Inflammopharmacology https://doi.org/10.1007/s10787-019-00672-8 Inflammopharmacology ORIGINAL ARTICLE Brachychiton populneus as a novel source of bioactive ingredients with therapeutic effects: antioxidant, enzyme inhibitory, anti‑inflammatory properties and LC–ESI‑MS profile Ilhem Rjeibi1 · Anouar Ben Saad2 · Sana Ncib3 · Sami Souid1 · Mohamed Salah Allagui4 · Najla Hfaiedh1 Received: 13 September 2019 / Accepted: 16 November 2019 © Springer Nature Switzerland AG 2019 Abstract Brachychiton populneus is one of the unexploited Tunisian plants, traditionally eaten as food and used for medicinal purposes. The present study aimed to investigate the phytochemical components of the seeds, leaves and flowers from B. populneus using three different solvents and to explore their antioxidant, anti-inflammatory and neuroprotective effects. Further, this study was focused on the identification of phenolic compounds from the most active extract. In vitro, all extracts showed strong antioxidant property by DPPH, ferrous ion chelating and lipid peroxidation-inhibiting assays, noticeable anti-inflammatory activity by protein denaturation and membrane stabilization methods and important neuroprotective effects by acetylcholinesterase inhibitory test. In vivo, B. populneus (50, 100 and 200 mg/kg, i.p.) showed significant dose–response anti-inflammatory effects against carrageenan-induced paw edema. With respect to the phenolic profile, the leaf methanol extract presented eight phenolic acids, one flavone and four flavonoids, with salvianolic acid B (820.3 mg/kg), caffeic acid (224.03 mg/kg), syringic acid (100.2 mg/kg) and trans-ferulic acid (60.02 mg/kg) as the major compounds. The results of the current study suggested that B. populneus could be a precious source of health-benefitting biomolecules and may be developed as new antioxidant, anti-inflammatory and AChE inhibitors. * Ilhem Rjeibi rjeibii@yahoo.fr 1 Research Unit of Macromolecular Biochemistry and Genetics, Faculty of Sciences of Gafsa, 2112 Gafsa, Tunisia 2 Faculty of Sciences of Gafsa, University of Gafsa, 2112 Gafsa, Tunisia 3 Unit of Common Services, Faculty of Sciences Gafsa, University of Gafsa, 2112 Gafsa, Tunisia 4 Laboratory of Animal Ecophysiology, Faculty of Science of Sfax, University of Sfax, 3018 Sfax, Tunisia 13 Vol.:(0123456789) I. Rjeibi et al. Graphic abstract Keywords Brachychiton populneus · Anti-inflammatory · Antioxidant activity · Neuroprotective activity · LC–ESI-MS Introduction For many years, plants have been used as the main source of medical treatment (Sofowora 1996). Currently, numerous studies are developed into exploring the potential properties and biologically active ingredients from herbal extracts to treat or prevent the development of many diseases (e.g., diabetes, Alzheimer’s and inflammation) (Pandey and Rizvi 2009; Altunkaya and Gökmen 2008; Uysal et al. 2018). The choice of these natural products is because of their lower toxicity and higher safety as compared to synthetic constituents, which have shown more side effects. The Sterculiaceae is a family of flowering plants, including more than 68 genera and 700 species worldwide. Among them, the genus Brachychiton is represented by about 30 species of trees and great shrubs with a widespread distribution. Members of this genus have been traditionally used as herbal tea. For example, Brachychiton diversifolius R.Br. has been eaten as a domestic food by Australian Aborigines (Rao et al. 1989; Abdel-Megeed et al. 2013). The aerial part of B. diversifolius has been reported for their antimicrobial and antioxidant activities (Mokbli et al. 2018; Salem et al. 2014). The ethanolic extracts from the leaves, flowers, and 13 seeds of B. acerifolius have shown considerable antioxidant activities (Farag et al. 2015). Besides, Zeid et al. (2017) have described the anti-diabetic effects of the ethyl acetate extract from B. acerifolius. Recently, flavonoid-rich fractions isolated from B. rupestris were found to have anti-diabetic and hepatoprotective activities (Thabet et al. 2018). Brachychiton populneus is known to possess wide range of medicinal properties including anti-inflammatory and antipyretic activities (Khan 2017; Thabet et al. 2018). The authors have used this plant to treat bacterial skin infections. Phytochemical studies have shown that Brachychiton species are important sources of bioactive ingredients such as essential oils, polyphenols and alkaloids. However, few reports were found regarding the phytochemical and biological properties of B. populneus. Abdul-Hafeez et al. (2014) have demonstrated the antioxidant activity of its aqueous extract. Recent pharmacological investigation of Mokbli et al. (2018) evidenced that the seed oil has a marked antioxidant activity in vitro. This plant has also been demonstrated as potent hepatoprotective and anti-inflammatory agents (Batool et al. 2018). These effects were associated with the presence of phenolic components, namely catechin, rutin and myricetin. Despite, researches associated with the therapeutic uses of B. populneus Brachychiton populneus as a novel source of bioactive ingredients with therapeutic effects:… leaves, detailed information regarding the phytopharmaceutical properties of its various plant parts are limited. This work could be considered as a new investigation on the anti-inflammatory activity, antioxidant effect and enzymatic inhibitory potential of B. populneus, as well as its HPLC analysis. ascorbic acid and butylated hydroxytoluene (BHT) as standards. A volume of 1 mL of each tested sample (10–600 µg/ mL) was mixed with 2 mL of 0.06 mM DPPH and kept in the dark for 30 min. The absorbance of the mixture was read at 517 nm. RSA