Bangladesh Journal of Pharmacology Volume: 18; Number 3; Year 2023 Cite this article as: Maher RA, Alombro NC, de Vera Peter JD. Anti-angiogenic activity of Centrosema molle leaf aqueous extract. Bangladesh J Pharmacol. 2023; 18: 116-18. Anti-angiogenic activity of Centrosema molle leaf aqueous extract Sir, Angiogenesis is a process in which new blood vessels are formed from pre-existing vessels. This process is a crucial factor associated with tumor growth, progre- ssion, and metastasis (Teleanu et al., 2019). Plants such as Teucrium stocksianum (Tabassum et al., 2015), Gynura segetum (Seow et al., 2011), and Nelumbo nucifera (Lee et al., 2015) have been reported to have anti-angiogenic properties. Centrosema molle Mart. ex Benth (butterfly pea) is a climbing, trailing, and twining perennial plant belon- ging to the family Fabaceae. It has no recorded folkloric medicinal use in the Philippines. However, in other countries, this plant has been used for treating snake and scorpion bites and womb cleansing (Ariwaodo et al., 2012). Several studies had already been conducted regarding the biological activities of this plant, such as cytotoxic (Interino et al., 2023) and wound-healing (Ekpo et al., 2011) activities. One of the most common and feasible ways of preliminary investigating the anti-angiogenic activity of plant extracts is the chick embryo chorioallantoic mem- brane (CAM) assay. This assay enables the researchers to study the angiogenic effect and metastasis of plant extracts. This in vivo model is a relatively simple and low-cost and quick estimation of anti-angiogenic poten- tial. Moreover, this assay does not require the approval of any institutional animal research ethics committee. Through this assay, the anti-angiogenic activity of the plant extract to be tested was established by counting the disrupted blood vessels around the disc that were submerged in the plant extract (Ribatti, 2014). The present study determined the anti-angiogenic acti- vity of the C. molle leaf aqueous extract through CAM assay and hopefully will contribute to the discovery and development of new anti-cancer molecules and drugs in the future. Prior to the collection of the C. molle leaves for aqueous extraction, plant samples were first sent for authentica- tion to the Biology Department of Ateneo de Davao University, Davao City. After authentication, plant leaf samples were collected from Pinaring, Sultan Kudarat, Maguindanao del Norte, Bangsamoro Autonomous Region in Muslim Mindanao. Aqueous extraction of C. molle leaf was done by pulverizing the dried leaf sam- ples and 30 g of it was suspended in 100 mL of deio- nized water. The solution was heated at 60°C using a water bath and was regularly monitored to obtain the appropriate temperature. Filtration of the heated solu- tion using cheesecloth was done after 1 hour of heating and was centrifuged at 3,000 rpm for 5 min. The obtain- ed supernatant liquid was kept in a clean amber glass bottle. Serial dilution was done to prepare the different concentrations (5, 250, 500, 750, and 1000 µg/mL) of C. molle aqueous extract. The presence of secondary meta- bolites in the extract was studies by the standard method (Harborne, 1993). The CAM assay was used to determine the anti-angio- genic activity of the different concentrations of C. molle leaf aqueous extract. The method was described else- where (Chen et al., 2013). A four-day-old 63 fertilized chicken eggs were bought from a nearby hatchery. These fertilized eggs were sprayed with 70% ethanol to prevent any contamination. Incubation was done at 37° C and moisture of about 60%-70% was provided. Incu- bation was done in 5 days. The experiment was done in three replicates and in three trials. Distilled water was used as negative control while retinoic acid for positive control. Perforation of eggs was done carefully to avoid possible contamination. Perforated windows in the eggs were as small as 2 cm and this was done by removing the outer and inner covering of the egg at the air-space location. Then, perforated windows were covered with decontaminated parafilm and incubated. After the 6th day of incubation, perforated windows of incubated eggs were opened and a sterile filter paper disc which was initially soaked with the different concentrations of the C. molle leaf aqueous extract, positive and negative controls were placed at the junction of two large blood vessels of the CAM