Clinical Study Tualang Honey Supplementation Reduces Blood Oxidative Stress Levels/Activities in Postmenopausal Women

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Introduction
Studies have shown that oxidative stress is increased in postmenopausal women, and it was reversed by antioxidant action of estrogen [1][2][3].he antioxidant action of estrogens in vivo is due not to their chemical phenolic structure but rather to their interaction with estrogen receptors in cells which eventually lead to the activation of mitogen activated protein kinases (MAPK) and nuclear factor kappa B (NFB) [ , 5].Despite the proven beneit of hormone replacement therapy (HRT), beliefs about side efects and concerns about safety hinder the acceptance of HRT among patients even in recent researches [6] .H e n c e ,th e r ei san eedt oi n dac o mparable alternative treatment to alleviate postmenopausal symptoms.
Honey contains various kinds of phytochemicals with high phenolic and lavonoid content which contribute to its high antioxidant activity [7,8].Flavonoids and other polyphenols, common constituents of honey, have the potential to impound metal ions in complexes, preventing the formation of free radicals [9].Malaysian Tualang honey (TH) is reported to have the highest antiradical activity compared to Gelam honey, Indian forest honey, and Pineapple honey [10] and a total of six phenolic acids and ive lavonoids [10][11][12].Recently, our research group has reported the beneicial efects of estrogen progestin therapy (EPT) and TH on verbal memory of postmenopausal women [13].hese led us to hypothesize that both EPT and TH improved verbal memory of postmenopausal women by reducing blood oxidative stress levels/activities.herefore, the aim of this study was to compare the blood oxidative stress levels/activities of postmenopausal women receiving EPT and TH supplementation.

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. Study Subjects.his study involved 78 postmenopausal women aged between 5 and 60 years, recruited from the 2 ISRN Oxidative Medicine Family Medicine and the Obstetrics and Gynecology Clinic, Hospital Universiti Sains Malaysia (USM).Participants were excluded if they took any other herbal or hormone replacement therapy 3 months prior to the study, smoked, had a history of drug or alcohol abuse, or had a history of serious medical, surgical, mental, or gynecological diseases.Selected participants underwent physical examinations including pelvic ultrasonography to exclude any gynecological problems.

Study
Procedure and Design. he study protocol was approved by the Research and Ethics Committee of USM.Participants were briefed on the nature of the study, and informed consent was obtained at the initial visit.Randomization was computer generated, and participants were identiied by their hospital registration numbers and were randomly assigned to one of two groups: TH and EPT groups.heTHgroupreceivedoralTH(AgroMas)atadailydoseof 20 g. he EPT group received oral Femoston 1/5 (1 mg 17 estradiol and 5 mg dydrogesterone) daily.Participants took the treatment for 16 weeks, and their health status, compliance, and possible adverse efects were monitored every 8 weeks.

Determination of Blood Oxidative
Stress Levels/Activities. he blood GSH to GSSG ratio, plasma catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were determined using a commercially available kit (Calbiochem, Darmstadt, Germany).Plasma -hydroxynonenal ( -HNE) and total protein were determined using the OxiSelect HNE-His adduct Elisa kit (Cell Biolabs, Inc., San Diego, USA) and Protein Assay kit (Bio-Rad, Hercules, CA, USA), respectively.Procedures for the veriication of antioxidant enzymes lipid oxidative damage and total protein were in accordance with the manufacturer's protocol.

Statistical Analysis
. h er e s u l t sw e r ea n a l y z e du s i n g PASW Statistics version 20 sotware.Values of outcome data are expressed as mean and standard error of mean (SEM).Demographic data were characterized using descriptive statistics and group comparisons were conducted using repeated measures ANOVA. he diferences in blood oxidative stress values between pre-and postinterventions were analyzed using paired -test when within-group diference in repeated measures ANOVA was signiicant.< 0.05 was considered statistically signiicant.

Results
A total of 78 postmenopausal women successfully completed this study and the participants' characteristics are summarized in Table 1.here was no signiicant diference in the mean age of participant, mean age of menarche, mean age of menopause, and duration of menopause between EPT and TH groups.EPT was used as active comparator since it is the standard-of-care therapy.In the present study, there were no group diferences in the preintervention values of blood oxidative markers.hese data relect that the baseline values Values are expressed as mean (SEM).TH: Tualang honey; EPT: estrogen progestin therapy.Statistical comparisons were made between groups using independent -test.a <0.05 was considered statistically signiicant.
were similar in both groups at the start of the study and were used as control values.he level/activity of oxidative stress markers between the two groups was analyzed using repeated measures ANOVA.here were signiicant diferences between the two groups in mean CAT activity ( 1,76 = 36.73and ≤ 0.001), GPx activity ( 1,76 = 10.309 and = 0.02), and -HNE level ( 1,76 = 7.587 and = 0.007).However, there were no signiicant diferences between the two groups in mean SOD activity ( 1,76 = 1.045 and = 0.310) and GSH/GSSG ratio ( 1,76 = 0.062 and = 0.804).he diferences in mean CAT activity, GPx activity, and -HNE level between pre-and postinterventions were further analyzed using paired -test.Differences in the levels/activities of oxidative stress marker between pre-and postinterventions were summarized in Table 2.

