Journal of Food Bioactives, ISSN 2637-8752 print, 2637-8779 online
Journal website www.isnff-jfb.com

Original Research

Volume 16, December 2021, pages 34-47

Blackberry phenolic and volatile extracts inhibit cytokine secretion in LPS-inflamed RAW264.7 cells

Figures

Figure 1.
Figure 1. Percentage of viability of RAW264.7 cells after a preventive treatment of one, two and three hrs with phenolic extracts (“Phe”) diluted 10-, 20-, or 40-fold or volatile extracts (“Vol”) diluted 2-, 4-, or 8-fold. Values represent mean ± SEM (n = 3). * Denotes significant difference from cells that received no treatment (P<0.05).
Figure 2.
Figure 2. Average concentration of nitric oxide (NO, μmol/L) produced by RAW264.7 cells after a preventive treatment of one hr with phenolic extracts (“Phe”) diluted 10-, 20-, or 40-fold or volatile extracts (“Vol”) diluted 2-, 4-, or 8-fold, followed by LPS stimulation for 24 hr. The negative control (“NC”) corresponds to cells without treatment nor LPS stimulation. The positive control (“LPS”) corresponds to cells with LPS stimulation, but no treatment. The “Mix” is a lab-made solution of standard volatiles based on the average composition of the three blackberry volatile extracts. Conditions not connected by the same letter are significantly different (P<0.05).
Figure 3.
Figure 3. Average concentration of IL-6 (pg/ml) produced by RAW264.7 cells after a preventive treatment of one hr with phenolic extracts (“Phe”) diluted 10-, 20-, or 40-fold or volatile extracts (“Vol”) diluted 2-, 4-, or 8-fold, followed by LPS stimulation for 24 hr. The negative control (“NC”) corresponds to cells without treatment nor LPS stimulation. The positive control (“LPS”) corresponds to cells with LPS stimulation, but no treatment. The “Mix” is a lab-made solution of standard volatiles based on the average composition of the three blackberry volatile extracts. Conditions not connected by the same letter are significantly different (P<0.05).
Figure 4.
Figure 4. Average concentration of TNF-α (pg/ml) produced by RAW264.7 cells after a preventive treatment of one hr with phenolic extracts (“Phe”) diluted 10-, 20-, or 40-fold or volatile extracts (“Vol”) diluted 2-, 4-, or 8-fold, followed by LPS stimulation for 24 hr. The negative control (“NC”) corresponds to cells without treatment nor LPS stimulation. The positive control (“LPS”) corresponds to cells with LPS stimulation, but no treatment. The “Mix” is a lab-made solution of standard volatiles based on the average composition of the three blackberry volatile extracts. Conditions not connected by the same letter are significantly different (P<0.05).

Tables

Table 1. Volatile composition (ng/mL) of low temperature vacuum distillates obtained from three blackberry genotypes
 
