Saraca indica Bark Extract Shows In Vitro Antioxidant, Antibreast Cancer Activity and Does Not Exhibit Toxicological Effects

Medicinal plants are used as a complementary and alternative medicine in treatment of various diseases including cancer worldwide, because of their ease of accessibility and cost effectiveness. Multicomposed mixture of compounds present in a plant extract has synergistic activity, increases the therapeutic potential many folds, compensates toxicity, and increases bioavailability. Saraca indica (family Caesalpiniaceae) is one of the most ancient sacred plants with medicinal properties, exhibiting a number of pharmacological effects. Antioxidant, antibreast cancer activity and toxicological evaluation of Saraca indica bark extract (SIE) were carried out in the present study. The results of the study indicated that this herbal preparation has antioxidant and antibreast cancer activity. Toxicological studies suggest that SIE is safer to use and may have a potential to be used as complementary and alternative medicine for breast cancer therapy.


Introduction
In recent years, large numbers of research studies are conducted, which stabilised the therapeutic use of antioxidants in treatment of various diseases such as cardiovascular diseases, diabetes, neurodegeneration, inflammation, and cancer [1][2][3]. Free radicals like hydroxyl, peroxyl, and superoxide radicals can be produced during normal metabolic function, are very transient and highly reactive, cause damage to the biomolecules, leading to adverse effects on human health, and cause severe diseases [4,5]. Several studies showed that elevated level of free radicals is associated with carcinogenesis [6][7][8][9]. ROS is a double edge sword while ROS generation is essential for cell survival, proliferation, and progression of cancer cells. In contrast increased level of ROS also induces the apoptosis and hence plays a crucial role in cancer chemotherapy [10][11][12]. Damage caused by free radicals can result in formation of single and double strand breaks of DNA and oxidation of purine and pyrimidine bases, leading to genome instability and subsequent carcinogenesis [13][14][15]. Therefore, protection of cell from oxidative damage by antioxidant supplements is very helpful in prevention and treatment of cancer [16][17][18].
Since ancient time, medicinal plants were used as key therapeutic agents all over the globe, especially among 2 Oxidative Medicine and Cellular Longevity the rural communities of developing countries due to the unavailability of an accessible and affordable primary health care system [19,20]. According to World Health Organization (WHO), 80% people across the globe used medicinal plants. A wide range of biological and pharmacological properties of medicinal plants manifest their therapeutic potential, for the treatment of various diseases [21][22][23].
Cancer is responsible for the majority of the death all over the world, out of which breast cancer is the most commonly occurring cancer in women. It is estimated that approximately 25% of all cancers diagnosed in women cause 0.52 million deaths worldwide, out of which approximately 62.13% of deaths occur in less developed regions of the world [39,40].
Surgery, radiation and chemotherapy are the standard methods for the treatment of cancer including breast cancer [41]. These therapies showed success to a varying extent to give relief from symptoms and enhance the survival time of patients; but they are also associated with severe side effects, as in case of chemotherapy drugs, they have very narrow therapeutic indexes in terms of nonselective toxic effects on normal tissues and they are also associated with many unwanted side effects such as nausea, vomiting, anaemia, loss of hair, pain in joints, lymphedema, and even the development of secondary cancers [41][42][43][44][45].
Uses of herbal medicine in the treatment of breast cancer and other types of cancers are well substantiated in the literature [46][47][48][49][50]. Hartwell (1982) described more than 3000 medicinal plants, possessing anticancer properties and subsequently used as potent anticancer drugs [51][52][53]. Medicinal plants have the ability to provide accessible, cost effective, and also a relatively safe treatment, in comparison to the standard method [43,45,50]. Although medicinal plants are considered nontoxic, a number of safety studies reported that they can cause various side effects; hence safety evaluation of medicinal plants is also required [54,55].
The present study was carried out to evaluate antioxidant and anticancer activity of Saraca indica bark extract (SIE) in breast cancer cell lines (MCF-7 and MDA-MB-231). In vivo repeated dose toxicity study was conducted to evaluate the safety of the oral administration of SIE. Results from this study will be helpful to understand the use of Saraca indica stem bark extracts in prevention and treatment of cancer as well as to evaluate any adverse effects associated with use of SIE for health benefits.

