Journal of Food Measurement and Characterization https://doi.org/10.1007/s11694-017-9697-9

ORIGINAL PAPER

Borago officinalis L. : a comprehensive study on bioactive compounds and its health-promoting properties

Ehsan Karimi1 · Ehsan Oskoueian2 · Afshin Karimi3 · Reza Noura4 · Mahdi Ebrahimi5,6

Received: 15 September 2017 / Accepted: 22 November 2017 © Springer Science+Business Media, LLC, part of Springer Nature 2017

Abstract The herbal Borago officinalis L. flower were analysed for its bioactive compounds and antioxidant, antibacterial, anti-inflammatory and anticancer activities using different solvent polarities (methanol, ethanol and water). The RP-HPLC analyses of the methanolic extract confirmed the presence of phenolics (gallic acid, pyrogallol, salicylic acid, caffeic acid), flavonoids (myricetin, rutin) and isoflavonoid (daidzein). Besides, the major individual fatty acids of methanolic extract were α-linolenic, stearidonic, palmitic, linoleic and γ-linolenic acids. The methanolic extract possessed the highest antioxidant properties as compared to the ethanolic and water extracts. The flower methanolic, ethanolic and water extracts showed high, moderate and weak antibacterial activities against common human and foodborne pathogenic bacteria, respectively. Furthermore, the flower extracts showed weak anti-inflammatory in murine RAW 264.7 macrophage cells and low anticancer properties against human hepatic, prostate and colon cancer cells. This high-value flower could be considered as a source of putative antioxidant and antibacterial compounds to improve the human health and to be used as biopreservative in food and cosmetic industries.

Keywords Antibacterial · Anticancer · Anti-inflammatory · Antioxidant · Phenolics

Introduction

Several research studies have been carried out on the advan- tages of various phytochemicals in many , fruits and vegetables and their significant effects on human healthcare * Ehsan Karimi [1]. The importance of phytochemicals in scavenging free [email protected]; [email protected] radicals and thus preventing from deadly diseases including * Ehsan Oskoueian cancer, stroke, and heart-related disorders has been recog- [email protected] nized [2]. In this regards, the phenolic and flavonoid com- 1 Department of Biology, Faculty of Sciences, Mashhad pounds are found to be the most important phytochemicals Branch, Islamic Azad University, Mashhad, Iran [3, 4]. Flavonoids are hydroxylated phenolic substances 2 Mashhad Branch, Agricultural Biotechnology Research occur as a C6–C3 unit linked to an aromatic ring [5]. Many Institute of Iran (ABRII), Agricultural Research, Education, of compounds in these groups exert low toxicity in mam- and Extension Organization (AREEO), Mashhad, Iran mals and some of them are widely employed in medicine 3 Quality Department of Nutricia, Mashhad Milk Powder for various purposes such as arteriosclerosis treatment [5]. Industrial, Mashhad, Iran These bioactive compounds were also demonstrated to have 4 Department of Agriculture, Payame Noor University (PNU), antioxidant potential and because of which they exhibit sev- Tehran, Iran eral beneficial effects, including antimicrobial, anti-allergic 5 Department of Plant Science and Biotechnology, Faculty and anti-inflammatory as well as anti-cancer activities [6, of Life Science and Biotechnology, Shahid Beheshti 7]. These biological activities are attributed to the ease with University, Tehran, Iran which a hydrogen atom from an aromatic hydroxyl group 6 Department of Veterinary Preclinical Sciences, Faculty can be donated to a free radical and the ability of an aro- of Veterinary Medicine, Universiti Putra Malaysia, matic compound to support an unpaired electron [8]. From 43400 Serdang, Selangor, Malaysia

