TiO2 nanotube immobilised 5-lipoxygenase-mediated screening and isolation of anti-inflammatory active compounds from the leaves of lonicera japonica thunb

Abstract In this work, a highly effective separation approach mediated by 5-Lipoxygenase (5-LOX) was established for screening and isolation of anti-inflammatory ingredients from leaves of Lonicera japonica Thunb. (LLJT). Using 5-LOX immobilised on TiO2 nanotubes as a microreactor, the targeted screening was exploited by combining with HPLC-MS system. Four compounds confirmed as luteolin, luteoside, lonicerin, and isochlorogenic acid C and a fraction (M1) were screened out to be potent inhibitors of 5-LOX. Their anti-inflammatory activities were further investigated and confirmed by RAW 264.7 cells inflammation model and rat foot swelling model. Furthermore, M1 was prepared by MCI GEL CHP20P column chromatography, and further separated by Pre-HPLC. One new compound confirmed to be 5,7,3′,4′-tetrahydroxyflavone-7-O-sambubioside was first isolated from LLJT. The results provide a new method for the effective separation of active components derived from natural products. Highlights A 5-LOX mediated separation method was established for isolation of anti-inflammatory compounds. An anti-inflammatory ingredient was separated by MCI GEL CHP20P column chromatography. One new compound was first isolated from leaves of Lonicera japonica Thunb. 5-LOX was immobilised on TiO2 nanotubes and exploited by combining with HPLC-MS system. The anti-inflammatory activity of screened components was evaluated.

by mixing 25 mL double distilled water+225 mL methanol+340 μL concentrated sulfuric acid+24.5 mg ferrous ammonium sulfate hexahydrate+19 mg xylenol orange), and incubated at 25 °C for 10 min. And finally the absorbance of the system at 590 nm was determined.
Each concentration was replicated 6 times. The formula for calculating the inhibition rate of 5-LOX activity is as follows: where Ablank is the absorption resulting from the enzymatic catalyzing without the inhibitor, and Atested is the absorption resulting from the enzymatic catalyzing in the presence of the inhibitor (sample).
IC50 is the concentration of the test sample at which the inhibition rate reaches 50%.

Cell experiment
The RAW264.7 mouse macrophage cell line (RAW264.7) was cultured in DMEM (Dulbecco's modified eagle medium) supplemented with 10% (v/v) Fetal bovine serum (FBS), 1×10 5 U·L -1 penicillin and 100 mg·L -1 streptomycin under an atmosphere of humidified 5% CO2 at 37 °C to the logarithmic phase. The samples were dissolved in DMSO and diluted with medium to the desired concentration. The content of DMSO should not exceed 1‰.
There are blank group (no test sample, no LPS), model group (100 ng/mL LPS), experimental group (60 μM test sample + LPS) and negative control group (60 μM test sample) were tested.
For cell morphology observation, the cells were seeded into 6-well plates at an inoculation amount of 6×10 5 /well, cultured at 37 °C and 5% CO2 for 24 h, and then the medium was changed. After 2 h incubation with different groups, LPS stimulation with a final concentration of 100 ng/mL were added into the model group and experimental group. The

Rat foot swelling model
All the animals were acclimated under standard laboratory conditions and had free access to standard water and food. All procedures were conducted in accordance with the "Guiding Principles in the Care and Use of Animals" (China) and were approved by the Ethics Committee of Biomedical Scientific Research of Henan University.
Thirty two male and female rats were divided into four groups with eight rats in each group.
They were normal saline negative control group, dexamethasone positive control group, n-butanol phase of Lonicera japonica Thunb. leaves (RDY nB) administration group and the RDY M1 administration group, respectively. The intraperitoneal injection method was used for administration, and the detailed information of administration is shown in Table S1.
[ Table S1 near here] One hour after injection the corresponding drugs in each group, 0.1 mL of fresh egg white was injected into the right postal paw of each rat. Before and after the injection of egg white at 10, 20, 30, 40, 50, and 60 min, the volume of the right postal paw of rat was measured by a toe volume meter, and the swelling rate was calculated.

Statistical analysis
Statistical analysis was analyzed by one-way ANOVA through SPSS 17.0 software, and all data expressed as mean ± SD. Meanwhile, the significant difference between groups was evaluated by Tukey post-hoc test, P<0.05 and P<0.01 were considered statistically significant.

Anti-inflammatory effect research results
In order to explore the anti-inflammatory applications of the screened components, RAW264.7 cells was used to investigate.

Effect of screened components (60 μM) on morphology of RAW264.7 cells
As shown in Figure S1, Isochlorogenic acid C, lonicerin, and M1 had no significant effect on normal cells, but they can improve the deformation of the inflammatory cells, thereby protect cells.

Effect of the test substance on the viability of RAW264.7 cells
From Table S2 and Table S3, it can be seen that the three test concentrations ( [ Based on the above results, it can be seen that isochlorogenic acid C, lonicerin and M1 had low toxicity on RAW264.7 cells and had anti-inflammatory effects, which can be expected to be applied as anti-inflammatory drugs derived from natural products for further research.

Anti-inflammatory effect of M1 on foot swelling rat
Based on the superior anti-inflammatory effect of M1 on the mouse macrophage cell line RAW264.7, we used the rat foot swelling model to further research its anti-inflammatory activity. Because M1 were prepared from the n-butanol phase (RDY nB), so the anti-inflammatory activity of RDY nB were also investigated.
Dexamethasone, a commonly used drug for treating inflammation, was employed as a positive control, and normal saline was used as a negative control. The anti-inflammatory effect was investigated by the swelling rate. The lower the swelling rate, the better the anti-inflammatory activity. The result was shown in Figure S2.
It can be seen that the swelling rate of the negative control group was much higher than that of the other administration groups. In addition, the swelling rate reached the highest at 30 min, and then it gradually decreased, indicating that the inflammation caused by the injection of egg white would subside after 30 min due to the autoimmunity of the mice. Therefore, if the inflammation could be suppressed within 30 minutes, the drug can be expected to be used in the treatment of inflammation.
[ Figure S2 near here] Dexamethasone, as a positive control group, showed a good anti-inflammatory effect relative to the negative control. The foot swelling rate of rats treated with n-butanol phase (RDY n-B) was between the negative control group and the positive control group, indicating that it had a certain anti-inflammatory effect, but the anti-inflammatory effect was weaker than that of dexamethasone. The foot swelling rate of in the group administered with M1 (RDY M1) was lower than that of dexamethasone in the first 30 min, which indicated that M1 could better inhibit the production of inflammation from the initial stage to the peak period of inflammation, and its effect was better than that of dexamethasone, which is a hormone drug.
After the peak of inflammation (30 min later), its anti-inflammatory effect was slightly lower Information Classification: General than that of dexamethasone, and still better than that of n-butanol group, indicating that its anti-inflammatory effect was excellent. The experimental results further indicated that M1 was the main anti-inflammatory active ingredient in n-butanol phase of LLJT extract. In conclusion, the active ingredient of M1 in LLJT isolated by MCI GEL CHP20P column chromatography had excellent anti-inflammatory effect. Although dexamethasone is widely used as anti-inflammatory drug, it is a hormone drug, which has adverse effects on the body for long-term use. Therefore, M1 with excellent anti-inflammatory effect is expected to be applied to the research and development of natural anti-inflammatory drugs.