Oxidative Medicine and Cellular Longevity

Oxidative Medicine and Cellular Longevity / 2018 / Article
Special Issue

Bioactive Compounds of Food: Their Role in the Prevention and Treatment of Diseases

View this Special Issue

Clinical Study | Open Access

Volume 2018 |Article ID 2091872 | 11 pages | https://doi.org/10.1155/2018/2091872

A Double-Blind Placebo-Controlled Randomized Trial Evaluating the Effect of Polyphenol-Rich Herbal Congee on Bone Turnover Markers of the Perimenopausal and Menopausal Women

Academic Editor: Anderson J. Teodoro
Received13 Mar 2018
Revised24 Sep 2018
Accepted16 Oct 2018
Published21 Nov 2018

Abstract

Based on the benefit of polyphenolic compounds on osteoporosis, we hypothesized that the polyphenol-rich herbal congee containing the combined extract of Morus alba and Polygonum odoratum leaves should improve bone turnover markers in menopausal women. To test this hypothesis, a randomized double-blind placebo-controlled study was performed. A total of 45 menopausal participants were recruited in this study. They were randomly divided into placebo, D1, and D2 groups, respectively. The subjects in D1 and D2 groups must consume the congee containing the combined extract of M. alba and P. odoratum leaves at doses of 50 and 1500 mg/day, respectively. At the end of an 8-week consumption period, all subjects were determined serum bone markers including calcium, alkaline phosphatase, osteocalcin, and beta CTX. In addition, the hematological and blood clinical chemistry changes, and total phenolic content in the serum were also determined. The results showed that the menopausal women in D2 group increased serum alkaline phosphatase, osteocalcin, and total phenolic compounds content but decreased CTX level. Clinical safety assessment failed to show toxicity and adverse effects. Therefore, herbal congee containing the combined extract of M. alba and P. odoratum leaves is the potential functional food that can decrease the risk of osteoporosis.

1. Introduction

Bone is a dynamic organ that undergoes continuous remodeling by the coordination and balance between resorption and the formation activities of osteoclast and osteoblast cells [1]. It is well established that women are vulnerable to bone loss especially during and after menopause. Postmenopausal women lose trabecular bone mineral density (BMD) rapidly in their vertebrae, pelvis, and ultradistal wrist. After menopause, the cortical bone loss in the long bones and vertebrae also occurs, but the rate of bone loss is slower than that in trabecular bone [2]. It has been reported that bone resorption assessed by using the bone resorption markers as indicator increases around 90% during this period [3]. Therefore, this situation increases bone fracture risk.

Currently, a noninvasive instrument such as dual energy X-ray absorptiometry (DXA) can be used as a tool to measure the body composition including bone density. However, it still has a limitation, and bone markers are more sensitive for detecting the change of bone dynamic [4]. Therefore, the application of bone turnover markers (BTM) as the indices indicating bone dynamic has gained much attention. BTM are classified as the formation or resorption markers depending on their origins. It has been demonstrated that BTM changes occur in parallel with bone turnover process, so their alterations can reflect bone turnover state. Among various BTM, osteocalcin and alkaline phosphatase are regarded as bone formation markers, whereas C-telopeptide of type I collagen (CTX) and N-telopeptide of type I collagen (NTX) and tartrate-resistant acid phosphatase (TR-ACP) are regarded as bone resorption markers [58]. Recent studies have clearly demonstrated that polyphenolic compounds provide beneficial effect to bone health by decreasing oxidative stress and inflammation. In addition, they can also modulate osteoblastogenesis and osteoclastogenesis [9]. Based on the previous finding in experimental menopause that the combined extract of Morus alba and Polygonum odoratum showed the antiosteoporotic effect [10], the positive modulation effect of the congee containing the combined extract of P. odoratum and M. alba on bone turnover has been raised.

However, recent study has demonstrated that polyphenol substance can exert prooxidant activity [11] and interactions with other agents which in turn produces numerous detrimental effects [12]. Therefore, the adverse effects and toxicities can possibly occur especially in the repetitive administration of the polyphenol-rich product. To assure that the repetitive administration of the congee containing the combined extract of P. odoratum and M. alba exert the positive modulation effect on the bone turnover and safe for repetitive consumption, the effect of the congee mentioned earlier on bone formation and bone resorption markers together with the hematological and clinical chemistry values in the perimenopausal and menopausal women were determined.

2. Materials and Methods

This study was designed as the randomized controlled trial. All experiments were conducted at the Integrative Complementary Alternative Medicine Research and Development Center, Faculty of Medicine, Khon Kaen University. The study (code number UAPSBS201401) was conducted in accordance with the International Conference of Harmonization (ICH) for Good Clinical Practice (GCP) and in compliance with the Declaration of Helsinki and its further amendments. All protocols were approved by the Khon Kaen University Ethical Committee on Human Research (HE571373); ClinicalTrials.gov was approved ID NCT02562274.

