1. INTRODUCTION:

Angiogenesis is the formation of new blood vessels from a pre-existing vasculature and is a complex process involving an extensive interaction between cells, the extracellular matrix, and soluble factors¹. Angiogenesis plays an important role in pathologic processes such as the growth and metastasis of tumors. Therefore, newly designed anti-angiogenic agents have the potential for treatment of a broad spectrum of diseases, including cancer. Angiogenesis relies on the proliferation and migration of endothelial cells, which are linked to the expression of growth factors by tumor cells^2-4.

It also has important roles in the etiology of many diseases, such as chronic inflammatory disorders and some pregnancy-related diseases like intrauterine growth restriction and preeclampsia. The remarkable diversity in angiogenesis signaling pathways provides many options for therapeutic intervention, and since angiogenesis plays an essential role in tumor growth and invasion, anti-angiogenesis is currently a major area of oncologic research. A tumor is unable to grow more than 2 mm in diameter unless there is the development of new vessels by angiogenesis⁵. Capillaries are needed in all tissues for diffusion exchange of nutrients and metabolites. Changes in metabolic activity lead to proportional changes in angiogenesis, and hence, proportional changes in capillary perfusion. Oxygen plays a pivotal role in this regulation. Hemodynamic factors are critical for the survival of vascular networks and for structural adaptations of vessel walls. Drugs targeting pathologic angiogenesis have been designed to interfere with any of these steps and some of them are currently undergoing evaluation in clinical studies⁶. It has hypothesized that these drugs help normalize the blood vessels that supply the tumor. Angiogenesis inhibitor therapy does not necessarily kill tumors, but instead, may prevent tumors from growing; therefore, long therapy is required. Consequently, anti-angiogenesis is regarded as a promising strategy in cancer therapy^7–11.

Recent studies have shown that vascular cells can produce reactive oxygen species (ROS) through NADPH oxidase. It has been suggested that these free radicals play concentration-dependent roles in neo-vascularisation since high concentrations of ROS can cause oxidative stress that leads to apoptosis, while low levels function as signaling molecules that mediate the proliferation and migration of endothelial cells, assisting in angiogenesis in vivo^12-14. According to research, ROS levels may be determined by the mechanisms that control the production of oxidant species associated with the activation of enzyme systems that perform an antioxidant function^14,15.

Understanding the role of ROS in triggering the formation of new blood vessels may enable the components to be classified as potential targets for the treatment of angiogenesis-dependent diseases. The conclusion can then be drawn that substances which modulate the action of oxidants in tissues could be important mediators of neo-vascularization in pathological processes^16,17. Moreover, it is observed that many natural products contain diverse chemical combinations, which adds an additional layer of complexity to observed angiogenic properties. Assortments of chemicals can be seen assisting in the modulation of effects produced by oxidation substrates in biological systems^{18- 23}.

The study at hand was undertaken to analyze the anti-angiogenic, antioxidant and cytotoxic properties of marketed extracts derived from the fever few plants compared with amlodipine. In addition, we investigated the cytotoxic properties of the extracts to identify potential sources of antineoplastic agents.

2. MATERIALS AND METHODS:

2.1 Preparation of Tanacetum parthenium:

The highest content of parthenolide was found in flower heads (1.38%) followed by the leaves (0.95%). Tanacetum parthenium extract solution was diluted with different pH buffers to study the solution stability and separation of parthenolide in feverfew. It was then diluted with distilled water to 1mg/ml of parthenolide. Solid phase extraction and mass spectroscopy were used to evaluate parthenolide concentration in the diluted drug formulation. It was subsequently diluted to10^-4, 10^-5, or 10^-6M.

2.2 Preparation of amlodipine besylate:

2.2.1 Tablet sample:

Tablet samples were grounded to fine powder and the powder was weighed to ensure mass equivalence to one tablet. Distilled water was added to a mortar and triturated with finely powdered amlodipine. Sonication extraction was performed for 5 minutes to ensure uniform mixing and distribution of the drug, which was then diluted to 1mg/ml with distilled water. The drug solution was then filtered through a 0.45μm filter to remove the excess and foreign particles from the extract. Amlodipine concentrations were adjusted accordingly using distilled water.

2.3 Materials and reagents:

2.3.1Fertilized E6 chicken eggs:

Fertilized E6 chicken eggs were collected from poultry farm near SRMIST and were allowed to incubate for 3days at 37^oC and 60% relative humidity to ensure proper aeration and to maintain the growth of the embryos. The incubator was checked at regular intervals. The eggs were not allowed to move or shake to prevent air bubble formation or death of embryos.

