Propolis is an important bee product which has been applied to the treatment of several diseases. The aim of this study was to understand the material basis of Chinese propolis on pain relief; different Chinese propolis fractions (40W, 40E, 70E, and 95E raw propolis extracted followed by 40%, 70%, or 95% ethanol) were prepared, and their antinociceptive effects were evaluated. By analyzing using UPLC-Q-TOF-MS, we showed that 40W was rich in phenolic acids, like caffeic acid, while 40E, 70E, and 95E have relatively high levels in flavonoids, like galangin, pinocembrin, and chrysin. Notably, chrysin amounts in 70E and 95E are much higher than those in 40E fraction. Antinociceptive effects by these propolis fractions were evaluated in mice using acetic acid-induced writhing test, hot plate test, and tail immersion test, respectively. We noticed that only 40E fraction showed a significant reduction on acetic acid-induced writhing test. Importantly, in the hot plate test, all groups showed their effectiveness, except for the 70E group. We also noticed that 40W, 40E, and 95E administration caused an increase in the tail withdrawal latency of the mice. These data suggested that the different antinociceptive effects of different fractions from Chinese propolis extracts are directly link to their flavonoid composition.
The inflammation displays with classical symptoms of pain, swelling, and heat. Pains happened in the freakishness of any part of the body, including spine joints, tendons, muscles, internal organs, and ligaments. Relief of pain is required for the quality and normal life activities [
The usage of herbal medicine in the treatment for various kinds of diseases has a long history [
Propolis is an important hive product collected by the honeybees (
It has been recognized as a critical issue for understanding the material basis of herbal medicine, which is necessary for the modern medicine development as well as clinical applications. Here, we used different Chinese propolis fractions and several in vivo animal models were applied to provide systemic data for elucidating the potential antinociceptive effects of Chinese propolis.
The abundant polyphenolic compounds are known as key contributors to the therapeutic effects of propolis. It has been known that different solvents will affect the yield of these bioactive constituents. We first performed the phytochemical analysis on the bioactive fractions of CP (40W, 40E, 75E, and 95E). As shown in Table
Total phenolic content (TPC), total flavonoid content (TFC), reducing power, and DPPH free radical scavenging activities of different fractions of CP.
TPC (mg GAE/g) | TFC (mg QE/g) | Reducing power ( |
DPPH-scavenging activity IC50 ( | |
---|---|---|---|---|
BHT | / | / | / | 82.42 ± 3.7 |
40W | 175.5 ± 0.8 | 9.3 ± 0.1 | 118.7 ± 7.8 | 27.7 ± 1.2 |
40E | 515.8 ± 4.0 | 142.7 ± 0.6 | 128.6 ± 3.9 | 19.5 ± 2.0 |
75E | 270.5 ± 28.9 | 125.9 ± 18.0 | 87.1 ± 2.1 | 52.5 ± 3.5 |
95E | 280.9 ± 0.3 | 126.9 ± 3.4 | 68.2 ± 4.0 | 78.8 ± 6.2 |
DPPH free radical scavenging activity assay and FRAP assay are widely used in the screening of natural antioxidant as well as plant extracts. Previous studies have been performed exhaustively on the free radical scavenging of propolis [
Polyphenolic constitutions, including phenolic acids and flavonoids, are regarded as major contributors to the antioxidant activities. Generally, our results showed to be in parallel with other studies in which all CP fractions have potent antioxidant properties. However, we noticed that the 40W group showed the best
HPLC-DAD/Q-TOF-MS analysis on Chinese propolis.
Peak | Compounds | RT (min) | ( |
Content (mg/g) | |||
---|---|---|---|---|---|---|---|
40E | 40W | 70E | 95E | ||||
1 | Protocatechuic acid | 9.525 | 155.0339 | / | / | / | / |
2 | Vanillic acid | 16.445 | 169.0495 | / | / | / | / |
3 | Caffeic acid | 16.996 | 181.0495 | 7.24 | 44.03 | 1.84 | 1.66 |
4 | Syringic acid | 18.13 | 199.0601 | / | / | / | / |
5 | 7-Hydroxycoumarin | 19.751 | 163.0389 | / | / | / | / |
6 | P-Coumaric acid | 19.929 | 165.0546 | 7.59 | 15.62 | 1.77 | 1.41 |
7 | Ferulic acid | 21.063 | 195.0652 | 3.90 | 5.71 | 0.76 | 0.53 |
8 | Isoferulic acid | 22.003 | 195.0652 | 7.79 | 10.28 | 1.43 | 1.97 |
9 | Rutin | 23.85 | 611.1607 | / | / | / | / |
10 | 3,4-Dimethoxycinnamic acid | 24.337 | 209.0808 | 17.20 | 8.53 | 4.78 | 3.62 |
11 | Myricetin | 24.839 | 319.0448 | / | / | / | / |
12 | Trans-cinnamic | 26.135 | 149.0597 | / | / | / | / |
13 | Quercetin | 26.865 | 303.0499 | 0.09 | / | 0.03 | 0.05 |
14 | Pinobanksin | 27.043 | 273.0757 | 12.87 | 4.18 | 6.69 | 2.99 |
15 | Luteolin | 27.529 | 287.055 | 0.76 | 0.23 | 0.23 | 0.18 |
16 | Kaempferol | 28.501 | 287.055 | 4.77 | 1.14 | 2.03 | 1.51 |
17 | Apigenin | 28.793 | 271.0601 | 9.18 | 2.10 | 2.02 | 1.06 |
18 | Pinocembrin | 30.43 | 257.0808 | 9.30 | 9.40 | 34.34 | 27.52 |
19 | Chrysin | 31.11 | 255.0652 | 24.37 | 4.85 | 37.66 | 55.03 |
20 | CAPE | 31.305 | 285.1121 | 10.65 | 2.68 | 5.28 | 2.15 |
21 | Galangin | 31.661 | 271.0601 | 37.77 | 10.62 | 39.00 | 31.06 |
22 | Curcumin | 31.823 | 369.1333 | / | / | / | / |
23 | Artepillin C | 34.675 | 301.1798 | / | / | / | / |
24 | 41.854 | 411.1802 | / | / | / | / |
mg/g means the content of compounds per g extract of different fractions.
