[Application strategy of artificial intelligence technology in feed-based development of medicinal and edible homologous traditional Chinese medicine(TCM)resources in era of the "antibiotic ban"].

In the context of the "antibiotic ban" era, the feed conversion of medicinal and edible traditional Chinese medicine(TCM) resources is a research hotspot in the field of antibiotic alternatives development. How to develop feed products that are beneficial to agriculture and livestock while ensuring nutrient balance and precision using medicinal and edible TCM resources as raw materials has become a challenge. Artificial intelligence(AI) technology has unique advantages in feed production and improving the efficiency of intelligent breeding. If AI technology is applied to the feed development of medicinal and edible TCM resources, it is possible to realize feeding and antibiotic-replacement value while ensuring precise nutrition. In order to better apply AI technology in the field of feed development of medicinal and edible TCM resources, this article used CiteSpace software to carry out literature visualization analysis and found that AI technology had a good application in the field of feed formulation optimization in recent years. However, there is still a gap in the research on the intelligent utilization of medicinal and edible TCM resources. Nonetheless, it is feasible for AI technology to be applied to the feed conversion of medicinal and edible TCM resources. Therefore, this article proposed for the first time an intelligent formulation system framework for feed materials derived from medicinal and edible TCM resources to provide new ideas for research in the field of feed development of medicinal and edible TCM resources and the research on the development of antibiotic alternatives. At the same time, it can pave the way for a new green industry chain for contemporary animal husbandry and the TCM industry.

[Inhibitory effect of paeonol on aortic endothelial inflammation in atherosclerotic rats by up-regulation of caveolin-1 expression and suppression of NF-κB pathway].

To explore whether paeonol can play an anti-atherosclerotic role by regulating the expression of aortic caveolin-1 and affecting NF-κB pathway, so as to inhibit the inflammatory response of vascular endothelium in atherosclerotic rats. The atherosclerotic model of rats was induced by high-fat diet and vitamin D_2. The primary culture of vascular endothelial cells(VECs) was carried out by tissue block pre-digestion and adherent method. The injury model of VECs was induced by lipopolysaccharide(LPS), and filipin, a small concave protein inhibitor, was added for control. HE staining was used to observe pathological changes of aorta. TNF-α, IL-6 and VCAM-1 were detected by ELISA. Western blot assay was used to detect the protein expression levels of caveolin-1 and p65 in aorta and VECs. The results showed that as compared with model group, paeonol significantly reduced aortic plaque area and lesion degree in rats, decreased the level of serum TNF-α, IL-6 and VCAM-1 in the rats and enhanced the relative expression level of caveolin-1, decreased p65 expression conversely(P<0.05 or P<0.01). In vitro, as compared to model group, paeonol obviously improved cell morphology, decreased the secretion of TNF-α, IL-6 and VCAM-1 in VECs, increased caveolin-1 expression, and decreased p65 protein expression(P<0.05 or P<0.01). Furthermore, filipin could reverse the effect of paeonol on expression of inflammatory factors and proteins(P<0.05 or P<0.01). According to the results, it was found that paeonol could play the role of anti-atherosclerosis by up-regulating the expression of caveolin-1 and inhibiting the activation of NF-κB pathway to reduce vascular inflammation in atherosclerotic rats.

Monoterpenoid indole alkaloids from the bark of Melodinus henryi.

Four new alkaloids, melodinines W1-W4 (1-4), together with twenty one known alkaloids (5-25) were isolated from Melodinus henryi. The structures with absolute configurations were elucidated by extensive MS and NMR spectroscopic methods, as well as the single crystal X-ray diffraction and ECD calculations. All compounds were evaluated for their cytotoxicities to five human cancer cell lines. Many compounds showed certain cytotoxicities to five human cancer cell lines with an IC50 range of 1.4-29.4 µM.

A general gas-assisted three-liquid-phase extraction method for separation and concentration of puerarin, 3'-methoxydaidzin, puerarinxyloside, daidzin and daidzein from puerariae extract.

