Dual inhibitory drug, which is the drug that blocks the pathways of both Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) is considered as anti-inflammatory intervention strategy that attracts interest due to their ability to inhibit two key enzymes involved in the arachidonic acid metabolism. COX-2 and 5-LOX have not only been implicated in inflammation but also in angiogenesis, as COX-2 and 5-LOX are pro-angiogenic with a convergent targeting of VEGF expression and release. COX-2 and 5-LOX stimulate inflammatory mediators that in turn promote angiogenesis. Prostaglandin E1 and E2 that have been shown to induce angiogenesis in vivo, which in turn stimulate tumorigenesis (Remmers et al, 1991). Leukotriene in the other hand, such as LTC4 has been shown to promote tube formation in vitro which is essential in angiogenesis while LTC4 and LTD4 are shown to increase vessel growth in the chorioallantoic membrane assay (Grauson et al, 1993; Nissen et al, 1996).
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Angiogenesis is a process of new blood vessel formation by endothelial cells that plays a critical role in normal physiology, such as development and wound healing. Although angiogenesis is a crucial process that is needed to maintain body homeostasis, it also contributes significantly in the initiation, progression, and prognosis of numerous diseases, such as cancer, arthritis, and cardiovascular diseases (Woo et al, 2007; Folkman, 1971; Folkman, 1995; Khurana et al, 2005; Nakano et al, 2005; Chen et al, 2005; Hayden & Tyagi, 2004). Angiogenesis is a critical factor that contributes to development of malignant tumors, by allowing the tumor to increase in size, as well as by providing a route for metastasis to the distal sites of the body (Fang et al, 2003).
Since essential tumor-promoting functions involve interplays of various stimuli such as pro-angiogenic growth factors, cytokines, chemokines and proteinases that are secreted by tumor-infiltrating leukocytes, anti-inflammatory drugs are used as anti-cancer therapeutic alternative to inhibit angiogenesis and metastasis and further arrest tumor development. The rationale is strongly supported by the significant correlation between long-term use of aspirin and non-steroidal anti-inflammatory drugs (NSAIDs) and the reduction of cancer risks. NSAIDs are shown to inhibit tumor angiogenesis by their ability to attenuate COX-2 activity, a key enzyme converting arachidonic acid to prostaglandin, which in turn induce inflammatory reactions (Williams et al, 1999). Despite the advantage of using NSAIDs as molecular targeting approach to tackle tumor angiogenesis, non-specific inhibitors such as aspirin chronic use is associated with significant side effects including gastrointestinal ulceration and bleeding, as well as renal dysfunction (Thun et al, 2002; Hawkey & Jones, 2001).
Drug development from natural products has emerges rapidly with a highly promising approach to identify novel anti-angiogenic and anticancer agents (Weipeng et al, 2008). A considerable literature bodies consisting of experimental and clinical studies have shown that several bioactive compounds from functional and medicinal foods are the most efficacious and investigated anti-angiogenic agents. There are several naturally-occurring bioactive compounds, which have been identified as inhibitors of angiogenesis, including those which inhibit angiogenesis via COX-2 and 5-LOX pathway, such as Curcumin in turmeric, Naringenin in citrus, Humulone in beer hop, Betulinic acid in almond hull, as well as Capsaicin in pepper and Resveratrol in grapes (Shishodia et al, 2005; Raso et al, 2001; Shimamura et al, 2001; Kwon et al, 2002; Surh, 2002; Brakenhielm et al, 2001; Delmas et al, 2006).
Ardisia crispa (AC) belongs to the family of Myrsinaceae, and it can be found in the undergrowth of jungle fringes, dappled shades and shady edges in Malaysia (Heyne, 1987). Several studies conducted by using AC indicated the existence of bioactive substance such as triterpenoid saponin, n-peptide, and benzoquinone which exert utercontraction in mice, anti-hypertensive anti-platelet aggregating properties in vitro, and anti-metastatic effect respectively (Chaweewan et al, 1987; Yoshida et al, 1987; Kang et al, 2001; Roslida & Kim, 2008). Study has also revealed AC possessing anti-inflammatory and anti-hyperalgesic effect (Roslida & Kim, 2008). AC is shown to possess anti-ulcerogenic effect as well (Sri Nurestri & Kim, 2007). As AC is suggested to exhibit its anti-inflammatory effect by affecting the regulation of COX-2, it is postulated that AC might be having the potential to act on COX-2 and 5-LOX pathway to suppress angiogenesis and consequently further arrest cancer progression. Thus, the study of investigating anti-angiogenic properties of AC is intended to be done to create an understanding of the extent of the potential outcome that AC might display in inhibiting dual pathway of COX-2/5-LOX, as far as tumor angiogenesis is concerned.
