determine cell proliferation inhibitory activity of plum extracts on Colon cancer cells.

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In next page I have collected many results from different articles and all of them determined that plum has significant effect to inhibit colon cancer cells.

1. I want you to write two pages single space discussion about my result which mean you summarize all the results and include a literature review which mean find other fruit or vegetables have the same effect to inhibit colon cancer cells.

2. Write one page single space conclusion.

A comparative study of laboratory and commercially extracted plum extract on Colon-26 adenocarcinoma cells

Anti-Cancer Effects of Plum

With high antioxidants, plum can prevent an individual from developing cancer. Antioxidants work by combating the free radicals which are the one of the causes of cancers. Furthermore, anthocyanin and phytonutrients present in plums have shown to induce powerful preventive effects against cancers (Byrne et. al. 2009). Studies have proved that plum components such as epicatechin aids in inhibiting the proliferation and growth of malignant cancer cells and promotes apoptosis in human liver cancer (Watson & Preedy, 2010). Studies have also proved that antioxidants such as neo-chlorogenic and chlorogenic acids are effective remedies for breast cancer cells and have no side effects on the normal healthy cells.

The fact that dried plums can reduce weight loss can be associated with the reduction of colorectal cancer whose risk factors include obesity (Byrne et. al. 2009). Prunes also encourage microbial metabolism and the retention of beneficial microbiota throughout the colon hence reduces chances of developing precancerous lesions within the colon. Studies have also shown that the reddish pigment in plums known as anthocyanins can protect the body against respiratory and gastrointestinal tract cancer. Plums are also rich in vitamin A which have been proved to be useful in protecting the body against cavity and oral cancers.

The above observations suggest that plums can be very effective in preventing cancer from different origins, including colon cancer. The present investigation was undertaken to study the effect plum’s extract on colon cancer.

Hypothesis:

As discussed above, plums are rich in polyphenols which have been shown to exert anti-cancer effects. Based on this rationale, my hypothesis is that polyphenols in plums are good bioactive agents to inhibit proliferation in colon cancer cells.

Objectives: We have tested our hypothesis using following objectives:

1. To characterize the phytochemical profile and ant oxidation properties of plum extract.

We have used commercially available standardized preparation of plum extract named “PE60” and laboratory prepared extracts from plum’s skin and pulp. We have determined total polyphenolic content (TPC), total flavonoid content (TFC), ant oxidation capacity (DPPH) and oxygen scavenging activity (ABTS) of these extracts

2. To determine cell proliferation inhibitory activity of plum extracts on Colon cancer cells.

The plum extracts described in objective #1 were used to determine their anti-proliferation activity on colon-26 adenocarcinoma cells using MTT assay. We also performed nuclear staining to determine the morphological changes induced by plum extracts in colon-26 cells.

The results that we found from different articles is

Determination of Total Phenolic Content (TPC):

In different extracts of plum, the amount of TPC was determined to find out if they are better dissolved in any particular solvent. The data is shown in Figure 21. We found that the commercial preparation of PE60 supposed to contain 600 mg of TPC in 1000 mg of the PE extract (60%). The data indicates that water, and DMSO extract contained TPC in range 490 – 500 mg/g of dry extract, while the methanol of skin extract has around 200 mg/g of TPC. However, the methanol extract of plum’s pulp has significantly less TPC around 50 mg/g of dry weight.

3.1.2 Determination of Total Flavonoid Content (TFC):

The amounts of TFC in different plum extract is shown in Figure 22. The PE60 in DMSO preparation contained TFC in 190 mg/g of dry weight, whereas no significantly different between the methanol and water extract of plum’s skin which contained around 50-55 mg/g of TFC. Similar to TPC the methanol extract of plum’s pulp has significantly less TFC around 10 mg/g of dry weight compared to that of other preparations

3.1.3 Anti-oxidation activity (DPPH):

The anti-oxidation activity in different extracts of plum is shown in Figure 23. The data indicates that only the commercial preparation of PE60 in water extract has the highest extent of anti-oxidation activity and represented about 4000 mM equivalent of Trolox activity. whereas nonsignificant differences in all others plum extract which have less extent of anti-oxidation activity.

3.1.4 Oxygen scavenging capacity (ABTS):

The oxygen scavenging activity of different plum extracts is shown in Figure 24. Similar to Anti-oxidation activity (DPPH) it is evident from data that PE60 in water extract contained the highest capacity for scavenging free oxygen. The total oxygen scavenging activity in PE60 in water extract is 3670.625 mM Trolox equivalent, while the methanol extract of plum’s skin contained 904.083 mM Trolox equivalent. whereas nonsignificant differences between methanol extract of plum’s pulp and water extract of plum’s skin.

