SUPPLEMENTARY MATERIAL
Title: Atheroprotective potentials of curcuminoids against ginger extract in hypercholesterolemic rabbits
Authors: Elseweidy M.Ma*, Younis N.Na, Elswefy S.Ea, Abdallah F.Ra, El-Dahmy S.Ib, Elnagar Ga, Kassem H.Ma
Affilliation: aBiochemistry department, bPharmacognosy department, Faculty of Pharmacy, Zagazig
University, Zagazig, 44519, Egypt.
*Corresponding author: Professor Dr Mohamed M Elseweidy. E-mail: mmelseweidy@yahoo.com
Abstract
The antiatherogenic potentials of total ginger (Zingiber officinale) extract (TGE) or curcumenoids
extracted from turmeric (Curcuma Longa); members of family Zingiberacea were compared in
hypercholesterolemia. Rabbits were fed either normal or atherogenic diet. The rabbits on atherogenic
diet received treatments of TGE or curcumenoids and placebo concurrently for 6 weeks (n=6). The antiatherogenic effects of curcuminoids and ginger are mediated via multiple mechanisms. This effect was
correlated to their ability to lower CETP. Ginger extract exerted preferential effects on plasma lipids,
reverse cholesterol transport, cholesterol synthesis and inflammatory status. Curcuminoids, however,
showed superior antioxidant activity.
Keywords: hypercholesterolemia, ginger, curcuminoids, HDL-c, CETP, ox-LDL, cardiovascular risk.
Experimental
1. Plant material and preparation of extracts
The fresh rhizomes of ginger and turmeric were purchased from Egyptian herbal market, Cairo, Egypt on
January 2012. The identification of rhizomes was verified by Dr. A. el Hadad, Faculty of Science, Cairo
University.
Voucher specimen (No.201 and 202, respectively) were deposited in Pharmacognosy
Department, Faculty of Pharmacy, Zagazig University. Methanolic extract of fresh ginger rhizomes was
filtered and evaporated under reduced pressure at 45˚C (Choi et al. 2011). Curcumenoids were
extracted from Curcuma roots as previously prescribed (Piper et al. 1998). The active fraction
curcumenoids stored at 2-8°C.
2. Animals and experimental design
Twenty four New Zealand male rabbits weight 1.75 ± 0.25 kg were purchased from the farms of faculty
of Agriculture at Zagazig University. Rabbits were housed in stainless steel rodent cages under
environmentally controlled conditions and allowed one week for acclimatization with a 12 hours
dark/light cycle before experimental work. Rabbits were fed commercially available rabbit normal pellet
diet and water ad libitum.
Rabbits were randomly divided into four groups (n=6 each). Three groups were fed a high cholesterol
diet (HCD) diet for 6 weeks which consists of normal diet supplemented with 0.2% cholesterol dissolved
in coconut oil (Madhumathi et al. 2006). The first group received no treatment and served as
hypercholesterolemic control (placebo). The other two groups received either curcumenoids (50 mg/kg
body weight) (Piper, Singhal, Salameh, Torman, Awasthi and Awasthi 1998) or total ginger extract (TGE;
200 mg/kg body weight) (Bhandari et al. 1998) daily for 6 weeks. Both extracts were individually
suspended in distilled water using gum acacia as suspending agent and freshly delivered to animals via
oral gavages. Rabbits from normal group were fed normal diet contains 18% pure protein, 2.88% pure
fats, and 10.5% pure fibers, supplied from the faculty of Agriculture, Zagazig University, Zagazig, Egypt.
Experimental design and animal handling were performed according to the guidelines of the Ethical
Committee of the Faculty of Pharmacy, Zagazig University, for Animal Use and in accordance with the
recommendations of the Weather all report. The study was approved by the ethical committee of the
Faculty of Pharmacy, Zagazig University (study approval number: P8/2/2013).
3. Blood sampling
Fasting blood samples were collected from a marginal ear vein, centrifuged at 5000xg for 10 min to
separate serum. Serum total cholesterol (TC), triglycerides (TG), and HDL-C were determined
immediately on fresh serum samples and the remaining serum was aliquoted and stored at -20°C for
further determination of high sensitivity C-reactive protein (hsCRP), oxidized LDL (Ox-LDL), matrix
metalloproteinase-9 (MMP9), homocysteine (Hcy), lipoprotein(a) (Lp(a)), and CETP mass.
