Abstract:
The litchi pericarp extract was subjected to partition by hexane, ethyl acetate and water. Epicatechin, proanthocyanidin B2 and proanthocyanidin B4 were isolated and purified from the ethyl acetate fraction by reverse-phase high performance liquid chromatography. The immunomodulatory activities of epicatechin, proanthocyanidin B2, proanthocyanidin B4 and the ethyl acetate fraction were examined using proliferation of mouse splenocytes. The results showed all these samples had much higher stimulatory effects on splenocyte proliferation than that of the reference, rutin. Epicatechin and the ethyl acetate fraction showed a significantly (P<0.05) stimulatory effect when the concentration was up to 12.5 micro g/ml. Proanthocyanidin B2 and proanthocyanidin B4 exhibited little lower stimulatory effects than epicatechin and the ethyl acetate fraction. The anti-breast cancer activities of epicatechin, proanthocyanidin B2, proanthocyanidin B4 and the ethyl acetate fraction were also evaluated. Epicatechin and proanthocyanidin B2 had lower cytotoxicities to human breast cancer cell MCF-7 and human embryolic lung fibroblast than paclitaxel.
Introduction
Litchi (Litchi chinensis Sonn.) is an exotic fruit in Southeast Asia, especially in China. As litchi fruit is accepted gradually by consumers for its delicious taste and attractive shape, the fruit is planted in other semitropical regions [1] and [2]. Litchi pericarp tissues account for approximately 15% of the total weight of fresh fruit, and comprise of significant amounts of flavonoids [3]. Thus, it can be taken as an important source of flavonoids.
Flavonoids play some important pharmacological roles against diseases, such as cardiovascular disease, cancer, inflammation and allergy [4] and [5]. The role of the immune system has become increasingly important in the understanding of the mechanisms that are involved in disease prevention. Isoflavone daidzein could stimulate murine nonspecific immunity, activate humoral immunity and enhance cell-mediated immunity [6] and [7]. Epidemiological studies have indicated the relationship between flavonoid intake and reduced risk of certain cancers. In many studies of dietary prevention of cancer, model of breast cancer has been prominent in assessing the impact of a wide variety of flavonoids for their efficacy in inhibiting cancer [8] and [9]. A reduced risk of breast cancer incidence has been associated with a high intake of genistein and moderate consumption of red wine [10] and [11]. The suggested mechanisms for breast cancer prevention effect of flavonoids are multifaceted, including effects on signal transduction pathways involved in cell proliferation, antioxidant activities, and modulations of enzyme activity associated with estrogen biosynthesis and metabolic pathways of carcinogens [12].
Wang et al. [13] and [14] reported the anticancer activity of water-soluble ethanolic extract of litchi fruit pericarp against human breast cancer and hepatocellular carcinoma in vitro and in vivo, but the compositions of the water-soluble extract were not identified. Our previous work indicated that the major flavonoids in ethyl acetate fraction of litchi pericarp extract (LPE) were identified as epicatechin, proanthocyanidin B2 and proanthocyanidin B4 [15]. For a better exploration of the litchi fruit pericarp tissues, it was necessary to understand the biological activities of these compounds and ethyl acetate fraction. In the present study, immunomodulatory and anti-breast cancer activities of epicatechin, proanthocyanidin B2, proanthocyanidin B4 and the ethyl acetate fraction on mouse splenocytes and human breast cancer cells MCF-7 were investigated, respectively.
2. Materials and methods
2.1. Plant materials
Fresh fruits of litchi (L. chinensis Sonn.) at the commercially mature stage were picked from a commercial orchard in Guangzhou, China. Fruits were selected for uniformity of shape and colour prior to storage at 4 °C in refrigerator.
2.2. Chemicals
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), β-mercaptoethanol, l-glutamine, paclitaxel and rutin were purchased from Sigma Chemical Co (St Louis, MO, USA), while RPMI-1640 medium was purchased from GIBCO/BRL Invitrogen (Caithershurg, MD). Fetal calf serum (FCS), penicillin and streptomycin were obtained from Zhongshan University (Guangzhou, China). All other chemicals used were of analytical grade.
2.3. Animals
Normal female BALB/c mice (2–3 months old) from Zhongshan University (Guangzhou, China) were housed under standard environmental conditions and fed with standard pellets and tap water.
2.4. Extraction of flavonoids
Pericarp tissues (5 g, dry weight) were extracted for 2 h at 30 °C in 400 ml 85% ethanol by the methods of Lee and Wicker, and Argolo et al. with a minor modification [16] and [17]. After filtering the extract through Whatman No. 1 paper, the residue was re-extracted and filtered a second time. Filtrates were combined and dried using a rotary evaporator at 40 °C. The dried extract was re-dissolved in 100 ml water and then partitioned with 300 ml of hexane and ethyl acetate sequentially. The ethyl acetate fraction was dried using a rotary evaporator at 40 °C, prior to further purification.
