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Cardamom supplementation improves inflammatory and oxidative stress biomarkers in hyperlipidemic, overweight, and obese pre-diabetic women

This study is published here

Insulin resistance, which is the key feature of pre-diabetes and T2DM, results mainly from low physical activity and obesity, which are associated with the repletion of lipid into adipocytes and the accumulation of adipose tissue.3-5 An increase in insulin, free fatty acid (FFA), and/or glucose levels can increase reactive oxygen species (ROS) production and oxidative stress.6

Oxidative stress is a direct outcome of hyperglycaemia and may be involved in metabolic complications in these subjects.7
 The release of additional acute phase reactants, including TNF-α, IL-6, and CRP, by white adipose tissue has been shown in obese subjects.4 By diminishing insulin receptor signalling and increasing insulin resistance, IL-6 and TNF-α can cause chronic hyperglycaemia in these subjects, leading to the development of diabetes.8
An increased oxidative stress is present in pre-diabetes stages9, which may result in endothelial damage in these subjects.10 Therefore, it can be assumed that interventions to reduce oxidative stress and inflammation could improve the condition of pre-diabetes and prevent its complications and development to T2DM.

Lately, herbal remedies like spices have been considered because of their phytochemical content, which has a beneficial potent.11 Spices may somewhat have the same effects as functional foods to improve health or reduce risks of diseases. 12,13 Like vegetables and fruits, spices also have antioxidant effects.14 Several studies have shown that some spices have great potential to inhibit chronic inflammation and oxidative stress because of their phytochemicals and free radical scavengers like polyphenols, flavonoids, and phenolic compounds.15,16

It has been suggested that by increasing the consumption of spices, chronic diseases morbidity can be prevented.17
Green cardamom, also known as the Queen of Spices consists of the whole or ground dried fruit of Elettaria Cardamomum (Linn.) Maton, which belongs to the ginger family (Zingiberaceae).18,19

So far, several investigations have shown some advantages of cardamom for teeth, gum and throat infection, lung congestion, pulmonary tuberculosis, and gastrointestinal disorders.20 Various studies suggest that cardamom extracts display antimicrobial, anti-cancer, anti-inflammatory, anti-proliferative, pro-apoptotic and anti-oxidative activities.18,20,21-25 Several animal and cellular model studies have shown anti-inflammatory and anti-oxidative activities of cardamom.18,24-30 A few human studies have been conducted to investigate the effects of cardamom on inflammation and oxidative stress status, which have shown conflicting results.11,25,31In one study, supplementation with Greater cardamom or E. Cardamomum improved the antioxidant status in patients with ischemic heart disease or hypertension.25,31 However, the results of another study did not show any significant effect of cardamom on blood oxidative stress and inflammation in T2DM patients.11

According to the results of an in-vitro study, the key components of the essential oil in cardamom (i.e. 1.8-cineol [eucalyptol], beta-pinene, geraniol), by binding with TNF, IL-1 β; IL-4 and IL-5, show anti-inflammatory activities.18In an animal study, cardamom supplementation improved oxidative stress markers and ameliorated the inflammatory cell infiltration and fibrosis in the liver of rats fed on a high-carbohydrate high-fat (HCHF) diet.27

In a human study, 3g cardamom powder intake for 12 weeks in individuals with primary hypertension significantly increased the total blood antioxidant capacity.25 However, no study has been done yet to evaluate the effects of cardamom supplementation in pre-diabetic subjects who are at risk of oxidative stress and inflammation. Previous studies have suggested the need for well-controlled clinical trials of spices.17 Therefore, there is a hypothesis that cardamom may have beneficial effects on pre-diabetes. So, the present study was designed to investigate the effects of cardamom on blood inflammatory and oxidative stress biomarkers in pre-diabetic subjects.
EXPERIMENTAL STATE
Study design and subjects

