Preparation and Antioxidant Activities of Phenylethanoids from Dracocephalum heterophyllum

The health benefits of Dracocephalum heterophyllum are widely reported in traditional Tibetan medicines, but the reported chemical composition is limited, probably due to difficulties in separating and purifying compounds. In this study, antioxidative phenylethanoids were isolated from an extract of Dracocephalum heterophyllum using medium- and high-pressure liquid chromatography, coupled with on-line HPLC–1,1-diphenyl-2-picrylhydrazyl recognition. Firstly, crude samples (1.3 kg) of Dracocephalum heterophyllum were pretreated via silica gel medium-pressure liquid chromatography to yield 994.0 g of Fr2, of which 10.8 g was then pretreated via MCI GEL®CHP20P medium-pressure liquid chromatography. The resulting Fr23 and Fr25 were further separated and purified using high-pressure liquid chromatography, and yielded 8.08 mg of Fr2391, 9.76 mg of Fr2551, 16.09 mg of Fr2581, and 8.75 mg of Fr2582. Furthermore, analysis of the purity and structures of the phenylethanoids suggested that Fr2391, Fr2551, Fr2581, and Fr2582 corresponded to decaffeoylverbascoside, rosmarinic acid, acteoside, and 2′-O-acetylplantamajoside, respectively, with all being over 95% pure. Finally, the antioxidant potential of the compounds was explored based on their ability to scavenge 1,1-diphenyl-2-picrylhydrazine, as well as through molecular docking of proteins related to antioxidant pathways. Altogether, our findings revealed that the proposed method is promising for separating pure antioxidative phenylethanoids from other natural compounds

Preparation and Antioxidant Activities of Phenylethanoids from <i>Dracocephalum heterophyllum</i>

The health benefits of Dracocephalum heterophyllum are widely reported in traditional Tibetan medicines, but the reported chemical composition is limited, probably due to difficulties in separating and purifying compounds. In this study, antioxidative phenylethanoids were isolated from an extract of Dracocephalum heterophyllum using medium- and high-pressure liquid chromatography, coupled with on-line HPLC–1,1-diphenyl-2-picrylhydrazyl recognition. Firstly, crude samples (1.3 kg) of Dracocephalum heterophyllum were pretreated via silica gel medium-pressure liquid chromatography to yield 994.0 g of Fr2, of which 10.8 g was then pretreated via MCI GEL®CHP20P medium-pressure liquid chromatography. The resulting Fr23 and Fr25 were further separated and purified using high-pressure liquid chromatography, and yielded 8.08 mg of Fr2391, 9.76 mg of Fr2551, 16.09 mg of Fr2581, and 8.75 mg of Fr2582. Furthermore, analysis of the purity and structures of the phenylethanoids suggested that Fr2391, Fr2551, Fr2581, and Fr2582 corresponded to decaffeoylverbascoside, rosmarinic acid, acteoside, and 2′-O-acetylplantamajoside, respectively, with all being over 95% pure. Finally, the antioxidant potential of the compounds was explored based on their ability to scavenge 1,1-diphenyl-2-picrylhydrazine, as well as through molecular docking of proteins related to antioxidant pathways. Altogether, our findings revealed that the proposed method is promising for separating pure antioxidative phenylethanoids from other natural compounds

rLj-RGD3, a Novel Recombinant Toxin Protein from <i>Lampetra japonica</i>, Protects against Cerebral Reperfusion Injury Following Middle Cerebral Artery Occlusion Involving the Integrin-PI3K/Akt Pathway in Rats