3.2: Effect of plum extract on colon-26 cancer cells 24 hours

For treatment, cells were washed once with serum free media and then treated with different concentration of extracts in serum free media for 24 or 48 hours as described in the “Materials and Methods” section. The viability of cell was determined using MTT assay. The results of plum extract from skin, pulp and PE60 after 24 hr treatment are shown in Figures 25 to 33.

The cellular morphology after 24 hr of treatment with water extract of plum’s skin is shown in Figure 25. The viable cells as stained using MTT dyes are shown in Figure 26 whereas the quantified cell viability data is shown in Figure 33. It is evident from cell morphology, staining of viable cells and the quantified data that the cells viability was not significantly different.

The cellular morphology after 24 hr of treatment with methanol extract of plum’s skin is shown in Figure 27, whereas the viable cells as stained using MTT dye is shown in Figure 28. The quantified cell viability data is shown in Figure 33. Similar to data from water plum’s skin extract methanol extract of plum’s skin almost has no significant effect except at the highest concentration causing a significant reduction by 30% (P<0.05) at 300 g/ml.

The effect methanol extract of plum’s pulp on cellular morphology after 24 hr of treatment with shown in Figure 29 whereas the viable cells as stained using MTT dyes are shown in Figure 30 The quantified data id presented in Figure 33. Data consistently show that methanol extract of plum’s pulp has no significant effect at any tested concentration.

The effect water extract of PE60 on cellular morphology after 24 hr of treatment is shown in Figure 31 whereas the viable cells as stained using MTT dyes are shown in Figure 32 The quantified data is presented in Figure 33. On treatment with water extract of PE60 cell viability is reduced in a dose-dependent reaching a significant reduction by 25% (P<0.05) at 10 g/ml. On further increasing the concentration of extract, the cells viability was further reduced reaching to a maximum inhibition by 80% (P<0.05) at 300 g/ml.

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3.3: Effect of plum extract on colon-26 cancer cells 48 hours

Effect of plum extract from skin, pulp and PE60 after 48 hours of treatment are presented in Figures 34 – 42.

The cellular morphology after 48 hr of treatment with water extract of plum’s skin is shown in Figure 34. The viable cells as stained using MTT dyes are shown in Figure 35 whereas the quantified cell viability data is shown in Figure 42. Similar to 24 hours treatment, it is evident from cell morphology, staining of viable cells, and the quantified data that the cells viability was also not affected on longer incubation at 48 hrs. .

The effect of methanol extract of plum’s skin on cellular morphology after 48 hr of treatment is shown in Figure 36, whereas the viable cells as stained using MTT dye is shown in Figure 37. The quantified cell viability data is shown in Figure 42. Similar to data from water plum’s skin extract, methanol extract of plum’s skin almost has no significant effect except at the highest concentration causing a significant reduction by 30% (P<0.05) at 300 g/ml.

The effect om cellular morphology after 48 hr of treatment with methanol extract of plum’s pulp is shown in Figure 38. The viable cells as stained using MTT dyes are shown in Figure 39 and the quantified data sd presented in Figure 42. Data consistently show that methanol extract of plum’s pulp also has no significant effect at any tested concentration.

../Desktop/Screen%20Shot%202017-09-15%20at%204.16.41%20PM.pngThe quantitative data for the effects of water extract of PE60 on cellular morphology after 48 hr of treatment is shown in Figure 40. The appearances of the viable cells as stained using MTT dye are shown in Figure 41. The quantified data is reported in figure 42. The water extract of PE60 reduced colon cell viability in a dose-dependent reaching a significant reduction by 40% (P<0.05) at 50 g/ml. On further increasing the concentration of extract, the cells viability was further reduced by 60% at 100 g/ml; however, it reached at a plateau and furthering increasing the concentration of extract caused no more reduction in cell viability.

3. 4: Effect of crude polyphenols from plum skin extracts on Colon-26 cancer cells on 24 hours treatment

From these initial experiments it was evident that the laboratory preparation of water or methanol extract of plup’ skin and methanol extract of plum’s pulp has very little effect on colon cell viability. However, the commercial preparation of plum extract (PE60) significantly inhibited colon 26 cells proliferation. This difference in effect between laboratory plum extracts and PE60 was due to higher content of polyphenols in the PE60 preparation. Further experiments were, therefore, performed using plum extract based on their polyphenol contents as determined earlier Figure 21. However, we did not further test pulp extracts because it contained substantially low amounts of polyphenols as shown in Figure 21.