This double-blind, placebo-controlled trial study was conducted on 79 newly diagnosed pre- diabetic women from two health care centres of Karaj city in Iran from February to April 2014. The aim of the study was to determine the effect of cardamom supplementation on serum lipids, glycemic indices, blood pressure, oxidative stress, and inflammatory biomarkers in overweight and obese pre-diabetic women. Since the majority of these individuals were female, the investigation was limited to female subjects. This study was performed based on the guidelines laid down in the Declaration of Helsinki. All procedures involving human subjects were approved by the Ethics Committee of Tehran University of Medical Sciences. A written informed consent form was signed and dated by the subjects and investigators. This study was registered on the Iranian Registry of Clinical Trials website (http://www.irct.ir/, IRCT2014060817254N2).
A sample sizeof at least 36 in each group was calculated according to the standard deviation of hs-CRP in a similar study30 in the following order: (α=0.05, power=80%)
= 2.18 / 
d=μ −μ =1.47 =0.47
σ√2 3.07
[(z ∝/ ) + (z )] (1.96 + 0.84)
= d = (0.47) = 36

Considering the loss to follow-up, 40 subjects in each group joined the study. The age of the subjects was in the range of 30 to 70 years with a body mass index (BMI) of 25–39.9 kgm-2and had at least one of the following criteria: Fasting blood sugar (FBS) 100-125 mgdl-1, HbA1c 5.7– 6.4%, two-hour blood glucose 140–199 mgdl-1 identified in the last two months. In addition, they had at least one of these risk factors: 300>triglyceride>150 mgdl-1, total cholesterol>200 mgdl-1, 160>low-density lipoprotein-cholesterol (LDL-c) >100 mgdl-1, high-density lipoprotein- cholesterol (HDL-c)<50 mgdl-1. Exclusion criteria were BMI<25 or ≥40 kgm-2, pregnancy or lactation, professional athletes, allergy to cardamom, smoking, clinical history of peptic ulcers, gall or kidney stones, clinical history of inflammatory diseases like diabetes, cardiovascular diseases, multiple sclerosis (MS), rheumatoid arthritis, cancer, inflammatory diseases of the gastrointestinal tract and respiratory (e.g. asthma, allergies), multivitamin or antioxidant supplement consumption at least two days a week in the past month, taking medications for dyslipidemia, blood glucose disturbance, hypertension, psychiatric disease, thyroid and hormonal disease, following a specific diet over the last three months, having LDL-c≥160 mgdl-1 or triglyceride ≥300 mgdl-1, blood pressure>130/80 mmHg, developing diabetes during the study, and not taking the prescribed supplements more than 10%.

Randomization and intervention
The participants were randomized into two groups using block randomization. The stratified randomization method was used to control the age and BMI (age in the range of ≤40 and 41–70 years, BMI in the range of 25–29.9 and 30–39.9 kgm-2).
Forty subjects were classified into each group to receive one capsule of either cardamom or placebo powder three times a day with meal for eight weeks.

The dose of 3.0 g used in the study was chosen based on the two previous human studies investigating the effects of cardamom supplementation 25, 31. Also, 3.0 g of cardamom powder can be a reasonably large amount to be consumed through a diet. Fruits of E. Cardamom were provided by the Traditional Medicine and Research Centre (TMRC), Shahid Beheshti University of Medical Sciences, Tehran, Iran. Fruits of cardamom and dried breadcrumbs are transferred to this centre. After grinding and sifting the whole green cardamom and breadcrumbs, the capsules were filled with these materials.
Each capsule contained one gram of green cardamom powder or breadcrumbs. They were exactly similar in shape, size, and appearance. All placebo capsules were placed near cardamom so that they could take its smell. Sixty capsules were placed in similar jars, which were labelled A and B to be delivered to the individuals. Each person in the study got three jars every 20 days. To confirm the compliance of subjects, they were called up every week. To evaluate compliance, the remaining capsules in the jars were counted.

Dietary analysis
A 24-hour food recall questionnaire of a typical day was completed by interview to get information on food intake at the beginning and the end of the study. Then, the N4 software was used to estimate the daily dietary intake of nutrients.
Anthropometry and physical activity assessments
Anthropometric characteristics, including height and weight, were measured at the beginning and end of the study. Body weight was measured with minimal clothing and without shoes by using a digital scale with a measurement accuracy of 100g. Standing height was measured without shoes to the nearest 0.5cm using a tape measure. BMI was calculated by dividing the weight by the square of height. Physical activity was measured by a short form of the International Physical Activity Questionnaire (IPAQ).33