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To separate, isolate, and identify bioactive constituent(s) in the crude extract Ardisia crispa root.
To determine anti-angiogenic effect of Ardisia crispa root crude extract and its bioactive constituent(s), in vitro and in vivo.
To investigate whether the anti-angiogenic effect exhibited is due to the dual COX-2/5-LOX inhibiting properties.
Animal- ICR mice (6-8 weeks), Day 0 fertilized egg of chick
Drugs- Indomethacin, Licofelone
Chemical and reagent- Carboxymethylcellulose (CMC), dexamethasone (DMSO), phosphate-buffered saline (PBS), ethanol, n-hexane, ethyl acetate, Evans blue dye, carmine red, gelatine, agarose, formamide, hematoxylin and eosin, croton oil
Growth factor- VEGF 165
Collection, extraction and fractionation of Ardisia crispa root (ACR).
Ardisia crispa root (ACR) is obtained from Port Dickson. ACRs are dried in the oven for 72 hours at 400c of temperature. These roots are then pulverized into powder by using Mill grinder (Retsch SM100). Powdered form ACR is extracted with 80% ethanol exhaustively and further concentrated under reduced pressure. ACR ethanol extract (ACRE) is then fractionated successively with n-hexane for 72 hours before being evaporated to dryness using rotary vacuum evaporator.
Separation, isolation and identification of ACR bioactive constituent(s)
Bioactive constituents of ACR are isolated using the means of normal phase column chromatography. Fractions are eluted with increasing polarity of mobile phases which are prepared by adding increased percentage of ethyl acetate in hexane. Fractions are collected in 20 ml vials. Identification of ACR bioactive constituents are done by using thin layer chromatography for qualitative measurement, and Gas Chromatography-Mass Spectrometer (GC/MS) for quantitative measurement. TLC is used extensively throughout the study. Bioactive compounds are detected via UV lamp 254/365 nm wavelength and spraying of 10 % Sulphuric acid and heating of TLC plate for compound confirmation. Individual Retention factors (Rf) of the compounds are determined.
In vivo study
Vascular permeability assay, mouse sponge implantation method and murine air pouch granuloma
Eight-week of male ICR mice weighing between 20- 30g will be used. 8 mice will be housed per cage, with water and pellet will be provided ad libitum. Light and dark cycle will be 12 hours each. Acclimatization period is allowed for one week before the test is being carried out. All experiments are done in accordance to ACUC, Faculty of Medicine and Health Sciences, UPM.
Chorioallantoic membrane assay
Day 0 fertilized eggs are used in this assay. Three eggs are used per group. The eggs are incubated at 37oc with constant humidity throughout the experiment. The eggs are candled in each experimental day to ensure egg viability and to inspect the position of embryo.
Vascular permeability test
The assay is carried out as described by Pakneshan et al (2008). Mice are pre-treated for 5 days with AC extract of various dosages (10, 30, 100 mg/kg), bioactive compounds, indomethacin (100 mg/kg) as positive control and carboxy methylcellulose (0.5%) as negative control. Mice are anesthetized prior to the assay by diethyl ether inhalation. 100Âµl of Evans blue dye is administered intravenously via tail vein (1% in 0.9% saline). 10 minutes later, 50Âµl of VEGF (1ng/Âµl) is administered intradermally at the dorsal skin of mice. 20 minutes later, mice are sacrificed and the dorsal skins are harvested. The skin sample is extracted in 1 ml of formamide over 5 days at room temperature. The absorbance is determined spectrophotometrically at 620 nm. Eight mice are assigned per group.
Mouse sponge implantation method
As described by McCarty (2002), absorbable gelfoam will be cut into 5mm x 5mm x 5mm, hydrated in sterile PBS and strengthen by 0.4% agarose. VEGF will be impregnated in control group, while combination of VEGF and extract (100 ng) or VEGF and bioactive compounds are being impregnated simultaneously for the test groups. Mice will be anaesthetized with sodium pentobarbitone (30 mg/kg, i.p.). An incision will be given along the midline and one gel piece will be inserted into each subcutaneous pocket created laterally. The animals are allowed to recuperate for 14 days. On the 14th day, they will be sacrificed and the gel foam is harvested. The gels will be fixed in formalin and sectioned (<4Âµm) and stained with H&E. The number of vessels will be counted in 15 consecutive fields using a 20Ã- objective and the mean microvessel density (MVD will be calculated). Eight gels were evaluated per group.
