|Ahead of print publication
Evaluation of antiangiogenic and antiproliferative potential of ethanolic extracts of Andrographis echioides using in vitro and in ovo assays
Karthiga Muralidharan, Punnagai Kumaravelu, Darling Chellathai David
Department of Pharmacology, Sri Ramachandra Medical College, Sri Ramachandra Institute of Higher Education And Research (Deemed University), Porur, Chennai, Tamil Nadu, India
|Date of Submission||08-May-2019|
|Date of Decision||17-Jul-2019|
|Date of Acceptance||23-Oct-2019|
|Date of Web Publication||09-Oct-2020|
Department of Pharmacology, Sri Ramachandra Medical College, Sri Ramachandra Institute of Higher Education And Research (Deemed University), Porur, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Introduction: Andrographis echioides is a prevalently used medicinal herb in South Asian countries. Scientific researches with the extracts of A. echioides revealed its antipyretic, anti-inflammatory, antimicrobial, ulceroprotective, and hepatoprotective properties. This study was done to elucidate antiproliferative and antiangiogenic potential of ethanolic extracts of A. echioides (EEAE) by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and chorioallantoic membrane (CAM) assay.
Materials and Methods: EEAE was prepared using Soxhlet apparatus with ethanol after being sun-dried and powdered. MCF 7 (human invasive breast ductal carcinoma) cell lines retaining attributes of differentiated mammary epithelium with both estrogen and progesterone receptors were treated with EEAE, and antiproliferative effect was seen using Mosmann method of MTT assay using 5-fluorouracil (5-FU) as a comparator. The evaluation of antiangiogenic potential of EEAE was done by comparing mean vessel density (MVD) in chick CAM after treatment with EEAE, thalidomide, and vascular endothelial growth factor (VEGF) using CAM assay, an in ovo assay.
Results: EEAE displayed antiproliferative activity from low to high concentrations with MTT assay. The IC50 of EEAE and 5-FU was 62.5 and 15.6 μg/ml, respectively (P < 0.05). The exhibition of its antiangiogenic activity increased proportionately with increasing concentration. VEGF increased MVD by 45.94%; thalidomide decreased it by 53.76%. There was a decrease of MVD by 5.91%, 20.46%, and 35.95% at concentrations of 25, 50, and 100 μg of EEAE, respectively.
Conclusion: EEAE possessed significant antiangiogenic and antiproliferative activity, making them a promising substrate in the development of a novel anticancer drug and can be successfully used in the therapy of various cancers after establishment of the anticancer effects in animal models and subsequently in clinical trials.
Keywords: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide assay, Andrographis echioides, chick chorioallantoic membrane assay
|How to cite this URL:|
Muralidharan K, Kumaravelu P, David DC. Evaluation of antiangiogenic and antiproliferative potential of ethanolic extracts of Andrographis echioides using in vitro and in ovo assays. J Can Res Ther [Epub ahead of print] [cited 2020 Oct 31]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=297618
| > Introduction|| |
Cancer is the second foremost cause of death worldwide. It was estimated to be responsible for 9.6 million deaths in 2018. Globally, one in six deaths is accountable for cancer. The pathology of cancer includes uninhibited cell proliferation that gradually ceases to be controlled by normal regulators of cell kinetics as a result of changes at the cellular, genetic, and epigenetic levels. The key steps of carcinogenesis include initiation, promotion, and progression. Tumor angiogenesis defined as the formation of new blood vessels via a complex mechanism involving a number of stimulating and inhibiting factors on the endothelial cells remains a crucial prerequisite for carcinogenesis., The primary goal of cancer therapy is generally to cure cancer or to considerably prolong life through chemotherapy, radiotherapy, and palliative treatment. There are innumerable drugs available for the treatment of cancer and even more number of them in the pipeline waiting to be discovered. However, the toxic profile affiliated with such drugs remains an unfortunate constraint for its widespread use. Hence, the present scientific scenario has started to incline toward naturally derived compounds found abundantly in plants. The ubiquitous presence of plants and the search for new anticancer compounds such as alkaloids, flavonoids, quinones, tannins, phenols, and terpenoids in them prove to be a promising strategy., The vinca alkaloids, vincristine, and vinblastine are also plant-derived chemotherapeutic agents obtained from Madagascar periwinkle, Catharanthus roseus (L.) that is successful in the therapy of conditions such as leukemias, lymphomas, solid cancers, and sarcomas.,