In this work, a general and novel separation technique gas-assisted three-liquid-phase extraction was established and applied in separating and concentrating isoflavonoids from the actual sample of puerariae extract by one step. For the gas-assisted three-liquid-phase extraction method, optimal conditions were selected: polyethylene glycol 2000 and ethyl acetate as the flotation solvent, pH 5, (NH4 )2 SO4 concentration 350 g/L in aqueous phase, N2 flow rate 30 mL/min, flotation time 50 min, and flotation twice. Five isoflavonoids compounds puerarin, 3'-methoxydaidzin, puerarinxyloside, daidzin and daidzein were separated with recoveries of 82, 84, 80, 88 and 89%, respectively. The separated products were purified by preparative high-performance liquid chromatography, and the purity of the final products was >96%. The established general gas-assisted three-liquid-phase extraction was used to separate anthraquinones from Cassiae Semen under the optimal conditions, and the recoveries were >75%. The experimental results showed that the established gas-assisted three-liquid-phase extraction method is a general technique for separating active compounds from herb extract.

Transcutaneous electric nerve stimulation over acupoints for patients with diarrhea-predominant irritable bowel syndrome: Protocol for systematic review and meta-analysis.

BACKGROUND: At present, drug therapy for diarrhea-predominant irritable bowel syndrome (IBS-D) has made great progress; however, it does not often produce a satisfying curative effect. Transcutaneous electric nerve stimulation over acupoints (Acu-TENS) might be more effective in improving patient's symptoms and producing fewer side-effects as a result.Although with a great progress of the drug therapy for IBS-D, it is often hard to achieve its satisfactory curative effect. Acu-TENS that may be effective to improve patients' symptoms and fewer side-effects will be sought. There is no systematic review concerning the efficacy of Acu-TENS for IBS-D published. Therefore, this review aims to systematically evaluate the efficacy of Acu-TENS on IBS-D. METHODS: Four English (PubMed, EMBASE, The Cochrane Library, Web of Science) and 4 Chinese electronic databases (Biomedical Literature Database, CNKI, VIP, Wanfang Database) will be searched from their inception to November 26, 2018. Randomized controlled trials that evaluated the effect of Acu-TENS on patients with IBS-D will be included. The primary outcome measures will include average weekly stool frequency, visual analog scale (VAS), and the Bristol scale. The secondary outcome measures will include the MOS 36-item short-form health survey (SF-36), IBS Quality of Life Questionnaire (IBS-QOL), severity of IBS symptoms (IBS-SSS), and rectal perception. Quality evaluation and data extraction will be independently undertaken, respectively. The data from the eligible trials will be analyzed by RevMan5.3. RESULTS: For patients with IBS-D, this systematic review will provide evidences related to the efficacy of Acu-TENS in these evaluation aspects, stool frequency, VAS and the Bristol scale, SF-36, IBS-QOL, IBS-SSS, and rectal perception. CONCLUSION: This evidence may be useful to medical workers with regard to the use of Acu-TENS in the treatment of IBS-D.PROSPERO registration number: PROSPERO CRD442018109294.

Cordyceps sinensis Promotes the Growth of Prostate Cancer Cells.

BACKGROUND: This study aims to test whether Cordyceps sinensis (CS), the most expensive Asian nutrient supplement might stimulate growth of prostate cancer cells. METHODS: Impact of CS on growth of prostate cancer was determined in vivo and in vitro. RESULTS: Firstly, the serum testosterone level was significantly elevated in mice fed CS. Prostate glands were significantly enlarged (weight index 0.53 ± 0.04 mg/g vs. 0.31 ± 0.04 mg/g, P = 0.006). Furthermore, cell viability was increased twofold in the androgen-responsive prostate cancer cell line (VCaP) after CS treatment. This promoting effect disappeared after bicalutamide was added. In addition, serum prostate-specific antigen (PSA) in mice bearing VCaP xenografts was significantly elevated (0.66 ± 0.04 ng/ml vs. 0.26 ± 0.06 ng/ml, P < 0.001) after treatment with CS. Finally, VCaP tumors in mice treated with CS grew much faster (479.2 ± 78.74 mm3 vs. 283 ± 58.97 mm3, P = 0.074). However, the above promoting effects of CS were not observed in parallel studies using the PC-3 cell line which lacks AR expression. CONCLUSIONS: These results suggest that CS promotes growth of prostate cancer cells by increasing production of testosterone and stimulating the AR-dependent pathway. Additional studies are required to see whether CS is safely consumed by patients with prostate cancer.

