(Thunb.) Lindl. (Loquat) (EJ) is a member of the Rosaceae family and has been used as a medicinal plant in China and Japan since ancient times. All parts of the plant are traditionally used for different ailments; e.g., pharyngolaryngitis, nosebleed, cough, bronchitis, constipation, diarrhea, depression and skin diseases [1
]. The astringent leaves of EJ have been used for a long time to treat chronic bronchitis, coughs, phlegm, high fever and gastro-enteric disorders [2
]. In Jordan, it has a long history of use in traditional medicine to treat diabetes [3
]. Such traditional usage encouraged researchers to study the pharmacological effect of EJ. For instance, previous studies on the triterpene acids isolated from EJ showed a significant anti-viral [5
], anti-inflammatory [5
], anti-mutagenic [11
] and anti-tumor properties [12
Since the traditional use of EJ is related to modulating inflammation processes, our earlier studies on EJ leaves were performed on the water extract to investigate specific cytokines’ modulation. These earlier studies have shown that EJ leaf water extract (WE) induces cytokines’ production from unstimulated human blood cells in a dose-dependent manner. The response was in favor of pro-inflammatory cytokines’ production, such as interleukin (IL)-12, interferon (IFN)-γ and tumor necrosis factor-alpha (TNF)-α, more than an anti-inflammatory cytokine, IL-10 [15
]. Further fractionation of WE with butanol resulted in a water-phase termed WP that produced higher amounts of pro-inflammatory cytokines in vitro
] and in vivo
]. WP also modulated cytokines toward IFN-γ in the tumor microenvironment and enhanced the survival of mouse bearing fibrosarcoma [16
]. Such pharmacological behavior reflects the type of initial extraction and the solvent used. Similarly, Sun et al.
showed that oral administration of EJ seed extracts in rats decreased IL-4 in the allergic dermatitis lesion and elevated IFN-γ, IL-2 and IL-10 levels, suggesting balancing of T helper 1 and 2 cytokines’ response in allergic dermatitis lesions [17
The aim of this study is to specify the groups of compounds from the WP extract that are causing this specific cytokine induction. Therefore, further fractionation of the WP was performed in order to identify the sub-fractions and their constituents that have this immunomodulatory activity. The long-term goal, however, is finding an immunomodulator that can polarize immune cells and balance immune responses in T helper 2 immune-mediated diseases, such as allergies and systemic lupus erythematosus.
A recent study has indicated that EJ water extract (WE) exhibits a significant immunomodulatory effect by inducing IL-12, TNF-α and IFN-γ [15
]. Such an effect was thought to be related to the presence of polysaccharides and polar phenolic compounds, like procyanidins and flavonoid glycosides, in the extract. Extraction of HE by n-butanol to yield EJ water residue (WP) was previously performed to concentrate polysaccharides, high molecular weight oligomeric procyanidins and related polyphenols and to exclude low molecular weight compounds [12
]. This WP extract was proven to induce the release of IFN-γ from mouse spleen cells and NK cells [16
]. In the current investigation, however, further fractionation of WP was performed using methanol:acetone to yield the MAU fraction and MAL. The MAU fraction induced IFN-γ production from unstimulated, as well as PMA-stimulated mouse spleen cells better than the original extract, WP and MAL fraction, suggesting that the chemical compounds, such as polyphenols, tannins and flavonoids, were possibly the ones that could be responsible for the immunomodulatory effects of EJ leaves [12
To understand such a mechanism, the extracts were tested on isolated T cells, NK cells and macrophages. Both MAU and MAL fractions significantly enhanced IFN-γ production from T and NK cells, with better enhancement shown by the MAU fraction. These effects were less effective on IL-12 production when the extracts were tested on isolated macrophages. In addition, when the anti-IL-12 antibody was added to cultures of splenocytes simultaneously with the addition of WP, IFN-γ induction levels were partially affected. This led to the conclusion that WP extract induces IFN-γ partially through the IL-12-mediated pathway. However, further fractionation with methanol:acetone reduced the IL-12-mediated pathway, but increased the IFN-γ activation pathway. Furthermore, JAK-STAT inhibitors reversed and blocked MAU-AW sub-fraction-induced IFN-γ production, indicating that JAK-STAT is the main signaling pathway of MAU-AW [30
Most of the Eriobotrya
extract-related studies revealed the anti-inflammatory action of such extracts. The anti-inflammatory action is mediated mainly by the triterpene acids and epicatechin isolated from Eriobotrya
]. However, studies by us and another group showed that EJ leaves polarize T helper 1 cytokines over T helper 2 cytokines [15
]. Therefore, to identify the compounds or group of compounds that are responsible for the induction of IFN-γ, further sub-fractionation of MAU fraction was conducted by column chromatography using eluents with different polarities. The highest sub-fraction inducing IFN-γ production from unstimulated mouse spleen cells was by the acetone followed by methanol and, lastly, the ethanol sub-fraction. This indicates that the more polar compounds in the HE, as in the MAU-AW sub-fraction, are better at inducing IFN-γ. Furthermore, the MADLI-TOF analysis revealed compounds of more than 500 Daltons in each of the extracts and sub-fractions, indicating that the water extract does not contain the known anti-inflammatory compounds, triterpene acids and epicatechin [31
While comparing our MALDI-TOF results to the literature on EJ, naringenin-8-C rhamnoglucoside, quercetin 3-sambubioside and cinchonain glucoside were interpreted to be present in all of the sub-fractions [12
]. In addition and except for MAU-EW, quercetin-3-O
-glucosyl-rhamnosyl-glucoside was also identified [23
]. Furthermore, nerolidol 3-O
-rhamnopyranosyl glucopyranoside was only identified in MAU-AW [7
]. Furthermore and to the authors’ knowledge, very limited studies pointed out cytokines’ induction or modulation by the EJ constituents revealed herein. Amongst these studies, Mackenzie et al.