Vol.:(0123456789)1 3 E. Karimi et al. a biological standpoint, this is critical since compounds Plant material with antioxidant activity could protect cellular systems from harmful effects of metabolic processes that cause excessive Borago officinalis plant was harvested in spring from oxidation. Moreover, the antioxidant could interrupt free Tandoureh National Park, Khorasan Razavi, Iran in April radical chain reactions and scavenge free radicals [9]. These 2014. The GPS location details were 37°30′57.0″N latitude properties are important in preventing cancer, heart, vascu- and 58°36′24.2″E longitude. The voucher specimen was lar, and neurodegenerative diseases [10]. deposited in the Herbarium of the Department of Botany, Medicinal plants have been used for several purposes, Shahid Beheshti University, Tehran, Iran. The B. officinalis including medicine, nutrition, flavorings, fragrances, cos- were freeze-dried and then ground to a powder using metics, charms, repellents, beverages, dyeing, smoking and a household grinder. The ground powder was transferred into other industrial applications. Nowadays, the extracts from glass container at kept at − 20 °C for further experiments. medicinal plants are attractive not only in the modern phy- topharmacology and phytotherapy but also for food and feed Extract preparation industries [11]. The Borago officinalis L. aerial parts have been used since the ancient times in traditional medicine in Borago officinalis flowers were extracted using methanol, Iran as atonic, tranquillizer, treatment of cough, sore throat, ethanol and boiling water as solvents. The methanol and pneumonia, swelling and inflammatory diseases. The ethanol extraction were performed based on the method and seeds indicated biological activities in cancer and heart described by Crozier et al. [16]. Briefly, a freeze-dried sam- diseases prevention [12] and possessed antibiotic proper- ple (1 g) was weighed and placed in a 250 ml conical flask, ties [13], reduced cardiovascular diseases [14] and provided and aqueous ethanol or methanol (80 ml, 80% (v/v)) were health-improving benefits due to their various biological added, followed by an addition of 6 M HCl (10 ml). The activities [15]. In Iranian traditional medicine, the decoc- mixture was then refluxed for 120 min at 90 °C and then tion of B. officinalis flower is reportedly used for the treat- the extract was filtered using Whatman No. 1 filter paper ment of a variety of ailments including cough, sore throat, (Whatman, UK). The filtrate was evaporated using a rotary skin sores and as tranquilizer, however, much of the infor- evaporator (Buchi, Switzerland). The boiling water extract mation is empirical at best and lacking in scientific valida- was obtained as described earlier by Gulcin et al. [17]. 5 g tion. Moreover, the industrial and potential applications of of ground sample were added to the boiling water (100 ml) the flower still require more investigations. Therefore, this in a glass beaker and mixed by magnetic stirring for 20 min. comprehensive study was designed to evaluate the bioactive The extract was then filtered and evaporated as mentioned compounds, antioxidant, antibacterial, anti-inflammatory above. The dried crude extract was weighed and dissolved and anticancer activities of the B. officinalis flower using in DMSO and stored at − 20 °C for further evaluations. The different solvent polarities (methanol, ethanol, and water). extraction process was repeated three times.

Materials and methods Total phenolics assay

Chemicals and reagents The total phenolic compounds (TPC) of the extracts were determined as described earlier by Hendra et al. [18]. The The reagents including 1,1-diphenyl-2-picrylhydrazyl 0.5 ml of extract was mixed with 2.5 ml Folin–Ciocalteu (DPPH), acetonitrile HPLC grade, alpha-tocopherol, alu- reagent and 2 ml of 7.5% (w/v) Na­ 2CO3. The mixture was minium chloride, ascorbic acid, butylated hydroxy tolu- vortexed for 15 s and incubated in dark at room temperature ene (BHT), dimethyl sulfoxide (DMSO), Folin–Ciocalteu for 90 min. The absorbances of the mixtures were measured reagent, hydrochloric acid, methanol, sodium carbonate, using a visible spectrophotometer (Novaspec II Visiblespec- sodium hydroxide, purchased from Fisher Scientific, USA. tro) at 765 nm. The results were expressed as mg gallic acid Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine equivalents/g dry weight (DW). serum (FBS), 3-4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide thiazolyl blue (MTT), phosphoric acid, naphthyl Total flavonoid assay ethylenediamine dihydrochloride, sulfanilamide, Nω-nitro-l- arginine methyl ester (L-NAME), lipopolysaccharide (LPS) In each extract, the total flavonoid compounds (TFC) and all phenolics and flavonoids standard were purchased was determined according to Ismail et al. [19] An aliquot from Sigma-Aldrich and Interferon gamma (IFN-γ) was pur- (0.1 ml) of each extract was added to 0.3 ml 5% (w/v) chased from eBioscience, Inc. All other chemicals used in ­NaNO2 followed by incubation for 5 min in dark at room this experiment were purchased from Merck.

1 3 Borago officinalis L. flower: a comprehensive study on bioactive compounds and its…