Solvothermal synthesis, characterization and photocatalytic property of zirconium dioxide doped titanium dioxide spinous hollow microspheres with sunflower pollen as bio-templates.

Zirconium dioxide (ZrO2) doped titanium dioxide (TiO2) spinous hollow microspheres were successfully prepared through a facile solvothermal method using sunflower pollen as bio-templates. The products were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR), N2 adsorption-desorption isotherms and UV-Vis diffuse reflectance spectroscopy. It was found that the products have spinous microsphere morphology with an approximate diameter of 12 µm. The ZrO2 doped TiO2 hollow microspheres exhibited a higher photocatalytic activity in the degradation of Rhodamine B (RhB) in aqueous solutions under UV-light irradiation compared with TiO2 hollow microspheres and ZrO2-doped TiO2 particles. In particular, the removal of RhB followed pseudo-first-order kinetics, and 96.3% of RhB was degraded in 60 min under UV-light irradiation when ZrO2 doped TiO2 spinous hollow microspheres were used as the photocatalysts. Neutral and alkaline conditions were found to favor over acidic conditions for the photocatalytic degradation of RhB. Furthermore, scavenging experiments indicated that photogenerated holes (h+) and radicals (OH and O-2) were the main reactive species in the photocatalytic process using ZrO2 doped TiO2 hollow microspheres as the catalysts under UV light irradiation.

Separation of Four Flavonol Glycosides from Solanum rostratum Dunal Using Solvent Sublation Followed by HSCCC and Low Column Temperature Preparative HPLC.

Hyperoside, 3'-O-methylquercetin 3-O-ß-D-galactopyranoside, astragalin and 3'-O-methylquercetin 3-O-ß-D-glucopyranoside from an invasive weed Solanum rostratum Dunal were separated and purified successfully by high-speed counter-current chromatography (HSCCC) with a solvent system composed of n-hexane-ethyl acetate-methanol-water (1:7:1:7, v/v) and gradient elution mode preparative high-performance liquid chromatography (prep-HPLC) with low column temperature. In the sample pretreatment section, target compounds in aqueous extract of the weed were concentrated using solvent sublation. Two target fractions with purities of 93.75% and 93.68% were obtained from HSCCC. Their chemical structures were identified. The fraction 1 is a pure compound hyperoside and the fraction 2 is the mixture of astragalin, 3'-O-methylquercetin 3-O-ß-D-galactopyranoside and 3'-O-methylquercetin 3-O-ß-D-glucopyranoside by nuclear magnetic resonance and liquid chromatography-mass spectra. Then, the three flavonol glycosides in the fraction 2 were separated and purified successfully by prep-HPLC with low column temperature.

[Application of DNA-based electrochemical biosensor in rapid detection of Escherichia coli exist in licorice decoction].

A new method for detection of Escherichia coli exist in licorice decoction was developed by using DNA-based electrochemical biosensor. The thiolated capture probe was immobilized on a gold electrode at first. Then the aptamer for Escherichia coli was combined with the capture probe by hybridization. Due to the stronger interaction between the aptamer and the E. coli, the aptamer can dissociate from the capture probe in the presence of E. coli in licorice decoction. The biotinylated detection probe was hybridized with the single-strand capture probe. As a result, the electrochemical response to Escherichia coli can be measured by using differential pulse voltammetric in the presence of α-naphthyl phosphate. The plot of peak current vs. the logarithm of concentration in the range from 2.7×10² to 2.7×108 CFU·mL⁻¹ displayed a linear relationship with a detection limit of 50 CFU·mL⁻¹. The relative standard deviation of 3 successive scans was 2.5%，2.1%，4.6% for 2×10²ï¼Œ2×104，2×106：6 CFU·mL⁻¹ E. coli, respectively. The proposed procedure showed better specificity to E. coli in comparison to Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis. In the detection of the real extractum glycyrrhizae, the results between the proposed strategy and the GB assay showed high degree of agreement, demonstrating the designed biosensor could be utilized as a powerful tool for microbial examination for traditional Chinese medicine.

Comparative Proteomic Profiling and Biomarker Identification of Traditional Chinese Medicine-Based HIV/AIDS Syndromes.

