Dietary fatty acids from pomegranate seeds (Punica

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Received: 10 July 2018 Revised: 10 October 2018 Accepted: 4 November 2018 DOI: 10.1111/jfbc.12739

FULL ARTICLE

Dietary fatty acids from pomegranate seeds (Punica granatum) inhibit adipogenesis and impact the expression of the obesity‐ associated mRNA transcripts in human adipose‐derived mesenchymal stem cells Shamsiya Trichur Khabeer1,2 1

Department of Food Protectants & Infestation Control, Central Food Technological Research Institute (CSIR – CFTRI), Mysore, India 2

Academy of Scientific and Innovative Research (AcSIR), New Delhi, India

| Akila Prashant3 | Manonmani Haravey Krishnan1 Abstract Obesity is a metabolic disorder that manifests into various forms. Recent studies have indicated that the pomegranate (Punica granatum) seed oil (PSO) has many bio‐ logically active components that help in controlling diet‐induced obesity and insulin

Centre of Excellence in Molecular Biology and Regenerative Medicine, Department of Biochemistry, JSS Medical College, JSS Academy of Higher Education and Research, Mysore, India

resistance. However, its impact on the adipogenic differentiation of human adipose‐

Correspondence Manonmani Haravey Krishnan, Department of Food Protectants & Infestation Control, Central Food Technological Research Institute (CSIR – CFTRI), Mysore 570020, India. Email: [email protected]

[(9Z,11E,13Z)‐9,11,13‐Octadecatrienoic acid], oleic acid [Cis‐9‐Octadecenoic acid],

3

derived mesenchymal stem cells (HADMSC) remains unclear. Here we have at‐ tempted to study the anti‐obesity potential of SHAMstat3pg, a fatty acid composite extracted from PSO. It is composed of three dietary fatty acids: punicic acid and linoleic acid [(9Z,12Z)‐octadeca‐9,12‐dienoic acid]. In this study, we discuss the impact of the fatty acids on adipogenesis, inflammation, glucose uptake, and mito‐ chondrial ATP production. The impact of SHAMstat3pg on the expression of various obesity‐associated protein and mRNA transcripts in HADMSC was also analyzed. The results indicate that exposure to 10 µg/ml of SHAMstat3pg (24 hr) inhibited adi‐ pogenesis of HADMSC, ameliorated inflammation, attenuated ATP production, and glucose uptake. Also, the extract favorably regulated the mRNA expression of the studied obesity‐associated gene transcripts.

Practical applications SHAMstat3pg has the potential to serve as a multi‐targeted therapy for the manage‐ ment of obesity. This study demonstrated that the dietary fatty acids inhibited the differentiation of preadipocytes to adipocytes. SHAMstat3pg has also shown to have a favorable impact on the expression of the obesity‐linked proteins and genes in HADMSC that are associated with adipogenesis, inflammation, satiety, energy in‐ take/expenditure (central and peripheral signaling molecules). The study gives an overview of the vast number of genes impacted by the treatment with SHAMstat3pg paving the way for future studies to demonstrate the exact mode of action of how dietary fatty acids can help manage obesity, insulin resistance, and type 2 diabetes. KEYWORDS

adipogenesis, dietary fatty acids, inflammation, inhibition of differentiation, mesenchymal stem cells, obesity, pomegranate seed oil extract, Punica granatum, RT‐PCR

J Food Biochem. 2018;e12739. https://doi.org/10.1111/jfbc.12739

wileyonlinelibrary.com/journal/jfbc

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TRICHUR KHABEER et al.

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1 | I NTRO D U C TI O N

Pomegranate (Punica granatum), is a plant widely studied for its anti‐obesity properties (Bhutani & Gohil, 2010; González‐Castejón

