Anti-angiogenic activity of Calotropis gigantea leaf extract by chick chorioallantoic membrane (CAM) assay method

INTRODUCTION

Cancer is a condition in which a group of abnormal cells divide uncontrollably by breaking the normal rules of cell division. Normal cells are constantly subjected to signals that provide information whether the cell should divide, differentiate into another cell or die. Cancer cells are devoid of these signals, resulting in uncontrolled cell growth and proliferation.¹

The anti-cancer drugs are classified as follows:

A. Cytotoxic drugs

B. Targeted drugs

C. Hormonal drugs

The targeted drugs contain of Angiogenesis inhibitors. The formation of new blood vessels is called angiogenesis. It is a normal part of growth and healing. But it plays a major role in several diseases, including cancer. A tumour requires oxygen and nutrients for its growth and spreading. Blood contains all these ingredients. The tumour sends chemical signals that stimulates growth of blood vessels and the blood vessels carry blood to the tumour.

Angiogenesis inhibitors, also called antiangiogenics, are drugs that block angiogenesis. Blocking of the nutrients and oxygen from a tumour, “starves” it. Hence these drugs are an important part of treatment for some types of cancer.²

CAM assay is used to determine the antiangiogenetic properties of plants. CAM assays have been widely used to study angiogenesis, metastasis and tumour cell invasion. The CAM model has many advantages, such as (a) the highly vascularized nature of the CAM greatly promotes the efficiency of tumour cell grafting: (b) high reproducibility; (c) simplicity and cost effectiveness, and (d) as the CAM assay is a closed system, the half-life of many experimental molecules such as small peptides tends to be much longer in comparison to animal models, allowing experimental study of potential anti-metastatic compounds that are only available in small quantities. The CAM is composed of a multilayer epithelium; the ectoderm at the air interface, mesoderm (or stroma) and endoderm at the interface with the allantois sac. It also contains extracellular matrix proteins (ECM) such as laminin, collagen type I, fibronectin and integrin. The presence of these extracellular matrix proteins mimics the physiological cancer cell environment.³

The leaves of Calotropis gigantea possessed the anti- cancer activity which was conducted using breast cancer cells MCF-7. Hence these leaves belonging to the family Asclepiadaceae were selected to study for presence of the activity as it is commonly available.

MATERIAL AND METHODS

1. Collection and authentication of Calotropis gigantea:

The leaves were collected from Bengaluru region during the month of June 2019. The fresh sample of the leaves was subjected to authentication by Dr. Rama Rao at Regional Ayurveda Research Institute for metabolic disorders.

2. Pharmacokinetically evaluation of the crude drug:

A. Macroscopic characters: The leaves were studied for colour, odour, taste, and size.

B. Microscopic characters

a. Powder microscopy: The dried leaves were powdered, treated with chloral hydrate and slightly heated and equal volume of phloroglucinol and conc. hydrochloric was added and powder characters were studied.

b. Transverse section: Thin transverse section of fresh leaf was taken and treated with chloral hydrate and slightly heated and equal volume of phloroglucinol and conc. hydrochloric acid was added.

3. Successive solvent extraction

Hot successive extraction using Soxhlet assembly by using petroleum ether followed by chloroform, and methanol solvents. Extraction was carried out in each solvent until the solution in the siphon tube is colourless. After the effective extraction, the solvent was recovered using rotary flash evaporator and was distilled. The extract was then concentrated and weighed. The % yield was calculated on the basis of air-dried drug.

4. Phytochemical screening⁴

All the extracts were subjected to phytochemical screening to test for the presence of phyto constituents such as carbohydrates, proteins and amino acids, alkaloids, glycosides, phytosterols, triterpenes, saponins, phenolic compounds and flavonoids.

5. TLC for the extracts⁴

TLC for the chloroform and methanol extracts were carried out for the separation of phyto constituents present in the extract using different solvent systems as a mobile phase and the one which showed clear and maximum separation was finally selected.

Sample preparation: 2mg of the extracts were dissolved in 5ml of their respective solvents.

Stationary phase: Silica gel 60 F254 (0.25µ). This is activated by heating the plates at 1050C for 30 minutes. On these, the extracts were spotted maintain the distance.