Base peak chromatogram in the UV spectrum in the 280 nm of the extract of different fractions on Chinese propolis.
Rodent writhing test model induced by the acetic acid is a typical study for antinociceptive studies. This model has high sensibility for the screening of a number of diseases, like nonsteroidal anti-inflammatory drugs (NSAIDs), muscle relaxant, and depressant drugs [
Effects of oral administration of different fractions of Chinese propolis extracts on acetic acid-induced writhing test on acetic acid-induced visceral nociception in mice (
For assessing the opioidergic analgesic mechanisms as well as narcotic analgesia, the hot plate test is a classical model [
Effects of oral administration of different fractions of Chinese propolis extracts on hindpaw lick latency of the mice under hot plate test (
The antinociceptive effects of CP fractions as well as ibuprofen (60 mg/kg) were tested by the use of a thermal nociceptive stimulation (tail immersion in a 48°C water bath). As shown in Figure
Effects of oral administration of different fractions of Chinese propolis extracts on tail withdrawal latency of the mice under tail immersion test in mice (
In summary, our data suggested that different fractions from Chinese propolis extracts enriched in polyphenolic constitutions showed central and peripheral antinociceptive effects that can be linked with their antioxidant activities. These results support the clinical usage of propolis as an alternative approach for painful disease treatment. Since the antinociceptive properties are closely linked to the anti-inflammatory effects, which we have not fully understood, the modulating effect of these fractions on the inflammatory cytokine releases is still needed to be explored in the future.
Acetic acid, DPPH, and ABTS, as well as standards for the chemical analysis, were obtained from Sigma-Aldrich (St. Louis, Mo., USA). Methanol was obtained from Fisher Scientific (Pittsburgh, PA, USA). All other reagents were obtained from Sangon Biotechnology (Shanghai, China) or as indicated in the specified methods.
Chinese propolis (CP), which was originated from poplar (
Male ICR mice (20 ± 2 g) were purchased from the Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The mice were kept under controlled standard environment conditions. All experimental protocols were approved by the Animal Ethical Committee of Institute of Apicultural Research, CAAS.
The 0.85% acetic acid was intraperitoneally injected to the mice to induce peritoneal irritation, with typical symptoms of abdominal contortions as well as hind limb extensions [
The hot plate analgesia meter (Ugo Basil, Italy; Socrel DS-37) was used for generating a heated surface (55 ± 0.2°C). The mice were moved to the glass cylinder with a diameter of 20 cm and put on the heated surface. Treated mice received oral vehicle, drug standard (ibuprofen 60 mg/kg, GSK China), and 5 g/kg bioactive fractions of CP (40W, 40E, 75E, and 95E) for 7 d. On the 7th day, the latency to nociceptive response of the mice was recorded at 0.5, 1, 2, and 4 h after oral treatment with different samples. Paw licking and jumping were evaluated as the indices of the thermal reactions.
The tail immersion test involved immersing the extreme 4 cm of the mouse tail in a water bath containing water at a temperature of 45 ± 0.5°C [
Data are expressed as the means ± SD for the indicated number of independently performed experiments. Statistical comparison of the data was performed by using Student’s
The data used to support the findings of this study are available from the corresponding author upon request.
All the authors declare that they have no conflict of interests.
For this manuscript, Liping Sun provided the sample and identified its information, as well as wrote the paper. Lei Liao was responsible for extracting the samples and writing the part of extraction, and the analysis and writing of chromatography were done by Bei Wang. Liping Sun designed the experiment procedure and wrote Discussion.
This research was supported partly by grants from the Agricultural Science and Technology Innovation Program (no. CAAS-ASTIP-2018-IAR) and the National Natural Science Foundation of China (no. 31272509).