Mulberry Leaf Reduces Oxidation and C-Reactive Protein Level in Patients with Mild Dyslipidemia

C-reactive protein (CRP) is the inflammatory marker that could represent the inflammation in blood vessels resulted from dyslipidemia. The objective of this study was to evaluate the antioxidative activity of mulberry leaf powder using DPPH assay and the effect of mulberry leaf powder on lipid profile, CRP level, and antioxidative parameters in mild dyslipidemia patients. A within-subjects design was conducted and patients received three tablets of 280 mg mulberry leaf powder three times a day before meals for 12 weeks. Total of 25 patients were enrolled but one subject was excluded. After three months of mulberry leaf consumption, serum triglyceride and low-density lipoprotein (LDL) level were significantly reduced and more than half of all patients' CRP levels decreased every month as well as the mean CRP level but no statistically significant difference was found. The average erythrocyte glutathione peroxidase activity of patients was increased but not at significant level; however, the mean serum 8-isoprostane level was significantly lower after mulberry treatment for 12 weeks. It can be concluded that mulberry leaf powder exhibited antioxidant activity and mulberry leaf powder has potential to decrease serum triglyceride, LDL, and CRP levels in mild dyslipidemia patients without causing severe adverse reactions.


Introduction
Mulberries (Morus alba L., Moraceae) have been widely used in traditional Oriental medicine for several applications including prevention of diabetes [1]. It contains various nutritional components such as �avonoids and polyphenols, especially 1-deoxynojirimycin (DNJ), a potent glucosidase inhibitor, which shows hypoglycemic [2], hypolipidemic [3], and antiatherogenic effects [4,5] in certain animal models. Shibata et al. [6] reported that mulberry leaf-derived aqueous fractions inhibit tumor necrosis factor--induced nuclear factor B activation and lectin-like oxidized low-density lipoprotein receptor-1 expression in vascular endothelial cells. Harauma et al. [5] also found that mulberry leaf powder can prevent atherosclerosis in apolipoprotein E-de�cient mice. Our earlier research also found that mulberry leaf powder was effective in reducing lipid pro�le in mild hyperlipidemia patients [3]. Even though mulberry leaf powder seems to have several advantages in cardiovascular diseases, very few studies have been investigated in human subjects.
Dyslipidemia with high serum cholesterol, both total cholesterol and low-density lipoprotein (LDL), and low highdensity lipoprotein (HDL), is a critical cardiovascular risk factor [7,8]. It is now established that oxidation of LDL constitutes a key event in in�ammation and atherogenesis [9]. Mechanisms of LDL oxidation involve concerted mod-i�cation by oxidants produced by arterial wall cells, such as reactive nitrogen species, hydroxyl radicals, and lipid-soluble free radicals [10]. Because most mechanisms involve the oxidation process, antioxidants may be useful in preventing endothelium blood vessel related to atherosclerosis.
ere is extensive evidence that link hypercholesterolemia with increased lipid peroxidation and increased oxidative stress [11,12]. e oxidative modi�cation of lipoproteins, particularly LDL, has emerged as a fundamental process in the development of atherosclerosis [13]. Oxidative stress due to increased reactive oxygen species (ROS) generation and unbalanced oxidative/antioxidative equilibrium are also implicated in the development of coronary arteriosclerotic cardiovascular disease especially in patients with hyperlipidemia [14]. Glutathione peroxidase (Gpx) is considered as one of the primary defense systems which eliminate excess ROS and maintain equilibrium between oxidative and antioxidative activity under normal physiological conditions [15] while 8-isoprostane, the �nal esteri�ed product of oxidized arachidonic acid, seems to be a good marker of oxidative injury and is considered a gold standard for measuring oxidative stress in vivo.
C-reactive protein (CRP), generated from the liver, is a protein in the kind of acute phase reactant. It will respond immediately aer the rising of nonspeci�c in�ammation in the blood vessel [16,17]. CRP has been identi�ed in human atherosclerotic lesions and has been hypothesized to mediate endothelial dysfunction through induction of endothelial adhesion molecules, tissue factor, and proin�ammatory cytokine synthesis [18]. Accordingly, CRP in blood can re�ect the process involved with in�ammation and it is a strong independent predictor of future peripheral arterial disease [19], myocardial infarction, and stroke among apparently healthy men and women [20]. As a marker of in�ammation, CRP measurement has been recommended in cardiovascular risk strati�cation and clinical treatment guidelines, additional to traditional cardiovascular risk assessment [21,22]. When combined with lipid screening, CRP improves global risk prediction in patients who would not be identi�ed for primary prevention of cardiovascular events by lipid assessment alone [23].
e purposes of this study are to evaluate the antioxidant property of mulberry leaf-derived aqueous fraction in vitro; the effect of mulberry leaf on the serum lipid pro�le, erythrocyte glutathione peroxidase activity, and 8-isoprostane, and the effect of mulberry leaf in reducing the CRP levels in patients with mild dyslipidemia.