2.1. Participants

Forty-five healthy Thai perimenopausal and postmenopausal women at the age between 45–60 years old (<5 years menstruation cessation) who lived in northeastern of Thailand were recruited to participate this study via advertisements at menopause clinic at Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Thailand. All subjects in this study were subjected to a physical examination and interviewed using a structured questionnaire to obtain information concerning demographic data, smoking, alcohol intake, physical activity, and dietary status. Subjects were excluded from this study if they were diagnosed with hypertension, cancer, autoimmune diseases, gout or high uric acid, and disorders as described as follows: heart, liver, kidney, lung, mental, and endocrinological disorders such as thyroid, parathyroid, and diabetes. In addition, they should not have a history of drug use that could affect bone turnover and bone mineral density, pesticide exposure during one week before the test, hysterectomy and/or oophorectomy, alcohol or cigarettes addiction (cigarettes smoking >10 pieces/day), regular exercise (more than 3 time/weeks), unable to follow the study instructions during the trial and the participation in other projects.

2.2. A Polyphenol-Rich Congee Preparation

The aerial parts of Polygonum odoratum and M. alba leaves were collected from Amphoe Muaeng Khon Kaen, Thailand, during April 2013 and prepared as water extract by decoction method. Then, they were filtered and lyophilized as powder. The combination extract of P. odoratum and M. alba was prepared according to petty patent number 9314, Department of Intellectual Property, Thailand, and used as the functional ingredient in herbal porridge. Each package (25 g/package) contained rice (dried mashed) 90.71%, chicken (dried mashed) 7.26%, and green shallot and coriander (dried mashed) 0.73%. Both placebo and herbal congee were prepared with the same procedure and contained the same ingredients except that the herbal congee contained the combined extract of P. odoratum and M. alba at doses of 50 and 1500 mg/pack, respectively (the selected doses were obtained from our preclinical data [10] by calculating the human equivalent doses). The placebo, D1, and D2 had the same appearance and smell. The congee containing high and low doses of the combined extract of P. odoratum and M. alba failed to show the significant calorie from placebo (placebo provided 94.24 whereas the herbal congee containing low and high doses of the combined extract of P. odoratum and M. alba provided 95.50 and 97.25 Kcal, respectively). Each package contained carbohydrate 20 grams, protein 2 grams, total fat 0 gram, cholesterol 0 mg, and fiber less than 1 gram. In addition, the fingerprint chromatogram of the herbal congee was determined via high performance liquid chromatography which consisted of 515 HPLC pump and 2998 photodiode array detector (Water Company, USA). Chromatographic separation was performed using C-18 endcapped Purospher® STAR column (250 × 4 mm; particle size; 5 μm) and guard column HPLC-Cartridge, Sorbet Lot No. HX255346 (Merck, Germany). Two mobile phases consisting of 2.5% acetic acid in deionized (DI) water (B) and methanol (A) were used to induce gradient elution. The gradient elution was carried out at a flow rate of 1.0 mL/min with the following gradient: 0 min, 10% A; 17 min, 70% A; 18–22 min, 100% A; 25, 50% A; 26–30 min, 10% A. The sample was filtered (0.2 μm, Whatman), and a direct injection of tested sample at the volume of 20 μL on the column was performed. The chromatograms were recorded at 280 nm using UV detector, data analysis was performed using EmpowerTM3, and the results were shown in Figure 1.

2.3. Experimental Design

Eligibility was evaluated by using the semistructure questionnaire, physical examination, and the results of the prestudy laboratory tests. Then, they were randomly allocated into placebo group and D1- and D2-treated groups. Subjects in D1- and D2-treated groups must consume the herbal congee containing the combined extract of P. odoratum and M. alba at doses of 50 and 1500 mg/day, respectively. Prior to the study participation, the participants must fill the informed consent form.

On the experimental day, all participants should not drink tea or coffee or alcohol beverages at least 12 hours before their appointments. In addition, the participants must consume the assigned substance once daily in the morning before breakfast for 8 weeks in a double-blind fashion. All subjects collected blood for the determination of bone markers consisting of serum levels of calcium, osteocalcin, alkaline phosphatase, and beta CTX together with the safety parameters including hematological and clinical chemistry changes prior to the study and at the end of an 8-week study period.

All volunteers were kept blind about treatments. The data interpretation and analysis were performed by blinded investigators, and the code numbers and the group allocation were revealed after the assessment of the last subject. All volunteers were instructed to call the study center in case of any adverse effect occurred during the study. Volunteers had the opportunity to withdraw from the study at any time. All the volunteers were contacted at definite intervals to ensure that they consumed the assigned substance regularly.

2.4. Blood Collection

Blood samples were collected from the participants after an overnight fast by venipuncture and allowed to clot and spun at 3000 rpm for 10 minutes in order to separate cells from serum. Then, the serum was transferred into dry well-labeled specimen plastic tubes and analyzed.

2.5. Determination of Bone Markers

The assessment of serum osteocalcin concentration was carried out by using human Gla-OC High sensitive EIA Kit with the intra- and interassay coefficient of variations (CVs) <5%. In brief, an aliquot of 100 μL standard or sample was added to an appropriate well and incubated at room temperature for 2 hours. Then, the sample solution was removed, and the well was washed 3 times with 400 μL of PBS. 100 μL total of antibody-POD conjugate solution was added into the wells and incubated for 1 hour. At the end of the incubation period, the solution was aspirated from the wells, and the wells were washed again for 4 times with washing buffer (100 μL/well). After this step, the substrate solution at the volume of 100 μL was added to each well and incubated at room temperature for 15 minutes. At the end of incubation period, 100 μL of the stop solution was added into all wells and mixed gently. Then, an absorbance at 450 nm was recorded via microplate reader. All assays were performed in triplicate.