temperature. Then the 0.3 ml 10% (w/v) ­AlCl3 and 2 ml of the extracted fatty acids to their fatty acid methyl esters 1 N NaOH was added and the total volume was made up to (FAME) was carried out using KOH in methanol and 14% 5 ml with distilled water. The absorbance was measured at methanolic boron trifluoride (BF3) (Sigma Chemical) 510 nm by using visible spectrophotometer (Novaspec II according to methods by AOAC (1990). The FAME were Visiblespectro) at 510 nm. The results were expressed as mg separated by gas chromatography (Agilent 7890A), using rutin equivalents/g dry weight (DW). a Supelco SP 2560 capillary column of 100 m × 0.25 mm ID × 0.2-μm film thickness (Supelco, Bellefonte, PA, USA). Determination of phenolic and flavonoid 1 μl was injected by an auto sampler (Agilent Auto Analyzer compounds by HPLC 7683 B series, Agilent Technologies, Santa Clara, CA, USA) into the chromatograph and equipped with a split/splitless The phenolic and flavonoid compounds of B. officinalis flow- injector and a flame ionization detector. The carrier gas was ers were quantitatively measured by a reverse-phase HPLC nitrogen at a flow rate of 1.2 ml/min. The split ratio was 1:20 technique as reported previously by Crozier et al. [16] with after injection of 1 μl of the FAME. The injector temperature slight modifications. The phenolic compound standards used was programmed at 250 °C, and the detector temperature in this study were caffeic acid, gallic acid, pyrogallol and was 270 °C. The column temperature program started to run salicylic acid while the flavonoid and isoflavonoid com- at 150 °C, for 2 min, warmed to 158 °C at 1 °C/min, held pound standards were apigenin, daidzein, genistein, kaemp- for 28 min, warmed to 220 °C at 1 °C/min and then held for ferol, myricetin, naringin, quercetin and rutin. An aliquot 20 min to achieve satisfactory separation. The peaks of sam- of sample extract was loaded on an Agilent-1200 series ples were identified, and concentrations calculated based on high-performance liquid chromatography (HPLC) instru- the retention time and peak area of known standards (Sigma ment equipped with an analytical column (Intersil ODS-3 Chemical). The fatty acid concentrations are expressed as 5 μm 4.6 × 150 mm, GL Sciences Inc.), autosampler, binary g/100 g of the sum of identified peaks measured in each pump, vacuum degasser and ultravioletdiode array detector sample. The extraction and analysis of fatty acids was car- (DAD). The solvents used were deionized water (solvent ried out in triplicate. A) and acetonitrile (solvent B), whilst the pH of the water was adjusted with trifluoroacetic acid to 2.5. The phenolic Antioxidant activity and isoflavonoid compounds were detected at 280 nm while flavonoid compounds were detected at 350 nm. The column DPPH scavenging activity was equilibrated by 85% solvent A and 15% solvent B then the ratio of solvent B was increased up to 85% in 50 min The potential of the extracts for free radical scavenging followed by reducing solvent B to 15% in 55 min. This ratio activities were determined as described earlier by Gulcin continued for another 5 min for the next analysis at a flow et al. [17]. All measurements were performed in triplicates. rate at 0.6 ml/min. The reaction mixture with lower absorbance values repre- sents higher free radical scavenging activity. The free radi- Fatty acid analysis by gas chromatography cal scavenging activities of the extracts were expressed as a percentage of inhibition and were calculated according to The total fatty acids were extracted from flower based the following equation [22]. on the method of Folch et al. [20] modified by Ebrahimi Percent (%) inhibition of DPPH = A − A ∕A  × 100% et al. [21] using chloroform/methanol 2:1 (v/v) contain- 0 1 0 ing butylated hydroxyl toluene to prevent oxidation during The ­A0 was the absorbance value of the blank sample or sample preparation. The 1-g of B. officinalis flowers were control reaction and A­ 1 was the absorbance value of the test homogenized in 40 ml chloroform/methanol (2:1 v/v) using sample. A curve of percent inhibition or percent scaveng- an Ultra-Turrax T5 FU homogenizer (IKA Analysentechnik ing effect against samples concentrations was plotted and GmBH, Heidolph, Viertrieb, Germany) in a 50-ml stop- the concentration of the sample required for 50% inhibition pered ground-glass extraction tubes. After filtration of the was determined. The value for each of the test sample was mixture, 10 ml of normal saline solution was added to ease presented as inhibition curve at 50% or ­IC50. phase separation. After complete separation, the lower phase was collected in a round bottom flask and rotary-evaporated Nitric oxide scavenging activity (Laborota 4000-efficient; Heidolph, Germany) at 70 °C. An internal standard, heneicosanoic acid (C21:0) (Sigma The extracts were tested for nitric oxide (NO) scaveng- Chemical, St. Louis, MO, USA), was added to each sample ing activity according to method described by Tsai et al. before transmethylation to determine the individual fatty [23] Briefly, sixty microliters of two-fold diluted extract acid concentration within the sample. Transmethylation were mixed with 60 μl of 10 mM sodium nitroprusside in