pointed out that type-A dimeric procyanidin inhibits NF-kB and, thus, might have an anti-inflammatory activity [32
]. In addition, gallic acid, which is a constituent of some of the Eriobotrya
compounds, inhibited T helper-2 cytokines’ production (IL-4, IL-5 and IL-13) [34
]. Therefore, it would be difficult to further interpret our results based on the data shown. However, it can be speculated that the compounds identified in MAU-AW (see above) are responsible for the IFN induction.
In conclusion, further fractionation of WP extract and the concentration of active constituents induced better IFN-γ production from mouse spleen cells. Fractionating WP extract with methanol:acetone enhanced IFN-γ production from both stimulated and unstimulated spleen cells, isolated NK and T cells. This induction was partially mediated by the IL-12-IFN-γ pathway. Such induction was inhibited completely by JAK-STAT inhibitors. Since these EJ sub-fractions were not toxic to spleen cells and increase the production of IFN-γ, further chemical elucidation is warranted to lead to a specific IFN-γ inducer that will have a clinical potential as an immunomodulator.
4. Materials and Methods
4.1. Chemicals and Reagents
RPMI-1640 media supplemented with fetal bovine serum (FBS), trypsin, penicillin/streptomycin and amphotericin B were obtained from Biochrom AB (Berlin, Germany). Aurintricarboxylic acid (ATA), tyrphostin AG 490 (AG490), perhexiline maleate (PM), 4-aminopyridine (4-AP), SB-203580 (SB), PD169316 (PD), Igepal CA-630, phytohemagglutinin (PHA) and lipopolysaccharide (LPS, L-6143) were obtained from Sigma (St. Louis, MO, USA). The mouse erythrocytes lysing kit was purchased from R&D systems (Minneapolis, MN, USA). Endotoxin-free Dulbecco’s PBS without calcium and magnesium were obtained from EuroClone S.P.A. (Siziano, Italy). Tissue culture 96-well flat bottom plates were purchased from Nalge Nunc International (Rochester, NY, USA).
4.2. Plant Material
Fresh EJ leaves were collected from a cultivated garden in the Tarek area (East Amman, Jordan; 9/2013), dried for 10 days at room temperature and identified in comparison with authentic EJ obtained from the Botanical Institute, University of Cologne (Cologne, Germany) and deposited under UOP039013.
4.3. Plant Material Extraction
One hundred and thirty seven (137) grams of dried leaves were crushed by hand into small pieces, washed and extracted three times using 2 L of boiling distilled water for five minutes, then filtered. This extraction process yielded 1700 mL of EJ water extract (WE). One thousand four hundred fifty milliliters of WE were partitioned three times with butanol in a 1:1 ratio (v:v). The aqueous phase was collected to yield a 625-mL EJ water phase (WP). Three hundred seventy five milliliters of WP were partitioned three times with the same volumes of the methanol:acetone (7:3) mixture to yield two phases: the lower (MAL) and the upper (MAU). A 250-mL volume of each extract and sub-fraction was freeze dried, and the percentage of yield was calculated.
4.4. Column Chromatography
Five grams of MAU dissolved in ethanol were subjected to column chromatography (130 cm length and 5 cm width) on Sephadex®
LH-20 using 1.5 L of each of the following eluting solvent systems: ethanol:water (70:30), methanol:ethanol (50:50) and acetone:water (70:30) [3
]. The collected sub-fractions were named MAU-EW, MAU-ME and MAU-AW, respectively. The three sub-fractions were then subjected to volume reduction by a rotary vacuum evaporator followed by freeze drying.
Adult female C57Bl/6 mice were obtained from Taconic Farms Inc. (New York, NY, USA) and housed at the University of Petra’s Animal House facility. All animal experiments were performed in compliance with FELASA guidelines (Federation of European Laboratory Animal Science Association), and the study protocol was approved by the Deanship of Scientific Research at the University of Petra (10/3/2013).
