**3. Results**

### *3.1. Physical and Chemical Analysis on the Sa*ffl*ower Honey*

All data regarding the physical and chemical indicators of Xinjiang sa fflower honey (see Table 3), using the relevant methods in the EU standard, revealed better results than the standard values cited. Among them, the moisture content was 18.2%, which meets the EU standard for honey (not more than 20%). The acidity (1 mol/L sodium hydroxide titration) was 25.0 mL/kg, which is lower than the EU standard limit of 50 mL/kg. Hydroxymethylfurfural, sucrose, and ash were not detected; the three were far below the relevant EU standard limits. The fructose content was 36.9%, the glucose content was 25.2%, and the total of the two was 62.1%, higher than the EU standard (not less than 60%); the amylase value was 21.1 mL/(g·h), which is much higher than the EU standard (not less than 8), which is twice the EU requirements.



Note: ND means not detected.

#### *3.2. Preliminary Analysis of Phenolic Flavonoids in ECH by HPLC-QTOF-MS*

HPLC-QTOF-MS detection of ECH was carried out with 24 phenolic flavonoid standards. As shown in Table 4, eight phenolic acids were detected, with a total content of 7.197 mg/kg honey. Vanillic acid and p-hydroxybenzoic acid were the highest phenolic acids, with 3.196 mg/kg honey and 1.524 mg/kg honey, respectively. Protocatechualdehyde was not detected. Seven flavonoids

were detected, with a total content of 7.633 mg/kg honey, with quercetin and myricetin being the highest, with 3.196 mg/kg honey and 1.524 mg/kg honey, respectively. Rutin, quercetin-3-O-glucoside, kaempferol, naringenin and pinobanksin were low in content. Morin, luteolin, diosmetin, pinocembrin, galanin, caffeic acid phenethyl ester, chrysin and kaempferol-3-O-glucoside were not detected. As far as the content of safflower honey phenolic acid flavonoids is concerned, the phenolic acid components were characterized by vanillic acid and p-hydroxybenzoic acid, and the flavonoids were characterized by quercetin and myricetin. The diversity and content of phenolic acid and flavonoids play a positive role in the antioxidant and anti-inflammatory activities of safflower honey.


**Table 4.** High-performance liquid chromatography combined with a quadrupole time-of-flight mass. spectrometry (HPLC-QTOF/MS) analysis of phenolic flavonoids in safflower.

> Note: ND means not detected.

#### *3.3. In Vitro Antioxidant Free Radical Scavenging Capacity of ECH*

DPPH and ABTS+ free radical scavenging experiments are commonly used to evaluate natural antioxidants [15]. It can be seen from Table 5 that the concentration of ECH inhibiting 50% DPPH free radical was 68.23 ± 0.40 μg/mL, and the concentration of ECH inhibiting 50% ABTS+ free radical was 81.88 ± 0.54 μg/mL.

**Table 5.** In vitro ECH (extract from *Carthamus tinctorius* L. honey) free radical scavenging activity.


### *3.4. In Vitro Antioxidant, Anti-Inflammatory Activies by ECH*

#### 3.4.1. Effects of ECH on Raw 264.7 Cell Survival

By treating Raw 264.7 cells with ECH in concentrations from 2.5 to 20 μg/mL and using the CCK-8 reagen<sup>t</sup> to detect cell viability, we found the most suitable ECH concentration to ensure that adding ECH concentration to Raw 264.7 cells produced no obvious toxic effects (see Figure 1). Compared with the control group, there was no significant difference in the growth of Raw 264.7 cells when the ECH

concentration were 2.5 μg/mL and 5 μg/mL. When the ECH concentration was 10 μg/mL, the Raw 264.7 cells growth was highly significantly inhibited. When the ECH concentrations were 15 μg/mL and 20 μg/mL, the inhibitory effect on the growth of Raw 264.7 cells was highly significant. Therefore, the safe concentration of ECH available in this experiment was 2.5–5 μg/mL.

**Figure 1.** The effects of various concentrations of ECH on RAW 264.7 cell viability. Raw 264.7 cells were pretreated with ECH (2.5 to 20 μg/mL) or not for 24 h, cell viability was tested using the CCK-8 method. \*\* (*p* < 0.01) and \*\*\* (*p* < 0.001) indicates significant difference compared with the control group.

