Background
Methods
Materials
Propolis samples and extraction
Sample no. | Apiary site (Province) | Coordinates |
---|---|---|
P1 | Anseong (Gyeonggi) | 37°00'30"N, 127°16'30"E |
P2 | Icheon (Gyeonggi) | 37°15'50"N, 127°29'03"E |
P3 | Yangpyeong (Gyeonggi) | 37°29'32"N, 127°29'16"E |
P4 | Goyang (Gyeonggi) | 37°39'30"N, 126°49'50"E |
P5 | Wonju (Gangwon) | 37°20'15"N, 127°56'47"E |
P6 | Goesan (Chungbuk) | 36°48'45"N, 127°47'20"E |
P7 | Chungju (Chungbuk) | 36°58'12"N, 127°57'09"E |
P8 | Daejeon | 36°22'08"N, 127°22'27"E |
P9 | Dangjin (Chungnam) | 36°53'54"N, 126°37'51"E |
P10 | Goryeong (Gyeongbuk) | 35°43'36"N, 128°15'56"E |
P11 | Uljin (Gyeongbuk) | 36°59'30"N, 129°24'46"E |
P12 | Jinju (Gyeongnam) | 35°09'49"N, 128°02'24"E |
P13 | Changnyeong (Gyeongnam) | 35°32'52"N, 128°29'35"E |
P14 | Geochang (Gyeongnam) | 35°41'19"N, 127°54'44"E |
P15 | Imsil (Jeonbuk) | 35°36'44"N, 127°17'07"E |
P16 | Buan (Jeonbuk) | 35°43'46"N, 126°42'59"E |
P17 | Jeonju (Jeonbuk) | 35°49'17"N, 127°09'17"E |
P18 | Gunsan (Jeonbuk) | 35°58'06"N, 126°44'14"E |
P19 | Gwangju | 35°09'35"N, 126°51'11"E |
P20 | Jeju (Jeju) | 33°14'46"N, 126°33'55"E |
Determination of total polyphenol and flavonoid contents
Fluorescence resonance energy transfer enzyme assay
Acetylcholinesterase inhibition assay
DPPH radical scavenging assay
Cancer cell lines and cell proliferation assay
Human rhinovirus serotypes and antiviral assay
Intestinal bacterial strains and growth inhibitory assay
Harmful or nonpathogenic bacteria | Beneficial bacteria |
---|---|
Gram-positive | Gram-positive lactic acid-producing bacteria |
Clostridium difficile ATCC 9689 |
Bifidobacterium bifidum ATCC 29521 |
Clostridium paraputrificum ATCC 25780 |
Bifidobacterium breve ATCC 15700 |
Clostridium perfringens ATCC 13124 |
Bifidobacterium infantis ATCC 25962 |
Staphylococcus aureus ATCC 12600 |
Bifidobacterium longum ATCC 15707 |
Lactobacillus acidophilus ATCC 4356 | |
Lactobacillus casei ATCC 393 | |
Gram-negative | Gram-positive acidulating bacterium |
Bacteroides fragilis ATCC 25285a
|
Clostridium butyricum ATCC 25779 |
Escherichia coli ATCC 11775a
| |
Salmonella typhimurium ATCC 13311 |
Data analysis
Results
Total polyphenol and flavonoid contents of propolis samples
Sample | Total polyphenol (mg GAE/g EEP) | Total flavonoid (mg QE /g EEP) |
---|---|---|
P1 | 219.5 ± 5.31a–c
