My Blog

Effect of Blueberry Polyphenols on 3T3-F442A Preadipocyte Differentiation

To cite this article:
Shiwani S. Moghe, Shanil Juma, Victorine Imrhan, and Parakat Vijayagopal. Journal of Medicinal Food. May 2012, 15(5): 448-452. doi:10.1089/jmf.2011.0234.

Published in Volume: 15 Issue 5: May 2, 2012

Abstract

Today obesity is an epidemic, and its prevalence has increased significantly over the last few decades. To avoid excessive accumulation of fat, optimum energy intake along with regular exercise is mandatory. Polyphenols present in green tea, grape seeds, orange, and grapefruit combat adipogenesis at the molecular level and also induce lipolysis. However, very little is known regarding the role of blueberry polyphenols on adipocyte differentiation. Hence we tested the dose-dependent effects of blueberry polyphenols on mouse 3T3-F442A preadipocyte differentiation and lipolysis. 3T3-F442A preadipocytes were incubated with three doses of blueberry polyphenols (150, 200, and 250 μg/mL [BB-150, BB-200, and BB-250, respectively]), and intracellular lipid content, cell proliferation, and lipolysis were assayed. Blueberry polyphenols suppressed adipocyte differentiation determined by Oil Red-O staining and AdipoRed assay. Intracellular lipid content in control (11,385.51±1,169.6 relative fluorescence units) was significantly higher (P<.05) than with the three doses of blueberry polyphenols (8336.86±503.57, 4235.67±323.17, and 3027.97±346.61, respectively). This corresponds to a reduction of 27%, 63%, and 74%, respectively. Cell proliferation was observed to be significantly higher in the control (0.744±0.035 optical density units) than with BB-150 (0.517±0.031), BB-200 (0.491±0.023), and BB-250 (0.455±0.012). However, when tested for lipolysis, there was no significant difference observed among the groups. We conclude that blueberry polyphenols may play an effective role in inhibiting adipogenesis and cell proliferation.

fulltext content

Introduction

obesity is becoming an epidemic in both children and adults. Its prevalence has increased significantly over the last few decades, and it is the major risk factor for cardiovascular diseases and type 2 diabetes mellitus. Surplus energy from a well-fed state is stored as triglycerides in the adipocytes. Combating unnecessary adipogenesis at the molecular level can be beneficial to prevent diseases at a very early stage. Therefore, in vitrostudies to identify compounds that will interfere and inhibit adipogenesis are warranted. Fruits, vegetables, and legumes are not only rich in fiber but also abundant in antioxidants like polyphenols. Some polyphenols improve endothelial function and reduce total cholesterol and triglycerides and thereby are effective in the treatment of cardiovascular diseases.

They also demonstrate anti-inflammatory effects. Anthocyanins, compounds present in polyphenols, have antioxidant effects and suppress development of obesity in mice fed a high fat diet. Polyphenols stimulate adipokine secretion and attenuate gene expression of adipocyte-specific genes like peroxisome proliferator-activated receptor γ and CCAAT/enhancer binding proteins (α and β) in 3T3 L1 preadipocytes isolated from rats. Polyphenols present in green tea, grape seeds, orange, and grapefruit have been shown to inhibit adipogenesis and initiate lipolysis. Blueberry polyphenols have shown promising results in the treatment of cognitive impairment, ischemic heart disease, oxidative stress, and neurological degeneration. Blueberries have also been reported to attenuate diet-induced atherosclerosis in mice. Moreover, purified blueberry anthocyanins and blueberry juice are effective in preventing obesity in C57BL mice. However, to our knowledge, there has been no investigation of the effect of blueberry polyphenols on adipogenesis. Therefore, in this study we examined the effect of blueberry polyphenol extract on adipocyte differentiation and lipolysis.

Materials and Methods

Blueberry polyphenol extraction

Polyphenol extraction was performed by modification of a previously described procedure by Kim et al. Freeze-dried blueberry powder was provided by the US Highbush Blueberry Council (Folsom, CA, USA) and stored at −20°C in the dark. The blueberry powder consisted of a blend of Tifblue and Rubel blueberries in a 1:7 ratio. In brief, blueberry polyphenols were extracted with 80% ethanol in subdued light. After sonication of the extract for 20 min at room temperature under nitrogen, the extract was filtered through a Buchner funnel. The filtrate was then concentrated by rotary evaporation and lyophilized. The lyophilized polyphenol extract was stored in an amber-colored bottle at −20°C.