Obesity has transpired as one of the foremost public health prob‐

& Rodriguez‐Casado, 2011; Latha, Reddy, & Ismail, 2010; Verma &

lems in the recent years and is associated with an increased risk

Paraidathathu, 2014). Separate studies have shown pomegranate

of several metabolic disorders like atherosclerosis, hypertension,

seed oil (PSO) and its major fatty acid, punicic acid, to inhibit ad‐

insulin resistance, systemic inflammation, cardiovascular diseases,

ipocyte differentiation and suppress lipid accumulation in the adi‐

and type 2 diabetes (Poulos, Dodson, & Hausman, 2010; Suganami

pocytes and the hepatocytes (Kang, Kim, Tomás-Barberán, Espín, &

et al., 2007). The physical manifestation of obesity arises due to an

Chung, 2016; Lai et al., 2012; Les, Arbonés‐Mainar, Valero, & López,

increase in the adipocyte numbers as well as the size of the adipo‐

2018). However, there are no reports on the effect of PSO on the

cytes (Farmer, 2006). Adipogenic differentiation is controlled by various transcription factors. Peroxisome proliferator‐activated receptor gamma (PPARG)

differentiation of HADMSC. Also, there is a gap in the knowledge of the impact of the PSO on cell viability, molecular, and cellular mech‐ anisms in HADMSC.

and CCAAT/enhancer binding protein‐a (C/EBPα) are considered the

In an earlier study, we have presented in detail the purifica‐

master regulators of adipogenesis (Rosen & MacDougald, 2006). The

tion, characterization and in vitro lipase inhibitory effect of the

storage of excess energy as fat in the adipose tissue results due to an

Punica granatum extract, SHAMstat3pg (Khabeer, Manjunatha, &

imbalance in the physiological energy homeostasis (Shawky & Sadik,

Manonmani, 2016). SHAMstat3pg was solvent extracted from the

2012). The energy balance (intake/expenditure) under normal physio‐

seeds of Punica granatum (SHAM: name of first author, stat: lipase

logical conditions is controlled by environmental factors like diet and

inhibitor, 3: constitutes 3 fatty acids, pg: source is Punica grana‐

physical activity. Genetics and biological chemicals like hormones,

tum). The

receptors of orexigenic/anorectic peptides and neuro‐transmitters

ester carboxyl signals at 178.92 and 172.93 ppm compared to the

(central and peripheral signaling molecules) play a key role in regulat‐

signal at 172.51 ppm. This characteristic

13

C NMR spectrum has shown the presence of stronger 13

C NMR spectrum is at‐

ing the physiological energy balance. Malfunctions in the working of

tributed to the presence of triglycerides. The GC–MS/MS analysis

the genes associated with the energy homeostasis can lead to severe

of SHAMstat3pg (gas chromatography‐mass spectrometry eval‐

obesity (monogenic/common obesity) (Chawla et al., 2013).

uation) was performed to elucidate its complete fatty acid profile

There is growing scientific research validating the use of human

(Khabeer et al., 2016). SHAMstat3pg was found to be a triglyceride

adipose‐derived mesenchymal stem cells (HADMSC) in cell‐based

composed of three fatty acids: punicic acid [(9Z,11E,13Z)‐9,11,13‐

regenerative therapies for the treatment of various neurological,

Octadecatrienoic acid], oleic acid [Cis‐9‐Octadecenoic acid], and

cardiovascular and metabolic disorders like osteoarthritis, diabe‐

linoleic acid [(9Z,12Z)‐octadeca‐9,12‐dienoic acid] (Khabeer et al.,

tes, etc. (Toma, Pittenger, Cahill, Byrne, & Kessler, 2002; Zhang,

2016).

Marsboom, Toth, & Rehman, 2013). Mesenchymal stem cells have

In this study, however, we have attempted to assess the effect

the ability to differentiate into various cell types: mature adipocytes,

of SHAMstat3pg on the inhibition of adipogenesis and on the vari‐

chondrocytes, osteocytes, and myocytes and neurons (Cristancho &

ous neurotrophic, obesogenic, inflammatory and cellular respiratory

Lazar, 2011). The understanding of the precise mechanisms that reg‐

mRNA transcripts in the human adipose‐derived mesenchymal stem.

ulate the growth, expansion and differentiation of the HADMSC is important to develop effective therapies against various disorders. Dietary phytochemicals are being studied extensively for their use as multi‐target, anti‐obesity agents (Chawla et al., 2013). The phytochemicals could control obesity by: inhibiting of adipogene‐ sis (Prathapan, Krishna, Lekshmi, Raghu, & Menon, 2012), inhibiting

2 | M ATE R I A L S A N D M E TH O DS 2.1 | Materials Punica granatum of the Indian cultivar, Bhagwa (Kesar) were pur‐

fatty acid biosynthesis (Zhao, Mao, et al., 2014), increasing energy

chased from the local market in Mysore. EZXpand™ Human

expenditure and satiety (Mohamed, Ibrahim, Elkhayat, & El Dine,

Adipose‐derived Mesenchymal Stem Cell Culture Kit (HiMedia,

2014), decreasing inflammation and oxidative stress (Abdel‐Zaher,

India) (CL007‐T25) constituting three parts; A: HiFi™ HADMSC,

Abdel‐Rahman, & ELwasei, 2011; Pinent et al., 2011).