| 29.7 ± 0.77f–h
|
P2 | 148.0 ± 4.32f–i
| 40.5 ± 0.77b,c
|
P3 | 229.0 ± 4.82a,b
| 23.5 ± 0.15i,j
|
P4 | 202.4 ± 4.11b–d
| 30.4 ± 0.36e–g
|
P5 | 196.5 ± 4.28c–e
| 26.3 ± 0.57g–i
|
P6 | 135.9 ± 5.70hi
| 23.6 ± 0.38i,j
|
P7 | 205.6 ± 5.48b–d
| 35.9 ± 0.57c–e
|
P8 | 151.9 ± 5.01f–h
| 26.5 ± 0.56g–i
|
P9 | 205.0 ± 5.98b–d
| 49.8 ± 0.79a
|
P10 | 238.9 ± 4.61a
| 36.5 ± 0.61b–d
|
P11 | 132.3 ± 2.78h,i
| 25.3 ± 1.27g–j
|
P12 | 125.1 ± 5.37i
| 28.7 ± 0.17f–i
|
P13 | 233.5 ± 4.39a,b
| 28.3 ± 2.13f–i
|
P14 | 181.7 ± 4.20d,e
| 27.6 ± 0.29f–i
|
P15 | 183.2 ± 5.75d,e
| 26.6 ± 0.07g–i
|
P16 | 168.7 ± 5.68e–g
| 37.8 ± 2.02b–d
|
P17 | 190.3 ± 5.45c–e
| 20.8 ± 0.17j
|
P18 | 171.6 ± 6.21e,f
| 26.6 ± 0.20g–i
|
P19 | 148.3 ± 5.77f–i
| 24.4 ± 2.01h–j
|
P20 | 48.5 ± 4.08j
| 42.2 ± 1.75b
|
Australian | 142.4 ± 3.61g–i
| 38.0 ± 0.90b–d
|
Brazilian | 126.8 ± 4.12h,i
| 53.0 ± 0.22a
|
Chinese | 132.1 ± 3.28h,i
| 32.5 ± 0.53d–f
|
In vitro BACE-1 inhibitory activity
Sample | IC50, μg/mL (95 % CL) | Slope ± SE | χ2a
|
P-value |
---|---|---|---|---|
P1 | 55.8 (47.7–63.9) | 1.8 ± 0.15 | 2.67 | 0.996 |
P2 | 36.3 (25.2–52.3) | 0.9 ± 0.10 | 7.32 | 0.961 |
P3 | 70.0 (55.9–87.5) | 1.1 ± 0.10 | 5.07 | 0.983 |
P4 | 101.1 (84.7–120.6) | 1.6 ± 0.29 | 5.74 | 0.981 |
P5 | 52.9 (37.3–75.0) | 0.9 ± 0.12 | 8.74 | 0.931 |
P6 | 58.2 (48.8–69.5) | 1.5 ± 0.15 | 3.98 | 0.991 |
P7 | 25.7 (21.8–30.4) | 1.2 ± 0.07 | 3.56 | 0.989 |
P8 | 99.1 (91.4–107.5) | 1.6 ± 0.12 | 2.55 | 0.997 |
P9 | 115.4 (99.7–133.6) | 3.4 ± 1.50 | 2.77 | 0.997 |
P10 | 61.4 (49.1–76.8) | 1.4 ± 0.15 | 6.90 | 0.974 |
P11 | 291.9 (278.0–306.6) | 1.3 ± 0.03 | 1.41 | 0.999 |
P12 | 140.0 (122.0–160.6) | 1.2 ± 0.08 | 3.56 | 0.992 |
P13 | 122.5 (110.9–135.4) | 2.5 ± 0.42 | 3.36 | 0.995 |
P14 | 140.5 (125.5–157.2) | 2.2 ± 0.24 | 3.65 | 0.994 |
P15 | 141.1 (127.8–155.8) | 2.0 ± 0.18 | 3.27 | 0.995 |
P16 | 64.6 (52.1–80.2) | 1.7 ± 0.26 | 4.87 | 0.987 |
P17 | 96.7 (91.4–102.2) | 2.3 ± 0.39 | 2.40 | 0.997 |
P18 | 117.5 (108.7–127.1) | 2.0 ± 0.21 | 2.91 | 0.996 |
P19 | 97.8 (92.3–103.6) | 2.3 ± 0.41 | 2.57 | 0.997 |
P20 | 128.7 (112.6–147.2) | 1.3 ± 0.09 | 3.99 | 0.997 |
Australian | 127.5 (114.2–142.3) | 1.7 ± 0.15 | 3.60 | 0.993 |
Brazilian | 476.5 (458.5–495.2) | 1.6 ± 0.05 | 1.30 | 0.999 |
Chinese | 116.4 (105.4–128.6) | 2.7 ± 0.66 | 3.23 | 0.995 |
Human acetylcholinesterase inhibitory activity