was calculated using the following equation: Materials and methods Inhibition (% ) absorbance of control − absorbance of sample = absorbance of control × 100. Plant material Seeds, flowers and leaves of Brachychiton populneus (Schott & Endl.) R.Br. were collected from National Park of Djebel Orbata (34°23′N, 9°03′ E) in December 2016. Dr. Elkadri Lefi, Faculty of Sciences of Gafsa, identified the species and the voucher specimen (MSE 0787) was deposited in the herbarium of the Faculty of Sciences Gafsa, Tunisia. Mineral content Mineral profiles of each plant parts were quantified by flame atomic absorption spectrometry according to the procedure described by Rjeibi et al. (2018). First, samples were dried to ash using the muffle furnace, and then digested with hydrochloric acid. Results were expressed as mg/100 g. (1) Ferrous ion chelating assay In this test, 100 µL of 2 mM FeSO4 was mixed with different concentration of extract (1 mL, 10–600 µg/mL) and incubated for 5 min at 25 °C. Then the reaction mixtures were allowed to react with ferrozine solution (0.2 mL, 5 mM) for 10 min at 25 °C (Carter 1971). EDTA was used as the positive control. The absorbance was measured at 562 nm. The Fe2+ chelating activity was calculated using the following equation: Fe2+ chelating rate (% ) = 1 − absorbance of sample × 100. absorbance of control (2) Sample preparation and phytochemical contents Liver lipid peroxidation‑inhibiting assay Fifty gram of each powdered plant material were stirred for 24 h with 500 mL of methanol or ethyl acetate, then filtrated and concentrated using a rotary evaporator to obtain methanol and ethyl acetate extracts, respectively. However, the water extracts were obtained by boiling 50 g of powdered plant parts in 500 mL of distillated water for 25 min. The dried extracts were stored after the calculation of yields. The content of total phenolic was determined via Folin–Ciocalteu reagent method with gallic acid as the standard (Wolfe et al. 2003). The different extracts (100 µL) were mixed with sodium carbonate solution (750 µL, 6%) and the Folin reagent and then incubated in the dark for 90 min. The absorbance was recorded at 765 nm. The content of total flavonoid was assayed through aluminum chloride colorimetric procedure using quercetin as the standard (Jia et al. 1999). One hundred microliter of each sample was mixed with AlCl3 (1 mL, 2%) and incubated at room temperature for 10 min. The absorbance was measured at 517 nm. The effects of extracts on liver lipid peroxidation were evaluated as per methods described by Su et al. (2009) using FeCl2–H2O2 as an inducer and ascorbic acid as the standard. The mice liver tissues were cut into small pieces and homogenized in phosphate buffer. The reaction mixture composed of liver homogenate (1 mL, 1%), FeCl2 (50 µL, 0.5 mM), H2O2 (50 µL, 0.5 mM) and 200 µL of various extracts (0.5–6 mg/mL) was incubated for 60 min at 37 °C. Then trichloroacetic acid (1 mL, 15%) and thiobarbituric acid (1 mL, 7%) were added to the reaction and heated in boiling water for 15 min. The absorbance was read at 532 nm. The inhibition was calculated using the following equation: Inhibition rate (% ) = 1 − (A1 − A2) × 100, A0 (3) where A0 and A1 were, respectively, the absorbance without and with the test sample and A2 was the absorbance without liver homogenate. Antioxidant activities Acetylcholinesterase (AChE) inhibitory activity DPPH assay AChE activity was assayed using the modified method of Ellman et al. (1961) and Bekir et al. (2013). In this procedure, AChE served as the enzyme, acetylthiocholine The method of Yıldırım et al. (2001) was used to determine the DPPH radical-scavenging ability (RSA) of extracts using 13 I. Rjeibi et al. iodide (ATCI) as the substrate and galanthamine (10–50 µg/ mL) as the positive test. Briefly, 560 µL of Ellman’s reagent DTNB (5,5-dithio-bis(2-nitrobenzoic) acid), 80 µL of extract (25–300 µg/mL), 25 µL of AChE prepared in sodium phosphate buffer (pH 8.0) and 210 µL of buffer (50 mM Tris–HCl, pH 8.0, containing 0.1% bovine serum albumin) were incubated for 15 min at 25 °C. Then 125 µL of ATCI was added to start the reaction. Eventually, the enzymatic hydrolysis of AChE was interpreted at 412 nm. The inhibitory activity (IA) was calculated using the following equation: Membrane stabilization (% ) = IA (% ) = blood cells (RBCs) were diluted in PBS (pH 7.4) to make a 10% (v/v) suspension. The buffer mixed with RBC was used as a control, while indomethacin mixed with RBC was used as a control drug. Tested samples were prepared at concentrations of 25, 50, 100, 150, 200, and 250 µg/ mL in PBS. Then 1 mL of each one was mixed with 1 mL RBC and incubated at 54 °C for 20 min. After cooling, the tubes were centrifuged at 2100 rpm for 5 min and the absorbance of the supernatant was read at 560 nm (Sadique et al. 1989). The % inhibition was calculated using the following equation: Absorbance of control − Asborbance of sample × 100. Absorbance of control 1 − ES × 100. E (6) Anti‑inflammatory activity in vivo (4) ES and E were the respective activity of enzyme with and without the test sample. Anti‑inflammatory activity in vitro Inhibition/enhancement of protein denaturation The reaction mixture consisted of 0.5 mL bovine serum albumin (1%, w/v), 2.5 mL of Tris buffer (pH 6.4) and 2 mL of plant extract (50–500 µg/mL) (Ullah et al. 2014). The control tube was prepared using all reagents without sample; the latter was replaced by 2 mL of distilled water. The positive control (Indomethacin, Ind) was prepared using the same procedure. The mixtures were incubated at 36 °C for 10 min and then heated to 70 °C for 6 min. The absorbance was measured at 660 nm. The % inhibition