Zerumbone là gì

Hassan Othman Hemn,1,2 Muhammad Mustapha Noordin,1 Heshu Sulaiman Rahman,1,2 Hamza Hazilawati,1 Abubakr Zuki,1 Max Stanley Chartrand3  

1Department of Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia; 2College of Veterinary Medicine, University of Sulaimani, Sulaimani City, Kurdistan, Republic of Iraq; 3DigiCare, Behavioral Research, Casa Grande, AZ, USA

Abstract: Owing to the high incidence of cholesterol-induced cardiovascular disease, particularly atherosclerosis, the current study was designed to investigate the preventive and therapeutic efficacies of dietary zerumbone [ZER] supplementation on the formation and development of atherosclerosis in rabbits fed with a high cholesterol diet. A total of 72 New Zealand white rabbits were divided randomly on two experimental studies carried out 8 weeks apart. The first experiment was designed to investigate the prophylactic efficacy of ZER in preventing early developed atheromatous lesion. The second experimental trial was aimed at investigating the therapeutic effect of ZER in reducing the atherosclerotic lesion progression and establishment. Sudanophilia, histopathological, and ultrastructural changes showed pronounced reduction in the plaque size in ZER-medicated aortas. On the other hand, dietary supplementation of ZER for almost 10 weeks as a prophylactic measure indicated substantially decreasing lipid profile values, and similarly, plaque size in comparison with high-cholesterol non-supplemented rabbits. Furthermore, the results of oxidative stress and antioxidant biomarker evaluation indicated that ZER is a potent antioxidant in suppressing the generation of free radicals in terms of atherosclerosis prevention and treatment. ZER significantly reduced the value of malondialdehyde and augmented the value of superoxide dismutase. In conclusion, our data indicated that dietary supplementation of ZER at doses of 8, 16, and 20 mg/kg alone as a prophylactic measure, and as a supplementary treatment with simvastatin, significantly reduced early plague formation, development, and establishment via significant reduction in serum lipid profile, together with suppression of oxidative damage, and therefore alleviated atherosclerosis lesions.

Keywords: zerumbone, antihypercholesterolemic, antioxidant, atherosclerosis, rabbit model

Introduction

Hypercholesterolemia refers to elevated levels of cholesterol in the blood and is considered to be a critical step in the initiation and development of atherosclerosis.1 In hypercholesterolemia, elevated levels of plasma low-density lipoprotein [LDL] will contribute to the development of circulating oxidized LDL [oxLDL], which is considered a useful marker to identify patients with acute coronary heart disease [CHD].2,3 In fact, oxLDL is thought to play a key role in the pathogenesis of atherosclerosis.4 Zingiber zerumbet [Linnaeus] Smith, belonging to the family Zingiberaceae, is an edible ginger originating in South-East Asia.5 This herbal plant is believed to possess high medical prominence.6 A recent investigation revealed that supplementary treatment with Z. zerumbet rhizome’s ethanolic extract [EE] can suppress body weight gain and reduce fat accumulation in high-fat diet-induced rats via upregulation of hepatic peroxisome proliferator-activated receptor alpha, thus modulating lipid metabolism.7

Different from other substances extracted from the essential oil of Z. zerumbet rhizomes, zerumbone [ZER] is a crystalline, monocyclic, sesquiterpene, phytochemical substance that was first isolated as a major natural compound in 1960 from the essential volatile oil of rhizomes of Z. zerumbet.8,9 Several studies have shown that ZER exerts a broad spectrum of pharmacological activities, and more importantly, is an effective antioxidant in suppressing the generation of free radicals;10 this is especially important because oxidative stress plays a key role in the process of atherogenesis.11 Furthermore, ZER attenuates free radical generation by chronic inflammatory cells [macrophages and lymphocytes] as an antioxidant, cancer-preventive agent.12 It is a known fact that these cells have a pivotal role in fatty streaks formation, plaque accumulation, and in early lesion development.13,14