Sample | IC50, μg/mL (95 % CL) | Slope ± SE | χ2a
|
P-value |
---|---|---|---|---|
P1 | 70.0 (56.9–86.1) | 1.0 ± 0.12 | 7.33 | 0.9113 |
P2 | 15.6 (11.2–21.6) | 0.6 ± 0.05 | 3.04 | 0.9321 |
P3 | 55.7 (44.7–69.4) | 1.1 ± 0.14 | 7.71 | 0.9018 |
P4 | 47.9 (39.0–59.0) | 1.1 ± 0.14 | 6.96 | 0.9128 |
P5 | 119.9 (98.2–146.4) | 2.4 ± 0.48 | 12.2 | 0.9066 |
P6 | 89.8 (80.1–100.6) | 2.2 ± 0.23 | 6.75 | 0.9685 |
P7 | 48.0 (40.8–56.55) | 1.2 ± 0.12 | 5.88 | 0.9406 |
P8 | 79.3 (72.6–86.6) | 4.8 ± 0.67 | 6.67 | 0.9760 |
P9 | 103.2 (91.8–116.1) | 2.6 ± 0.33 | 7.56 | 0.9665 |
P10 | 104.2 (89.2–121.7) | 2.0 ± 0.26 | 8.59 | 0.9459 |
P11 | 123.4 (115.1–132.2) | 2.6 ± 0.20 | 4.49 | 0.9878 |
P12 | 61.6 (56.4–67.2) | 2.5 ± 0.25 | 5.57 | 0.9724 |
P13 | 101.6 (84.8–121.7) | 2.0 ± 0.32 | 10.29 | 0.9242 |
P14 | 102.1 (84.8–122.8) | 1.8 ± 0.26 | 9.87 | 0.9245 |
P15 | 104.9 (85.6–128.6) | 1.8 ± 0.30 | 11.04 | 0.9070 |
P16 | 33.9 (26.5–43.2) | 1.1 ± 0.13 | 6.39 | 0.9012 |
P17 | 130.8 (122.5–139.6) | 4.1 ± 0.60 | 5.58 | 0.9842 |
P18 | 124.8 (101.7–153.2) | 1.6 ± 0.22 | 9.94 | 0.9094 |
P19 | 327.3 (297.7–359.7) | 2.6 ± 0.26 | 6.04 | 0.9785 |
P20 | 26.7 (20.7–34.5) | 0.9 ± 0.09 | 4.85 | 0.9213 |
Australian | 242.9 (231.9–254.5) | 3.2 ± 0.22 | 3.39 | 0.9940 |
Brazilian | 147.0 (139.4–155.1) | 2.9 ± 0.19 | 3.66 | 0.9930 |
Chinese | 230.9 (208.7–255.5) | 3.6 ± 0.62 | 7.88 | 0.9696 |
Tacrine | 1.2 (0.9–1.6) | 0.4 ± 0.02 | 3.87 | 0.9712 |
Huperzine A | 0.2 (0.1–0.3) | 0.4 ± 0.03 | 5.33 | 0.9246 |
DH | 0.4 (0.3–0.6) | 0.3 ± 0.02 | 4.78 | 0.9466 |
DPPH free radical scavenging activity
Sample | IC50, μg/mL (95 % CL) | Slope ± SE | χ2a
|
P-value |
---|---|---|---|---|
P1 | 43.4 (36.6–51.4) | 1.7 ± 0.16 | 5.32 | 0.983 |
P2 | 115.3 (100.1–132.9) | 1.3 ± 0.11 | 4.29 | 0.989 |
P3 | 52.7 (44.0–63.3) | 1.6 ± 0.17 | 5.71 | 0.981 |
P4 | 80.2(69.4–92.7) | 1.3 ± 0.10 | 4.28 | 0.989 |
P5 | 56.1 (47.6–66.1) | 1.5 ± 0.14 | 5.10 | 0.985 |
P6 | 97.4 (79.9–118.7) | 1.2 ± 0.13 | 5.76 | 0.980 |
P7 | 73.5 (62.7–86.2) | 1.4 ± 0.13 | 4.87 | 0.986 |
P8 | 159.9 (139.7–183.1) | 1.9 ± 0.18 | 4.44 | 0.990 |
P9 | 81.7 (69.8–95.6) | 1.9 ± 0.31 | 5.76 | 0.983 |
P10 | 58.3 (47.1–72.1) | 2.2 ± 0.32 | 6.73 | 0.976 |
P11 | 161.1 (141.8–183.3) | 1.9 ± 0.16 | 4.20 | 0.991 |
P12 | 235.0 (219.6–251.5) | 1.3 ± 0.04 | 1.96 | 0.998 |
P13 | 67.2 (56.0–80.6) | 1.7 ± 0.24 | 6.03 | 0.979 |
