Angiogenesis is a multiphase process that begins with pre-existing functioning vessels and ends with the formation of new blood capillaries.1 The balance of several positive and negative angiogenic modulators in the vascular microenvironment tightly controls angiogenesis.2 A growing body of research indicates that angiogenesis is a pre-requisite for many diseases. Numerous malignancies, diabetic retinopathy, autoimmune disorders, rheumatoid arthritis, atherosclerosis, cerebral ischemia, cardiovascular disorders, and delayed wound healing are all characterized by pathological angiogenesis.3,4 Numerous studies in this field are revealing novel pharmacological targets, metabolic pathways, and pro and anti-angiogenic compounds that may be used as therapeutic targets in these diseases. Chemicals or herbal remedies derived from the plant may have an impact on the angiogenesis process.5 One of the key advantageous mechanisms in the wound-healing process is angiogenesis. Some plant extracts promote angiogenesis, such as leaves extract of Ficus religiosa has angiogenic potential and can be used clinically.6 With this in mind, a lot of herbal medicines include Angiogenesis, which is the process of creating new blood vessels from preexisting ones. This process is crucial for cell growth and development and is also involved in the advancement of cancer (excess angiogenesis) and wound healing (deficient angiogenesis). It is significant in a wide range of clinical and physiological disorders. Angiogenesis can be therapeutically modulated. A variety of substances from diverse sources can be used to manipulate the angiogenesis process, which can then be used to treat a variety of disorders that result from either excessive or insufficient angiogenesis. Due to numerous angiogenic inhibitory substances have already been studied, compounds with the potential to promote angiogenesis, however, have not been well studied. Therefore, by finding the substances that show pro-angiogenic potential, conditions such as wound healing, ischemic heart disease, non-union of bone fractures etc. can be treated. In the current study, plant-based products were chosen for their pro-angiogenic potential due to their natural, herbal properties and minimal negative effects. A high number of angiogenic inhibiting compounds is already researched upon. However, compounds with pro angiogenic potential have not been researched to that extent. So by identifying the agents exhibiting pro angiogenic potential they can be used for the treatment of insufficient angiogenesis for e.g. wound healing, ischemic heart disease, bone fracture nonreunion etc. Plant based products were selected as the source for pro-angiogenic potential because of their minimal side effects and natural, herbal effects.

The work done during this research can be divided in two main categories: 1. Phytochemical studies involving Yield of extract, Ash content of dry leaves, IR and UV spectrophotometric studies, Thin Layer Chromatography profile of plant extracts. 2. Chick Chorioallantoic membrane (CAM) study as a biological model for studying the angiogenic potential using Focal application method and AbGel sponge method. PART 1: PHYTOCHEMICAL STUDIES The hot methanolic extract of Ficus religiosa leaves was prepared using Soxhlet extraction. Extraction process was carried out by repeated maceration with agitation, percolation and by continuous extraction using Soxhlet extractor. The extract's colour and consistency were observed. The Soxhlet extraction procedure was used to create the hot methanolic extract. A small portion of the extracted material was also dried to determine the yield of each extract. and extract was stored for further use. Yield of Extract - Yield of hot methanolic extract is determined with reference to dry weight of leaves. Ash Content - Ash content was determined by using incineration method. This ash was further taken for Acid Soluble & Acid Insoluble Ash Content. The values are represented with reference to dry weight of leaves. Total ash usually is of inorganic salts consist of carbonates, oxides, phosphates, silicates, & silica. It is a criterion to judge the identity or purity of drug. Adhering dirt and sand may be determined by acid insoluble ash. Further phytochemical analysis of extract was done by IR and UV spectrophotometric