B: HiMesoXL™ Mesenchymal Stem Cell Expansion Medium, and

Dietary omega‐3 fatty help in managing obesity by increasing

C: Antibiotic‐Antimycotic Solution 100X; HiAdipoXLTM Adipocyte

levels of leptin, inducing mitochondrial biogenesis and suppressing

Differentiation Medium (HiMedia, India) which is a composite of

obesity‐induced low‐grade inflammation within the adipose tissue

mesenchymal stem cell expansion media and 250 nm dexametha‐

(Awad, Begdache, & Fink, 2000; Puglisi, Hasty, & Saraswathi, 2011).

sone, 0.5 mM 3‐isobutyl‐1‐methylxanthine (IBMX) and 1 µg/ml in‐

Also, Xanthigen, a source of fucoxanthin and punicic acid extracted

sulin (MDI) was used for differentiation of stem cells to adipocytes.

from pomegranate seed and brown seaweed has shown to inhibit

The cell culture plates were purchased from Eppendorf, India.

differentiation of preadipocytes by the downregulation of C/EBPs,

Dulbecco’s Phosphate Buffered Saline (TL1006), Fetal Bovine

PPARG and modulation of SIRT‐1 (sirtuin 1), AMPK (AMP‐activated

Serum (FBS) (RM1112/RM10432), Trypsin‐EDTA solution (TCL007),

protein kinase), and FOXO (forkhead box, sub‐group O) pathways

Antibiotic/Antimycotic solution (A002), dimethyl sulfoxide (DMSO),

(Lai et al., 2012).

3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT),

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Trypan Blue 0.5% solution, Oil red O stain, Nile red stain and 2′,7′‐di‐

were trypsinized, collected and seeded at a cell density of 1.5 × 10 4

chlorofluorescein diacetate (DCFDA) were purchased from HiMedia,

to 2.5 × 10 4 cells/well in a fresh 96 well plate. The cells were incu‐

India. T25 or T75 cell culture treated flasks and pipettes were pur‐

bated at 37°C, 5% CO2 humidified incubator for 48 hr. After 2 days

chased from Eppendorf, India.

of growth, the cells were induced to differentiate. For the adipocyte

TaqMan® Array Human Obesity 96‐well plate, Maxima First

differentiation, cells were placed in 10% FBS in HiAdipoXLTM adi‐

Strand cDNA Synthesis Kit for RT‐PCR, Thermo Scientific Gene JET

pocyte differentiation medium, to which 2% antibiotic‐antimycotic

RNA Purification Kit was purchased from Thermo Scientific, India.

solution was added (day 1 of adipogenic differentiation). The plate

Human Obesity antibody array was procured from Abcam, India.

was incubated at 37°C in a 5% CO2 humidified incubator for 48 hr.

Mitochondrial ToxGlo™ Assay kit was purchased from Promega,

After 2 days of induction (i.e., day 3), the medium was replaced with

India. Glucose Uptake Fluorometric Assay Kit was purchased from

fresh complete differentiation medium which was replaced every

Sigma Aldrich, India.

48–72 hr. The cells were consistently incubated at 37°C in a 5% CO2

The research presented in this paper was conducted in the Center

humidified incubator This procedure was continued for 18–21 days

of Excellence in Molecular Biology and Regenerative Medicine, at

for complete differentiation after which the microscopically visible

JSS Medical College Mysuru, India.

lipid vesicles were observed (Fink & Zachar, 2011; Zhang et al., 2013).

FBS was used as the vehicle for the delivery of the fatty acid‐ based PSO extract, SHAMstat3pg to cells in culture. The control used for this study is cells treated with FBS only.