The most substantial and remarkable in vitro findings proposed that ZER has a distinct ability to suppress the expression of multiple scavenger receptors on human leukocytes, via downregulation of nuclear factor kappa beta [NF-κB], which in turn blocks the uptake of oxLDL by macrophages, and, as a result, reduces foam cell formation.15 NF-κB activation is possibly induced by tumor necrosis factor alpha, which is involved in various pathological mechanisms associated with the development of atherosclerosis.16 As a result, from that point of view, ZER is expected to have a significant antiatherogenic effect via its antihypercholesterolemic property. On the other hand, the search for nutraceutical compounds that reduce serum cholesterol and prevent hypercholesterolemic atherosclerosis would be an even more important achievement.

Therefore, the purpose of the present study is to evaluate the antihypercholesterolemic effects of dietary ZER supplementation on the initiation and propagation of atherosclerosis in cholesterol-fed rabbits as a preventive and therapeutic measure, through assessment of serum lipid profiles, and histopathological, ultrastructural, and gross evaluation of atherosclerotic lesions in the aorta.

Materials and methods

ZER extraction

ZER was extracted using the hydrodistillation [steam distillation] method according to a previously described protocol.17 In brief, fresh rhizomes of Z. zerumbet were obtained from a local market in Kuala Lumpur, Malaysia. Initially, the rhizomes were cleaned, washed, sliced, and later placed in a steam distiller containing tap water and heated immediately using a heating mantel. The channel of the device was connected to a special container in order to collect the steamed volatile oil mixed with water. The collected solution was mixed with absolute hexane [100%] [Sigma-Aldrich Co, St Louis, MO, USA] in a separator funnel to separate water from volatile oil. Later, the oil–hexane solution was placed in a glass baker and left standing in a fume hood to allow hexane evaporation and ZER crystallization. The crystals were collected and used. To obtain highly pure ZER, recrystallization was performed using hexane, and the solution was left standing to evaporate overnight. Crystals of ZER were kept at 4°C until further use. We could extract 1.3 g of ZER crystal from one kg of ginger rhizome.

Preparation of ZER solution

ZER solution was prepared fresh every day by dissolving pure ZER crystals with the appropriate amount of corn oil in a glass container [McCartney tube] by the aid of a sonicator [Emerson Industrial Group, Danbury, CT, USA], through induction of ultrasonic vibrating movement for 15 minutes with 40°C heat. The 0.4% [8 mg/kg bodyweight], 0.8% [16 mg/kg bodyweight], and 1% [20 mg/kg bodyweight] ZER solutions were prepared by dissolving 100, 200, and 250 mg ZER crystals in 25 mL corn oil. The protocol used was described previously.18

Preparation of simvastatin solution

Simvastatin [SIM] solution was prepared fresh every day by dissolving one tablet with 15 mg SIM [Zocor®; Merck & Co, Inc, Whitehouse Station, NJ, USA] per one rabbit with 2 mL of sterilized distilled water in a Bijou bottle. The solution was obtained via gentle shaking at room temperature for a few minutes. Similarly, to get 5 mg SIM solution, one tablet of 5 mg SIM was dissolved in 2 mL sterilized distilled water. Dosages were calculated according to a previous study for the 15 mg regimen dose,19 whereas for the 5 mg regimen dose, calculations were done according to earlier research.20–22

Preparation of 1% high-cholesterol diet

The 1% high-cholesterol diet was designed as an atherogenic diet for induction of experimental atherosclerosis following previous investigations,23 and prepared according to methods described earlier but with slight modifications.24,25 Hypercholesterolemic diets [HCDs] were prepared initially by dissolving pure cholesterol powder isolated from sheep wool [Sigma-Aldrich] in chloroform [Avantor Performance Materials, Center Valley, PA, USA]. Briefly, 1.0 g cholesterol was added to 100 g of rabbit pellets; therefore, for every 1,000 g pellets, we utilized 10 g cholesterol. Furthermore, 10 g of cholesterol was suspended in 100 mL of chloroform, equivalent to 1 g in 10 mL; the mixture was stirred with the aid of a magnetic stirrer [LabTech Software, Tampa, FL, USA] for 30 minutes at room temperature. Subsequently, the 100 mL solution of cholesterol was mixed evenly with 1,000 g standard laboratory rabbit pellets in a special tray inside a fume hood [Frontier™; ESCO Corporation, Portland, OR, USA].

