In vitro antioxidant and cytotoxic potential of different parts of Tephrosia purpurea.

The antioxidant and cytotoxic properties of four major parts of methanolic extracts of Tephrosia purpurea including leaves, root, stem and seed were investigated and compared. In vitro antioxidant activity of T. purpurea extracts was evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), reducing power assay and antihemolytic assay. In vitro cytotoxic effect of T. purpurea extracts on SW620 colorectal cancer cell line was studied using 3-(4, 5-dimethylthiazolyl -2,5-diphenyl-tetrazolium bromide (MTT) assay. Folin-ciocalteu and aluminium chloride methods were used to determine the total phenolic and flavonoid contents respectively. Among the four extracts studied, leaves extract showed the highest antioxidant activity, DPPH: 186.3 ± 14.0 μg/mL, FRAP: 754.2 ± 50.9 μmol Fe(II)/mg and reducing power activity: 65.7 ± 4.2 μg/mg of quercetin equivalent (QE/mg) and there was no significant difference observed in antihemolytic activity. Leaves extract showed effective cytotoxicity on colorectal cancer cells (IC50: 95.73 ± 9.6 μg/mL) and also had the higher total phenolic (90.5 ± 6.7 μg/mg of gallic acid equivalent (GAE/mg) and flavonoid content (21.8 ± 5.4 μg QE/mg). These results suggest higher antioxidant and cytotoxic activities of leaves extract in comparison with other extracts and these activities could be due to the presence of rich phenolic and flavonoid content.

INTRODUCTION

Human beings are often exposed to free radicals which are generated from normal metabolic processes in the body. Free radicals are not always harmful; however, high level of free radicals may disturb the normal cellular mechanism by inducing oxidative damage to DNA, protein and lipids which ultimately result in chromosomal instability and mutation(12). Moreover, overproduction of free radicals may reduce the potency of the biological defence system to detoxify these radicals which result in oxidative stress. The oxidative stress might play a role in the risk of development of several metabolic diseases like cancer, cardiovascular diseases, diabetes, obesity, neurodegenerative and aging-related disorders(34).

Antioxidant mechanism delays or prevents free radicals formation and thereby suppresses the life-threatening diseases. Besides, it can prevent cancer progression by maintaining normal cell cycle regulation, inhibiting cell proliferation and inducing apoptosis(56). Numerous plants and their active compounds are reported to possess the antioxidant activity which controls many disease conditions. Therefore, natural antioxidant compounds have been investigated for various types of treatment modalities including cancer(78). In this context, we have selected the traditional Indian medicinal plant Tephrosia purpurea for our study. T. purpurea belongs to Fabaceae family, and it is distributed throughout India, popularly known as “Sharpunkha” in Sanskrit and “Wild Indigo” in English(9). In Ayurvedic medication, different parts of this plant are used to treat various diseases like asthma, diarrhoea, jaundice, rheumatism and kidney disorders(10).

T. purpurea has also been reported to possess many pharmacological properties like anticancer, anti-inflammatory, anti-diabetic, wound healing, hepatoprotective, antiulcer, antimalarial, antimicrobial and antioxidant activities(11121314151617). Despite the existence of few studies, there is no adequate knowledge about the antioxidant and cytotoxic properties of individual major parts of T. purpurea. Therefore, in the present study, we aimed to evaluate and compare the in vitro antioxidant and cytotoxic properties of methanolic extracts of T. purpurea leaves, root, stem and seed. We have also estimated the total phenolic and flavonoid content of these extracts by using standard methods.

MATERIALS AND METHODS

Plant collection and extraction

T. purpurea was collected at flowering stage from JIPMER Campus, Pondicherry, India. The plant was authenticated and deposited in Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India with voucher No. BSI/SRC/5/23/2012-13/Tech. 368. Sequentially the collected plant was cleaned, washed and air-dried. Then, 100 g of different parts of T. purpurea such as leaves, root, stem and seed were extracted separately with methanol using soxhlet apparatus and the concentrated extracts were stored at 4 °C until further use. All the chemicals used were of molecular and analytical grades.