Materials.
Mulberry leaf powder and tablets were derived from Kitayamakit Co. Ltd. (Kyoto, Japan). ey were produced from chemical-free mulberry leaves. Each tablet weighed 280 mg and contained 254.8 mg mulberry leaf powder, which had 0.367 mg of the active ingredient DNJ.

Analysis of Antioxidant
Activity. Antioxidant activity of mulberry leaf was determined by free radical scavenging activity using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay [24]. Mulberry leaf powder (0.3 g) was extracted in 3 mL of hot water (95 ∘ C) with continuous shaking for 30 minutes. Aer getting cool at room temperature, it was centrifuged at 5,000 rpm for 20 minutes then �ltered through 0.45 m �lter. e supernatant obtained was considered as undiluted mulberry leaf extract (100%). Serial dilution of undiluted extract was performed in order to obtain extract at 50, 25, 12.5, and 6.25%, respectively. en 100 L of different concentrations of mulberry leaf extracts were mixed with 150 L of 0.1 mM DPPH methanolic solution. e samples were shaken vigorously and allowed to stand for 20 minutes at room temperature. A control was prepared without any sample and puri�ed water was used for baseline correction. e decrease in the absorbance of the formed blue to violet reagent was determined aer 20 minutes at 550 nm and the percentage inhibition activity was calculated from the following equation: where 0 = Absorbance of the control and 1 = Absorbance of the extract/standard. Experiments were carried out 5 times. Data were expressed as the half maximal inhibitory concentration (IC 50 ), which is the concentration of an antioxidant at which 50% inhibition of free radical activity is observed.

