1. INTRODUCTION

Developing cancer therapeutics is an ever demanding field of modern science due to the failure of existing therapies that in spite of providing several advantages, pose disadvantages, contributing to clinical complications. Angiogenesis is a key factor in cancer progression and targeting angiogenesis is one of the promising strategies in cancer therapy [1]. Vascular endothelial growth factor (VEGF), a signaling molecule, is highly expressed in cancers and plays a critical role in angiogenesis. However, targeting VEGF is insufficient to prevent the progression of cancers [2]. Many physiological processes are involved in tumorigenesis including abnormal interactions between cell adhesion molecules and extracellular matrix (ECM) [3]. MMPs, calcium dependent zinc endopeptidases, are responsible for the degradation of basement membrane and ECM components. Under normal physiological conditions, MMPs are minimally expressed and over-expression of MMPs is associated with extensive angiogenesis [4]. Since MMPs are implicated in tumor progression and metastasis, inhibiting them is of potential value in preventing angiogenesis.

The therapeutic drugs avastin, suramin and linomide inhibit angiogenesis by interfering with VEGF pathway. Clinical complications of these drugs restrict their therapeutic use [5]. Plant derived compounds such as xanthohumol, genistein and resveratrol are proven to be effective angiogenesis inhibitors by targeting MMP expression [6,7,8]. Nyctanthes arbor–tristis Linn., commonly known as night jasmine, is a medicinal and ornamental plant belonging to the family Oleaceae. The plant is widely distributed in Indo-Pak subcontinent, Bangladesh, Thailand, and South East Asia. In traditional system of medicine, the plant is used for the treatment of cancer among an indigenous tribe from Chittoor district of Andhra Pradesh (India) [9], arthritis, and oxidative damage [10]. Phytochemicals of biological significance present in this plant are beta sitosterol, astragaline, nicotiflorin, oleanolic acid, ascorbic acid, lupeol, anthocyanins, iridroid glycosides and methyl salicylate [11]. Among these compounds, lupeol derived from Bambax ceiba was reported to inhibit angiogenesis in human umbilical vein endothelial cells [12] and be cytotoxic against hepatocellular carcinoma [13]. Glioblastoma is an aggressive form of glial cell cancer characterized by extensive angiogenesis, evolving in cerebral hemispheres with a median survival time of 15 months. In this study, we investigated into the antiangiogenic and cytotoxic potential of phytocompounds of Nyctanthes arbor-tristis in the form of its ethanolic extract (ENA) on CAM angiogenesis and glioma cells.

2. RESULTS AND DISCUSSION

In the present investigation, antiangiogenic activity of ENA evaluated using CAM assay revealed significant reduction in the capillary formation at concentrations of 160 and 320 μg (Table 1). In the presence of 320 _g ENA, the capillary formation was inhibited by 98.07 %. CAM assay is most widely used method for angiogenesis studies as it mimics the tumor induced angiogenesis due to the lack of a mature immune system in 7 day old chick embryo. Results indicated that ENA changed vascularization pattern by inhibiting the new blood vessel formation in the treated CAMs as well as distortion of the existing vasculature. Several signaling pathways inclusive of VEGF and FGF are known to play a key role in angiogenic process [14]. It appears that ENA influences the suppression of secondary and tertiary blood vessel proliferation and inhibits CAM angiogenesis by interfering with VEGF and FGF pathways. Based on these preliminary findings, further purification of ENA was carried out to study the mechanism of action.

Table 1. Effect of Ethanolic Extract of Nyctanthes arbor-tristis (ENA) on CAM Angiogenesis
Full size table

Following incubation of various concentrations of ENA and its fractions with bacterial collagenase, the gelatinolytic activity was compared with the control to assess its MMP inhibitory potential. Highest gelatinolytic activity represented by the halo zone was observed in the control, representing maximum enzyme activity. Diameter of the gelatinolytic zone decreased upon the treatment with ENA in a concentration dependent manner (Fig. 1A). Among the solvent fractions of ENA, chloroform fraction exerted maximum MMP inhibitory activity, followed by ethanol, ethyl acetate, and hexane fractions (Fig. 1B). Among the seven subfractions isolated from preparative TLC of chloroform fraction, NACS-5 was found to inhibit MMP activity to maximum degree at a concentration of 2 μg/disc (Fig. 1C).

