In Vitro Anticancer MCF-7, Anti-Inflammatory, and Brine Shrimp Lethality Assay (BSLA) and GC-MS Analysis of Whole Plant Butanol Fraction of Rheum ribes (WBFRR)

2.2. Extraction

The plant rheum ribes was collected from the mountains of Chaman, Balochistan, Pakistan, and identified by a taxonomist Prof. Dr. Rasool Bakhsh Tareen, Department of Botany, University of Balochistan, Quetta, Pakistan. 3kg whole plant of rheum ribes was dried in shade and then grinded, soaked in 5 liter of methanol, kept for 7 days, and shaken daily. After a 7-day period, methanol containing whole plant of rheum ribes was filtered and vaporized with the help of rotary evaporator. Dried semisolid whole plant methanolic extract of rheum ribes (WMERR) was 48.6 gm. This crude extract 11.5gm was examined for anticancer MCF-7, anti-inflammatory, and Brine Shrimp Lethality Assay (BSLA) while the remaining extract was fractionated with solvents, for instance, n-hexane, aqueous, and butanol.

2.3. Fractionation of Crude Extract

In a separatory funnel, with crude extract two solvents were added such as aqueous and n-hexane. The separatory funnel was shaken thoroughly to create two layers such as aqueous layer and n-hexane layer. Both solvents were separately vaporized with the help of rotary evaporator to form whole plant n-hexane fraction of rheum ribes (WHFRR) 0.1 gm and whole plant aqueous fraction of rheum ribes (WAFRR) 23.6 gm. Both fractions were examined for anticancer MCF-7, anti-inflammatory, and Brine Shrimp Lethality Assay (BSLA) while whole plant aqueous fraction of rheum ribes (WAFRR) was further fractionated with a solvent such as butanol [11, 12].

2.4. Fractionation of Whole Plant Aqueous Fraction of rheum ribes (WAFRR)

Whole plant aqueous fraction of rheum ribes (WAFRR) was fractionated with solvent such as butanol.

2.5. Formation of Whole Plant Butanol Fraction of rheum ribes (WBFRR)

In a separatory funnel, with whole plant aqueous fraction of rheum ribes (WAFRR), two solvents were added such as butanol and aqueous. The separatory funnel was shaken thoroughly to create two layers such as butanol layer and aqueous layer. Both solvents were separately vaporized with the help of rotary evaporator to form whole plant butanol fraction of rheum ribes (WBFRR) 2.5gm and whole plant aqueous fraction of rheum ribes (LAFRR). Whole plant butanol fraction of rheum ribes (WBFRR) was examined for anticancer MCF-7, anti-inflammatory, and Brine Shrimp Lethality Assay (BSLA) [11, 12].

2.6. MTT Assay (MCF-7 Cell Lines)

In this assay, Dulbecco's Eagle modified medium, containing ten percent fetal bovine serum, was used for the culturing of MCF-7 cell lines which were then kept in five percent CO₂ and incubated at 37°C. MCF-7 cells were collected when confluency was developed and 8000 cells per well were plated in 96 well flat. After 24 hours, the extracts and fractions with 50 ug/ml were added and then incubated for 48 hours. After incubation, extracts and fractions were removed. To each well, 0.5mg/ml MTT was added and kept in incubator for 3 hours at 37°C. MTT was reduced into Formazan crystals which was then dissolved in 100ul DMSO and at 570 nm absorbance was taken using micro-plate reader (Spectra Max plus, Molecular Devices, CA, USA). In this assay, doxorubicin was used as a standard drug. The decrease in viable cells or percent inhibition was calculated with the help of following formula:

% Inhibition = 100 - (mean of O.D. of test compound - mean of O.D. of negative control)/(mean of O.D. of positive control - mean of O.D. of negative control) x 100)

For the calculation of IC₅₀ 20 mM stock solution of extracts and fractions was diluted into working solution with 50 uM and then in order to get less than 50 percent inhibition, working solution was further diluted in serial dilutions. With the help of EZ-fit5 software, IC50 was calculated [13].

2.7. Anti-Inflammatory Assay

Oxidative Burst Assay Using Chemiluminescence Technique. In this technique, 25 ul diluted whole blood HBSS⁺⁺ and 25 ul of extracts and fractions were incubated for 15 min at 37°C and then plated in 96 well plates. Control wells contain HBSS⁺⁺ and cells while blank wells contain HBSS⁺⁺. 25 ul luminol and 25 ul serum opsonized zymosan were added into each well. In terms of relative light units, the level of ROS was recorded in luminometer. In this assay, Ibuprofen with IC₅₀ 11.2 ± 1.9 is used as a standard drug [14].

2.8. Brine Shrimp Lethality Assay

B-Hatching Techniques. Scatter 50 mg of brine shrimp eggs in a hatching tray which was already half filled with filtered brine solution. Put it in incubator at 23°C. Take 20 mg of extracts and fraction and dissolve it in 2ml of solvent. Transfer 5, 50, and 500 ul from this solution to 3 vials. Leave them overnight to allow the solvent to evaporate. With the help of Pasteur pipette put 10 larvae per each vial. Add 5ml seawater. Under illumination, for 24 hours, incubate them at 25-27°C. For positive and negative controls, add reference cytotoxic drug along with solvent in other vials. For the determination of LD₅₀, Finney computer program was used [15–17].

2.9. Gas Chromatography Mass Spectrometry (GC-MS) Analysis: Triple Quadrupole Acquisition Method MS Parameters

For identification and quantification of rheum ribes compounds, 2 ul of rheum ribes extract or fraction was directly injected into the gas chromatograph mod.6890N Network GC System (Agilent Technologies Palo Alto, CA) together in the presence of mass spectrometer mod. 5973 Network Mass Selective Detector (Agilent Technologies Palo Alto, CA) and furnished in the presence of a column HP-5MS (30 m length, 0.25 mm interior diameter, 0.25 um film width Agilent Technologies, Palo Alto, CA). Helium gas was off. Injection was made into a split-splitless injector (split ratio 30:1) at 250°C. The oven program was as follows: 70°C for 3 min, then 6°C/min to 180 for 5 min, then 6°C/min to 280°C for 10 min, then 8°C/min to 290°C for 20 min. The MSD transfer line was set at a temperature of 250°C; MSD temperature quadrupole was of 150°C and ionization temperature was 230°C; mass spectra were seventy electrovolts and scan achievement was accomplished in the series between thirty five and 300m/z. The identification of the components of the rheum ribes extract or fraction was assigned by matching their mass spectra with those available in the libraries NIST 02 and WILEY [18].