Increases in Phenolic, Fatty Acid, and Phytosterol Contents and


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Bioactive Constituents, Metabolites, and Functions

Increases of Phenolics, Fatty Acids, Phytosterols, and Anticancer Activity of Sweet Potato after Fermented by Lactobacillus acidophilus Yixiao Shen, Haiyan Sun, Haiying Zeng, Witoon Prinyawiwatkul, Wenqing Xu, and Zhimin Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05414 • Publication Date (Web): 03 Mar 2018 Downloaded from http://pubs.acs.org on March 5, 2018

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Journal of Agricultural and Food Chemistry

Increases of Phenolics, Fatty Acids, Phytosterols, and Anticancer Activity of Sweet Potato after Fermented by Lactobacillus acidophilus Yixiao Shen†, Haiyan Sun‡, Haiying Zeng∥, Witoon Prinyawiwatukul†, Wenqing Xu† and Zhimin Xu†,*

Running title: Bioactives and anti-cancer of fermented sweet potato †

School of Nutrition and Food Sciences

Louisiana State University Agricultural Center Baton Rouge, USA ‡

Key Laboratory of Life Resources of Shaanxi Province,

Shaanxi University of Technology, Hanzhong, China ∥

School of Liquor and Food Engineering

Guizhou University Guiyang, China.

Authors Shen and Sun contributed equally to this work. *Corresponding author Tel: 225-578-5280. Fax: 225-578-5300. E-mail:[email protected]

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Abbreviations:

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DMSO, Dimethyl sulfoxide;

3

DW, dry weight;

4

FBS, fetal bovine serum;

5

FID, flame ionization detector;

6

FW, fresh weight;

7

GC, gas chromatography;

8

HPLC, high performance liquid chromatography;

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MS, mass spectrometry;

10

PBS, phosphate buffered saline;

11

LR, lipophilic extract of raw sweet potato;

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LF, lipophilic extract of fermented sweet potato;

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HR, hydrophilic extract of raw sweet potato;

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HF, hydrophilic extract of fermented sweet potato

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Abstract

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Changes of phenolics, fatty acids, and phytosterols in the sweet potato (SP) fermented

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by Lactobacillus acidophilus were evaluated and compared with its raw and boiled SPs.

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The differences of profiles and levels of phenolics between raw and boiled SPs were not

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as significant as their differences from fermented SP. The level of caffeic acid or 3,5-

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dicaffeoylquinic acids in fermented SP increased to more than 4 times higher than raw or

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boiled SP. Two phenolics, p-coumaric acid and ferulic acid which were not detected in

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either raw or boiled SP were found in fermented SP. The level of each fatty acid or

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phytosterol increased in fermented SP, but decreased in boiled SP.

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hydrophilic and lipophilic extracts obtained from both of raw and fermented SPs, the

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hydrophilic extract of fermented SP exhibited the highest capability in inhibiting cancer

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cell PC-12 proliferation. However, each of the extracts had very low cytotoxicity to

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normal monkey kidney cell growth.

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Lactobacillus acidophilus significantly increased free antioxidant-rich phenolics and

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enhanced inhibit cancer cell proliferation activity without cytotoxicity to normal cells.

Among the

The results indicated that fermented SP by

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Key words: sweet potato; fermentation; phenolics; phytosterol; fatty acids; proliferation

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inhibition.

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INTRODUCTION

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Sweet potato (Ipomoea batatas (L.)) is an important agricultural crop rich in dietary

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fiber and vitamins. It is becoming one of the most promising economic commodities due

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to its versatility, high production yield and strong resistance in various environmental

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conditions.1 The compositions of sweet potatoes after being boiled, steamed, baked or

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roasted have been well evaluated.2 It can also be used for the production of ethanol.3

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However, composition changes, especially for phenolics, fatty acids, and phytosterols in

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fermented sweet potato by lactic acid bacteria Lactobacillus acidophilus have not been

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documented. Fermentation is a popular food processing method which provides the

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preservation and organoleptic properties for foods such as cheese, vinegar, wine and

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soybean curd with special flavors, aromas and textures.4 Generally, most bioactive

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compounds in sweet potato are in bound form present in flesh and considered to be