Then, the mixture was placed overnight in an air-ventilated oven at 40°C [Sheldon Manufacturing, Inc, Cornelius, OR, USA], allowing the chloroform to evaporate and the pellets to be soaked with cholesterol completely. The cholesterol-rich diets were prepared fresh every day. Rabbits were given access to 100 g rabbit chow diet and water daily, and 1% cholesterol-rich diet was given as a diet/head/day according to a previous study.26 Any remaining pellets in the feeding pots were collected the next day, weighed, then discarded. The feeding pots together with the water bottles were washed and refilled again every day throughout the experimental period.

Animals and management

The experimental protocols were reviewed and approved by the Animal Care and Use Committee [ACUC] for the ethics of animal care, and experiments were assigned with a reference number [UPM/FPV/PS/3.2.1.551/AUP] from the Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor. The New Zealand white [NZW] rabbit is the strain commonly used as an animal model for spontaneous, as well as diet-induced atherosclerosis.27 Healthy male NZW albino rabbits 10–12 weeks old weighing 1.5–2 kg were used in the current investigation. Animals were purchased from A-Sapphire Enterprise [Selangor, Malaysia] and placed at the Experimental Animal Research Unit, Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor. Upon receipt, rabbits were acclimatized in an air-conditioned room at 22°C–25°C for 7 days and were provided with free access to food and water. All animals were individually caged under normal experimental laboratory conditions in a 12-hour-light/12-hour-dark cycle, and received 100 g rabbit chow diet and water ad libitum.

Experimental design

A total of 72 NZW rabbits were divided randomly between two experimental studies carried out according to a diversified timeline. The interval between the two experiments was at least 8 weeks. The first experiment was designed to investigate the prophylactic efficacy of ZER in preventing early development of atheromatous lesion. Thirty rabbits were equally allotted into five groups, namely control [CN], HCD, and three ZER preventive groups [ZPI, ZPII, and ZPIII]. Those in the HCD, ZPI, ZPII, and ZPII groups received a 1% high cholesterol diet. However, rabbits in the ZPI, ZPII, and ZPII groups were given ZER at a dose of 8, 16 and 20 mg, respectively. The second experimental study was aimed at investigating the therapeutic effect of ZER in reducing atherosclerotic lesion progression and establishment, wherein 42 rabbits were assigned in equal numbers to seven groups, namely CN, HCD, ZER treatment groups [ZI, ZII, and ZIII], SIM group [SG], and a ZER-SIM combination group [ZSG]. All rabbits, except those in the CN group, received the HCD [1%] for a period of 10 weeks. Following this 10-week period of feeding, the therapeutic regime was initiated for an additional period of 4 weeks. Six rabbits each were assigned to the ZI, ZII, and ZIII groups, which received ZER at the dose of 8, 16, and 20 mg, respectively. Likewise, another six rabbits each were assigned to the SG and ZSG groups that received 15 mg SIM or 5 mg SIM plus 8 mg ZER, respectively.

Body weight and feed intake

The body weight of all rabbits was recorded before the commencement of the experiments and weekly thereafter until the end of the trials by using a commercial weight scale. Leftover rabbit food was collected at the end of each day and also weighed.

Clinical signs

Rabbits were observed twice daily [morning and afternoon] for signs of aspiration pneumonia, oral gavage injuries, hypercholesterolemia, and also for ZER and SIM toxicity.