Given the challenges in exploring lifelong therapy with little side effect for human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) cases, there is increasing interest in developing traditional Chinese medicine (TCM) treatments based on specific TCM syndrome. However, there are few objective and biological evidences for classification and diagnosis of HIV/AIDS TCM syndromes to date. In this study, iTRAQ-2DLC-MS/MS coupled with bioinformatics were firstly employed for comparative proteomic profiling of top popular TCM syndromes of HIV/AIDS: accumulation of heat-toxicity (AHT) and Yang deficiency of spleen and kidney (YDSK). It was found that for the two TCM syndromes, the identified differential expressed proteins (DEPs) as well as their biological function distributions and participation in signaling pathways were significantly different, providing biological evidence for the classification of HIV/AIDS TCM syndromes. Furthermore, the TCM syndrome-specific DEPs were confirmed as biomarkers based on western blot analyses, including FN1, GPX3, KRT10 for AHT and RBP4, ApoE, KNG1 for YDSK. These biomarkers also biologically linked with the specific TCM syndrome closely. Thus the clinical and biological basis for differentiation and diagnosis of HIV/AIDs TCM syndromes were provided for the first time, providing more opportunities for stable exertion and better application of TCM efficacy and superiority in HIV/AIDS treatment.

Evolutionary history of Coleoptera revealed by extensive sampling of genes and species.

Beetles (Coleoptera) are the most diverse and species-rich group of insects, and a robust, time-calibrated phylogeny is fundamental to understanding macroevolutionary processes that underlie their diversity. Here we infer the phylogeny and divergence times of all major lineages of Coleoptera by analyzing 95 protein-coding genes in 373 beetle species, including ~67% of the currently recognized families. The subordinal relationships are strongly supported as Polyphaga (Adephaga (Archostemata, Myxophaga)). The series and superfamilies of Polyphaga are mostly monophyletic. The species-poor Nosodendridae is robustly recovered in a novel position sister to Staphyliniformia, Bostrichiformia, and Cucujiformia. Our divergence time analyses suggest that the crown group of extant beetles occurred ~297 million years ago (Mya) and that ~64% of families originated in the Cretaceous. Most of the herbivorous families experienced a significant increase in diversification rate during the Cretaceous, thus suggesting that the rise of angiosperms in the Cretaceous may have been an 'evolutionary impetus' driving the hyperdiversity of herbivorous beetles.

Magnetic nanoparticles and high-speed countercurrent chromatography coupled in-line and using the same solvent system for separation of quercetin-3-O-rutinoside, luteoloside and astragalin from a Mikania micrantha extract.

A new in-line method of magnetic nanoparticles (MNPs) coupled with high-speed countercurrent chromatography (HSCCC) using a same solvent system during the whole separation process was established to achieve the rapid separation of flavonoids from Mikania micrantha. The adsorption and desorption capacities of five different MNPs for flavonoid standards and Mikania micrantha crude extract were compared and the most suitable magnetic nanoparticle Fe3O4@SiO2@DIH@EMIMLpro was selected as the in-line MNP column. An in-line separation system was established by combining this MNP column with HSCCC through a six-way valve. The comparison between two solvent systems n-hexane-ethyl acetate-methanol-water (3:5:3:5, v/v) and ethyl acetate-methanol-water (25:1:25, v/v) showed that the latter solvent system was more suitable for simultaneously in-line separating three flavonoids quercetin-3-O-rutinoside, luteoloside and astragalin from Mikania micrantha. The purities of these three compounds with the ethyl acetate-methanol-water solvent system were 95.13%, 98.54% and 98.19% respectively. Results showed the established in-line separation system of MNP-HSCCC was efficient, recyclable and served to isolate potential flavonoids with similar polarities from natural complex mixtures. The in-line combination of magnetic nanoparticles with high-speed countercurrent chromatography eluting with the same solvent system during the whole separation process was established for the first time.

A general ionic liquid pH-zone-refining countercurrent chromatography method for separation of alkaloids from Nelumbo nucifera Gaertn.