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gradually metabolized in the intestine which contains different varieties of probiotic

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floras.5

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compounds to increase their bioavailability via fermentation, meanwhile they could

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reduce sugar content in the fermented foods. It has been reported that a number of people

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with digestion problems such as small intestinal bowel infection or colonic infection are

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not able to metabolize and absorb those nutrients in sweet potato.6 Therefore, the

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fermentation by Lactobacillus acidophilus could be considered as a pre-digestion

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processing method which could increase free form bioactive compounds in sweet potato

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and improve their bioavailability with reducing of sugar content.7

The floras assist in releasing and bio-transforming those health benefit

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Lactobacillus acidophilus is one of the dominant probiotic floras in the human

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gastrointestinal tract.8 It plays an important role in generating antimicrobial peptides,

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improving gastrointestinal barrier function and competing with pathogens for epithelial

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adherence.9 Lactobacillus acidophilus is mainly used in dairy products and reported to

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have health beneficial effects including the prevention of diarrhea, stimulation of the

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immune system, anti-inflammation of intestinal disorders, etc.8

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acidophilus can also convert some carbohydrates to organic acids and has cinnamoyl

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esterase and decarboxylase activities for releasing phenolic compounds from the

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fermented food matrix.10 Sweet potato is rich in free sugar and other carbohydrates and

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an ideal substrate to be fermented by Lactobacillus acidophilus. The fermentation could

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release the bound bioactive compounds and produce free secondary metabolites with high

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bioactivity and bioavailability. Therefore, the levels and profiles of antioxidant phenolics,

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fatty acids, and phytosterols in the fermented sweet potato were evaluated and compared

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with the original raw sweet potatoes.

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inhibiting pheochromocytoma derived cancer cell (PC-12) and its cytotoxicity to normal

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monkey kidney cell (CV-1) were studied in this study as well. The results of this study

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could provide a new utilization method for sweet potato through the fermentation by

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Lactobacillus acidophilus. Compared with raw sweet potato, the fermented sweet potato

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could have a higher level of free bioactives with high bioavailability which enhances the

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overall health promoting and anticancer capabilities.

Lactobacillus

The capability of fermented sweet potato in

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MATERIALS AND METHODS

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Chemicals and materials

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purchased from Fisher Chemicals (Fair Lawn, NJ, USA). Acetone and ethyl acetate were

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purchased from Macron (Charlotte, NC, USA) and EM Science (Gibbstown, NJ, USA),

HPLC grade acetonitrile, acetic acid and hexane were

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respectively.

Standards of phenolic acids, fatty acids, and phytosterols as well as

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derivatization reagents trimethylsilyl imidazole (TMSIM) and BCl3-methanol were

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purchased from Sigma-Aldrich (St. Louis, MO, USA). Frozen cultures of Lactobacillus

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acidophilus LA-K (Chr. Hansen’s Laboratory, Milwaukee, WI) were stored at -35°C

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before use. Pheochromocytoma derived cancer cell (PC-12) and normal monkey kidney

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cell (CV-1) lines were purchased from ATCC® (Manassas, VA, USA). Fetal bovine

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serum (FBS), dimethyl sulfoxide (DMSO), phosphate buffered saline (PBS), cell titer

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blue and antibiotic (penicillin–streptomycin) were ordered from Invitrogen (Grand Island,

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NY, US).

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Garnet sweet

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Fermentation of sweet potato mash and microbiological analysis

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potatoes (Ipomoea batatas (L.)) were purchased in a local grocery store (Baton Rouge,

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LA, USA). They were cleaned, peeled and cut into 1 cm cubes and then ground by a

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kitchen blender with sterilized water at the ratio of 2:1 (w/v). Then, the sweet potato

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mash (30 g) was added in each of nine flasks (250 mL). Three of the flasks were used for

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each of the raw, boiled, or fermented sweet potato group. For the boiled sweet potato

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group, the mash in flask was boiled on a hot plate stirrer (PC-351 Corning, Corning, NY,

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USA) for 15 min with stirring by a magnetic stirring bar.

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The three flasks used as the fermented sweet potato group were autoclaved before inoculation of Lactobacillus acidophilus solution.