CompoundQuantified asRI1Genotype
A2528TA2587TNatchez
1RI = retention index. 2Means (N=3 ± Std.Dev.). 3Means within rows with different letters are significantly different (p < 0.05).
1-Butanol1-Butanol642ND1.53 ±0 .622a36.53 ± 4.16a
2-Methylbutan-1-ol1-Butanol, 2-methyl-7230.71 ± 0.12aND0.92 ± 0.28a
Trans-3-hexen-1-olTrans-3-hexen-1-ol8461.79 ± 0.13a0.31 ± 0.08b0.79 ± 0.13c
Cis-3-hexen-1-olCis-3-hexen-1-ol8518.90 ± 0.94a5.73 ± 0.88b9.07 ± 1.25a
2-hexen-1-ol,1-Hexanol86116.89 ± 1.73a1.85 ± 0.36b2.94 ± 1.08b
1-Hexanol1-Hexanol86326.36 ± 1.34a5.56 ± 0.57b34.68 ± 4.83c
2-Heptanol2-Heptanol8958.92 ± 1.32b190.43±28.11a1.68 ± 0.30b
1-Heptanol1-Heptanol9681.19 ± 0.49a1.25 ± 0.29a0.83 ± 0.22a
1-Octen-3-ol1-Octen-3-ol9780.94 ± 0.18a1.36 ± 0.69a0.76 ± 0.05a
1-Pentanol, 3-ethyl-4-methyl-1-Hexanol1,0190.21 ± 0.09NDND
2-Ethyl-1-hexanol1-Hexanol1,0283.74 ± 0.55a3.17 ± 1.31a3.96 ± 0.64a
2- Phenylethyl alcoholPhenylethyl alcohol1,117ND44.21 ± 2.78a23.47 ± 3.74b
1-Nonanol1-Nonanol1,1690.82 ± 0.24b2.56 ± 1.01a1.09 ± 0.17b
6-Camphenolα-Terpineol1,1772.49 ± 0.93bND40.42 ± 3.75a
1-Decanol1-Decanol1,2710.36 ± 0.19b1.45 ± 0.52a0.69 ± 0.08b
p-Mentha-1-en-9-olPerillic alcohol1,305ND32.72 ± 2.98a10.71 ± 1.36b
1-Undecanol1-Decanol1,3710.05 ± 0.09NDND
Dodecanol1-Decanol1,4740.67 ± 0.30b1.65 ± 0.13a0.28 ± 0.15b
Total alcohols74.04 ± 7.71b293.79 ± 40.33a138.84 ± 18.45b
2-Butenal (E)Butanal, 3-methyl-6290.37 ± 0.33NDND
2-Butenal (Z)Butanal, 3-methyl-6479.58 ± 2.01NDND
PentanalPentanal6774.32 ± 2.43a3.36 ± 1.60a1.46 ± 0.27a
2-Ethyl-2-butenalButanal, 3-methyl-7271.06 ± 0.20aND2.34 ± 0.53b
2-PentenalPentanal7441.30 ± 0.60a0.31 ± 0.27b0.33 ± 0.29b
3-Methyl-2-butenalButanal, 3-methyl-7760.63 ± 0.64a1.13 ± 0.38a0.82 ± 0.19a
HexanalHexanal7983.46 ± 0.43a4.20 ± 1.23a4.08 ± 0.68a
HeptanalHeptanal9020.05 ± 0.01a0.09 ± 0.03a0.06 ± 0.02a
BenzaldehydeBenzaldehyde9652.20 ± 0.51b1.57 ± 0.03b4.85 ± 1.13a
OctanalOctanal1,0031.64 ± 0.35b4.30 ± 2.04a1.31 ± 0.50b
NonanalNonanal1,1044.48 ± 4.18a8.48 ± 3.74a2.26 ± 0.47a
DecanalDecanal1,2062.85 ± 1.02a9.08 ± 5.78a7.02 ± 1.30a
2-Decenal2-Decenal1,2650.71 ± 0.41b1.35 ± 0.75b2.55 ± 0.43a
Perilla aldehyde2-Decenal1,289ND0.67 ± 0.10ND
UndecanalUndecenal1,3080.29 ± 0.16a0.87 ± 0.59aND
DodecanalDecanal1,4110.34 ± 0.21ab0.63 ± 0.24a0.14 ± 0.09b
Total aldehydes33.85 ± 13.80a36.04 ± 16.78a27.22 ± 5.89a
α,p-Dimethylstyreneα,p-Dimethylstyrene1,0959.80 ± 5.23a45.74 ± 6.70b27.83 ± 3.63c
Total aromatic hydrocarbons9.80 ± 5.23c45.74 ± 6.70a27.83 ± 3.63b
Ethyl acetateEthyl acetate59376.29 ± 11.89a50.07 ± 38.26a67.25 ± 13.08a
Methyl propionateMethyl butanoate604ND0.23 ± 0.21ND
Ethyl propanoateMethyl butanoate6990.26 ± 0.18a0.25 ± 0.12aND
Methyl butanoateMethyl butanoate7110.30 ± 0.19a0.25 ± 0.09aND