Mobile phase: Different solvent systems were used and finally one is selected which gives maximum and clear separation.

Visualization:

UV at 254 nm and 366 nm: Plates were air dried and observed under UV at 254nm and 366nm and spots were marked. The solvent systems tried for the optimization of TLC solvent system are given in the below table.

6. Anti-angiogenic activity:

In vivo evaluation of anti-angiogenic activity by CAM assay method:

1. Select fresh eggs (not older than 7 days), and clean.

2. Place eggs in an incubator. Incubate eggs at 38.3 ± 0.2°C and 58 ± 2% relative humidity. Hand rotate eggs five times per day until day 8.

3. Candle the eggs on incubation day 8 and remove any nonviable or defective eggs.

4. Eggs are returned to the incubator.

5. Remove eggs from the incubator on day 9 for use in the assay. Candle eggs. Discard any nonviable or defective eggs.

6. Mark the air cell of the egg. Cut it. Care should be taken when removing the egg shell to ensure that the inner membrane is not injured.

7. Apply liquid substances or diluted substances directly onto the CAM surface.

8. Check for blood vessel lysis.

Preparation of DMSO solvent:

Stock A: 1ml of DMSO is diluted to 100ml

Stock B: Pipette 1.5ml of Stock A and make up to 50ml

Preparation of sample solution:

Dissolve 25mg of chloroform, methanol and aqueous extract using DMSO solvent and make up the volume to 25ml, which gives the conc. of 1mg/ ml (1000µg/ml). 1ml of this is added to the CAM surface.

RESULT

1. Collection and authentication of Calotropis gigantea: The leaves were authenticated as Calotropis gigantea by Dr. Rama Rao at Regional Ayurveda Research Institute for metabolic disorders (Fig.1).

2. Pharmacognostical evaluation of the crude drug: A. Macroscopic characters:

• Simple, opposite, sub-sessile, slightly thick, fleshy, coriaceous,

• Length: 10-15 cm.; Width: 4.5 to 6.5 cm.

• broadly cuneate, obovate or obovate oblong, slightly cordate and auricled at base with tuff of short simple hairs on the upper side near place of the attachment to the petiole.

• The tender leaves are covered with ashy grey pubescence.

• Mature leaves are nearly smooth or even globous and pale green.

B. Microscopic characters: The powder microscopy of leaves showed the presence of xylem vessels, phloem fibres and parenchyma cells.

3. Successive solvent extraction:

After the completion of soxhlation, the extracts were completely dried. Colour, nature and percentage yield of the extract was noted.

5. TLC for the extracts:

Chloroform extract showed 7 spots in the solvent system Hexane: Ethylacetate in the ratio 1:1. Whereas the methanol extract showed 4 spots in the solvent system Benzene: Chloroform in the ratio 1:1

6. Anti- angiogenic activity:

The chloroform extract showed the maximum activity when compared to the methanol and aqueous extracts.

The egg treated with DMSO solvent remained the same even after 24 hours. Thus, it proved that the solvent does not interfere in the activity (Fig. 2-4).

DISCUSSION

Calotropis gigantea plant is commonly available throughout and is a fast growing one. Hence, this became the one of the criteria for selection of this plant. The collected leaves were dried, powdered and was analysed for all the proximate values, macroscopic and microscopic characters.

The successive solvent extraction was done using petroleum ether, chloroform, methanol and water which gave the percentage yield of 4.3%w/w, 3.1%w/w, 12.46%w/w and 8.9%w/w respectively.

The phytochemical screening of the extract revealed the different components in each extract. Chloroform extract contained triterpenes and saponins, whereas methanolic extract contained phenols, alkaloids and glycosides. Then the separation of components was done by TLC. In this chloroform extract showed 7 spots in the solvent system Hexane: Ethyl acetate in the ratio 1:1.

Whereas the methanol extract showed 4 spots in the solvent system Benzene: Chloroform in the ratio 1:1. Then anti- angiogenic activity was performed by CAM assay method where chloroform extract showed the maximum activity.

CONCLUSION

By this investigation, we can conclude that the Calotropis gigantea leaves contained antiangiogenic activity and further investigation can be carried out for the isolation of the component responsible for the activity.

Conflict of interest: Nil

Financial support: nil