Evaluation of pharmacological effect of Teucrium stocksianum extract on angiogenesis using chorioallantoic membrane assay

The present study was aimed to evaluate the effect of Teucrium stocksianum on angiogenesis by using chorioallantoic membrane (CAM) assay. Fertilized eggs were incubated on the 5 th day and dose of different dilutions 0.03%, 0.05%, 0.1%, and 0.5% of the plant extract was applied on 6 th day. Evaluation of primary, secondary and tertiary blood vessels diameter and CAM area on 7 th day by SPIP software. T. stocksianum showed antiangiogenic effect by reducing the diameter of CAM of blood vessels by applying the dilutions while significant results were obtained at dilution of 0.5%.


Introduction
Tumor cells development can be restrained by angiogenesis inhibition.Judah Folkman suggested first time that anti-angiogenesis was used as a target to treat tumor and reduce metastasis (Folkman, 1971).Many in vitro (endothelial cells migration assay, endothelial cells proliferation assay and tube formation assay) and in vivo [corneal angiogenesis assay and chorioallantoic membrane (CAM) assay] methods are adapted to assess anti-angiogenic and pro-angiogenic potentials of molecules (Auerbach et al., 2003).
Vascular endothelial growth factor (VEGF) and angiogenic factors IL production are accelerated with production of free radicals in cells (Brown et al., 2000).VEGF is a vital angiogenesis mediator which increases the expression of eNOS resulting VEGF-induced angiogenesis (Bouloumie et al., 1999).After binding on VEGF receptors promotes cellular events like migration, endothelial proliferation and extracellular matrix component (ECM) degradation (Ferrara and Davis-Smyth, 1997).Oxidative stress causes MMP-1 secretion resulting in tumor growth due to angiogenesis (Brown et al., 2000).
De novo development blood vessels perform vital role in tumor (Pepper, 1997).
The aim of study was to evaluate anti-angiogenic effect of different dilutions of Teucrium stocksianum using in vivo CAM assay.
were purchased from BDH Laboratory Supplies, England.Buffered solution of 0.9% sodium hydroxide was purchased from E-Merck.

Preparation of CAM
Fifty fresh fertilized chicken eggs of 4 th day were obtained from a local hatchery (big bird).All the eggs were sprayed with 70% ethanol to reduce contamination from eggs surface and were air dried.Then eggs were incubated at 37ºC at humidity 60-70% for 5 days (Lin et al., 2008).Eggs were divided into two groups.One was control and second was treated group.Treated group was further divided into 1, 2, 3 and 4 sub-groups.Ten eggs per group were taken.Then CAM assay was performed in the laminar flow hood on each chicken egg one by one.On day 5 of incubation, eggs were windowed aseptically (Ejaz et al., 2005).Briefly, a small window (approximately 2 cm in diameter) was made by removing the shell and inner shell membrane from the air-space site.On the same day, 4-5 mL of albumin was aspirated with a sterile syringe to allow the embryos to develop in a way accessible to quantification and enhance its manipulating properties and let the embryo to grow in a way easy for assessment.The windows were then sealed with sterile parafilm and eggs were returned to the incubator at 37ºC (humidity 55-60%) till day 6 of incubation.

Plant material
The aerial parts T. stocksianum were collected from hills of Talash District Dir (Lower), Malakand Division KPK (Pakistan), in the month of April 2011.The plant was identified and authenticated by the taxonomist Prof. Jehandar Shah, Vice Chancellor of Shaheed Benazir Bhutto University, Sheringle Dir (Upper).

Preparation of extracts
Aqueous methanolic (70:30) extract of T. stocksianum was prepared by using the cold maceration process.The grounded plant material (3 kg) was soaked in 7 liters of an aqueous methanolic mixture (70:30) for 72 hours at room temperature.After three days of occasional shaking, whole material was filtered and the solvent was evaporated under reduced pressure using rotary evaporator.The crude extract was then air-dried to obtain a solid mass.

Samples administration
On sixth day of incubation, windows of eggs of each group were opened and 150 μL of dilution was applied on treated developing CAMs.Windows were sealed again with sterile parafilm and chicken eggs were returned to the incubator for 24 hours.On 7 th day, windows were opened and pattern of CAMs and CAMs area were evaluated by taking images with digital Lebecca cam at 30 frames/sec using a camera shutter speed of 1/2000 sec.Scanning probe image processor (SPIP) was used for quantification of results.All images were converted into grayscale for improved the contrast by black and white inversion with the help of Adobe Photoshop 6.0 so that every image possible to discern anatomical structures and to facilitate precise quantification of angiogenesis.