Methyl 3-methylbutanoateMethyl butanoate7700.19 ± 0.21NDND
Ethyl butanoateEthyl butanoate7941.70 ± 0.35NDND
Butyl acetateButyl acetate8150.84 ± 0.27NDND
Methyl hexanoateMethyl hexanoate9211.38 ± 1.01a0.33 ± 0.25a0.57 ± 0.18a
Hexyl formateMethyl butanoate9290.27 ± 0.11NDND
Ethyl 3-hydroxybutyrateMethyl butanoate939NDND1.05 ± 0.17
Ethyl hexanoateEthyl hexanoate9982.36 ± 1.44a1.56 ± 0.49a2.47 ± 0.36a
Methyl octanoateMethyl octanoate1,1220.44 ± 0.21a0.36 ± 0.20a0.22 ± 0.11a
Total esters84.05 ± 15.85a53.06 ± 39.61a71.56 ± 13.89a
2-Heptanone2-Heptanone8900.88 ± 0.35ab0.84 ± 0.04a1.37 ± 0.24b
5-Hepten-2-one, 6-methyl-5-Hepten-2-one, 6-methyl-9870.50 ± 0.11a0.52 ± 0.14a0.23 ± 0.03b
AcetophenoneAcetophenone1,076ND0.73 ± 0.68a0.71 ± 1.23a
Total ketones1.38 ± 0.46a2.09 ± 0.86a2.31 ± 1.50a
β-Myrceneβ-Myrcene9924.25 ± 0.92a14.21 ± 1.01b6.82 ± 0.52c
D-LimoneneD-Limonene1,0362.05 ± 0.82a10.50 ± 1.87b6.96 ± 1.67c
p-Cymenep-Cymene1,0400.22 ± 0.10a2.60 ± 0.41b1.04 ± 0.003c
m-Cymenep-Cymene1,045NDNDND
β-Ocimeneβ-Myrcene1,0500.56 ± 0.15b2.74 ± 0.54a1.13 ± 0.17b
Dihydro myrcenolMyrtenol1,0720.52 ± 0.11a0.47 ± 0.35a0.14 ± 0.07a
Linalool oxideLinalool1,0780.48 ± 0.11NDND
LinaloolLinalool1,1005.09 ± 1.30b11.59 ± 0.97a4.47 ± 0.29b
6-CamphenoneAcetophenone1,1300.47 ± 0.07aND2.36 ± 0.11b
Cis-p-mentha-2,8-dien-1-ol1-Nonanol1,142ND0.56 ± 0.20ND
VerbenolTerpinen-4-ol1,158ND0.88 ± 0.16a2.53 ± 1.16a
IsopulegolIsopulegol1,1640.37 ± 0.12NDND
α-Phellandrene-8-olα-Terpineol1,175ND29.54 ± 6.83ND
BorneolBorneol1,1870.96 ± 0.26a4.03 ± 0.50b7.45 ± 0.79c
Terpinen-4-olTerpinen-4-ol1,1884.91 ± 2.67a52.89 ± 4.20b35.44 ± 4.86c
p-Cymene-8-olTerpinen-4-ol1,1915.78 ± 3.21a32.37 ± 5.65b19.75 ± 4.94c
α-Terpineolα-Terpineol1,20024.55 ± 13.61b68.60 ± 13.80a36.21 ± 4.45b
MyrtenolMyrtenol1,2081.67 ± 0.64b18.58 ± 6.97a1.88 ± 0.46b
NerolNerol1,2245.80 ± 2.31a7.20 ± 1.02a5.12 ± 0.92a
VerbenoneD-Carvone1,2282.54 ± 1.11b35.07 ± 5.02a7.67 ± 1.37b
CitronellolCitronellol1,2330.42 ± 0.28a6.32 ± 0.86b4.81 ± 0.78c
D-CarvoneD-Carvone1,256ND51.87 ± 8.33ND
GeraniolGeraniol1,2585.59 ± 1.36b5.02 ± 0.67b16.19 ± 3.86a
Geranial2-Decenal1,2740.53 ± 0.31b1.97 ± 0.42a0.88 ± 0.22b
Perillic alcoholPerillic alcohol1,2991.87 ± 1.22a14.38 ± 1.03b7.30 ± 1.47c
β-Damascenoneβ-Damascenone1,39811.08 ± 6.85a21.85 ± 1.85aND
Total monoterpenes79.72 ± 36.52c393.23 ± 61.23a168.15 ± 27.58b
β-Iononeβ-Ionone1,5020.42 ± 0.16a0.26 ± 0.01ab0.10 ± 0.01b
Total Noisoprenoids0.42 ± 0.16b0.26 ± 0.01a0.10 ± 0.01b
EpicubenolPerillic alcohol1,657ND19.73 ± 3.45a5.23 ± 1.56b
α-CadinolPerillic alcohol1,669ND7.78 ± 3.78a2.39 ± 0.83a
Total sesquiterpenes0.42 ± 0.16b27.77 ± 7.24a7.72 ± 2.40b
Total volatiles283.25 ± 45.39c851.73 ± 49.23a443.62 ± 34.98b