was calculated using the following equation: The in vivo assays were performed according to the Tunisian code of practice for the Care and Use of Animals of the European convention for the protection of animals used for experimental (Council of European Communities 1985). The anti-inflammatory effect of B. populneus was evaluated using carrageenan-induced paw edema (Winter et al. 1962). Briefly, six groups of male Swiss albinos of about 35–40 g (n = 6) were fasted for 24 h prior to receiving intraperitoneal (ip) doses of the leaf methanol extract of B. populneus (50, 100 and 200 mg/kg, body weight, i.p.), vehicle (NaCl 0.9%, 2.5 mL/kg) or the reference drug (indomethacin, 10 mg/kg, i.p.). One hour later, all groups received sub-plantar injection of carrageenan (1% carrageenan in 0.9% NaCl). Afterward, the thickness of paw was measured using the plethysmometer every hour until 5 h. The percentage of inhibition was calculated using the following equation: Inhibition (% ) = Absorbance of control − Absorbance of sample × 100. Absorbance of control (5) Inhibition (% ) = Increase in paw edema (control) − Increase in paw edema (test) × 100. Increase in paw edema (control) (7) Membrane stabilization The blood was collected from Regional Hospital, Gafsa, Tunisia, from healthy donors who had not consumed any anti-inflammatory medicament for at least 1 week. Ten milliliter of blood was centrifuged at 2500 rpm for 10 min and then washed several times with saline solution. Red 13 Characterization and quantification of phenolic compounds from B. populneus by LC–ESI‑MS The separation and quantification of phenolic compounds of leaf methanol extract from B. populneus were performed on LCMS-2020 quadrupole mass spectrometer (Shimadzu, Brachychiton populneus as a novel source of bioactive ingredients with therapeutic effects:… Kyoto, Japan) supplied with electrospray ionization source (ESI) according to the procedure previously reported by Chahbani et al. (2018). The column used was AQUASIL C18 (150 m × 3 μm × 3 mm) with a temperature of 40° and a flow rate of 0.6 mL/min. The parameters of the mass spectrometer were negative ion mode with a nebulizing gas flow of 1.5 L/ min, drying gas flow of 12 L/min, heat block temperature of 400 °C and dissolving line temperature of 250 °C. The identification of phenolic compounds was performed by comparing their UV–Vis, retention times and mass spectra with those of standard compounds. Quantitative analysis was done by comparison with the calibration curve of each phenolic standard. Statistical analysis Statistical analysis was accomplished using the SPSS version 18.0 software. All the obtained data were analyzed using Analysis of Variance technique followed by Student’s t test. All values are expressed as mean ± SD. Results and discussion Mineral content The mineral content of B. populneus varied significantly among the three plant organs (leaves, seeds and flowers) and potassium was the most important mineral followed by calcium, magnesium, sodium, iron, manganese and zinc (Table 1). These elements have been reported as cofactor of manifold enzyme systems and protein synthesis. They are also basic for many physiological procedures such as the muscle contraction (Bhatta et al. 2018). Leaves indicated the highest value of potassium 395.81 mg/100 g, when compared to the flowers (301.01 mg/100 g) and seeds (200.9 mg/100 g). This value was higher than those obtained for other edible plants like Calligonum comosum (220.15 mg/100 g) and Senna siamea (257.01 mg/100 g) (Mohammed et al. 2013; Gasmi et al. 2019). In addition, the Table 1 Mineral profile of Brachychiton populneus organs Mineral (mg/100 g) Leaves Potassium (K) Sodium (Na) Calcium (Ca) Magnesium (Mg) Iron (Fe) Zinc (Zn) Manganese (Mn) Seeds 395.81 ± 0.75a 200.9 ± 0.30c 85.91 ± 0.68b 41.03 ± 0.15c 337.9 ± 1.91a 170.48 ± 0.62c 62.65 ± 0.01b 56.63 ± 0.31c 10.97 ± 0.24b 5.03 ± 0.15c a 1.19 ± 0.01 0.75 ± 0.01c b 0.69 ± 0.01 1.39 ± 0.01a Flowers 301.01 ± 1.21b 103.65 ± 0.61a 202.18 ± 0.02b 156.47 ± 0.19a 12.27 ± 0.03a 0.93 ± 0.01b 0.59 ± 0.01c The data are presented as mean values (n = 3); means with letters (a,b,c,d) indicate significant differences at p < 0.05 among plant organs Na/K ratio was < 1, which highlights the importance of B. populneus in preventing from cardiovascular disease (Perez and Chang 2014). With regard to the microelement contents, iron was found with significantly higher concentration in flowers than the other plant parts of B. populneus. Phytochemical contents and extraction yields Numerous investigations have demonstrated that the extraction of bioactive molecules depends on the nature of the solvent. In fact, a wide choice of solvents including methanol and water (Elfalleh et al. 2019), acetone, butanol, methanol and water (Botsaris et al. 2015), water, ethyl acetate and methanol (Tlili et al. 2019) have been used to extract bioactive molecules from natural sources. The extraction yield of different organs obtained using three solvents (water, methanol and ethyl acetate) is presented in Table 2. Results showed that the methanol extract has the highest extraction yields (30.27, 20.67 and 9.95% of extract from leaves, flowers and seeds, respectively), whereas the ethyl acetate extracts indicate the lowest extraction yields (16.38, 14.14 and 2.11% of extract from leaves, flowers and seeds, respectively). These results denote that the extraction yield rises with the rising of the polarity of solvents which corroborates with previous studies. For instance, Abdel-Megeed et al. (2013) reported that the methanol is the most effective solvent to obtain the maximum extraction yield of wood branches from B. diversifolius. Data from