Research Articles

2018  |  Vol: 4(6)  |  Issue: 6 (November-December)  |
Comparative study of anti-angiogenic and cytotoxic activity of leaf and leaf callus silver nanoparticles of Tephrosia villosa Pers.

V. Ranjitha1, K. Kalimuthu1, Chinnadurai Vajjiram1, Y. Sharmila Juliet1, M. Saraswathy2

1Plant Tissue Culture Division, PG and Research Department of Botany, Government Arts College (Autonomous), Coimbatore -641018, India.

2Department of biological science, Vidhyasagar Women’s College of Education,     Chengalpattu, Tamilnadu, India

*Address for Corresponding Author  

Dr. K. Kalimuthu, Assistant Professor,

PG and Research Department of Botany, 

Government Arts College (Autonomous), Coimbatore ‑ 641018, Tamil Nadu, India. 


Objective: The present study aimed to synthesis silver nanoparticle (SNPs) from leaves and leaf callus of Tephrosia villosa and its anti-angiogenesis and cytotoxicity activity. Materials and methods: Antiangiogenic activity was conducted on fertilized eggs by modified in vivo CAM method and also in vitro cytotoxicity activity was studied by MTT assay at different concentration. Results: Both samples showed the higher angiogenic activity 94.5±0.5, 92.0±0.1 at the concentration 125 µg/ml. The CAM treated samples displayed distorted vascularation as well as perturbation on existing vasculature. The percentage of vessels inhibition in treated CAM was 13.5±1.5 and 12.5±1.5 at 125 µg/ml concentrations of leaf and leaf callus SNPs respectively. The in-vitro anticancer activity of leaf and leaf callus SNPs carried out against MCF-7 cell line by MTT assay showed 74.46 %, 74.35 % cell death at 250 μg/ml followed by 125 μg/ml concentrations with 70.23 and 74.35 μg/ml respectively. The IC50 value of leaf and leaf callus SNPs was 63.20 μg/ml, 12.15 μg/ml respectively. When compared the leaf SNPs, the callus SNPs activity was more in the degree of cell mortality. Conclusion: This study confirmed that both the leaf and leaf callus SNPs revealed of anti-angioenic and anticancer activity in in vitro system. For the commercial and medicinal purpose instead of using wild plant parts, we can use callus. This will reduce the pressure on wild plant collection and also conserve the plant species.

Keywords: Silver nanoparticle, angiogenesis, MTT assay and cytotoxicity

Abbreviations: SNPs: Silver nanoparticle, MTT: 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.


Cancer, as an increasingly up-to-date health problem during the last two decades, is now ranked the second cause among the 10 top leading of death around the world. Fortunately, the loss of life has been significantly inhibited by the rapid developments of different diagnostic devices and therapeutic method (Liu et al., 2012). However, among all the current clinical cancer treatments, there is none methodology can only selectively bind and target cancer cells to avoid the toxicity and unwanted side effects of the patients. A new contribution to control this problem is the emergence of nanoparticles synthesis, which opens a new interdisciplinary research area to analyze potential alternative nano-sized materials for cancer diagnosis and treatment (Mollick et al., 2014).

Silver, a noble metal commonly used for preparing jewelry over 200 years ago since discovered, has long been used as a potential anti- bacterial agent with lower toxicity and bacterial resistance properties (Loo et al., 2016; Dipankar and Murugan, 2012). Silver nanoparticles are the greatest importance to expand their biomedical applications and it focuses on describes therapeutic applications of nanoparticles based on their mechanical agents in biocompatible nanocomposites such as micellar nanoparticles; nano capsules (Panyan et al., 2003). Silver nanoparticles have been proved to have great potencial in anti-tumor activity in recent years (Chang et al., 2017; Guo et al., 2015).

The biosynthesis of silver nanoparticles using the plant Tephrosia villosa belonging to the family Fabaceae. Tephrosia villosa is widely used in traditional Indian medicine as a treatment for dropsy and diabetes (Madhusudhanaa et al., 2010). The taxon is also used as green manure in Coffee and Hevea Plantations and as a shade crop in tea plantations (Bosman and De Haas, 1983). Roots, leaves, fruits and twigs of Tephrosia villosa showed significant activity against Culex quinquefasciatus larvae (Nondo et al., 2011). Tephrosia villosa leaves showed reduction in glucose level and pancreatic cells regeneration in alloxan induced diabetes in presence of 20 (29)-lupen-3-ones a compound (Prashant and Krupadanam, 1993; Kim et al., 2001). Four new rotenoid were isolated from seeds and dehydroxyrotenoid and lupenone were isolated from whole plant. The present study aimed to synthesis silver nanoparticle (SNPs) from leaf and leaf callus of Tephrosia villosa and its anti-angiogenesis and cytotoxicity activity.

Materials and Methods

Preparation of plant extract

The powdered Tephrosia villosa leaf and callus was extracted using 100 ml of ethanol for each sample by using the Soxhlet extractor for 14 hrs (Gafner et al., 1998). The extracts was filtered through Whatmann No.1 filter paper to remove all undissolved matter including cellular materials and other constitutions that are insoluble in the extraction solvent and stored at 4° C used for further experiments.