In this study, the concentration of beta-carboxy-terminal cross-linking telopeptide of type I collagen (CTX) levels was measured by using commercial human Micro ELISA Kit with intra- and interassay coefficients of variations 8 and 10%, respectively. In brief, all reagents and samples were left at room temperature before used. After thawing, samples were centrifuged again before the assay. All the reagents were mixed thoroughly by gently swirling before pipetting. An aliquot of 50 L of biotinylated detection Ab working solution was added to each well, covered with plate sealer, gently tapped to mix a solution, and incubated at 37°C for 45 minutes. Each well was aspirated and washed with wash buffer for three times. After washing, the remaining wash buffer was removed by aspirating, and the plate was inverted and patted against thick clean absorbent paper. Then, 100 μL of HRP conjugate working solution was added to each well, covered with a new plate sealer, and incubated at 37°C for 30 minutes. After the incubation, washing was performed for five times. Then, 90 μL of substrate solution was added to each well, covered with a new plate sealer, and incubated at 37°C for 15 minutes with light protection. The reaction time can be shortened or extended according to the actual color change, but not more than 30 minutes. Then, add 50 μL of stop solution to each well and the optical density at 450 nm was monitored via microplate reader.

2.6. Determination of Total Phenolic Compounds

The total phenolics compounds of vegetables were measured by using Folin-Ciocalteu colorimetric method (Quettier-Deleu et al., 2000). Briefly, 20 μL of each plant extracts was mixed with 0.2 mL of Folin-Ciocalteu reagent and 2 mL of distilled water and incubated at room temperature for 5 minutes. Then, 1 mL of 20% sodium carbonate was added and incubated at room temperature for 2 hours. The total polyphenolic compounds were determined by measuring the absorbance at 765 nm with spectrophotometer. Gallic acid was used as a standard, and the total phenolics were expressed as gallic acid equivalents (mg/L GAE/mg extract). All determinations were performed in triplicate.

2.7. Determination of Calcium and Alkaline Phosphatase in Serum

Blood was collected, and the determinations of calcium and alkaline phosphatase changes were performed at Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. The coefficient of variation (% CV) of calcium (Ca2+) and alkaline phosphatase (ALP) was 1.7% and 0.6%, respectively.

2.8. Blood Collection and Toxicity Assessment

Blood was taken from each volunteer and prepared as plasma in order to assess the changes of hematological and clinical chemistry parameters. All parameters were assessed at Srinagarind Hospital.

2.9. Statistical Analysis

All data were expressed as mean ± SD. Comparisons between groups were performed using one-way analysis of variance (ANOVA) followed by post hoc (LSD) multiple comparison tests and Kruskal-Wallis one-way analysis of variance test by using SPSS statistical software. value < 0.05 was considered significant.

3. Results

3.1. Demographic Data of Subjects

Seventy-five menopause women were enrolled to participate in this study via the advertisements at menopause clinic, Srinagarind Hospital, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand. After the interview and the screening of health status by the physician, it was found that only 45 subjects met the inclusion criteria and were allocated to placebo, D1, and D2 groups as shown in Figure 2. The baseline demographic data of all participants were presented in Table 1. No significant differences in all parameters were observed.


ParametersPlaceboMP 50 mg/dayMP1500 mg/day value

Age (years)51.41 ± 4.2150.47 ± 3.2050.47 ± 3.640.697
Education (years)7.73 ± 4.897.73 ± 4.137.47 ± 4.500.983
Body mass index (kg/m2)24.27 ± 2.9125.23 ± 3.5224.91 ± 3.810.742
Blood sugar (mg/dL)87.33 ± 13.5084.67 ± 6.8988.93 ± 15.040.635
Uric acid (mg/dL)5.34 ± 0.775.05 ± 0.765.43 ± 0.920.422
Heart rate (beats/min)77.00 ± 12.8673.20 ± 9.2074.27 ± 8.660.455
Respiratory rate (times/min)19.80 ± 2.4018.00 ± 1.9818.80 ± 1.820.070
Systolic blood pressure (mmHg)115.80 ± 11.48118.67 ± 13.84119.13 ± 13.060.745
Diastolic blood pressure (mmHg)75.87 ± 8.9378.00 ± 9.3876.33 ± 8.470.791

3.2. Effect of Herbal Congee on Bone Makers

The effect of various doses of the herbal congee containing the combined extract of M. alba leaves and P. odoratum on the level of osteocalcin, alkaline phosphatase, calcium, and beta CTX in serum was shown in Figures 36. Subjects who consumed the herbal congee containing the combined extract of M. alba leaves and P. odoratum showed the elevations of serum osteocalcin and alkaline phosphatase levels ( value < 0.01and 0.05; compared to placebo group) as shown in Figures 3 and 4. However, the serum calcium level failed to show the significant change, while the serum beta CTX showed the significant reduction ( value < 0.01; compared to placebo group) as shown in Figures 5 and 6, respectively.

