Selection and collection of plant materials
Catharanthus roseus is a flowering plant species, local and endemic to Madagascar, yet developed somewhere else as a fancy and restorative plant. The plant materials were cultivated and followed by using the standard guidelines of plant nurseries of Karnataka State Forest Department, Karnataka State. One such nurseries is well-known as “Dhanavantri Vana”, and generally recognized as ‘Medicinal Plants of Paradise’. Fresh and healthy cultivated leaves of C. roseus plants (Fig. 1) were gathered from the nurseries of the ‘Medicinal Plants of Paradise’, Jnana Jyothi Nagar, Jnana Bharathi, Bengaluru (12° 56′ 30.67“ N and 77° 29’ 53.85” E with an elevation of 800.41 m asl), Karnataka 560,056, India. Congregated leaves were presented to the affirmation process and identified by Dr. H. R. Raveesha, Professor in the Department of Botany, Bangalore University, Bengaluru 560,056, Karnataka, India. Also, a voucher specimen of this material was deposited and now, it is made available in an herbarium of the same Botany Department, Bangalore University. A vouchering reference number CRDVSR00VII0B021 has been provided by the Botany Department.
Preparation of plant leaf extract using methanolic technique
Methanolic technique for the C. roseus plant leaves extractions (CrPLE) were experimented with as described by Kumar et al. [28], Koel et al. [26], and Syeda and Riazunnisa [59]. Weighed 20 g of desiccated sample powder and disintegrated in 100 ml of methanol in a 500 ml container with covering by aluminum foil. Then, at that point, the container was placed in a hot steam bath at 50 ° C for 4 h. After keeping aside, the extract was separated using Whatman strain removing paper and the remainder was gathered in a 50 ml container. Filtrate present over the strain-removing paper was disposed of and the remainder was engaged for additional utilization. Then the remainder was kept at 50 °C for a few hours until the extract got completely dehydrated and turned into semi-solid form. This semi solid sample was weighed and the yield was observed. The methanolic leaf extractions of C. roseus were determined utilizing the formula:
$$\%\;\text{yield}=\left(\text{Dry}\;\text{extract}\;\text{weight}\right)/\left(\text{Total}\;\text{sample}\;\text{weight}\right)\;\text{X}\;100$$
Characterization of the CrPLE
Determination of phytochemicals screening
The phytochemical screening of achieved methanolic leaf extracts of the C. roseus plants was experimented with as endorsed by van Der Heijden et al. [60], Nayak and Pereira [39], Jaleel et al. [18], and Govindasamy and Srinivasan [17]. The screening was performed for the sign (whether present or absent) of phytochemicals through the diagnostic tests by the color concentration or the precipitate formation. The results were qualitatively determined and stated as a positive ‘+’ sign for the appearance and a negative ‘−’ sign for the nonappearance of phytochemicals [17, 49].
Thin-layer Chromatography (TLC) analysis of the methanolic leaf extracts of C. roseus plants
Thin-layer chromatography (TLC) was accomplished the survey to the known purity of C. roseus plant leaves extracts (CrPLE) [28, 63]. 10 mg/ml samples were ready for experimentation. 2.5 μl of the sample was marked on a TLC plate and permitted to dry. A TLC plate is comprised of a slim layer of Silica gel (0.25 mm) with fluorescent indicator F254 with the dissolvable system. Chloroform: methanol (9.5: 0.5) was employed as a solvent system for TLC analysis. The strip or plate is then positioned with this end dunking into the dissolvable blend, taking into consideration that the example spot/zone isn’t drenched in the dissolvable. As the dissolvable moves towards the opposite finish of the strip, the test combination isolates it into different parts. This is called as improvement of TLC plates. The division relies upon a few elements. The plate is taken out after an ideal advancement time and dried, and the spots/zones are identified utilizing a UV chamber and the Rf value is determined utilizing the accompanying formulae:
$$\textrm{Retention}\ \textrm{factor}\ \left(\textrm{Rf}\right)\kern0.5em \textrm{value}=\frac{\textrm{Distance}\ \textrm{moved}\ \textrm{by}\ \textrm{the}\ \textrm{compound}}{\textrm{Distance}\ \textrm{moved}\ \textrm{by}\ \textrm{the}\ \textrm{solvent}}$$
In a silica sheet, the mark is made with a pencil about 1 cm range from the lower edge of the end. The fluid concentrate sample of C. roseus leaves was marked online, with the assistance of a fine capillary. The distance moved by the solvent is set apart with a pencil and the spots are seen by a UV lamp and set apart as the shape and color they make. The distance traveled by the spots and the distance traveled by the solvent are noted and the retention factor (Rf) value of each spot is determined [63]. The solvent ascent was fixed at 10 cm in all cases [28]. The Rf values of all the TLC plates were then determined.