Polyphenol assay

Total polyphenol content of blueberry extract was determined using gallic acid as a standard and expressed as gallic acid equivalents using a previously described procedure.

Cell culture

3T3-F442A cells were obtained from Dr. Howard Green (Harvard Medical School) and cultured in Dulbecco's modified Eagle's medium containing 10% calf serum. After 24 h, that is, on Day 0, differentiation was initiated with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 167 nM insulin in the presence and absence of different concentrations (150, 200, and 250 μg/mL of medium) of blueberry polyphenol (BB-150, BB-200, BB-250, respectively). After 48 h (Day 2), the cells were switched to Dulbecco's modified Eagle's medium and 10% fetal bovine serum with or without blueberry polyphenol. Thereafter, the culture medium was replaced every 48 h with fresh medium of identical constitution. Analyses were performed on Day 8.

Oil Red-O staining

Cells were plated in a six-well plate at a density of 4×10 cells per well. They were allowed to differentiate in the presence and absence of blueberry polyphenols. On Day 8, cells were fixed with 10% formalin and then stained with Oil Red-O by modification of a previously described procedure.

AdipoRed assay

Cells were plated in six-well plates at a density of 4×10 cells per well and allowed to differentiate in the presence and absence of blueberry polyphenols. On Day 8 of differentiation, cells were treated with AdipoRed (Lonza, Houston, TX, USA), and total lipid content was determined as per the manufacturer's instructions.

Cell titer

The effect of blueberry extract on cell proliferation was determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay (Promega, Madison, WI, USA) as per the manufacturer's instructions. In brief, 4×10 cells were plated per well of a six-well plate. Cells were allowed to differentiate in the presence and absence of blueberry extract as mentioned in the above procedure. Cell viability was determined on Day 8. The cells were washed with Hanks' buffered salt solution and incubated with MTS reagent for 1–4 h at 37°C in a CO (5%) incubator. Absorbance was then measured at 490 nm in a plate reader.

Fatty acid assay

Cells were plated in six-well plates at a density of 4×10 cells per well and allowed to undergo differentiation without blueberry. On Day 8, varying doses of blueberry polyphenols were added to the wells, and the cells were placed at 37°C in a 10% humidified CO incubator for 3 days. On Day 11, free fatty acid content of the media was determined using the free fatty acid assay kit from BioVision (San Francisco, CA, USA). Values were corrected for the absorbance obtained for cell-free incubations.

Statistics

One-way analysis of variance along with Tukey's post hoc test was performed to assess the significance of differences among the groups. Data are mean±SE values. Results were considered significant when P<.05.

Results

When the cells were examined on Day 8 of differentiation, blueberry polyphenols caused a dose-dependent suppression of intracellular lipid accumulation within the 3T3-F442A cells. In order to highlight actual lipid accumulation, cells were fixed with 10% formalin and then stained with Oil Red-O. The result showed the dose-dependent decrease in triacylglycerol content with increasing concentrations of blueberry polyphenols. When the BB-250 group was observed under a high-resolution microscope, the cells had fine red-colored lipid droplets surrounding the inner lining of the cell membrane. To quantify the qualitative results obtained with Oil Red-O staining, we performed the AdipoRed assay. AdipoRed is used to quantify the intracellular lipid droplets formed during adipogenesis. The results confirmed that control cells had the highest amount of lipid concentration measured in relative fluorescence units. All blueberry polyphenol concentrations had significantly reduced cellular lipid content compared with the control (P<.05). The reduction was 26.8%, 62.8%, and 73.4% for blueberry doses of 150, 200, and 250 μg/mL, respectively. When the three blueberry doses were compared among themselves, BB-150 had significantly higher intracellular lipid content than BB-200 and BB-250 (P<.05); however, there was no significant difference between BB-200 and BB-250 (P>.05).
Cell viability was analyzed by MTS assay to determine the dose-dependent effect of blueberry polyphenols on cell proliferation. Compared with all the blueberry groups, cell proliferation was observed to be significantly higher in the control (P<.05). However, there was no significant difference among the three doses of blueberry polyphenols. Thus, blueberry polyphenol seems to inhibit cell proliferation.
The free fatty acid assay was performed to determine the effect of blueberry polyphenols on adipocyte lipolysis. There were no significant differences among the three doses of polyphenols .

