Obesity is caused by excess body weight and presents significant social and economic problems worldwide¹⁾. Obesity is characterized by adipogenesis, the differentiation of preadipocytes into adipocytes, which results in the accumulation of intracellular triglycerides²⁾. Many studies have reported a close association between obesity and inflammation, with overweight and obese individuals exhibiting elevated inflammatory markers and responses^3-5). The obese individuals increased blood levels of inflammatory cytokines such interlukine-6 (IL-6) and tumor necrosis factor-α (TNF-α), and the others³⁾. In addition, the consumption of high fat diet increased the blood levels of lipopolysaccharides (LPS) in the gut compared with a low fat diet-fed group⁴⁾. In particular, obesity-induced increases in LPS contribute to intestinal inflammatory diseases by promoting gut leakage^4,5). Therefore, many studies focus on the development of natural products from dietary sources to improvement of obesity and intestinal inflammation^6,7).

Vegetable sprouts are widely consumed worldwide, and are used in the preparation of various functional food products⁸⁾. The vegetable sprouts are well-known for being rich in bioactive compounds such as vitamins, minerals, and polyphenols⁸⁾. In addition, many studies have reported various biological activities, such as anti-obesity, anti-diabetics, anti-oxidant, anti-cancer, and anti-inflammatory effects⁹⁾. Several studies have shown that long-term consumption of broccoli sprouts in overweight individuals improves obesity and inflammation by inhibition of blood LPS and inflammatory cytokines^10,11). In addition, broccoli sprouts have been demonstrated attenuate intestinal inflammation under cellular and in vivo inflammatory bowel disease models^12,13). However, the studies on obesity and intestinal inflammation related to vegetable sprouts has primarily focused on broccoli sprouts, while studies on other species of vegetable sprouts in relation to obesity and intestinal inflammation have not been investigated. Moreover, comparisons of the anti-adipogenesis and anti-inflammatory effects of commonly consumed vegetable sprouts in Korea remain insufficient.

Therefore, the aim of this study was compare the anti-adipogenesis and anti-inflammatory effects of commonly consumed 12 species of vegetable sprouts in Korea. We investigated the anti-adipogenesis activities of 12 vegetable sprouts species in differentiated 3T3-L1 cells. In addition, we confirmed the protective effects of 12 species from vegetable sprouts extract against intestinal inflammation in the LPS-induced HT-29 cells.

1. Sample preparation

The sprouts of green broccoli, kohlrabi, rape, and red radish were purchased from Nanumgongdongchae (Andong, Korea). The sprouts of pea, red buckwheat, yellow buckwheat were obtained from Hankyung University Industry-Academic Cooperation Foundation (Uiwang, Korea). The sprouts of barley and wheat were purchased from Daegwallyeong Clean Farm (Pyeongchang, Korea) and sprouts of alfalfa, clover, yellow broccoli were purchased by K Farm (Gwangju, Korea). The 12 species of vegetable sprouts were freeze-dried at -20 ℃, ground, and extracted for 24 h by adding ethanol to 20 times this amount. The extract was filtered using filter paper (185 mm, Advantec, Tokyo, Japan), and the extraction procedure was repeated three times. The combined filtrate was evaporated in a vacuum at temperatures below 35 ℃ using a rotary evaporator. The extraction yield of the 12 species of vegetable sprout extracts was examined in Table 1. The sprout extract were stored at -20 ℃ and dissolved in dimethyl sulfoxide at a concentration of 0.1 g/mL for use in the experiment.

Table 1

Information on the 12 Species of Vegetable Sprouts Extracts

Common name	Scientific species name	Yield (% w/w)
Alfalfa	Medicago sativa	19.0
Barley	Hordeum vulgare	34.5
Clover	Trifolium repens	13.5
Green broccoli	Brassica oleracea var. italica	22.5
Kohlrabi	Brassica oleracea var. gongylodes	16.5
Pea	Pisum sativum	34.0
Rape	Brassica napus	37.0
Red buckwheat	Fagopyrum esculentum	38.0
Red radish	Raphanus sativus L.	11.5
Wheat	Triticum aestivum	14.0
Yellow broccoli	Brassica oleracea var. italica	33.0
Yellow buckwheat	Fagopyrum esculentum	29.0

2. Reagents

Dulbeccós modified eagel's medium (DMEM), Roswell Park Memorial Institute 1640 medium (RPMI), fetal bovine serum (FBS), bovine calf serum (BCS), and trypsin-EDTA solution used in cell culture were purchased by Welgene (Gyeongsan, Korea). The 100 units/ml penicillin-streptomycin, LPS, and Oil Red O reagent were purchased from Sigma Aldrich Co. (Seoul, Korea). The 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) (MTT) was purchased from Invitrogen (Carlsbad, CA, USA), cytotoxicity detection kit measuring for lactate dehydrogenase (LDH) was obtained from Roche Co. (Seoul, Korea).