Table 2 indicated that the total phenolic contents were considerably affected by the nature of the solvent. The highest contents were observed in the Table 2 Yields and phytochemical composition of the Brachychiton populneus organs Extracts Leaves Methanol Water Ethyl acetate Seeds Methanol Water Ethyl acetate Flowers Methanol Water Ethyl acetate Yield (%) Total polyphenols (mg GAE/g) Total flavonoids (mg QE/g) 30.27 ± 0.04a,A 24.67 ± 0.01a,B 16.38 ± 0.08a,C 42.67 ± 0.77a,A 37.96 ± 1.24a,B 16.38 ± 1.15a,C 7.77 ± 0.06a,A 4.50 ± 0.33a,A 3.16 ± 0.76a,A 9.95 ± 0.09c,A 4.76 ± 0.04c,B 2.11 ± 0.07c,C 29.64 ± 0.47b,A 14.98 ± 0.99b,B 10.95 ± 1.12b,C 3.92 ± 0.14c,B 2.52 ± 0.25c,B 2.63 ± 0.07b,B 20.67 ± 0.04b,A 17.46 ± 0.01b,B 14.14 ± 0.02b,C 16.68 ± 0.68c,A 8.95 ± 0.94c,A 6.34 ± 0.16c,B 5.07 ± 0.11b,C 3.70 ± 0.08b,C 2.94 ± 0.02b,B The data are presented as mean values (n = 3); means with letters in minuscule (a,b,c,d) and capital (A,B,C) indicate significant differences at p < 0.05 among extract organs and solvent, respectively QE quercetin equivalent, GAE gallic acid equivalent 13 I. Rjeibi et al. methanol leaves extract with 42.67 mg GAE/g extract, followed by water leaves extract (37.96 mg GAE/g extract) and ethyl acetate extract (16.38 mg GAE/g extract). In the same way, the highest total flavonoid contents were detected in the methanol leaves extract (7.77 mg QE/g extract), followed by the water then ethyl acetate extracts (4.50 and 3.16 mg QE/g extract, respectively). The observed findings corroborate with previous reports in which authors indicated higher methanol extracts contents when compared to other solvents (Tlili et al. 2019; Yakoub et al. 2018). Based on our results, leaves from B. populneus exhibited more phenolic compounds that can be ideally soluble in methanol than other solvents. In vitro bioactivities of B. populneus Antioxidant activity Several previous studies have reported that phenolic compounds extracted from medicinal plants can be used as antioxidant agents by hydrogen donation or as free radical acceptors to interrupt chain oxidation or by metal chelation reactions (Irawaty and Ayucitra 2018; Říha et al. 2014). In this investigation, the antioxidant activity of various extracts from B. populneus was assessed using three methods, including Fe2+ chelating, lipid peroxidation, and DPPH assays. Data from Table 3 showed that the antioxidant effect of B. Table 3 Antioxidant activity and acetylcholinesterase inhibitory potential of different parts from Brachychiton populneus extracts Extracts populneus depends on both the nature of the solvent and plant organs. In fact, the methanol extract had the highest DPPH radical-scavenging activity while ethyl acetate extract represents the lowest activity. In the methanol extract, leaves showed greater inhibition potential (IC 50 = 148.13 µg/ mL) when compared to seeds and flowers (IC50 = 199.67 and 404.77 µg/mL, respectively). Likewise, the highest Fe2+ chelating and liver lipid peroxidation activities were found in leaf methanol extract with respective IC50 values of 196.65 and 52.96 µg/mL. These findings support previous studies conducted by Farag et al. (2015), showing that leaves from B. acerifolius exhibited the higher antioxidant potential toward DPPH radical (IC50 = 0.015 mg/mL) followed by flowers and seeds extract (IC50 = 6.5 and 56.46 mg/ mL, respectively). In the same context, Abdel-Megeed et al. (2013) reported that the antioxidant activity of B. diversifolius was significantly higher in the methanol extract than the other extracts. These differences in the antioxidant effects of Brachychiton species can be related to the presence of polyphenolic compounds with various chemical structures and polarities; the latter may be solubilized differently in each specific solvent (Chan et al. 2009; Farag et al. 2015). In addition, the synergetic effect of polyphenolic compounds and other constituents present in the extract may also influence the antioxidant capacity of medicinal plant (Irawaty and Ayucitra 2018). IC50 (µg/mL) DPPH radical scavenging Liver lipid peroxidation Fe2+ chelating Leaves Methanol Water Ethyl acetate Seeds Methanol Water Ethyl acetate Flowers Methanol Water Ethyl acetate AA BHT EDTA GAL AChE 148.13 ± 1.27c,C 302.03 ± 1.18c,B 313.73 ± 1.27c,A 52.96 ± 2.90c,C 102.82 ± 2.08c,B 153.55 ± 0.54c,A 196.65 ± 0.75c,C 109.33 ± 0.25c,C 352.82 ± 3.25c,A 202.37 ± 1.29c,B 261.46 ± 6.70c,B 276.56 ± 0.39c,A 199.67 ± 1.93b,C 315.39 ± 0.64b,B 400.67 ± 0.78b,A 61.89 ± 0.26b,C 112.95 ± 0.99b,B 167.92 ± 2.18b,A 356.48 ± 7.40b,C 208.69 ± 3.16b,C 421.55 ± 2.23b,B 240.81 ± 2.80b,B 500.09 ± 7.85b,A 333.79 ± 6.65b,A 404.77 ± 0.79a,C 419.79 ± 1.71a,B 496.66 ± 1.91a,A 28.34 ± 2.30d,D 159.46 ± 0.24c,C – – 176.91 ± 3.15a,C 203.70 ± 1.92a,B 275.25 ± 4.57a,A 44.14 ± 0.45c,C – – – 480.07 ± 6.78a,C 270.04 ± 3.68a,B 532.63 ± 4.45a,B 251.22 ± 1.06a,C 606.19 ± 2.40a,A 380.67 ± 12.29a,A – – – – – 128.11 ± 0.96d,D – 96.54 ± 0.68d,D The data are presented as mean values (n = 3); means with letters in minuscule (a,b,c,d) and capital (A,B,C,D) indicate significant differences at p < 0.05 among extract organs and solvent, respectively AA, BHT, EDTA and GAL; ascorbic acid, butylated hydroxytoluene, ethylenediaminetetraacetic acid and galantamine, respectively AChE acetylcholinesterase inhibitory activity 13 Brachychiton populneus as a novel source of bioactive ingredients with therapeutic effects:… AChE inhibitory activity It is recognized that the brain cholinesterase inhibitors (e.g., Donepezil and Galantamine) play an important role in the control of acetylcholine biosynthesis and contribute to enhancing cognitive function and memory capacities (Goh et al. 2011). The use of these common