Discussion

Our study demonstrated that polyphenols extracted from blueberry inhibit 3T3-F442A preadipocyte differentiation. Cells treated with blueberry polyphenols showed a dose-dependent reduction in intracellular lipid accumulation as demonstrated by Oil Red-O staining and AdipoRed assay. Oil Red-O stains only the fat droplets and gives a qualitative estimate of triacylglycerol accumulation in the cell. When cells start accumulating triacylglycerol, they become spherical in shape. Although we did not directly measure cell size, visual comparison of cell sizes and fat content between the control cells and blueberry polyphenol-treated cells indicated that both the cell size and cellular lipid content decreased with increasing concentrations of blueberry polyphenols. Previous studies have reported that polyphenols from green tea, as wells other polyphenolic compounds, such as resveratrol, genistein, and quercetin, also inhibited adipogenesis in 3T3-L1 cells.

Green tea polyphenols belong primarily to the catechin family, but blueberry polyphenols are a mixture of different phenolic compounds, including flavonoids, anthocyanins, proanthocyanidins, and hydrocinnamic acids. Thus, polyphenols as a whole appear to inhibit adipogenesis regardless of the nature of the individual compound. Preadipocyte differentiation involves the activation of several adipocyte genes, including peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, and differentiation-dependent factor 1/sterol regulatory element binding protein isoform.

Collectively, they regulate the sequence of adipocyte differentiation. Polyphenolic compounds such as epigallocatechin gallate from green tea, resveratrol from red grapes, and curcumin from turmeric have been shown to suppress one or more of these genes and thus inhibit differentiation of 3T3-L1 cells.

Although in this study we did not investigate the mechanism of blueberry polyphenol-induced adipocyte differentiation, it is likely that blueberry polyphenols also inhibit adipocyte differentiation by one of the above mechanisms. Blueberry polyphenols decreased cell viability as determined by the MTS assay.

In this procedure, 3T3-F442 preadipocytes were differentiated in the presence and absence of varying doses of blueberry polyphenols, and cell proliferation was determined on Day 8 of differentiation. Although cell proliferation was reduced significantly in the blueberry-treated cells compared with control cells, cell proliferation did not differ across the three doses of blueberry polyphenols. We also observed inhibition of cell proliferation in cultures incubated with a polyphenol concentration as low as 75 μg/mL (data not shown).

Recent studies have shown that epigallocatechin gallate from green tea and a combination of genistein, quercetin, and resveratrol also inhibited proliferation of 3T3-L1 preadipocytes in culture. Inhibition of preadipocyte proliferation by blueberry polyphenols could reduce the number of differentiated adipocytes. Thus, it appears that polyphenols reduce adipocyte lipid accumulation not only by inhibiting intracellular lipid content but also by reducing adipocyte number. The net intracellular lipid content in differentiated adipocytes represents a balance between total triacylglycerol synthesis and lipolysis. Increased triacylglycerol synthesis accompanied by reduced β-oxidation will result in substantial intracellular lipid accumulation. Therefore, in our study in order to determine whether blueberry polyphenols affect adipocyte lipolysis, we performed lipolysis assay in mature adipocytes exposed to different polyphenol doses. The result indicates that blueberry polyphenols have no effect on adipocyte lipolysis.

However, it is possible that the assay was not sensitive enough to detect very small changes in lipolysis. The sensitivity limit of the current assay was 2 μM free fatty acid. Use of a more sensitive method might have produced different results. The blueberry polyphenol doses used in our study were selected based on preliminary results of cell viability and adipogenesis. Concentrations lower than 150 μg/mL had very little inhibitory effect on adipogenesis.

Similarly, polyphenol doses higher than 250 μg/mL appeared to cause excessive cell loss. Our test dose of 250 μg/mL blueberry polyphenols is equivalent to approximately 8 g of freeze-dried blueberry powder or about one-half cup of fresh blueberry. Although this dose of blueberry consumption is easily achievable in humans, we cannot extrapolate the results of cell culture studies to humans.