The alkaloids from lotus (Nelumbo nucifera Gaertn) are effective in lowering hyperlipemia and level of cholesterol. However, there is not a general method for their separation. In this work, a general ionic liquid pH-zone-refining countercurrent chromatography method for isolation and purification of six alkaloids from the whole lotus plant was successfully established by using ionic liquids as the modifier of the two-phase solvent system. The conditions of ionic liquid pH-zone-refining countercurrent chromatography, involving solvent systems, concentration of retainer and eluter, types of ionic liquids, the content of ionic liquids as well as ionic liquids posttreatment, were optimized to improve extraction efficiency. Finally, the separation of these six alkaloids was performed with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water-[C4mim][PF6] at a volume ratio of 5:2:2:8:0.1, where 10mM TEA was added to the organic stationary phase as a retainer and 3mM HCl was added to the aqueous mobile phase as an eluter. As a result, six alkaloids including N-nornuciferine, liensinine, nuciferine, isoliensinine, roemerine and neferine were successfully separated with the purities of 97.0%, 90.2%, 94.7%, 92.8%, 90.4% and 95.9%, respectively. The established general method has been respectively applied to the crude samples of lotus leaves and lotus plumules. A total of 37.3mg of liensinine, 57.7mg of isoliensinine and 179.9mg of neferine were successfully purified in one run from 1.00g crude extract of lotus plumule with the purities of 93.2%, 96.5% and 98.8%, respectively. Amount of 45.6mg N-nornuciferine, 21.6mg nuciferine and 11.7mg roemerine was obtained in one step separation from 1.05g crude extract of lotus leaves with the purity of 96.9%, 95.6% and 91.33%, respectively.

The effects of Chinese medicines on cAMP/PKA signaling in central nervous system dysfunction.

Neuropathological injury in the mammalian adult central nervous system (CNS) may cause axon disruption, neuronal death and lasting neurological deficits. Failure of axon regeneration is one of the major challenges for CNS functional recovery. Recently, the cAMP/PKA signaling pathway has been proven to be a critical regulator for neuronal regeneration, neuroplasticity, learning and memory. Also, previous studies have shown the effects of Chinese medicines on the prevention and treatment of CNS dysfunction mediated in part by cAMP/PKA signaling. In this review, the authors discuss current knowledge of the role of cAMP/PKA signaling pathway in neuronal regeneration and provide an overview of the Chinese medicines that may enable CNS functional recovery via this signaling pathway.

Isolation of four phenolic compounds from Mangifera indica L. flowers by using normal phase combined with elution extrusion two-step high speed countercurrent chromatography.

Two-step high speed countercurrent chromatography method, following normal phase and elution-extrusion mode of operation by using selected solvent systems, was introduced for phenolic compounds separation. Phenolic compounds including gallic acid, ethyl gallate, ethyl digallate and ellagic acid were separated from the ethanol extract of mango (Mangifera indica L.) flowers for the first time. In the first step, gallic acid of 3.7mg and ethyl gallate of 3.9mg with the purities of 98.87% and 99.55%, respectively, were isolated by using hexane-ethylacetate-methanol-water (4:6:4:6, v/v) in normal phase high speed countercurrent chromatography from 200mg of crude extract, while ethyl digallate and ellagic acid were collected in the form of mixture fraction. In the second step, further purification of the mixture was carried out with the help of another selected solvent system of dichloromethane-methanol-water (4:3:2, v/v) following elusion-extrusion mode of operation. Ethyl digallate of 3.8mg and ellagic acid of 5.7mg were separated well with high purities of 98.68% and 99.71%, respectively. The separated phenolic compounds were identified and confirmed by HPLC, UPLC-QTOF/ESI-MS, 1H and 13C NMR spectrometric analysis.

Separation of four flavonol glycosides from Solanum rostratum Dunal using aqueous two-phase flotation followed by preparative high-performance liquid chromatography.

