Extraction Of Β-Caryophyllene Lab Report

Extraction of β-caryophyllene

Ground cloves will be used to isolate β-caryophyllene via steam distillation. 5g of ground cloves will be taken in a 500mL RBF(Round bottom flask) with boiling stones, 40mL of dH2O, and 3 to 4 drops of an antifoaming agent (to prevent violent boiling). Then, the contents of the flask will be heated on a heating mantle for an hour and followed by condensation of the distillate through a water jacket. Then allow it to collect in a graduated cylinder. Steam distillation will allow the clove oil to co-distill with the water, which take place at a least temperature than the boiling temperature of the individual solutions. This is desirable, because the components of clove oils get decompose at high temperatures. Then

500gms of fresh beetroot or its hairy root has to be homogenized with sand continuously for three times in 70% ethanol (100mg/L).Then extracts will be centrifuged at 10xg for half an hour and supernatants will be allowed to vaporise at 40°C under vacuum till they get dried. Then, the residues will be dissolve in 0.5L of 70% of methanol and this sample-methanol mixtures keep in refrigerator at -20 degrees for 24hr, consequently thawed and supernatants are carefully collected from the precipitate. Under vacuum, methanol will be evaporated, at 40°C, from the supernatants and following betalains are lyophilized from aqueous fractions and finally 6.5gms of dry betalains will be obtained (Christ, Alpha 1-2,

Exponentially growing cells, 1x104, will be added to each well in 96 well plate and followed by treatment with different concentrations of drug and allow them to incubation for some period. Upon addition of MTT, the plates will be allowed for incubation at 37°C for 4 h. Then the samples will be read at 570 nm, upon dissolution of formazan salt in DMSO to calculate the 50% inhibitory concentration (IC50) of drug.

b. Trypan Blue exclusion technique By Trypan Blue exclusion technique, the effect of drug on the MCF-7 Cancer cell will be determined. Briefly, 3x104 cells will be added to each well in 24 well plates and then expose with different concentrations of drug. Then plates will be allowed for incubation at 37°C and by using hemocytometer, after staining with 0.4% Trypan Blue every 24 h, number of cultured cells in the different wells will be counted to calculate the doubling time.

d. Cell viability in presence of caspase-3 inhibitor

The viability of MCF-7 cells treated with caspase-3 inhibitor and evaluation of drug will be done by using MTT assay. After incubation with caspase-3 inhibitor for 1 h, cells will be treated with different concentrations of drug(s) for upto 48 h after which viability will be