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Phenolic Compounds in Organic and Aqueous Extracts from Acacia farnesiana Pods Analyzed by ULPS-ESI-Q-oa/TOF-MS. In Vitro Antioxidant Activity and Anti-Inflammatory Response in CD-1 Mice Delgadillo Puga Claudia 1, * , Cuchillo-Hilario Mario 1 , Navarro Ocaña Arturo 2 , Medina-Campos Omar Noel 3 , Nieto Camacho Antonio 4 , Ramírez Apan Teresa 4 , López-Tecpoyotl Zenón Gerardo 5, Díaz Martínez Margarita 1 , Álvarez-Izazaga Marsela Alejandra 6 , Cruz Martínez Yessica Rosalina 7 , Sánchez-Quezada Vanessa 8 , Gómez Francisco Enrique 9 , Torre-Villalvazo Iván 9 , Furuzawa Carballeda Janette 10 , Camacho-Corona María del Rayo 11 and Pedraza-Chaverri José 3 1

2 3 4 5 6 7 8 9 10 11

*

Departamento de Nutrición Animal Dr. Fernando Pérez-Gil Romo, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), CDMX 14080, Mexico; [email protected] (C.-H.M.); [email protected] (D.M.M.) Departamento de Alimentos y Biotecnología, Facultad de Química, Universidad Nacional Autónoma de México (UNAM), CDMX 04510, Mexico; [email protected] Facultad de Química, Departamento de Biología, Universidad Nacional Autónoma de México (UNAM), CDMX 04510, Mexico; [email protected] (M.-C.O.N.); [email protected] (P.-C.J.) Instituto de Química, Universidad Nacional Autónoma de México (UNAM), CDMX 04510, Mexico; [email protected] (N.C.A.); [email protected] (R.A.T.) Colegio de Posgraduados en Ciencias Agrícolas, Puebla 72760, Mexico; [email protected] Departamento de Nutrición Aplicada y Educación Nutricional, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), CDMX 14080, Mexico; [email protected] Facultad de Química, Universidad Nacional Autónoma de México (UNAM), CDMX 04510, Mexico; [email protected] Facultad de Química, Universidad Autónoma de Zacatecas, Zacatecas 98500, Mexico; [email protected] Departamento de Fisiología de la Nutrición, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), CDMX 14080, Mexico; [email protected] (G.F.E.); [email protected] (T.-V.I.) Departamento de Inmunología y Reumatología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), CDMX 14080, Mexico; [email protected] Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, Monterrey 64570, Mexico; [email protected] Correspondence: [email protected]; Tel.: +52-5554-870900

Received: 22 August 2018; Accepted: 10 September 2018; Published: 18 September 2018

Abstract: Background: Acacia farnesiana (AF) pods have been traditionally used to treat dyspepsia, diarrhea and topically for dermal inflammation. Main objectives: (1) investigate the antioxidant activity and protection against oxidative-induced damage of six extracts from AF pods and (2) their capacity to curb the inflammation process as well as to down-regulate the pro-inflammatory mediators. Methods: Five organic extracts (chloroformic, hexanic, ketonic, methanolic, methanolic:aqueous and one aqueous extract) were obtained and analyzed by UPLC-ESI-Q-oa/TOF-MS. Antioxidant activity (DPPH•, ORAC and FRAP assays) and lipid peroxidation (TBARS assay) were performed. Assessment of anti-inflammatory properties was made by the ear edema induced model in CD-1 mice and MPO activity assay. Likewise, histological analysis, IL-1β, IL-6, IL-10, TNF-α, COX measurements plus nitrite and immunohistochemistry analysis were carried out. Results: Methyl gallate, gallic acid,

Molecules 2018, 23, 2386; doi:10.3390/molecules23092386

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galloyl glucose isomer 1, galloyl glucose isomer 2, galloyl glucose isomer 3, digalloyl glucose isomer 1, digalloyl glucose isomer 2, digalloyl glucose isomer 3, digalloyl glucose isomer 4, hydroxytyrosol acetate, quinic acid, and caffeoylmalic acid were identified. Both organic and aqueous extracts displayed antioxidant activity. All extracts exhibited a positive effect on the interleukins, COX and immunohistochemistry assays. Conclusion: All AF pod extracts can be effective as antioxidant and topical anti-inflammatory agents. Keywords: Acacia farnesiana pods; antioxidant and anti-inflammatory activities; bioactive compounds; polyphenols