2.5 | Cell viability using MTT assay Cell viability was evaluated by a modified 3‐(4,5‐dimethylthiazol‐

2.2 | Preparation of Punica granatum seed extract The Punica granatum fruits were used for the extraction of the bioac‐

2yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay (Kang, Nam, Kim, Huh, & Lee, 2013). HADMSC fibroblasts were seeded at a den‐ sity of 2 × 10 4 cells/well in a 96 well plate. After incubation for 24 hr

tive, SHAMstat3pg. The standardized protocol for the purification

(37°C, 5% CO2), the cells were treated with different concentrations

of SHAMstat3pg has been recorded in detail in an earlier report

(0.1–1,000 µg/ml) of Punica granatum seed extract, SHAMstat3pg,

(Khabeer et al., 2016). In brief, Punica granatum fruit was processed

for 24 hr. This was followed by the addition of 30 µl of MTT (5 mg/

to separate the pulpy arils from the inner seeds. SHAMstat3pg, the

ml) to each well for 4 hr. After incubation, the culture supernatant of

PSO extract, was extracted from the dried seeds using solvent ex‐

each well was carefully replaced with 100 µl of DMSO to dissolve

traction. 1 kg of blended seeds was extracted with 99% ethanol (6 L)

the MTT formazan crystals. Controls were cells not treated with

four times at 37°C for 24 hr. The extract was subjected to prepara‐

SHAMstat3pg. The optical density (OD) of the plate was read at a

tive HPLC using water: methanol: ethyl acetate (80:15:5) as the sol‐

wavelength of 570 nm.

vent phase. Further, the purified active fraction was TLC purified using hexane: diethyl ether: acetic acid (6.8:3:0.2).

2.3 | Characterization of HADMSC

2.6 | Inhibition of adipogenesis of fibroblast by SHAMstat3pg HADMSC fibroblasts were grown to 70% confluence in a 24‐well

Proliferating mesenchymal stem cells isolated from adipose‐rich tis‐

plate and treated with (0.1–200 µg/ml) of SHAMstat3pg. This was

sue were supplied in liquid cell culture medium by HiMedia, India.

followed by the induction of cells to differentiate. The differential

HADMSC are pluripotent cells having the potential to differenti‐

change in adipogenesis was compared between control cells and

ate into different lineages. This particular batch of HADMSC was

SHAMstat3pg treated cells. The maturation of the adipocytes was

assessed for positive surface markers of mesenchymal stem cells.

confirmed by Oil red O staining of lipid droplets on day 15. The cells

70% confluent cells were trypsinized, centrifuged at 200 g for

were washed with PBS, fixed with 4% paraformaldehyde in 0.1 M

10 min at 4°C, washed twice with phosphate‐buffered saline (PBS)

phosphate buffer, pH 7.4 for 15 min at room temperature, and

and fixed with cold 2% formaldehyde for 4 hr at 56°C (Yamamoto et

washed 3 times with deionized water. A mixture of Oil red O (0.6%

al., 2015). These cells were then analyzed for the surface markers

Oil red O dye in isopropanol) and water at a 6:4 ratio was layered

CD90, CD105, CD34, and CD45 by flow cytometry using the marker

on the cells for 10 min, (Wang et al., 2011). After staining, excess

analysis assay. The data were generated on 8 parameter 3 laser

Oil red O was removed from cells by washing three times with 1 ml

®

PartecCyFlow Cube 8 Flow Cytometer (HiMedia Labs India, 2017).

of PBS. Images were acquired using an inverted microscope (Varinli, Osmond‐McLeod, Molloy, & Vallotton, 2015).

2.4 | Cell culture and differentiation of HADMSC HADMSC pre‐adipocytes were grown, sub‐cultured, and differen‐ tiated as per the protocol described by HiMedia, India. HADMSC

2.7 | Inhibition of lipid accumulation by SHAMstat3pg

were cultured in HiMesoXL™ Mesenchymal Stem Cell Expansion

The effect of SHAMstat3pg on adipogenesis and lipid accumulation

Medium enriched with 10% FBS until 70% confluent. These cells

in adipocytes was assessed in two steps.