The solution was prepared by

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suspending 2 milliliters of freshly thawed Lactobacillus acidophilus (LA-K) culture in 20

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mL of sterilized phosphate buffer (10 mM, pH 7.0). Then, the flasks were incubated at

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37oC for 24 h fermentation. The pH of the fermented sweet potato mash was measured

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using a pH meter (Orion 3 star Benchtop; Thermo Orion, Beverly, MA, USA). The

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populations (CFU/mL) of Lactobacillus acidophilus before and after the fermentation

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were enumerated using direct plate count on Lactobacilli MRS agar.

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Extraction and determination of phenolics, fatty acids, and phytosterols in sweet

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potatoes

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potato mash was extracted with 20 mL of methanol three times at 60°C for 20 min. After

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the methanol layers were combined, the methanol solvent was evaporated by a vacuum

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centrifuge evaporator (Labconco, Kansas City, MO, USA).

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dissolved in acetone (20 mL) twice to precipitate possible protein and carbohydrate

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contaminants. Then, the supernatants were collected and evaporated again to obtain the

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dried hydrophilic extract for determination of phenolics. For the lipophilic extract, ten

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grams of each of those sweet potato mesh samples was extracted by a mixture of hexane

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and ethyl acetate (50:50; v/v) and followed the same procedure as the methanol

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extraction above to obtain the dried extracts for determination of lipophilic fatty acids,

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and phytosterols. A working solution (10 mg/mL) of each hydrophilic or lipophilic

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extract was prepared by using methanol and hexane, respectively.

For the hydrophilic extract, ten grams of the raw, boiled or fermented sweet

The dried extract was

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Phenolics were determined by an HPLC system (2690, Waters, Torrance, USA) with a

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reversed phase column (C18, id 250×4.60 mm, 5 µm, Phenomenex, Torrance, USA) and

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a diode array detector based on the method of Du et al.11 The hydrophilic extract

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working solution (2 mL) was filtered by 0.45 µm microporous film before injection. The

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concentration of each compound was calculated by its corresponding standard calibration

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curve.

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The lipophilic extract working solution (2 mL) was mixed with an internal standard

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solution (C17:0). After hexane solvent was evaporated, 2 mL of BCl3-methanol was

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added to carry out esterification derivatization. After incubation at 60°C for 30 min, 1

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mL of hexane was added to the reaction solution and vortexed. The upper hexane layer

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was collected and transferred to a clean test tube containing 0.1 g of anhydrous NaSO4 for

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removing any moisture and then transferred to a GC vial. A GC system equipped with

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FID detector and Supelco SP2380 (30 m × 0.25 mm) column (Bellefonte, PA, USA) was

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employed to determine fatty acids in the samples according to the method in the study of

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Du et al.11

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The determination method of phytosterols in the samples was based on the study of

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Xu and Godber.12 Two milliliter of the lipophilic extract working solution was transferred

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to a clean test tube and dried again. Then, 200 µL of TMSIM and 50 µL of acetonitrile

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were mixed with the extract and incubated at 65 ˚C for 30 min for derivatization. Then it

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was extracted by 200 µL of hexane. The supernatant was transferred to a GC vial. A

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Varian CP-3800 GC (Valnut Creek, CA, USA) was interfaced with a Saturn 2200 MS

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and a DB-5 column (60 m × i.d. 0.25 mm and 0.25 µm thin coating film) (Supelco,

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Bellefonate, PA, USA). The oven temperature increased from 200˚C to 280 ˚C at a rate

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of 10 ˚C /min and was held at the final temperature for 62 min. Helium was used as a

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carrier gas at a constant flow rate of 1.5 mL/min with a split injection mode (1: 50). The

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injection port temperature was set at 280˚C.

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Determination of effect of raw and fermented sweet potato extracts on

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Pheochromocytoma derived cancer (PC-12) cell line and normal monkey kidney

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(CV-1) cell line Pheochromocytoma derived cancer cell (PC-12) cell line of rat adrenal

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medulla and normal monkey kidney (CV-1) cell line were used to determine the effect on

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cancer cell and normal cell proliferations of each extract, respectively. The cells were

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maintained in a CO2 incubator with 5% CO2 and 95% humidity and supplemented with

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Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine

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serum (FBS) and 1 % antibiotic (penicillin–streptomycin). The PC-12 or CV-1 cells

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were plated in a 96-well plate at a density of 3.0 × 103 cells/well and incubated for 48 h.