The plant, Andrographis echioides L. [Figure 1] belonging to Acanthaceae family, locally called False water willow, is an annual herb present in parts of South Asia, especially South India and Sri Lanka. The phytoconstituents found abundantly in the plant are androechioidins, dihydroechoin, phenols, flavonoids, alkaloids, and tannins.,, It has been found to possess antioxidative, anti-inflammatory, antipyretic, antimicrobial, antihelminthic, antiulcerative, and diuretic properties as gathered from various studies., While conducting the literature survey about the Acanthacaeae family, it was revealed that Andrographis paniculata, sharing similar phytoconstituents and biological activity, was better studied and found to be a prospective candidate with respect to anticancer effect, unlike A. echioides. Owing to the bitter taste and ability to impair liver functions by A. paniculata, the compliance rate of the same has become low in spite of various therapeutic properties.,,, Furthermore, the flavonoids and glucosides are well known to inhibit cell proliferation by apoptosis induction, DNA fragmentation, nuclear condensation, cell shrinkage, and ultimately cell death.,, It is also said to hinder angiogenesis by blocking formation of angiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hypoxia-inducible factor 1. Hence, we wanted to explore the antiproliferative attribute of ethanolic extract of whole plant of A. echioides in vitro using human breast adenocarcinoma cancer cell lines (MCF 7) and compared it with 5-fluorouracil (5-FU), a standard anticancer drug that acts by inhibiting thymidylate synthase (TS), the rate-limiting enzyme in the pyrimidine nucleotide synthesis. The antiangiogenic potential was also explored using chick chorioallantoic membrane (CAM) assay, an in ovo method.
| > Materials and Methods|| |
Plant material and its extraction
The whole plant of A. echioides was obtained from Kumaragiri Hills, Salem, Tamil Nadu, and was authenticated by Prof. Sasikala, Research Officer and Botanist, and a specimen voucher (dated 17.01.2014) was deposited in the Pharmacognosy Museum of Siddha Institute of Research, Chennai. After taking the whole plant, it was cut into pieces, dried in air, and was pulverized. Later, 25 g of powdered sample was taken and extracted with 300 ml of ethanol using Soxhlet apparatus for 12 h of time. Then, the crude extract was posed to filtration and the solvents were condensed with rotary evaporator followed by storage of the extract in an airtight container at room temperature till further analysis.
The reagents Dulbecco modified Eagle medium (DMEM), thalidomide, and VEGF were purchased from Sigma Aldrich, India.
Cell lines and culture
Human breast cancer cell line (MCF 7) was bought from National Centre for Cell Sciences, Pune. The cells were kept in DMEM to which 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml) was added and maintained at a humidified atmosphere of 50 μg/ml CO2 at a temperature of 37°C.
Evaluation of antiproliferative effect using in vitro 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide assay
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, anin vitro method to measure the nonviable cells in a given population, is based on the principle that mitochondrial dehydrogenase enzyme of the viable cells reduces the yellow-colored tetrazolium dye (MTT) into insoluble formazan crystals that are purple. By supplementing detergent, the crystals are solubilized and the color of the treatment and control group can be differentiated using a spectrophotometer, and percentage killing can be calculated. This is a sensitive method for screening cytotoxicity of test samples.
To perform the MTT assay, the cells were plated in 24-well plates with each well containing 1 × 105/well). The plates were incubated at 37°C with 5% CO2. Subsequently, the extracted samples were added at various concentrations once cells start reaching the confluence followed by incubation for 24 h. After removing the cells from the well, they were washed with phosphate-buffered saline (pH 7.4) or DMEM without serum. Later, 100 μl (5 mg/ml) of 0.5% MTT was added to each well and further incubated for 4 h. Later, DMSO (1 ml) was added to all the wells. The absorbance at 570 nm was determined with ultraviolet spectrophotometer using DMSO as blank. The percentage cell viability is calculated using the formula.
% Cell viability = (Average A570 of control cells – Average A570 treated cells)/A570 of control cells) ×100
IC50, defined as the concentration of drug that produces 50% inhibition, is calculated after plotting the graph with the concentration of the sample at X-axis and % cell viability at Y-axis. The point at which there is 50% cell viability is the IC50 value.
