Cytotoxicity and Mitochondrial-Mediated Apoptosis Induced by Ethanolic Leaf Extract of Barleria lupulina Lindl. in Human Leukemia Cells Via Reactive Oxygen Species Generation

Background: Barleria lupulina Lindl. (Hop-headed) is a small shrub, possess potent anti-inflammatory, analgesic, anti-leukemic, antitumor, anti-hyperglycemic, anti-amoebic, virucidal, diuretic, bactericidal and antibiotic properties. Methods: Cytotoxicity, bioactive assay and genetic analysis of B. lupulina were investigated in the present communication. The leaf extract was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Neutral red uptake (NRU), DNA fragment, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP) assay, gene expression analysis and cDNA synthesis to evaluate anti-cancerous potency using cancerous THP-1 cell lines in vitro and in vivo. Results: HPTLC analysis reveals four spots and GC-MS analysis displayed the presence of eleven bioactive compounds among which benzofuranon, hexadecanoic acid, ethyl 9,12,15-octadecatrienoate, and 3,7,11,15-tetramethyl-2-hexadecanoic acid were the most prominent compounds. The ethanolic extract showed significant cytotoxicity (P<0.5) against THP-1 cell line at a concentration of 1mg/mL. The cells were also observed for apoptosis through DNA fragmentation in B. lupulina treated cells. Conclusions: It can be concluded that if the dose range was further refined within the range of 100-1000 µg/mL there could be dose at which the entire population of the THP-1 cell line would be apoptosis induced. The extract induced ROS in the cells after 30 minutes of exposure displaying cytotoxic effects and DNA fragmentation assay.


Background
Oncogenes stimulated the uncontrolled growth of cells resulting in tumor that is the causing cancer leading the death of the sufferers. About one-half of all men and one-third of all women in the world develop cancer. Now a days, millions of people are living with cancer or have cancer. It is quite dangerous for all people and an easy task to treat the ailment. Using herbals for the treatment of malignancies is common in many cultures especially in India, because some herbal products contain abundant anti-cancerous compound. In addition, useful compounds from these herbals are being used in production of various modern drugs. Herbal medicines constitute a major substitute for cancer prevention and treatment around the globe. The effect of plant extracts as anticancerous was widely studied owing to their low toxicity and side effects [1]. Hence, such studies investigating medicinal herbs have been steadily held with interests.  [4,5,6], Anti-inflammatory [2,7], Analgesic, Antiulcerogenic [7], Antidiabetic [8], Neuropharmacological [9], Toothache [10], Antibacterial [11], Anticancer [12], Antiartheritis, Acute and sub-chronic diuretic [13], Anti-viral [14]. However, very little is known regarding the molecular mechanisms by which they may exert their  [12] and trypan blue exclusion dye method as per Jayashree and Thenmozhi [16].

DNA fragment assay
The cells were cultured (1×10 5 cells/mL) in the 6-well microtitre plate. After incubation for 24h, different concentrations of extracts were treated followed by pipetting out the confluent cells in the plate without scratching. Lysing buffer was added once about 100 µL for extracting the cells through scratching. Same step was followed again and collected in labeled Eppendroffs. Then 2µL RNAse was added in each Eppendroffs tube and kept for incubation for 1 hr. Then 2µL proteinase K was added and the Eppendroff incubated for 1 hr. Buffered phenol/chloroform/isomyl propanol was added followed by centrifugation at 10,000 rpm for about 10 minutes. The supernatant was isolated to which 50 µL sodium acetate and 200 µL isopropanol were added followed by centrifugation. The pellets were isolated, 70% ethanol was added and the contents were centrifuged. The collected pellets were kept for air drying. Thereafter, Tris-EDTA buffer was added to the dried pellets and was used for quantifying DNA using nanodrop.
The loading samples were created according to amount of DNA in Eppendroffs and dye was added. Gel electrophoresis unit was prepared. The gel was casted and the samples and the ladder (as a control) were loaded into gel at 80-100 volt. Further, the gel was analyzed under gel documentation system.

Reactive oxygen species generation
Cell suspension (100 μL) was seeded in each well of 96 well plate (20, The medium was removed and 100 μL of 1× PBS was added in each well and 5 μL of Rhodamine 123 (dissolved in 1000 μL of DMSO and 1000 μL of 1 × PBS) was added in the control and treatment wells and incubated for 30 minutes. Thereafter, the plate was read at 500 nm (excitation) and 526 nm (emission) with the help of microplate reader [17].

Gene expression analysis with quantitative PCR
The cells were cultured (1×10 5 cells/mL) in the 6 well plate and was treated with 100µg/mL concentration of B. lupulina in each well. The control and the treated cells were pooled from the plate and was treated with Trizol reagent and was stored at -20º C.
After the removal of growth medium from the culture dish, 1ml Trizol reagent was added to the cells. 0.2 ml chloroform was added per 1ml of Trizol reagent used for homogenization. It was incubated for 3 min at room temperature followed by centrifugation at 1200g for 15 minute at 4°C. The aqueous phase of the sample was removed by angling at 45 degree and pipetting the solution out. The aqueous phase was placed into the new tube and the spots for RNA isolation was proceeded including RNA precipitation, RNA wash, and RNA resuspension [18].

cDNA Synthesis
After isolation of total RNA, 1μg RNA was used for synthesis of cDNA with the help of Thermo Scientific Verso cDNA kit. 1μg RNA was first mixed with the primers of interest and was heated at 65°C for 5 mins and then 5X cDNA synthesis buffer, dNTP

Statistical analysis
Data were expressed as mean standard deviation (SD). One-way analysis of variance ANOVA were performed by using GraphPad Prism5 and statistical significance of results measured by using Duncan's multiple range, significance test (P < 0.05).

Trypan blue exclusion dye
The effect of ethanalic leaf extract was very effective after the treatment of Trypan blue dye on THP-1 cell line. The highest percentage of viable cells was 90.09 at 0.1 µg/mL and the lowest was 25.13 µg/mL after 24 h treatment while, the number of viable cells were 95.25 in control well at 0 h shown in (Fig. 3 and Fig. 4).
The morphological changes in the cells were observed in treated cells which showed extensive cell death. The cell viability of the cancerous cells exposed to B.
lupilina extracts decrease in the cells.
The methods, temperature and time of extraction, solvent type, concentration of solvent, etc. affect the extraction of phytochemical constituents. Ethanolic extract of B.
lupulina exhibited the highest selective index (SI) (781.5) with lowest (IC50) 50% inhibitory concentration dose (0.02 µg/mL) against HSV-2 cells [21]. It was the lowest IC50 value. In contrast, IC50 value was higher because cell lines and solvent system were different and extraction procedure was also different. However, it has been proved that every cell line contains ability to survive in stress condition as well as exposure of treatment of extracts.
Clinacanthus nutans did not show any activity against these virus strains. Jayavasu [22] also Apoptosis has been studied with the ROS production and mitochondrial membrane potential measurements. ROS production induced the intracellular damages including the DNA damage. During apoptosis, mitochondrial membrane became more permeable and thus released caspase and membrane activity was lost. In relation to

Availability of data and materials
The data sets that support the conclusions of this article are included within the article.