Subjects and Study Design.
A within-subjects research design was conducted at the outpatient internal medicine clinic, Phramongkutklao Hospital, ailand. All patients were screened at the beginning of the study. Eligibility criteria for entry into the study included the following: (1) age between 20 and 60, (2) met the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria for dyslipidemia [9,25], (3) must have serum LDL level in the range of ≥ 140 and < 190 mg/dL and fasting plasma glucose < 126 mg/dL, (4) should not have more than one major cardiovascular disease (CVD) risk factor according to NCEP ATP III guidelines, (5) must not be receiving lipid lowering drugs except for diet control, only diuretics were allowed for patients with hypertension and their blood pressure had to be controlled at level < 140/90 mmHg, and (6) should have liver enzymes (alanine aminotransferase and aspartate aminotransferase) < 40 U/L and blood urea nitrogen < 20 mg/dL, with serum creatinine in the range of 0.6-1.2 mg/dL. e patients with the following criteria were excluded from the study: (1) patients needing to receive lipid lowering drugs according to NCEP ATP III guidelines and if they had a high risk factor for CVD or equivalent, (2) had a Framingham risk score greater than 20%, (3) had abnormal liver or renal function tests, (4) had severe complications or had been admitted to the hospital for cardiovascular events in the three months prior to enrollment in this study, and (5) patients with cancer and those who were pregnant or breastfeeding. Written informed consent was obtained from all study participants aer a thorough discussion of the protocol, its rationale, and potential risks. e protocol was approved by the Ethics Committee of the Institutional Review Board of Phramongkutklao Hospital.
Prior to the enrollment, all subjects were asked about underlying diseases, current medications, and personal pro-�le. On the �rst visit of the screening period, blood samples were collected from all patients and a dietician advised them during the four-week period of diet control. is advice included the diet consumed, food exchange, and a diet that was appropriate for each patient, and they were carefully instructed on how to record their total oral intake using household measures. Each patient was requested to make a three-day food record by recording all of the food and beverages that they consumed over two working days and one day of the weekend. Blood samples were collected to determine the lipid pro�le aer four weeks of diet control. Patients who reached the target lipid pro�le aer diet control were withdrawn from the study while patients who could not reach the target according to NCEP ATP III guidelines continued in this study and the lipid pro�le before receiving mulberry leaf tablet therapy was examined. All included subjects were assigned to receive three tablets of 280 mg mulberry leaf powder three times a day before meals, the dose which has been proven to reduce the LDL in mild dyslipidemia patients [3], for 12 weeks. CRP measured by high-sensitivity methods was used in order to measure low levels of CRP more accurately. Brie�y, blood samples were completely coagulated and centrifuged as serum and then stored frozen at −80 ∘ C. e CRP levels were determined by particle-enhanced immunonephelometry. e serum sample of 200 L was diluted and reacted with CRP reagent (suspension of polystyrene particles coated with mouse monoclonal antibodies to CRP) to form an immune complex. A beam of light at the wavelength of 840 nm was then passed through the sample; the amount of light scattering was measured by photodetector. e intensity of the scattered light was proportional to the concentration of the relevant protein in the samples. e result was evaluated by comparing with a standard of known concentration. Routine blood analyses including lipid parameters, CRP, fasting plasma glucose, and liver function tests of all subjects were performed every four weeks. Clinical evaluation for side effects and pill counts to determine compliance were also performed every four weeks at the follow-up visits. If total pill count indicated more than 20% of mulberry leaf tablets untaken, subjects were excluded from the study.
Glutathione peroxidases (Gpx) was measured by the modi�ed method of Paglia and Valentine [26] as described by Jacobson et al. [27]. e rate of glutathione oxidation was measured by monitoring the disappearance of NADPH+H + in the reaction medium (decrease of absorbance at 340 nm), since NADPH+H + was consumed for the reduction of oxidized glutathione by glutathione reductase. 8-isoprostane concentrations were measured in duplicate using a speci�c enzyme immunoassay kit (Cayman Chemicals, Ann Arbor, MI, USA). e detection limit was 5 pg/mL and the intraassay and interassay variabilities were 6 and 7%, respectively.

Statistical Analysis.
Results were expressed as mean and standard deviations. e t-test was used to evaluate the difference between pre-and posttreatment. Repeatmeasures one-way ANOVA by Bonferroni was used to test the differences in CRP levels in each period. A was considered statistically signi�cant.