Materials and Methods
Ethics Statement. All animal procedures have been approved and prior permission from the Institutional Animal Ethical Committee was obtained as per the prescribed guidelines (IAEC Approval No. IAEC/2012/86).

Plant
Material. Saraca indica bark was collected and the sample was authenticated by Dr. K. R. Arya, Principal Scientist, Botany Division, CSIR-Central Drug Research Institute Lucknow (U.P.), India. Specimen sample of Saraca indica has been allotted a voucher sample specimen No. KRA/23998 and kept at the medicinal plant repository of the institute.

Preparation of Saraca indica Bark Extracts (SIE). The
Saraca indica Bark was dried in an oven at 40 ∘ C for 5 days and then grounded in an electric blender. The powder was suspended in 80% alcohol and left at room temperature for 24 h. The crude extract was filtered using 125 mm Whatman qualitative filter paper under sterile condition. This process was repeated 5 times and then the solvent (alcoholic extract of Saraca indica Bark), thus collected, was evaporated to dryness under reduced pressure using a rotary evaporator below 50 ∘ C. The residue was further subjected to dryness by incubating them for 8 days at 37 ∘ C. The extract was kept at 4 ∘ C until use. The yield of the extract was 9.7% (w/w).

Determination of Total Phenolic Contents in the Plant
Extracts. The concentration of phenolic compounds in SIE was determined by spectrophotometric method. Methanolic solution of the extract in the concentration of 1 mg/mL was used in the analysis. Briefly the reaction mixture was obtained by mixing 0.5 mL of methanolic solution of extract, 2.5 mL of 10% Folin-Ciocalteu's reagent dissolved in water, and 2.5 mL 7.5% NaHCO 3 . Blank was solution containing 0.5 mL methanol, 2.5 mL 10% Folin-Ciocalteu's reagent dissolved in water, and 2.5 mL of 7.5% of NaHCO 3 and absorbance was determined using spectrophotometer at max = 765 nm. Same method was used for solution of gallic acid and the calibration line was drawn. All experiment was performed in triplet. Phenolic content of extract was expressed in terms of gallic acid equivalent (mg of GA/g of extract) [56].

Nitric Oxide Free Radical Scavenging Activity.
To measure the nitric oxide free radical scavenging activity, 50 L of plant extract of different concentrations, dissolved in DMSO, was taken and then methanol was added to make the volume 150 L. 2.0 mL of sodium nitroprusside (10 mM) in phosphate buffer saline was added in each tube and they Oxidative Medicine and Cellular Longevity 3 were incubated at room temperature for 150 min. After the incubation, 5 mL of Griess reagent was added to each tube and the absorbance of chromophore formed was measured at 546 nm on spectrophotometer. Same procedure was repeated with ascorbic acid (positive control) and methanol (blank which served as control) [58,59] (1)

Lipid Peroxidation Inhibition
Activity. MDA assay was used to determine the lipid peroxidation inhibition effect of SIE as described by Baharum et al. [60]. Briefly rat liver tissue (2.0 g) was sliced and homogenized in 10 mL 15 mM KCl-Tris-HCl buffer (pH 7.2). The reaction solution (0.25 mL liver homogenate, 0.1 mL Tris-HCl buffer (pH 7.2), 0.05 mL 1 mM ascorbic acid, 0.05 mL 4 mM FeCl 2 ) and 0.05 mL of plant extract was taken in tube. The reaction tube was incubated at 37 ∘ C for 1 h. After incubation 0.5 mL 0.1 N HCl, 0.2 mL 9.8% sodium dodecyl sulfate, 0.9 mL distilled water, and 2 mL 0.6% TBA were added to each tube and vigorously shaken. Then, the tubes were placed in a boiling water bath at 100 ∘ C for 30 min. After cooling, the flocculent precipitate was removed by adding 5 mL -butanol, mixed well, and centrifuged at 9,000 rpm for 10 min. The absorbance (Abs) of the supernatant was measured at 532 nm [61]. The percentage of lipid peroxidation inhibition was measured using the following equation: Lipid peroxidation inhibition (%) = (Abs control − Abs sample) Abs control × 100%.
(2) Rats were observed for toxicity symptoms as defined in the Common Toxicity Criteria developed by the Cancer Therapy Evaluation Program with some modification if needed (National Cancer Institute, 1999, Common Toxicity Criteria Version 2.0, Cancer Therapy Evaluation Program). Their body weight changes and food and water intake were recorded on alternate days.
At the end of the study, the animals were fasted overnight, although water was made available ad libitum. They were then anesthetized using diethyl ether for necropsy and blood collection. Blood was collected in two different tubes: one tube containing the anticoagulant EDTA and one tube without anticoagulant for hematological and biochemical examination, respectively. The vital organs of animals were dissected and removed with care. Weight of each organ was taken and examined for macroscopic features.