Blueberry polyphenols have been shown to improve insulin sensitivity in 3T3-L1 adipocytes and obese, insulin-resistant men and women. Furthermore, consumption of whole blueberries decreased cardiovascular risk factors in people with metabolic syndrome. Our study demonstrates for the first time that blueberry polyphenols also inhibit adipocyte differentiation in 3T3-F442 preadipocytes. Further studies are needed to elucidate the physiological significance of these findings.

Acknowledgments

Our sincere thanks to Dr. Barney Venables and Mr. Andrew Barker, University of North Texas, Denton, TX, for their assistance with the use of a lyophilizer. This study was supported by the Human Nutrition Research Fund, Texas Woman's University.

High Temperature- and High Pressure-Processed Garlic Improves Lipid Profiles in Rats Fed High Cholesterol Diets

Chan Wok Sohn,1,* Hyunae Kim,1,* Bo Ram You,1 Min Jee Kim,1 Hyo Jin Kim,1 Ji Yeon Lee,1Dai-Eun Sok,2 Jin Hee Kim,3 Kun Jong Lee,4 and Mee Ree Kim1

1Department of Food & Nutrition and 2College of Pharmacy, Chungnam National University, Daejeon, Korea.3Department of Herbal Skin Care, Daegu Hanny University, Gyeongsan, Korea.

4Department of Food & Nutrition, Seoil College, Seoul, Korea.

ABSTRACT

Garlic protects against degenerative diseases such as hyperlipidemia and cardiovascular diseases. However,raw garlic has a strong pungency, which is unpleasant. In this study, we examined the effect of high temperature/high pressure-processed garlic on plasma lipid profiles in rats. Sprague–Dawley rats were fed a normal control diet, a highcholesterol (0.5% cholesterol) diet (HCD) only, or a high cholesterol diet supplemented with 0.5% high temperature/highpressure-processed garlic (HCP) or raw garlic (HCR) for 10 weeks. The body weights of the rats fed the garlic-supplementeddiets decreased, mostly because of reduced fat pad weights. Plasma levels of total cholesterol (TC), low-density lipoproteincholesterol, and triglyceride (TG) in the HCP and HCR groups decreased significantly compared with those in the HCD group.Additionally, fecal TC and TG increased significantly in the HCP and HCR groups. It is notable that no significant differencesin plasma or fecal lipid profiles were observed between the HCP and HCR groups. High temperature/high pressure-processedgarlic contained a higher amount of S-allyl cysteine than raw garlic (P < .05).

The results suggest that high temperature/high pressure-processed garlic may be useful as a functional food to improve lipid profiles

KEY WORDS: S-allyl cysteine high temperature/high pressure-processed garlic plasma lipid profiles

INTRODUCTION

Dyslipidemia associated with elevated cholesterol and triglycerides (TGs) is a leading cause of cardiovasculardisease. Accordingly, attempts have been made to identify

natural products that reduce blood lipid levels. Garlic (Allium sativum L.) has been consumed as a medicinal plant and as a food seasoning for millennia.

Consuming garlic is very helpful for regulating plasma lipidlevels and plasma anticoagulant activity as well as for preventingatherosclerosis.

Additionally, hepatoprotective, anticancer, immune-enhancing, antioxidant, and chemopreventive activities of garlic have been reported, but the cardioprotective effects of garlic may be the most studiedhealth-promoting effects. There is no doubt that garlic and garlic preparations possess anticoagulant ability. However, Kerckhoffs et al. reported that it is still uncertain whether garlic or garlic preparations can be used as lipid-lowering agents. Controversy exists regarding the plasma lipid regulating and antioxidant properties of garlic.The health-promoting effects of garlic are derived from many bioactive compounds.

Most bioactive substances in garlic are affected by cooking or other processing methods.In particular, the main bioactive sulfur-containing compound in fresh garlic is allicin, which is very unstable underheat and is unpleasant to eat.

Accordingly, the optimal conditions for preparing processed garlic are very important.However, knowledge about the influence of processingmethods on bioactive properties of garlic is limited.

In a preliminary experiment, the pungency of high temperature/high pressure-processed garlic was much weaker than that of raw garlic. However, as pungency still remains in processed garlic, green tea leaves were added to the garlic during high pressure cooking and removed after processing.