This procedure was done in a triplicate manner to maintain accuracy and reliability of measurement of cell viability. A similar procedure was followed for the control drug, i.e., 5-FU. The extracts and control were screened at the concentrations of 1000, 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 μg/ml for theirin vitro anticancer activity.
Evaluation of antiangiogenic effect of Andrographis echioides extracts using in ovo chick chorioallantoic membrane assay
The chicken CAM, being a highly vascularized extra-embryonic structure, is used nowadays as a promising preclinicalin vivo model to study blood vessels in scientific researches owing to its simpler structure, easier accessibility, and ability to be studied after being treated with various agonists and antagonists of angiogenesis., The fertilized eggs for CAM assay were procured from Government Poultry Research Station, Nandanam, Chennai, Tamil Nadu, and were weighed and sterilized with ethanol. They were incubated at 37°C in 85% humidity for 2 days. A hole was drilled carefully over the air sac using a nipper without breaking the shell after marking it with a pen for easy identification of vascular zone on chick CAM. To moisten the inner shell membrane next to CAM so as to make it easily separable, two drops of 0.9% sodium chloride was added. A window of size 1 cm × 1 cm was made on the membrane after clamping and raising it using an ophthalmic forceps for the exposure of the vascular zone. Sample concentrations and controls were loaded on a 5 mm × 5 mm sterilized disks that acted as a carrier. It was then stuck to the vascular zone with sterile flexible packing film. The eggs were then placed in an incubator for 9 days. The disc area of CAM was observed under light photomicroscopy on the12th day. The analysis of the digital images in the outlined area of the disc was done using Aphelion imaging software [Figure 2]. The concentrations of ethanolic extracts of A. echioides (EEAE) used were 25, 50, and 100 μg in each egg and the positive control egg had 50 ng VEGF and negative control had 10 μg of thalidomide. The concentrations of the extracts used were derived from toxicity studies done with the plant by Basu et al. in accordance with OECD 423 guidelines. Each experiment was done in three eggs to ensure reproducibility and the mean blood vessel density was calculated in terms of mm2 area.
|Figure 2: Procedure of chick chorioallantoic membrane assay. (a) Sterilized eggs, (b) Made a 1 cm × 1 cm window. (c) Samples were loaded on sterile disc. (d) Loaded samples were placed on the membrane. (e) Treated eggs were sealed|
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The percentage cell viability at each concentration was expressed as mean ± standard deviation. ANOVA method was used to compare the mean values, and a P < 0.05 was considered statistically significant.
| > Results|| |
Antiproliferative effect of ethanolic extracts of Andrographis echioides in 3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-tetrazolium bromide assay
The extracts of A. echioides were tested for antiproliferative effect in MCF 7 cell lines at concentrations of 1000, 500, 250, 125, 62.5, 31.2, 15.6, and 7.8 μg/ml. It was found that it exhibited antiproliferative activity starting from low to high concentration with IC50 found at 62.5 μg/ml [Table 1] and [Figure 3]. Meanwhile, IC50 of 5-FU, a standard anticancer drug studied in same cell lines, was found at 15.6 μg/ml, thereby implying the extracts though possessed antiproliferative potential was less compared to 5-FU [Table 2] and [Figure 4]. Moreover, the P value calculated by ANOVA method was 0.00016 and 0.0001 for EEAE and 5-FU, respectively, depicting the results are statistically significant [Figure 5].