2.3.2 0.1% benzalkoniumbromide:

0.1% benzalkonium bromide was used as a disinfectant. To maintain aseptic conditions, the tops of the eggs were sterilizedprior to drilling.

2.3.3 Methanol and acetone:

These chemicals at 70% concentration were also as a disinfectant.

2.3.4 Filter-paper disc (Whatman, catalog number: 1441150)

The filter paper was cut into a small sized circular disc of size 3mm diameter for the impregnation of the drug.

2.3.5 Packing film (Parafilm):

Paraffin film was used as a packing material for the eggs after the placement of drug pellets.

2.3.6 Drugs used:

Amlodipine 5mg tablets (marketed formulation- AMLONG - 5mg) were purchased from Micro Labs Limited (Bangalore, Chennai).

Feverfew tablets (marketed formulation from feverfew leaves-380mg; 0.7% parthenolide) were purchased from Nature’s Way (England).

2.4 Chick embryo chorio Allantoic Membrane (CAM) assay:

The CAM assay was performed according to the traditional method with some modifications^24-28. The fertilized chicken eggs were incubated for 3 days at 37^◦C under a constant relative humidity of 60%. The eggs were positioned in a horizontally and rotated several times in a day. On day 3, the eggs were swabbed with 0.1% benzalkonium bromide under a laminar flow hood. Albumin (2–3 ml) was injected using a 21-gauge needle through the pointer end of the egg in order to allow detachment of the developing CAM from the egg shell. A window was then cut in the shell using a sterile surgical blade and the shell was removed with sterile forceps. This window served as a portal of access for the CAM. Any non-viable eggs were then disposed of at this stage, and the remaining eggs were sealed with parafilm. The eggs were returned to the incubator and kept horizontal with the windows facing upwards. On day 7, photographs of the embryos were captured to show the blood vessels where the drug was to be administered. Filter paper discs with the test substances were placed directly over a blood vessel on the growing CAM under sterile conditions. A second photograph was taken to record the actual positioning of the disk on the CAM. The eggs were resealed with parafilm and returned to the incubator^29-31. The final evaluations were carried out at time points of 20 minutes, 30 minutes, 3 hours, and 24 hours after the addition of each drug. At each stage, the filter paper disc was gently removed from the CAM which was then examined for anti-angiogenic effects at the site of sample application. A third photograph was taken to obtain the image of the CAM after treatment with extracts or standard drug. Surface images of corresponding CAMs within the same test sample (before and after treatment) were compared to quantify anti-angiogenic effects within the area of CAM covered by the filter paper disc^32-34. A modified semi-quantitative scoring system with a scale of 0-2 was used for grading. Two-blinded; independent observers recorded any anti-angiogenic effects present. Ten eggs per sample were prepared to allow for the 30–40% mortality rate inherent to the procedure, yielding a minimum of 4-6 eggs available for use³⁵. For every sample, the average score was calculated and interpreted for anti-angiogenic effects (Table 1) as follows:

After scoring, an equation was used for the determination of the average mean score for each drug concentration. The equation is as follows.

Average score = [Egg number (Score 2) X 2 + Egg number (Score 1) X 1] / [Total egg numbers (Score 0, 1, 2)]

Average scores < 0.5 were taken to indicate no anti-angiogenic effect, between 0.5 and 1 were weak in anti-angiogenic activity, 1 to 1.5 indicated moderate anti-angiogenic effect and greater than1.5 was strong in anti-angiogenic activity.

Table 1: Scoring of Anti-Angiogenic Activity

Score	Effect	Definition
0	Absent	Normal embryo, no difference with respect to surrounding capillaries.
0.5	Weak	There is no area lacking capillary vessels. The density of the capillaries is decreased but not larger than the pellet.
1	Moderate	The area lacking capillaries is small or capillary density is decreased in a certain area. The effects are not more than twice of the pellet area.
2	Strong	The area lacking capillaries is at least as twice as pellet area.

2.5 Antioxidant activity:

2.5.1 DPPH free radical scavenging activity:

The ability of the extracts to annihilate the DPPH radical (1, 1-diphenyl-2-picrylhydrazyl) was investigated using methods described by Marsden S. Blois³⁶. A stock solution of fewer few extract was prepared at 1mg/ml. 100, 150 or 200µg of each extract was added in a normalized volume to 0.1mM DPPH resuspended in methanol. The reaction mixture was incubated for 30minutes at room temperature and the absorbance values were recorded at 517nm. Butylated Hydroxyl Toluene (BHT) was used as a standard control. The annihilation activity of free radicals was calculated as the percentage of inhibition according to the formula³⁷:

% Inhibition = [(absorbance of control – absorbance of test)/absorbance of control] X 100.

