**1. Introduction**

Honey used widely as a food and medicine [1]. It is often studied due to its biological activities, among them the anti-inflammatory, anti-bacterial, and anti-oxidation properties are the most published [2–5]. Others include the resistance to bromobenzene-induced liver injury in mice [6] and potential treatment for colitis [7]. However, there are some special honeys, which come from particular floral sources, for example, Manuka, which can play an anti-ulcer role through antioxidant and anti-inflammatory effects [4,8], or Gelam honey, which was able to effectively inhibit airway inflammation in an ovalbumin-induced allergic asthma mouse model [9]. There are significant differences in the antioxidant activities among different honeys [10]. Safflower (*Carthamus tinctorius* L.) honey is brewed by honeybees collecting safflower nectar, which is a local speciality honey type of Xinjiang Providence, China. This honey is mainly produced in the Balruk Mountains of Xinjiang (82◦12–83◦30 longitude in the East and 45◦24–46◦3 latitude in the north), and contains a variety of natural sweeteners with bioactive components [11–13], which is the largest safflower planting base in China. Our recent preliminary results suggested that the sa fflower honey has several superiorities, such as comprehensive and rich nutrients, and a large number of bioactive substances [14]. Nevertheless, detailed information is still unavailable regarding its chemical composition and biological activities, which limits the application and development of this special agriproducts.

Recent decades, several studies reported on the anti-inflammatory and antioxidant activities of honey, which are mainly attribute to its abundant phenolic and flavonoid contents [15,16]. Honey contains rich phenolic acids and flavonoids, which contribute to the antioxidant, antimicrobial, anti-inflammatory, anti-proliferation, anti-cancer, and anti-metastasis e ffects [17]. Studies showed that these compounds in honey were able to inhibit the pro-inflammatory activity of nitric oxide synthase (iNOS) and had anti-inflammatory e ffects [17]. Bangladesh honey samples are rich in phenolic acids and flavonoids with high antioxidant potential [18]. Honeys from specific floral sources or collected by di fferent bee species, given their special and rare compounds, such as certain phenolic acids and flavonoids, for instance, in stingless honey, can show anti-inflammatory and antioxidant effects [19]. Data published with Camellia honey presented an antioxidant e ffect closely related to its phenolic content [20]. We therefore inferred that the sa fflower honey also some activities, such as the anti-inflammatory and antioxidant properties. In our previous studies, we evaluated the anti-inflammatory activities by the bee products using the bacterial lipopolysaccharides (LPS) challenged murine macrophage model (RAW 264.7 cells), in which model a number of typical inflammatory responses can be mimicked in vitro, including the releases of inflammatory mediators, accompanying with the oxidative stress to the cells. LPS induced RAW 264.7 cells were shown to be with an increase in nitric oxide (NO) release [21,22], which is mediated via the activation of the inflammation related-nuclear factor kappa-B (NF-κB) signaling pathway [23], and thereafter induced oxidative stress. NO has a significant correlation with oxidative stress [24], and its release depends on the expression of inducible NO synthase (iNOS). LPS-activated Raw 267.4 cells also lead to the rapid phosphorylation and degradation of IκBα [25]. The phosphorylation of IκBα or IκB-β can activate the NF-κB signaling pathway and promote NF-κB-p65 protein in the nucleus. However, the NF-κB-p65 protein can mediate the synthesis/releases of tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β), monocyte chemoattractant protein 1 (MCP-1), which further regulate the transcription of other inflammatory mediators [26–28]. In addition, the NF-E2-related factor 2 (Nrf-2) signaling activation regulates the expression of a series of downstream antioxidant factors (4-nitroquinoline-N-oxide (NQO), HO-1 as well as the thioredoxin reductase(TXNRD)) [29,30]. Activation of these internal anti-oxidant enzymes alleviated the cells against oxidative stress [31].

In the current study, *Carthamus tinctorius* L. sa fflower honey were collected to carry out a screening of physical and chemical indicators and the extract from *Carthamus tinctorius* L. sa fflower honey (ECH) were obtained for preliminary characterizations on major main phenolic acids and flavonoid components based on the high-performance liquid chromatography–quadrupole-time of flight mass spectrometry (HPLC-QTOF-MS). Furthermore, we tested the in vitro free radical scavenging activities by ECH and its anti-inflammatory and antioxidant potentials were evaluated in in LPS-activated Raw 264.7 murine macrophages.

### **2. Materials and Methods**

### *2.1. Chemicals and Reagents*

Primary antibodies including IκBα, p-IκBα, and NF-κB-p65 were purchased from Cell Signaling Technology (Danvers, MA, USA). Nrf-2, HO-1, and β-actin were purchased from Cambridge Bio (Boston, MA, USA). The NanoDrop 2000 Ultra Micro Spectrophotometer was purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). Gallic acid, sodium nitrite, the Prime Script TM RT Master Mix kit and TB Green ® Premix Ex Taq TM were purchased from Biotech Co. Ltd. (Shanghai, China). Protein loading bu ffer, 20% SDS, electroporation solution and Tris-HCl bu ffer were purchased from Solarbio (Beijing, China). BCIP/NBT alkaline phosphatase developer and 40% Acr-Bis were purchased from Beyotime Biotechnology (Shanghai, China). The CCK-8 kit was purchased from Dojindo

Laboratories (Kumamoto, Japan). Amberlite XAD-2 resin was purchased from Sigma-Aldrich Trading Co. Ltd. (Shanghai, China). Other chemicals, including the LPS (*Escherichia coli* 0127: B8) and alkaline phosphatase-conjugated secondary antibody (anti-rabbit IgG) and the standards applied in the chemical analysis were purchased from Sigma (St. Louis, MO, USA).