1. Introduction The loss of the prooxidant-antioxidant balance in biological systems is responsible of many disorders resulting in oxidative damage [1]. Phytochemicals from plants help to maintain this equilibrium by enhancing the scavenging properties against reactive oxygen species (ROS) [2]. Also, inflammation is a biological process that responds to several internal and external agents and aggressors as infection or injuries which can turn into pathological and chronic stages affecting to the host [3]. Plant bioactive compounds are capable of down-regulating the activation of the mediators involved in the inflammatory cascade [4–6]. Bioactivity of phytochemicals to counteract inflammation is enhanced if plant bioactive compounds are tested as extracts [7–9]. Phenols are the frequent chemical group closely related to these effects [10,11]. Acacia genus contains phenols and some other phytochemicals like amines, alcaloids, essential oils, non-protein aminoacids, cumarines, diterpenes, fatty acids, triterpenes, phytosterols, saponines, flavonoids, gums and tannins. Bioactivity and health promoting properties of these phytochemicals include the abatement of some chronical disorders e.g., cancer, obesity, aging and diabetes [12]. In pods, leaves, stems, barks, flowers, and roots of Acacia farnesiana (L.) Willd (family: Fabeceae), commonly named as sweet acacia or Huizache, it has been described the occurrence of phytochemicals like albumin, gallic acid, glutelins, kaempferol, quercetin, methyl gallate, myricetin, naringenin, diosmetin, apigenin, catechin, ellagic acid, lupeol, α-amyrin, β-amyrin, β-sitosterol, ferulic and caffeic acids, among others. In this way, recently Hernández [13], isolated and described the structural characteristic of three new compounds in hexanic-cloroformic extract from AF pods: (3β,22E)-stigmasta-5,22-dien-3-ol, β-D-glucopyranoside, (3β,22E)-stigmasta-5,22-dien-3-yl and (2S)-2,3-dihydroxypropyl tetracosanoate. Some of which have been pointed out as protectors against ROS-induced damage [14]. Particularly, A. farnesiana (AF) pods have been previously recognized as resource of phytochemicals with effective protective properties against oxidative stress and antibacterial activity [13,15,16]. The consumption of AF as infusions and/or decoctions includes antidiarrheal, antispasmodic and anti-hyperglycemic uses and alleviation of dyspepsia [17,18]. The use of edible plants and their recommendations to alleviate health disorders, require exhaustive investigation to support the benefits or limitations. The aims of this study were: (1) to investigate the antioxidant activity and protection against oxidative induced damage of six extracts [chloroformic (CE), hexanic (HE), ketonic (KE), methanolic (ME), methanolic:aqueous (80:20 v/v); (MEAE) and one aqueous (AE)] from AF pod and (2) to test the same six extracts for their capacity to curb the inflammation process as well as to down-regulate the pro-inflammatory mediators. 2. Results 2.1. Total Polyphenols Content Total polyphenols content from CE, HE, KE, ME, MAE and AE from AF pods were 506, 620, 594, 378, 399 and 565 mg of equivalents of gallic acid/100 g of dry matter, respectively. These results keep

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up a correspondence with non-polar and polar properties of solvents as well as their ability to extract phenolic units from AF pods. 2.2. AF Pod Extracts Analyzed by UPLC-ESI-Q-oa-/TOF-MS In the six extracts, 12 phenolic compounds were identified by UPLC-ESI-Q-oa-/TOF-MS analysis which include: methyl gallate, gallic acid, galloyl glucose isomer 1, galloyl glucose isomer 2, galloyl glucose isomer 3, digalloyl glucose isomer 1, digalloyl glucose isomer 2, digalloyl glucose isomer 3, digalloyl glucose isomer 4, hydroxytyrosol acetate, quinic acid, and caffeoylmalic acid (Table 1). It is worth mentioning that the methyl gallate was identified in the six extracts and the gallic acid was found in the chloroformic, methanolic:aqueous and aqueous extracts (see all chromatographic spectra extracts in Figure S1). Table 1. Compounds identified in six extracts from Acacia farnesiana (AF) pods. Rt (min)

[M − H]− (m/z)

Molecular Formula

Dominant Ion Fragment

Assignment

Reference

0.866 0.935 1.826 1.912 2.169 2.512 4.673 4.776 4.982

387.0918 331.0602 169.0484 331.0612 331.0610 493.0616 183.0608 483.0230 483.0224

C13 H16 O10 C7 H6 O5 C13 H16 O10 C13 H16 O10 C8 H8 O C20 H20 O14 C20 H20 O14

341.1011 (100) 271.0561 (100) 125.0678 (100) 271.0566 (100) 271.0572 (100) 271.0562 (100) 168.9363 (100) -

Unknow Galloyl glucose isomer 1 Gallic acid Galloyl glucose isomer 2 Galloyl glucose isomer 3 Unknow Methyl gallate Digalloyl glucose isomer 1 Digalloyl glucose isomer 2

[19] [20] [19] [19] [21] [22] [22]

0.866 0.935 1.826 1.912 2.169 2.512 4.673 4.776 4.982

387.0918 331.0602 169.0484 331.0612 331.0610 493.0616 183.0608 483.0230 483.0224

C13 H16 O10 C7 H6 O5 C13 H16 O10 C13 H16 O10 C8 H8 O C20 H20 O14 C20 H20 O14

341.1011 (100) 271.0561 (100) 125.0678 (100) 271.0566 (100) 271.0572 (100) 271.0562 (100) 168.9363 (100) -