3.3. Effect of Herbal Congee on the Total Phenolic Compounds

The effect of various doses of the herbal congee containing the combined extract of M. alba leaves and P. odoratum on the level of total phenolic compounds was shown in Figure 7. It was found that after 2 months of administration, subjects who consumed the herbal congee containing the combined extract of M. alba leaves and P. odoratum at dose of 1500 mg per day showed the significant elevation of total phenolic compound ( value < 0.001; compared to placebo, value < 0.001; compared to baseline level).

3.4. Effect of Herbal Congee on the Blood Chemistry Parameters

Table 2 showed the effect of various doses of herbal congee containing the combined extract of M. alba leaves and P. odoratum on blood chemistry values. It was found that no significant differences of the parameters were observed at baseline level. At the end of an 8-week consumption period, subjects who consumed herbal congee containing the combined extract of M. alba leaves and P. odoratum at doses of 50 and 1500 mg per day showed the significant reduction of triglyceride ( value < 0.01and 0.05, respectively; compared to placebo group) together with the elevations of albumin ( value < 0.05; compared to placebo-treated group). In addition, the elevation of high-density lipoprotein cholesterol (HDL-C) was also observed in subjects who consumed the high dose of the herbal congee at the end of experimental period ( value < 0.05; compared to placebo-treated group).


Blood chemistryNormal valueGroupBaseline8 weeks
Mean ± SDMean ± SD

Glucose (mg/dL)70–100 mg/dLPlacebo87.33 ± 13.5088.27 ± 12.19
MP 50 mg/day84.67 ± 6.89 F (1,28) = 0.00, 81.47 ± 7.79 F (1,28) = 2.777,
MP 1500 mg/day88.93 ± 15.04 F (1,28) = 0.23, 88.60 ± 15.80 F (1,28) = 2.814,

Uric acid (mg/dL)2.7–7.0 mg/dLPlacebo5.34 ± 0.774.96 ± 0.81
MP 50 mg/day5.05 ± 0.76 F (1,28) = 1.097, 4.53 ± 1.08 F (1,28) = 0.656,
MP 1500 mg/day5.43 ± 0.92 F (1,28) = 0.078, 4.74 ± 1.17 F (1,28) = 0.622,

Bicarbonate (mEq/L)20.6–28.2 mEq/LPlacebo26.41 ± 1.7825.07 ± 1.34
MP 50 mg/day27.68 ± 2.56 F (1,28) = 0.520, 24.79 ± 1.99 F (1,28) = 0.124,
MP 1500 mg/day27.07 ± 2.42 F = (1,28) = 0.741 24.11 ± 3.24 F = (1,28) = 0.149

Blood urea nitrogen (mg/dL)20.6–28.2 mEq/LPlacebo11.40 ± 2.7311.53 ± 2.57
MP 50 mg/day11.61 ± 3.18 F (1,28) = 0.039, 12.19 ± 2.57 F (1,28) = 0.496,
MP 1500 mg/day10.89 ± 2.39 F (1,28) = 0.292, 11.41 ± 2.87 F (1,28) = 0.013,

Creatinine (mg/dL)0.5–1.5 mg/dLPlacebo0.66 ± 0.090.69 ± 0.10
MP 50 mg/day0.68 ± 0.11 F (1,28) = 0.538, 0.68 ± 0.08 F (1,28) = 0.072,
MP 1500 mg/day0.65 ± 0.09 F (1,28) = 0.905, 0.66 ± 0.12 F (1,28) = 0.195,

Sodium (mEq/L)130–147 mEq/LPlacebo140.47 ± 2.00138.20 ± 2.60
MP 50 mg/day140.80 ± 1.66 F (1,28) = 0.400, 133.40 ± 18.73 F (1,28) = 0.256,
MP 1500 mg/day139.67 ± 2.13 F (1,28) = 0.303, 138.27 ± 2.46 F (1,28) = 0.319,

Potassium (mEq/L)3.4–4.7 mEq/LPlacebo4.67 ± 0.244.78 ± 0.57
MP 50 mg/day4.69 ± 0.38 F (1,28) = 0.421, 5.03 ± 0.68 F (1,28) = 1.216,
MP 1500 mg/day4.71 ± 0.62 F (1,28) = 0.822, 4.97 ± 0.55 F (1,28) = 1.444,

Chloride (mEq/L)96–107 mEq/LPlacebo94.70 ± 23.3494.32 ± 23.24
MP 50 mg/day100.73 ± 1.22 F (1,28) = 0.550, 100.47 ± 1.51 F (1,28) = 0.542,
MP 1500 mg/day100.27 ± 2.79 F = (1,28) = 0.514 101.33 ± 2.38 F = (1,28) = 1.872

Cholesterol (mg/dL)127–262 mg/dLPlacebo214.00 ± 50.96195.53 ± 48.86
MP 50 mg/day217.27 ± 48.77 F (1,28) = 0.032, 208.07 ± 42.55 F (1,28) = 0.362,
MP 1500 mg/day266.80 ± 32.03 F (1,28) = 0.215, 203.44 ± 24.42 F (1,28) = 0.650,