Table 1: Antiproliferative effect of ethanolic extracts of Andrographis echioides in breast cancer (MCF 7) cell line using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide assay
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|Figure 3: Antiproliferative effect of various concentrations of ethanolic extract of Andrographis echioides on MCF 7 cell lines. (a) Normal MCF 7 cell line. (b) Antiproliferative effect at 1000 μg/ml. (c) Antiproliferative effect at 62.5 μg/ml. (d) Antiproliferative effect at 7.8 μg/ml|
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|Table 2: Antiproliferative effect of 5-fluorouracil in breast cancer (MCF 7) cell line using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide assay|
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|Figure 4: Antiproliferative effect of various concentrations of 5-fluorouracil on MCF 7 Cell lines. (a) Normal MCF 7 cell line. (b) Antiproliferative effect at 1000 μg/ml. (c) Antiproliferative effect at 15.6 μg/ml. (d) Antiproliferative effect at 7.8 μg/ml|
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|Figure 5: Graphical representation comparing the antiproliferative effects of ethanolic extract of Andrographis echioides and 5-fluorouracil on MCF 7 cell lines in 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazolium bromide assay with concentration of both test and control on X axis and percentage viability on Y axis. IC50of the samples is defined as the concentration at which there is 50% cell viability of cells|
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Antiangiogenic effect of ethanolic extracts of Andrographis echioides in chick chorioallantoic membrane assay
There was an ample growth of new blood vessels observed on the CAM treated with VEGF on the 12th day of incubation. Furthermore, a higher vascular density with a mean vessel density (MVD) of 91.70 mm2 with an increase of 45.94% was found with CAM treated with VEGF. Meanwhile, the observed MVD was 59.12, 49.97, and 40.24 mm2 with CAM treated with EEAE at concentrations of 25, 50, and 100 μg corresponding to a decrease in MVD by 5.91%, 20.46%, and 35.95%, respectively. The CAM that was treated with thalidomide 10 μg showed MVD of 29.05 mm2 and a decrease by 53.76%. From the above findings, we can infer that EEAE possessed antiangiogenic effect that increased proportionately with increasing concentration [Table 3] and [Figure 6] and [Figure 7].
|Table 3: Antiangiogenic effect by ethanolic extracts of Andrographis echioides in comparison with vascular endothelial growth factor and thalidomide in the chick chorioallantoic membrane assay|
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|Figure 6: Chorioallantoic membrane of a 12-day-old chick embryo incubated for 4 days with a coverslip showing (a) normal, (b) vascular endothelial growth factor (50 ng), (c-e) ethanolic extract of Andrographis echioides (25, 50, and 100 μg, respectively), (f) thalidomide (10 μg)|
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|Figure 7: Graphical representation of antiangiogenic response of the ethanolic extract of Andrographis echioides (25, 50, and 100 μg/egg), vascular endothelial growth factor (50 ng), normal and thalidomide (10 μg) in the chorioallantoic membrane assay on the 12th-day incubation. The concentration of the samples was plotted in X axis and mean blood vessel density (mm2) was plotted on Y axis|
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| > Discussion|| |
The primary requisites for any lead compound to be developed as an anticancer drug include ability to induce apoptosis, inhibit secretion of protein kinases and matrix metalloproteinases (MMPs), and stop tumor cell adhesion, invasion, and angiogenesis. Among all angiogeneses is one of the cardinal aspects responsible for metastases of tumor cells., Hence, products inhibiting angiogenesis combined with other classes of chemotherapeutic drugs have been the recent interest of the study of pharmacotherapy of various cancers. The extracts of the whole plant of A. echioides studied here were characterized by the presence of mainly flavonoids such as dihydroechioidinin, flavones – echioidinin, echioidin, a chalcone glucoside, androechin and terpenoids, tannins, and sterols. The presence of such remarkable phytoconstituents in the plant of A. echioides already proved to possess antioxidant, antiulcer, and hepatoprotective properties.,,, A study was also done to manifest its analgesic, anti-inflammatory, and antipyretic effect, and it was proved to be as efficacious as paracetamol and phenylbutazone. It also proved its antimicrobial potential against Gram-negative and Gram-positive bacteria and fungi as well.,,, The ethanol extract from the leaves of A. echioides showed larvicidal activity against various blood-sucking parasites. Although it was widely studied for its antioxidant and antimicrobial activities, there was not much research done to evaluate its anticancer property. Elaiyaraja and Chandramohan showed cytotoxic effect of A. echioides in oral cancer (KB cell line) whose IC50 was found at 68.5 μg/ml. When antiproliferative effect of methanolic extracts of A. echioides was studied in skin melanoma cell lines by Guroji et al., they observed the IC50 was 270 μg/mL. De Yang Sheen et al. concluded that it is flavonoids that constitute the major phytoconstituent of A. echioides and because of which it exhibited an appreciable antioxidant activity.