P14 | 135.4 (117.1–156.3) | 1.7 ± 0.18 | 4.80 | 0.988 |
P15 | 98.7 (87.1–111.8) | 1.4 ± 0.11 | 3.89 | 0.991 |
P16 | 81.6 (71.8–92.8) | 1.4 ± 0.11 | 4.00 | 0.991 |
P17 | 74.0 (63.7–86.0) | 1.6 ± 0.16 | 4.89 | 0.987 |
P18 | 96.2 (85.0–109.0) | 1.5 ± 0.13 | 4.05 | 0.991 |
P19 | 202.0 (175.0–233.1) | 1.5 ± 0.11 | 4.39 | 0.989 |
P20 | 269.0 (248.3–291.4) | 1.5 ± 0.06 | 2.44 | 0.996 |
Australian | 73.8 (49.3–110.5) | 0.9 ± 0.12 | 9.76 | 0.922 |
Brazilian | 148.1 (121.2–180.9) | 1.6 ± 0.22 | 6.51 | 0.976 |
Chinese | 179.0 (136.8–234.2) | 1.8 ± 0.29 | 8.59 | 0.958 |
Ascorbic acid | 14.0 (12.4–15.8) | 1.8 ± 0.17 | 4.45 | 0.983 |
Antiproliferative effect on cancer cell lines
Sample | ||||||
---|---|---|---|---|---|---|
PC-3 cell | MCF-7 cell | A549 cell | ||||
IC50, μg/mL (95 % CL) | Slope ± SE | IC50, μg/mL (95 % CL) | Slope ± SE | IC50, μg/mL (95 % CL) | Slope ± SE | |
P1 | 54.0 (44.6–65.4) | 0.7 ± 0.07 | 58.0 (50.6–66.4) | 1.5 ± 0.14 | 6.5 (4.4–9.5) | 0.8 ± 0.10 |
P2 | 15.9 (13.7–18.4) | 1.2 ± 0.10 | 38.6 (33.6–44.3) | 2.5 ± 0.35 | 20.2 (17.1–23.8) | 0.7 ± 0.05 |
P3 | 49.8 (42.1–59.0) | 0.9 ± 0.08 | 87.9 (71.0–108.8) | 1.0 ± 0.13 | 164.5 (141.6–191.2) | 0.7 ± 0.05 |
P4 | 69.7 (62.8–77.3) | 0.9 ± 0.05 | 218.2 (184.9–257.4) | 1.4 ± 0.14 | 61.1 (49.1–76.1) | 0.9 ± 0.09 |
P5 | 44.2 (37.0–53.0) | 1.1 ± 0.11 | 134.2 (106.1–169..6) | 1.1 ± 0.14 | 40.9 (33.6–49.8) | 0.9 ± 0.08 |
P6 | 58.2 (49.5–68.5) | 1.1 ± 0.10 | 56.4 (49.7–64.0) | 2.0 ± 0.22 | 16.3 (12.5-21.2) | 0.9 ± 0.11 |
P7 | 29.9 (24.3–36.8) | 1.2 ± 0.13 | 31.1 (28.2–34.4) | 1.5 ± 0.09 | 9.8 (7.4–13.1) | 0.7 ± 0.07 |
P8 | 59.6 (47.3–75.0) | 1.1 ± 0.14 | 78.8 (73.6–84.4) | 4.4 ± 0.42 | 11.0 (8.5–14.2) | 0.7 ± 0.08 |
P9 | 77.9 (68.3–88.8) | 0.9 ± 0.07 | 47.7 (41.8–54.4) | 1.8 ± 0.19 | 9.9 (8.0–12.2) | 1.0 ± 0.12 |
P10 | 228.8 (189.5–276.3) | 0.7 ± 0.06 | 109.5 (90.2–132.9) | 1.2 ± 0.15 | 365.3 (298.5–447.1) | 1.2 ± 0.15 |
P11 | 44.0 (35.1–55.1) | 1.4 ± 0.19 | 98.3 (82.6–116.9) | 1.3 ± 0.15 | 65.3 (61.1-69.8) | 4.6 ± 0.91 |
P12 | 36.7 (29.5–45.6) | 1.1 ± 0.14 | 17.7 (15.5–20.2) | 2.0 ± 0.23 | 70.7 (57.9–86.3) | 0.5 ± 0.05 |
P13 | 106.6 (90.9–125.0) | 0.7 ± 0.05 | 75.9 (68.3–84.5) | 1.2 ± 0.08 | 262.1 (225.9–304.2) | 1.2 ± 0.11 |
P14 | 75.3 (68.9–82.3) | 1.4 ± 0.21 | 26.0 (22.8–29.6) | 4.5 ± 1.17 | 176.1 (143.5–215.9) | 1.2 ± 0.14 |
P15 | 70.2 (56.8–86.9) | 0.7 ± 0.06 | 104.1 (91.3–118.7) | 0.8 ± 0.05 | 73.5 (60.6-89.1) | 5.9 ± 2.76 |