1 3 E. Karimi et al. phosphate buffered saline (PBS) in a 96-well flat-bottomed Anti‑inflammatory assay plate and incubated under light at room temperature for 150 min. Then, an equal volume of Griess reagent was The anti-inflammatory activity of each extract was deter- added to the reacting mixture to determine the free NO mined using macrophage cell culture model (RAW 264.7 content. Ascorbic acid and butylated hydroxyl toluene murine macrophages). Cells were cultured in Dulbecco’s (BHT) were used as reference antioxidants. The NO scav- Modified Eagle Media (DMEM; 2 mM l-glutamine, 45 g/l enging activity was calculated according to the formula: glucose, 1 mM sodium pyruvate, 50 U/ml penicillin; 50 μg/ [(A0 − A1)/A0] × 100%; where ­A0 was the absorbance of ml streptomycin) supplemented with 10% FBS and incu- the control reaction and A­ 1 was the absorbance in the pres- bated at 37 °C with 5% ­CO2. The RAW 264.7 cells (100 µl, ence of extract. 1 × 106cells/ml) were seeded in 96-well tissue culture plate and incubated for 24 h at 37 °C with 5% CO­ 2. The 100 µl of each extract dissolved in DMSO was serially diluted in Ferric‑reducing antioxidant power (FRAP) assay DMEM to obtain final concentration of 100 µg/ml in 0.1% DMSO. The stimulation of the cells was then performed The ferric reducing property of the extracts was deter- with 10 µg/ml of LPS and 200 U/ml of IFN-γ and cells were mined by using assay described by Yen and Chen [24]. incubated for another 17 h. The nitrite content of cell culture 1 ml (concentration of 100, 150, 200, 250, and 300 μg/ media was determined using Griess reagent and absorbance ml) of flower extract was mixed with 2.5 ml of potassium was read at 550 nm using a microplate reader (Spectra Max phosphate buffer (0.2 M, pH 6.6) and 2.5 ml of potassium Plus 384, Molecular Devices Inc., Sunnyvale, CA, USA). ferricyanide (1 g/100 ml). The mixture was incubated at Subsequently, the RAW 264.7 cells viability was detected by 50 °C for 25 min. Then, to stop the reaction the trichlo- MTT cytotoxicity assay. The L-NAME was used as ananti- roacetic acid (10%) was added to the mixture. An equal inflammatory agent (positive control) at the concentration volume of distilled water was added followed by 0.5 ml of 250 µM [26]. ferum chlorate (0.1 g/100 ml) ­(FeCl3). The procedure was carried out in triplicate and allowed to stand for 30 min Anticancer activity assay before measuring the absorbance at 700 nm. The above procedures were repeated with ascorbic acid, and BHT Human cancer cell lines including liver (HPG2), prostate as reference antioxidants. The percentage of antioxidant (LNCaP) and colon (HT-29) cancer cells together with activity in FRAP assay of the samples was calculated human hepatocytes (Chang liver cells) were purchased according to the below formula: from the American Type Culture Collection (ATCC). Cells Antioxidant activity (%) = A A ∕A were cultured in DMEM and grown at 37 °C in humidi- 1 0 1 3 fied 5% ­CO2 incubator. The 100 µl of cells (5 × 10 /100 µl) A0 absorbance of the control (potassium phosphate were seeded in 96-well cell culture plate and incubated for buffer + FRAP reagent), ­A absorbance of sample. 1 24 h at 37 °C with 5% ­CO2. The 100 µl of each extract dis- solved in DMSO was serially diluted in DMEM to obtain final concentration of 200 µg/ml in 0.5% DMSO. Cells were Antibacterial activity assay then incubated for 3 days at 37 °C in humidified 5% ­CO2 incubator. The cytotoxicity of the extracts were determined The B. officinalis flower extracts were evaluated for their by using MTT assay according to Ahmad et al. [26]. The antibacterial activity against Escherichia coli E256, Mic- anticancer drug (Tamoxifen) was used as a positive control rococcus luteus, Enterococcus aerogenes, Pseudomonas in the present study. aeruginosa PI96, Staphylococcus aureus S1431, Klib- siella pneumonia K36, Bacillus cereus B43, Bacillus Statistical analysis subtilis B145 using disc diffusion method as described earlier by Boussaada et al. [25]. All the bacteria were pur- The data were analysed using a complete randomized design chased from the Institute of Malaysian Research (IMR). following the model: Yi = µ + Ti + ei, where µ is the mean, The positive control without extracts (solvent) and refer- Ti is the treatment effect and ei is the experimental error, ence antibiotic (kanamycin) were used in this assay. The respectively. The means were compared using Duncan’s inhibitions of extracts were corrected based on inhibition multiple range test and considered significant when p < 0.05. caused by positive control and compared to the inhibition The data obtained from anti-inflammatory and anticancer of reference antibiotic. All measurements were performed evaluation tests were statistically analysed using GraphPad in triplicate. Prism 6 software (GraphPad Software Inc., San Diego, CA, USA).

1 3 Borago officinalis L. flower: a comprehensive study on bioactive compounds and its…

Results and discussion able to extract more phenolic and flavonoid compounds. In line with the study, the results of Zadernowskia et al. [29], Total phenolic and flavonoid contents Mhamdi et al. [30] and Zemmouri et al. [29–31] confirmed the presence of phenolic and flavonoid compounds in other The TPC and TFC contents of different extracts are pre- aerial parts of the B. officinalis including seeds and leaves. sented in Table 1. The methanolic, ethanolic and boiling The results of Zemmouri et al. [31] showed that the high- water extracts showed significant differences (p < 0.05) est contents of TPC and TFC were presented in the leaves in the TPC and TFC contents. The highest TPC and TFC with the values of 94.1 ± 1.7 mg gallic acid eq./g dry extract contents were observed in the methanolic extract with the and 20.8 ± 2.95 mg quercetin eq./g dry extract, respectively. values of 3.56 mg GAE eq./g DW and 2.75 mg RE eq./g Another recent study conducted by Pilerood and Prakash DW, respectively. These results were consistent with the [32] has also confirmed the presence of polyphenols, flavo- previous studies that reported higher TPC and TFC values noids, carotenoids, anthocyanin and vitamin C in B. offici- in the methanolic extract of different plants [27, 28]. This nalis flower. high content of TPC and TFC in the methanolic extract is probably attributed to the high polarity index of metha- Determination of phenolic and flavonoid nol as compared to the ethanol and water and thus, it was compounds by HPLC