Blood sampling

Approximately 3–5 mL of blood was sampled via ear lobe vein using a 21G hypodermic needle, while the rabbits fasting for the previous 12 hours were placed in a restrainer. The sampling was done at week 0 [W0], week 5 [W5], and week 10 [W10] in both experiments, and also at week 14 [W14] in the therapeutic trial. The collected blood was put into either heparinized or plain vacutainer tubes placed on ice. All blood samples were processed within 2–4 hours post-collection.

Serum lipid profile analysis

The degree of hypercholesterolemia in all rabbits was measured at W0, W5, and W10 in all experiments, with an additional point of collection at W14 in the therapeutic experiment. Animals fasted for approximately 12 hours prior to blood collection. The collected blood from each rabbit was centrifuged at 3,500 rpm, 4°C for 10 minutes [Hettich GmbH & Co, Tuttlingen, Germany]. The total cholesterol [TC], triglyceride [TG], low-density lipoprotein cholesterol [LDL-C], and high-density lipoprotein cholesterol [HDL-C] were then analyzed in an automated biochemical analyzer [Hitachi 902; Hitachi Ltd, Tokyo, Japan]. All serum lipid profiles were estimated spectrophotometrically using a commercial rapid test kit [Hoffman-La Roche Ltd, Basel, Switzerland].

Histopathology

The collected organs including, aorta, liver, kidneys, spleen, adrenals, and brain samples were sliced into small sections of about 0.5–2 cm thickness and were fixed in 10% formalin for at least 48 hours. The fixed samples were placed in plastic cassettes and dehydrated using an automated tissue processor [ASP300; Leica Microsystems, Wetzlar, Germany]. The tissues were embedded in paraffin wax [EG1160; Leica Microsystems], and the blocks were trimmed and sectioned to 4 μm using a semiautomated microtome [RM2155; Leica Microsystems]. Then, the tissue sections were mounted on glass slides using a hot plate [HI1220; Leica Microsystems].

Subsequently, the tissue sections were deparafinized by two changes of xylene for 5 minutes each and rehydrated by three changes of different graded ethanol dilution [100%, 90%, and 70%] for 5 minutes each, respectively. Afterward, the sections were stained with Harris’s hematoxylin and eosin [HE] method, as described previously.28 Finally, tissue sections were mounted with glass cover slips using mounting medium distyrene-plasticizer xylene and were examined using a light microscope image analyzer [BX51TF; Olympus Corporation, Tokyo, Japan].

Assessment of atherosclerotic plaques [sudanophilia]

Macroscopic assessment of atherosclerotic plaque distribution was estimated and calculated according to previous published works within the same field.26,29 After dissection, the ascending parts, including the aortic arch [first experiment] and thoracic-abdominal aorta [second experiment], were removed with any remaining blood rinsed away with ice-cold physiological saline, cut, and opened longitudinally, and were fixed in 10% formalin for 24 hours at room temperature. Then, a sample was prepared for detection and quantification of the sudanophilic lesion area.30

Thereafter, the fixating aortic strips were pinned in a tissue-embedding cassette and enclosed with a plastic lid to avoid sample movement. Then, the aortas were rinsed briefly in 70% ethyl alcohol and immersed in for 20 minutes at room temperature in Herxheimer’s solution that contained 5 g Sudan IV dye, 500 mL of 70% ethyl alcohol, and 500 mL acetone. The aortas were then transferred to 80% alcohol for 20 minutes for de-staining. Thereafter, the segments were consecutively washed in running tap water for 1 hour. The strength of the running water stream was noted to avoid tissue damage. The Sudan IV stain is lipophilic; therefore, it will stain the lipid deposition on the inner surface of the aorta a deep red color, allowing a clear illustration of the plaques.31

Macroscopic atheromatous plaque quantification

The inner surface of the aorta segments were flattened, and pinned on a green rubber cobble to provide high-quality contrast. The images of the aortas were photographed using a notebook [iPad 3; Apple, Inc, Cupertino, CA, USA]. The extent of aortic atherosclerosis was evaluated as a lesion area relative to the total inner surface and was measured in mm2 using graph paper. The severity of atheromatous plaque was expressed as a percentage of the luminal surface that was covered by macroscopic atherosclerotic lesion.