The aim of this investigation was to determine the effect of high temperature/high pressure-processed garlic on the major bioactive sulfur-containing compounds in garlic as well as their bioactivity, particularly on lipid profiles of rats fed a high-cholesterol diet (HCD).

MATERIALS AND METHODS

Garlic processing

Fresh garlic samples were obtained from a local market in Daejeon, Korea. The garlic was peeled, washed in water, and processed by two different methods. Raw garlic was prepared by crushing the garlic samples in a blender followed by freeze-drying. The high temperature/high pressure-processed garlic was processed at 120C and high pressure of 1.5 kgf/m2 with 1% green tea leaves for 20 min in an autoclave(model HB-506-6, Hanbaek Scientific Co., Seoul, Korea).

The green tea leaves were removed, and the garlic was freeze-dried and powdered in a mill (particle size, < 25 lm). All garlic samples were stored at - 70C until the experiment.

Hypercholesterolemic rat model and diets

Male Sprague–Dawley rats (weight, 120–140 g; 4–5weeks of age) were obtained from Animal Husbandry ofDamul Science (Daejeon) and housed in polycarbonate cages with controlled temperature (23 – 3C) and humidity(55 – 10%) under a 12-h light:dark cycle. The rats were fed apelleted commercial diet (Samyang Co., Seoul) for the first 2 weeks. After acclimation, the rats were randomly dividedi nto four treatment groups of 10 rats each and allowed free access to water and the assigned diets for 10 weeks. The normal control (NC) diet was the AIN 93 diet. The HCD was 97.5% AIN 93 G diet with 0.5% cholesterol and 2% soybean oil. The crushed raw garlic (HCR) diet was the HCD plus0.5% crushed garlic, and the HCP diet was the HCD diet with 0.5% high temperature/pressure-processed garlic. The controland experimental diets were isoenergenic. Diet composition was as follows: NC diet contained fat at 16.0% of kcal,protein at 20.3% of kcal, and carbohydrate at 63.8% of kcal;HCD diet contained fat at 20.1% of kcal, protein at 20.3% ofkcal, and carbohydrate at 60.0% of kcal; HCR (raw garlic)diet contained fat at 20.3% of kcal, protein at 20.0% of kcal,and carbohydrate at 59.8% of kcal; and HCP (high temperature/high pressure-processed garlic) diet contained fat at20.2% of kcal, protein at 20.0% of kcal, and carbohydrate at 59.8% of kcal. Energy content was calculated using 4 kcal/gfor protein and carbohydrate and 9 kcal/g for fat.The body weights of the rats, feces, and food were measured daily, and the feed efficiency ratio (FER) was calculated throughout the experiment. Animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

After 10 weeks, rats were fasted for 12 h and sacrificed by cervical decapitation, and fasting blood samples were collected in heparinized tubes. Heart, kidneys,spleen, and liver were immediately removed, rapidly washed in saline buffer, collected into cryovials, weighed, andimmediately stored in liquid nitrogen for lipid peroxidationand antioxidant marker assays.Plasma lipid profile measurementsPlasma TG, total cholesterol (TC), and high-density lipoprotein cholesterol concentrations were determined enzymatically using immuno assay kits (Asan Pharmaceuticals,Seoul) and an enzyme-lined immunosorbent assay reader(Pharmacia Biotech, Cambridge, United Kingdom) according to the manufacturer’s protocol. Plasma low-density lipoproteincholesterol (LDL-C) was calculated using the Friedewald equation.

Measurements of fecal lipid profile (TC and TG)

Feces powder was homogenized with chloroform:methanol(2:1) to a final volume 20 times the volume of the feces sample (0.2 g in 4mL of solvent mixture). After dispersion,the whole mixture was agitated for 15–20 min on a shaker at room temperature. The solvent was washed with water (1mL). After vortex-mixing, the mixture was centrifuged at low speed (1,500 g) to separate the two phases. After centrifugation, the upper phase was siphoned off, and the lower chloroform phase containing lipids was evaporated under vacuum in a rotary evaporator.Then, the residue was dissolved in ethanol and analyzed at 500 and 550 nm using TC and TG kits purchased from Asan Pharmaceuticals.

Histopathological examination of liver

Livers were excised, fixed in formalin solution, stained with hematoxylin–eosin, and examined under a light microscope to assess fatty changes.