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2.7.1 | Quantification of adipogenesis using Oil red O

by treatment with 100 pM TNFa in a serum‐free medium and incu‐ bated for 5 hr (Kabayama et al., 2005). Vehicle‐treated cells were maintained as controls. Glucose transport was assayed by measuring

To measure the degree of lipid accumulation in the HADMSC mon‐

the 2‐DG uptake as per the manufacturer’s protocol (Fluorometric

olayers, the uptake of the Oil red O stain by the differentiated cells

Glucose uptake assay kit, Sigma Aldrich India). The cells were later

was quantified. The Oil red O dye was extracted from the stained

stimulated with 100 nM insulin for 30 min. The cells were treated

cells using 500 µl of 100% isopropanol per well. 200 µl was trans‐

with 100 μl/well of 2‐NBDG solution (2‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐

ferred to a black 96‐well plate. The OD reading of Oil red O was

diazol‐4‐yl) amino)‐2‐deoxyglucose) made in KRH (Krebs‐Ringer‐

measured at 490 nm using a multimode plate reader (Thermo

Hepes‐bicarbonate) buffer and incubated for 30 min, 5% CO2 at

Scientific, Varioskan™). The OD is directly proportional to lipid ac‐

37°C. The 2‐NBDG efflux was stopped by washing the cells with

cumulated in the cells (Varinli et al., 2015).

ice cold PBS. The fluorescence (excitation at 460 nm and emission at 530 nm) was measured using a multimode plate reader (Thermo

2.7.2 | Visualization of differentiated cells using Nile Red staining

Scientific, Varioskan™). The fluorescence is directly proportional to the uptake of 2‐deoxyglucose (2‐DG) which is an estimate of the glu‐ cose uptake potential of the cells (Kabayama et al., 2005).

HADMSC fibroblasts were grown in a 24‐well plate to 70% conflu‐ ence and treated with 10 µg/ml of SHAMstat3pg. The cells were then induced to differentiate. The differential change in adipogen‐ esis was compared between control cells and SHAMstat3pg‐treated

2.10 | Human Obesity Antibody Array The Human Obesity Antibody Array Membrane from Abcams

cells. The level of adipogenesis was assessed at regular intervals by

(ab169819) is a multiplex protein detection system. The antibody

the fluorometric staining of the lipid droplets using Nile red dye.

array, based on the principle of enzyme‐linked immunosorbent

The spent media was aspirated out and the cells were washed with

assay was used for the simultaneous detection of 62 obesity‐re‐

200 μl of PBS once. These cells were then fixed with 100 μl of 4%

lated markers. HADMSC fibroblasts were grown to 70% confluence

paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 and incubated

in 25 T flasks incubated at 37°C, 5% CO2. HADMSC fibroblasts

for 5 min at RT. The cells were then washed twice with 200 μl of PBS

were then treated with 10 µg/ml of SHAMsta3pg for 24 hr at

and treated with 1 µg/ml Nile red, 400 μl/well (dissolved in minute

37°C, 5% CO2. The vehicle‐treated cells were used as controls.

amounts of acetone and diluted with PBS). The cells were incubated

SHAMstat3pg‐treated and control cells were induced to differen‐

for 15 min in the dark to prevent reaction of the dye with light. The

tiate to mature adipocytes. On day 21 of differentiation, the cells

cells were microscopically observed for the fluorescent stained

were separated from the media. 1 ml of the spent media from the

lipid droplets. The degree of adipogenesis was recorded at regular

treated and the control cells was added to individual membranes

intervals of 1, 3, 7, 15, and 21 days (excitation at 450 nm and emis‐

separately and it was incubated on a rocker for proper coating.

sion at 540 nm) using a multimode plate reader (Thermo Scientific,

The coated membranes were then washed with the buffer after

Varioskan™) (Aldridge et al., 2013; Su et al., 2013).

which the membranes were flooded with the biotinylated detec‐ tor antibody followed by streptavidin HRP conjugated secondary

2.8 | Cellular ATP assay HADMSC seeded at a density of 10,000 cells/well in a 96‐well plate

antibody. The membranes were then visualized and documented in a Bio Rad Gel imaging and documentation system. The blots were analyzed using the Image J software and the relative change in the

were treated with 10 µg/ml of SHAMstat3pg for 24 hr. The cells were

expression of the obesity markers was quantified. The relative fold

induced to differentiate and the intracellular ATP was measured at

increase or decrease was calculated with respect to the expression

regular intervals throughout the process of differentiation using the

level of the respective protein in the control group. Of the 62 tar‐

Mitochondrial ToxGlo™ Assay as per the manufacturer’s directions.

gets, only the significantly different results have been presented in

Prior to the assay, the cells were exposed to glucose‐free media for

this study (Table 1).