1,1-diphenyl-2-picrylhydrazyl radical scavenging assay

1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was used to study the ability of T. purpurea extracts to trap free radicals using previously described method with slight modifications(18). Different concentrations (50 to 500 μg/mL) of 50 μL of plant extracts was mixed with 200 μL of 0.1 mM DPPH and incubated at room temperature for 30 min in dark and the absorbance was measured at 517 nm. Ascorbic acid was used as a positive control. Conclusively, the free radical scavenging activity of extracts was calculated using the formula:

DPPH scavenging effect (%) = (control – test / control) × 100. (1)

where, control is absorbance of vehicle at 517 nm, test is absorbance of extract at 517 nm. The concentration of plant extract required to scavenge 50% of DPPH free radicals termed as IC50 value.

Ferric reducing antioxidant power

The ferric reducing antioxidant power (FRAP) assay was performed to measure the ferric tripyridyltriazine to ferrous tripyridyltriazine (Fe2+ -TPTZ) iron reduction by extracts according to a method suggested by Benzie and Strain(19). The FRAP was expressed as micromoles of ferrous equivalents, Fe(II) per mg of T. purpurea extracts (μmol Fe(II)/mg).

The reducing power assay

Reducing power of an extract correlated with antioxidant activity, is determined by the ability to reduce Fe3+ to the Fe2+ and was described by Yen and Chen(20). The reducing power of T. purpurea extracts was expressed as μg/mg of quercetin equivalent (QE/mg).

Anti-hemolytic assay

The antihemolytic activity of T. purpurea extracts was analysed using a previously reported method with some modifications(21). The red blood cells (RBC) were resuspended in phosphate-buffered saline (PBS) (20% cell suspension) and oxidative hemolysis was induced by addition of 2,2’-azo-bis(2-amidinopropane) hydrochloride (AAPH) which is a peroxyl radicals initiator. RBC suspension (0.5 mL) was mixed with 0.5 mL of 100 μg/mL of T. purpurea extracts in PBS along with 250 μL AAPH (400 mM). Further, the solution was incubated at 37° C for 1 h in a water bath, followed by centrifugation at 2500 rpm for 15 min. The supernatant was collected and the absorbance was measured at 540 nm. Gallic acid was used as a positive control and AAPH without plant extracts or positive control was expressed as 100% hemolysis. The percentage of hemolytic inhibition was calculated using the following equation:

Hemolytic inhibition (%) = (control – test/ control) × 100 (2)

where, control is the absorbance of control and test is absorbance of test (extracts or positive control).

Cell viability assay

The cytotoxic property of T. purpurea extracts was assessed on SW620 colorectal cancer cells and these cells were obtained from National Center for Cell Science, Pune, India. Cancer cells were treated with 20–200 μg/mL of leaves extract and 100–1000 μg/mL of root, stem and seed extracts. 3-(4, 5-dimethylthiazolyl)-2,5 – diphenyl – tetrazolium bromide (MTT) colorimetric assay was used to study the cytotoxicity of the extracts (22).

Determination of total phenolic content

Total phenolic content (TPC) of extracts was determined by Folin Ciocalteu's method(23). Gallic acid (20 – 100 μg/mL) was used as a standard to plot the curve and the T. purpurea extracts were expressed as μg/mg of gallic acid equivalent (GAE/mg).

Determination of total flavonoid content

The presence of total flavonoid content (TFC) in T. purpurea extracts was measured using aluminium chloride method, as stated by Zou, et al.(24). The total flavonoid content of each extract was expressed as μg/mg of quercetin equivalent (QE/mg), calculations were performed based on the calibration curve obtained from quercetin.

Statistical analysis

Numerical data obtained from the three independent experiments were expressed as mean ± standard deviation (SD). Statistically significant differences among the four groups were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni multiple comparisons test using GraphPad (Version 3.06). A P-value < 0.05 was considered statistically significant.

RESULTS

The DPPH free radical scavenging (IC50) activity of T. purpurea extracts is shown in Table 1. The leaves extract significantly (P < 0.05) quenched the DPPH free radicals at a concentration of 186.3 ± 14.0 μg/mL as compared to other extracts which had antiradical activity at the concentrations of 276.7 ± 27.5 μg/mL (root), 269.6 ± 24.5 μg/mL (stem) and 341.6 ± 36.8 μg/mL (seed). Ascorbic acid as the positive control showed the IC50 value at a concentration of 9.6 μg/mL.