3. Cell culture

The 3 T 3-L1 preadipocytes and HT-29 human intestinal epithelial cells were purchased from the Korean cell line bank (Seoul, Korea). The 3T3-L1 cells were grown in DMEM supplemented with 10% BCS, 1% penicillin-streptomycin (100 units/ml). The HT-29 cells were incubated in RPMI supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were cultured at 37 °C in a 5% CO₂ incubator. When cells were 80% confluence, they were sub-cultured using 0.05% trypsin-EDTA.

4. Differentiation of 3T3-L1 cells

The differentiation of 3T3-L1 cells was induced using 0.5 M 3-isobutyl-1-methylxanthine, 10 mM dexamethasone, 10 μg/mL insulin (MDI) in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin for 2 days. Subsequently, the cells were maintained in DMEM supplemented with 10 μg/mL insulin for 8 days. The culture media were replaced with fresh media every 2 days.

On the first day of differentiation, 12 species of vegetable sprout extract at a concentration of 25 μg/mL treated to cells for 2 days.

5. Cell viability

Cell viability was determined by MTT assay¹⁴⁾. The 3T3-L1 cells were seeded at 1 × 10⁵ cells/mL in 96-well cell culture plates. After 2 days, vegetable sprout ethanol extracts added to each well and incubated for 3 days. The HT-29 cells were seeded at 5 × 10⁵ cells/mL in 96-well plate and incubated for 24 h. And then, 25 μg/mL vegetable sprout ethanol extracts and 500 μg/mL LPS were treated to each well. After 24 h, these cells were treated 0.25 mg/mL MTT solution and incubated for 4 h. The purple insoluble formazan was then dissolved in dimethyl sulfoxide. The absorbance was measured at 560 nm using microplate spectrophotometer (Multiskan skyhigh, Themo Fisher Scientific, Seoul, Korea).

6. Oil Red O staining

Effect of vegetable sprouts on liquid accumulation in 3T3-L1 cells was measured by Oil Red O staining¹⁵⁾. After differentiation, cell culture medium was removed, and the cells were washed with phosphate buffered saline (PBS). And then, cells were fixed with 10% formalin, washed with 60% isopropyl alcohol, and dried at 40 ℃. After drying, lipid droplet were stained with 0.6% Oil Red O solution for 20 min, and then washed 4 times with PBS. The cells were then washed with 60% isopropyl alcohol, and stained lipid droplet were confirmed using a microscope (Invitrogen EVOS M7000, Carlsbad, CA). To quantitative analysis of lipid droplet, Oil Red O stain was dissolved in 100% isopropyl alcohol. The absorbance was measured at 500 nm using microplate spectrophotometer.

7. LDH release

The LDH release was determined by using Cytotoxicity Detection KitPLUS commercial kit (Roche, Basel, Switzerland). The HT-29 cells seeded at 5 × 10⁵ cells/mL in a 96-well plate. After 24 h, 25 μg/mL of 12 species from vegetable sprout ethanol extracts and 500 μg/mL LPS added to the cells and incubated for 24 h. The 100 μL of cell culture supernatant from each well was mixed with 100 μL of cytotoxicity detection kit mixture, and then incubated for 30 min. The absorbance of each well was measured at 490 nm using a microplate spectrophotometer.

8. Inflammatory cytokines

The HT-29 cells were seeded at a density of 5 × 10⁵ cells/mL in 96-well plates. After incubation for 24 h, the cells were treated with 25 μg/mL of vegetable sprout extracts and 500 μg/mL of LPS. After 24 h, production of TNF-α and IL-6 in the cell culture supernatant was measured using ELISA kit (Thermo Fisher Scientific, USA) following the manufacturer’s protocols.

9. Statistical analysis

The values are presented as the means±standard deviation. The data were analyzed using SPSS software ver. 23.0 (SPSS Inc., USA). The significant difference was analyzed by one-way analysis of variance followed by Duncan's multiple range test. Value of P<0.05 was considered statistically significant.