Albumin (g/dL)3.8–5.4 g/dLPlacebo3.90 ± 1.414.46 ± 040
MP 50 mg/day3.52 ± 1.59 F (1,28) = 0.789, 4.19 ± 0.02 F (1,28) = 6.404,
MP 1500 mg/day3.13 ± 1.63 F (1,28) = 0.050, 4.41 ± 26 F (1,28) = 8.348,

Globulin (g/dL)2.6–3.4 g/dLPlacebo2.69 ± 0.902.83 ± 0.35
MP 50 mg/day2.42 ± 1.05 F (1,28) = 0.520, 3.01 ± 0.44 F (1,28) = 2.718,
MP 1500 mg/day2.14 ± 1.08 F (1,28) = 0.058, 2.95 ± 0.28 F (1,28) = 3.079,

Bilirubin total (mg/dL)0.3–1.5 mg/dLPlacebo0.61 ± 0.390.51 ± 0.12
MP 50 mg/day0.67 ± 0.35 F (1,28) = 0.000, 0.49 ± 0.16 F (1,28) = 0.776,
MP 1500 mg/day0.66 ± 0.36 F (1,28) = 0.246, 0.51 ± 0.24 F (1,28) = 0.566,

Bilirubin direct (mg/dL)0–0.5 mg/dLPlacebo0.21 ± 0.090.19 ± 0.09
MP 50 mg/day0.21 ± 0.09 F (1,28) = 0.178, 0.16 ± 0.07 F (1,28) = 0.741,
MP 1500 mg/day0.20 ± 0.10 F (1,28) = 0.276, 0.15 ± 0.09 F (1,28) = 1.233,

Alanine aminotransferase or ALT (U/L)4–36 U/LPlacebo21.93 ± 10.8526.33 ± 13.71
MP 50 mg/day16.67 ± 7.21 F (1,28) = 2.005, 18.53 ± 9.04 F (1,28) = 3.492,
MP 1500 mg/day17.00 ± 5.54 F (1,28) = 2.129, 18.87 ± 5.60 F (1,28) = 4.192,

Aspartate aminotransferase or AST (U/L)12–32 U/LPlacebo22.00 ± 5.0124.93 ± 7.27
MP 50 mg/day19.93 ± 3.79 F (1,28) = 0.919, 22.20 ± 7.31 F (1,28) = 1.716,
MP 1500 mg/day24.40 ± 6.45 F (1,28) = 0.934, 23.93 ± 7.45 F (1,28) = 2.329,

Lactic acid dehydrogenase or LDH (U/L)89–221 U/LPlacebo214.07 ± 42.39218.50 ± 49.20
MP 50 mg/day202.40 ± 37.44 F (1,28) = 0.990, 219.54 ± 36.49 F (1,28) = 0.097,
MP 1500 mg/day197.38 ± 24.93 F (1,28) = 0.879, 219.67 ± 47.83 F (1,28) = 0.586,

Creatine kinase–MB or CK-MB (U/L)0–25 U/LPlacebo19.30 ± 5.1218.53 ± 5.05
MP 50 mg/day18.91 ± 6.34 F (1,28) = 1.781, 21.53 ± 5.90 F (1,28) = 1.883,
MP 1500 mg/day17.71 ± 7.51 F (1,28) = 1.879, 20.53 ± 9.20 F (1,28) = 1.657,

Triglyceride (mg/dL)10–200 mg/dLPlacebo141.53 ± 57.48148.60 ± 54.76
MP 50 mg/day137.53 ± 62.04 F (1,28) = 2.755, 97.20 ± 45.88 F (1,28) = 8.434,
MP 1500 mg/day117.93 ± 48 F (1,28) = 3.092, 119.07 ± 50.23 F (1,28) = 8.792,

High density lipoprotein-cholesterol or HDL-C (mg/dL)>35 mg/dLPlacebo48.79 ± 17.4052.07 ± 9.18
MP 50 mg/day41.95 ± 16.44 F (1,28) = 2.90, 60.27 ± 12.06 F (1,28) = 0.491,
MP 1500 mg/day36.93 ± 16.46 F (1,28) = 2.571, 64.47 ± 18.80 F (1,28) = 5.267,

Low density lipoprotein-cholesterol or LDL-cholesterol (mg/dL)0–150 mg/dLPlacebo121.49 ± 40.8141.80 ± 24.29
MP 50 mg/day109.19 ± 47.47 F (1,28) = 0.007, 156.20 ± 47.27 F (1,28) = 0.001,
MP 1500 mg/day100.82 ± 51.02 F (1,28) = 0.107, 141.47 ± 24.23 F (1,28) = 0.001,

3.5. Effect of Herbal Congee on the Hematological Changes

Table 3 showed the effect of various doses of herbal congee containing the combined extract of M. alba leaves and P. odoratum on the hematological values. It was found that at baseline consumption period, no significant changes of all parameters were observed. At the end of an 8-week consumption period, subjects who consumed herbal congee containing the combined extract of M. alba leaves and P. odoratum at dose of 1500 mg per day showed the significant reduction of platelets ( value < 0.05; compared to placebo group). However, the changes of all parameters were also in the normal range. No changes of other parameters were observed.