2.5. Separation and purification of flavonoids by reverse-phase high performance liquid chromatography
The ethyl acetate fraction was loaded to a reverse-phase high performance liquid chromatography (RPHPLC) procedure on a polystyrene/divinyl column (100 × 6.4 mm i.d.) (Pharmacia, Swiss) for isolation of epicatechin, proanthocyanidin B2 and B4. Elution conditions were as follows: solvent A, water/formic acid (98:2, v/v); solvent B, acetonitrile/water/formic acid (80:19:1, v/v/v); isocratic for 4 min with 3% B following from 3 to 50% B within 14 min at a flow rate of 1 ml/min. The RPHPLC profiles were recorded at 280 nm. The RPHPLC can collect about 150 μg for each compound at one time. More than one hundred times were used repeatedly to collect enough samples. The purity of these three compounds was higher than 99%, by HPLC analysis with standards.
2.6. Cell line and culture
Human breast cancer MCF-7 and human embryonic lung fibroblast (HELF) cell lines were obtained from immune system analysis laboratory of Zhongshan University (Guangzhou, China). The cells were maintained in RPMI-1640 complete medium in a humidified 5% CO2 atmosphere at 37 °C.
Mice were killed by cervical dislocation and then spleens were removed aseptically. Single cells were prepared by mincing spleen fragments and pressing through a stainless 200-mesh screen in RPMI complete medium [18]. An amount of 1 × 107 cells was placed in a 16-mm well and incubated for 3 h in 5% CO2 at 37 °C. The supernatant together with the non-adherent cells was collected by centrifugation at 1000 rpm for 10 min. The cell pellets were re-suspended in RPMI complete medium and then adjusted to 1 × 106 cells/ml [19].
RPMI-1640 complete medium was supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamine, 50 μM β-mercaptoethanol, penicillin (100 U/ml) and streptomycin (100 μg/ml).
2.7. In vitro proliferation assay
The proliferation stimulatory effect of various litchi pericarp components on splenocytes and the cytotoxicity to both MCF-7 and HELF cells were determined by the MTT assay [20]. All cells were seeded into 4 wells of a 96-well flat-bottom microtiter plate at 1 × 106 cell/ml in 100 μl RPMI complete medium. Various litchi pericarp components (0–500 μg/ml), paclitaxel (0–100 μg/ml) and rutin (0–200 μg/ml) were added to the medium, respectively, giving a final volume of 200 μl. The plate was incubated at 37 °C in a humidified atmosphere with 5% CO2. After 44 h, 50 μl of MTT solution (5 mg/ml) was added to each well and incubated for 4 h. The supernatant was removed carefully by pipetting. DMSO solution (100 μl) was added to each well and shaken for 15 min. The absorbance at 570 nm was measured with a microplate reader (Bio-Rad, Richmond, CA), using wells but without cells as blanks. All experiments were performed in triplicate. The cytotoxicity of various litchi pericarp components to the proliferation of MCF-7 and HELF was calculated as cytotoxicity (%) = (A570 of control cells − A570 of treated cells) / A570 of control cells × 100%. The stimulatory effect of various litchi pericarp components on the proliferation of splenocytes was calculated as the percentage = (A570 of treated cells − A570 of control cells) / A570 of control cells × 100%.
2.8. Statistical analyses
All the data were expressed as means ± standard deviation (SD) of three replicated determinations. Statistical calculations by OriginPro Version 7.5 software (OriginLab Corporation, USA) were carried out. One way of variance analysis was applied for determining differences between results of samples. Values of P < 0.05 were considered as significantly different.
3. Results and discussion
3.1. Separation and purification of flavonoids
Ethyl acetate exhibited a good dissolving capacity to most of flavonoids and can be used to classify flavonoids in plant extract [21] and [22]. Our previous study indicated that the ethyl acetate fraction of flavonoids accounted for 83.1% of the total quantity in LPE. The RPHPLC profile showed that three major flavonoids in the ethyl acetate fraction of LPE were identified as epicatechin, proanthocyanidin B2 and proanthocyanidin B4 [15]. They were collected and used for evaluation of immunomodulatory and anti-breast cancer activity.
3.2. Immunomodulatory activity of flavonoids
Splenocyte proliferation from female BALB/c mice species cultured in the presence of different samples was used to evaluate cell stimulatory effects on the splenocyte growth. As shown in Fig. 1, all samples used in this study had good stimulatory effects. When the concentrations of epicatechin and the ethyl acetate fraction were up to 12.5 μg/ml, the lowest concentration used in this study, significantly (P < 0.05) stimulatory effect on splenocyte proliferation was observed. The concentration of epicatechin, proanthocyanidin B2, proanthocyanidin B4 and the ethyl acetate fraction to obtain 50% of stimulatory effect was 84, 99, 120 and 67 μg/ml, respectively. There was a 110.5 ± 5.2% of stimulatory effect when 200 μg/ml ethyl acetate fraction was used. Proanthocyanidin B2 and proanthocyanidin B4 had significantly (P < 0.05) stimulatory effects when the concentration used was up to 25 μg/ml, and exhibited little lower stimulatory effects compared with epicatechin and the ethyl acetate fraction. The reference, rutin, had a significantly (P < 0.05) stimulatory effect when the concentration was up to 50 μg/ml. Previous studies indicated that many plant extracts could stimulate the proliferation of splenocyte [23] and [24]. Kong et al. reported that propolis flavone could promote lymphocyte proliferation [23]. In the present study, strong stimulatory effects of these flavonoids on splenocyte proliferation suggested that the litchi pericarp components could be used as an additional drug constituents against some inflammations or other diseases.