2.5.2 Nitric oxide scavenging activity:

Nitric oxide radical inhibition was estimated by the use of Griess Illosvoy reaction^38.A stock solution of fewer few extract and ascorbic acid (used as standard) were prepared at 1mg/ml. The reaction buffer was prepared using 2ml of sodium nitroprusside (10mM) and 0.1ml of phosphate buffered saline.100, 150 or 200µg of plant extract or ascorbic acid standard was added to the buffer and incubated at 25ºC for 150 minutes. After incubation, 0.5ml of the reaction cocktail was added to 1ml of 1% sulphanilamide and allowed to stand for 5 minutes to complete diazotization. Then, 1ml of 0.1% naphthyl ethylenediamine dihydrochloride was added, mixed and allowed to stand for 30minutes at 25ºC. The absorbance of these solutions was measured at 540nm and normalized against a blank control. The blank consisted of all necessary reagents, except water was substituted for extract solution. The annihilation activity of free radicals was calculated as the percentage of inhibition according to the same equation as mentioned above.

2.5.3 Ferric reducing antioxidant power assay:

Total antioxidant activity is traditionally measured by the ferric reducing antioxidant power (FRAP) assay according to Benzie and Strain³⁹. They showed that change in absorbance is directly related to the total reducing power of electron- donating antioxidants present in the reaction mixture. The samples of extract were made up to 1ml with distilled water and were mixed with 1.5ml of working FRAP reagent and incubated at 37°C for 4 minutes. After incubation, absorbances were measured at 593nm. Ferrous sulfate standard was processed in the same way and calibration curve was generated using various concentrations of ferrous sulfate (200 - 1000µg). A blank for eliminating background consisted of all the reagents, except the extract was substituted with water³⁹.

2.5.4 Glutathione-S-Transferase assay:

The GST assay was carried out with the GST Assay Kit (Sigma; St. Louis, Missouri) (Cat#: CS0410) according to the manufacturer's protocol. The required amount of L-Glutathione reduced was dissolved in water and the solution was kept on ice and was used the same day. Samples were diluted with sample buffer to a concentration within the activity range of the assay. 196µl of substrate master mix (9.8ml of Dulbecco’s phosphate buffered saline, 0.1ml of 200mM reduced L-glutathione, and 0.1ml of 100mM 1-Chloro-2,4-dinitrobenzene (CDNB) were added to 10µl of the sample and the contents were mixed⁴⁰. Absorbance values were measured at 340nm every minute for 5 minutes. GST specific activity (µM/min/mL) was calculated according to the following:

(ẚ A₃₄₀)/min= [A₃₄₀(Final read)- A₃₄₀(Initial read)]/ Reaction time (min)

[(ẚ A₃₄₀)/min*V (ml)*dil/ᵠ_mM*V_enz(ml)]Ṫ≠mol/ml/min

Where,

‘dil’ is the dilution factor, ᵠ_Mm is the extinction coefficient for CDNB conjugate at 340nm (0.53mM for 96-well plate) and V_enz (ml) is the volume of enzyme sample.

2.6 Cytotoxicity analysis:

MTT assay for cell viability and toxicity:

We adopted a form of the MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay described by Mossman⁴¹. Culture media consisted of Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum (DMEM)⁴². Cells were maintained in DMEM at 37^oC in a humidified atmosphere with 5% CO₂⁴³. Cells were plated in 1.2 X 10⁴cells/well in 96-well flat bottom tissue culture plates and allowed to adhere overnight at 37^oC^44-47. The fresh DMEM was replaced and cells were incubated with different concentrations for 48 hours⁴⁸. After the incubation, 100µl of new DMEM was added with 5mg/mL of MTT and was allowed to incubate for 4 more hours⁴⁹. The media was then discarded and 100µl of DMSO was added to dissolve the formazan crystals⁵⁰. Absorbance of the supernatant was read at 570nm in a microtiter plate reader. Cell survival was evaluated by calculating the following formulas:

% Viability = [(Test OD/Control OD)] X 100

% Cytotoxicity = 100 – % Viability.