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The dried hydrophilic and lipophilic extracts of raw sweet potato and fermented sweet

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potato were dissolved with 0.2 % DMSO in PBS culture media. Then, the PC-12 or CV-

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1 cells were incubated with a series of the extracts with different concentrations for 24 h

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at 37 °C. The cells mixed only with 0.2 % DMSO in PBS culture media were used as a

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blank group. After incubation, the media was discarded. The cells were stained by 100

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µL of fresh media containing 20% cell titer blue (Invitrogen, Grand Island, NY, US).

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The fluorescence intensity of each well was obtained by a FluoStar Optima micro-plate

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reader (BMG, Germany) at excitation/emission wavelengths of 570/615 nm to calculate

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the cell viability in the well. The percentage of survived cells of sample group relative to

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the blank group was used to indicate the anti-cancer capability or cytotoxicity of each

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treatment.

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Data analysis Determinations of phenolics, fatty acids, and phytosterols in the samples

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were carried out in triplicates and expressed as means ± standard deviation by using

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Microsoft Excel (Redmond, WA). The significant differences among treatments were

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conducted by one-way ANOVA at P< 0.05 (SAS, 9.1.3, Cary, NY, US). The results

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from the cell culture studies were obtained from five replications for each group and

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analyzed by GraphPad Prism (Version 6.0, GraphPad Software Inc., USA).

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significant difference of the results between two groups was considered at p < 0.05.

The

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RESULTS AND DISCUSSIONS

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Profiles and levels of phenolics in raw, boiled and fermented sweet potatoes As

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shown in Table 1, the pH of sweet potato mash after fermentation drop to 3.45 from 6.20,

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because of the production of organic acids through the enzymatic conversion of

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carbohydrates by Lactobacillus acidophilus.3 The bacteria can take advantage of glucose

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in sweet potato to produce lactic and acetic acids which were mainly responsible for

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lowering the pH after fermentation.13 The protein and carbohydrates in sweet potato also

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provided a sufficient source of nitrogen and carbon for the bacteria proliferation. The

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viable cell counts of Lactobacillus acidophilus in the fermented sweet potato increased to

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7.48 ± 0.67

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indicated that sweet potato mash could be a suitable substrate for the fermentation by

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Lactobacillus acidophilus.

×

108 from 3.80 ± 0.01

×

106 CFU/mL after incubation at 37oC for 24 h. It

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The main phenolics in sweet potato are chlorogenic acid and its related derivatives.

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The raw sweet potato contained five major phenolics including chlorogenic acid, caffeic

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acid, and 4,5-dicaffeoylquinic, 3,5-dicaffeoylquinic and 3,4-dicaffeoylquinic acids. The

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concentrations of the phenolics in raw, boiled, and fermented sweet potatoes are shown in

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Table 2. In raw sweet potato, 3,4-dicaffeoylquinic acid (29.12 ± 1.68 mg/100g DW) was

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the dominant phenolic, while the other phenolics were in a range of 2.39 mg/100g DW

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for caffeic acid to 5.00 mg/100g DW for 4,5-dicaffeoylquinic acid. With the involvement

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of fiber and starch in raw sweet potato flesh, some phenolics could be in bound and

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conjugated form which usually have lower bioavailability than free form.14 Generally, the

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traditional cooking method, boiling, is believed to be able to disrupt the fiber or starch

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matrix to release bound or conjugated form phenolics.15 As shown in Table 2, the levels

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of most phenolics in the boiled sweet potato were significantly higher than those in raw

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sweet potato, except 3,4-dicaffeoylquinic acid.