4. Tissue collection
Following blood collection, animals were scarified by exsanguination under ketamine anesthesia; livers
were removed, rinsed with cold saline and dried with filter paper. Liver specimen was quickly frozen in
liquid nitrogen (-170⁰C) and stored at -20⁰C for determination of hepatic cholesterol and the gene
expressions of APO A-I, APO A-II, APO B and CETP.
5. Analytical methods
Serum TC, TG and HDL-C were determined colorimetrically using assay kits supplied by Spinreact Co.,
Spain. LDL-C was calculated by Friedewald formula: LDL-C (mg/dl) = TC–[HDL-C+TAG/5] (Friedewald et al.
1972) and atherogenic index was calculated from the ratio LDL-C/HDL-C.
hsCRP, MMP9 and CETP mass were determined by sandwitch ELISA using rabbit ELISA kits (Uscn Life
Science Inc, China). Ox-LDL was measured by ELISA using rabbit Ox-LDL ELISA kit (NovaTeinBio, Inc.,
Cambridge, USA). Hcy was measured by ELISA using rabbit Homocysteine ELISA kit (Abnova Taiwan).
Lp(a) was measured by ELISA using rabbit lp(a) ELISA kit (Wuhan Eiaab science co. Ltd, Wuhan, China). In
all assays we followed manufacturers’ instructions.
The hepatic cholesterol was extracted as previously described (Bligh and Dyer 1959) then determined
colorimetrically using the same enzymatic kit as used for the serum.
6. RNA isolation and RT-PCR assay for APO A-I, APO A-II, CETP and APO B genes
For the detection of APO A-I, APO A-II, CETP and APO B genes by real-time polymerase chain reaction
(RT-PCR), RNA was extracted using SV Total RNA isolation system (Promega, Madison, WI, USA), reverse
transcribed into cDNA and amplified by PCR using RT-PCR kit (Stratagene, USA). The oligonucleotide
sequences of forward and reverse primers are as shown in table S1. The amplification reactions were
performed in a 50µl final volume, with thermal cycling conditions of 2min at 50⁰C, 10min at 95⁰C, and
40 cycles of 15s at 95⁰C, 30s at 60⁰C, 30s at 72⁰C, and 10min at 72⁰C. Cycle threshold (Ct) data were
normalized using GAPDH, which was stably expressed across all experimental groups. Relative gene
expression was calculated using the -2ΔΔCt method (Schmittgen and Livak 2008).
7. Statistical analyses
Statistical analyses of data were done by Prism 5, Graph pad, CA, USA. Results were expressed as mean
± SD. Results of placebo group were also expressed as a fold change over the normal group. Statistical
differences were sought using Student’s t-test or one way analysis of variance (ANOVA) followed by
Fisher’s least significant difference (LSD) post hoc test, taking p<0.05 as statistically significant.
Table S1: Sequence of the primers used for real-time PCR
Gene
Primer sequence
Annealing
Product size
Temp. (⁰C)
(bp)
61
172
60
141
60
445
61
297
61
215
Forward primer: 5'-TGTGTATGTGGATGCGGTCA-3'
Apo A-I
Reverse primer: 5'-ATCCCAGAAGTCCCGAGTCA-3'
Forward primer: 5'-AATGGTCGCACTGCTGGTAA-3'
Apo A-II
Reverse primer: 5'-TTGGCCTTCTCCACCAAATC-3'
Forward primer: 5'-GGTTGGGCATCAATCAGTCT-3'
CETP
Reverse primer: 5′-CAGCCATGATGTTGGAGATG-3′
Forward primer: 5′-TCCTCAGCAGATTCATGATTATCT-3′
Apo B
Reverse primer: 5′-AGCATTTTTAGCTTTTCAATGATT-3′
Forward primer: 5′- GTCGGTGTGAACGGATTTG-3′
GAPDH
Reverse primer: 5′- AAGATGGTGATGGGCTTCC-3′
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