Table 1

Antioxidant activity of Tephrosia purpura extracts.

Ferric reducing activity

The leaves extract showed significantly higher ferric reducing activity (754.2 ± 50.9 μmol Fe(II)/mg) followed by the stem (484.2 ± 32.3 μmol Fe(II)/mg), seed (477.6 ± 21.4 μmol Fe(II)/mg) and root (239.5 ± 13.5 μmol Fe(II)/mg) (Table 1).

Reducing power assay

The reducing power of T. purpurea extracts decreased as follows: Leaves (65.7 ± 4.2 μg QE/mg) > stem (48.1 ± 5.6 μg QE/mg) > seed (43.2 ± 3.3 μg QE/mg) > root (28.3 ± 3.0 μg QE/mg) (Table 1).

Antihemolytic activity

All four extracts showed effective antihemolytic activity on human erythrocytes against AAPH (Table 1). Both extracts and positive control (gallic acid: 93 ± 2) showed more than 90% of antihemolytic activity. Statistically significant difference was not observed between the four groups.

Cytotoxicity on colorectal cancer cells

The leaves extract showed potent (p < 0.05) cytotoxic effect against SW620 colorectal cancer cells at a concentration (IC50) of 95.73 ± 9.6 μg/mL. All other extracts showed the IC50 values > 300 μg/mL for example, 382.33 ± 18.78 μg/mL (root), 324.80 ± 21.20 μg/mL (stem) and 303.97 ± 24.31 μg/mL (seed) (Table 2; Fig. 1).

Table 2

Cytotoxic activity, phenolic and flavonoid content of Tephrosia purpurea extracts.

Fig. 1

The cytotoxic effect of (A) leaves, (B) root, (C) stem and (D) seed extracts of Tephrosia purpurea on SW620 colorectal cancer cells after exposure for 24 h. Percentage of inhibition of cancer cell proliferation was determined by MTT assay. Values are expressed as mean ± SD of three independent experiments (n = 3).

The concentration of T. purpurea extract required to inhibit 50% of the SW620 cancer cell growth was termed as IC50 value.

Total phenolic and flavonoid content

Total phenolic and flavonoid content in four methanolic extracts of T. purpurea are given in Table 2. The TPC varies from 46.8 ± 4.0 μg GAE/mg to 90.5 ± 6.7 μg GAE/mg of the different parts of the plant. Also it was higher in the leaves extract.

The leaves and the seed extracts had the maximum amount of TFC (21.8 ± 5.4 μg QE/mg and 21.4 ± 4.2 μg QE/mg, respectively), followed by root (14.3 ± 2.9 μg QE/mg) and stem extracts (12.9 ± 3.2 μg QE/mg). However, the significant difference was not found between the studied groups (Table 2).

DISCUSSION

The present study evaluated and compared the antioxidant and cytotoxic properties of methanolic extracts of four parts of T. purpurea such as leaves, root, stem and seed and also estimated their total phenolic and flavonoid content. The leaves extract showed more antioxidant and cytotoxic activities when compared to other parts.

To determine the antioxidant potential of T. purpurea extracts, DPPH, FRAP, reducing power assay and anti-hemolytic assay were carried out. These assays, in general, were used as in vitro methods to evaluate the antioxidant activity of plant extracts and compounds. The antioxidant activity indicates the capacity to quench more reactive oxygen species (ROS) leading to decreased oxidative stress. It is a well-known fact that the medicinal plants are important sources of natural antioxidants. The leaves of T. purpurea are rich in phenolic and flavonoid content and this could be the reason for the higher antioxidant activity. DPPH assay evaluated the potential of test compounds to quench DPPH radicals via hydrogen donating ability. The antioxidant agents convert DPPH into a stable diamagnetic molecule, 1-1diphenyl-2-picryl hydrazine by transferring electron or hydrogen. The colour change from purple to yellow indicates the increasing radical scavenging activity of the test compound(25). The DPPH free radical scavenging activity of four different parts was evaluated and compared. The leaves extract showed higher activity (Table 1). The DPPH radical scavenging activity of root extract is in agreement with similar results reported by a previous study by Nile, et al.(26). Both FRAP and reducing power assay are based on the reduction of Fe3+ complex to Fe2+ form. The reducing power of extracts was directly correlated with the antioxidant activity. In both the assays, leaves extract exhibited the highest antioxidant activity by reducing ferric iron to ferrous form. Previous studies also observed the leaves and root extract of T. purpurea possessed potent antioxidant activity(1727).