Chorioallantoic membrane (CAM) assay
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As described by Wong et al (2009), fertilized eggs are purchased at Day 0 and incubated at 37oc in constant humidity. The eggs are candled in each experimental day to assess egg viability and the position of embryo. At day 4, small hole is made at the pointed end of the egg to aspirate 1.5 ml of albumin using 21 gauge needles to render enough space between CAM and the egg shell. 2cm x 2cm window is created onto the shell to expose central vein and will be sealed with parafilm. The eggs are incubated with licofelone as positive control (100 Âµg/ml) and AC extract at various concentrations (10, 50, 100 Âµg/ml) and bioactive compounds respectively, on a filter paper disc for 24 hours. After that, anti-angiogenic effects are measured using scoring method via stereomicroscope.
Murine air pouch granuloma
Granuloma tissue will be induced in mice by the injection of 3 ml of air into dorsal subcutaneous tissue on day 1, followed by intra-pouch injection of 0.5 ml of 0.1% v/v croton oil in Freund's complete adjuvant on day 0. Then 0.5 ml of test sample (20 mg/kg) and dexamethasone (2 mg/kg) or vehicle will be administered orally twice daily on days 0-5. The degree of granulomatous inflammation and vascular density will be assessed after 6 days. On day 6, a vascular cast will be made by intravenous injection of 1 ml of a solution of 10% carmine red in 5% gelatine. The granulomatous air pouch linings will be dissected and treated as described by Farndale et al (1986). The dye content of samples will be assayed spectrophotometrically at 490 nm against a carmine red standard curve. The results will be expressed as a vascular index (V.I = mg dye/ g dry tissue).
In vitro study
Analysis of arachidonic acid metabolism via HPLC
Human peripheral venous blood samples will be taken from healthy volunteers and collected into heparinized tubes. Aliquots will be then transferred to tubes containing either vehicle (DMSO) or test compound and incubated for 15 min at 370C under continuous agitation. Calcium Ionophore, A23187 (40 mM) alone or in combination with LPS (500 mg) will be then added and incubation continued for either 30 min in the case of A23187 alone, or 24 h for samples stimulated with A23187+LPS. Eicosanoids will be extracted from the samples into ethyl acetate. Samples will be then evaporated to dryness under nitrogen, re-suspended in the mobile phase to precipitate proteins and then centrifuged. The supernatant will be then directly analyzed by HPLC using Hypersil ODS 3 mM columns (12.5_0.46 mm), a methanol/water/0.08% acetic acid mobile phase at a flow rate of 1 ml/min and UV detection firstly at 270 nm and 12 min later at 245 nm. Eicosanoids will be separated on the following gradients: 70-80% methanol for10 min, 100% methanol on 19 min for 5 min and 70% methanol for 7 min (Pommery et al., 2005).
In the CAM and mouse sponge implantation assay, the statistical analysis of MVD scores of control versus test group will performed by unpaired two-tailed Student's t-test using the Sigma stat and sigma plot statistical software. The results will be expressed as mean Â± standard deviation (S.D.) and P value is either P < 0.0001, P < 0.001, P < 0.005 or P < 0.05. For granuloma test, results are presented as means + s.e.m. Effective dose 50% (ED50) values will be calculated from at least three doses (n=6-12). When appropriate, 95% of confidence limits will be calculated. The level of statistical significance will determined by analysis of variant (ANOVA), followed by Dunnett's t -test for multiple comparisons. The statistical software used for the data analysis is SigmaStat.
Following treatment with ACR crude extract and its bioactive constituents, it is expected that vascular permeability, microvascular density (MVD) and vascular index (VI) in mice will significantly be reduced. Apart from that, vascular network seen in chorioallantoic membrane in chick embryo will expected to be significantly reduced as well.
WORKING PLAN/ GANTT CHART
Collection, extraction, fractionation
Isolation and identification of active compound(s)
Vascular permeability test
Mouse sponge implantation method
Murine air pouch granuloma
Analysis of arachidonic acid via HPLC
Discussion and conclusion