5 hr after which 100 µl of the ATP detection reagent was added. The fluorescence (excitation at 485 nm and emission at 525 nm) was measured after a 5 min incubation period using a multimode plate reader (Thermo Scientific, Varioskan™). The luminescence is directly proportional to the amount of intracellular ATP present.

2.11 | Human obesity TaqMan® 96‐well Plate Array HADMSC fibroblasts were treated with 10 µg/ml of SHAMsta3pg for 24 hr at 37°C, 5% CO2. The vehicle‐treated cells were used as controls. Treated and control cells were induced to differentiate to

2.9 | Glucose uptake assay Completely differentiated cells cultured in a 96‐well black, clear‐

mature adipocytes. On day 21 of differentiation, SHAMstat3pg‐ treated and vehicle‐treated HADMSC were subjected to total RNA extraction using Thermo Scientific GeneJET RNA Purification Kit.

bottom culture plate were treated with 10 µg/ml of SHAMstat3pg

The purified RNA was quantified using ND‐1000 spectropho‐

for 24 hr. The cells were then induced to become insulin resistant

tometer (NanoDrop Technologies) in ng/μl units. Complementary

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TA B L E 1 The effect of SHAMstat3pg on the protein expression levels of various obesity‐associated markers was assessed by ELISA‐ based paper array Protein code

Protein name

Fold change ± SD

Effect

AGRP

Agouti related protein

0.41 ± 0.3

↓

IGFBP‐3

Insulin like growth factor binding protein 3

0.63 ± 0.4

↓

IGF‐1

Insulin like growth factor‐1

0.64 ± 0.5

↓

IGF‐1SR

Insulin like growth factor‐1 sub receptor

0.75 ± 0.2

↓

Leptin

Leptin

1.82 ± 0.3

↑

VEGF

Vascular endothelial growth factor

0.78 ± 0.3

↓

Resistin

Resistin

0.45 ± 0.03

↓

Serum Amyloid A

Serum Amyloid A

0.62 ± 0.04

↓

IGFBP‐1


1.98 ± 0.4

↑

IGFBP‐2


1.81 ± 0.1

↑

Insulin

Insulin

1.62 ± 0.2

↑

Growth hormone

Growth hormone

2.62 ± 0.1

↑

TECK

Thymus expressed chemokine

0.82 ± 0.2

↓

TGF‐B

Transforming growth factor beta

0.76 ± 0.2

↓

TIMP‐1

TIMP metallopeptidase inhibitor 1

0.98 ± 0.3

↓

TIMP‐2

TIMP metallopeptidase inhibitor 2

0.88 ± 0.04

↓

IL‐1R4/ST2

Interleukin 1 Soluble IL‐1 Receptor 4/ST2

0.59 ± 0.3

↓

IL‐1sR1

soluble interleukin‐1 receptor 1

0.46 ± 0.03

↓

IL‐10

Interleukin 10

0.59 ± 0.04

↓

1L‐11

Interleukin 11

0.54 ± 0.4

↓

IP‐10

Interferon gamma‐induced protein 10

0.67 ± 0.1

↓

TNF‐a

Tumor necrosis factor alpha

0.76 ± 0.2

↓

IFN G

Interferon gamma

0.39 ± 0.1

↓

IL‐12

Interleukin 12

0.47 ± 0.2

↓

IL‐1a

Interleukin 1alpha

0.39 ± 0.2

↓

IL‐1ß

Interleukin 1 beta

0.79 ± 0.3

↓

IL‐6

Interleukin 6

1.68 ± 0.4

↑

IL‐6sR

Interleukin 6 soluble receptor

1.31 ± 0.3

↑

IL‐8

Interleukin 8

0.51 ± 0.03

↓

s TNFR11

Tumor necrosis factor receptor 11

0.66 ± 0.1

↓

s TNFR1

Tumor necrosis factor receptor 1

1.43 ± 0.1

↑

Notes. HADMSC were induced to differentiate post treatment with the extract. Vehicle‐treated cells were taken as controls. The results were ex‐ pressed as fold change (±SD), n = 2 with respect to the control. The results were statistically analyzed using ANOVA and Bonferroni post‐tests (p