Short Chain Fatty Acid Derived from Type III Resistant StarchFermentation of Sweet Potato (Ipomoea batatas) Inhibited

Proliferation of Colon Carcinoma Cell Line HCT-1161* 2 3E.Y. Purwani , S. Marya , M. T. Suhartono

1 Indonesian Center for Agricultural Postharvest Research and Development, Jln. Tentara Pelajar No.12, Bogor 16114, Indonesia

2 Primate Research Center, Bogor Agricultural University, Bogor, Indonesia3 Department of Food Science, Bogor Agricultural University,

Darmaga Campus Bogor, Bogor, Indonesia*eyplab@gmail.com

Badan Penelitian dan Pengembangan PertanianKementerian Pertanian

INTRODUCTION Resistant starch (RS) is defined as the sum of starch and starch

products of starch degradation that is not absorb in the intestine of healthy person. It is fermented by the bacteria in large intestine, which will ferment the resistant starch into short chain fatty acids (SCFA), particularly acetate, propionate and butyrate as well as producing gasses such as hydrogen, carbon dioxide and methane [1]. The SCFA especially butyrate is the main energy substrate for the colonocytes and has been implicated in the prevention of colitis and colorectal cancer [2].

Colorectal cancer (CRC) is cancer cell that grows in colon/rectum. Around 15% of CRC is inhirited (direct and indirect), the others is acquired. Up to 80% of CRC cases have been attributed to diet [3]. It is suggesting that CRC is preventable diseseases. In the current study, RS of sweet potato origin was prepared. Sweet potato was selected due to their great potential to be developed into valuable product such as functional food ingredient.

The objective of this research was to investigate the effect of SCFAs produced from RS fermentation with Eubacterium rectale DSM 17629 on proliferation inhibition and apoptosis induction of colon carcinoma cell line HCT-116.

Figure 1. Effect on VERO and HCT-116. Left-right: VERO treated with CFS containing 625 uM of butyrate, VERO non-treated, HCT-116 treated with CFS containing 650 uM of butyrate, HCT-116 non-treated. Magnification 160 x

Figure 2. Proliferation inhibition of HCT-116 treated with cell free supernatant containing SCFA

Apoptosis Detection:• HCT-116 treated with CFS containing SCFA undergo nuclear fragmentation which is one of characteristics of apoptosis cell.

Fig 2. Hoescht staining of HCT-116 (a) not treated (100x), (b) treated with 325 µM (100x)), (c) treated with 650 µM (100x), (d) treated with 650 µM (200x).

ConclusionSCFA produced from type-3 resistant starch of sweet potato with the concentration around 625-650 μM butyrate was toxic to HCT-116 but not to VERO. HCT-116 treated with the cell free supernatant containing SCFA as butyrate at 650 μM undergo apoptosis resulting in an observed characteristic of DNA fragmentation in cells.

References[1] Louis P, Scott KP, Duncan SH, Flint HJ. Review article: understanding theeffects of diet on

bacterial metabolism in the large intestine. J Appl Microbiol2007;102:1197e208.[2] Augenlicht LH, Mariadason JM, Wilson A, Arango D, Yang WC, Heerdt BG, et al.Short chain

fatty acids and colon cancer. Am Soc Nutr Sci 2002;132:3804Se8S.[3] Le Leu RK, Brown IL, Yu H, Morita T, Esteman A, Young GP. Effect of dietary resistant starch

and protein on colonic fermentation and intestinal tumorigenesis in rat. Carcinogenesis. 2007; 28(2): 240-245.

[4] Purwani EY, Purwadaria P, Suhartono MT. Fermentation RS3 derived from sago and rice starch with Clostridium butyricumBCC B2571 or Eubacterium rectale DSM 17629. Anaerobe 2012; 18 : 55-61.

[5] Ruemmele FM, Dionne S, Qureshi I, Sarma DSR, Levy E, Seidman EG. Butyrate Mediates Caco-2 Cell Apoptosis via Up-Regulation of Pro-Apoptotic BAK and Inducing Caspase-3 Mediated Cleavage of Poly-(ADP-Ribose) Polymerase (PARP). Cell Death and Differentiation 1999; 6:729-735.

[6] Purwani EY, Iskandriati D, Suhartono MT. Fermentation product of RS3 inhibited proliferation and induced apoptosis in colon cancer cell HCT-116.Adv Biosci Biotech 2012; 3: 1189-1198 ABBdoi:10.4236/abb.2012.38145 Published Online December 2012.

Aknowledgement: This study was financially supported by KKP3T Grant No. 82/LB.620/I.1/3/2011 from the Indonesia Agency for Agricultural Research and Development.

MATERIAL & METHODSPreparation of RS and Cell Free Supernatan [4]

Cell Culture and Analysis

HCT-116 was treated with cell free supernatant. Non cancerous cell line VERO was also treated as control. Cell morphology was observed by inverted microscope, cell numbers were counted and proliferation inhibition value was calculated. Apoptosis was observed with fluorescence microscope using Hoescht 33258 dye.

RESULTToxicity and Proliferation Inhibition to VERO and HCT-116.• Cell free supernatant (CFS) with butyrate of 625 uM was found to

be toxic on VERO. There was no toxic effect observed on HCT-116 treated with CFS containing 650 uM of butyrate (Figure 1).

• Proliferation of HCT-116 was also inhibited (Figure 2), and it was in agreement with other study [5,6]

SCIENCE, INNOVATION, NETWORK