using sterile forceps. The treated eggs were covered with decontaminated parafilm and were re-incubated for 24 hours. The sealed windows were unlocked during the 7th day of incubation. The embryos were sacrificed and the CAMs were harvested by removing the hard shell and were placed on a petri dish. The CAM at the site of application of the filter disc was examined. Any changes such as inhibition of blood vessel formation were noted in terms of their number and were compared with the eggs treated with negative A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2023; 18: 116-118 Journal homepage: www.banglajol.info; www.bdpsjournal.org Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088; DOI: 10.3329/bjp.v18i3.67311 Letter to the Editor This work is licensed under a Creative Commons Attribution 4.0 International License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor and positive controls. The number of CAM vessel branch points at the area of the filter paper disc was counted. Inhibition of angio- genesis by the extracts would result in the absence or lack of new blood vessel formation. The number of branch points was noted by counting all the intersec- tions of branching blood vessels. Table I showed the results of phytochemical screening. Alkaloids, carbohydrates, flavonoids, reducing sugar, and tannins were found to be present in the C. molle leaf aqueous extract while saponins were found to be absent. CAM assay tested C. molle leaf aqueous extract concen- trations (5, 250, 500, 750, and 1000 µg/mL) for anti- angiogenic activity and was compared against the positive and negative controls. Results showed that all the concentrations of C. molle leaf aqueous extract except 50 µg/mL were able to inhibit the branching points of blood vessels in CAM during the experiment (Figure 1). The negative control (distilled water) yielded the highest branching points while no branching points were noted in the eggs treated with positive control (retinoic acid). The calculated IC50 for the C. molle leaf aqueous extract concentrations tested in this study is 79.17 µg/mL. The results of this study may indicate the potential of the C. molle leaf aqueous extract concentra- tions’ anti-angiogenic activity. Several studies had already indicated the anti-angio- genic potential of some secondary metabolites present in plant extracts that may interfere with angiogenesis and may affect the branching of blood vessels (Dai et al. 2015). Thus, future studies identifying bioactive and isolating bioactive compounds present in the C. molle leaf aqueous extract concentration are recommended to determine their respective contribution to the anti- angiogenic activity. Since this study is only limited to the branching points of blood vessels in the CAM, it is also recommended that in future studies, other para- meters in the CAM assay such as the diameter and roughness of blood vessels should be considered. Financial support: Self-funded Conflict of Interest: The authors declare that they have no conflict of interest. Rawan A. Maher1, Nancy C. Alombro1 and Peter Jan D. de Vera2 1 Natural Sciences Department, Notre Dame University, Cotabato City, Philippines; 2 Natural Sciences Department, College of Arts and Sciences, Mindanao State University – Maguindanao, Dalican, Datu Odin Sinsuat, Maguindanao del Norte, Bangsamoro Autonomous Region in Muslim Mindanao (BARMM), Philippines. Corresponding author: email: peterjandevera0302@gmail.com References Ariwaodo JO, Chukwuma EC, Adeniji KA. Some medicinal plant species of Asamagbe stream bank vegetation, forestry Bangladesh J Pharmacol 2023; 18: 116-118 117 Figure 1: Anti-angiogenic effects of different C. molle leaf aqueous extract using CAM assay (n = 63) Table I Phytochemical screening of C. Molle leaf aqueous extract Phytochemicals Types of test Results Alkaloids Dragendorff’s and Mayer’s + Carbohydrates Molisch’s + Flavonoids Wilstatter + Reducing sugar Benedict’s + Saponins Froth - Tannins Ferric chloride + N u m b e r o f B ra n c h in g p o in ts 100 80 60 40 20 0 Negative control Positive control 50 250 500 750 1000 µg/mL y = -10.444x + 57.498 R 2 = 0.44 mailto:mohanrajupu62@gmail.com mailto:peterjandevera0302@gmail.com research institute of Nigeria, Ibadan. Ethnobot Res Appl. 2012; 10: 541-49. Chen Z, Wen Z, Bai X. In vivo chick chorioallantoic membrane (CAM) angiogenesis assays. 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