Results
Free radical scavenging activities at room temperature from mulberry leaf extracts, indicated as IC , are shown in Figure 1. e results showed that mulberry leaf extracted by hot water exhibits good antioxidant activity. Undiluted mulberry leaf extracts exhibit the highest free radical scavenging activities (the lowest IC value) while the most diluted mulberry leaf extracts show the lowest free radical scavenging activities. e concentration of mulberry leaf extracts correlates well with their antioxidant activities. ere is a signi�cant difference in IC of 100% and 6.25% mulberry leaf extract. Twenty-�ve subjects have enrolled in the study. Table 1 summarizes the demographic characteristics and baseline laboratory data of all participants in this study. None of the participants had underlying disease or current medications. e mean age was 35.88 years with the majority being female. Generally, it can be concluded that the subjects with mild dyslipidemia and having normal body weight were at low risk of coronary heart disease.
Most subjects complied with their medication regimens. e percentage of compliance ranged from 89.63 to 98.52% with the mean of 95.00%. One subject was eliminated from the study since she discontinued her lifestyle modi�cation by consuming a high fat diet routinely aer starting the  mulberry leaf tablet regimen. us, her lipid level could increase and affect the CRP level to be higher than usual. Table 2 represents the serum lipid pro�le in patients with mild dyslipidemia aer 12 weeks of treatment with mulberry leaf. Aer 12 weeks of treatment, serum triglyceride and LDL level were signi�cantly reduced by approximately 10.6% and 8.2%, respectively ( ), from baseline, while diet control did not improve lipid pro�le. Moreover, the HDL was increased by 6.3% even though no signi�cant difference was found.
According to the monthly CRP blood level, more than half of all patients' CRP levels decreased every month compared to the CRP level before the intake of mulberry leaf tablets as indicated in Table 3. e mean CRP level of subjects also decreased every month as shown in Figure 2. Aer three months of mulberry leaf tablet consumption, 16 patients (66.67%) had lower CRP levels compared to baseline aer lifestyle modi�cation. However, there is no statistically signi�cant difference of CRP level between each month. e average erythrocyte glutathione peroxidase activity of patients at baseline (aer diet control) was 1 16 ± 3 U/g Hb while this parameter increased to 1 ± 86 U/g Hb aer mulberry treatment for 12 weeks but no signi�cant difference was found. e level of 8-isoprostane showed the same trend� however, the signi�cance difference between T 3: Mean difference of CRP level and number of patients whose CRP level decreased comparing to baseline (month 0, aer diet therapy) ( baseline and aer mulberry treatment was found. At baseline the average 8-isoprostane level was 63 1 ± 1 16 pg/mL and the value decreased to 68 ± 17 pg/mL aer mulberry treatment.
Twelve adverse reactions from mulberry leaf tablet consumption were observed. Details are shown in Table 4. Most common adverse reaction was diarrhea, which could occur on the �rst day of the mulberry consumption. However, the diarrhea, as well as other symptoms, was considered as minor and the patients were able to well tolerate mulberry leaf consumption aer one week. All conditions disappeared aer the patients followed the advice by taking mulberry leaf tablets immediately aer meals. No severe adverse reaction was found in this study.