Unknow Galloyl glucose isomer 1 Gallic acid Galloyl glucose isomer 2 Galloyl glucose isomer 3 Unknow Methyl gallate Digalloyl glucose isomer 1 Digalloyl glucose isomer 2

[19] [20] [19] [19] [21] [22] [22]

1 2 3

0.917 1.826 2.084

331.0600 331.0605 331.0597

C13 H16 O10 C13 H16 O10 C13 H16 O10

Galloyl glucose isomer 1 Galloyl glucose isomer 2 Galloyl glucose isomer 3

[19] [19] [19]

4

2.392

493.0606

-

Unknow

-

5 6

4.279 4.588

483.0223 183.0610

C20 H20 O14 C8 H8 O

Digalloyl glucose isomer 1 Methyl gallate

[23] [21]

7

4.691

483.0228

C20 H20 O14

Digalloyl glucose isomer 2

[22]

8

4.914

483.0219

C20 H20 O14

Digalloyl glucose isomer 3

[22]

9 10

5.051 5.651

483.0226 401.0812

C20 H20 O14 -

271.0560 (45) 211.0483 (1) 271.0565 (100) 271.0559 (100) 271.0557 (17) 313.0538 (1) 169.0465 (1) 183.0624 (100) 168.9363 (100) 271.0562 (15) 211.0498 (8) 169.0481 (49) 125.0660 (1) 271.0562 (36) 211.0494 (3) 169.0481 (28) 125.0646 (1) -

Digalloyl glucose isomer 4 Unknow

[22] -

1

0.832

331.0601

C13 H16 O10

Galloyl glucose isomer 1

[19]

2 3 4 5

1.072 1.603 1.912 2.581

331.0606 483.0223 483.0223 183.0606

C13 H16 O10 C20 H20 O14 C20 H20 O14 C8 H8 O

Galloyl glucose isomer 2 Digalloyl glucose isomer 1 Digalloyl glucose isomer 2 Methyl gallate

[19] [19] [19] [21]

Peak Chloroformic 1 2 3 4 5 6 7 8 9 Hexanic 1 2 3 4 5 6 7 8 9 Ketonic

Methanolic 271.0561 (40) 169.0482 (1) 133.0557 (1) 271.0565 (100) 271.0487 (100) 271.0566 (100) 168.9363 (100)

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Table 1. Cont. Peak

Rt (min)

[M − H]− (m/z)

Molecular Formula

Dominant Ion Fragment

Assignment

Methanolic: Aqueous Molecules 2018, 23, x FOR PEER REVIEW 1 2 3 4 5 6 7 8

0.9 1.02 1.946 2.118 2.41 2.821 4.965 5.171

2 3 4 5 6 7 Aqueous 8 1 Aqueous 0.8666 2 1 1.02 3 1.26 2 4 1.398 3 5 1.929 4 4.879 6 5 6

Reference

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387.0913 377.0659 (40) 341.1000 (18) Unknow 331.0603 C13 H16 O10 271.0562 Galloyl glucose isomer 1 341.1000(100) (18) 125.0661 (100) (100) Gallicisomer acid 1 1.02169.0483331.0603 C7 H6CO135H16O10 271.0562 Galloyl glucose O710 271.0565 (100) (100) Galloyl H6 O 5 125.0661 Gallicglucose acid isomer 2 1.946331.0606169.0483 C13 H16C 331.0611 C H O10 271.0569 (100) Galloyl glucose isomer 3 271.0565 (100) Galloyl glucose isomer 2 2.118 331.0606 13 16 C13H16O10 493.0619 271.0559 (100) Unknow 271.0569 (100) Galloyl glucose isomer 3 2.41 331.0611 C13H16O10 183.0604 C8 H8 O 168.9363 (100) Methyl gallate 2.821483.0225493.0619 C H O 271.0559 Unknow 271.0564 (100) (100) Digalloyl glucose isomer 1 20 20 14 168.9363 (100) Methyl gallate 4.965 183.0604 C8H8O 271.0564 (100) Digalloyl glucose isomer 1 5.171 483.0225 C20H20O14 195.0799 165.0759 (100) Hydroxytyrosol acetate 133.0546195.0799 115.0463 (100) (100) unknow 0.8666 165.0759 Hydroxytyrosol acetate 191.0494 C H O 111.0524 (100) Quinic acid 1.02 133.0546 7 12 6 115.0463 (100) unknow 295.0470 C13 H12 O8 Caffeoylmalic acid 111.0524 (100) Quinic acid 1.26 191.0494 C7H12O6 169.0478 C7 H6 O5 125.0657 (100) Gallic acid 13H12O8 - (100) Caffeoylmalic acid 1.398183.0609295.0470 C HCO 168.9363 Methyl gallate 8 8 125.0657 (100) Gallic acid 1.929 169.0478 C7H6O5 168.9363 (100) Methyl gallate 4.879 183.0609 C8H8O