The antiproliferative activity of ethanolic extracts of A. echioides was investigated on human breast adenocarcinoma cancer cell lines (MCF 7) using MTT assay and was compared with 5-FU in this study. The cells with different concentrations of the extracts ranging from 7.8 to 1000 μg/ml were incubated, and the percentage of cell viability was measured. The observed IC50 of the extracts was at a concentration of 62.5 μg/ml, indicating that EEAE can induce noteworthy and dose-dependent antiproliferative activity against MCF 7 cell lines. AlSalhi et al. demonstrated inhibition of proliferation of human breast adenocarcinoma cancer cell line (MCF 7) by silver nanoparticles of A. echioides with IC50 at 31.5 μg/mL. The reason for the IC50 to be lower than IC50 obtained from our study can be explained by the fact that perusal of nanoparticle-mediated delivery of the extract has advantages such as high stability (i. e., long shelf life); high carrier capacity (i.e., many drug molecules can be incorporated in the particle-matrix); and feasibility of incorporation of both hydrophilic and hydrophobic substances. The measured IC50 of any presumable agent must always be low enough to produce more therapeutic effects than on specific side effects. Going by the guidelines laid by The American National Cancer Institute that mentions if a prospective anticancer product exhibits significant antiproliferative effect with value IC50≤30 μg/ml, it can be proceeded to further bio guided research for the development of a new anticancer drug. The antiproliferative activity demonstrated by EEAE in this study could be due to the presence of active products, especially flavonoids in the ethanolic extract. Few of the various mechanisms postulated for the potential anticarcinogenic effects of flavonoids are their ability to inhibit protein kinases, especially the mitogen-associated protein kinases (MAPK) which inhibits signal transduction of cell proliferation, to inhibit prooxidant enzymes responsible for generation of secondary messengers in several pathways that lead to increase in cell proliferation, resistance to apoptosis, and activation of proto-oncogenes such as cFOS, cJUN, and cMyc and by activating caspases through Bax/Bcl-2 pathway they cause apoptosis of cancer cells.
There is an array of pro-angiogenic factors hypothesized to play a key role in the process of angiogenesis, namely VEGF and its receptor VEGFR, MMPs, tissue inhibitors of metalloproteinases, and bFGF participating in every step of the angiogenesis and vasculogenesis. They also regulate tumor-related angiogenesis required for tumor invasion and metastases. VEGF is believed to be the major component of angiogenesis because of its ability to initiate the endothelial cells to form sprouts and hence formation of new blood vessels., Therefore, drugs that are intended to breach the cascade of pathological angiogenesis target mainly VEGF and its receptors are postulated to be ideal antiangiogenic drugs used in cancer chemotherapy. Various antiangiogenic drugs including monoclonal antibodies in use are notorious in causing varied and severe side effects necessitating the need for efficacious and safer compounds.
In this study on chick CAM, the blood vessels showed a well-structured branching pattern. The MVD (mm2) measured with ethanolic extracts of A. echioides at the concentrations of 25, 50, and 100 μg were 59.12, 49.17, and 40.24, respectively. While MVD of CAM treated with VEGF (50 ng) was 91.70 mm2, it was 29.05 mm2 for thalidomide (10 μg), a known antiangiogenic drug. Dai et al. showed A. paniculata belonging to the same family as A. echioides inhibited angiogenesis in ovo using CAM assay and possible mechanism mentioned could be blocking of Mir-21-5p/TIMP3 signaling pathway an important pathway of angiogenesis., The dose-dependent exhibition of antiangiogenic activity by EAEE could also be by a similar mechanism affecting VEGF signaling pathway that has to be explored in further studies.
Large-scale screening programs inclusive of newer scientific strategies are usually preferred for the detection of biological activity of various natural products. Extracts from plants that are used in field of allopathy are considered to be cardinal sources of biologically active compounds. It is also postulated that screening procedures for various crude extracts rather than pure compounds that are excerpted from natural products turn out to be more successful in its initial steps.
Limitations of the study include this study being only the primary step, investigation into characterization and mechanism of antiangiogenic and antiproliferative activity of A. echioides has to be entailed in further research works. Furthermore, the testing of the antiproliferative and antiangiogenic potential of extracts of A. echioides needs to be carried on in animals using variousin vivo models to confirm its efficacy and potency.
| > Conclusion|| |
This study elucidated the antiproliferative and antiangiogenic activity of the EEAEin vitro and in ovo using MTT assay and chick CAM assay, respectively. More detailed analysis is required to comprehend the phytochemical compounds and to appreciate the molecular mechanisms involved in cancer cell inhibition.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3]