#### 3.4.2. Effect of ECH on LPS-Induced Nitric Oxidase (NO) Release in RAW 264.7 Cells

A typical Griess method was used to determine the amount of NO released by different treatment cells (see Figure 2). Dexamethasone (DXMS) treatment at 100 μg/mL (referred as DXMS-100) to the LPS-activated cells was used as the positive control group. Compared with the Control group, the concentrations of NO releases in Raw 264.7 cells was increased about nine times following LPS treatment, indicating the model of Raw 264.7 cells inflammation model was successfully established. After co-treatment with ECH and LPS, the NO release decreased by about two-thirds compared with the LPS group, and the decreasing effects even better than the DXMS-100 group. Therefore, ECH was shown to effectively reduce the amount of NO released during macrophage inflammation induced by LPS.

**Figure 2.** The effects of ECH on the nitric oxide (NO) release in LPS-activated macrophages. RAW 264.7 cells were pretreated with/without indicated concentrations of ECH or dexamethasone (100 μg/mL, positive control) for 1 h then stimulated with LPS (1 μg/mL) for 24 h. NO concentrations in the cell culture medium were measured using the Griess method. \*\* (*p* < 0.01) indicates significant difference compared with the LPS group.

3.4.3. Effect of ECH on LPS-Induced Inflammation and Oxidation-Related Gene Expression in RAW 264.7 Cells

Next, RT-qPCR was applied to detect the expression levels of inflammation and oxidation-related genes in LPS-induced Raw 264.7 cells (see Figure 3). After LPS treatment, the expression of NQO decreased significantly, the expression of iNOS, IL-1β, TNF-α and MCP-1 increased significantly. Compared with cells treated with LPS alone, the expressions of iNOS, IL-1β, TNF-α and MCP-1 in ECH-treated groups were decreased, and the inhibitory effect was stronger with the increase of the ECH concentration. For HO-1, TXNRD, and NQO, ECH significantly increased these antioxidant-related gens expressions.

**Figure 3.** Effects of ECH on the expressions of antioxidant and inflammatory genes in LPS stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with/without indicated concentrations of ECH or dexamethasone (100 μg/mL, positive control) for 1 h then stimulated with LPS (1 μg/mL) for 6 h. mRNA expression in RAW 264.7 cells were measured using RT-qPCR. \*\* (*p* < 0.01) and \*\*\* (*p* < 0.001) indicates significant difference compared with the LPS group. ## (*p* < 0.01) and ### (*p* < 0.001), compared with the normal control group.

3.4.4. Effect of ECH on the Expressions of Inflammation and Anti-Oxidant Signaling Related Proteins in LPS-Activated RAW 264.7 Cells

The expression of inflammatory and anti-oxidation-related proteins in LPS-induced Raw 264.7 cells was detected by western blot (Figure 4). The expression level of P-IκBαin the LPS group was significantly higher than that of IκBα, however, after ECH treatment, the expression of P-IκBα in the LPS induced group was significantly reduced compared to the LPS treatment group only. IκBα phosphorylation was inhibited, and 5 μg/mL was found with a more potent inhibitive effects thant the low dosage group (2.5 μg/mL). The expression levels of Nrf-2 and HO-1 increased significantly in cells induced by LPS after ECH pretreatment. When the concentration of ECH increased, the expression levels of Nrf-2 and HO-1 increased more obviously.

**Figure 4.** Effect of ECH on the expressions of inflammation and anti-oxidant signaling related proteins in LPS-induced activated RAW 264.7 cells RAW 264.7 cells were pretreated or not with indicated concentrations of ECH for 1 h then were activated with LPS (1 μg/mL) for 30 min (left) or 6 h (right). Whole cell lysates were analyzed by Western blotting analysis using specific antibodies.

3.4.5. Effect of ECH on the Nuclear Localization of NF-κB-p65 Induced by LPS

A laser confocal scanning microscope was used explore the effect of 5 μg/mL ECH on the nuclear localization of NF-κB-p65 in LPS-induced inflammatory cells (see Figure 5). Compared with the control group, NF-κB-p65 entry into the nucleus was significantly increased during LPS treatment alone, indicating with the activation of NF-κB. After 5 μg/mL ECH incubation and LPS treatment, NF-κB-p65 entry into the nucleus was significantly inhibited; DXMS-100 group treatment of NF-κB-p65 into the nucleus was also significantly inhibited.

**Figure 5.** The inhibited effects of ECH on the transport from the cytoplasm to the nucleus of NF-κB-p65 proteins.
