4%-24.1%.

Production of flavonoids in organogenic cultures of Alpinia zerumbet.

Alpinia zerumbet plantlets were cultured in vitro in MS medium supplemented with growth regulators, including IAA, TDZ and BAP. Using high performance liquid chromatography (HPLC), the production of rutin, kaempferol-3-O-glucuronide, and kaempferol-3-O-rutinoside was evaluated, based on leaf hydroalcoholic extracts of three-month-old plantlets. The relative concentration of phenolics from the hydroalcoholic extracts of plantlets cultured in control medium reached 100% compared with plantlets treated with growth regulators and donor plants (80%). The in vitro rutin production was more pronounced than the other flavonoids. While no direct relation between the content of phenolic compounds and increased flavonoid production was observed, the combination of IAA + TDZ enhanced the production of rutin (83.2 microg/g dried leaves) and kaempferol-3-O-glucuronide (29 microg/g dried leaves), compared with growth regulators used alone. Overall, these findings suggest the value of in vitro cultivation as a means of enriching phenolic and flavonoid production in medicinal plants.

New Rev-export inhibitor from Alpinia galanga and structure-activity relationship.

Bioassay-guided separation by use of the fission yeast expressing NES of Rev, an HIV-1 viral regulatory protein, disclosed 1'-acetoxychavicol acetate (ACA, 1) as a new inhibitor for nuclear export of Rev from the roots of Alpinia galanga. Both analysis for mechanism of action with biotinylated probe (2) and several synthesized analogs established crucial portions in 1 for Rev-export inhibitory activity.

Synergistic effects of the combination of galangin with gentamicin against methicillin-resistant Staphylococcus aureus.

The antimicrobial killing activity toward methicillin-resistant Staphylococcus aureus (MRSA) has been a serious emerging global issue. New effective antimicrobials and/or new approaches to settle this issue are urgently needed. The oriental herb, Alpinia officinarum, has been used in Korea for several hundreds of years to treat various infectious diseases. As it is well known, one of the active constituents of Alpinia officinarum is galangin. Against the 17 strains, the minimum inhibitory concentrations (MICs) of galangin (GAL) were in the range of 62.5 ~ 125 microg/ml, and the MICs of gentamicin (GEN) ranged from 1.9 microg/ml to 2,000 microg/ml. The fractional inhibitory concentrations (FICs) of GAL, in combination with GEN, against 3 test strains were 0.4, 3.9, and 250 microg/ml, and were all 15.62 microg/ml in GEN. The FIC index showed marked synergism in the value range of 0.19 to 0.25. By determining time-kill curves, also confirmed the low synergism of the GAL and GEN combination against 4 h, 8 h, 12 h, and 24 h cultured MRSA. The time-kill study results indicated a low synergistic effect against 3 test strains. Thus, the mixture of GAL and GEN could lead to the development of new combination antibiotics against MRSA infection.