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,
3,
4,
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,
12,
13], which is the largest safflower planting base in China. Our recent preliminary results suggested that the safflower 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 effects [
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 effects [
17]. Bangladesh honey samples are rich in phenolic acids and flavonoids with high antioxidant potential [
18]. Honeys from specific floral sources or collected by different 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 effect closely related to its phenolic content [
20]. We therefore inferred that the safflower 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 IκB-α 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 (TNF-α), interleukin-1β (IL-1β), monocyte chemoattractant protein 1 (MCP-1), which further regulate the transcription of other inflammatory mediators [
26,
27,
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. safflower honey were collected to carry out a screening of physical and chemical indicators and the extract from Carthamus tinctorius L. safflower 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.
4. Discussion
Using the methods recommended in the European Community Directives, we found that all the physical and chemical indexes results of Xinjiang safflower honey samples were in line with the requirements. Hydroxymethylfurfural is a substance produced by honey during storage or heating. It is an important criterion for evaluating the quality of honey [
43,
44]. As the honey storage time becomes longer or the heating temperature rises higher, the more its content increase. Amylase is an important indicator of freshness in honey [
1]. Honey enzymes will inactivate (or the value will drop) when honey is heated to a temperature higher than 40 °C. The amylase value measured in our experiment was 21.1 ± 0.3 mL/g honey, which is much higher than the 8 mL/g honey of EU standard (2001/110/EC). We did not detect hydroxymethylfurfural nor sucrose in the safflower honey samples. The moisture complied with EU standard 2001/110/EC to not be higher than 20 g/100 g. The safflower honey results for moisture were better than carob honey from Morocco as studied by Redouan et al. [
45], The carob honey moisture was 19.5 g/100 g. Therefore, the Xinjiang safflower honey samples were fresh and mature honey.
As polyphenols endowed honey with distinct color, taste, flavor and biological activities. Polyphenol analysis is therefore considered as an important tool for determining the quality of honey. A total number of 14 polyphenolic compounds were preliminary characterized using the HPLC-Q-TOF/MS analysis, and the sum of each component content was 14.83 mg/kg honey. Solid phase extraction (SPE) using the Amberlite XAD-2 resin is one of the most widely used extraction methods for isolating individual phenolic and flavonoid compounds in honey [
46]. In the present study, we didn’t measure the phenolic compounds recovery, which is a major limitation. Nevertheless, the honey was reported with a mean recovery of 43.7% (ranging from 6.7%, gallic acid, to 65.2%, naringin) [
47]. In addition, the discrepancies of the extraction recoveries have been found among different types of honey, which might be attributed to sugars as well as some other matrix constitutions. Among them, eight phenolic acid components, and the vanillic acid and p-hydroxybenzoic acid contents were higher, which were 3.196 mg/kg honey and 1.524 mg/kg honey, respectively, accounting for 65.6% of the total phenolic acid content. Protocatechuic acid, gallic acid, caffeic acid, ferulic acid, and other active components were also detected. In addition, seven flavonoid components were detected, among which quercetin and myricetin were higher, at 5.342 mg/kg honey and 1.021 mg/kg honey, respectively, accounting for 83.4% of the total flavonoid content. Rutin, quercetin-3-O-glucoside, kaempferol and naringenin were lower. Protocatechualdehyde, kaempferol-3-O-glucoside, morin, luteolin, diosmetin, chrysin, pinocembrin, galanin, and CAPE, were not detected. We propose the first reason is that different honey plants, different regions, and other environments have a great impact on the polyphenol composition. Secondly, our polyphenol extraction method has a low extraction rate of flavonoids. In a previous study, Bangladesh honey samples (Mustard flower honey, Kalijira honey, Padma flower honey, and Teel/sesame honey) had higher levels of caffeic acid and benzoic acid in phenolic acid substances, while gallic acid, kaempferol, myricetin, and naringenin were also detected [
18]. Sundarban, Bangladesh, which is a multifloral honey collected from the largest mangrove forest in the world, showed various phenolic compounds, including gallic acid, vanillic acid, pyrogallol, trans-cinnamic acid, and other phenolic substances, as well as naringin, rutin, and quercetin flavonoids. Among all these phenolic compounds, trans-cinnamic acid and naringin had the highest contents [
48]. There are more than 30 kinds of polyphenols in honey. Naringenin and hesperetin typically only appear in specific honeys, such as orange honey [
17].
The DPPH· and ABTS
+ free radical scavenging capacity experiments used the IC
50 (semi-inhibitory rate, i.e., the concentration of the analyte required to scavenge 50% of free radicals) as the criterion for evaluating the antioxidant activity. The literature showed that due to the rich polyphenol content, honey has a very significant antioxidant effect. Five types of honey in Croatia showed high DPPH· free radical scavenging activity (black locust honey 125.48 mg/mL, lime honey 42.77 mg/mL, sage honey 25.04 mg/mL, chestnut honey 16.02 mg/mL, and honeydew honey 8.69 mg/mL [
49]), In eight kinds of carob honey from different areas of Morocco, the DPPH free radical IC
50 values ranged from 12.54 mg/mL to 23.52 mg/mL [
45] and seven kinds of Turkish single flower honey (Heather, Oak, Chestnut, Pine, Astragalus, Acacia, Lavender) demonstrated, ABTS
+·free radical IC
50 values from 0.06 g/mL–3.68 g/mL [
50]. In our experiment, the DPPH and ABTS
+·free radical IC
50 values of ECH were 68.23 ± 0.40 µg/mL and 81.88 ± 0.54 µg/mL, respectively, and they had high antioxidant activity.