[19] [19] [19] [19] [19] [19] [19] [19] [21] - [22] [21] [22] [24] [24][25] [26] [25] [23] [26] [21] [23] [21]

2.3. The Effect of the Extracts on the Elimination of Radicals Is Concentration-Dependent 2.3. The Effect of the Extracts on the Elimination of Radicals Is Concentration-Dependent

As the concentration of the extracts increased, the radical scavenging capacity tended to increase As the concentration of the extracts increased, the radicalamong scavenging capacity tended to we as well (Figure 1A–G). No differences (p > 0.05) were observed the six extracts when increase as well (Figure 1A–G). No differences (p > 0.05) were observed among the six extracts when compared the same concentrations (1.2–300 µg/mL) against quercetin. However, quercetin increased we compared the same concentrations (1.2–300 μg/mL) against quercetin. However, quercetin the scavenging capacity of DPPH• radical in the first five concentrations (from 1 to 30 µg/mL) and increased the scavenging capacity of DPPH• radical in the first five concentrations (from 1 to 30 towards the end the assay 1A). In (Figure contrast, HE, ME and MEAE showed a much lower μg/mL) andoftowards the (Figure end of the assay 1A). In KE, contrast, HE, KE, ME and MEAE showed a response to DPPH discoloration at initial concentration equated the extracts at 300 µg/mL. much lower • response to DPPH• discoloration at initialand concentration andother equated the other extracts In the same CE and AEsame responded 120AE andresponded 300 µg/mL. However, µg/mL no favorable at 300line, μg/mL. In the line, CEtoand to 120 and 300below μg/mL.60However, below 60 μg/mL favorablefor response wastwo observed for the latest two In relation to IC 50 value,the response was no observed the latest extracts. In relation to extracts. IC50 value, quercetin showed quercetin showed while the best result μg/mL), while with AE, CE and HE it was to best result (22 µg/mL), with KE,(22 AE, CE and HE it wasKE, necessary to increase thenecessary concentration increase the concentration around to one, two, three and seven-fold more (respectively) than around to one, two, three and seven-fold more (respectively) than quercetin to scavenge 50 percent of to scavenge 50 percent of DPPH• radical (Figure 1H). DPPH•quercetin radical (Figure 1H).

Figure 1. Cont.

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Figure 1. Scavenging capacity of the DPPH• radical (A–G) and inhibitory concentration 50% Figure 1. Scavenging capacity of the DPPH• radical (A–G) and inhibitory concentration 50% (IC50) (IC50 ) values (H)ofofdifferent different extracts Acacia farnesiana pods. data are with the mean with the values (H) extracts fromfrom Acacia farnesiana pods. The dataThe are the mean the standard standard deviation of three independent repetitions. CE = chloroformic HE = hexanic extract; deviation of three independent repetitions. CE = chloroformic extract; extract; HE = hexanic extract; KE = KE = ketonic extract; MEAE MEAE==methanolic:aqueous methanolic:aqueous extract and = aqueous ketonicextract; extract; ME ME == methanolic methanolic extract; extract and AEAE = aqueous a,b,c,d,e a,b,c,d,e extract;extract; Q = quercetin. Different letters difference(p(p< 0.05). Similarly, when we CE, AEextract, and HE, which needed 1.7, 1.6, 1.5statistically and 1.4 mmoles of (p Trolox equivalents/g of extract, evaluated the six extracts concentrations (60,(p120 and 300 mmoles of Trolox respectively. However, this is at nothigher statistically different > 0.05). Similarly, when weequivalents/g evaluated the of extract), no differences were observed. When evaluated a particular extract at different six extracts at higher concentrations (60, 120 and 300wemmoles of Trolox equivalents/g of extract), concentration, we observed significant differences (p < 0.05). However, the highest concentration no differences were observed. When we evaluated a particular extract at different concentration, (300 mmoles of Trolox equivalents/g of extract) resulted in the best ferric-reducing antioxidant we observed significant differences (p < 0.05). However, the highest concentration (300 mmoles of power. The values for this activity were: ME, 8.4; MEAE, 6.6; KE, 6.1; AE, 5.4; CE, 4.9 and HE, 3.4 μg Trolox Trolox equivalents/g of extract) resulted in the best ferric-reducing antioxidant power. values equivalents/g, respectively. Regarding to FRAP, we observed IC50 values of 1.8, The 1.5, 2.0, 2.6, for this activity were: ME, 8.4; MEAE, 6.6; KE, 6.1; AE, 5.4; CE, 4.9 and HE, 3.4 µg Trolox equivalents/g, 2.0 and 1.9 for CE, HE, KE, ME, MEAE and AE compared to 1.4 of quercetin. respectively. Regarding to FRAP, we observed IC50 values of 1.8, 1.5, 2.0, 2.6, 2.0 and 1.9 for CE, HE, KE, ME, MEAE and AE compared to 1.4 of quercetin.