Laboratory measurements
After 12 hours of fasting, 10ml of venous blood was drawn by a trained nurse before and after the intervention period. Blood samples were centrifuged at 3000 rpm for 10 minutes to separate blood cells and serum. Blood cells were washed three times with 0.9 gl-1NaCl solution. Cell membranes were removed by centrifugation at 1200g for five minutes at 4°C. The haemolysates were then used to determine erythrocyte antioxidant enzyme activity. All samples were then stored at -80°C. Serum levels of hs-CRP (Diagnostics Biochem Canada Inc., Canada), IL-6, and TNF-α (Diaclone, France) and PC (ZellBio GmbH Ulm, Germany) were measured using ELISA kits. The inter- and intra-assay coefficients of variation for hs-CRP, IL-6, TNF-α, and PC were 9.1% and 9.5%, 7.7% and 4.2%, 9% and 3.3%, and <12% and <10%, respectively.

Serum TAC and MDA levels and erythrocyte activities ofSOD and GR were measured using assay kits (ZellBio GmbH Ulm, Germany). The inter- and intra-assay coefficients of variation for TAC, MDA, SOD, and GR were <4.2% and <3.4%, 7.6% and 5.8%, 7.2% and 5.8%, and 6.6% and 5.2%, respectively. At the time of laboratory assessment, the serum of one subject in the cardamom group was not available. In addition, the number of available haemolysates for measuring erythrocyte SOD and GR activity in the cardamom group was 39 and in the placebo group, 40.
Statistical analysis Analysis was done by intention to treat (ITT) using SPSS version 16 (SPSS Inc., Chicago, IL, USA). The ITT population included all the enrolled and randomized participants. The missing observations were accounted for by using the Last Observation Carried Forward (LOCF) and Last Observation Carried Backward (LOCB) methods. Normality distribution of data was evaluated using the Kolmogorov-Smirnov test. Non-parametric tests were used for TNF-α, TAC, and PC, which were non-normally distributed after transformation. The hs-CRPIL-6-1 ratio was calculated by dividing hs-CRP to IL-6 levels. To compare the data between the two groups, the independent sample t-test and Mann-Whitney was used for normal and non-normal data respectively, considering the normality of data. The analysis of covariance (ANCOVA) was used to identify any differences between the two groups after intervention, adjusting for baseline measurements and covariates. Differences of P<0.05 were considered to be statistically significant.

RESULTS
To evaluate the effects of cardamom supplementation on blood oxidative stress and inflammation status,80 participants were enrolled in the study. All participants consumed 90– 100% of the prescribed supplements and all of them completed the study.

Demographic and anthropometric measurements and dietary intakes
The distribution of weight, BMI, and mean duration of pre-diabetes and physical activity were almost similar between the intervention and the control groups (Table 1). In addition, no significant difference was observed in weight, BMI and physical activity between the two groups at the end of the study. The mean±SD ages of the participants in the cardamom and placebo groups were 48.3±10.4 and 47.5±10.3 years, respectively, while no significant differences were found between the two groups. The mean ± SD energy for the cardamom group was 2107.5 ± 317.0 and 2153.4 ± 198.9 kcald-1 before and after the study respectively. These figures for the placebo group were 2157.7±242.2 and 2181.1±212.2 kcald-1, respectively. Comparison between the two groups showed that after intervention, saturated fatty acid (SFA) intake was significantly higher (p=0.005) and poly unsaturated fatty acid (PUFA) intake was lower (p=0.02) in the cardamom group (Table 2). No significant difference was found between the two groups in daily energy, vitamin A, C, E, and selenium intake.

Effect of cardamom supplementation on inflammatory and oxidative stress biomarkers
After intervention, between-group comparisons showed that in the cardamom group, the mean TNF-α (p<0.001) was significantly higher, and hs-CRP (p=0.04), hs-CRP IL-6-1 ratio (p=0.01), PC(p<0.001) and MDA (p=0.003) were lower than the placebo group. However, after adjustment for SFA and PUFA intake changes and baseline values, between-group differences of hs-CRP (p=0.02), hs-CRP IL-6-1 ratio (p=0.008), and MDA (p=0.009) remained significant (Table 3). DISCUSSION
This randomized clinical trial is the first investigation of green cardamom effects on inflammatory and oxidative stress indices in overweight and obese pre-diabetic women who are at risk of cardiovascular diseases. After eight weeks, cardamom supplementation reduced serum hs-CRP, and hs-CRP IL-6-1 ratio. In addition, cardamom supplementation reduced serum MDA levels. However, a complete improvement of inflammatory and oxidative stress parameters was not achieved. Different results may be obtained with a higher supplement dose and duration of intervention.