Discussion
here was a signiicant increase in CAT activity ater 16 weeks ofEPT ,andtheseresultswereincontrastwithpreviousstudies conducted on postmenopausal women.Previous studies reported that CAT activity displayed minute diferences between treated and nontreated postmenopausal women [1 ] and between premenopausal and postmenopausal women [15]. he discrepancy could be due to diferent age groups and duration of HRT used in the studies.
he increased CAT activity in TH group could be explained by its antioxidant contents such as glucose oxidase, diastase, invertase, catalase, and peroxidase and other bioactive constituents [16].Our inding was also supported by previous animal studies that showed restoration of CAT activity in the erythrocytes of young and middle-aged rats [17], pancreas of diabetic rats [18], and liver of male BALB/c mice administered trichlorfon [19] following honey supplementation.
GPxactivitywasfoundtobesigniicantlyincreasedater 16 weeks of EPT and was consistent with previous studies [1,20].here was a signiicant increase in GPx activity ater 3 and 6 months of Premarin and Provera therapy in postmenopausal women [1].In addition, the erythrocytes GPx were positively correlated with serum estrogen levels in both premenopausal women and HRT-treated postmenopausal women [20].Estrogen has been suggested to exert its antioxidant efects via the modulation of intracellular GPx activity [20].Values are expressed as mean (SEM).TH: Tualang honey; EPT: estrogen progestin therapy; GSH : GSSG: ratio of reduced to oxidized glutathione; GPx: glutathione peroxidase; -HNE: -hydroxynonenal; CAT: catalase; SOD: superoxide dismutase.Statistical comparisons were made between pre-and postinterventions using paired t-test, and a < 0.05 was considered statistically signiicant.
he increase in GPx activity following TH supplementation could be explained by its vitamin contents which reduce the imbalance between uncontrolled ROS generation and scavenging enzyme activities [21].his inding was also supported by studies in the erythrocytes of young and middleaged rats [17] as well as in male BALB/c mice administered trichlorfon [19] which showed honey supplementation restored GPx activity.
GSH : GSSG ratio represents the glutathione redox status, an index of oxidative status in all type of cells.here were no signiicant changes in blood GSH to GSSG ratios ater EPT and TH supplementation.his could be explained by inadequate antioxidant capability of EPT and TH to restore intracellular GSH [18] or improper sample handling resulting in GSH which is available in RBC to be oxidized to GSSG.
h ep l a s m aS O Dl e v e lw a sa l m o s tu n c h a n g e da t e r1 6 weeks of EPT or TH as supported by previous inding [22].Our results are inconsistent, however, with those of previous studies who showed either increased SOD levels in postmenopausal women with HRT [1 ] or decreased SOD levels in pancreas of diabetic rats [18].hese inconsistencies could be explained by diference in response between animal and human following honey supplementation.
he present study showed a reduction in plasma level of -HNE, a marker for lipid peroxidation, ater 16 weeks of EPT and TH supplementation.his could be explained by the capability of both EPT and TH supplementation to increase the blood antioxidant capacity and ofset oxidative stress as evidenced by the increased CAT and GPx activities.
he level of -HNE was earlier reported to be higher in postmenopausal women compared to fertile or premenopausal women [23]. he reduction in oxidative damage was evident by a decrease in -HNE level in the EPT group and this could be explained by direct efect of estrogen as antioxidant or its indirect efect via induction of GPx and CAT activities.
A study on buckwheat honey discovered that phenolic antioxidants from processed honey are bioavailable and could increase plasma antioxidant activity and augment defenses against oxidative stress [27].hese indings suggest that phenolic content of TH may also be bioavailable and responsible for its observed antioxidant efects as supported by a recent inding by Khalil and colleague [11]whorevealedthat irradiated TH had the highest total phenolic content among other types of Malaysian honey, the nearest to the total phenolic content in Manuka honey.In addition to its phenolic compounds, other constituents in honey may act synergistically and contribute to its antioxidant efects [28].hese include vitamins (C and E), enzymes (catalase, peroxidase, and glucose oxidase), lavonoids, carotenoids, and products of the Maillard reaction [28,29].herefore, the results of the present study were the irst to demonstrate that honey supplementation reduces blood oxidative stress comparable to that of EPT.

Table 1 :
Characteristics of the participants.

Table 2 :
Blood oxidative stress level/activity of the participants at pre-and postinterventions.