CBCNormal valueGroupBaseline8 weeks
Mean ± SDMean ± SD

Red blood cell or RBC (x 106/μL)4.0–5.20 (x 106/μL)Placebo3.92 ± 1.714.13 ± 1.33
MP 50 mg/day3.15 ± 1.97 F (1,28) = 1.284, 3.65 ± 1.62 F (1,28) = 2.048,
MP 1500 mg/day2.69 ± 1.03 F (1,28) = 2.138, 3.19 ± 1.77 F (1,28) = 1.845,

Hemoglobin or HGB (g/dL)12.0–14.3 g/dLPlacebo10.87 ± 4.9110.99 ± 3.69
MP 50 mg/day8.41 ± 5.19 F (1,28) = 0.208, 10.12 ± 4.49 F (1,28) = 0.761,
MP 1500 mg/day7.35 ± 2.84 F (1,28) = 1.215, 8.34 ± 4.49 F (1,28) = 0.947,

Hematocrit or HCT (%)36.0–47.7%Placebo32.34 ± 14.7036.58 ± 12.48
MP 50 mg/day25.10 ± 15.77 F (1,28) = 1.399, 33.95 ± 15.42 F (1,28) = 0.291,
MP 1500 mg/day21.98 ± 8.34 F (1,28) = 0.862, 29.48 ± 16.79 F (1,28) = 0.323,

White blood cells or WBC (x 103/μL)4.60–10.60 x 103/μLPlacebo6.19 ± 3.255.41 ± 1.91
MP 50 mg/day4.65 ± 1.99 F (1,28) = 0.950, 5.66 ± 1.15 F (1,28) = 0.692,
MP 1500 mg/day4.02 ± 1.875.94 ± 1.13

Platelet or PLT (x 103/μL)173–383 x 103/μLPlacebo248.01 ± 105.23 F (1,28) = 1.049, 274.20 ± 85.65 F (1,28) = 4.229,
MP 50 mg/day183.86 ± 95.34 F (1,28) = 1.304, 245.65 ± 104.49 F (1,28) = 0.071,
MP 1500 mg/day158.11 ± 71.84 F (1,28) = 0.134, 206.37 ± 108.42 F (1,28) = 6.935,

Mean platelet volume or MPV (fl)8.5–12.8 flPlacebo7.28 ± 3.078.10 ± 2.55
MP 50 mg/day5.65 ± 3.39 F (1,28) = 0.000, 7.07 ± 3.02 F (1,28) = 0.070,
MP 1500 mg/day4.85 ± 1.99 F (1,28) = 0.218, 5.96 ± 3.20 F (1,28) = 0.008,

Neutrophil or NE (%)43.7–70.9%Placebo48.36 ± 21.5042.54 ± 14.71
MP 50 mg/day37.54 ± 20.67 F F (1,28) = 0.001, 37.56 ± 16.97 F (1,28) = 0.292,
MP 1500 mg/day32.02 ± 13.38 F (1,28) = 0.002, 33.72 ± 17.88 F (1,28) = 0.155,

Lymphocyte or LY (%)20.1–44.5%Placebo29.21 ± 13.6137.29 ± 12.32
MP 50 mg/day23.96 ± 12.03 F (1,28) = 0.035, 33.91 ± 15.01 F (1,28) = 0.108,
MP 1500 mg/day19.70 ± 8.25 F (1,28) = 0.032, 27.70 ± 14.12 F (1,28) = 0.267,

Monocyte or MO (%)3.4–9.8%Placebo5.78 ± 1.956.10 ± 1.92
MP 50 mg/day4.88 ± 2.71 F (1,28) = 1.00, 5.26 ± 2.06 F (1,28) = 0.466,
MP 1500 mg/day3.83 ± 1.80 F (1,28) = 0.037, 4.44 ± 2.12 F (1,28) = 0.005,

Eosinophil or EO (%)0.7–9.2%Placebo3.49 ± 1.975.16 ± 3.23
MP 50 mg/day3.24 ± 0.89 F (1,28) = 1.765 4.72 ± 3.02 F (1,28) = 1.255,
MP 1500 mg/day3.40 ± 1.21 F (1,28) = 2.131, 3.82 ± 8.09 F (1,28) = 1.788,

Basophil or BA (%)0.0–2.6%Placebo0.44 ± 0.21 F (1,28) = 0.757, 0.78 ± 0.47 F (1,28) = 0.373,
MP 50 mg/day0.38 ± 0.17 F (1,28) = 0.519, 0.68 ± 0.42 F (1,28) = 373,
MP 1500 mg/day0.29 ± 0.12 F (1,28) = 0.911, 0.62 ± 0.29 F (1,28) = 0.794,

Mean corpuscular volume or MCV (fl)80.0–97.8 flPlacebo71.33 ± 31.1178.58 ± 25.49
MP 50 mg/day54.06 ± 31.73 F (1,28) = 0.363, 70.17 ± 31.05 F (1,28) = 0.450,
MP 1500 mg/day47.06 ± 19.38 F (1,28) = 0.517, 56.01 ± 28.70 F (1,28) = 0.553,

Mean corpuscular hemoglobin MCH (pg)25.2–32.0 pgPlacebo23.98 ± 10.4325.39 ± 8.20
MP 50 mg/day18.10 ± 10.34 F (1,28) = 2.559, 22.81 ± 10.10 F (1,28) = 0.100,
MP 1500 mg/day15.71 ± 6.60 F (1,28) = 0.273, 18.25 ± 9.39 F (1,28) = 0.219,