Determination of alliin and S-allyl cysteineby high-performance liquid chromatography

Dry garlic powder extraction under alliinase-inhibiting conditions. Dry garlic powder (1 g) was extracted at room temperature using 10mL of methanol:water (80:20, vol/vol)and 0.05% formic acid (pH 3). An aliquot was diluted fivetimes and filtered (pore size, 0.2 lm). Then, 10 lL was injected into the high-performance liquid chromatography(HPLC) apparatus.

Dry garlic powder extraction under alliinase-activatingconditions.

Dry garlic powder (1 g) was extracted at roomtemperature using 10mL of water (pH 6–8). An aliquot was diluted five times and filtered (pore size, 0.2 lm), and 10 lL was injected.

HPLC instrumentation and method.

Garlic extracts were subjected to HPLC using a Varian Prostar 210 pump and a Prostar 325 ultraviolet-visible detector (Varian Inc.,Santa Clara, CA, USA). Compounds were separated on a150- · 3-mm (i.d.) (3 lm particle size) Thermo Quest FortisC18 column at 20C (Fortis Technologies Ltd., Neston,Cheshire, United Kingdom) and an ultraviolet detector operatedat 208 nm. The column flow rate was 0.4 mL/min.The mobile phase consisted of (A) 20mM sodium dihydrogen phosphate and 10mM heptanesulfonic acid, pH 2.1(adjusted with 85% orthophosphoric acid) and (B) acetonitrile:20mM sodium dihydrogen phosphate and 10mM heptanesulfonic acid, pH 2.1 (50:50, vol/vol). Data were acquired using Galaxie software from Varian. Alliin, allicin, and S-allylcysteine standards were purchased from LKT Labs (St. Paul,MN, USA). Quantitative HPLC determinations were performedbased on standard curves.

Statistical analysis

All data are presented as mean – SD values. An analysis of variance followed by Duncan’s multiple range test was performed to evaluate differences among the groups, using SAS version 6.0 (SAS Institute, Cary, NC, USA). Statistical significance was defined as P < .05.

RESULTS

Body and organ weights

After 10 weeks of treatment, rat body weights increased significantly in the HCD group(554.7 – 29.0 g), compared with that in the NC group (525.0 –24.9 g) group

(P < .05). Treatment with HCR (511.4 – 26.1 g) or HCP (521.2 – 26.2 g) restored body weights to control levels. The FER in both the HCP and HCR groups decreased significantly, whereas the fecal weight in the HCP and HCR groups increased significantly, compared with those of the HCD group. The weights of liver, kidney, spleen, and heart increased in all groups, but the liver and heart weights in the HCP group were significantly lower than those in the HCDgroup (P < .05) . Visible fat deposition in the retroperitoneal,mesenteric, epididymal, spleen, and total fat pad areas of rats decreased significantly in the HCR and HCPgroups (P < .05) . The amount of visible fat, except

inguinal fat, was not significantly different in the HCR and HCP groups from that of the NC group.

Plasma and fecal lipid profiles.

Rats fed a diet enriched with high cholesterol for 10 weeks developed hypercholesterolemia; plasma TC was 192.8 mg/dL, versus 70.8 mg/dL in the NC group. The levels of TC,TG, and LDL-C in plasma decreased significantly in groups fed the processed garlic-supplemented diet compared withthe HCD group (P < .05), demonstrating that dietary supplementation with processed garlic improved lipid profiles.

In contrast, fecal levels of TC and TG (in lmol/day) increased significantly in the HCP group compared with those in the HCD group, demonstrating that high temperature/highpressure-processed garlic supplementation results in cholesterolexcretion via the feces .

Serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase,and lactate dehydrogenase

The levels of aspartate aminotransferase, alanine aminotransferase,alkaline phosphatase, and lactate dehydrogenasein serum were significantly higher in the HCD group than in the NC group (P < .05), whereas these values decreased in the processed garlic-supplemented diet group. Aspartate andalanine aminotransferase levels decreased remarkably in the HCR and HCP groups compared with those in the HCDgroup . Furthermore, serum glucose increased significantly in groups treated with garlic compared with theHCD group (P < .05), although albumin and protein levels were not significantly different .