Fig. 1.
figure 1

MMPinhibitory activity of (A) ENAat various concentrations, (B) solvent fractions at 2 μg/disc concentration, and (C) chloroform subfractions at 2 μg/disc concentration. Data present mean ± SD (n = 3 for * p < 0.01 compared to control.

Full size image

MMPs play a critical role in the activation of gelatinase A and subsequent degradation of extracellular matrix, eventually leading to angiogenesis, tumor cell migration and invasion [15]. This investigation revealed inhibition of MMP activity by ENA on gelatin gels. The antiangiogenic activity of ENA, as indicated by MMP inhibition, may be attributed to the presence of terpenoids in the extract and this attribute was further supported by the maximum MMP inhibition by chloroform fraction of ENA, which is rich in terpenoid content. Triterpenoids isolated from Ginkgo biloba inhibited angiogenic activity by downregulating VEGF [16] and triterpenoids from Rabdosia rubescens exhibited significant antiangiogenic activity [17]. However, reports on the terpenoids of Nyctanthes arbor-tristis on angiogenesis inhibition are not available.

ENA exhibited dose dependent cytotoxic effect on U87MG cells with an IC50 value of 149.63 μg/mL, as evaluated by MTT assay. Among the different solvent fractions, chloroform fraction had the most potent cytotoxic activity on these cells. Further, NACS-5 possessed maximum cytotoxic activity among the subfractions isolated from chloroform fraction with an IC50 value of 10.75 μg/mL in MTT assay. Results of trypan blue assay also indicated the similar trend with NACS-5 being the most cytotoxic on glioma cells with an IC50 value of 11.93 μg/ml. The IC50 values for ENA, fractions, and chloroform subfractions are given in Fig. 2.

Fig. 2.
figure 2

Cytotoxic activity (IC50) of ENA, solvent fractions, and active fraction NACS-5 on U87MG cells by MTT and trypan blue dye exclusion assays. Data present mean ± SD (n = 3); ENA = ethanolic extract of Nyctanthes arbor-tristis; HF = hexane fraction; CF = chloroform fraction; EAF = ethyl acetate fraction; EF = ethanol fraction of NACS-5.

Full size image

From the results of MTT assay, it is evident that treatment with ENA was potentially able to change the mitochondrial enzyme activity and initiate a preliminary injury leading to cell death. From the preparative TLC of chloroform fraction, the cytotoxic activity of separated spots individually revealed that one of subfractions, viz., NACS-5 exhibited potent cytotoxicity on glioma cells at a very low concentration of 10.75 μg/mL and elicited a minimum cytotoxicity (10.58% at a concentration of 20 μg/mL) on dermal fibroblasts. This compound was found to be a terpene, as indicated by qualitative screening. Doll-Boscardin, et al. [18] reported the in vitro cytotoxic potential of terpene á pinene isolated from Eucalyptus benthamii against T cell leukemia and cervical cancer cells with IC50 values of 186.09 and 84.24 μg/mL, respectively.

TLC of chloroform fraction from ENA yielded seven spots with Rf values of 0.44, 0.55, 0.64, 0.70, 0.79, 0.85 and 0.97 respectively. Bioactivity results indicated that the fifth subfraction (NACS-5) exhibited potential MMP inhibitory and cytotoxic activities. Repeated recrystallization of this spot with chloroform yielded a white powder (NACS-5, 23 mg). The FT-IR spectrum revealed prominent peaks at 3938, 3778, 3433, 2956, 2924, 2328, 2115, 1656, 1600, 1575, 1442, 1406, 1120, 1047, 744 and 650 cm -1. MS analysis indicated major ion [M+H]+ peak at m/z = 429.2. By comparison of the obtained spectral data with those reported in literature [19,20,21], NACS-5 was characterized as lupeol with molecular formula C30H50O.