Hematological and Biochemical Analysis of Blood.
Blood collected in EDTA coated vials was analyzed using MS-9 automatic hematology analyzer (Melet Schloesing Ltd., France), shortly after its collection. Blood samples were collected for serum chemistry analysis in tubes lacking anticoagulant and placed at room temperature for at least 90 min prior to centrifugation; after centrifugation at 1600 g for 10 min, serum was collected and biochemical parameters were measured using fully automated random access clinical chemistry analyzer (Beckman Synchron CX5, USA).  RNAse A (30 g/sample) and incubated for 30 min at room temperature in dark [68]. Samples were acquired by BD FACS Calibur flow cytometer and analysed by using a software BD FACSuite Software.

Apoptosis Analysis.
Apoptosis induced by SIE was measured by Annexin-V-FITC-PI staining using flow cytometer. MCF-7 cells (1 × 10 6 /well) were seeded in 6-well plate and allowed to grow for 24 h. Cells were treated with different concentration of SIE for 72 h. At the end of the treatment, all cells including floating cells were harvested, washed with PBS, and stained with Annexin-V-FITC and propidium iodide (Sigma-Aldrich) for 10 min at RT [68,69]. Samples were acquired by flow cytometer FACS caliber (BD biosciences).

Microscopic Analysis by Hoechst Staining.
Morphological changes in the nucleus induced by SIE treatment were studied by Hoechst 33258 staining. MCF-7 cells (2 × 10 4 /well) were seeded in 24-well plate and after 24 h of growth, cells were treated with different concentration of SIE for 72 h and cells were fixed with 4% paraformaldehyde for 10 min and then washed with PBS and permeabilised with 3% paraformaldehyde containing 0.5% triton X-100 for 30 min and then stained with Hoechst 33258 stain (Invitrogen 3 mg/mL) for 30 min and images were captured by Microscope (Leica). The extract was scanned both in positive and negative ion mode within a range of 100 to 800 m/z, where the positive ion mode showed greater ionization and sensitivity. Data profiling was recorded at a speed of 0.15 s/scan and the scanning delay of 0.01 s during analysis. The main working parameters of the mass spectrometer were (i) ion spray voltage (ISV)-5500, (ii) curtain gas (CUR)-25, and (iii) ion source gas one (GS1) and two (GS2)-10 and quadruple set on unit resolution. Data processing was performed using Analyst version 1.5 software package (SCIEX).

Chemical Analysis by Mass
2.14. Statistical Analysis. The data generated during the study was analyzed using one-way ANOVA test and the value less than 0.05 was considered to be significant.

Total Phenolic Contents.
Phenolic phytocompounds of plants show powerful free radical scavengers activity. They have potential to inhibit the lipid peroxidation by neutralizing peroxyl radicals generated during the oxidation of lipids [70]. The total phenolic content of SIE accessed using the Folin-Ciocalteu's reagent is expressed in terms of gallic acid equivalent. The values obtained for the concentration of total phenols are expressed as mg of GA/g of extract. The total phenolic content of SIE was calculated to be 55 mg GA/g.

Antioxidant Activity
3.2.1. DPPH Radical Scavenging Assay. The antioxidant activity of SIE was evaluated using the DPPH free radical scavenging method. Ascorbic acid was used as standard compound. The SIE exhibited strong antioxidant activity in the DPPH inhibition assay as evidenced by the low IC 50 values. The IC 50 value obtained was 38.5 g/mL in the DPPH inhibition assays.

Nitric
Oxide Scavenging Activity. Nitric oxide scavenging activity was performed with SIE using ascorbic acid as standard compound. In this study it was observed that SIE has ability to scavenge nitric oxide radical in dose dependent manner. The IC 50 value of SIE was found to be 29.1 g/mL in nitric oxide radical scavenging assay.

