In Vitro Anti Angiogenetic Activity of Traditional Plants Saraca asoca, Glycyrrhizia glabra and Curcuma longa Beneficial for Endometriosis Treatment

Plant materials: The bark of S. asoca (Fabaceae) and rhizome of C. longa (Zingiberaceae) were obtained from the Ayurvedic Research Centre (ARI), Poojappura, Kerala and roots of G. glabra (Fabaceae) from Foundation for Revitalisation of Local Health Traditions (FRLHT), Bangalore respectively. All the plant materials were washed thoroughly with distilled water for removing dust particles. All these plant materials were oven dried at 40° for 1 w and then powdered using a mixer grinder and stored in the dark at room temperature for experiments. Preparation of plant extract: Powdered plant materials of 24 g were used for extraction by the Soxhlet method in 250 ml of ethanol and concentrated under reduced pressure with rotavapor (Buchi Labor Technik AG, Flawil, Switzerland) R-210 set at 40°. All crude extracts obtained were stored at 4° until use. Total phenol: Folin-Ciocalteu (FC) method with minor modifications was used to determine the Total Phenolic Content (TPC) of different extracts using gallic acid as standard [15] . The FC reagent was mixed with test extracts, mixed thoroughly and incubated in the dark for 5 min. 20 % sodium carbonate solution was added to the solution and incubated for 30 min at room temperature. The absorbance was measured at 760 nm using a 1700 Shimadzu Ultraviolet (UV)-visible spectrophotometer. The calibration curve was prepared by employing gallic acid at a concentration of 10-60 µg/ml. The TPC was determined by using a linear regression equation from the standard gallic acid graph plot and was expressed as Gallic Acid Equivalent (GAE) per gram of the sample. The average of the triplicates was analyzed. Total flavonoid: Aluminum chloride (AlCl 3) method was used to determine the Total Flavonoid Content (TFC) using quercetin as standard [16,17] . Briefly, the test extract was mixed with 0.1 ml AlCl 3 and 0.1 ml Potassium acetate (CH 3CO2K), incubate at 30° for 10 min. The absorbance was measured at 415 nm and TFC was expressed as quercetin per gram (Quality Assurance Evaluator (QAE)) of the sample. The average of triplicates was analyzed. Chorio-Allantoic Membrane (CAM) assay: The chicken CAM is a highly vascularized embryonic membrane that offers clear advantages to studying vascular functions. The anti- angiogenic activity was evaluated using the CAM model according to the previously reported

with minor modifications [18] . Fertilized eggs were purchased from Kerala State Poultry Development Corporation (KEPCO), Kerala. The development of the embryo was ensured with the help of an egg candler. Eggs were carefully surface sterilized with 70 % isopropanol and incubated at 37° with 80 % relative humidity. After then the eggs were randomly divided to carry out the experiment. Positive and negative controls used were Dienogest (50 µg) and Phosphate-Buffered Saline (PBS) respectively. S. asoca , G. glabra , and C. longa ethanolic extracts (250 µg) were the test groups. www.ijpsonline.com Indian Journal of Pharmaceutical Sciences 478 March-April 2024 Drug administration: On the 5 th d of incubation, control and test extracts of 20 µl were inoculated into the CAM of fertilized eggs and sealed with parafilm. Tilted the egg gently and all eggs were kept in a vertical position with air sack upwards and were incubated not touching the eggshell. Drabkin’s reagent test: Determination of Hemoglobin (Hb) in the CAM was performed as explained by Drabkin and Austin with slight modification [19] . On the 12 th d of incubation, the eggs were broken gently from the side of the air sac and the inner content was removed without disturbing the membrane. The CAM was scrapped and dispersed carefully into a tube and centrifuged at 3000 rpm for 5 min. 20 µl of supernatant was added to 5 ml of Drabkin’s reagent. The reaction was incubated at room temperature (37°) for 5 min. Hb level was measured at 546 nm in a spectrophotometer. The level of Hb in the sample is compared to a standard and results are expressed as anti-angiogenesis percentage. All the tests were performed in triplicates to ensure the reproducibility of the result. The percentage of inhibition of angiogenesis was calculated by the following equation: % Anti-angiogenisis=Optical Density (OD)/OD sample×100