Discussion
Our present study indicated that mulberry leaf tablet therapy is more effective than diet control alone for controlling lipid pro�le in mild dyslipidemia patients as shown by a signi�cant fall in serum triglycerides and LDL as well as total cholesterol/HDL ratio. It also showed a rise in HDL in all patients and it is well known that improving the lipid pro�le potentially reduces the risk of major cardiovascular events.
C-reactive protein can be used as a marker of acute in�ammation and it also has been widely used for monitoring disease activity in cardiovascular disease and diabetes [28][29][30], which emphasizes the likely role of chronic in�ammation in the aetiology. C-reactive protein also rises with vascular insufficiency and damage of most types, which includes acute myocardial injury or infarction, stroke, and peripheral vascular compromise. Elevation of the CRP level has predictive value for an increased risk of an acute coronary event compared to very low CRP levels [31]. Regarding to gender, women normally have signi�cantly higher CRP levels than men [32] and stronger correlation was found in women between the association of obesity and CRP level [33]. Our results indicated that mulberry powder extracted exhibited strong antioxidant property, which is similar to results found by others [5,34]. Even though earlier report showed that mulberry leaf extracted by water had very little effect on cell cycle progression and had less 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities compared to mulberry leaf extracted by methanol or butanol [35], it still shows strong free radical scavenging activity in vitro. From our result, it could be stated that lifestyle modi�cation can prevent more in�ammation in blood vessel that occurs from dyslipidemia resulting in the decreased CRP levels especially when the intake of the mulberry leaf tablet was included. We found that CRP mean level in subjects receiving mulberry leaf tablets had a tendency to decrease every month, especially aer 12 weeks of mulberry leaf consumption. It could be stated that mulberry leaf tablet consumption can prevent more in�ammation of blood vessels that occurs from dyslipidemia. However, the difference of CRP levels in each month was not statistically signi�cant which may be due to a small number of the participants and the initial CRP levels in all subjects were rather low. Even though most of subjects in this study were female who has high tendency of elevated CRP, most subjects were considered as normal (healthy weight) according to their body mass index resulting in small-elevated CRP. Despite its high sensitivity, it has only 19-hour half-life which might have caused deviation [36]. However, according to patient interviews, there was no illness, in�ammation or infection, injury, or medication use during the study period, which could relate to their particular CRP levels.
Maximum CRP levels > 3 mg/dL had positive predictive values > 20% for proven or probable early-onset infections or in�ammation [37]. Only 5 out of 25 patients in our study had CRP levels higher than 3 mg/dL at initial stage with no acute phase reaction of in�ammation or infection reported in their patient pro�les and their CRP levels aer 3 months of mulberry consuming reduced signi�cantly in all patients and the levels are within normal range (data not shown). In month 3, the average CRP levels decreased only slightly, possibly due to the fact that most subjects had mild dyslipidemia with low risk of coronary heart disease and their initial CRP levels were rather low. e intake of mulberry leaf tablet could, thereby, reduce CRP levels to a certain extent.
Taking other factors into consideration, one patient who took oral contraceptives during the research participation exhibited a high initial CRP level. is might be due to the estrogen/progestogen hormone use. Aer changing her lifestyle and consuming mulberry leaf tablets, her CRP level continued to decrease and reduced by 77% in the third month (data not shown). is might, therefore, be an indication that the decrease in CRP level will be obvious when the patient's initial CRP level was high.
Glutathione peroxidase activity is a key antioxidant enzyme within most cells including endothelial cells. Due to its important role in the prevention of oxidative stress, it is considered to be an antiatherogenic enzyme [38].
Guo et al. [39] showed that reduced expression of Gpx resulted in an increase of cell-mediated oxidation of LDL in mice. In human, the lack of Gpx activity in atherosclerotic lesions appeared to be associated with the development of more severe lesions [40]. Our results indicate that mulberry leaf enhances the antioxidant activity in human by increasing Gpx activity even though no signi�cant difference between before and aer mulberry consumption was found which may possibly due to; again, most subjects had only mild dyslipidemia with no severe life-threatening condition.
e isoprostanes are a unique series of prostaglandinlike compounds formed in vivo via a nonenzymatic mechanism involving the free radical-initiated peroxidation of arachidonic acid [41]. 8-isoprostane has been focused due to its stability, speci�city for lipid peroxidation, and relative abundance in biological �uid [42]. In this present study, the mean serum 8-isoprostane level was signi�cant lower aer mulberry treatment for 12 weeks despite that its initial serum level at the baseline was much higher. is observation may verify the notion that mulberry leaves possess antioxidative properties in clinical application.
During this study, there was no severe adverse reaction found. A minor diarrhea occurred, however, possibly due to the fact that mulberry leaf tablets are rich in dietary �ber, which could stimulate defecation. Accordingly, constipation is also possible when there is insufficient water in the bowels' contents. An active ingredient in mulberry leaf, DNJ, also acts as alpha-glucosidase inhibitor, which prevents disaccharide digestion, thereby causing gastrointestinal side effects such as �atulence or abdominal distention. Moreover, mulberryinduced reduction in blood glucose might increase appetite or cause dizziness.

Conclusions
Extraction of mulberry leaf powder by hot water exhibited strong antioxidative activity. is research also reveals the tendency of mulberry leaf powder in reducing serum LDL and triglyceride as well as blood vessel in�ammation stemmed from dyslipidemia, by the measurement of decreased CRP levels. Moreover, mulberry leaf powder can increase the erythrocyte glutathione peroxidase activity and decrease 8-isoprostane in serum. No severe adverse reaction was found and minor side effects can be relieved by taking mulberry leaf tablets immediately aer meals.

Con�ict o� �nte�ests
All authors have indicated that they have no �nancial/ commercial con�ict of interests regarding the content of this paper.