1. Effects of vegetable sprout ethanol extracts on cell viability in 3T3-L1 cells

To confirm the cytotoxicity of the 12 species of vegetable sprout ethanol extracts on 3T3-L1 cells, cell viability was evaluated using MTT assay. The 3T3-L1 cells were evaluated by treating 12 species of vegetable sprout ethanol extracts at a various concentrations of 10~100 μg/mL for 72 h. As shown in Fig. 1, treatment of 12 species from vegetable sprout ethanol extracts at a concentration of 25 μg/mL showed a cell viability of more than 80% (Fig. 1). Therefore, 12 species of vegetable sprout extract at concentration of 25 μg/mL were used for further experiments.

Fig. 1. Effects of 12 species from vegetable sprouts extract on cell viability in 3T3-L1 cells. Values are mean±standard deviation. Different letters (a-d) are significantly different (P<0.05) among groups by Duncan’s multiple range test.

2. Effects of vegetable sprout ethanol extracts on lipid accumulation in 3T3-L1 cells

To investigate the effects of 12 species from vegetable sprout extract on the inhibition of adipocyte differentiation, we investigated the treatment of 12 species from vegetable sprout extract at concentration of 25 μg/mL in the differentiated 3T3-L1 cells using Oil Red O staining. As shown in Fig. 2, differentiated 3T3-L1 cells showed an increased number of lipid droplets, compared with the non-differentiated normal group. However, treatment of 12 species of vegetable sprouts extract showed a decreased number of lipid droplets.

Fig. 2. Effects of 12 species from vegetable sprouts extract on lipid droplet in the differentiated 3T3-L1 cells by light microscopy.

As shown in Fig. 3, quantification of the lipid droplets revealed that the triglyceride (TG) content increased to 168.45% in differentiated 3T3-L1 cells, compared to nondifferentiated 3T3-L1 cells (100.00%). The 12 species of vegetable sprout extract showed reduced TG content in the differentiated 3T3-L1 cells. The following order of TG content was observed, from lowest to highest: red radish (124.63%)

Fig. 3. Effects of 12 species from vegetable sprouts extract on TG accumulation in differentiated 3T3-L1 cells. Values are mean±standard deviation. TG: triglyceride. Different letters (a-f) are significantly different (P<0.05) among groups by Duncan’s multiple range test.

3. Effects of vegetable sprout ethanol extracts on cell viability in LPS-induced HT-29 cells

To evaluate the protective effects of 12 species from vegetable sprouts against intestinal inflammation, we confirmed the effects of vegetable sprouts on the cell viability of HT-29 cells induced by LPS. As shown in Fig. 4, treatment with 500 μg/mL of LPS decreased the cell viability to 53.64% relative to that of non-treated normal group. However, treatment with 12 species of vegetable sprouts extract at concentration of 25 μg/mL significantly increased the cell viability. The cell viability of 12 species from vegetable sprouts was high in the order of red radish (109.61%) >green broccoli (101.15%) >pea (97.97%), yellow buckwheat (95.50%), clover (93.13%), barley (91.35%) >yellow broccoli (90.16%) >rape (90.07%), kohlrabi (88.54%), red buckwheat (88.38%), wheat (88.36%), alfalfa (87.56%).

Fig. 4. Effects of 12 species from vegetable sprouts extract on cell viability in LPS-induced HT-29 cells. Values are mean±standard deviation. LPS: lipopolysaccharide. Different letters (a-e) are significantly different (P<0.05) among groups by Duncan’s multiple range test.

4. Effects of vegetable sprout ethanol extracts on LDH release in LPS-induced HT-29 cells

The LDH release of 12 species from vegetable sprouts ethanol extracts in LPS-induced HT-29 cells is shown in Fig. 5. The cells were treated with 500 μg/mL LPS elevated LDH release compared to that in non-treated cells. However, 25 μg/mL of 12 species from vegetable sprouts ethanol extracts-treated group significantly suppressed the LDH release in LPS-induced HT-29 cells than LPS-treated control group. In addition, treatment of alfalfa sprout extract showed the lowest LDH release at 103.31%, followed by the red radish sprout extract-treated group, which had a lower LDH release of 104.64%. Therefore, these results demonstrated the protective effects of 12 species from vegetable sprouts ethanol extracts against intestinal inflammation in LPS-induced HT-29 cells.

Fig. 5. Effects of 12 species from vegetable sprouts extract on LDH release in LPS-induced HT-29 cells. Values are mean±standard deviation. LDH: lactate dehydrogenase, LPS: lipopolysaccharide. Different letters (a-g) are significantly different (P<0.05) among groups by Duncan’s multiple range test.