Lipid Peroxidation Inhibition Activity.
Lipid peroxidation inhibition activity was measured in vitro by determining the malondialdehyde (MDA) and related compounds in rat liver homogenate [71]. Lipid peroxidation is one of the reasons of occurrence of various diseases including cancer [72]. So, inhibition of lipid peroxidation is an indicator of therapeutic property of plant extract. The SIE exhibited lipid peroxidation inhibition activity and the IC 50 value was 66 g/mL.

General
Observations. The effects of oral administration of SIE are summarized in Table 1. The results showed that oral administration of SIE 2000 mg/Kg B.Wt. does not produce any sign of toxicity in both sex. There was no significant difference in body weight of control and treated groups in both sexes (Figures 1 and 2).

Biochemical and Hematological
Analysis. The effect of SIE on biochemical and hematological parameters was summarized in Tables 2, 3, 4, and 5. Statistical analysis of the results shows that the SIE does not produce any sign of toxicity. Biochemical parameters which include markers of hepatotoxicity (ALT, AST, ALP, and TBIL) and nephrotoxicity (CREA and BUN) indicate nontoxic effects of SIE on liver and kidney. Blood parameters were statistically similar in control and treated groups, and any shape related abnormalities in RBCs were not observed.
Macroscopic analysis of major vital organs did not show any significant change in colour texture and size when compared with the control in male and female. Reproductive organ weight does not show any significant difference between control and treated groups in case of both sexes (Tables 6 and 7).    Figure 3). In vitro safety evaluation was done in HEK-293 cells and the SIE does not induce significant cytotoxicity up to the concentration of 200 g/mL ( Table 8). Distribution of cells in different phases of cell cycle followed by SIE treatment in MCF-7 cells for 72 h, cell cycle analysis was carried out using propidium iodide (PI) staining by flow cytometry. SIE treatment arrested cells at S phase of cell cycle (Figure 4) probably by interfering with DNA replication [73]. Furthermore, morphological changes in the nucleus induced by SIE were studied with Hoechst 33258 staining, a popular nuclear counter stain that emits blue fluorescence when bound to two dsDNA, which stain nucleus of the live cells with uniform blue fluorescence while apoptotic cells had bright blue nuclei due to karyopyknosis and chromatin condensation [74]. Our results showed increase in fluorescence and chromatin condensation in MCF-7 cells followed treatment with SIE as compared to vehicle control in dose dependent manner ( Figure 5). We also confirmed if the inhibition of cell growth induced by SIE is associated with physiological apoptosis (programmed cell death) or nonspecific necrosis. We stained the MCF-7 cells with Annexin-V-FITC-PI followed by SIE treatment. 6 Oxidative Medicine and Cellular Longevity        Oxidative Medicine and Cellular Longevity   Flow cytometric data showed that SIE induces significant increase in the late apoptotic cells population and induction of apoptosis dose dependently ( Figure 6). These data indicate that SIE inhibit proliferation of MCF-7 cells by arresting cells in S-phase which ultimately induces programme cell death by apoptosis.  [26,29,75,76]. The combined result of detected compounds from the mass spectrometric analysis and literature is shown in Table 9 ( Figure 7).

