The Changes Of Anti-oxidant And Anti-colon Cancer Activity To HCT-116 Cells Of The Mulberry Soluble Extract After Thermal Treatment

Colon cancer is a common malignant tumor that is severely a threatening to human health.Mulberries rich in bioactive substances have many health functions such as lowering blood pressure,protecting liver,protecting nerves,anti-oxidation anti-inflammation and anti-cancer effect.They are the classic members in the list of daily foods and medicines in China.The people eat fresh fruits and vegetables,but also eat their processed products.For example,most vegetables need a cooking treatment at home before their intake.However,polyphenols in fruits and vegetables are sensitive to many environmental factors such as high temperature,oxygen,and alkaline p H.Thus,it is necessary to assess whether the composition and biological activity of mulberry extract are affected by the used heat treatment.In this study,the freeze-dried and ground black mulberry?Morus nigra L.?and white mulberry?Morus alba L.?were extracted with water and 75%ethanol,respectively;while the obtained soluble extracts by centrifugal separation was subjected into various treatments like ethanol removal,activated carbon-mediated dephenolization,or 100°C thermal treatment of 15-45 min.Finally,the total phenol,flavonoid or anthocyanin content and anti-oxidant activity of the treated extracts were evaluatedfor their anti-cancer effects using model cells?human colon cancer HCT-116 cells?.The results showed that the used thermal treatment increased the values of total phenols and flavonoids contents,but decreased the values of anthocyanin contents.After the thermal treatment of 45 min,the total phenol contents of the water extracts of both black and white mulberries increased from 267.6 and 146.5 mg/L to 297.8 and 161.3 mg/L?gallate equivalent?,respectively;however,their flavonoid contents increased from 66.2 and 23.4 mg/L to 71.3 and 30.4 mg/L?quercetin equivalent?,respectively.The anthocyanin contents of the water and ethanol extracts of black mulberry decreased from 27.6 and 31.6 mg/L to 18.6 and 21.3 mg/L?Cyanidin-3-O-glucoside equivalent?,respectively.In addition,the thermal treatment also increased the values for total phenols and flavonoids contents of the dephenolized extracts.Based on the assaying results of endogenous fluorescence intensity,UV-absorption,and browning index,it was preliminarily concluded that the Maillard reaction occurred in the dephenolized extract with the formation of unknown aromatic compounds;moreover,it is preliminarily believed that this reaction led to increased total phenol and flavonoid contents.Based on the results from assaying free radical scavenging activity and reducing power,the extracts with thermal treatment were also observed with decreased activities to scavenge DPPH·,ABTS⁺,and·OH,or lower abilities to reduce Fe³⁺.CCK-8 and DCFH-DA analyses were used to determine proliferation inhibition and pro-oxidative effects of these extracts on HCT-116 cells.The results showed that these extract had inhibition effect on the cells and could induce the production of intracellular reactive oxygen species?ROS?;however,the heated extracts showed stronger cytotoxicity and pro-oxidation effect on the cells.In addition,the heaed dephenolized extracts also gave stronger proliferation inhibition and pro-oxidation effect on the cells than the unheated dephenolized extracts.The results from cell morphology observation showed that when the heated extracts acted on the cells for 24 h,the cell showed changed morphology clearly,which was regarded as typical apoptotic morphology.The mitochondrial membrane potential?MMP?and Ca²⁺concentration of the treated cells were also investigated.It was found that these extracts increased the MMP loss and enhanced the intracellular Ca²⁺concentration?P<0.05?;however,the heated extracts were also higher activity.Fluorescence quantitative RT-PCR results also indicated that these extracts could promote the expression of several apoptosis-related genes;however,the heated extracts were verified with higher abilities to regulate gene expression.In summary,the used thermal treatment of mulberry extracts at 100°C caused Maillard reaction or other unidentified reactions,resulting in changed composition,decreased anti-oxidant activity,but increased anti-colon cancer activity to HCT-116 cells.This study thus provides a scientific basis for the development of mulberry-based functional foods.The results of this study also give necessary evidence for the control of processing conditions of these functional foods.