High-Performance Liquid Chromatography (HPLC) analysis of the methanolic leaf extracts of C. roseus plants
Raw extract (10 mg/ mL), methanol chloroform, and hexane (based on solubility) from a stock of C. roseus leaves were liquified to the concentration of 1.5 mg/mL, then sieved through a 0.45 μm nylon Phenex syringe strain remover and investigated utilizing a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) fixed with a reversed-phase column C18 column (Synergi 4u Polar-RP80A Column 250 x 4.6 mm 5um) (Torrance, USA) utilizing UV detection (UV-VIS detector SPD-10A) set at 210 nm and 280 nm. The portable phase consisted of 3% (v/v) acetonitrile in 0.2% (v/v) orthophosphoric acid (Solvent A) and absolute acetonitrile (Solvent B). An auto-injector (SIL-10A HT) was utilized to inject 20 μL sample volumes onto the HPLC at a flow rate of 1 mL/min. with a incline elution plan as follows: 0–10 min., 0% B; 10–30 min., 50% B; 30–45 min., 50% B; 45–55 min., 60% B; 55–60 min., 0% B [49]. Chemical mixtures that existed in various extracts of C. roseus were resolved trailed by utilizing a technique of Bramati et al. [4], Pan et al. [42], Wagay et al. [63], Lin et al. [32], Liu et al. [34] and Deguchi et al. [9].
Reference guidelines including caffeic acid and catechin were manufactured in methanol at the concentration of 200 μg/mL for recognition and measurement of the specific substances confined in the extracts. Peaks were recognized in light of the retention time of caffeic acid and catechin standards. Complete coloring matters as a percentage of content was determined from the total peak area by adding from the adjustment diagram [33]. Unidentified individual mixtures were evaluated involving Caffeic acid and Catechin as a source of the perspective norm at 250 and 278 nm respectively and the result were expressed as μg equivalents per ml of extract (μg/ml) [33, 49]. The diagnostic signs were observed at 2–20 mV latent functional. The following equation was utilized to regulate the concentrations of the substances present in the extracts
$$\textrm{X}\frac{mg}{L}=\left\{\textrm{Area}\ \textrm{of}\ \textrm{sample}\div \textrm{Area}\ \textrm{of}\ \textrm{standard}\right\}20\ \textrm{mg}/\textrm{L}$$
Along these lines, a purified solution was examined by a high-performance liquid chromatography system.
Cell line culture and preparation
Human breast cancer MDA-MB-231 strain -CRM-HTB-26 cell line was acquired from the American Type Culture Collection (ATCC) stock cells, then this was cultivated in Dulbecco’s modified eagle’s medium (DMEM) enhanced with 10% deactivated Fetal Bovine Serum (FBS), penicillin (100 IU/ ml), streptomycin (100 μg/ ml) in a moistened atmosphere of 5% CO2 at 37 °C pending blended. The cell lines were separated with the cell separating solution (0.2% trypsin, 0.02% EDTA, and 0.05% glucose in PBS). The feasibility of the cells was monitored and centrifuged. Additionally, the 50,000 cells/ well were cultivated in a 96 well plate and nurtured for 24 h at 37 °C, a 5% CO2 incubator.