Discussion
Various scientific studies show that aberrance in redox balance with elevated level of oxygen-free radicals, reactive oxygen species (ROS), and reactive nitrogen species (RNS) plays an important role in the origin and progression of most human diseases including cancer [77][78][79][80][81].
Reactive oxygen species (ROS) act as secondary messenger in intracellular signalling cascades and elevated level of ROS associated with carcinogenesis by promoting initiation, progression, and metastasis of cancer cells. It also induced DNA damage leading to genetic lesions that initiate tumorigenicity and subsequent tumor progression [8,[82][83][84]. However, many studies also suggested that free radicals are essential mediators of apoptotic pathway for triggering cell death and therefore function as anticancer agents. Thus, free Oxidative Medicine and Cellular Longevity radicals production approach is used in nonsurgical therapeutic methods for cancer therapy, including chemotherapy, radiotherapy, and photodynamic therapy [82,85,86]. Free radicals produced in cancer therapy are associated with serious side effects. Furthermore, elevated level of ROS in cancer cell leads to intercellular transfer of hydrogen peroxide (H 2 O 2 ) to neighbouring cells, and stimulates them to acquire uncontrolled ROS production [83]. Free radical scavenger activity plays a protective role in normal healthy cells. They prevent the ROS from spreading and ultimately protect the adjacent cells from oxidative DNA damage and check the cancer progression. Many clinical trials have also suggested that intake of exogenous antioxidants can protect the healthy cells from oxidative stress as well as ameliorate toxic side effects of cancer therapy without affecting therapeutic efficacy [85].
Extracts of medicinal plants have been used for the treatment of various diseases, including cancer all over the globe, as they are easily prepared, standardized, and stored. Herbal extracts are also cost effective which increase their accessibility to the patients of all economic status [87,88]. Global health policies promote the therapeutic use of herbal Control 50 g/mL 150 g/mL 75 g/mL extract. World Health Organization (WHO) also encourages the use of medicinal plants in the treatment of disease [21,89,90].
Medicinal plants used as therapeutic agents are considered nontoxic for human consumption, while many studies reported the various side effects of medicinal plant [54,55]. Medicinal plants uses for health benefit are not taken under the appropriate instruction and consultant of physician. Although people are using medicinal plants from ancient time, safety evaluation of these medicinal plants are required [49].
The modern approach to discover a new drug molecule involves either isolation from a natural source or the synthesis of a particular compound responsible for a therapeutic effect [46,91,92]. However, a complex interplay of various signalling pathways is responsible for carcinogenesis and cancer progression, which limit the efficacy of a single drug to provide a desired therapeutic result. As of now, inability of single drug to produce most effective results in breast cancer treatment enhances the future prospective of medicinal plants as complementary and alternative medicines in cancer therapy [93][94][95].
Extracts of medicinal plants are multicomposed mixtures of active components; they show their synergistic effect by acting at the same or different nodes of a cancer signalling network resulting in increase of therapeutic potential many folds, in comparison to a single drug-target therapy, and also compensate the toxicity and increased bioavailability of active compounds [96][97][98][99][100][101]. Ability to target the multiple nodes of cancer signalling network may restrict the cancerous cells to develop the resistance against medicinal plant extracts [102].
In present study, SIE showed growth inhibition in both ER positive (MCF-7) and ER negative (MDA-MB-231) breast cancer cells. SIE induced significant growth inhibition in MCF-7 cells as compared to MDA-MB-231 cells by inducing apoptosis mediated cell death. Furthermore safety evaluation was done in HEK-293 cells and CF rat. Repeated dose toxicity study was carried out to find the possible toxic effect of SIE. In this study no significant change in body weight, food and water intake, behaviour, or mortality was observed as compared with control group. No significant changes were observed in organ weight and macroscopic parameters of vital organs when compared with control group.
In present study, biochemical parameters varied widely between different dose groups and sex, but these changes were not significant and altered values fall within normal ranges. Changed biochemical parameters do not show a dose response. Liver function was evaluated by using ALT, AST, ALP, and total bilirubin level, because they are liver function markers. In this study no significant change was observed in the level of liver function markers. The serum levels of triglycerides (TG), total cholesterol (TCHO), total protein (TP), albumin (ALB), total Glucose (GLU), calcium (Ca), and inorganic phosphorus (IP) were assessed to find out the general metabolic changes. Their values show no significant change and linear profile in various groups. CRTEA, BUN, and UREA level were observed to evaluate the kidney function. They also show no significant difference and dose response [103].
All haematological parameters lie within normal range and did not show any significant changes between different dose groups and sex. These results suggested that SIE does not produce any adverse effect on blood under these experimental conditions. The present toxicological study suggests that SIE does not show signs of toxicity and safer to be used as an alternate therapeutic agent.    Mass spectrometry method was used to detect the chemical constituent of SIE. Further research studies are still required to find out the mechanism of action of specific bioactive compounds responsible for antibreast cancer activity. Phenol and other bioactive compounds present in SIE are responsible for higher radical scavenger activity. Result of the present study shows that these bioactive components could exert anticancer activity due to their antioxidant potential; as well as they are also involved in modulation of signalling pathways.

Conclusion
Together, the findings of in vitro cytotoxicity on normal cell line and in vivo repeated dose toxicity study shows that SIE does not induce significant toxicity. SIE also show a potent in vitro antioxidant and antitumor activity. Antibreast cancer, antioxidant and toxicological evaluation of Saraca indica bark extract are promising and indicate that this herbal preparation may have a potential to be used in complementary and alternative medicine for breast cancer therapy.