The oxidative stress is known to induce erythrocyte membrane lysis. The antioxidant compounds from plants are believed to inhibit RBC membrane lysis by decreasing lipid peroxidation(2829). In our study, all four extracts showed relatively similar antihemolytic activity against AAPH. This finding suggests the protective role of T. purpurea extracts towards RBCs by preventing the free radical-induced hemolysis. Also, it indicates nil or less toxicity to blood cells while it strongly protects the RBC cells from AAPH induced toxicity. Earlier this plant was used as a blood purifier and the reason for the same could be its protective role on RBC cells against hemolysis. A similar report where Thymus satureioides extract exhibited protective activity against AAPH induced hemolysis due to the presence of antioxidants(3031).

The main obstacle to current anticancer therapy is chemotherapy-induced toxicity. Whereas herbal medicines are considered safe as they produce minimal or no adverse effects over modern synthetic drugs(32). In the current study, the anticancer activity of extracts of T. purpurea on SW620 colorectal cancer cell lines was evaluated. The leaves extract exhibited high growth inhibitory activity on colorectal cancer cells at < 100 μg/mL of IC50 concentration. While the remaining three extracts showed more than 300 μg/mL of IC50 values. Gulecha, et al. reported the cytotoxic activity of T. purpurea leaves fractions on MCF7 cell lines. Hussain, et al. reported the chemopreventive potential of T. purpurea on N-nitrosodiethylamine-induced hepatocarcino-genesis by virtue of its antioxidant property(1133). These findings are in concordance with the results of the present study where we observed that all the parts of T. purpurea extract (leaves, root, stem, and seed) exhibited cytotoxicity and this effect was more pronounced with leaves extract. The cytotoxic effect of the plant extracts could be attributed to the apoptotic activity. However, further molecular mechanistic studies are required to delineate the exact role of these extracts on the anticancer and apoptotic activity.

Our study demonstrated that the amount of TPC was higher in the extracts of all the parts of T. purpurea; however, it is significantly more in leaves and seed extracts. On the other hand, statistically significant difference was not observed in the flavonoid content among the four groups. A previous study has reported the presence of total phenolic and flavonoid content in aqueous and ethanolic extracts of T. purpurea(17). The study by Nile, et al. assayed the TPC and TFC of methanolic root extract of T. purpurea and the phenolic content reported in the study is in agreement with that reported in our study though the results of flavonoids in these studies did not agree(26). Flavonoids are the important major group of polyphenols, over 4000 varieties of flavonoids have been identified. These flavonoids often accumulate in the leaves due to the biosynthesis and also in other aerial parts of plant. This could be the reason for the presence of higher phenolic and flavonoid content in the leaves extract of T. purpurea in comparison with the other parts(34). Several previous studies have clearly proven that the polyphenolic compounds are excellent antioxidants and play a crucial role in the defence against free radicals due to hydrogen donating ability or potential to scavenge oxidizing species. Also, these phenols and flavonoids are reported to act as effective anticancer agents by inducing cell cycle arrest, apoptosis and anti-angiogenic activities(353637). As reported in previous studies, our study also showed the degree of antioxidant and cytotoxic effects vary with the extent of phenolic and flavonoid content in T. purpurea extracts. However, the root extract though rich in phenolic and flavonoid content showed the least antioxidant and cytotoxic activities and we could not explain this exception. Further studies are needed to identify the active compounds responsible for the above activities.

CONCLUSION

The leaves extract of T. purpurea has higher antioxidant and cytotoxic activities when compared to the extracts of root, stem and seed. This could be attributed to the presence of rich phenolic and flavonoid content in the leaves. It may be prudent to further explore the leaves extract for its usefulness in the oxidative stress induced disorders and cancer. Currently, we are in the process of isolating from the leaves of T. purpurea the active compounds which are responsible for the antioxidant and anticancer activity.