Medicinal practices of the biologically synthesized methanolic leaf extracts of C. roseus plants
Assessment for anti-microbial action, cell cycle studies by flow cytometry, in vitro assay for cytotoxicity, and anticancer action by apoptosis tests utilizing prepared methanolic leaf extracts of C. roseus plants were experimented by succeeding the beneath referenced events.
Anti-microbial activity
Minimum inhibitory concentration (MICs) is the minimum dosage of a compound prevent microorganism development and was calculated based on cultures comprising a changeable dosage of the methanolic leaf extracts of C. roseus plants (CrPLE) utilizing the agar well diffusion technique [11, 49, 59].
The anti-microbial present in the plant extract is permitted to diffuse out into the medium and communicate in a plate newly cultivated with the test organisms. The resulting zones of inhibition would be consistently roundabout and the distance across of zone of inhibition can be measured in millimeters. The agar utilized was Meuller-Hinton agar for bacteria, Potato Dextrose agar for fungi, and Yeast Peptone Glucose (YEDP) Agar for yeast that is thoroughly tried for configuration and pH. This strategy is fine reported as the standard sectors of inhibition have been calculated powerless in addition to safe qualities.
Petri plates (with a diameter of 90 mm) comprising 20 mL Meuller-Hinton agar were cultivated utilizing cotton swabs with 24 h (old) culture of the microbial strains. Wells were cut (10 mm diameter) and 50 μL of various concentrations of a test sample. The plates were then nurtured at 37 °C for 24 h. The anti-microbial action was examined by estimating the diameter of the inhibition zone shaped around the well against the test microorganisms [51, 53]. Moreover, Staphylococcus aureus - ATCC 6538 and Escherichia coli - ATCC 8739 cell suspension were ready and grown on tryptone soup, and cultures were nurtured for 24 h at 37 °C. The cell suspensions of the cultures were acclimated to 1–2 x 106 cells/ ml. The methanolic leaf extracts of C. roseus plants are utilized as the test compounds with samples (20 μl and 10 μl), standard as Ciprofloxacin (20 μl) for S. aureus and E. coli were added to the 5 mm well on agar plates [57]. Besides, Aspergillus niger ATCC 6275 and Candida albicans ATCC 10231 spore suspension were ready and developed on potato dextrose broth, and cultures were nurtured for 5 to 7 days at room temperature (27 °C). Once more, the methanolic leaf extracts of C. roseus plants are utilized as the test compounds with samples (20 μl and 10 μl), standard as Itraconazole (20 μl) for A. niger and C. albicans were added to the 5 mm well on agar plates.
After incubation, the experimented plates were detected for a zone of inhibition around the wells. The antimicrobial action was assessed by estimating the diameter of the inhibition zone of the tested microorganisms [3, 45, 51, 63].
Cytotoxicity studies
Levels of cells in different phases of the cell cycle with the mixtures of treated and untreated (controlled) populations were estimated through flow cytometry utilizing the FACS Caliber (BD Biosciences, San Jose, CA).
An in vitro assay typically experimented with the monolayer cell culture was trypsinized and the total of cells was acclimated to 5.0 x 105 cells/ ml utilizing separate media encompassing 10% FBS in half-area flat-bottomed microtiter plates. To each well of the 96-well microtiter plate, 100 μl of the watery cell suspension (50,000 cells/ well) was supplemented. Later 24 h, when a partial monolayer was shaped, the supernatant was spun off, splashed the monolayer once with medium and 100 μl of various trial dosages of trial samples were supplementary to the fractional monolayer in microtiter plates.
The plates were then nurtured at 37 °C for 24 h in a 5% CO2 atmosphere. The decrease of tetrazolium salts was broadly acknowledged as a solid method for inspecting cell expansion. The yellow tetrazolium MTT (3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide) was diminished through metabolically dynamic cells, partially employing the activity of dehydrogenase catalysts, to create decreasing counterparts, for instance, NADH and NADPH. The resultant intracellular purple formazan can be solubilized and evaluated thru spectrophotometric analysis. Test estimates the cell multiplication amount and then again, when metabolic occasions lead to programmed cell death or corruption, the decrease in cell viability.