3. RESULTS AND DISCUSSION:

Amlodipine is a calcium channel blocker that aids in the treatment of hypertension⁵¹. It effectively lowers blood pressure which reduces the risk of fatal and nonfatal cardiovascular events primarily strokes and myocardial infarctions⁵². Diltiazem, which is also a Calcium Channel Blocker, acts as a tyrosine kinase inhibitor where the study has already been done for its anti-angiogenic effects. The CAM is treated with varied concentrations of Amlodipine and Tanacetum parthenium (Feverfew) (Figure 1). The findings presented here indicate that amlodipine has anti-angiogenic properties in addition to its anti-hypertensive effects. The CAM treated with amlodipine concentration (10^-5M) showed a significantly greater anti-angiogenic effect compared to that of other concentrations (10^-4M and 10^-6M) at different time intervals (Figure 1). When the time of contact increased, the number of blood vessels decreased, and this effect was estimated by the scoring system (Figure 2). These drugs act by blocking the VEGF signaling pathway, which reduces the number of blood vessels. The anti-angiogenic scoring done for Tanacetum parthenium (Feverfew) as compared to amlodipine also demonstrated a reduction in the number of blood vessels (Figure 3). We believe this to be due to the presence of parthenolide. The chemical constituent parthenolide has demonstrated antioxidant (Tables 2, 3, 4, 5) and cytotoxic activity (Table 6) as well. Despite this, Tanacetum parthenium exhibited anti-angiogenic effects to a lesser extent than did amlodipine.

RESULTS:

Table 2: Scavenging potential of leaf extracts

Solvent	Concentration (µg)	Absorbance of sample	% of inhibition
Hexane	100	0.3228	6.026200873
	150	0.3609	19.62138085
	200	0.3758	22.86535304
Ethyl acetate	100	0.3678	24.50738916
	150	0.3109	36.1863711
	200	0.256	47.45484401
Ethanol	100	0.2745	43.65763547
	150	0.26	46.63382594
	200	0.2345	51.86781609
BHT	100	0.1133	76.74466338
	150	0.108	77.83251232
	200	0.091	81.32183908

Table 3: Nitric Oxide Scavenging Activity

Solvent	Concentration (µg)	Absorbance of sample	% of inhibition
Hexane	100	1.456	21.50097046
	150	1.411	23.92710804
	200	1.354	27.00021566
Ethyl acetate	100	1.214	34.54819927
	150	1.089	41.28747035
	200	0.986	46.84062972
Ethanol	100	0.8762	52.76040543
	150	0.7797	57.96312271
	200	0.7401	60.09812379

Table 4: Reducing ability (FRAP assay)

Solvent	Concentration (µg)	Absorbance of sample	µM of Fe(II)/g of dry mass
Hexane	100	0.456	110
	150	0.492	120
	200	0.512	135
Ethyl acetate	100	0.700	180
	150	1.010	250
	200	1.128	320
Ethanol	100	2.140	530
	150	2.178	570
	200	2.197	600

Table 5: GST specific activity

Sample	1 min	5 min	Average	A340/min	GST specific activity	Average
Hexane	0.593	0.769	0.681	0.0352	0.026566038	0.027019
Hexane	0.62	0.802	0.711	0.0364	0.027471698	0.027019
Ethyl acetate	1.788	1.98	1.884	0.0384	0.028981132	0.023774
Ethyl acetate	2.03	2.153	2.0915	0.0246	0.018566038	0.023774
Ethanol	0.35	0.61	0.48	0.052	0.039245283	0.038038
Ethanol	0.343	0.587	0.465	0.0488	0.036830189	0.038038

Table 6: Cytotoxicity effect of the sample

	Control	100 µg	150 µg	200 µg
Absorbance at 570nm	1.235	1.239	1.195	1.012
Absorbance at 570nm	1.423	1.116	1.054	1.024
Average	1.329	1.1775	1.1245	1.018
% of viability	100	88.60045	84.61249	76.59895
% of toxicity	0	11.39955	15.38751	23.40105

4. CONCLUSION:

In conclusion, the amlodipine and Tanacetum parthenium exhibited some anti-angiogenic activity in the CAM assay. There was a significant reduction in the number of blood vessels when these drugs were administered under different concentrations and at different time intervals. It is believed that amlodipine acts as a tyrosine kinase inhibitor that blocks the VEGF (Vascular Endothelial Growth Factor) signaling pathway, which is responsible for the prolongation of the progression-free survival in cancer. Also, Tanacetum parthenium formulation contains parthenolide as one of the main chemical constituents, where it is responsible in suppressing the expression of biomarker protein VEGF (Vascular Endothelial Growth Factor) and VEGF receptors besides having antioxidant and cytotoxic properties. Hence, we can conclude that both of these drugs may be used as a potential source for protection against cancer.

5. CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

6. ACKNOWLEDGMENTS:

We are highly indebted to SRM College of Pharmacy for their guidance and constant supervision, as well as for providing necessary information regarding the project and their support throughout the duration of the study. We owe our thanks to Prof. V. Chitra, M. Pharm., Ph. D., Vice Principal, SRM College of Pharmacy for her moral support in carrying out our work and innovative ideas.

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Received on 18.08.2019 Modified on 12.11.2019

Research J. Pharm. and Tech. 2020; 13(4):1665-1671.

DOI: 10.5958/0974-360X.2020.00302.9