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dicaffeoylquinic acid had been present in free form in raw sweet potato and degraded

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during boiling treatment. The increase of other phenolics may be due to a significant

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amount of their bound phenolics were released and convert to free form by heat treatment,

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although some of their free form phenolics could also be degraded at the same time.16

It was suggested that most of 3,4-

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In this study, it was found that the increases of most of the phenolics in fermented

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sweet potato are much higher than boiled sweet potato after the fermentation by

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Lactobacillus acidophilus (Figure 1 & Table 2). For example, the level of caffeic acid or

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3,5-dicaffeoylquinic acid in fermented sample was three times higher that in raw sweet

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potato or boiled sweet potato (Table 2). Furthermore, p-coumaric acid and ferulic acid

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which were not detected in either raw or boiled sweet potato were produced to 26.42 ±

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2.01 and 19.10 ± 1.87 g/100g DW in the fermented sweet potato, respectively.

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indicated that the fermentation effectively released bound phenolics through the enzymes

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of Lactobacillus acidophilus which are able to utilize or break down the fiber or starch

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matrix of sweet potato. It was reported that lower pH of fermented sweet potato could

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also assist in the release of phenolics which have ester and ether bonds with plant cell

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wall.17 Compared with boiling treatment, undesirable thermal degradation of phenolics is

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totally avoided during the fermentation. Therefore, the bound phenolics were largely

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converted to free form phenolics and retained in the fermented sweet potato without

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thermal degradation.

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balancing gut microbiota, inhibiting pathogenic infection, lowering blood cholesterol and

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reducing the risks of colon cancer.18 The fermentation of Lactobacillus acidophilus

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occurring in the intestine of humans may also improve the release and bioavailability of

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bound phenolics in consumed food fiber.

There are evidences that Lactobacillus acidophilus help in

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The biotransformation of chlorogenic acid to caffeic acid by Lactobacillus acidophilus

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during fermentation was confirmed by using cholorgenic acid only in MSR media for 24

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h in this study. Similar result has been reported in the study of Bel-Rhlid et al, in which,

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Lactobacillus johnsonii NCC 533 could convert chlorogenic acid to caffeic acid in green

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coffee extract.19 The biotransformation might be due to that Lactobacillus acidophilus

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contains chlorogenate esterase and hydroxycinnamate decarboxylase which exhibit high

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catalytic activity on aromatic compounds such as chlorogenic acid.19

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Generally, ferulic and p-coumaric acid are hydroxycinnamic acids which are mostly in

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bound form as ester-linked with polysaccharides, organic acids, lipids or protein of plants

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in the cell wall.20 After the fermentation, the two bound phenolics significantly changed

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to free form and became the major phenolics in fermented sweet potato. It has been

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reported that Lactobacillus species produces active extracellular cellulolytic enzymes

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such as feruloyl esterases.21 Thus, it could de-esterify dietary fiber and release free ferulic

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acid and p-coumaric acid from plant cell wall. Some clinic studies have demonstrated

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free caffeic acid was more readily absorbed in the stomach and small intestine than

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chlorogenic acid, while ferulic acid and p-coumaric could be bioavailable in blood

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plasma.22 Therefore, the releasing of bound phenolics in sweet potato by fermentation,

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especially caffeic acid, ferulic acid and p-coumaric acid would enhance their

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bioavailability to the human body.

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Profiles and levels of fatty acids and phytosterols in raw, boiled, and fermented

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sweet potatoes The fatty acid compositions of the three sweet potatoes are listed in Table

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3. In raw sweet potato, the most abundant fatty acid was linoleic acid (C18:2n-6) (85.67±

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5.56 mg/100g DW), then followed by palmitic acid (C16:0), myristic acid (C14:0),

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linolenic acid (C18:3n-3) and stearic acid (C18:0) which were a range from 47.18 ± 3.24

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to 17.96 ± 0.61 mg/100g DW. However, the level of each fatty acid significantly

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decreased in boiled sweet potato, especially for unsaturated fatty acids, linoleic and

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linolenic acid. Similar results were reported in the study of Longo et al. that heat

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treatment (60-100 °C) resulted in degradation of unsaturated fatty acids in tomatoes.23

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After the fermentation, linolenic acid (171.07 ± 12.77 mg/100g DW) or each of other

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fatty acids which ranged from 42.86 ± 0.87 to 109.29 ± 3.74 mg/100g DW in fermented

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sweet potato was approximately more than two times higher, compared with raw sweet

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potato. In addition to free form fatty acids, some fatty acids also bind with phytosterols,

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glycerols, phospholipids and sugar-containing glycolipids in plants as important