<div><p>Background</p><p>The RGD-toxin protein Lj-RGD3 is a naturally occurring 118 amino acid peptide that can be obtained from the salivary gland of the <i>Lampetra japonica</i> fish. This unique peptide contains 3 RGD (Arg-Gly-Asp) motifs in its primary structure. Lj-RGD3 is available in recombinant form (rLj-RGD3) and can be produced in large quantities using DNA recombination techniques. The pharmacology of the three RGD motif-containing peptides has not been studied. This study investigated the protective effects of rLj-RGD3, a novel polypeptide, against ischemia/reperfusion-induced damage to the brain caused by middle cerebral artery occlusion (MCAO) in a rat stroke model. We also explored the mechanism by which rLj-RGD3 acts by measuring protein and mRNA expression levels, with an emphasis on the FAK and integrin-PI3K/Akt anti-apoptosis pathways.</p><p>Methods</p><p>rLj-RGD3 was obtained from the buccal secretions of <i>Lampetra japonica</i> using gene recombination technology. Sprague Dawley (SD) rats were randomly divided into the following seven groups: a sham group; a vehicle-treated (VT) group; 100.0 μg·kg^-1, 50.0 μg·kg^-1 and 25.0 μg·kg^-1 dose rLj-RGD3 groups; and two positive controls, including 1.5 mg·kg^-1 Edaravone (ED) and 100.0 μg·kg^-1 Eptifibatide (EP). MCAO was induced using a model consisting of 2 h of ischemia and 24 h of reperfusion. Behavioral changes were observed in the normal and operation groups after focal cerebral ischemia/reperfusion was applied. In addition, behavioral scores were evaluated at 4 and 24 h after reperfusion. Brain infarct volumes were determined based on 2,3,5-triphenyltetrazolium chloride (TTC) staining. Pathological changes in brain tissues were observed using hematoxylin and eosin (H&E) staining. Moreover, neuronal apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) assays. We determined the expression levels of focal adhesion kinase (FAK), phosphatidyl inositol 3-kinase (PI3K), protein kinase B (Akt, PKB), caspase-3 and Bcl-2 in the brain using western blot analysis and RT-PCR assays. The research protocol was approved by the Institutional Ethics Committee of Dalian Medical University.</p><p>Results</p><p>The behavioral scores and cerebral infarct volumes of the rLj-RGD3 groups were markedly lower at 4 and 24 h/RF. The rLj-RGD3 protein significantly ameliorated pathological changes in the brain and reduced the number of apoptotic neurons. Furthermore, the FAK and PI3K/Akt pathways were activated. rLj-RGD3 significantly increased the expression of FAK, p-FAK and Bcl-2 proteins. In contrast, caspase-3 expression was inhibited.</p><p>Conclusion/Significance</p><p>We conclude that recombinant <i>Lampetra japonica</i> RGD-peptide (rLj-RGD3) exerts a protective effect against cerebral ischemia/reperfusion injury in the brain. In addition, the mechanism of this protection is associated with the activation of the integrin-PI3K/Akt pathway. These results provide a theoretical foundation and an experimental basis for using RGD peptides as novel drugs for treating ischemic cerebral vascular diseases in addition to promoting the research and development of marine biotechnology drugs.</p></div

The role of PI3K/Akt signaling in the neuroprotective effects of rLj-RGD3 against OGD/R-induced PC12 cells apoptosis.

<p>P-Akt, caspase-3 and Bcl-2 expression levels in PC12 cells were assessed by western blotting. Our results show that OGD/R significantly inhibited Akt phosphorylation, but pretreatment with rLj-RGD3 attenuated the effects of OGD/R, indicating that rLj-RGD3 activates the PI3K/Akt pathway. MK-2206 significantly inhibited the effects of rLj-RGD3 treatment. The data are shown as the means ± SD, n = 3. ^## <i>p</i><0.01 <i>vs</i>. the control group, **<i>p</i> <0.01 <i>vs</i>. the OGD/R group, ^& <i>p</i><0.05 <i>vs</i>. the rLj-RGD3 group, ^&& <i>p</i><0.01 <i>vs</i>. the rLj-RGD3 group.</p

PI3K, caspase-3 and Bcl-2 mRNA levels detected using RT-PCR analysis in focal cerebral ischemia/reperfusion rats.