anti-Alzheimer’s drugs is associated with adverse effects; therefore, the synthesis or discovery of new AChE inhibitors has become urgent. The inhibitory activity of B. populneus has not previously been reported elsewhere. Moreover, this study could be considered as the first report on the AChE inhibitory potential of this species and results are summarized in Table 3. Results revealed that the AChE inhibitory activity of B. populneus was significantly influenced by the extraction solvent (p < 0.05). The methanol extracts exhibited the highest inhibitory action (IC50 = 109.33, 208.69 and 270.04 µg/ mL of extract from leaves, seeds and flowers, respectively), whereas ethyl acetate extracts showed the lowest inhibitory action (IC50 = 276.56, 333.79 and 380.67 µg/mL of extract from leaves, seeds and flowers, respectively). In this activity, the high inhibitory effect of the methanol extract could be related to its high amount of phytochemical constituents. Anti‑inflammatory activity in vitro The majority of biological proteins can lose their biological function when denatured under the effect of external stimuli. Such process in the tissues may be observed in autoimmune diseases namely rheumatoid arthritis. Many studies have suggested that the denaturation of protein tissues is a marker for inflammation of arthritis (Ojha et al. 2014; Ullah et al. 2014; Ruiz-Ruiz et al. 2017). In this study, the ability of B. populneus extracts to inhibit thermal denaturation of albumin was investigated. As shown in Fig. 1, all extracts inhibited the heat-induced albumin denaturation in a concentration-dependent manner. The maximum inhibition of 83.22% was observed with the leaf methanol extracts at a concentration of 500 μg/mL. Meanwhile, the standard acetylsalicylic acid showed an inhibition of 91.14% for the same dose. The anti-inflammatory potential of B. populneus extracts was further confirmed using the membrane stabilization method. Since the morphology of the lysosomal membrane is similar to that of the erythrocyte membrane, the latter has Fig. 1 Effect of Brachychiton populneus extracts on heat-induced albumin denaturation. a) Leaf; b seed; c flower. ASA acetyl salicylic acid. The data are presented as mean values (n = 3) 13 I. Rjeibi et al. been used to evaluate the effects of anti-inflammatory agents on the lysosome (Ferrali et al. 1992). Many studies have demonstrated that the thermal stimuli can cause the rupture of erythrocyte membrane (Ferrali et al. 1992; Škerget et al. 2005). As shown in Fig. 2, B. populneus extracts were able to protect red blood cells (RBC) from heat-induced erythrocyte hemolysis as compared to the standard indomethacin. The leaf methanol extract displayed the highest antihemolytic activity of RBC with IC50 value of 22.96 µg/mL in a manner similar to indomethacin (IC50 = 21.19 µg/mL). However, ethyl acetate extract exhibited the lowest activity (IC50 = 446.59 µg/mL). In vitro, our results anticipated that the B. populneus extracts can be assessed as natural therapeutics for the management of inflammatory disorders. Moreover, our findings demonstrated that the leaf methanol extract from B. populneus may be more advantageous than Fig. 2 Effect of Brachychiton populneus extracts on heat-induced hemolysis of erythrocyte membrane. The data are presented as mean values (n = 3). Means with letters in minuscule (a,b,c) and capital (A,B,C) indicate significant differences at p < 0.05 among extract organs and solvent, respectively the other extracts to confirm the anti-inflammatory property in vivo since it revealed the most effective pharmacological effects in vitro. In vivo anti‑inflammatory activity of B. populneus Carrageenan is well known to investigate the anti-edematous capacities of new biomolecules. The development of edema occurs in two phases during which a diversity of chemical mediators are liberated (Garcıa et al. 2004). The initial phase (0–1 h) is characterized by the release of 5-hydroxytryptamine, prostaglandin (PG), serotonin and histamine. The late phase (1–5 h) is associated with the migration of neutrophils and elevated production of leukotrienes, cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α) and PG (Loram et al. 2007). In this study, results of the anti-inflammatory activity are depicted in Fig. 3. Subplantar injection of carrageenan promotes the development of edema, with a peak at 3 h, which is assigned to PG release (Seibert et al. 1994). However, B. populneus pretreatment induced a dose-dependent reduction in paw edema thickness when compared to the positive group. Figure 3B shows that the anti-edematous effect became noteworthy at the last phase of carrageenan injection and the highest inhibition was spotted after 5 h. At this time, B. populneus at the doses of 50 and 100 mg/kg reduced the paw edema thickness up to 48.95% and 56.18%, respectively. Furthermore, the inhibitory effect of B. populneus at the dose of 200 mg/kg was much closer to the effect of indomethacin. Our findings demonstrated that B. populneus was efficient in both phases of edema development and this suggests that the extract acts as an inhibitor of kinin and PG (Okpo et al. 2001; Xu et al. 2014). Our results are concomitant with the findings Fig. 3 Anti-inflammatory effect of Brachychiton populneus using carrageenan-induced paw edema. Data are represented as mean ± SD (6 animals/group) 13 Brachychiton populneus as a novel source of bioactive ingredients with therapeutic effects:… Fig. 4 Chromatogram of the leaf methanol extract of Brachychiton populneus obtained by LC–ESI-MS of Batool et al. (2018) and Rao et al. (1989). These latter revealed that leaves from B. populneus were able to inhibit the expression