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membrane constituents in the chloroplasts and mitochondria.24 The increase of the fatty

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acids in fermented sweet potato might result from the enzymatic action based on

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desertification of bound fatty acids through the fermentation to release palmitic, myristic

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and stearic acids.25 Since unsaturated fatty acids are important to brain cells growth,

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neurological development and cognitive function, the accumulation of those fatty acids in

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fermented sweet potato would have higher health promoting function than raw or boiled

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sweet potato.26

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Although three phytosterols, campesterol, stigmasterol and β-sitosterol slightly

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decreased in sweet potato after boiled due to thermal degradation, they significantly

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increased in the fermented sweet potato (Table 3). As phytosterols have been reported to

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inhibit intestinal cholesterol absorptions and control cholesterol concentrations in plasma

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due to they have similar chemical structure of cholesterol, the increases of the

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phytosterols in fermented sweet potato would also improve the function of lowering

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serum cholesterol level.27

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In general, as Lactobacillus acidophilus is a probiotic bacteria species which could

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help in reducing overgrowth of pathogens, stimulating an immune response, improving

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the blood lipid metabolism or potentially killing cancer cells in the human gastrointestinal

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tract.28 The fermented sweet potato could be considered as an ideal processed food source

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to enhance bioavailability of the bioactive compounds of sweet potato and deliver

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probiotic Lactobacillus acidophilus at the same time. Based on the results above, the

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hydrophilic and lipophilic raw (HR and LR) and fermented sweet potato extracts (HF and

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LF) were selected to evaluate their effects on the pheochromocytoma derived cell (PC-12)

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and normal monkey kidney cell (CV-1) proliferations.

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Effect of raw and fermented sweet potato extracts on proliferation of

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pheochromocytoma cells (PC-12) and normal monkey kidney cells (CV-1) of raw

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and fermented sweet potato extracts The inhibitory effect on cancer cell proliferation

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of hydrophilic and lipophilic extracts of raw sweet potato versus fermented sweet potato

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were evaluated by using pheochromocytoma cancer cell (PC-12) model.

Figure 2a

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demonstrates that the hydrophilic extracts of raw (HR) and fermented (HF) sweet potato

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had a dose-dependent inhibition mode on PC-12 cell proliferation.

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concentration increased to 0.2 mg/mL, the survival rate of the cancer cells dropped to

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9.5% in HF treatment, while it was 63.2% in HR treatment (Figure 2a). When the

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concentration of HF increased to 0.5 mg/mL, only 2.1% of the cancer cells was survived,

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compared with the control, while the survival rate in HR treatment was 30.1% (Figure 2a).

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Thus, the greater inhibitory capability of HF may be contributed by the higher levels of

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free form phenolics or other bioactives generated by the fermentation. For example, the

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total level of phenolics including caffeic acid, p-coumaric acid, ferulic acid and 3,5-

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dicaffeoylquinic acid in fermented sweet potato was much higher than that in raw sweet

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potato (Table 2). Similar result was found in the study of Rocha et al., which reported

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that caffeic acid, 3,4-, 3,5- and 4,5- dicaffeoylquinic acid as well as ferulic and p-

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coumaric acid could inhibit colon cancer cell lines including RKO, HT-29 and Caco-2.29

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However, the difference between the lipophilic extracts of raw (LR) and fermented (LF)

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sweet potatoes in inhibiting PC-12 cell proliferation was not as significant as the

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hydrophilic extracts (Figure 2b). Although the concentration of fatty acids concentration

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in fermented sweet potato was much higher than that in raw sweet potato, they were not

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the primary factors responsible for the anticancer potential. Thus, the LR or LF extract

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treatment exhibited relatively weaker anticancer potential than either HR or HF extract

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treatment.