5. Effects of vegetable sprout ethanol extracts on inflammatory cytokines in LPS-induced HT-29 cells

The anti-inflammatory activity of 12 species from vegetable sprout ethanol extract was evaluated by measuring the concentration of inflammatory cytokines such as IL-6 and TNF-α released from HT-29 cells treated with LPS (Fig. 6). It was found that HT-29 cells treated with 500 μg/mL of LPS significantly increased the levels of IL-6 and TNF-α than those of LPS untreated cells. As shown in Fig. 6A, cells treated with 12 species from vegetable sprout ethanol extract, except wheat sprout extract, showed significant reduction in the IL-6 levels in the LPS-treated HT-29 cells. In particular, the IL-6 production of the kohlrabi and red radish sprout-treated group showed 77.41% and 77.59%, respectively, resulting in the highest inhibitory effects of IL-6 levels among other vegetable sprouts extract. In addition, all of the vegetable sprout ethanol extract-treated group showed a significant reduced TNF-α levels, compared with LPS-treated control group (Fig. 6B). Especially, TNF-α production of the kohlrabi sprout-treated group showed the lowest values at 84.25%. In addition, red radish and pea sprout-treated group showed reductions of 85.08% and 85.15% in TNF-α production, respectively. Therefore, these results indicated the anti-inflammatory effects of 12 species from vegetable sprout ethanol extract through the inhibition of inflammatory cytokines such as IL-6 and TNF-α.

Fig. 6. Effects of 12 species from vegetable sprouts extract on (A) IL-6 and (B) TNF-α in LPS-induced HT-29 cells. Values are mean±standard deviation. IL-6: interleukin-6, TNF-α: tumor necrosis factor-α, LPS: lipopolysaccharide. Different letters (a-i) are significantly different (P<0.05) among groups by Duncan’s multiple range test.

Consumption of high fat diet increased blood LPS levels compared with the consumption of normal diet group, it called metabolic endotoxemia^4,16). The endotoxemia induced by consumption of high fat diet leads to inflammatory responses in the various tissues, including liver, adipose, and gut¹⁷⁾. Although the cellular pathway leading the mechanisms of endotoxemia are not yet fully understood, several studies have demonstrated that the gut microtbiota dysfunction contributes to endotoxemia^17-19). The consumption of high fat diet increased LPS production from gut microbiota, which increased gut permeability and the circulation of LPS in the body^18,19). Therefore, obesity induced by high fat diet is closely associated with intestinal inflammation due to endotoxemia.

The red radish has long been used for its medicinal properties, such as antidiarrheal, anti-inflammatory, antioxidant, and gastroprotective effects worldwide²⁰⁾. In addition, buckwheat has been widely used in traditional medicine for the treatment of ulcerative gastrointestinal diseases, intestinal bleeding, and boosting body energy²¹⁾. Barley is used in traditional medicine to treat various inflammatory and cardiovascular diseases, despite a lack of pharmacological understanding behind its actions²²⁾. This traditional evidence suggests a correlation with the potential of several vegetable sprouts in offering protective effects against obesity and intestinal inflammation in this study. Recent studies have reported that dietary factors such as grape seed, flavonoids, and probiotics inhibited metabolic endotoxemia in the intestinal inflammation of obese model^5,23,24). In addition, many studies focused on the development of therapeutic agent derived from natural product without side effects and any toxicity for both obesity and intestinal inflammation. Therefore, we investigated the effects of 12 species from vegetable sprouts extract on obesity and intestinal inflammation under cellular system.

To evaluate the anti-obesity effects of 12 species from vegetable sprouts extract, we investigated effects of 12 species from vegetable sprouts on adipogenesis in 3T3-L1 cells. The cytotoxicity of ethanol extracts from 12 species of vegetable sprouts was evaluated using the MTT assay, with cells treated 12 species of vegetable sprouts at a concentration of 25 μg/ml for 3 days. The results indicated that the extracts maintained cell viability of over 80%, confirming that this concentration did not exhibit any cytotoxicity. Therefore, we performed further experiments using this concentration.

Oil Red O staining was used to assess the inhibition of preadipocyte differentiation by vegetable sprout ethanol extracts. The Oil Red O staining reagent binds to intracellular lipids, resulting appears red color²⁵⁾. Differentiated 3T3-L1 cells were stained with Oil Red O, observed through a microscope, and quantified (Figs. 2, 3). Cells treated only MDI actively induced adipocyte formation compared to cells not treated with MDI, leading to an increase in the lipid droplets and TG accumulation. However, cells treated with 12 species of vegetable sprout ethanol extracts showed a significant decrease in lipid droplet, confirming the reduction of the TG production. In particular, the TG decrease was noticeable in treatment of red radish sprout. These results indicate that sprouts, in particular red radish sprout, can effectively reduce adipocyte differentiation and lipid accumulation. In a recent study, wheat, barley and broccoli sprouts have inhibitory effects on lipid accumulation in 3T3-L1 cells^26-28). Similar to our study, Lee et al.²⁹⁾ reported that treatment with red radish coral spout extract decreased body weight in the high fat diet-induced obese mice and Kim et al.³⁰⁾ reported that red radish sprout extract decreased lipid accumulation by regulation of adipogenic transcription factors in 3T3-L1 cells.