The results of polyphenol concentration in the methanol or water extracts of plum’s skin is shown in Figure 43. The data show that a concentration of polyphenols in methanol extract of plum’s skin at 25 g/ml inhibited colon cell viability significantly by 55% (P<0.05). The cell viability was further decreased by 70% at 75 g/ml reaching a plateau of a maximum inhibition by 80% at 500 g/ml. The polyphenols in water extract also inhibited Colon-26 cell viability but to a lesser extent than that of methanol extract. The polyphenols at 25 g/ml inhibited cell viability significantly by 45% (P< 0.05); however, the cell variability remained at 45% – 50% inhibition on further increasing the concentration of polyphenols up to 500 g/ml.

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3.5: Effect of polyphenols in PE60 extract on colon-26 cancer cells after 24 hr of incubation.

The effect of polyphenols in PE60 extract on colon-26 cancer cells after 24 hours incubation is shown in Figure 44. It is evident from the data that a significant inhibition of colon cell growth by 20% (P< 0.05) was observed at 25 g/ml of polyphenols in the water extract of PE60. On further increasing the polyphenol concentration of the water extract of PE60, the cell viability decreased in a dose-dependent manner reaching a plateau of almost a significant 50% (P< 0.05) inhibition at 75 g/ml. No further decrease in cell viability was observed on further increasing the concentration of polyphenols up to 150 g/ml.

3.6: Effect of crude polyphenols from plum skin extracts on Colon-26 cancer cells on 48 hours treatment.

The results of polyphenol concentration in the methanol or water extracts of plum’s skin after 48 hr of treatment is shown in Figure 45. The data show that a concentration of polyphenols in methanol extract of plum’s skin at 25 g/ml inhibited colon cell viability significantly by 25 % (P<0.05). The cell viability was further decreased by 80% in a dose –dependent manner up to 150 g/ml and then reached a plateau. Similar to 24 hr. incubation, the polyphenols in water extract also inhibited Colon-26 cell viability but to a lesser extent than that of methanol extract. The polyphenols at 25 g/ml inhibited cell viability significantly by 40% (P< 0.05), and then caused a gradual reduction in cell viability by 50% at 300 g/ml. However, the cell viability reduced by almost 80% at 500 g/ml, which was similar to the methanolic extract.

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3.7: Effect of PE60 extract on colon-26 cancer cells 48 hours

The effect polyphenols in the water extract of PE60 on colon-26 cancer cells after 48 hours incubation is shown in Figure 46. The viability of Colon-26 cells were effectively inhibited by polyphenols in PE60 extract on 48 incubation. The cell viability inhibited significantly by 85% (P< 0.05) at 12.5 g/ml. There as only a slight improvement in inhibition and a maximum inhibition of 90% (P< 0.05) was observed on further increasing the concentration over 12.5 g/ml.

ABTS

uM Trolox Eq./g sample

2.194927865177626 3.307189138830754 7.216878364870322 129.1498644985739 2.194927865177626 3.307189138830754 7.216878364870322 129.1498644985739 SKIN-H2O PULP-ME-OH SKIN-ME-OH PE-60-H2O 154.7708333333333 145.8125 904.0833333333335 3670.625

µM Trolox Eq./g sample

DPPH

5.504430169084356 4.530033654928407 10.85043164234533 175.9936402122661 5.504430169084356 4.530033654928407 10.85043164234533 175.9936402122661 SKIN-H2O PULP-ME-OH SKIN-ME-OH PE–60-H2O 145.5172413793103 112.3850574712643 198.448275862069 3785.977011494253

µM Trolox eq./g sample

Polyphenols in PE60 Extract (g/ml)

020406080100120140160

Cell Viability (%)

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PE60-Water

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*

*

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Polyphennols in PE60 (g/ml)

0306090120150

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PE60-Water

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Total Polyphenolic Content (TPC) in PlumExtracts

Pulp-MeOH

Skin-MeOH

Skin-Water

PE60/DMSO

PE60/water

Polypheolic content

(Gallic Acid,

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Total Polyphenolic Content (TPC) in PlumExtracts

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g/mg dry extract)

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Total Flavonoids in Plum Samples

Pulp-MeOH

Skin-Water

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PE60-Water

PE60-DMSO

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g/mg Dry Extract)

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Total Flavonoids in Plum Samples

Pulp-MeOH

Skin-Water

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PE60-Water

PE60-DMSO

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