of proinflammatory cytokines (TNF-α and IL-6) in rats treated with carbon tetrachloride. Moreover, the activity of the extract could be associated with the scavenging effect of its bioactive secondary metabolites. Identification of bioactive metabolites LC–ESI-MS analysis of the leaf methanol extract revealed the presence of 13 compounds distributed into 8 phenolic acids, one flavone and 4 flavonoids (Fig. 4). The major phenolic acid found in B. populneus was quinic acid (988.02 mg/ Table 4 Concentration and analytical characteristics of selected phytochemicals in the leaf methanol extract from Brachychiton populneus Peaks RT (min) [M–H]− (m/z) MS2 (m/z) λmax (nm) Compounds Concentration (mg/kg) Identification type 1 2 3 4 2.60 3.71 5.14 7.12 191 169 153 289 198 230, 270 260 280 Quinic acid Gallic acid Protocatchuic acid Catechin (+) 988.02 ± 0.28 10.02 ± 0.02 5.03 ± 0.01 8.56 ± 0.03 DAD, MS, [1,2] DAD, MS, [2] DAD, MS, [1,2] DAD, MS, [3] 5 6 7 8 7.86 7.91 10.01 12.56 179 197 193 515 295, 323 267 236, 324 324 DAD, MS, [1,2] DAD, MS, [2] DAD, MS, [2] DAD, MS, [1] 12.71 12.78 447 717 Caffeic acid Syringic acid Trans-ferulic acid 3,4-Di-O-caffeoylquinic acid Luteolin-7-O-glucoside Salvianolic acid B 224.03 ± 0.69 100.2 ± 0.12 60.02 ± 0.10 2.98 ± 0.01 9 10 4.31 ± 0.01 820.3 ± 0.89 DAD, MS DAD, MS, [4] 11 15.23 447 173 (4), 127 (5) 125 (100) 109 (100) 245 (40), 203 (30), 187 (40), 161 (30) 135 (100) 167 (12), 153 (30) 178 (73), 134 (60) 353 (80), 191 (100), 179 (47), 173 (5), 135 (7) 285 (100) 519 (100), 493 (7), 339 (84), 321 (5), 295 (12), 197 (9) 301 (100) 11.05 ± 0.02 DAD, MS, [5] 12 16.50 271 282; 328 5.02 ± 0.01 DAD, MS, [6] 13 31.78 283 199 (100), 200 (70), 228 (47), 271 (20) 268, 240, 151 Quercetin-3-o-rhamnoside Naringenin 268, 330 Acacetin 8.09 ± 0.02 DAD, MS, [7] 345 278, 334 350 [1] Zhang et al. (2018), [2] Fang et al. (2002), [3] Chen et al. (2017), [4] Corina et al. (2019), [5] Gori et al. (2016), [6] Figueroa et al. (2018), [7] Parejo et al. (2004. The data are presented as mean values (n = 3) 13 I. Rjeibi et al. kg) followed by salvianolic acid B (820.3 mg/kg), caffeic acid (224.03 mg/kg), syringic acid (100.2 mg/kg), transferulic acid (60.02 mg/kg), gallic acid (10.02 mg/kg), protocatchuic acid (5.03 mg/kg) and 3,4-di-O-caffeoylquinic (2.98 mg/kg). However, quercetin-3-O-rhamnoside (11.05 mg/kg) was the major flavonoid followed by catechin (+) (8.56 mg/kg), luteolin-7-O-glucoside (4.31 mg/kg) and naringenin (5.02 mg/kg) (Table 4). The unique flavone was acacetin (8.09 mg/kg). Batool et al. (2018) reported the presence of rutin, catechin and myricetin in the leaf of B. populneus from Pakistan. Numerous studies have reported that all these compounds were involved in the prevention and treatment of various diseases. Oboh et al. (2013) have described the neuroprotective property of the caffeic acid and quinic acid. The anti-inflammatory effects of luteolin-7-O-glucoside and salvianolic acid B have been already demonstrated (Chen et al. 2008, 2011). Recently, Wu et al. (2019) have demonstrated that the anti-inflammatory effect of salvianolic acid was related to the inhibition of Keap-1/Nrf2/HO-1. Furthermore, caffeic acid, gallic acid, luteolin-7-O-glucoside and 3,4-di-O-caffeoylquinic acid were found to present a broad spectrum of anticancer and antioxidant activities (Bufalo et al. 2013; Ooi et al. 2011; Verma et al. 2013). Acacetin also showed anti-neuroinflammatory effect in mice model of ischemia (Ha et al. 2012). The presence of these promising bioactive molecules in B. populneus could efficiently explain all the highlighted biological properties. Conclusion In this study, various plant parts from B. populneus served as a source of bioactive molecules. Significant differences between plant parts and solvent extracts were observed. The leaf methanol extract showed the highest activities and the most important phenolic and flavonoid contents. The HPLC–MS analysis revealed that quinic acid, salvianolic acid B, caffeic acid, syringic acid and trans-ferulic acid were the most abundant compounds. The results revealed that all the extracts were endowed with an important antioxidant capacity, an interesting neuroprotective potential and a wide spectrum of anti-inflammatory activity. These activities have been attributed to the scavenging radical capacity of polyphenolic compounds, but the mechanisms involved in the obtained pharmacological properties deserve to be studied further. Acknowledgements The authors would like to thank Mr. Zied Tlili, a teacher of English for Specific Purposes at the Higher Institute of Business Administration of Gafsa, Tunisia, for high-quality language editing which significantly contributed to the completion of this work. 13 Compliance with ethical standards Conflict of interest The authors declare that there are no conflict of interest. References Abdel-Megeed A, Salem MZ, Ali HM, Gohar YM (2013) Brachychiton diversifolius as a source of natural products: antibacterial and antioxidant evaluation of extracts of wood branches. J Pure Appl Microbiol 7:1843–1850 Abdul-Hafeez EY, Karamova NS, Ilinskaya ON (2014) Antioxidant activity and total phenolic compound content of certain medicinal plants. Int J Biosci 5:213–222 Altunkaya A, Gökmen V (2008) Effect of various inhibitors on enzymatic browning, antioxidant activity and total phenol content of fresh lettuce (Lactuca sativa). Food Chem 107:1173–1179 Batool R, Khan MR, Zai JA, Ali S, Maryam S, Naz I, Bibi S (2018) Brachychiton populneus (Schott & Endl.) R. Br. ameliorate carbon tetrachloride induced oxidative stress through regulation of endoplasmic reticulum stress markers and inflammatory mediators in Sprague–Dawley male rats. Biomed Pharmacother 