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Asian Pacific Journal of Reproduction 2015; 4(2): 147-152

Asian Pacific Journal of Reproduction Journal homepage: www.apjr.net

doi:

Document heading

Ethanol and supercritical fluid extracts of hemp seed (Cannabis sativa L.) increase gene expression of antioxidant enzymes in HepG2 cells 1

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Sunghyun Hong , Kandhasamy Sowndhararajan , Taewoo Joo , Chanmook Lim , Haeme Cho , 3 1 1 Songmun Kim , Gur-Yoo Kim , Jin-Woo Jhoo * Kangwon National University, Department of Animal Products and Food Science, Chuncheon, Gangwon, Republic of Korea, 200-701

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Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, Serdang, Selangor, Malaysia, 43400

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Kangwon National University, Department of Biological Environment, Chuncheon, Gangwon, Republic of Korea, 200-701

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ARTICLE INFO

ABSTRACT

Article history: Received 15 November 2014 Received in revised form 20 February 2015 Accepted 25 March 2015 Available online 20 June 2015

Objective: To determine the gene expression of antioxidant enzymes by hemp seed extracts in human hepatoma (HepG2) cells. Methods: Ethanol and supercritical fluid (SF) extracts obtained from de-hulled hemp seed were used for the evaluation of in vitro antioxidant activity and gene expression of antioxidant enzymes. In vitro antioxidant activities of the samples evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical scavenging assays. The expression of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in HepG2 cells was evaluated by real-time PCR. Results: In the antioxidant assay, SF extract of hemp seed exhibited higher ABTS and DPPH radical scavenging activities (IC50 of 66.6 µg/mL and 9.2 mg/mL, respectively) than ethanol extract. The results of antioxidant enzyme expression in real-time PCR study revealed the H2O2 (200 µM) challenged HepG2 cells reduced the expression of enzymes such as SOD, GPx and CAT. However, the cells treated with ethanol and SF extracts were up-regulated the expression of antioxidant enzymes in concentration dependent manner. When compared to ethanol extract, the SF extract exhibited higher activity in the expression of all the antioxidant enzymes at the concentration of 500 µg/mL. Conclusion: In conclusion, the findings of our study demonstrated that the hemp seed effectively inhibited H2O2 mediated oxidative stress and may be useful as a therapeutic agent in preventing oxidative stress mediated diseases.

Keywords:

Antioxidant enzyme Cannabis sativa Hemp seed HepG2 cells Catalase Superoxide dismutase

1. Introduction Free radicals, generated in oxidation processes, are essential for the production of energy to fuel biological processes in most of the living organisms. However, the excessive productions of free radicals such as superoxide, hydroxyl and peroxy radicals etc., which responsible for the damage of lipids, proteins and *Corresponding author: Jin-Woo Jhoo, Department of Animal Products and Food Science, Kangwon National University, KNU Ave 1, Chuncheon, Gangwon, Republic of Korea, 200-701. Tel: +82-33-250-8649 Fax: +82-33-251-7719 E-mail: [email protected] Sunghyun Hong and Kandhasamy Sowndhararajan contributed equally. Foundation project: This research was funded by Pyeongchang County, Gangwon, Republic of Korea.

DNA in cells, leading to several degenerative diseases, including inflammation, cardiovascular diseases, cancer, diabetes, and neurological disorders[1]. Generally, all the organisms are well

protected against free radical damage by endogenous oxidative enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and catalase (CAT). However, these enzymes are commonly insufficient when it comes to completely preventing degenerative diseases and other health problems[2, 3].

In addition, several non-enzymatic antioxidant compounds such as

ascorbic acid, tocopherol, glutathione and other dietary compounds play an important role in defending the body against free radicals damage by scavenge or neutralize the oxidizing molecules and maintaining redox balance[4]. The plant kingdom offers a wide

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Sunghyun Hong et al./ Asian Pacific Journal of Reproduction (2015)147-152

range of natural antioxidant molecules including phenolic acids,

ethylbenzothiazoline-6-sulphonic acid) (ABTS), 3-(4,5-

flavonoids, and other secondary metabolites. These metabolites are

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT),

commonly found in a variety of fruits, vegetables, herbs, cereals,

trypan blue, hydrogen peroxide (H2O2) and penicillin-streptomycin

sprouts and seeds[5]. In recent years, there has been increasing

solution purchased from Sigma-Aldrich Chemical Co. (St. Louis,

interest in obtaining natural dietary antioxidants especially from

MO, USA). Dulbecco’s modified eagle medium (DMEM) and fetal

plants. Previous studies have been reported that the compounds

bovine serum (FBS) were purchased from HyClone Laboratories,

from plants provide potential health benefits, such as antioxidant,

Inc.(Utah, USA). RNeasy Mini kit and SYBR green master mix

anti-inflammatory, antitumor, anticarcinogenic, and antimicrobial

were purchased from Qiagen-GmbH (Hilden, Germany). SuperScript

activities. They can also be used for the treatment of various ailments

III First-Strand synthesis system was purchased from Invitrogen

including, atherosclerosis, arthritis and diabetes[6-9].

Hemp (Cannabis sativa L.) is an annual herbaceous plant belongs

to Cannabaceae family and originated in Central Asia. The plant has been grown cultivated widely for the purposes of fiber, food and medicine[10]. The seed of hemp is a rich source of nutrition, containing

25%–35% of lipid, 20%–25% of protein, 20%–30% of carbohydrate, 10%–15% of insoluble fiber and a rich array of minerals[11, 12]. The

hemp seed oil contains higher level of polyunsaturated fatty acid

(70%–80%), particularly linoleic (氊-6) and 毩-linolenic (氊-3) acids. Most of essential amino acids contained in hemp protein are sufficient

for the FAO/WHO suggested requirements of children[13, 14]. The cannabinoids are the most studied constituents from the hemp seed,

in particular delta-9-tetrahydrocannabinol is the main psychoactive component. In addition, several other bioactive compounds have been reported from Cannabis include terpenes, sugars, steroids, flavonoids, nitrogenous compounds and non-cannabinoid phenols[15].

(Carlsbad, CA, USA). The hemp seeds (Cannabis sativa L.) were collected from the cultivated field in Sudong area (Pyungchang, Republic of Korea). The seed sample was authenticated and deposited at Sudong Agricultural Association (Pyungchang County, Republic of Korea) with batch No. PH-2012-011-S01.

2.2. Ethanol extraction of hemp seed About 250 g of de-hulled hemp seed powder was macerated thrice over 48 h with ethanol (food grade 95%, Daehan Ethanol Life Co., Ltd., Seoul, Republic of Korea) at room temperature. The combined ethanol extract was concentrated by low-pressure evaporation (