studies, Thin Layer Chromatography profile of plant extracts Infra-Red Spectroscopy study - Infra-Red studies were done using Potassium bromide (KBr) pellets, which were charged with whole extract at different month of the year to determine the stability. Ultraviolet Spectroscopy UV studies were done using methanol as a diluent 1:5000 dilution at different month of the year to determine the stability of the extract. Thin Layer Chromatography (TLC) a) The TLC is done to establish the Phytochemical constituents of plant extracts. b) The method for performing TLC is taken from Wagner et al. local citation c) TLC was done using Silica gel S254 coated on a glass plate d) The following table enlists the solvent system used to separate the constituents and lists the developer used for a particular Phytochemical. e) The Rf values were recorded for cross referencing and identification of present constituents. f) The photograph of TLC plates was shot for record purpose. The standard solvent system, and developer used for the specific phytochemical in plant extract is shown in Table 1. PART 2: CHICK CHORIOALLANTOIC MEMBRANE (CAM) STUDY For determining angiogenic potential the Chick CAM used as an animal model because of its simplicity, validity, and high reproducibility and is easy to handle. Also, the study on Chick CAM does not require ethical committee approval as it is mentioned by NIH. National Institute of Health1991. The Public Health Service Responds to Commonly asked Questions.” ILaR news 33.4: 68–70. Office of Laboratory Animal Welfare. http://grants.nih.gov/grants/olaw/references/ilar91.htm. The flowchart of day wise procedure in eggs as per EDD from day 0 to day 14 day of chicken embryo for CAM study is shown in Table 2. METHODOLOGY FOR CAM ASSAY: Focal Application The Whatman filter paper disc was loaded with 10 microliters of extract air dried in sterile condition and were inoculated on CAM on 8th EDD as per standard procedure to observe the increase in growth of blood vessel the advantage is visual change in the growth pattern of angiogenesis can be observed by this method. The scoring is done by counting the blood vessel emerging towards the extract or spoke wheel pattern, or blood vessels by passing the extract taking an obvious turn towards the extract. ABGelTM Application In the AbGel application, the sponge containing extract were inoculated on CAM on 8th EDD as per standard procedure to observe the blood vessels of neovascularization penetrating the AbGel and number of vessels can be assessed histopathological studies. This method is an excellent technique because only the pro-angiogenic compound will attract the blood vessels in sponge and they must grow out of the tissue in the sponge block which is an indication of pro-angiogenic activity of drug. Both the above methods were simultaneously supported by using a positive control and a blank control and distribution of eggs used in two methods for evaluation of angiogenesis is shown in Table 3. It shows that total 29 eggs were distributed accordingly in two groups for focal application method and AbGel Sponge method including blank control and positive control.

The angiogenic potential of Ficus Religiosa was less as compared to the angiogenic potential of Plermin which is a positive standard. The average growth of angiogenic potential in the chorioallantoic membrane (CAM) model with F. religiosa was 43.5 % whereas with the Plermin standard was 85%. The average value of increase in percentage growth of F. religiosa was within the range of control 40% to 66%. This indicates that the growth of blood vessels in F. religiosa samples can be due to the natural cause of chorioallantoic membrane and not because of leaf extracts of F. religiosa. This result indicates that hot methanolic extracts of leaf of F. religiosa didn’t poses significant increase in angiogenic potential as compared to positive control Plermin. Hence the leaf extracts did not pose angiogenic potential. Table 4 shows results for phytochemical studies of yield of hot methanolic extract, which is found to be 10.99 % determined with reference to 100 gm dry weight of leaves. Ash content was determined by using incineration method. This ash was further taken for Acid Soluble & Acid Insoluble Ash Content. The values are represented with reference