Image acquision and quantification using SPIP software
After the image acquisition, SPIP (IBM, Denmark) was used to evaluate the images which work on the specific algorithm (Garnaes et al., 2006).The length and diameter of different blood vessels were measured through the calibration and measurement command.The CAMs surface angiogenesis was precisely quantified by measuring the 3D surface roughness (14 parameters) which is an important parameter in the 3D image analysis.Vascular area, angular spectrum and abbot curve of CAMs were measured.The blood vessels were quantified in micrometer scale to evaluate the in depth effects of T. stocksianum extract on angiogenesis.
The essential parameters of 3D surface roughness are following: arithmetic average roughness (Sa) is the mean of absolute values of roughness profile.It is region between mean line and its roughness profile.This is simple and efficient parameter which facilitates us to describe the surface roughness.It can also be defined as the average height of analyzed area.Skewness of topography height distribution (Ssk) is the measure of asymmetry of surface deviations about the mean plane.It can be successfully used to illustrate the shape of topography height distribution.For a Gaussian surface which has symmetrical shape for surface height distribution, the skewness is zero.For an asymmetric distribution of surface heights, the skewness may be negative, if distribution has longer tail at lower side of mean plane or positive, if distribution has longer tail at upper side of mean plane.It can give some indication of existence of spiky features.
By combination of Sku and Ssk values, it can be achievable to identify surfaces having relatively flat, top and deep valleys.
Hybrid properties are combination of spacing and amplitude.Modification in either spacing or amplitude can modify the hybrid property.Two Hybrid parameters are measured in this study.Arithmetic mean summit curvature of surface (Ssc) is the average of the principal curvatures of summit within the sampling area.Whereas, the sum of curvatures of surface at point are equal to the sum of principal curvatures.
Developed interfacial area ratio (Sdr) is the ratio of the increment of the interfacial area of surface over the sampling area.It reveals the hybrid surface property.The larger the Sdr value signifies the importance of either amplitude or spacing or both.
The functional parameters was observed in this study is core fluid retention (Sci).Core fluid retention index is the ratio of void volume of unit sampling area at the core zone over the root mean square deviation.
Maximum peak-to-valley roughness (Sy) is the vertical distance between the top of highest peak and bottom of deepest valley within sampling length.It is the maximum of all the peak-to-valley values.

Statistical analysis
All data was presented as mean ± SD. Analysis of variance (ANOVA) was performed to evaluate different parameters between controlled and treated samples; statistical significance was set at p<0.05.

Diameter of blood vessels
The diameters of PBVs, SBVs and TBVs of treated groups (0.03%, 0.05%, 0.1% and 0.5%) and control were measured by using the SPIP.The pronounced effect was seen at 0.5% (Figure 2).

3D surface roughness of blood vessels
The parameters of control CAM vasculature were greater than the treated CAMs vasculature parameters (Figure 1).
Ten point height of the surface is the mean of absolute heights of five highest peaks and the depths of five deepest pits or valleys of sampling area.It is extreme parameter.The Sz value of control was 282.5 ± 2.2 and in treated groups of T. stocksianum, Sz was 264.0 ± 7.2 with 0.03%, 261.0 ± 1 with 0.05%, 231.0 ± 1.5 with 0.1%, 225.0 ± 10.1 with 0.5% for T. stocksianum (Table II).
According to the 3D surface roughness parameters, heights and pits were less in the treated CAMs as compared to control CAMs because of less developed blood vessels.Similarly, other parameters such as root mean square slope, arithmetic mean summits, reduced    summit height, texture index, reduced valley depth, and core roughness depth were less the treated CAMs as compared to control CAMs.
The result showed that values of 3D roughness parameters of T. stocksianum were decreased than control (Figure 3).Moreover, graphical representation by abbott curve and angular spectrum showed that in T. stocksianum height of abbott curve (Figure 4) and angular spectrum (Figure 5) was decreased than in control.
Similarly, control CAMs angular spectrum and treated CAMs angular spectrum were measured.The angular spectrum refers to amplitude of regular intensity variation with angle.The result showed that height of curve for control CAM in Abbott curve and angular spectrum was increased in comparison with values of height of curve with T. stocksianum.

Discussion
CAM area and average surface roughness parameters were quantified to determine the anti-angiogenic Four parameters are utilized to describe the surface amplitude.They are categorized into four groups i.e., a) extreme, b) dispersion, c) height distribution sharpness and d) asymmetry of the height distribution.The Sq is root mean square value of surface within sampling area.It is dispersion parameter.

Figure 1 :
Figure 1: Macroscopic evaluation of chicken CAM.Well-defined vascular architecture of CAM vessels i.e.PBVs, SBVs, TBVs and well develop CAM area in (A) control while extensive deterioration of CAM vessels and reduction in CAM area representing anti-angiogenic activities in treated dilutions of T. stocksianum i.e. (B) 0.03% (C) 0.05% (D) 0.1% (E) 0.5%