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Figure 2. Total antioxidant activity of the extracts from Acacia farnesiana pods by oxygen radical Figure 2. Total antioxidant activity of the extracts from Acacia farnesiana pods by oxygen radical absorbance and ferric-reducing antioxidant power (FRAP) assays. Q = quercetin; Figurecapacity 2. Total (ORAC) antioxidant activity of the extracts from Acacia farnesiana pods by oxygen absorbance capacity (ORAC) and ferric-reducing antioxidant power (FRAP) assays. Q =radical quercetin; CE = chloroformic extract; HE = hexanic extract; KE = ketonic extract; ME = methanolic extract; absorbance capacity (ORAC) and ferric-reducing power (FRAP) assays. Q =extract; quercetin; CE = chloroformic extract; HE = hexanic extract; KEantioxidant = ketonic extract; ME = methanolic MEAE a,b,c,d,e,f Different letters showed MEAE CE = methanolic:aqueous extract and AE = aqueous extract. = chloroformic extract; HE = hexanic extract; KE = ketonic extract; ME = methanolic extract; MEAE a,b,c,d,e,f Different letters showed significant = methanolic:aqueous extract and AE = aqueous extract. a,b,c,d,e,f significant difference (p < 0.05) in the at extract. different concentrations (Kruskal–Wallis test). Different letters showed significant = methanolic:aqueous extract andsame AE =extract aqueous difference (p < 0.05) in the same extract at different concentrations (Kruskal–Wallis test). difference (p < 0.05) in the same extract at different concentrations (Kruskal–Wallis test).

Figure 3 compares the quantitative and qualitative basis the protective effect of extracts. We made Figure 3 compares thethe quantitative and basisthe the protective effect of extracts. Figure 3did compares quantitative and qualitative qualitative of concentrations. extracts. We We sure that extracts not cause any pro-oxidative effect onbasis cells at protective any of theeffect tested made sure sure that extracts diddidnot cause any effectononcells cells at of the tested extracts not any pro-oxidative pro-oxidative at ppm anyany of the tested Figure made 3A shows athat picture selection of cause cell cultures treated witheffect extracts at 200 since they were concentrations. Figure 3A 3A shows a picture selection of cellcultures culturestreated treated with extracts at ppm 200 ppm concentrations. Figure shows a picture selection of cell with extracts at 200 the most representative to observe the contrasting effects of extracts over ROS production. An evident most representativetoto observe observe the of of extracts overover ROS ROS since since they they werewere the the most representative the contrasting contrastingeffects effects extracts difference between cells treated with DMEM alone or with H2 O2 was observed in the cellular lysis production. An evident difference between cells treated with DMEM alone or with H 2 O 2 production. An evident difference between cells treated with DMEM alone or with Hwas 2O2 was detected as brilliant green fluorescent spots. Quercetin showed the best protective capacity than observed in cellular the cellular lysis detectedasasbrilliant brilliant green green fluorescent Quercetin showed the best observed in the lysis detected fluorescentspots. spots. Quercetin showed the best extractsprotective to inhibitcapacity the production of ROS (Figure 3B). In contrast, CE and KE demonstrated poorest than extracts inhibit theproduction production of 3B). In contrast, CEthe and protective capacity than extracts toto inhibit the ofROS ROS(Figure (Figure 3B). In contrast, CE KE and KE inhibition of ROS production. demonstrated the poorest inhibition of ROS production. demonstrated the poorest inhibition of ROS production.

Figure 3. Cont.


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Figure 3. Representative images of reactive oxygen species (ROS) produced on porcine kidney Figure 3. Representative images of reactive oxygen species (ROS) produced on porcine kidney cells cells exposed to H2 O2 and to 200 ppm of extracts of Acacia farnesiana pods (A) and ROS exposed to H2O2 and to 200 ppm of extracts of Acacia farnesiana pods (A) and ROS quantification quantification using 50, 100 and 200 ppm of extracts (B). H2 O2 = hydrogen peroxide; Q = quercetin; using 50, 100 and 200 ppm of extracts (B). H2O2 = hydrogen peroxide; Q = quercetin; CE = CE = chloroformic HE= =hexanic hexanic extract; = ketonic ME = methanolic extract; chloroformic extract; extract; HE extract; KE =KE ketonic extract; extract; ME = methanolic extract; MEAE = a,b,c,d Different letters showed significant MEAE methanolic:aqueous = methanolic:aqueous extract; AE = aqueous a,b,c,d Different letters showed significant difference extract; AE = aqueous extract.extract. difference < 0.05) among extracts and queretin at the same concentration (Kruskal–Wallis test). (p < (p 0.05) among extracts and queretin at the same concentration (Kruskal–Wallis test).