to dry weight of leaves in Table 5. Ficus religiosa has high acid soluble ash because of large number of inorganic contents in it. SPECTROPHOTOMETRIC STUDIES IR and UV The spectrophotometric studies indicated that the chemical properties of extract did not change over the course of study. The peak absorbance were found to be at the same wavelength during the course of study. Infra-Red studies using Potassium bromide (KBr) pellets are shown in Fig 1, which were charged with whole extract at different month of the year to determine the stability. UV studies were done using methanol is shown in Fig 2. as a diluent 1:5000 dilution at different month of the year to determine the stability of the extract. The constituents present in leaf extracts of Ficus religiosa is showed in Table 6 and Rf values of chromatogram is shown in Table 7. The photograph was taken for the cross reference and is showed in Fig 3. Fig 4. Shows formation of blood vessels at 40x slide , stain Hematoxylene & Eosin. A-Tissue with blood vessels in it, B-AbGel sponge with no blood vessels in it, C-Sponge-tissue interphase. No blood vessels can be seen in sponge. Photograph of Focal application method on CAM is shown in Fig 5 using Whatman filter paper containing leaf extract of F. religiosa on 8th EDD and 11th EDD showing relatively less growth of blood vessels around Filter paper. The Table 8 shows the growth of blood vessels as counted by photographic evaluation photos were taken on 8th EDD and 11th EDD to see the angiogenic effect of Ficus religiosa. TABLES & FIGURES Table 1. The standard solvent system, and developer used for the specific phytochemical in plant extract: CONTENTS SOLVENT SYSTEM RATIO DEVELOPER Alkaloids Ethyl acetate: MeOH: H2O 100:13.5:10 Dragendorff Anthroglycosides Ethyl acetate: MeOH: H2O 100:13.5:10 10%Ethanolic KOH Arbutin Ethyl acetate: MeOH: H2O 100:13.5:10 10%Ethanolic KOH Cardiac glycosides Ethyl acetate: MeOH: H2O 100:13.5:10 Kedde reagent Bitter Principles Ethyl acetate: MeOH: H2O 100:13.5:10 VS Flavonoids Ethyl acetate: MeOH: H2O 100:13.5:10 FBS Saponins Ethyl acetate: MeOH: H2O 100:13.5:10 VS Didrovaltrate Toluene: Ethyl acetate 93:7 HCL: acetic acid Valepotriates Toluene: Ethyl acetate 93:7 HCL: acetic acid Table 2. The flowchart of day wise procedure in eggs as per EDD from day 0 to day 14 day of chicken embryo for CAM study: Embryonic Development Day, EDD Procedure Action after Procedure 0 Cleaning of eggs transfer to incubator Discard any cracked eggs. 1 Identification of fertilized Eggs Candling method Mark the unfertilized eggs 2 Identification of fertilized Eggs Candling method 2 ml of albumin is removed from narrow end 3 Creating window Discard dead embryos 4 Observation for any mortality in embryo Discard dead embryos 5,6,7 Observation for any mortality in embryo Discard dead embryos 8 Loading test sample Only viable eggs are loaded with sample 9,10 Keep the eggs undisturbed for three days ---- 11,12,13 Observation for angiogenesis & mortality Discard dead embryos 14 Observation for angiogenesis & mortality Discard dead embryos Table 3. Distribution of eggs for evaluation of angiogenesis: Eggs- Category/quantity of extract (Microgram/disc) Number of Eggs for Disc Method Eggs- Category/concentration of extract (Microgram/ml) Number of Eggs for AbGel Method Blank Disc (Only Whatman filter paper) 2 Blank Gel (Only Whatman filter paper) 2 Blank Disc + Methanol 2 Blank Gel + Methanol 2 Blank Control 2 Blank Control 2 F. religiosa at 165 mcg/disc 5 F. religiosa at 330 mcg/ml 5 F. religiosa at 330 mcg/disc 5 F. religiosa at 495 mcg/ml 5 F. religiosa at 495 mcg/disc 5 F. religiosa at 660 mcg/ml 5 F. religiosa at 660 mcg/disc 5 F. religiosa at 825 mcg/ml 5 Plermin control at 20, 40 & 60 mcg/disc 3 Plermin control at 20, 40 & 60 mcg/ml 3 Total number of eggs 29 Total number of eggs 29 Table 4. Yield of extract of Ficus religiosa: Name of plant Wt. of dried leaves (gm) Total Vol. of extract (ml) Wt. of extract in 25ml Yield of extract% Ficus religiosa 100 330 0.833 10.99 Table 5. Ash Content: Name of plant Acid Soluble ash % Acid Insoluble ash % Total Ash Content % Ficus religiosa 10.11 1.11 11.22 Table 6. Constituents present in leaf extracts of Ficus religiosa: Contents Ficus religiosa Alkaloids + Anthraglycosides + Arbutin + Cardiac glycosides + Bitter Principles + Flavonoids + Saponins + Didrovaltrate + Valepotriates + Table 7. Rf values of chromatogram: Contents Ficus religiosa Alkaloids 0.34 Cardiac glycosides 0.4 Bitter Principles 0.71 Flavonoids 0.76 Saponins 0.93 Table 8. Number of blood vessels on the membrane in Ficus religiosa, Plermin and control group (Focal application method) Eggs- Category/quantity of extract (Microgram/disc) Mean number of blood vessels on 8th Embryonic development day Mean number of blood vessels on 11th Embryonic development day % Increase in Growth CONTROL Blank Disc (Only Whatman filter paper) 3 5 66% Blank Disc + Methanol 5 7 40 % Blank Control 3 3 0 % Ficus religiosa F. religiosa at 165 mcg/disc 4 6 50 % F. religiosa at 330 mcg/disc 3 4 33 % F. religiosa 495 mcg/disc 5 7 40 % F. religiosa 660 mcg/disc 4 6 50 % PLERMIN Plermin Control 1 (20 mcg) 5 10 100 % Plermin Control 2 (40 mcg) 5 9 80 % Plermin Control 3 (60 mcg) 4 7 75 %

Ficus religiosa also known as bodhi tree, is a tree of religious importance in Hindus and Buddhists. The tree is also known for its huge medicinal usage in folklore medicine. Its leaf, bark, roots, fruits all the parts have been used in medicine since ancient times. It has been used in treatment of epilepsy, as an anticonvulsant, asthma, diarrhoea, gastric problems, haematuria, scabies etc.7 Its bark is found to have been used in treatment of bone fracture, wound healing, and has anticancer, anti-inflammatory as well as astringent property.8-9 There can be many pathologies which arise due to insufficient angiogenesis like impaired wound healing, cerebral ischemia, ulcers etc. The Ficus religiosa due its wound healing property might possess the proangiogenic property. In the current study, we tried to identify the angiogenic potential of leaves of Ficus religiosa using Chicken chorioallantoic membrane as a model due to its specificity, easy of usage and reliable model for angiogenic studies in pre-clinical trials.10 The evaluation of angiogenic property is done by using focal application method and AbGel method, using Plermin (contains BB-PDGF as an active ingredient for proangiogenic), both the method revealed insignificant angiogenic property of hot methanolic extracts of leaves of Ficus religiosa as compared to Plermin. The phytochemical studies of Ficus religiosa indicated higher amount of organic compounds then inorganic contents. The Thin Layer Chromatography indicated presence of Alkaloids, cardiac glycosides, Bitter Principles, Flavonoids, Saponins in hot methanolic leaf extracts. Which might pose the angiogenic potential in synergism when occurs in natural extracts. The IR and UV studies performed during different months of the year, of hot methanolic extracts indicated the stability of the extracts during study. The literature on the study of plant extracts on CAM model is yet not clearly understood. The synergistic effects of all the components of leaf extracts may pose a limitation to the evaluation of angiogenic property at laboratory level. Although the ethnobotanical evidence suggests that F. religiosa has angiogenic property the evaluation at preclinical remains a research question. The evaluation of F. religiosa in clinical trials will be required to prove its usability in clinical practice. The research on plant extracts for pre-clinical and clinical trials is in developing stage. Our study indicated the limitations of angiogenic potential of plant extracts in CAM model. Further studies are required with other extraction methods of various parts of F. religiosa to prove its angiogenic property. The crude extract contains all the chemical components of plant, a fractionated form of crude samples is required to further establish the specific component of plant to establish its angiogenic property.

The hot methanolic extracts of leaves of Ficus religiosa did not show angiogenic properties in the chorioallantoic membrane (CAM) model when compared to Plermin. Although the extract maintained the phytochemical properties over the course of study. The study can be further investigated by using other extraction method which may or may not show the angiogenic potential of F. religiosa on CAM model. Acknowledgment: Nil Conflicts Of Interest The authors have declared that there is no conflict of interest Funding: Nil

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