4.6. Mouse Spleen Cells Culture and Cytokines Analysis
Spleen tissues from mice were squeezed between two slides forming a suspension in RPMI culture media, centrifuged for 10 min, diluted with lysing buffer (2 mL) and incubated for 10 min at room temperature. The tube was filled with diluted washing buffer, centrifuged for 10 min and re-suspended in culture media. Spleen cells was adjusted by dilution to yield 106
cells/mL, placed (105
/100 µL) into a sterile 96-well culture plates and incubated at 37 °C and 5% CO2
in a humidified incubator for 1 h before adding the extracts. Each extract was dissolved in culture media, sterilized by filtration with sterile filters (0.2 μm) and diluted to the required concentration. The plate was incubated for 48 h. In the stimulation culture experiments, a volume of 20 μL/well containing LPS and PHA at concentrations of 1 and 5 μg/mL, respectively, was added after 24 h [36
]. The well contents were collected in 1-mL Eppendorfs followed by the addition of 50 μL of 0.1% igepal, incubated for 10 min and then stored at −30 °C. Cytokine (IFN-γ, IL-12) levels were analyzed using DuoSet ELISA development kits acquired from R&D Systems (Minnesota, MN, USA). Maxisorb 96-well flat bottom plates were purchased from Nalge Nunc International (Rochester, NY, USA). In addition, a rat monoclonal anti-mouse IL-12 p70 (IgG1) was acquired from R&D Systems. At the end of the ELISA immunoassay, the absorbance was read at 450 nm and 600 nm by the GloMax®
-Multi Detection System (Promega Corporation, Madison, WI, USA).
4.7. Macrophage Cells Isolation
A volume of 15 mL of spleen cells suspension was added to 75 cm2
tissue culture flask, incubated for 1 h at 37 °C and 5% CO2
in a humidified incubator (Wahl and Smith, 2011) [37
]. The non-adherent cells were decanted, and the flask was washed twice with 10 mL RPMI-1460 media to get rid of any residual non-adherent cells. The adherent macrophage cells were collected by scrapping and re-suspended in RPMI.
4.8. NK and T Cells Isolation
MagCellect Magnet, mouse CD3+ T cell isolation kit and MagCellect mouse NK cell isolation kit were obtained from R&D Systems. As the manufacturer recommendation, the procedures for isolating T and NK cells were followed. Collected cells were re-suspended in culture media and adjusted to the desired concentration for further applications.
4.9. MCA-Induced Tumors, Cell Lines Preparation and Inoculation
Mice (2–4 weeks old) were inoculated subcutaneously (s.c.) into the right hind flank with 1 mg/mouse of MCA dissolved in olive oil. Mice were inspected weekly for tumor development. When tumors reached 1–2 cm in diameter, mice were injected intra-peritoneally with MAU-AW (10 µg) for three successive days [16
]. Twenty four hours post the last injection, mice were sacrificed, and tumor and spleen tissues were collected, weighed, placed into a pre-chilled tube and incubated with 2 mL of ice-cold endotoxin-free PBS containing 0.1% Igepal CA-630 under ice [36
]. The tissues were then homogenized with a tissue disrupter (Janke and Kundle) and centrifuged (6000 rpm for 6 min), and the supernatant was transferred to labeled microcentrifuge tubes and stored at −30 °C until the cytokine assays.
4.10. MultiTox-Fluor Multiplex Cytotoxicity Assay
The spleen cell suspension at a concentration of 5 × 105 was added to a 96-well plate with a black base (Corning Costar, New York, NY, USA) at volumes of 100μL/ well and incubated for 1 h. A volume of 100 μL culture media was added to the control wells and 75 μL culture media to the rest of the wells. Then, a 25 μL volume of the required concentration of (WP extract, MAU-EW, MAU-ME and MAU-AW) was added, and the plate was incubated for 48 h. At the end of incubation, a volume of (100 μL/well) containing two mixed reagents: glycyl-phenylalanylamino fluorocoumarin (GF-AFC, live-cell substrate) and [bisalanyl-alanyl-phenylalanyl-rhodamine 110 (bis-AAF-R110, dead-cell substrate), was added to all wells, mixed briefly on an orbital shaker and incubated for 30 min at 37 °C. The fluorescence was measured by the GloMax®-Multi Detection System for viability (excitation 495 nm; emission 505 nm), and (cytotoxicity: excitation 510 nm; emission 570 nm).
4.11. MALDI-TOF-MS Analysis
To obtain structural information, a MALDI- TOF-MS (Comstock Inc., Oak Ridge, TN, USA) analysis was performed using the LAZARUS II (home built), N2-laser (LSI VSL337ND) 337 nm, a 3-ns pulse width, focus diameter 0.1 mm, 16-kV acceleration voltage, 1-m drift length, data logging with LeCroy9450A, 2.5-ns sampling time and expected mass accuracy ±0.1%. Before the analysis, each sample was deposited from a solution in acetonitrile (ACN)/H2O (50/50) on a thin layer of 2,5-dihydroxybenzoic acid (DHB) crystals.
4.12. Statistical Analysis
The data were analyzed by one-way ANOVA followed by Tukey’s test (95% confidence) for multiple comparisons using SPSS (Version 20.0, IBM Corporation, New York, NY, USA). A p-value of <0.05 is considered statistically significant.