Preparation of silver nanoparticles

Aqueous solution of 1mM AgNO₃ was prepared and used for the synthesis of silver nanoparticles. 10 ml of T. villosa ethanol extract of leaf and callus is mixed with 90ml of AgNO₃ for the synthesis of silver nanoparticles. The formation of silver nanoparticles of leaf extract was confirmed by UV-visible spectroscopy, Fourier‑transform infrared (FT-IR), Energy‑dispersive X‑ray (EDX), Scanning electron microscopic (SEM), X‑ray diffraction (XRD) (Ranjitha et al., 2018).

Anti-angiogenesis assay

Antiangiogenic activity of leaf and leaf callus nanoparticle synthesis sample of T. villosa was conducted on fertilized eggs by modified in vitro CAM assay method (Parivash Seyfi et al., 2010). Five days incubated fertile white Leghorn chicken eggs were obtained from a local hatchery and they are incubated placed in horizontal position and rotated several times at 37º C in humified 3 days. The eggs were grouped as per type of SNPS and sprayed with 70% ethanol and air-dried to reduce contamination from the egg surface. On day 6, 26- gauze needle was used to puncture a small hole in the air sac of the egg, and 2-3 mL of albumen was sucked and sealed. This allows separation of vascularized CAM from the vitelline membrane and the shell. A window was then cut in the shell using a sterile blade and shell was removed with sterile forceps, under Laminar air flow. The window is closed with a cellophane tape after capturing the photographs of the embryo. The eggs were returned to the incubator after the filter paper discs (100 micrograms of SNPS) of SNPS are placed on blood vessels of embryo using sterile forceps. After 48 h incubation on 8th day photographs of embryos were taken to obtain the image of CAM after treatment with both SNPS. At least six eggs were used for each sample dose. The percentage inhibition was calculated using the following equation.

% inhibition = {(vessel number of untreated CAM-vessel number of CAM treated  with herbal extract)/vessel number of untreated CAM} x 100.

Cytotoxicity activity

Cell line

Human, Breast Adeno Carcinoma (MCF-7) was obtained from National Centre for Cell Science (NCCS), Pune and grown in Eagles Minimum Essential Medium containing 10% fetal bovine serum (FBS). The cells were maintained at 37°C, 5% CO2, 95% air and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week.

Cell treatment procedure

The monolayer cells were detached with trypsin-ethylene di amine tetra acetic acid (EDTA) to make single cell suspensions and viable cells were counted using a hemocytometer and diluted with medium containing 5% FBS to give final density of 1x105 cells/ml. One hundred microliters per well of cell suspension were seeded into 96-well plates at plating density of 10,000 cells/well and incubated to allow for cell attachment at 37°C, 5% CO2, 95% air and 100% relative humidity. After 24 h the cells were treated with serial concentrations of the test samples. They were initially dissolved in dimethyl sulfoxide (DMSO) and an aliquot of the sample solution was diluted to twice the desired final maximum test concentration with serum free medium. Additional four serial dilutions were made to provide a total of five sample concentrations. Aliquots of 100 µl of these different sample dilutions were added to the appropriate wells already containing 100 µl of medium, resulting in the required final sample concentrations. Following sample addition, the plates were incubated for an additional 48 h at 37°C, 5% CO2, 95% air and 100% relative humidity. The medium containing without samples were served as control and triplicate was maintained for all concentrations.

MTT assay

3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) is a yellow water soluble tetrazolium salt. A mitochondrial enzyme in living cells, succinate-dehydrogenase, cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Therefore,the amount of formazan produced is directly proportional to the number of viable cells.

After 48 h of incubation, 15µl of MTT (5mg/ml) in phosphate buffered saline (PBS) was added to each well and incubated at 37°C for 4h. The medium with MTT was then flicked off and the formed formazan crystals were solubilized in 100µl of DMSO and then measured the absorbance at 570 nm using micro plate reader. The percentage cell viability was then calculated with respect to control as follows

          % Cell viability = (A) Test / (A) control x 100

        % Cell inhibition = 100 - (A) Test / (A) control x 100


Characterization of silver nanoparticles

In our previous study, we performed Characterization of silver nanoparticles through UV-visible, FTIR, EDX, XRD, SEM and get published (Ranjitha et al., 2018).

Antiangiogenic activity

Antiangiogenic activity of SNPs of leaf, leaf callus samples were tested through in vivo CAM model. The 7th day old embryo after treatment for number of blood vessels and their reduction was examined. The analysis of blood vessel was based on evaluation of angiogenesis by measuring the area of inhibition surrounding the applied disc. The inhibition percentage is shown in the table 1. Both samples showed the higher angiogenic activity 94.5±0.5 and 92.0±0.1 at the concentration 125 µg/ml. The figure 1 and 2 respects normal vascularation in the treated CAM which consists of primary secondary and tertiary micro vessels. The CAM treated samples displayed distorted vascularation as well as perturbation on existing vasculature (Figure 1 and 2). The second best angiogenic activity was observed at 250 µg/ml as 86.0 ± 0.1 and 83.5±0.5% leaf and leaf callus against control (92 ± 0.1 and 58 ± 0.1) NaOH and DMSO respectively. The percentage of vessels inhibition in treated CAM was 13.5 ± 1.5 and 12.5 ± 1.5 at 125 µg/ml concentrations of leaf and leaf callus extracts SNPs.