The high-performanceliquid chromatography analysis was conducted on methanolic borage extract due to the high con- Table 1 Total phenolic and flavonoid compounds of Borago offici- tent of phenolic and flavonoid compounds as compared to nalis L. flower other extracts. The Figs. 1 and 2 illustrated the HPLC chro- Solvent Phenolic ­content1 Flavonoid ­content2 matogram of phenolic and flavonoid compounds presented in the flower of B. officinalis methanolic extract. The con- Methanol 3.56 ± 0.13a 2.75 ± 0.04a centrations of detected phenolics and flavonoids are shown Ethanol 3.14 ± 0.09b 2.33 ± 0.05b in the Tables 2 and 3. The analysis confirmed the presence Water 2.67 ± 0.05c 1.62 ± 0.11c of salicylic acid as the major phenolic and myricetin as SEM 0.07 0.05 the major flavonoid compounds with the values of 985.3 p value 0.01 0.01 and 912.5 µg/g DW, respectively. In addition, daidzein as Each value represents mean of three replicates an isoflavonoid was also detected at the concentration of Means in the same column with different superscripts are signifi- 691.2 µg/g DW of B. officinalis flower. This is the first report cantly different (p < 0.05) on HPLC analysis of individual phenolic and flavonoid com- SEM standard error of the mean pounds presence in the B. officinalis flower. The early studies 1 mg gallic acid equivalents/g DW reported the phenolic compounds present in the leaves and 2 mg rutin equivalents/g DW petioles (rosmarinic, syringic, sinapic, ferulic, cinnamic,

Fig. 1 The RP-HPLC chroma- togram of phenolic compounds (gallic acid, pyrogallol, caffeic acid, salicylic acid) and iso- flavonoid (daidzein) detected in the methanolic extract of Borago officinalis flower

1 3 E. Karimi et al.

Fig. 2 The RP-HPLC chroma- togram of flavonoid compounds (rutin and myricetin) detected in the methanolic extract of Borago officinalis L. flower

Table 2 Phenolic compounds presented in methanolic extract of polyunsaturated fatty acids. The rest of total fatty acids are Borago officinalis L. flower classified as monounsaturated (8%) and saturated fatty acids Phenolic contents (µg/g DW) (25%). The major individual fatty acids of the B. officinalis flower extract were α-linolenic acid (25.6%), stearidonic B. officinalis GA PG SA CA acid (16.1%), palmitic acid (13.2%), linoleic acid (13.1%) 705.3 523.6 985.3 267.18 and γ-linolenic acid (11.1%). These results were consistent GA gallic acid, PG pyrogallol, SA salicylic acid, CA caffeic acid, ND with the results of Ramandi et al. [34] who detected the not detected γ-linolenic acid, linoleic acid, palmitic acid and stearic acid in the flower. Moreover, Mhamdi et al. [35] has also reported the linoleic (35.4%), oleic (24.2%) and γ-linolenic (20.4%) and coumaric acids) [15, 30] seeds (gallic acid, chlorogenic acids as predominant fatty acids of B. officinalis seeds. acid, rosmarinic acid, syringic acid, coumaric acid and trans- cinnamic acid) [30] and seedcake (catechin, ferulic acid and Antioxidant activity gallic acid) [13]. The previous study conducted by Wettas- inghe et al. [33] showed that rosmarinic acid, syringic acid, The antioxidants in the body are responsible for scavenging and sinapic acid were the major phenolic compounds present and inhibiting the free radicals and resulted in protection of in the ethanolic extract of borage seed meal. humans against degenerative diseases and infections. At the presence of antioxidants, the radical chromogens such as Gas chromatography analysis of fatty acids DPPH, NO and FRAP are inhibited and disappeared [36]. In this process, these radicals are reduced by hydrogen- or The gas chromatography analysis was conducted on the electrondonation. Compounds which are able to perform this methanolic extract of B. officinalis L. flower. The fatty acid reduction can be considered as radical scavengers and anti- profile of methanolic extract is shown in Table 4. The result oxidants [37]. The comparing antioxidant activity in relation indicated that polyunsaturated fatty acids were the predomi- to reference antioxidants such as Vit C, Vit E, and BHT nant fatty acids (66.5%) from total fatty acids present in the provide useful information on the antioxidant potential of flower methanolic extract. The total polyunsaturated fatty the plant materials. The antioxidant activities of the extracts acids composed of 41.7% omega-3 and 24.7% omega-6 are presented in Figs. 3, 4 and 5. The antioxidant activity

Table 3 Flavonoid compounds Flavonoid and isoflavonoid compounds (µg/g DW) presented in the methanolic extract of Borago officinalis L. B. officinalis AP KF MYR NA QR RU DZ GN flower ND ND 912.5 ND ND 73.6 691.2 ND

AP apigenin, KF, kaempferol, MYR myricetin, NA naringin, QR quercetin, RU rutin, DZ daidzein, GN gen- istein, ND not detected

1 3 Borago officinalis L. flower: a comprehensive study on bioactive compounds and its…