We investigated the effects of 12 species from vegetable sprouts extract on intestinal inflammation in LPS-induced HT-29 cells. The treatment of LPS in the HT-29 cells significantly decreased cell viability and increased LDH release, indicating intestinal cell damage induced by LPS. However, treatment of 12 species from vegetable sprouts extract showed increase cell viability of over 80%. In particular, red radish sprout-treated group exhibited significantly higher cell viability compared to the other vegetable sprouts-treated groups.

In addition, treatment of 12 species from vegetable sprouts extract significantly decreased LDH release from HT-29 cells compared to the LPS-treated group. Especially, alfalfa and red radish-treated groups showed the lowest LDH release values among the other vegetable sprouts-treated groups. The LDH released into the culture medium is directly related to cell death and release, which can indicate changes in cell viability and toxicity³¹⁾. Therefore, we confirmed that the treatment of 12 species from vegetable sprouts extract attenuated LDH release, demonstrating protective effects of 12 species from vegetable sprouts extract, in particular alfalfa and red radish, against inflammatory response-induced intestinal cell damage in LPS-treated HT-29 cells.

LPS, produced by the outer membrane of Gram-negative bacteria, is known as an endotoxins³²⁾. The LPS is a potent activator of inflammatory response by activation of macrophages to release excessive amounts of inflammatory cytokines^32,33). Several studies have demonstrated that the exposure of LPS leads to intestinal tissue damage, compromising intestinal permeability and facilitating the transport of proinflammatory cytokines to other tissues³⁴⁾. In particular, obesity caused by over-consumption of high fat diet, is associated to systemic inflammation in various organs, including liver, adipose, and brain³⁵⁾. Therefore, recent research has focused on improving intestinal inflammation as a strategy to treat and prevent obesity^36,37). In this study, we examined the anti-inflammatory effects of 12 species from vegetable sprouts extract by measurement of inflammatory cytokines such as IL-6 and TNF-α. The LPS-treated control group exhibited a significant increase in both IL-6 and TNF-α, indicating an inflammatory response in the HT-29 cells induced by LPS. However, treatment of 12 species from vegetable sprouts, except for wheat sprouts, resulted in decrease of IL-6 production compared with LPS-treated control group. In addition, all vegetable sprouts-treated group showed inhibited TNF-α production than control group. Especially, treatment of kohlrabi and red radish sprouts showed the lowest levels of both IL-6 and TNF-α production among other vegetable sprouts-treated groups. Previous study reported that administration of radish sprout inhibited serum inflammatory cytokines, such as IL-6, IL-1β, and TNF-α, in the dextran sulfate sodium-induced colitis mouse model³⁸⁾. In addition, administration of buckwheat sprouts inhibited inflammatory cytokines, including IL-6, IL-8, and TNF-α in LPS-treated human intestinal epithelial cells and mice³⁹⁾. This study also demonstrated the protective effects of 12 species from vegetable sprouts, commonly consumed in Korea, against intestinal inflammation in LPS-induced HT-29 cells. In particular, the expression of inflammatory cytokines such as IL-6 and TNF-α is known to be up-regulated through the activation of nuclear factor kappa B (NF-κB) signaling⁴⁰⁾. To clarify these results, further studies on the anti-inflammatory mechanisms of 12 vegetable sprout species are needed, including the measurement of proteins and mRNA associated with the NF-κB signaling in LPS-induced HT-29 cells.

This study demonstrated the comparison of anti-adipogenesis and anti-inflammatory activities among 12 species of vegetable sprouts in the differentiated 3T3-L1 and LPS-treated HT-29 cells, respectively. Treatment of 12 species from vegetable sprouts inhibited lipid accumulation, with red radish sprout showed the most inhibition of lipid accumulation. The 12 species from vegetable sprouts suppressed inflammation by increasing cell viability, reducing LDH release, and decreasing inflammatory cytokine levels. These findings suggests that vegetable sprouts, particularly red radish sprouts, could be a promising natural products for improving obesity and intestinal inflammation.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2022R1I1A3069595).

No potential conflict of interest relevant to this article was reported.