Histological changes in liver

All rats in the NC group had normal liver histologica lfindings. However, the HCD group had prominent microvesicular steatosis and fatty changes around the portal triad in the liver. Livers from the processed garlic-treatedgroup showed only microvesicular steatosis, which was less extensive than that in livers from rats receiving HCD alone.Major sulfur compounds in high temperature/highpressure-processed garlic by HPLCMajor sulfur-containing compounds such as allicin, alliin,and

S-allyl cysteine in raw, high temperature, and high pressure-processed garlic were determined by HPLC. Allicin content in raw garlic was 2.6 mg/g,whereas that in high temperature/high pressure-processedgarlic was very low at 0.09 mg/g. Additionally, raw garliccontained 4.29 mg/g alliin, whereas high temperature/highpressure-processed garlic contained 1.76 mg/g alliin. However,S-allyl cysteine content was twofold higher in the high temperature/high pressure-processed garlic than in raw garlic.

DISCUSSION

Bioactive compounds in garlic are affected by cooking or other processing methods. Accordingly, the optimal conditions for preparing processed garlic are very important. However, knowledge about the influence of processing methods on bioactive properties of garlic is limited.

Therefore, the aims of this investigation were to determine the effects of high temperature and high pressure processingon the major bioactive sulfur-containing compounds of garlic as well as their bioactivity, particularly as they affected the lipid profiles of rats fed HCD. We demonstrated that plasma TC and LDL-C levels in rats fed HCD (0.5% cholesterol) for only 10 weeks increased by 2.7 and 5.7 times, respectively, compared with those in rats fed NC diet. HCD supplemented with processed garlic or high temperature/high pressure-processed garlic significantly reduced plasma TC, TG, and LDL-C levels inrats but significantly increased fecal TC and TG levels,compared with those in rats fed HCD only. This is consistent with previous findings that plasma lipid levels decreased significantly in rats fed a processed garlic product and HCD compared with those fed HCD only.

Previous studies have shown that raw garlic has profoundly decreases TC andTG levels without changing high-density lipoprotein cholesterol levels in rats fed HCD, whereas boiled garlic haslittle effect on these parameters.

In a human study, supplementation with raw garlic, powdered garlic, or aged garlic extract (4 g/day) for 6 months significantly lowered LDL-C and other plasma lipid levels in adults with moderate hypercholesterolemia. Our findings in rats provide further support for the antihyperlipidemic,antihypercholesterolemic, and anti-atherosclerotic actions of processed garlic products because both crushed raw garlic and high temperature/high pressure-processed garlic exhibited remarkable antihyperlipidemic action by decreasing the levels of TG, LDL-C, and TC in plasma and increasing the levels of TG and TC in feces.Very low amounts of allicin and alliin were detected in the high temperature/high pressure-processed garlic whereas the S-allyl cysteine level was approximately twofold greater in the high temperature/high pressure-processed garlic than in raw garlic. S-Allyl cysteine is the main component of aged garlic extract, which decreases TG and TC.In particular, S-allyl cysteine, a water-soluble organosulfur compound, is a potent inhibitor of cholesterol synthesis and hence may be the major component of garlic responsible for reducing plasma cholesterol level.The data from our study indicate that high temperature/high pressure-processed garlic improved lipid profiles,particularly decreases in TC and TG. Our studies further revealed that the lipid-lowering action of high temperature/high pressure-processed garlic originates, inpart, from the organo sulfur compounds, especially S-allylcysteine. Improved lipid profiles following ingestion of high temperature/high pressure-processed garlic might be derived from the sulfur-containing compounds such as Sallylcysteine, but not allicin. High temperature/highpressure-processed garlic is easily consumed by humans because of the lack of pungency without a loss in bioactivity.

In conclusion, high temperature/high pressure-processed garlic contained bioactive sulfur compounds in comparable amounts to those in raw garlic, particularly S-allyl cysteine.High temperature/high pressure-processed garlic improved plasma lipid profiles in rats fed a high cholesterol-containing diet, possibly by interrupting the enterohepatic circulation of cholesterol and cholesterol metabolites. These results suggest that high temperature/high pressure-processed garlic may have efficacy as a functional food for improving blood lipid profiles.

ACKNOWLEDGMENT

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology (grant NRF 2009-0077171).

Recent Posts

Categories

journal of medicinal food

blueberry for weight

garlic, good for lipid profiles?