Mean corpuscular hemoglobin concentration or MCHC (g/dL)31.3–33.4 g/dLPlacebo29.97 ± 13.7529.30 ± 10.07
MP 50 mg/day22.75 ± 13.62 F (1,28) = 3.604, 26.18 ± 12.08 F F (1,28) = 1.533,
MP 1500 mg/day20.02 ± 7.89 F (1,28) = 0.992, 22.02 ± 12.56 F (1,28) = 0.003,

Red blood cells distribution width or RDW (%)11.9–14.8%Placebo12.57 ± 3.4912.51 ± 3.96
MP 50 mg/day10.16 ± 5.27 F (1,28) = 0.011, 10.98 ± 4.60 F (1,28) = 3.822,
MP 1500 mg/day7.87 ± 4.22 F (1,28) = 2.650, 9.25 ± 4.89 F (1,28) = 0.170,

4. Discussion

This study clearly revealed that no major side effects or clinically significant symptoms were reported from any of the volunteers. No data of clinical chemistry and hematological values showed the significant toxicity. Therefore, these data support the consumption safety for healthy menopausal women. In addition, this study clearly revealed that subjects who consumed the herbal congee containing the combined extract of M. alba leaves and P. odoratum at dose of 1500 mg per day increased the total phenolic compounds in serum and improved bone formation markers including osteocalcin and alkaline phosphatase (ALP) but decreased bone resorption marker including serum collagen type 1 cross-linked C-telopeptide (beta CTx).

Osteoblasts which are responsible for bone formation are located on the bone surface. Bone formation is enhanced by the osteoproduction action of osteoblasts. During bone formation process, alkaline phosphatase is produced by osteoblasts during the synthesis of the collagen matrix [13]. In addition to alkaline phosphatase, osteocalcin, a 5.8 kDa hydroxyapatite-binding bone-specific protein, is also produced by osteoblasts [14]. In contrast to both parameters mentioned earlier, beta CTx is released into the bloodstream during bone resorption and serves as a specific marker for the degradation of mature type I collagen. Therefore, an elevated serum concentration of beta-CTx is reported to reflect bone resorption [15]. It has been reported that the rapid increase in serum beta CTX concentration is related to the secretion activity of osteoclasts [16]. The current data showed the elevation of alkaline phosphatase and osteocalcin but decreased beta-CTx level in serum of subjects who consumed MP at dose of 1500 mg/day. These results indicated the positive modulation effect of the herbal congee on the bone turn over which enhanced the bone formation but decreased bone resorption. Our data were in agreement with the previous study which showed the positive modulation effect of the combined extract of M. alba and P. odoratum (MP) on bone turnover in animal model of menopause [10].

Recent finding has demonstrated that polyphenolic compounds inhibit receptor activator of nuclear factor-κB ligand- (RANKL-) induced osteoclast formation [17]. Since the herbal congee containing the combination extract of P. odoratum and M. alba contained high concentration of polyphenol, this substance may possibly be active constituent which contributes a role on an antiosteoclastogenic effect of the herbal congee containing the combination extract of P. odoratum and M. alba. However, further researches concerning depth analysis for active ingredient and the detail mechanism of action are still essential.

Although the current results clearly showed that the congee containing P. odoratum and M. alba is safe for consumption, the cholesterol showed the increasing trends. Therefore, the long-term application of the product should be a caution about hypercholesterolemia. However, studies which focused on the effect of long-term treatment should also be performed in order to assure that no side effect on hypercholesterolemia occurs. In addition, drug interaction is also possible during long-term application especially the interaction with the drugs commonly used in daily life such as paracetamol. Thus, the information concerning this aspect still requires further researches. The limitation of this study is that no data concerning the bone density can be demonstrated because the treatment duration is not long enough to produce the changes of bone density.

5. Conclusion

The present study demonstrates the antiosteoporotic effect of the polyphenol-rich herbal congee which contained the combined extract of P. odoratum and M. alba. The possible underlying mechanism may occur via the improved bone turnover via the increased bone formation and the decreased bone resorption. Therefore, the herbal congee containing the combined extract of P. odoratum and M. alba may be useful for the prevention and treatment of osteoporosis in menopause. However, long-term treatment study is still required to assure that no side effect on hypercholesterolemia.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

No competing financial interests exist.

Acknowledgments

This study was supported by the National Research Council of Thailand, Research Division of Faculty of Medicine and the Integrative Complementary Alternative Medicine Research and Development Center, Khon Kaen University, Thailand (Research Fund Year 2014).