2.5. Lipid Is Inhibited by All Extracts in in a Dose-Dependent 2.5.Peroxidation Lipid Peroxidation Is Inhibited by All Extracts a Dose-Dependent Manner Manner ResultsResults of TBARS inhibition are shown in Figure 4. Inhibition of lipidofperoxidation was observed of TBARS inhibition are shown in Figure 4. Inhibition lipid peroxidation was observed in a dose-dependent manner inMethanolic all extracts.extract Methanolic extractTBARS decreased TBARS in in a dose-dependent manner in all extracts. decreased production production in 93%concentration with the smallest (10 μg/mL; to 1.0 nmol of TBARS/mg 93% with the smallest (10concentration µg/mL; equivalent toequivalent 1.0 nmol of TBARS/mg of protein). of protein). Moreover, 31.6 μg/mL equivalent to 0.5 nmol of TBARS/mg of protein of CE, KE and Moreover, 31.6 µg/mL equivalent to 0.5 nmol of TBARS/mg of protein of CE, KE and MEAE were MEAE were needed to reach over 90% of TBARS inhibition, while AE and HE resulted in the same needed to reach over 90% of TBARS inhibition, while AE and HE resulted in the same inhibition inhibition percentage with 56.2 μg/mL equivalent to 0.7 nmol of TBARS/mg of protein. In relation to percentage with 56.2 µg/mL equivalent to 0.7 nmol of TBARS/mg of protein. In relation to quercetin, quercetin, it is noteworthy that with only 5.6 μg/mL equivalent to 3.5 nmol of TBARS/mg of protein it is noteworthy that with only 5.6 µg/mL equivalent to 3.5 nmol of TBARS/mg of protein resulted in resulted in a TBARS inhibition over 60%. The 50% inhibitory concentration (IC50) values of the a TBARS inhibition over 60%. The 50% ) values of the on TBARS extracts on TBARS production are inhibitory showed in concentration Figure 4H. The(IC IC5050value of KE, MEextracts and MEAE not production are showed in Figure 4H. The IC value of KE, ME and MEAE not showed a statistical showed a statistical variation (p > 0.05) in50respect to the standard quercetin. MEAE registered 3.4 variation (p > 0.05) in respect the standard quercetin. MEAE registered nmol/mg, similar to ME; nmol/mg, similar to ME;to while CE, HE and AE IC50 were around to two, 3.4 three and five-folds of that concentration and lessaround active than standard Finally, and quercetin standard haveless while CE, HE and AE ICwere to two, threequercetin. and five-folds ofKE that concentration and were 50 were similar values ofquercetin. IC50. active than standard Finally, KE and quercetin standard have similar values of IC50 . 2.6. Acacia farnesiana Extracts Down Regulate Inflammation In the ear edema model, all extracts diminished the ear tissue weight after TPA (12-O-tetradecanoylphorbol-13-acetate) application. However, the extracts were less effective than indomethacin to prevent edema (Figure 5A). Likewise, all extracts showed an inhibition on MPO and IL-1β production (Figure 5B,C). Similar results were observed by IL-6 (Figure 5D), where the production of this interleukin was inhibited by all the extracts (except by HE). For IL-10, TPA group showed comparable values with respect to indomethacin, CE, HE and KE treatments. In contrast, ME, MEAE and AE reduced the value of this anti-inflammatory cytokine (Figure 5E). TNF-α production were inhibited by all groups treated with AF extracts, except in the groups treated with ME and MEAE (Figure 5F).


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Figure 4. Inhibition of lipid peroxidation (A–G) and inhibitory concentration 50% (IC50 ) values Figure 4. Inhibition of lipid peroxidation (A–G) and inhibitory concentration 50% (IC50) values (H) of (H) of the extracts from AF pods and standard quercetin, quantified by lipid peroxidation (TBARS) the extracts from AF pods and standard quercetin, quantified by lipid peroxidation (TBARS) production. CE = chloroformic extract; HE = hexanic extract; KE = ketonic extract; ME = methanolic production. CE = chloroformic extract; HE = hexanic extract; KE = ketonic extract; ME = methanolic extract; MEAE = methanolic:aqueous extract and AE = aqueous extract. Q = quercetin. The data are extract; MEAE = methanolic:aqueous extract and AE = aqueous extract. Q = quercetin. The data are the mean with the standard error of three independent repetitions. * p < 0.05 and ** p < 0.01 vs. the mean with the standard error of three independent repetitions. * p < 0.05 and ** p < 0.01 vs. control control group. group.