P16 | 331.5 (266.7–412.2) | 0.6 ± 0.05 | 79.2 (70.8–88.6) | 1.9 ± 0.017 | 127.1 (103.4–156.3) | 1.0 ± 0.12 |
P17 | 331.6 (279.8–392.9) | 0.8 ± 0.06 | 98.1 (81.3–118.3) | 1.3 ± 0.015 | 86.7 (73.4–102.4) | 2.2 ± 0.35 |
P18 | 76.4 (66.3–88.0) | 1.0 ± 0.07 | 145.1(122.6–171.8) | 1.2 ± 0.013 | 115.0 (97.6–135.6) | 2.3 ± 0.37 |
P19 | 161.0 (131.4–197.1) | 0.5 ± 0.03 | 49.8 (48.0–51.8) | 3.9 ± 0.23 | 64.9 (54.1–78.0) | 0.7 ± 0.07 |
P20 | 17.1 (13.9–21.1) | 1.0 ± 0.11 | 19.8 (17.6–22.3) | 2.2 ± 0.28 | 43.8 (38.8–49.3) | 2.7 ± 0.39 |
AU | 92.8 (78.5–109.7) | 2.0 ± 0.29 | 144.8 (134.3–156.1) | 3.2 ± 0.32 | 152.2 (126.3–183.3) | 1.9 ± 0.30 |
BA | 105.2 (96.8–114.3) | 2.1 ± 0.16 | 61.7 (54.7–69.7) | 1.8 ± 0.16 | 330.2 (287.7–379.1) | 4.9 ± 0.89 |
CN | 121.9 (115.1–129.1) | 1.5 ± 0.06 | 122.5 (108.1–138.7) | 1.7 ± 0.15 | 452.8 (361.8–566.5) | 1.1 ± 0.13 |
CPN | 61.5 (50.9–74.4) | 1.1 ± 0.11 | 17.6 (15.3–20.3) | 1.5 ± 0.15 | 6.2 (4.6–8.5) | 1.0 ± 0.14 |
Anti-human rhinovirus activity
Growth-inhibiting effect on intestinal bacteria
Test bacteria | No. active propolis | Propolis samplea (MIC, mg/mL) |
---|---|---|
C. difficile ATCC 9689b
| 3 | P6 (1.84), P9 (1.84), P11 (1.84), CFd (0.031) |
C. paraputrificum ATCC 25780b
| 1 | P12 (1.84), CFd (0.062) |
C. perfringens ATCC 13124b
| 1 | P9 (14.7), CFd (0.031) |
S. aureus ATCC 12600b
| 0 | CFd (0.031) |
B. fragilis ATCC 25285c
| 3 | P8 (3.7), P12 (1.84), P19 (1.84), CFd (0.062) |
E. coli ATCC 11775c
| 4 | P9 (1.84), P12 (1.84), P14 (1.84), P19 (1.84), CFd (0.062) |
S. enterica Typhimurium ATCC 13311c
| 1 | P19 (14.7), CFd (0.125) |
B. bifidum ATCC 29521b
| 1 | P8 (14.7), CFd (0.016) |
B. infantis ATCC 25962b
| 2 | P12 (14.7), P19 (14.7), CFd (0.031) |
B. breve ATCC 15700b
| 0 | CFd (0.031) |
B. longum ATCC 15707b
| 1 | P19 (14.7), CFd (0.031) |
L. acidophilus ATCC 4356b
| 0 | CFd (0.062) |
L. casei ATCC 393b
| 0 | CFd (0.031) |
C. butyricum ATCC 25779b
| 2 | P12 (14.7), P19 (7.4), CFd (0.031) |
Discussion
Activity | Correlation coefficient (r) | |
---|---|---|
Total polyphenol | Total flavonoid | |
BACE-1 inhibition | −0.337 | −0.194 |
Acetylcholinesterase inhibition | 0.056 | −0.413 |
DPPH free radical scavenging activity | −0.872 | 0.071 |
Antiproliferative activity | ||
PC-3 | 0.263 | −0.076 |
MCF-7 | 0.386 | −0.301 |
A549 | 0.453 | −0.078 |