References

  1. C. Cervellati, G. Bonaccorsi, E. Cremonini et al., “Oxidative stress and bone resorption interplay as a possible trigger for postmenopausal osteoporosis,” BioMed Research International, vol. 2014, Article ID 569563, 8 pages, 2014. View at: Publisher Site | Google Scholar
  2. B. L. Clarke and S. Khosla, “Physiology of bone loss,” Radiologic Clinics of North America, vol. 48, no. 3, pp. 483–495, 2010. View at: Publisher Site | Google Scholar
  3. P. Garnero, E. Sornay-Rendu, M. C. Chapuy, and P. D. Delmas, “Increased bone turnover in late postmenopausal women is a major determinant of osteoporosis,” Journal of Bone and Mineral Research, vol. 11, no. 3, pp. 337–349, 1996. View at: Publisher Site | Google Scholar
  4. D. C. Bauer, P. Garnero, J. P. Bilezikian et al., “Short-term changes in bone turnover markers and bone mineral density response to parathyroid hormone in postmenopausal women with osteoporosis,” The Journal of Clinical Endocrinology & Metabolism, vol. 91, no. 4, pp. 1370–1375, 2006. View at: Publisher Site | Google Scholar
  5. I. Cepelak and D. Cvoriscec, “Biochemical markers of bone remodeling – review,” Biochemia Medica, vol. 19, no. 1, pp. 17–35, 2009. View at: Publisher Site | Google Scholar
  6. H. Rosenbrock, V. Seifert-Klauss, S. Kaspar, R. Busch, and P. B. Luppa, “Changes of biochemical bone markers during the menopausal transition,” Clinical Chemistry and Laboratory Medicine, vol. 40, no. 2, pp. 143–151, 2002. View at: Publisher Site | Google Scholar
  7. C. Kleinhans, F. F. Schmid, F. V. Schmid, and P. J. Kluger, “Comparison of osteoclastogenesis and resorption activity of human osteoclasts on tissue culture polystyrene and on natural extracellular bone matrix in 2D and 3D,” Journal of Biotechnology, vol. 205, pp. 101–110, 2015. View at: Publisher Site | Google Scholar
  8. P. Dreyer and J. G. H. Vieira, “Bone turnover assessment: a good surrogate marker?” Arquivos Brasileiros de Endocrinologia & Metabologia, vol. 54, no. 2, pp. 99–105, 2010. View at: Publisher Site | Google Scholar
  9. L. Đudarić, A. Fužinac-Smojver, D. Muhvić, and J. Giacometti, “The role of polyphenols on bone metabolism in osteoporosis,” Food Research International, vol. 77, Part 2, pp. 290–298, 2015. View at: Publisher Site | Google Scholar
  10. S. Sungkamanee, J. Wattanathorn, S. Muchimapura, and W. Thukham-mee, “Antiosteoporotic effect of combined extract of Morus alba and Polygonum odoratum,” Oxidative Medicine and Cellular Longevity, vol. 2014, Article ID 579305, 9 pages, 2014. View at: Publisher Site | Google Scholar
  11. S. Eghbaliferiz and M. Iranshahi, “Prooxidant activity of polyphenols, flavonoids, anthocyanins and carotenoids: updated review of mechanisms and catalyzing metals,” Phytotherapy Research, vol. 30, no. 9, pp. 1379–1391, 2016. View at: Publisher Site | Google Scholar
  12. L. I. Mennen, R. Walker, C. Bennetau-Pelissero, and A. Scalbert, “Risks and safety of polyphenol consumption,” The American Journal of Clinical Nutrition, vol. 81, no. 1, pp. 326S–329S, 2005. View at: Publisher Site | Google Scholar
  13. E. J. Mackie, “Osteoblasts: novel roles in orchestration of skeletal architecture,” The International Journal of Biochemistry & Cell Biology, vol. 35, no. 9, pp. 1301–1305, 2003. View at: Publisher Site | Google Scholar
  14. C. M. Gundberg, J. B. Lian, and S. L. Booth, “Vitamin K-dependent carboxylation of osteocalcin: friend or foe?” Advances in Nutrition, vol. 3, no. 2, pp. 149–157, 2012. View at: Publisher Site | Google Scholar
  15. C. de la Piedra, M. L. Traba, C. D. Cabrera, and M. S. Henríquez, “New biochemical markers of bone resorption in the study of postmenopausal osteoporosis,” Clinica Chimica Acta, vol. 265, no. 2, pp. 225–234, 1997. View at: Publisher Site | Google Scholar
  16. S. A. Nesbitt and M. A. Horton, “Trafficking of matrix collagens through bone-resorbing osteoclasts,” Science, vol. 276, no. 5310, pp. 266–269, 1997. View at: Publisher Site | Google Scholar
  17. K. S. Shim, T. Kim, H. Ha et al., “Water extract of Magnolia officinalis cortex inhibits osteoclastogenesis and bone resorption by downregulation of nuclear factor of activated T cells cytoplasmic 1,” Integrative Medicine Research, vol. 4, no. 2, pp. 102–111, 2015. View at: Publisher Site | Google Scholar

Copyright © 2018 Jintanaporn Wattanathorn et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


More related articles

757 Views | 329 Downloads | 1 Citation
 PDF  Download Citation  Citation
 Download other formatsMore
 Order printed copiesOrder

Related articles

We are committed to sharing findings related to COVID-19 as quickly and safely as possible. Any author submitting a COVID-19 paper should notify us at help@hindawi.com to ensure their research is fast-tracked and made available on a preprint server as soon as possible. We will be providing unlimited waivers of publication charges for accepted articles related to COVID-19. Sign up here as a reviewer to help fast-track new submissions.