Table 4 Fatty acid composition (%) of Borago officinalis flower 100 Fatty acids Amount (%) 80 C16:0 (palmitic acid) 13.23 C16:1 (palmitoleic acid) 1.54 )

(% 60 C18:0 (stearic acid) 2.02 C18:1n-9 (oleic acid) 6.89 40

C18:2n-6 (linoleic acid) 13.15 Inhibition C18:3n-6 (γ-linolenic acid) 11.61 20 C18:3n-3 (α-linolenic acid) 25.63 C18:4n-3 (stearidonic acid) 16.15 0 C20:0 (arachidic acid) 9.78 0 50 100 150 200 250 300 SFA 25.03 Concentration (µg/ml ) MUFA 8.43 PUFA 66.54 Meoh Eoth Water BHT Vit C n-3PUFA 41.78 n-6PUFA 24.76 Fig. 4 Nitric oxide scavenging activity of Borago officinalis extracts n-6/n-3 0.59 and reference antioxidants at different concentrations. Each value rep- resents mean ± SEM of three replicates SFA saturated fatty acids, MUFA monounsaturated fatty acids, PUFA polyunsaturated fatty acids

120

120 100

100 80 ) (%

) 80 60 (%

60 40 Inhibition

Inhibition 40 20

20 0 0 50 100 150 200 250 300 0 Concentration (µg/ml) 0 50 100 150 200 250 300 350 Concentration (µg/ml) Meoh Eoth Water BHT Vit C

Meoh Eoth Water BHT Vit C Fig. 5 Ferric reducing antioxidant power activity of Borago offici- nalis extracts and reference antioxidants at different concentrations. Each value represents mean ± SEM of three replicates Fig. 3 DPPH scavenging activity of Borago officinalis extracts and reference antioxidants at different concentrations. Each value repre- sents mean ± SEM of three replicates

were lower than that of reference antioxidant. The results of the extract increased in the dose-dependent manner and obtained in this study augur well with the previous studies the extracts tested exhibited varying degrees of antioxidant which reported the antioxidant activity of the seed [13, activities. The methanolic, ethanolic and water extracts 38] and extracts [31, 39, 40] of B. officinalis plant. In showed the strongest, moderate and lowest antioxidant activ- the present study, the antioxidant activities observed in ity, respectively. the extracts were attributed to the presence of phenolics, The IC­ 50 concentrations were reported in Table 5. flavonoids, isoflavonoid and fatty acids. These results were In all the assays borage methanolic extracts exhibited consistent with early studies who reported the antioxidant higher antioxidant activities DPPH ­IC50 = 255.19, NO activity of the seed and leaves extracts due to the presence ­IC50 = 261.5, and FRAP ­IC50 = 206.2 µg/ml. The antioxi- of fatty acids (palmitic acid, oleic acid, linoleic acid, and dant activity of the extract was compared to the results of γ-linolenic acid) and phenolic compounds (gallic, chlo- vitamin C and BHT as reference antioxidants. The results rogenic, trans-cinnamic, rosmarinic, syringic, sinapic, indicated that antioxidant activities of the borage extracts ferulic, cinnamic, and coumaric acids) [13, 31, 33, 40].

1 3 E. Karimi et al.

Table 5 The ­IC50 values of extracts and reference antioxidants in pathogens [41–44]. The antibacterial potency of extracts DPPH, nitric oxide scavenging, and FRAP assays were assessed by the presence or absence of inhibition

Solvent IC50 (μg/ml) zones and zone diameters. As presented in Tables 6 and 7 the methanolic, ethanolic and hot water extracts showed DPPH assay NO assay FRAP assay antibacterial activity against Gram positive and Gram Ethanol 271.9b 283.7a 254.1b negative bacteria in different extents. The lower sensitiv- Water 290.3a ≥ 300 286.2a ity of Gram-negative bacteria against extracts was associ- Methanol 255.19c 261.5b 206.2c ated with the presence of an outer membrane as a unique Vitamin C 28.8d 19.99c 32.7d periplasmic space which is not found in Gram-positive BHT 25.1e 15.6d 23.5e bacteria [45]. SEM 7.52 7.21 7.36 The diameter of inhibition zones for methanolic, etha- p value p < 0.05 p < 0.05 p < 0.05 nolic and hot water extracts ranged from 0.77 to 1.46 cm, 0.34 to 1.39 cm and 0 to 0.76 cm at 0.5 mg/disc, respec- Means in the same column with the different superscripts are signifi- cantly different at p < 0.05 tively. Among Gram-positive bacteria, E.coli was the most P. aeruginosa Analyses were done in triplicate sensitive while was the most resistant strain. B. cereus SEM standard error of the mean While for Gram-negative bacteria the appeared to be the most sensitive and M. luteus was the most resist- ance bacteria against tested extracts. Overall, the antibac- Antibacterial activity terial activity of methanolic extract appeared to be more effective than ethanolic and hot water extract. The high The antibacterial activity of B. officinalis flower extracts antibacterial activity of methanol could be attributed to were evaluated for the first time against eight bacteria the potential of methanol in extracting bioactive com- (four Gram-negative bacteria and four Gram-positive pounds as compared to ethanol and hot water. No previous bacteria) which are bacterial known to be path- reports are available on antibacterial effects of B. offici- ogenic for human or considered as common foodborne nalis flower. However, other plants from the same family