As the extract

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The interaction of lipophilic and hydrophilic bioactive compounds in inhibiting cancer

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cell relies on their different accessibilities and inhibition pathways. Generally, lipophilic

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compounds could readily cross the lipid bilayers of cell membranes by simple diffusion

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or special membrane proteins for transferring such non-polar solutes, while most

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hydrophilic compounds have difficulties in penetrating the cell membrane.30 However, at

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a physiologic pH level, multiple hydroxyl groups in phenolics could interact with the

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polar head groups of phospholipids at membrane surface via hydrogen bonds formation.31

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It would then improve the penetration of the phenolics by deprotonation of hydroxyl

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groups and modify the membrane structure and fluidity.32 Compared with the control

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group shown in Figure 2c, the apoptotic progress of cancer cells was induced and

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accelerated by the hydrophilic fermented sweet potato extract. It resulted in the cell

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shrinkage and marginated nucleus indicated by the arrows in Figure 2d.

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The advantage of applying plant-derived bioactive extracts as anticancer medicines is

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that the extracts have lower cytotoxicity and side effect compared with other anticancer

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treatments such as chemotherapeutic agents or radiation. In this study, the monkey

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kidney cell lines (CV-1) were used for assessing the influences or toxicities of the sweet

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potato extracts on normal cells growth. At a relatively low concentration of 0.02 mg/mL

324

of HR or HF treatment, they could slightly increase the growth of CV-1 normal cells

325

(Figure 3). Even at the concentration of 0.5 mg/mL which HF had the most effective

326

inhibition to the PC-12 cancer cells, only 24.74% of CV-1 normal cells were inhibited,

327

while the levels of other treatments were all below that level (Figure 3). It indicated that

328

hydrophilic sweet potato extracts had high anticancer potential but low cytotoxicity on

329

normal cells. It could be explained by the different energy metabolisms between cancer

330

and normal cells. Generally, normal cells with competent mitochondria undergo Krebs

331

Cycle metabolic pathway and generate the majority of ATP for cell growth.33 However,

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332

most cancer cells have dysfunction in mitochondria and rely more on the glycolytic

333

pathway in cytosol to generate the metabolic intermediates and ATP.34 Thus, the key

334

enzymes in glycolytic pathway have been considered as the potential therapeutic targets

335

for inhibition of cancer cell proliferation. The non-covalent interactions with proteins

336

and phenolics have been reported as the inhibitors of the key enzymes in glycolytic

337

pathway, such as glucosidases, xanthine oxidase and PKM2 activities, to further perturb

338

proliferation of the cancer cells.33 For lipophilic bioactives, such as phytosterols, they

339

could interrupt balance between cancer cell proliferation and apoptosis by the

340

interference with protein phosphatase 2A (PP2A) in sphingomyelin cycle and block the

341

cell cycle at G0/G1 phase in prostate cancer, hepatocyte, and breast cancer cell lines.35, 36

342

In summary, this study revealed that increases of hydrophilic phenolics and lipophilic

343

fatty acids and phytosterols in the sweet potato after fermented by probiotic Lactobacillus

344

acidophilus LA-K. Also, the fermented sweet potato extracts, especially the hydrophilic

345

extract exhibited higher capability than raw sweet potato in inhibiting the

346

pheochromocytoma cancer cells (PC-12) proliferation, while it had very low cytotoxicity

347

to the normal monkey kidney cells (CV-1).

348

contributed by the high level of free form phenolics in fermented sweet potato, which

349

deactivate the key enzymes of glycolytic pathway in the energy metabolism of cancer

350

cells but had less influence on the Krebs Cycle pathway for normal cells. Therefore, the

351

fermentation by Lactobacillus acidophilus is a new biotechnological approach for sweet

352

potato to increase its nutritional value and bioavailability of various bioactive compounds.

353

The fermented sweet potato or its extract could be used as an assistant treatment with low

354

side effect to cooperate with chemotherapeutic drugs or other cancer remedy treatments.

The great anticancer potential may be

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355

Acknowledgements

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This study was supported by the Key Technologies Research and Development Program

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Agriculture of Shaanxi Province (2017NY-117) and Louisiana State University

358

Agricultural Center (NIFA Project No. LAB94334).

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Table 1 pH values and colony-forming units (CFU) of Lactobacillus acidophilus in sweet potatoes before (0 h) and after (24 h) fermentation Time pH Value 6

Visible cell (x10 CFU/mL)

0h

24 h

6.20 ± 0.15 a

3.45 ± 0.02 b

3.80 ± 0.01 a

7.48 ± 0.67 b

Values with different letters in a row are significantly different at p