Atheromatous lesions were graded semiquantitatively,32 based on a 4-point measuring scale [0, absent 0%–10%; 1, mild 10%–25%; 2, moderate 25%–50%; 3, severe 50%–75%; and 4, critical >75%], using the following formula as previously described.33

Semiquantitative evaluation of microscopic atherosclerotic lesions

Morphometric analysis of the histological sections from aorta was performed according to a previous study.34 Briefly, for each rabbit, four segments of 0.5–1.0 cm length from the aortic arch and thoracic aorta were fixed in 10% formalin for at least 48 hours at room temperature. Paraffin-embedded tissue sections [4–5 mm thick] were stained with HE. Tissue slides were examined under a light microscope with the aided image analyzer [BX51TF] for evaluation of the severity of the atherosclerosis. This evaluation was based on the presence of fatty streaks and atheromatous-fibrous plaques via estimation of intimal thickness, which was measured in μm and statistically expressed as a mean percentage. The following morphometric scales were considered for the semiquantitative evaluation of aortic histological sections: scale 0, scale +, scale ++, scale +++, and scale ++++. Finally, scoring of the lesion severity was graded, according to the percentage of intimal plaque thickness, as follows: 0%–10%, no lesion; 10%–25%, mild; 25%–50%, moderate; 50%–75%, severe; 75%–100%, highly severe.

Transmission electron microscopy

The harvested aortic tissue slices [1–3 mm2] were immediately fixed in 4% glutaraldehyde in 0.2 M cacodylate buffer 50 mL, 25% glutaraldehyde 16 mL, and 34 mL distilled water [pH 7.2–7.4] at 4°C for 24 hours. Then, the samples were washed with 0.1 M sodium cacodylate buffer for three changes of 15 minutes each, post-fixed in 1% freshly prepared osmium tetroxide for 2 hours at 4°C, and were subsequently dehydrated through a graded series of acetone solutions [35%, 50%, 70%, 95%, and 100%] for 15 minutes for each sample.

Aortic sections were cleared with propylene oxide and infiltrated with an acetone and resin mixture at a ratio of 1:1 for 2 hours and then were infiltrated with an acetone and resin mixture at a ratio of 1:3 overnight. Later, infiltration was done with 100% resin for an additional 2 hours, and sections were embedded in epoxy resin medium [dodecenyl succinic anhydride R1051, 7 mL; agar 100 resin R1034, 10 mL; methyl nadic anhydride R1081, 5 mL; and benzyl dimethyl aminutese, 0.5 mL] [all supplied by TAAB Laboratories Equipment Ltd, Aldermaston, UK]. Polymerization was accomplished in an oven [Memmert GmbH & Co, Schalksmühle, Germany] at 60°C for 48 hours. Plastic blocks were sectioned by ultramicrotome [Ultracut UCT M26; Leica Microsystems] into [1 μm] thick sections for orientation. The sections were stained by 1% toluidine blue in 1% borax for light microscopy. Ultrathin sections were collected on copper grids, and double-contrast staining was applied with uranyl acetate [100 mL methanol and 5 g uranyl acetate] and Reynold’s lead nitrate solution [1.76 g sodium citrate, 1.33 g lead nitrate, 50 mL distilled water, and 8 mL 1N NaOH]. Thin sections were examined under a transmission electron microscope [H7100 transmission electron microscope; Hitachi].