Aqueous two-phase flotation followed by preparative high-performance liquid chromatography was used to separate four flavonol glycosides from Solanum rostratum Dunal. In the aqueous two-phase flotation section, the effects of sublation solvent, solution pH, (NH4 )2 SO4 concentration in aqueous solution, cosolvent, N2 flow rate, flotation time, and volumes of the polyethylene glycol phase on the recovery were investigated in detail, and the optimal conditions were selected: 50 wt% polyethylene glycol 1000 ethanol solvent as the flotation solvent, pH 4, 350 g/L of (NH4 )2 SO4 concentration in aqueous phase, 40 mL/min of N2 flow rate, 30 min of flotation time, 10.0 mL of flotation solvent volume, and two times. After aqueous two-phase flotation concentration, the flotation products were purified by preparative high-performance liquid chromatography. The purities of the final products A and B were 98.1 and 99.0%. Product B was the mixture of three compounds based on the analysis of high-performance liquid chromatography at the temperature of 10°C, while product A was hyperoside after the identification by nuclear magnetic resonance. Astragalin, 3'-O-methylquercetin 3-O-ß-d-galactopyranoside, and 3'-O-methylquercetin 3-O-ß-d-glucopyranoside were obtained with the purity of 93.8, 97.1, and 99.2%, respectively, after the further separation of product B using preparative high-performance liquid chromatography.

Biological Activities of Ethanol Extracts of Phellinus baumii (Higher Basidiomycetes) Obtained by Different Fermentation Methods.

Phellinus baumii was used for fermentation, and 3 corresponding ethanol extracts were obtained by 3 different methods: extract I, liquid fermentation; extract II, solid fermentation in polypropylene plastic bags with medium mainly consisting of sawdust and wheat bran; and extract III, solid fermentation in culture bottles with medium mainly consisting of rice. Ethanol extract I presented the best inhibition ability on HepG2 cell growth; inhibiting rates were 48.2% and 65.0% at doses of 10 and 100 µg/mL, respectively. Ethanol extracts II and III had a better regeneration effect on injured PC12 neural cells than extract I. The superoxide radical, hydrogen peroxide radical, and DPPH radical scavenging activities of ethanol extract III was better than those of the other 2 extracts.

[The influence of further processing on Psoralea corylifolia alcohol extracts and the content of psoralen and isopsoralen].

OBJECTIVE: To improve the processing method of Psoralea corylifolia. METHODS: The self-made processing machine was used to flatten Psoralea corylifolia. Then it was extracted with water soluble and alcohol soluble with Psoralea corylifolia unflatten as the amount of Psoralea corylifolia and the content of psoralen and isopsoralen were factors. HPLC was used. The analytical column was a Supelco ODS-C18. The mobile phase consisted of methanol-water (43:57). The detector wavelength was 246 nm. The column temperature was 30 degrees C. RESULTS: Under the same condition, the conditioning treatment increased the water or ethanol extracting amount of Psoralea corylifolia and the content of psoralen and isopsoralen of the processing samples compared with the unconditioning treatment. The amount of water soluble extracts and erhanol soluble extracts increased 0.61 times and 1.53 times, respectively. And the content of psoralen and isopsoralen in water soluble and erhanol soluble extracts increased 1.22 times. CONCLUSION: The method to flatten Psoralea corylifolia can significantly improve the content of extraction and extraction rate of active components. It can also enhance the clinical effectiveness. The method is reasonable and deserves to be popularized.

Demethylbellidifolin preserves endothelial function by reduction of the endogenous nitric oxide synthase inhibitor level.

The present study examined the anti-oxidation and protective effects of demethylbellidifolin (DMB), a xanthone compound extracted from swertia davidi Franch, on endothelium. The relationship between the protective effects of DMB on endothelium and the level of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, was also determined in the present study. DMB significantly inhibited Cu(2+)-induced low-density lipoprotein (LDL) oxidation and scavenged DPPH radicals. DMB significantly attenuated the inhibition of endothelium-dependent vasodilator responses, induced by lysophosphatidycholine (LPC) in vitro and LDL in vivo, and increased release of lactate dehydrogenase induced by LDL in cultured endothelial cells. DMB significantly attenuated the increased concentration of malondialdehyde and ADMA, and the decreased level of nitric oxide induced by LDL in vivo and in cultured endothelial cells. DMB also significantly reduced the decreased activity of dimethylarginine dimethylaminohydrolase (DDAH) induced by LDL in cultured endothelial cells. In summary, the present results suggest that DMB protects endothelial damage induced by LPC in vitro and LDL in vivo or in endothelial cells, and the protective effect of DMB on the endothelium is related to reduction of ADMA concentration via an increase of DDAH activity by inhibition of lipid peroxidation.