Several animal studies have shown beneficial effects of cardamom on blood inflammation and oxidative stress indices. However, a few studies have investigated its effect on humans. In atherosclerotic rats, cardamom-rhizome-ethanolic-extract significantly increased SOD levels and decreased MDA, CRP and IL-6.30 By reducing the synthesis of eicosanoid mediators of inflammation, a dose-dependent anti-inflammatory effect of cardamom oil on rats has been shown.26 In addition, by decreasing cyclooxygenase-2 (COX-2) and inducible nitric oxidesynthase (iNOS) expression, the anti-inflammatory activity of cardamom has been reported.24 In another study, cardamom supplementation for eight weeks prevented oxidative stress and ameliorated the infiltration of inflammatory cell and fibrosis in the liver of rats fed on a HCHF-diet.27 A cellular study by Bhattacharjee et al. has shown that the key components of essential oil in cardamom (i.e. 1.8-cineol [eucalyptol], beta-pinene, geraniol) provide anti- inflammatory activity by binding with TNF-a, IL-1 beta; IL- 4; and IL-5.18

In individuals with stage-1 hypertension, Verma et al. found that the intake of 3g cardamom powder for 12 weeks significantly reduced blood pressure, enhanced fibrinolysis, and improved antioxidant status.25 Among the reasons that could lead to different results between Verma’s study and the present study is the lack of a control group in Verma’s study, a different intervention period (12 weeks vs. 8 weeks) and different target groups (hypertensive vs. pre- diabetic individuals).

In another human study by Verma et al., on patients with ischemic heart disease, the intake of G. Cardamom (Amomum subulatum Roxb.) fruit powder for 12 weeks showed an enhancement in the serum total antioxidant status.31 The conflicting results between the current study and that one may be, again, due to differences in the type of cardamom supplemented (E. Cardamom vs. G. Cardamom), different durations of intervention, and different target groups. However, in a study with 204 T2DM patients, intake of 3g cardamom with tea had no significant effect on serum hs-CRP and F2-isoprostan.11 Negative results observed in the study was explained by the combined effects of cardamom and black tea or the dominant effect of black tea.35

Weight gain and obesity, which increase oxidative stress and inflammatory mediators, are major risk factors for insulin resistance, pre-diabetes, and T2DM.4So, any agent with anti-inflammatory and antioxidant effects, such as cardamom, might interrupt this correlation. Cardamom presumably exerts its anti-inflammatory effect by reducing the synthesis of eicosanoid mediators of inflammation.26The antioxidant effect of cardamom also comes from the fact that it is a potent blocker of lipid peroxide formation and scavenger of superoxide anions and hydroxyl radicals.35 Recent studies have shown that the serum levels of CRP in IGT or IFG patients—in other words, pre-diabetic patients—is higher than that in normoglycaemic people.36-38 CRP is an indicator of systemic inflammation, and increased CRP during obesity is thought to be caused by IL-6 derived from adipose tissue.39Recent studies have shown that lower hs-CRP to IL-6 may reflect decreased inflammation.

So, it can be considered as a marker of the inflammation status.40-42Our results showed that cardamom significantly decreased the serum hs-CRP and hs-CRP IL-6-1 ratio. So, cardamom may have a function in decreasing hs-CRP in pre-diabetic individuals.

Tip:

In the present study, cardamom decreased MDA levels. However, our results did not show a significant influence of cardamom on PC, TAC, SOD, and GR levels. Overweight and obese subjects have more oxidative stress than normal-weight pre-diabetic subjects and may need a greater amount of cardamom or a longer duration of supplementation. So, this is perhaps the reason why, in our findings, cardamom consumption in overweight and obese subjects did not have the profound result on oxidative stress.

This study had some limitations. First, the sample size was small. Second, the intervention duration was too short to understand the real effects of cardamom supplementation. Third, a single 24-hour food recall questionnaire can result in measurement error. Fourth, as the population of the present study comprised only women, the results cannot be generalized to male pre-diabetic subjects. However, this study is one of the first investigations of green cardamom supplementation effects on inflammatory indices and oxidative stress in overweight and obese pre-diabetic subjects. The control group in the present study was another strength of this study.

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