107:1601–1610 Bekir J, Mars M, Souchard JP, Bouajila J (2013) Assessment of antioxidant, anti-inflammatory, anti-cholinesterase and cytotoxic activities of pomegranate (Punica granatum) leaves. Food Chem Toxicol 55:470–475 Bhatta S, Ratti C, Poubelle PE, Stevanovic T (2018) Nutrients, antioxidant capacity and safety of hot water extract from sugar maple (Acer saccharum M.) and red maple (Acer rubrum L.) bark. Plant Foods Hum Nutr 73:25–33 Botsaris G, Orphanides A, Yiannakou E, Gekas V, Goulas V (2015) Antioxidant and antimicrobial effects of Pistacia lentiscus L. extracts in pork sausages. Food Technol Biotechnol 53:472 Bufalo MC, Ferreira I, Costa G, Francisco V, Liberal J, Cruz MT, Sforcin JM (2013) Propolis and its constituent caffeic acid suppress LPS-stimulated pro-inflammatory response by blocking NFkappaB and MAPK activation in macrophages. J Ethnopharmacol 149:84–92 Carter P (1971) Spectrophotometric determination of serum iron at the submicrogram level with a new reagent (ferrozine). Anal Biochem 40:450–458 Chahbani A, Fakhfakh N, Balti MA, Mabrouk M, El-Hatmi H, Zouari N, Kechaou N (2018) Microwave drying effects on drying kinetics, bioactive compounds and antioxidant activity of green peas (Pisum sativum L.). Food Biosci 25:32–38 Chan SW, Lee CY, Yap CF, Mustapha WAW, Ho CW (2009) Optimisation of extraction conditions for phenolic compounds from limau purut (Citrus hystrix) peels. Int Food Res J 16:203–213 Chen HQ, Jin ZY, Wang XJ, Xu XM, Deng L, Zhao JW (2008) Luteolin protects dopaminergic neurons from inflammation-induced injury through inhibition of microglial activation. Neurosci Lett 448:175–179 Chen T, Liu W, Chao X, Zhang L, Qu Y, Huo J, Fei Z (2011) Salvianolic acid B attenuates brain damage and inflammation after traumatic brain injury in mice. Brain Res Bull 84:163–168 Chen PX, Zhang H, Marcone MF, Pauls KP, Liu R, Tang Y, Zhang B, Renaud JB, Tsao R (2017) Anti-inflammatory effects of phenolicrich cranberry bean (Phaseolus vulgaris L.) extracts and enhanced cellular antioxidant enzyme activities in Caco-2 cells. J Funct Foods 38:675–685 Corina D, Delia M, Ersilia A, Claudia F, Istvan O, Andrea B, Oana C (2019) Phytochemical characterization and evaluation of the Brachychiton populneus as a novel source of bioactive ingredients with therapeutic effects:… antimicrobial, antiproliferative and pro-apoptotic potential of Ephedra alata Decne. Hydroalcoholic extract against the MCF-7 breast cancer cell line. Molecules 24:13 Elfalleh W, Kirkan B, Sarikurkcu C (2019) Antioxidant potential and phenolic composition of extracts from Stachys tmolea: an endemic plant from Turkey. Ind Crop Prod 127:212–216 Ellman GL, Courtney KD, Andres V Jr, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95 Fang N, Yu S, Prior RL (2002) LC/MS/MS characterization of phenolic constituents in dried plums. J Agric Food Chem 50:3579–3585 Farag MA, Abou Zeid AH, Hamed MA, Kandeel Z, El-Rafie HM, ElAkad RH (2015) Metabolomic fingerprint classification of Brachychiton acerifolius organs via UPLC-qTOF-PDA-MS analysis and chemometrics. Nat Prod Res 29:116–124 Ferrali M, Signorini C, Ciccoli L, Comporti M (1992) Iron release and membrane damage in erythrocytes exposed to oxidizing agents, phenylhydrazine, divicine and isouramil. Biochem J 285:295–301 Figueroa JG, Borrás-Linares I, Lozano-Sánchez J, Segura-Carretero A (2018) Comprehensive characterization of phenolic and other polar compounds in the seed and seed coat of avocado by HPLCDAD-ESI-QTOF-MS. Food Res Int 105:752–763 Garcıa MD, Fernandez MA, Alvarez A, Saenz MT (2004) Antinociceptive and anti-inflammatory effect of the aqueous extract from leaves of Pimenta racemosa var. ozua (Mirtaceae). J Ethnopharmacol 91:69–73 Gasmi A, Benabderrahim MA, Guasmi F, Elfalleh W, Triki T, Zammouri T, Ferchichi A (2019) Phenolic profiling, sugar composition and antioxidant capacity of arta (Calligonum comosum L.), a wild Tunisian desert plant. Ind Crop Prod 130:436–442 Goh CW, Aw CC, Lee JH, Chen CP, Browne ER (2011) Pharmacokinetic and pharmacodynamic properties of cholinesterase inhibitors donepezil, tacrine, and galantamine in aged and young Lister hooded rats. Drug Metab Dispos 39:402–411 Gori A, Ferrini F, Marzano MC, Tattini M, Centritto M, Baratto MC, Pogni R, Brunetti C (2016) Characterisation and antioxidant activity of crude extract and polyphenolic rich fractions from C. incanus leaves. Int J Mol Sci 17:1344 Ha SK, Moon E, Lee P, Ryu JH, Oh MS, Kim SY (2012) Acacetin attenuates neuroinflammation via regulation the response to LPS stimuli in vitro and in vivo. Neurochem Res 37:1560–1567 Irawaty W, Ayucitra A (2018) Assessment on antioxidant and in vitro antidiabetes activities of different fractions of Citrus hystrix peel. Int Food Res J 25:2467–2477 Jia Z, Tang M, Wu J (1999) The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem 64:555–559 Khan AS (2017) Antipyretic and analgesic activities of some economically important woody plants. In: Medicinally important trees. Springer, Cham, pp 159–185 Loram LC, Fuller A, Fick LG, Cartmell T, Poole S, Mitchell D (2007) Cytokine profiles during carrageenan-induced inflammatory hyperalgesia in rat muscle and hind paw. J Pain 8:127–136 Mohammed A, Liman ML, Atiku MK (2013) Chemical composition of the methanolic leaf and stem bark extracts of Senna siamea Lam. J. Pharmacogn Phytother 5:98–100 Mokbli S, Sbihi HM, Nehdi IA, Romdhani-Younes M, Tan CP, AlResayes SI (2018) A comparative study of Brachychiton populneus seed and seed-fiber oils in Tunisia. Waste Biomass Valorization 9:635–643 Oboh G, Agunloye OM, Akinyemi AJ, Ademiluyi AO, Adefegha SA (2013) Comparative study on the inhibitory effect of caffeic and chlorogenic acids on key enzymes