AND DISCUSSION Being common ingredients in many Ayurvedic medicines, S. asoca , G. glabra , and C. longa have already demonstrated potent health benefits to humans. All the extracts were rich in total phenolic and flavonoid compounds. TPC and TFC

are simple, inexpensive and quick in estimation. The content of phenol was expressed in terms of GAE (standard curve equation: Y=0.0083X–0.0027, R 2= 0.9999) (fig. 1). It was observed from the analysis that the bark of S. asoca showed the highest phenolic content mg GAE/g of 483.5+0.001, G. glabra has 336+0.016 and C. longa has 164.14+0.002 in the sample (Table 1). The result of the present investigation indicates that flavonoid content was abundantly seen in C. longa when compared to G. glabra , and S. asoca . The content of flavonoid was expressed in terms of standard quercetin (Y=0.0237X–0.2081, R2=0.9895) (fig. 2). Rhizome of C. longa exhibited the highest flavonoid content of (873+0.0186) mg GAE/g than roots of G. glabra 106.56+0.0267 and bark of S. asoca 49.81+0.028 equivalence (Table 1). All data are presented as mean+Standard Deviation (SD). Fig. 1: Standard graph of gallic acid TPC (mg GAE/g) TFC (mg QE/g) S. asoca 483.5±0.001 873±0.0186 G. glabra 36±0.016 106.56±0.0267 C. longa 164.14±0.002 49.81±0.028 TABLE 1: ESTIMATION OF PHENOL AND FLAVONOID March-April 2024Indian Journal of Pharmaceutical Sciences 479 www.ijpsonline.com the impact of these plant extracts with reported Selective Estrogen Receptor Modulator (SERM) activity on the treatment of endometriosis can also be thoroughly investigated. The chick embryo CAM model was used to assess anti-angiogenesis activity. In this study, we have employed the same reliable method to study the anti-angiogenic effect of the selected plant extracts. The fertilized eggs were treated with ethanolic extract of S. asoca , G. glabra , and C. longa . The anti-angiogenic potentials of the extract were evaluated on the 12 th d of treatment. The in vitro quantitative determination of Hb in blood was expressed based on the cyanmethemoglobin method. The intensity of the color is proportional to the Hb concentration and is compared to known cyanmethemoglobin standard equivalence at 546 nm (standard curve equation: Y=0.0225X–0.0024, R 2=0.9995) (fig. 3). From the graph, the g per decilitre (dl) concentration of control and test extracts was calculated. It is clear and evident that all the extracts show effective anti-angiogenic activity in CAM assay with values (Hb/dl) of 2 for S. asoca , 3.07 for G. glabra , 2.49 for C. longa and extract combination shows 2.7 with respect to control of 7.92. The standard drug Dienogest shows an effect of 2.91 in the assay. The inhibitory potential of extracts was expressed as a percentage anti-angiogenesis. The study reveals that S. asoca showed the highest inhibition percentage of 73.65+0.021 followed by C. longa with 67.65+0.024 and G. glabra with 60.43+0.04 in the sample (Table 2 and fig. 4). The ethanolic extracts inhibited the growth of blood vessels on CAM were photographed and can be seen in fig. 5A-fig. 5E. The rich number of natural flavonoids and phenol contents in plants have positive effects on human health as antibacterial, antiviral and anti-inflammatory agents [20] . They may differ in concentration during their growth stages and are considered to have a very vital role in disease management. The potential benefits of these secondary metabolites derived from medicinal plants may act through multiple cell signaling pathways and may exert their potential effects by healing diseased cells without affecting normal cells [21] . S. asoca bark extract is a rich source of many polyphenols such as catechin groups and the estimation of total phenol and flavonoids are also in agreement with the previous reports demonstrating the plant with a variety of pharmacological properties [22] . Epicatechin is one of the primary components of this plant. Since-epicatechin strongly inhibits the Na +/H+ exchanger, it has been hypothesized that this can alter the fluidity of the cytosolic plasma membrane and inhibits the proliferation of cancer cells [23] . It was also reported that licorice also exhibits anti- inflammatory, antiviral, gastro protective and strong estrogen-like activities due to its wide range of flavonoid and phenolic content in the plant [24] . Significant flavonoid and phenolic content in C. longa also result in different medicinal properties like anti-bacterial, anti-inflammatory, anti- carcinogenic and anti-proliferative properties [25] . Although there is little evidence to support the use of estrogen-progestins for endometriosis, the European Society of Human Reproduction and Embryology (ESHRE) guidelines on endometriosis also noted that combined hormonal contraception is frequently used as a treatment for pain associated with endometriosis [26] . Therefore, Fig. 2: Standard graph of quercetin www.ijpsonline.com Indian Journal of Pharmaceutical Sciences 480 March-April 2024 Fig. 3: Standard graph of hemoglobin Fig. 4: Anti-angiogenic activity of selected plant extract Fig. 5: Inhibitory effect on angiogenesis and captured digital image of the CAM section Drugs Dose µg/ml Tested eggs % Anti-angiogenesis PBS (Negative control) - 3 - S. asoca 250 3 (73.65±0.021) % G. glabra 250 3 (60.43±0.04) % C. longa 250 3 (67.65±0.024) % Dienogest 50 3 (62.42±0.02) % TABLE 2: ANTI-ANGIOGENIC EFFECT OF EXTRACTS G. glabra Dienogest March-April 2024Indian Journal of Pharmaceutical Sciences 481 www.ijpsonline.com emphasize the beneficial use of conventional drugs in endometriosis therapy. Further research on the mechanism of action of the extracts has to be worked out in future studies to substantiate these findings on the basis of lower concentrations. The involvement of angiogenesis in preventing the growth of endometriosis has been indicated by several researches. However, many of them only provide circumstantial evidence, frequently based on the downregulation of anti-inflammatory factors like VEGF levels or Matrix Metalloproteinase-9 (MMP-9) activity [34] . According to preclinical investigations, the antiangiogenic treatment may have the largest initial impact on micro metastases and in general, smaller dosages of an angiogenesis inhibitor are needed to prevent neovascularization of microscopic metastases than to regress a primary tumour [35] . Our present study reveals that medicinal plants like S. asoca , G. glabra and C. longa with their major phenolic and flavonoid compounds may have a significant role in suppressing angiogenesis. It may be presumed that the collective contribution of phenol and flavonoid compounds in the extract provides anti-angiogenic properties. The study highlighted the activity of crude extract through CAM assay. This technique was well accepted and does not need so much of financial and technical support to perform. Although many aspects are relevant to the pathophysiologic process of endometriosis, it is clear that angiogenesis is necessary for the development and maintenance of ectopic implants in the tissue. We have investigated the antiangiogenic effect through a simple method and the analyzed plant extracts seem to be a promising therapeutic source for endometriosis treatment. Further, in vivo studies and gene expression studies are needed to investigate the role of these phyto metabolites for their molecular level of action.