In this experiment, ECH can significantly reduce the mRNA levels of the related inflammatory factors iNOS, IL-1β, TNF-α and MCP-1, while up-regulating the expression of the antioxidant genes HO-1, TXNRD and NQO. Due to the expression of iNOS when suppressed, the amount of NO released is reduced accordingly. Through the detection of IκBα phosphorylation and the NF-κB signaling pathway, we found that ECH was likely to inhibit the phosphorylation of IκBα to produce P-IκBα, thereby inhibiting the activation of the NF-κB signaling pathway and reducing inflammation. The Nrf-2/HO-1 signaling pathway demonstrated a protective effect against oxidative stress. Nrf-2 can regulate the gene expression of HO-1 at the transcription level, while HO-1 can inhibit the production of inflammatory factors in LPS-stimulated Raw 264.7 cells and protect the cells under oxidative stress [
27]. We measured the expression of Nrf-2 and HO-1, which increaseds in line with the improvement of the ECH concentration. The 2.5 µg/mL and 5 µg/mL ECH anti-inflammatory and anti-oxidant effects were superior to the positive control group Dexamethasone, indicating that ECH has good anti-inflammatory and anti-oxidant effects.
ECH is rich in phenolic acids and flavonoids and also has good anti-oxidation and anti-inflammatory effects. Thus, we speculated that the existence of these compounds provides a material basis for the antioxidant and anti-inflammatory activities of safflower honey extract. Vanillic acid can play an anti-inflammatory and analgesic role through anti-oxidation and inhibiting the production of pro-inflammatory cytokines associated with NF-κB [
51]. Protocatechuic acid and p-hydroxybenzoic acid showed significant antioxidant activity in thyme honey [
52]. The O
3-H
15 bond of gallic acid is easy to break to scavenge free radicals, providing antioxidant effects [
53]. Gallic acid can also block LPS-induced activation of TLR4/NF-κB (Toll-like receptor 4/nuclear factor-κB) to inhibit the inflammation of Raw 264.7 cells [
54]. Caffeic acid reduces LPS-induced neuroinflammation through the regulation of cytokine networks, down-regulates NF-κB-dependent pro-inflammatory genes, and reduces oxidative stress [
55]. Ferulic acid can exert anti-inflammatory activity by inhibiting nitroso-oxidative stress and pro-inflammatory cytokine production [
56]. We found that the greatest common denominator of these substances is that they all contain different amounts of hydroxyl groups; thus, we infer that the vast majority of the substances involved in antioxidant activities are all related to these hydroxyl groups. It was reported that quercetin can scavenge active oxygen, inhibit damage caused by oxidative stress, and prevent the development of TNF-α and the secretion of iNOS and IL-1β in Raw 264.7 cells induced by lipopolysaccharide (LPS) [
57]. Myricetin exerted anti-inflammatory effects by inhibiting NF-κB-p65 in the NF-κB pathway [
58]. Rutin [
59] and naringenin [
60] alleviate oxidative stress and exert anti-inflammatory effects by regulating mechanisms related to the Nrf-2/HO-1 signaling pathway. Quercetin-3-O-glucoside can inhibit the expression of TNF-α, IL-1β and IL-6 and the activation of NF-κB, and simultaneously up-regulate the expression of Nrf-2 and HO-1, and inhibit the inflammation and oxidation to resist the induction of cisplatin of acute kidney injury in mice [
61]. Kaempferol can up-regulate the expression of iNOS in rat articular chondrocytes stimulated by IL-1β, and can inhibit the degradation of IκBα and the activation of NF-κB in rat articular chondrocytes stimulated by IL-1β [
62]. Based on this, we propose that these polyphenolic compounds in ECH are the main contributors to the anti-oxidation and anti-inflammatory effects of safflower honey.
In the present study, we must explain the limitations of our experiment. First, as the identified polyphenols might had multiple isoforms and exist in the honey in a very complex form, their quantifications in our honey samples might have some bias. Second, phenolic compounds recovery from XAD-2 resin could to be estimated which can partly counteract the strong matrix effects in the honey. Third, in this study we only preliminary detected several polyphenolic compounds in our safflower honey samples, we still did not propose a “marker” compound in our honey samples, which is with great importance for the standardization and quality control on this special honey type. Nevertheless, our research group are keeping working on the isolation and identification on the marker compound of the safflower honey, which is helpful to discriminate from other commercial honey types. In such work accurate recovery rate will be examined on the single maker compound.