Scanning electron microscopy

Following the same primary fixation, washing, and post-fixation protocol used for transmission electron microscopy, aortic samples [0.5–1.0 cm2] were dehydrated through a gradually increasing acetone series [35%, 50%, 70%, 95%, and 100%] for 15 minutes per sample. The specimens were placed into a specimen basket and maintain in amyl acetate for critical point drying [Critical point dryer 030, BalTec, GmbH, Schalksmühle, Germany] for about 1.5 hours. Tissues were coated with gold-palladium particles with a sputter coater [sputter coating device 005; Baltec, Japan] and examined by scanning electron microscopy [SEM] [XL30 ESEM; Philips, Amsterdam, the Netherlands].

Estimation of plasma malondialdehyde concentration

Sample data of plasma malondialdehyde [MDA] were estimated in triplicates. Plasma MDA concentration was calculated by thiobarbituric acid reactive substances manual method as described in previous research,35 with slight modification. Briefly, 0.5 mL of plasma was added to a reaction mixture containing 2.0 mL of 1% phosphoric acid [pH 2.0], 2.5 mM butylated hydroxytoluene in absolute ethanol, 0.1 mL of 8.1% sodium dodecyl sulfate, and 1.0 mL aqueous solution of 0.6% thiobarbituric acid. The reactive mixture was mixed by slight vortexing in a Pyrex® centrifuge tube. The pH value of the analytical reactive mixture was about 0.9. Then, the reactive mixture was heated for 30 minutes at 95°C in boiling water inside a water bath [Sheldon Manufacturing, Inc]. After cooling at room temperature, 5.0 mL of n-butanol was added to the mixture and homogenized by vortexing, followed by centrifugation at 3,500 rpm for 15 minutes [Hettich GmbH & Co]. The n-butanol supernatant layer was collected in a plastic cuvette for fluorometric measurement at 532 nm using an electronic spectrophotometer [APEC-VC Korea, Daejeon, Republic of Korea].

The concentration of MDA was expressed as nmol/mL of plasma. The concentration of MDA was calculated by the following equation:36

where As is the absorbance of the sample, Ab is the absorbance of the blank, Vs is the volume of plasma sample, and VT is the volume of the total reactive mixture [mL] in the cuvette.

Estimation of erythrocyte-superoxide dismutase activity

All data were subjected to statistical analysis in triplicates. The erythrocyte-superoxide dismutase [E-SOD] activity was measured by pyrogallol oxidation inhibition assay as described earlier,37 with slight modification. Initially, 500 μL hemolysate [that was previously prepared by mixing with 3 parts of isotonic saline] was thawed at 25°C and added to 1.5 mL of ice-cold distilled water, followed by the addition of 0.5 mL ethanol, and then 0.3 mL chloroform. The solution was mixed after each addition, and finally, shaken vigorously for 1 minute by vortexing [Sheldon Manufacturing, Inc]. The well-mixed solution was centrifuged for 10 minutes at 3,000 rpm. The clear top supernatant layer was collected and stored at −20°C. Following an addition of 2.0 mL of deionized water into the mixture, a reactive solution was prepared by adding 75 μL chloroform-ethanol extract to 2.25 mL Tris-HCl [pH 8.0]. The mixture was then kept and pre-warmed at 25°C in a water bath for 10 minutes; then, 0.15 mL of 3 mM pyrogallol solution was added. The rate of spontaneous oxidation was measured immediately and spectrophotometrically at 420 nm. Standard controls were measured following the same procedure, but the sample was replaced with 20% ethanol. The enzyme activity was defined as unit per mL of hemolysate, following this equation:

where BΔA is the rate of spontaneous oxidation of blank control, SΔA is the rate of spontaneous oxidation of the sample, Ve is the final volume of extract, and Vs is the amount of the sample in the extract.

Statistical analysis

All descriptive statistical analysis was performed using SPSS [Statistical Package Social Sciences] version 15.0 [SPSS Inc, Chicago, IL, USA]. The data were analyzed and presented as mean ± standard errors [MSE] of six animals. Means of multiple groups were compared by Tukey’s HSD [honestly significant difference] post-hoc test, and only values of P