Table 6 Inhibition zones of Bacteria Zone of inhibition (cm) Borago officinalis extracts against Gram-negative Methanol Ethanol Hot water K S SEM pathogenic bacteria at the concentration of 0.5 mg/disc 0.50 mg/disc 1.0 μg/disc 1.0 μg/disc E. coli 1.46b 1.39b 0.76d 1.60a 1.13c 0.03 E .aerogenes 1.30b 0.97d 0.36e 1.44a 1.16c 0.04 K. pneumoniae 1.20b 0.96c 0.38d 1.34a 1.24ab 0.05 P. aeruginosa 0.77b 0.34d 0.13e 1.11a 0.55c 0.04

Means with different superscripts within rows are significantly different (p < 0.05) E. coli (Escherichia coli); E. aerogenes (Enterobacter aerogenes); K. pneumoniae (Klebsiella pneumo- niae); P. aeruginosa (Pseudomonas aeruginosa) K kanamycin, S streptomycin

Table 7 Inhibition zones of Bacteria Zone of inhibition (cm) Borago officinalis extracts against pathogenic Gram- Methanol Ethanol Hot Water K S SEM positive bacteria at the concentration of 0.5 mg/disc 0.50 mg/disc 1.0 μg/disc 1.0 μg/disc B. cereus 1.01c 1.25b 0.53d 1.45a 1.12bc 0.02 B. subtilis 1.16b 0.99c 0.61d 1.32a 0.91c 0.04 M. luteus 0.96c 1.11bc 0d 1.41a 1.20b 0.02 S. aureus 1.13b 1.09bc 0.51d 1.29a 1.05c 0.04

Means with different superscripts within rows are significantly different (p < 0.05) B. cereus (Bacillus cereus); B. subtilis (Bacillus subtilis); M. luteus (Micrococcus luteus); S aureus (Staph- ylococcus aureus) K kanamycin, S streptomycin

1 3 Borago officinalis L. flower: a comprehensive study on bioactive compounds and its… such as Echium amoenum, Cordia latifolia and Onosma antibacterial activity of flower together with its sensorial bracteatum exhibited antibacterial activity [46, 47]. preferences over the leave extract, it is recommended as suit- The phenolic and flavonoid compounds possess anti- able biopreservative to be used in the food, feed or cosmetic bacterial properties through induction of chemical barriers industries. against invading microorganisms, interrupting cytoplasmic membrane function, affecting energy metabolism, disorder- Anti‑inflammatory activity ing of nucleic acid synthesis, inactivation of adhesions and transport proteins, and non-specific reactions with carbohy- During inflammation, the inducible nitric oxide synthase drates in the cell wall [48–50]. Thus, the antibacterial activ- (iNOS) is up-regulated through secretion of pro-inflam- ity observed in the Borage flower extracts could be attrib- matory cytokines and the NO is produced from l-arginine. uted to the presence of phenolics, flavonoid and isoflavonoid Hence, the regulation of NO production is considered as an compounds (Tables 2, 3). The Borage extract is considered important target for inflammatory diseases. The anti-inflam- as a natural source of antibacterial compounds for the food matory activity of the B. officinalis extract was evaluated industry. The first aspect to be considered before the inclu- using LPS/INF-γ stimulated RAW 264.7 macrophage cells sion of a given plant extract to control the bacterial growth (Fig. 6). The cytotoxic effects of extracts were evaluated on in foods is its effect on the organoleptic profile of the final macrophage to ensure that the anti-inflammatory property products. Naturally, derived preservatives can alter the taste was not due to cytotoxicity effect from the extract (Fig. 7). of foods or exceed acceptable flavor thresholds [51, 52]. For The extracts inhibited the NO production in a dose-depend- this reason, low concentrations of active compounds limit ent manner. The methanolic and ethanolic extracts could the risk of changes in the sensory features of the treated inhibit the NO production similar to L-NAME at the con- foods. The results observed in this study was in agreement centration of 500 µg/ml. According to Kim et al. [55] classi- with the Aliakbarlu and Tajik [53] and Miceli et al. [54] who fication, the extract possessed weak anti-inflammatory activ- reported the antibacterial potential of leaf extract against ity. The cytotoxicity test revealed that the concentrations of several common foodborne and human pathogens such as 250 µg/ml and above could be toxic to the macrophage cells Salmonella enterica, Enterobacter spp., Staphylococcus as the cell viability decreased significantly (p < 0.05). aureus, Bacillus subtilis and Listeria monocytogenes. The results of Ratz-Lyko et al. [13] indicated the week antibacte- Anti‑cancer activity rial activity of the seedcake against P. aeruginosa, E. coli, and S. aureus. Although the leaves appeared to be a good Most of the anticancer drugs have been discovered through source of antibacterial compounds for food industry but the random screening of plant materials. Nowadays, identi- astringent taste limited its application. With regard to the fication of novel compounds with notable anticancer

Fig. 6 Effects of different concentrations of methanolic, ethanolic sents the mean ± standard error of triplicate analysis. ***p < 0.0001; and water extracts of Borago officinalis L. flower on inhibition of **p < 0.001, *p < 0.01 indicates significant difference as compared to nitric oxide production stimulated RAW 264.7 cells. Each bar repre- the control (L-NAME)

1 3 E. Karimi et al.