### *2.2. Sa*ffl*ower Honey Samples and Physical Deteriminations*

Sa fflower honey samples were obtained from three *Apis mellifera* L. colonies during the sa fflower (*C. tinctorius* L.) flower season in 2018. Each honey from the single hive was considered as one sample, 500 g each. The apiaries were located in the Yumin County, Tacheng City, China, which were belongs to bases of Jiangsu Rigao Bee Products Co., Ltd. (Xuyi, China). We collected the capped comb honeys in the day, and transfer them to the refrigerator at 4 ◦C, afterwards the honey samples were taken to the lab and storage at −20 ◦C until usage. The moisture, free acid, amylase, hydroxymethylfurfural, fructose, glucose, sucrose, and ash contents of the Xinjiang sa fflower honey samples were determined by the method specified in CODEX STAN 12-1981. Melissopalynology was applied to analyze the botanical origin of the pollens in the sa fflower honeys, following pervious published method [32]. The typical pollen grain in a sa fflower honey sample was shown in the Figure S1.

### *2.3. Extraction on the Sa*ffl*ower Honey*

The preparation of the extract from *C. tinctorius* sa fflower honey (ECH) refers to the method of Mu et al. [33] and was improved. XAD-2 resin was soaked in 95% ethanol for 24 h, washed two to three times with ultrapure water until there was no ethanol, and then placed for standby. We weighed 800 g of the Xinjiang sa fflower honey sample, mixed it with hydrochloric acid solution (adjusted using hydrochloric acid pH = 2 with ultrapure water) at 1:5 ( *w*/*v*), and used an ultrasonic instrument for 30 min until it dissolved. The activated XAD-2 resin was weighed to 800 g, treated with ultrasonic to make it free of air, then mixed with Xinjiang sa fflower honey aqueous solution, mixed evenly for 1 h, and left standing overnight. We discarded the supernatant, added XAD-2 resin into the glass column, washed with 2 volumes of hydrochloric acid water (pH = 2) and 3 volumes of ultra-pure water, and then eluted with 8 volumes of ethanol to collect the eluate, evaporated the ethanolic extract to a solid residue with a rotary evaporator with vacuum, and then dissolved the residue in 15 mL of pure water. This aqueous solution was extracted with 20 mL of ethyl acetate. We collected the ethyl acetate layer, repeated this four times, for each extraction for 20 min. The collected ethyl acetate layer was blown dry with nitrogen to obtain the resulting ECH, and placed in a −20 ◦C refrigerator for storage. We dissolved the ECH with an appropriate amount of ethanol for the further usages.

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

The HPLC-QTOF-MS analysis method of ECH was established by our laboratory [34]. Chromatographic conditions involved using a proshell 120 EC-C18 column (100 × 2.1 mm, particle size 2.7 μm), typical parameters of chromatographic were as follows: column temperature, 30 ◦C; injection volume, 5 μL; flow rate, 0.2 mL/min. The elution procedure is shown in Table 1.


**Table 1.** Mobile phase elution procedures.

Mass spectrometry conditions were conducted with an electrospray (ESI) ion source in negative ion mode The typical parameters of the mass spectrometer were as follows: drying gas temperature, 350 ◦C; drying gas flow rate, 6 L/min; sprayer pressure, 35 psi; capillary voltage 3500 V; atomizing gas temperature, 350 ◦C; and atomizing gas flow rate, 9 L/min. Qualitative and quantitative analysis were carried out by accuracy mass and extracted ion chromatography (EIC). Polyphenolic compounds ion chromatograms were extracted by Mass Hunter Qualitative Analysis software (Agilent Technologies) for ECH.

### *2.5. In Vitro Free Radical Scavenging Ability Determination Experiment*

### 2.5.1. DPPH Free Radical Scavenging Experiment

For the determination of the DPPH· clearance rate, we referred to WU et al.'s method [35], and made certain adjustments. We placed 100 μL each of DPPH working solution and ECH into a 1.5 mL centrifuge tube, shook and mixed, and react at room temperature in the dark for 30 min. We took 100 μL of the reaction liquid to a 96-well plate (100 μL/well), and measured the absorbance at 517 nm. For A1, the same method was used to determine the absorbance when adding 100 μL of 95% ethanol solution instead of DPPH working solution. The absorbance of the blank group (100 μL DPPH solution and 100 μL of 95% ethanol solution) was recorded as A0. The calculation formula of the clearance rate is:

$$\text{clearance rate}\% = 1 - \frac{\text{A1} - \text{A2}}{\text{A0}} \times 100\%$$

The removal ability of the sample is expressed by IC50.

### 2.5.2. ABTS+ Free Radical Scavenging Experiment

For the determination of the ABTS+ clearance rate, we referred to YANG et al. [36] and made certain adjustments. The ABTS solution was generated by the reaction of 15 mL 7 mM ATBS solution and 246 μL 140 mM potassium persulfate aqueous solution in the dark for 16 h. When used, it was diluted with methanol to the absorbance of 0.70 ± 0.02 at 734 nm. We placed 250 μL ABTS methanol working solution and 150 μL ECH (to dissolve) in a 1.5 mL centrifuge tube, shook and mixed, avoided light for the reaction for 10 min. We took 150 μL of the reaction liquid to a 96-well plate (150 μL/well), and measured the absorbance value, recorded as A1. The same method was used to determine the absorbance when adding 250 μL of methanol instead of ABTS methanol working solution. The absorbance of the blank group (250 μL ABTS solution and 150 μL (ECH solvent) solution) was recorded as A0, with parallel values. The calculation formula of the clearance rate is:

$$\text{clearance rate}\% = 1 - \frac{\text{A1} - \text{A2}}{\text{A0}} \times 100$$

The removal ability of the sample is expressed by IC50.