Lupeol is a triterpene, and there are reports on its isolation from Betula alnoides, Pterocarpus santalinus and Bombax ceiba [12, 22, 23]. It was known to possess inhibitory activity on DMBA induced skin carcinoma by modulating NF kB, PI3K/Akt pathway [24] and proved to inhibit ornithine decarboxylase protein, a biomarker of tumor promotion [25]. Cytotoxic activity of lupeol on human melanoma cells via modulating ratio of Bcl2 and Bax proteins at a concentration of 34 μM/L has also been reported [26].

As evidenced in the study, the yield of ENA was 12.3 % and it contained 28 mg of lupeol indicating the technical advantages for isolating lupeol from Nyctanthes arbor-tristis. Further, this study demonstrated that lupeol, a potent antiangiogenic compound, could be isolated from Nyctanthes arbor-tristis using relatively simpler techniques.

Synthesis of triterpenoids is considered as a complex process and, hence, their isolation from natural sources could be an effective approach. As indicated by results of the present investigation, Nyctanthes arbor-tristis Linn. leaves were found to be a rich source of lupeol having potential antiangiogenic and cytotoxic activity against U87MG cells. Exploration of pre-clinical and clinical efficacy of lupeol may afford a potent antiangiogenic and cytotoxic therapeutic agent.

3. MATERIALS AND METHODS

3.1. Materials

Chemicals and reactants. Gelatin, agarose, MTT reagent, Dulbecco’s Modified Eagle’e medium (DMEM), Fetal Bovine Serum (FBS), Trypsin-EDTA, Penicillin/streptomycin/amphotericin B (10000 U/10000 mg/ 25 μg), and trypan blue were procured from HiMedia (India). Bacterial collagenase type-1 and Doxycycline (purity 98%) were procured from Sigma Aldrich (USA). All other chemicals were of analytical grade and procured from Merck Millipore (India).

Plant material and extraction. Fresh leaves of Nyctanthes arbor-tristis Linn. were collected from a local garden in Mangalore, Karnataka. The plant was authenticated by a taxonomist Dr. Shobha, department of Botany, University College, Mangalore and voucher specimen (YU/YRC/MP/NA-01) was deposited in the institutional repository of Yenepoya University, Mangalore. The leaves were washed, shade dried, and powdered in an analytical mill (Ika, Germany), after which 100 g of the powder was subjected to hot percolation with ethanol in a soxhlet extractor for 7 h. The resultant extract was concentrated to dryness (ENA) and stored in a refrigerator at –20°C until use.

Cell lines and culture conditions. Glioblastoma multiforme (U87MG) cells were procured from National Centre for Cell Sciences (Pune, India). Cells were maintained in DMEM supplemented with 10 % FBS and antibiotic solution containing 10,000 units penicillin, 10 mg streptomycin and 25 μg amphotericin B. Cells were cultured at 37°C with 5% CO2 in a humidified atmosphere (Thermo Scientific, USA) and used for the experiments after 3 passages. Human dermal fibroblasts (HDFs), procured from HiMedia were maintained in DMEM supplemented with 15% FBS and antibiotic solution.

3.2. Methods

In ovoangiogenesis assay. Antiangiogenic activity of ENA was assessed using modified CAM assay [27]. Briefly, fertile white leghorn chicken (Gallus domesticus) eggs were obtained from a local hatchery. The shells were disinfected with 70% ethanol and incubated at 37.5°C for 72 h in an incubator. A small hole was created at the blunt end of the egg and 3 mL of albumen was withdrawn using a sterile 26 gauze needle so as to enable separation of vascularized CAM from the vitelline membrane and the shell. The eggs were further incubated up to 120 h. The shell was carefully cut to create a window (15 x 15 mm) under sterile conditions. The developing CAM was impregnated with sterile filter paper discs containing different concentrations (20, 40, 80, 160 and 320 μg) of ENA and incubated for 24 h. The capillaries were scored and percentage angiogenesis inhibition was calculated in comparison with control. Doxycycline (10 μg) was used as a positive control. Six eggs were used for each treatment regimen.