Figure 1. Antiangiogenesis activity of leaf SNPs of T. villosa






Figure 2. Antiangiogenesis activity of leaf callus SNPs of T. villosa





Table 1. Anti-antigenic activity of leaf and leaf callus extracts SNPs of T. villosa

S. No




No. of vessels in  treated CAM

% of vessels Inhibition

% Inhibition



Leaf SNPs








13.0 ± 01













Leaf callus   SNPs























12.5± 0.5









Three eggs used for each samples; Mean ± SD was calculated for % inhibition of each sample

Cytotoxicity activity

The in-vitro cytotoxicity activity of leaf and leaf callus SNPs carried out against MCF-7 cell line by MTT assay showed 74.46 %, 74.35 % cell death at 250 μg/ml followed by 125 μg/ml concentration with 70.23 and 74.35 μg/ml respectively (Table 2 and 3).  The IC50 value of leaf and leaf callus SNPs was 63.20 μg/ml, 12.15 μg/ml respectively as shown in the figure 3 and 4. From the results of MTT assay, it can be well predicted that the degree of cell mortality rate was directly proportional to the concentration (31.25, 62.5, 125 and 250 μg/ml) of the SNPs.

Figure 3. Cytotoxicity activity (MCF-7 cell line) of leaf SNPs of T. villosa


Figure 4. Cytotoxicity activity (MCF-7 cell line) of leaf callus SNPs of T. villosa


Table 2. Cytotoxicity activity (MCF-7 cell line) of leaf SNPs of T. villosa

S. No.


Test I

Test II

Test III


Cell Viability





























The normal MCF-7 breast cancer cell lines were spherical in shape which on treatment with SNPs, due to its cytotoxicity activity, cell growth was inhibited and eventually the cell death occurred and aggregated to form round dead cells (Figure 3 and 4). When compared the leaf SNPs the callus SNPs activity was more in the degree of cell mortality.

Table 3. Cytotoxicity activity (MCF-7 cell line) of leaf callus SNPs of T. villosa

S. No.


Test 1

Test II

Test III


Cell Viability






























Angiogenesis is essential in tumor growth and metastasis as the process provides necessary oxygen and nutrients for the growing tumor (Folkman, 1971). The present results showed that both leaf and leaf callus SNPs changed the vascularization pattern; both extracts inhibited the new blood vessels formation in the treated CAMs as well as distortion of existing vasculature.

The limitations of the available cancer management modalities create an urgent need to screen and generate novel molecules. Despite, well-documented illustrations of phytochemicals being used for prevention and treatment of cancer, their importance in modern medicine remains underestimated. Plants are the storehouse of “pre-synthesized” molecules that act as lead structure, which can be optimized for new drug development. In practice, a large number of cancer chemotherapeutic agents that are currently available in the market can be traced back to their plant resource (Heinrich et al., 2006). 

Cancer is one of the most common problems and serious health issue in this world. It has been observed that more than one in three people will develop some form of cancer in their entire lifetime. Based on the origin, there are variety of cancer exist, such as thyroid, prostate, bladder cancer, kidney cancer, pancreatic, breast cancer, melanoma, leukemia with all types, oral cancer, colon-rectal combined cancer, etc. In cancer, cells divide and grow uncontrollably, forming malignant tumors and invading nearby parts of the body. Till date a complete cure for this prevalent disease is yet to be discovered. Chemotherapy could reach all the body part including cancer cells, there may be possibility of occurrence of side effects during treatment. Biological synthesis of silver nanoparticles in nano-biotechnology area has increased its importance to create ecofriendly; cost effective, stable nanoparticles and their applications in medicines, agriculture and electronics are wider. From variety research on        nanotechnology for synthesis of silver nanoparticles it is found that it is safer and better by using natural plants. With the huge plant diversity much more plants are still not explored for the synthesis of nanoparticles and its applications in pharmaceutical and agricultural industries (Rath et al., 2014). In the present study both leaf and leaf callus SNPs showed very good inhibition percentage compare to leaf SNPs the cell inhibition percentage was more in callus SNPs. So it is conformed that both SNPs having very good cytotoxicity activity. In the present study results were compared by the earlier studies on cytotoxicity (Rath et al., 2014; Ramesh and Rajeshwari, 2015).


Based on our results it was clear that the silver nanoparticle of leaf and leaf callus of T. Villosa confirms the anti-angiogenesis and cytotoxicity properties. The confirmation of pharmacological properties of both the SNPs, establishing authenticity for the results. In addition, our results revealed that the leaf SNPs was effective in the same way as leaf callus SNPs. Due to the presence of almost similar activities of leaf callus, it can be used for medicinal purpose instead of wild plants. The various activities of SNPs suggest that it can be used as a source for new therapeutic compound development.


The author thanks the management of JSS College of Pharmacy, Ooty for necessary facilities.

Conflicts of interest: None


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