Fig. 7 Effects of different concentrations of methanolic, ethanolic and analysis. ***p < 0.0001; **p < 0.001, *p < 0.01 indicates significant water extracts of Borago officinalis L. flower on cell viability of RAW difference as compared to the control (L-NAME) 264.7 cells. Each bar represents the mean ± standard error of triplicate

Table 8 The ­IC50 values of methanolic, ethanolic and water extracts cells (non-tumor cells) showed the non-hepatotoxic effect of Borago officinalis L. and positive control on Chang liver, HPG2, of B. officinalis extract up to the concentration used in this LNCaP and HT-29 cell lines study. Results from this experiment revealed that metha-

Sample IC50 value (µg/ml) nolic extract with a higher phenolic and flavonoid content Chang liver HPG2 LNCaP HT-29 possesses stronger anticancer activity than ethanolic and water extracts which was in agreement with the previous a b d c Ethanol > 200 184.4 ± 4.53 91.3 ± 2.95 106.3 ± 4.89 reports [56]. Moreover, with regard to the report of US a a c b Water > 200 > 200 142.4 ± 6.37 161.7 ± 5.39 NCI plant screening program the crude extract with anti- Methanol > 200a 175.2 ± 3.83b 75.3 ± 2.95d 94.1 ± 3.15c cancer activity possesses IC­ 50 value (concentration causes a c b a Tamoxifen 43.4 ± 3.14 17.2 ± 1.65 33.6 ± 2.36 38.4 ± 3.12 the cell viability drop to 50%) of < 30 µg/ml [57], Since Means in the same column with the different superscripts are signifi- the IC­ 50 concentration of extracts in all cancer cells was cantly different at p < 0.05 > 30 µg/ml, therefore, they are not potent as an anticancer Analyses were done in triplicate therapeutic agent. Despite the weak anticancer potential of the flower extract observed in this study, a recent study conducted by Lozano-Baena et al. [15] reported the DNA properties has been an important part of the cancer protection and anti-carcinogenic effects of B. officinalis researchers for the development of potential anticancer leaves. These biological activities are associated with anti- drugs. For this reason, the cytotoxic effects of extracts oxidant potential and the presence of phenolic compounds were examined by using MTT assay against human liver mainly rosmarinic acid. (HPG2), prostate (LNCaP) and colon (HT-29) cancer Table 9 indicates the correlation analysis among all cells. The Chang liver cells were used as non-tumor cells parameters evaluated in this study. Based on the results, derived from normal liver tissue to determine the hepa- the phenolics and flavonoids contents showed high posi- totoxicity of the extracts used in this study. As shown tive correlations with antioxidant, antibacterial and cyto- 2 in Table 8 the extracts could inhibit the proliferation of toxic properties of extracts with correlation coefficientr ( ) cancer cells although their anticancer properties were values ranging from 0.74 to 0.95. Thereby the biological significantly lower than tamoxifen as an anticancer drug. activity of B. officinalis flower observed in this study could The methanolic extract exhibited stronger cytotoxic effect be attributed to the presence of phenolic and flavonoid than ethanolic and water extracts with the ­IC50 values of compounds. These results were in agreement with the pre- 175.2 ± 3.83, 75.3 ± 2.95 and 94.1 ± 3.15 µg/ml against vious results who demonstrated the positive high correla- HPG2, LNCaP and HT-29 cells for 72 h, respectively. The tions between phenolics and flavonoids with antioxidant high IC­ 50 concentration of methanolic extract for Chang activity [58, 59] antibacterial [60] anti-inflammatory [61] and cytotoxic [62] effects in various plants.

1 3 Borago officinalis L. flower: a comprehensive study on bioactive compounds and its…

Table 9 The relationship Correlation ­(r2) between TPC, TFC, DPPH, NO, AB, iNOS and CT of Parameters TPC TFC DPPH NO AB iNOS CT extracts obtained from Borago officinalis L. TPC – 0.95 0.78 0.89 0.74 0.92 0.85 TFC – – 0.82 0.94 0.89 0.93 0.88 DPPH – – – 0.74 0.66 0.94 0.83 NO – – – – 0.64 0.92 0.81 AB – – – – – 0.67 0.59 iNOS – – – – – – 0.77 CT – – – – – – –

TPC total phenolics, TFC total flavonoids,DPPH DPPH scavenging activity, NO nitric oxide scavenging activity, AB antibacterial activity, iNOS induced nitric oxide synthesis, CT cytotoxicity assay

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