We are gratefully acknowledging the facilities provided by JNTBGRI, Palode and CRDC, HLL Lifecare Limited, Trivandrum for supporting the study. This work was financially supported by ICMR for an SRF fellowship to Reshmi Nair. Conflict of interest: The authors declared no conflict of interests. Angiogenesis can be defined as the formation of new blood vessels from pre-existing vascular tissue [27] . Onishi et al. [28] have reported that the pathogenesis of endometriotic lesions includes major steps like blood vessel breakdown, membrane degradation, surrounding extracellular matrix and new blood vessel formation leading to angiogenesis. In these aspects, the long-term survival and proliferation of endometriotic lesions are critically reliant upon adequate blood supply via angiogenesis [29] . The goal of an anti-angiogenic therapy strategy for endometriosis illness is to successfully induce conception in infertile individuals while simultaneously relieving pelvic pain. Endometriotic lesions have the ability to create cytokines and growth factors that regulates their vascularization and proliferation [30] . It was also reported that the neovascularization of endometriotic lesions is significantly aided by the dominant Interleukin (IL)-1 released by active peritoneal macrophages, IL-1 [31] . Estradiol promotes endometrial Vascular Endothelial Cell Growth Factor (VEGF) production, and its levels are associated with neovascularization and increased vascular permeability during the late proliferative phase [32] . As endometriosis condition is heavily reliant on estrogen, hormonal therapies aim to reduce endogenous ovarian estrogen production The CAM method provides a simple and rapid evaluation of the anti-angiogenic effect of the extracts in well-developed vascular tissue [33] . Paradkar et al. [21] have described it as an efficient and widely used model for determining anti or pro-angiogenic activity in a sample. CAM assay model is an informative method used to study the process of endometriotic lesion formation in order to show that a viable endometrium is necessary to form an endometriotic lesion and the results indicate that S. asoca and C. longa extracts have strong anti-angiogenic activity than positive control dienogest, a potent drug currently used in endometriosis treatment. Thus, the studies are promising and clinically relevant for the therapy. The relevant amount of flavonoids and phenolic compounds in all the studied plants may be responsible for the bioactivity of the crude extracts and serve as an inhibitory agent for angiogenesis. Since the results show a proven anti-angiogenic effect in CAM, these extracts www.ijpsonline.com Indian Journal of Pharmaceutical Sciences 482 March-April 2024 activities of buckwheat ( Fagopyrum esculentum Moench) hulls and flour. J Ethnopharmacol 2000;72(1-2):35-42. 18. 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