2.6. Acacia farnesiana Extracts Down Regulate Inflammation In the ear edema model, all extracts diminished the ear tissue weight after TPA (12-O-tetradecanoylphorbol-13-acetate) application. However, the extracts were less effective than indomethacin to prevent edema (Figure 5A). Likewise, all extracts showed an inhibition on MPO and IL-1β production (Figure 5B,C). Similar results were observed by IL-6 (Figure 5D), where the production of this interleukin was inhibited by all the extracts (except by HE). For IL-10, TPA group showed comparable values with respect to indomethacin, CE, HE and KE treatments. In contrast,


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ME, MEAE and AE reduced the value of this anti-inflammatory cytokine (Figure 5E). TNF-α production were inhibited by all groups treated with AF extracts, except in the groups treated with Molecules 2018, 23, 2386 9 of 21 ME and MEAE (Figure 5F).

Figure 5. Effect of different extracts from Acacia farnesiana pods and the anti-inflammatory indomethacin Figure 5. Effect of different extracts from Acacia farnesiana pods and the anti-inflammatory (1 mg) on TPA-induced ear edema model. (A) ear edema measured at 4 h after TPA treatment indomethacin (1 mg) on TPA-induced ear edema model. (A) ear edema measured at 4 h after TPA and (B) oxidative enzyme myeloperoxidase (MPO) activity in supernatants of homogenates from treatment and (B) oxidative enzyme myeloperoxidase (MPO) activity in supernatants of TPA-treated ears. Levels of (C) interleukin-1β (D), interleukin-6 (E), interleukin-10 and (F) TNF-α homogenates from TPA-treated ears. Levels of (C) interleukin-1β (D), interleukin-6 (E), in supernatants of homogenates from ears after treatment with different extracts from AF pods. interleukin-10 and (F) TNF-α in supernatants of homogenates from ears after treatment with TPA = 12-O-tetradecanoylphorbol acetate; In = indomethacin; CE = chloroformic extract; HE = hexanic different extracts from AF pods. TPA = 12-O-tetradecanoylphorbol acetate; In = indomethacin; CE = extract; KE = ketonic extract; ME = methanolic extract; MEAE = methanolic:aqueous extract and chloroformic extract; HE = hexanic extract; KE = ketonic extract; ME = methanolic extract; MEAE = AE = aqueous extract. Each bar represents the mean ± standard deviation (n = 6). * p < 0.05 and methanolic:aqueous extract and AE = aqueous extract. Each bar represents the mean ± standard ** p < 0.001 with respect to TPA group. deviation (n = 6). * p < 0.05 and ** p < 0.001 with respect to TPA group.

2.7. Effect of Acacia farnesiana Extracts on Mice Ear Swelling 2.7. Effect of Acacia farnesiana Extracts on Mice Ear Swelling Figure 6 shows the mice ear tissue swelling as well as the ear thickness due to the effect of Figure shows the mice ear tissue swelling asevaluated well as the ear thickness due tofrom the control effect of extracts on the6 TPA-induced ear edema model. All the extracts were different TPA-inducedchange ear edema model. All the evaluated extracts werein different from control (pextracts < 0.001).onAthe morphological due to the aggressive agent (TPA) resulted a marked swelling (p < 0.001). A morphological change due to the aggressive agent (TPA) resulted in a marked swelling process, sub-epidermal layer and spongiosis (Figure 6A). After TPA application, MEAE decrease the process, of sub-epidermal and (Figure 6A). After TPA application, MEAE decrease the thickness the mice earslayer almost atspongiosis the same extent than indomethacin (323 and 356 µm; MEAE and thickness of the mice ears almost at theKE same than (323 and 356 μm;ofMEAE and indomethacin, respectively). In contrast andextent ME did notindomethacin show a satisfactory reduction the mice indomethacin, In contrast ears thickness in respectively). relation to control (Figure KE 6B).and ME did not show a satisfactory reduction of the mice ears thickness in relation to control (Figure 6B).


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Figure 6. Histological features of mice ear tissue (A) stained with hematoxylin-eosin (200× Figure 6. Histological features of mice ear tissue (A) stained with hematoxylin-eosin (200× magnification) on mice ear tissue thickness by the effect of different extracts of Acacia farnesiana pods on magnification) on mice ear tissue thickness by the effect of different extracts of Acacia farnesiana pods the TPA-induced ear edema model (B). TPA = 12-O-tetradecanoylphorbol acetate; In = indomethacin; on CE the= TPA-induced ear edema TPA KE = 12-O-tetradecanoylphorbol acetate; In = chloroformic extract; HE =model hexanic(B). extract; = ketonic extract; ME = methanolic extract; indomethacin; CE = chloroformic extract; HE = hexanic extract; KE = ketonic extract; ME = methanolic MEAE = methanolic:aqueous extract and AE = aqueous extract. Each bar represents the mean ± extract; MEAE = methanolic:aqueous extract and AE = to aqueous extract. Each bar represents the mean standard deviation (n = 6). ** p < 0.001 with respect TPA group. ± standard deviation (n = 6). ** p < 0.001 with respect to TPA group.