 

Table 2. Standard volatiles used to prepare the simulated blackberry volatile mixture
 
CompoundConcentration (ng/mL)CompoundConcentration (ng/mL)
2-Heptanol67.01Hexanal3.91
Ethyl acetate64.54Citronellol3.85
α-Terpineol43.12Pentanal3.04
Terpinen-4-ol31.08Benzaldehyde2.87
α, p-Dimethylstyrene27.791-Butanol, 3-methyl-2.59
1-Octanol22.30Octanal2.42
1-Hexanol22.20Ethyl hexanoate2.13
Geraniol8.93Toluene1.68
β-Myrcene8.432-Decenal1.54
3-Hexen-1-ol7.901-Nonanol1.49
Perillic alcohol7.85Cymene1.29
Myrtenol7.381-Heptanol1.09
Linalool7.052-Heptanone1.03
Thymol6.591-Octen-3-ol1.02
D-Limonene6.503-Hexen-1-ol0.97
Decanal6.321-Decanol0.83
2-Hexenal6.255-Hepten-2-one, 6-methyl-0.42
Nerol6.04Methyl octanoate0.34
Nonanal5.07β-Ionone0.26
α-Terpinene5.002-Octenal0.22
1-Pentanol4.32Heptanal0.07
Borneol4.15Total concentration407

 

Table 3. Composition and antioxidant activity of blackberry phenolic extracts
 
CompoundGenotype
A2528TA2587TNatchez
1Total anthocyanins and total flavonols were measured by HPLC-PDA and expressed as cyanidin 3-glucoside equivalents (C3GE) and rutin equivalents (RE), respectively. 2Total phenolics were measured using the Folin Ciocalteu assay and are expressed as gallic acid equivalents (GAE). 3Antioxidant activity was measured by the DPPH method and expressed as Trolox equivalents (TE). 4Results are expressed as mean ± SD (n = 3). 5Means within rows with different letters are significantly different (p < 0.05).
Total anthocyanins1 (µg C3GE/mL)1,484 ± 96b4,52,078 ± 28a2,263 ± 144a
Total flavonols1 (µg RE/mL)163 ± 13a105 ± 16b129 ± 13ab
Total phenolics2 (µg GAE/mL)4,315 ± 121a3,369 ± 92b3,680 ± 171c
Antioxidant activity3 (µmol TE/mL)30 ± 1a24 ± 1b25 ± 2b