<p>PI3K and Bcl-2 mRNA expression in the ischemic cortexes of the cerebral ischemia/reperfusion rats was higher in the rLj-RGD3 groups than in the VT group. The level of caspase-3 mRNA was significantly lower in the rLj-RGD3 groups. rLj-RGD3 up-regulates PI3K and Bcl-2 mRNA levels and down-regulates caspase-3 mRNA expression. We therefore conclude that rLj-RGD3 is able to activate the PI3K/AKT anti-apoptosis pathway. The drug Eptifibatide resulted in expression levels that were less significantly higher than those in the rLj-RGD3 100.0 μg·kg^-1 group (p <0.05). ^## <i>p</i><0.01 <i>vs</i>. the sham group, **<i>p</i> <0.01 <i>vs</i>. the VT group. The data are shown as the means ± SD. For RT-PCR, one-way ANOVA was used to compare DNA bands between groups after ischemia.</p

The effect of rLj-RGD3 on neurological deficit scores in focal cerebral ischemia/reperfusion rats.

<p>Neurological evaluations were used to assess rat behaviors after middle cerebral artery occlusion and reperfusion. The scores in the rLj-RGD3 groups were significantly lower (by 4 and 3) than that in the VT group (**<i>p</i> <0.01). rLj-RGD3 improved nerve functional deficits. ^##<i>p</i> <0.01 <i>vs</i>. the sham group. The data are shown as the means ± SD, n = 6.</p

The effect of rLj-RGD3 on infarct volume in the brains of focal cerebral ischemia/reperfusion rats.

<p>a, b, c, d, e, f and g in panel A represent the sham group, the VT group, the rLj-RGD3 100.0 μg·kg^-1 group, the rLj-RGD3 50.0 μg·kg^-1 group, the rLj-RGD3 25.0 μg·kg^-1 group, the Edaravone 1.5 mg·kg^-1 group, and the Eptifibatide 100.0 μg·kg^-1 group, respectively. TTC is a fat-soluble, light-sensitive complex. Normal tissue becomes red when dehydrogenase reacts with TTC. In contrast, ischemic tissue appears white because it lacks dehydrogenase. The greatest infarct volumes (approximately 40%) were observed in the VT group, and the infarct volumes were approximately 35% lower in the high-dose rLj-RGD3 group (**<i>p</i> <0.01 <i>vs</i>. the VT group). The data are shown as the means ± SD, n = 6.</p

The effects of rLj-RGD3 on caspase-3 and Bcl-2 protein expression in focal cerebral ischemia/reperfusion rats as assayed using western blot analysis.

<p>The expression of caspase-3 protein was significantly higher in the VT group than in the sham group (^##<i>p</i> <0.01). The rLj-RGD3 groups exhibited markedly lower levels of caspase-3 expression (**<i>p</i> <0.01 <i>vs</i>. the VT group). The anti-apoptosis gene Bcl-2 was markedly up-regulated in the rLj-RGD3 treatment groups, which demonstrated that rLj-RGD3 inhibits apoptosis. The data are shown as the means ± SD. For the western blotting analysis, one-way ANOVA was used to compare protein expression levels between the ischemia and sham groups.</p

Neuronal apoptosis in the brains of focal cerebral ischemia/reperfusion rats as shown by TUNEL assay.

<p>A, B, C, D, E, and F represent the sham group, the VT group, the rLj-RGD3 100.0 μg·kg^-1 group, the rLj-RGD3 50.0 μg·kg^-1 group, the Edaravone 1.5 mg·kg^-1 group, and the Eptifibatide 100.0 μg·kg^-1 group, respectively. Neuronal apoptosis was not observed in the sham group. In contrast, a significant amount of neuronal apoptosis was observed in the VT group. Neuronal apoptosis was lower in all drug administration groups than in the VT group. Moreover, neuronal apoptosis was markedly lower in the high-dose group (rLj-RGD3 100.0 μg·kg^-1) than in the other pretreatment groups.</p

The amino acid sequence of rLj-RGD3.

<p>Histidines are highlighted in yellow; arginines are highlighted in green.</p