2.8. Effect of Acacia farnesiana Extracts on TNF-α-Expressing Cells 2.8. Effect of Acacia farnesiana Extracts on TNF-α-Expressing Cells Figure 7 shows the inflammation post treatment with different extracts of Acacia farnesiana (AF) Figure 7 shows the inflammation treatment different of Acacia farnesiana (AF) pods on the TPA-induced ear edemapost model, by thewith expression of extracts TNF-α by immunohistochemistry. pods on the ear edema model, expression of TNF-α by immunohistochemistry. While TPATPA-induced induced conspicuous release of by thethe pro-inflammatory mediator in the ears, treatment with While TPAKE, induced conspicuous of thereduction pro-inflammatory mediator insimilar the ears, treatment CE, HE, ME and AE resultedrelease in an evident in TNF-α production to indomethacin, with CE,MEAE HE, KE, in an evident reduction in TNF-α production similar to while wasME the and only AE thatresulted did not reduce of the TNF-α expression. indomethacin, while MEAE was the only that did not reduce of the TNF-α expression.

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7. TNF-α expression (golden yellow color) in the mice ear cells due to the effect of different Figure 7. Acacia farnesiana farnesiana (AF) (AF) pods pods (3 (3 mg/ear) mg/ear) on the the TPA-induced TPA-induced ear ear edema edema model model (100 (100× extracts of Acacia × magnification). CE CE= chloroformic = chloroformic extract; = hexanic KE =extract; ketonicME extract; ME = magnification). extract; HE =HE hexanic extract;extract; KE = ketonic = methanolic methanolic extract; MEAE = methanolic:aqueous extract and AEextract. = aqueous extract. extract; MEAE = methanolic:aqueous extract and AE = aqueous

2.9. Effect of 2.9. Effect of Acacia Acacia farnesiana farnesiana Extracts Extracts on on COX COX Activity, Activity, Cell Cell Viability Viability and and Nitrite Nitrite Production Production The The inhibitory inhibitory activity activity of of cyclooxygenase cyclooxygenase (COX) (COX) is is shown shown in in Figure Figure 8. 8. CE, CE, HE HE and and KE KE evaluated evaluated at 10 and 30 µg/mL were found to have inhibitory activity similar to control group (Celecoxib). at 10 and 30 μg/mL were found to have inhibitory activity similar to control group (Celecoxib). In In contrast ME, MEAE demonstrated lower ability to inhibit the prostaglandin synthesis. contrast ME, MEAE andand AE AE demonstrated lower ability to inhibit the prostaglandin synthesis. The The viability of RAW treated with of lipopolysaccharides of E. coli is shown in viability of RAW 264.7264.7 cells cells treated with of lipopolysaccharides (LPS) (LPS) of E. coli is shown in Figure Figure 9A. The percentage of viability was over 70% in all groups evaluated and was similar to 9A. The percentage of viability was over 70% in all groups evaluated and was similar to the control the control group (treated withacid). oleanolic acid). Numerically, HEthe reported highest value (96.9%) group (treated with oleanolic Numerically, HE reported highestthe value (96.9%) while AE while AE the lowest percentage of viability (71.3%), however, no significant difference was the lowest percentage of viability (71.3%), however, no significant difference was found.found. With With respect to nitrite the nitrite production (Figure showed thebest bestinhibitory inhibitorypotential potential(7.4 (7.4 μM). µM). respect to the production (Figure 9B),9B), HEHE showed the Moreover, Moreover, nitrite nitrite production production of of cells cells treated treated with with any any of of the the left left extracts extracts (any (any but but HE), HE), were were between between oleic acid group (9.5 µM) and lipopolysaccharides group (13.5 µM) values. oleic acid group (9.5 μM) and lipopolysaccharides group (13.5 μM) values.


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Figure 8. Effect of different Acacia farnesiana extracts on prostaglandin production. Cx = Celecoxib; Figure 8. Effect of different Acacia farnesiana extracts production. Cx= extract; = Celecoxib; Figure 8. Effect of different farnesiana extracts onprostaglandin prostaglandin production. Cx Celecoxib; CE = chloroformic extract; HE =Acacia hexanic extract; KE =on ketonic extract; ME = methanolic MEAE CE = chloroformic extract; HE = hexanic extract; KE = ketonic extract; ME = methanolic extract; CE = chloroformic extract; HE = hexanic extract; KE = ketonic extract; ME = methanolic extract; = methanolic:aqueous extract and AE = aqueous extract. The data are the mean with theMEAE standard extract andand AE AE = aqueous extract.extract. The dataThe are data the mean withmean the standard MEAE == methanolic:aqueous methanolic:aqueous extract = aqueous are the with the deviation of three independent repetitions. Statistical difference was calculated in relation to COX at deviation of three independent repetitions. Statistical difference was calculated in relationin torelation COX at to standard deviation of three independent repetitions. Statistical difference was calculated 30 and 100 ppm. * p < 0.05. ** p < 0.001. ppm. * p *< p0.05. ** p