linked to Alzheimer’s disease and some pro-oxidant induced oxidative stress in rats’ brain-in vitro. Neurochem Res 38:413–419 Ojha D, Mukherjee H, Mondal S, Jena A, Dwivedi VP, Mondal KC, Malhotra B, Samanta A, Chattopadhyay D (2014) Anti-inflammatory activity of Odina wodier Roxb, an Indian folk remedy, through inhibition of toll-like receptor 4 signaling pathway. PLoS ONE 9:e104939 Okpo SO, Fatokun F, Adeyemi OO (2001) Analgesic and anti-inflammatory activity of Crinum glaucum aqueous extract. J Ethnopharmacol 78:207–211 Ooi KL, Muhammad TST, Tan ML, Sulaiman SF (2011) Cytotoxic, apoptotic and anti-α-glucosidase activities of 3,4-di-O-caffeoyl quinic acid, an antioxidant isolated from the polyphenolicrich extract of Elephantopus mollis Kunth. J Ethnopharmacol 135:685–695 Pandey KB, Rizvi SI (2009) Plant polyphenols as dietary antioxidants in human health and disease. Oxid Med Cell Longev 2:270–278 Parejo I, Jauregui O, Sánchez-Rabaneda F, Viladomat F, Bastida J, Codina C (2004) Separation and characterization of phenolic compounds in fennel (Foeniculum vulgare) using liquid chromatography–negative electrospray ionization tandem mass spectrometry. J Agric Food Chem 52:3679–3687 Perez V, Chang ET (2014) Sodium-to-potassium ratio and blood pressure, hypertension, and related factors. Adv Nutr 5:712–741 Rao KS, Jones GP, Rivett DE, Tucker DJ (1989) Fatty acid and amino acid compositions of Brachychiton discolor, Brachychiton diversifolius, and Brachychiton acerifolius seeds. J Agric Food Chem 37:916–917 Říha M, Karlíčková J, Filipský T, Macáková K, Rocha L, Bovicelli P, Mladěnka P (2014) In vitro evaluation of copper-chelating properties of flavonoids. RSC Adv 4:32628–32638 Rjeibi I, Feriani A, Ben Saad A, Sdayria J, Saidi I, Ncib S, Hfaiedh N (2018) Lycium europaeum extract: a new potential antioxidant source against cisplatin-induced liver and kidney injuries in mice. Oxid Med Cell Longev. https://doi.org/10.1155/2018/1630751 Ruiz-Ruiz JC, Matus-Basto AJ, Acereto-Escoffié P, Segura-Campos MR (2017) Antioxidant and anti-inflammatory activities of phenolic compounds isolated from Melipona beecheii honey. Food Agric Immunol 28:1424–1437 Sadique J, Al-Rqobahs WA, Bughaith EI, Gindi AR (1989) The bioactivity of certain medicinal plants on the stabilization of RBC membranesystem. Fitoterapia 60:525–532 Salem MZM, Ali HM, Mansour MM (2014) Fatty acid methyl esters from air-dried wood, bark, and leaves of Brachychiton diversifolius R. Br: antibacterial, antifungal, and antioxidant activities. BioResources 9:3835–3845 Seibert K, Zhang Y, Leahy K, Hauser S, Masferrer J, Perkins W, Lee L, Isakson P (1994) Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc Natl Acad Sci 91:12013–12017 Škerget M, Kotnik P, Hadolin M, Hraš AR, Simonič M, Knez Ž (2005) Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chem 89:191–198 Sofowora A (1996) Medicinal plants and traditional medicine in Africa. Karthala, Paris, p 380 Su XY, Wang ZY, Liu JR (2009) In vitro and in vivo antioxidant activity of Pinus koraiensis seed extract containing phenolic compounds. Food Chem 117:681–686 Thabet AA, Youssef FS, El-Shazly M, El-Beshbishy HA, Singab ANB (2018) Validation of the antihyperglycaemic and hepatoprotective activity of the flavonoid rich fraction of Brachychiton rupestris using in vivo experimental models and molecular modelling. Food Chem Toxicol 114:302–310 Tlili N, Kirkan B, Sarikurkcu C (2019) LC–ESI–MS/MS characterization, antioxidant power and inhibitory effects on α-amylase and tyrosinase of bioactive compounds from hulls of Amygdalus communis: the influence of the extracting solvents. Ind Crop Prod 128:147–152 13 I. Rjeibi et al. Ullah HA, Zaman S, Juhara F, Akter L, Tareq SM, Masum EH, Bhattacharjee R (2014) Evaluation of antinociceptive, in-vivo & invitro anti-inflammatory activity of ethanolic extract of Curcuma zedoaria rhizome. BMC Compl Altern Med 14:346 Uysal S, Aumeeruddy-Elalfi Z, Zengin G, Aktumsek A, Mocan A, Custodio L, Soković M (2018) Insight into the biological properties and phytochemical composition of Ballota macrodonta Boiss. et Balansa,—an endemic medicinal plant from Turkey. Ind Crop Prod 113:422–428 Verma S, Singh A, Mishra A (2013) Gallic acid: molecular rival of cancer. Environ Toxicol Pharmacol 35:473–485 Wolfe K, Wu X, Liu RH (2003) Antioxidant activity of apple peels. J Agric Food Chem 51:609–614 Wu CT, Deng JS, Huang WC, Shieh PC, Chung MI, Huang GJ (2019) Salvianolic acid C against acetaminophen-induced acute liver injury by attenuating inflammation, oxidative stress, and apoptosis through inhibition of the Keap1/Nrf2/HO-1 signaling. Oxid Med Cell Longev. https://doi.org/10.1155/2019/9056845 Xu Q, Wang Y, Guo S, Shen Z, Wang Y, Yang L (2014) Anti-inflammatory and analgesic activity of aqueous extract of Flos populi. J Ethnopharmacol 152:540–545 13 Yakoub ARB, Abdehedi O, Jridi M, Elfalleh W, Nasri M, Ferchichi A (2018) Flavonoids, phenols, antioxidant, and antimicrobial activities in various extracts from Tossa jute leave (Corchorus olitorus L.). Ind Crop Prod 118:206–213 Yıldırım A, Mavi A, Kara AA (2001) Determination of antioxidant and antimicrobial activities of Rumex crispus L. extracts. J Agric Food Chem 49:4083–4089 Zeid AHA, Farag MA, Hamed MAA, Kandil ZAA, El-Akad RH, ElRafie HM (2017) Flavonoid chemical composition and antidiabetic potential of Brachychiton acerifolius leaves extract. Asian Pac J Trop Biomed 7:389–396 Zhang Q, Hu XF, Xin MM, Liu HB, Sun LJ, Morris-Natschke SL, Lee KH (2018) Antidiabetic potential of the ethyl acetate extract of Physalis alkekengi and chemical constituents identified by HPLCESI-QTOF-MS. J Ethnopharmacol 225:202–210 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.