Cells cultivated in T-25 flasks were trypsinized and suctioned into a 5 mL centrifuge tube. The cell pellet was acquired by centrifugation at 300 x g. The total of the cell was changed, utilizing DMEM HG medium, with the end goal that 200 μl of suspension confined around 10,000 cells. To each well of the 96 well microtitre plate, 200 μl of the cell suspension was supplemented and the plate was nurtured at 37 °C and 5% CO2 atmosphere for 24 h.
Later 24 h, the spent medium was aspirated. 200 μl of various test dosages of test drugs were supplemented to the respective wells. The plate was then incubated at 37 °C and 5% CO2 atmosphere for 24 h. The plate was eliminated from the incubator and the drug-containing media was suctioned. 200 μl of medium encompassing 10% MTT reagent was then supplemented to each well to get a final concentration of 0.5 mg/mL and the plate was nurtured at 37 °C and 5% CO2 atmosphere for 3 h. The culture medium was eliminated deprived of upsetting the crystals designed. Then 100 μl of solubilization solution (DMSO) was supplementary and the plate was caringly shaken in a gyratory shaker to solubilize the formed blue formazan [37].
The optical density was estimated utilizing a microplate reader at a wavelength of 570 nm and 630 nm. The percentage growth inhibition was determined, after deducting the background and the absolute, and the dosage of the test drug expected to prevent cell growth by 50% (IC50) values was produced from the concentration-response curve for the cell line [10, 53].
The percentage of viability was calculated utilizing the formula:
$$\%\;\text{Cell}\;\text{viability}=\left[\left(\text{At}-\text{Ab}\right)/\left(\text{Ac}-\text{Ab}\right)\right]\;\text{X}\;100$$
where At = absorbance value of a trial substance,
Ab = absorbance value of absolute and
Ac = absorbance value of control.
Mechanism of cell death: apoptotic assay
Apoptosis is a cell death interaction considered through morphological and biochemical structures happening at numerous phases. As soon as set off, programmed cell death continues through various energy relying upon cell types and comes full circle with cell disturbance and arrangement. A basic phase of programmed cell death includes securing exterior fluctuations by kicking the bucket cells that at last outcomes in the acknowledgment as well as the take-up of these cells by phagocytes.
For programmed cell death induction, the MDA-MB-231 cell lines were splashed several times in DMEM cell culture medium and then cultivated in 6-well plates at 5 X 105 cells/ mL in DMEM cell culture with 1% FCS as defined by Koopman et al. [27]. To recognize cells that had lost membrane integrity, Propidium Iodide (PI) dye was supplementary to the last concentration of 10 txg/ ml before the examination [36].
The day preceding enlistment of apoptosis, plated 1 X 106 cells for every well for a 6-well plate utilizing DMEM cell culture medium. Later ~18 hours, the wells for drifting (dead) cells and dispensed with by pipette. Supplanted with a new culture medium to the original volume. Offered cells actuate apoptosis with 40 and 80 μg/ml of C. roseus leaves, and incubate for 24 h. Afterward, gathered cell culture medium into 15-mL tubes. Utilizing a policeman, the cells were confined from the dish and added 1 mL of medium to each well, and transported the contents to the 15-mL tubes. Centrifuged and disposed of the supernatant. Splashed the cells twice with cold PBS and afterward resuspend cells in 1 mL 1X Binding Buffer at a dosage of ~1 x 106 cells/mL. 500 μL of cell suspension is aliquoted and 10 μL of PI and 5 μL Annexin V is supplemented. The suspension is incubated for 15 minutes at RT in the dark. Post incubation, the cells were examined through a flow cytometer at the earliest opportunity (within 1 hour).