Bioactivity guided purification of ENA. ENA (10 g) was subjected to column chromatography on silica gel (60 – 120 mesh) column and eluted successively with n-hexane, chloroform, ethyl acetate and ethanol, to yield four respective solvent fractions. Preparative thin layer chromatography (TLC) was performed for the chloroform fraction (active) (SiO2; toluene-ethyl acetate-methanol-acetic acid mixture (80:10:5:3), UV, 365 nm) which yielded seven spots (NACS-1, NACS-2, NACS-3, NACS-4, NACS-5, NACS-6 and NACS-7). Based on the activity, recrystallization of NACS-5 in chloroform yielded 23 mg of the pure compound.

Gelatin digestion assay for MMP inhibition. Gelatin digestion assay was performed according to the protocol of Kim, et al. [28]. For this, 1% agarose solution with 0.15% porcine gelatin was prepared in collagenase buffer (50 mM Tris HCl, 10 mM CaCl2, 0.15 M NaCl, pH 7.8). Then, 3 mL of this solution was added to 6 well plates and allowed to solidify at room temperature for 1 h. Various concentrations of ENA (5 – 20 μg), solvent fractions (2 μg) and chloroform subfractions (2 μg) were incubated with bacterial collagenase 1 in collagenase buffer for 1 h, and 10 μL of each solution was loaded onto sterile filter paper discs (9 mm) and the discs were placed at the center of the solidified surface of agarose-gelatin medium. Doxycycline (2 μg/disc) was used as a positive control. Plates were incubated at 37°C for 18 h, discs were removed, and gels were stained with coomassie brilliant blue for 30 min. Gelatinase activity was assessed after destaining (10 min) by measuring the area of transluscent zone against dark blue background.

Cytotoxicity of ENA and its fractions against glioma cells. U87MG cells were seeded at a density of 5000 cells/well in 96 well microtiter plate. After 24 h incubation, various concentrations of the test compounds were added and further incubated for 48 h. Then, 100 μL MTT solution (1 mg/mL) was added to each well and, after 4 h, formazan crystals formed were solubilized in DMSO and optical density at 570 nm was recorded [29] to assess the cytotoxicity.

Trypan blue dye exclusion assay. The assay was performed according to the modified method of Strober [30]. U87MG cells were seeded at a density of 10,000 cells/well in 24 well plates, incubated for 24 h and were treated with different concentrations of the test compounds. The plates were further incubated for 48 h, cells were trypsinized, cell suspension was mixed with trypan blue (1:1) and the cell counts were taken using a hemocytometer.

Cytotoxicity of NACS-5 on HDFs. In order to evaluate the toxicity of the active purified fraction, viz., NACS-5 on normal cells, HDFs were seeded onto 96 well microtiter plates at a seeding density of 5000 cells/well. NACS-5 was added to the wells at 5, 10, 15 and 20 μg/mL concentrations. After 48 h, cytotoxicity of the test compound was assessed using MTT assay as described previously.

Characterization of the active compound. The compound with most potent MMP inhibitory and cytotoxic activity (NACS-5) was recovered from preparative TLC plate. The FT-IR spectrum was recorded using Shimadzu IR Prestige-21 spectrophotometer. For identification of the compound, mass spectrum was recorded by turbo spray method (Applied Biosystems, API 4000) in [M +H] + ion mode.

Statistical analysis. All experiments were performed in triplicates and the data were represented as mean ± SD. The significance of differences between groups was analyzed by Student’s t-test, wherever appropriate.