***Please annotate the research article (provided below) please and very details comments directly on the article and attach with the annotations***And then answer these questions below (they can be in paragraph form):1.What are the methods used in obtaining the data? Describe what type of data is presented. 2. What is the sample size of the study?3. What types of outside factors should have been limited in the study and how did the researchers explain what they did to limit those factors? 4. What forms of experimental controls were used?5. what were the results of the study? Were the findings significant? 6. How many times has the study been repeated? 7. A brief summary of what the article is about. 8. Was it a good or bad experimental design (support opinion with specific examples). Characteristics of a good experimental design include: Repition and Repeatability: it will be possible to do the same experiment and use the same materials under the same conditions again Experimental Controls: Need to have a value you measure to compare your experimental values to Adequate sample size: Has a large amount of # in participants (30 or more individuals) Limitation of Factors: Limit the things that will affect the result Measurable Results: Data
antioxidant_and_physiological_effects_of_si_wu_tang_on_skin_and_liver_a_randomized__double_blind__placebo_controlled_clinical_trial.pdf

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Chiu et al. Chin Med (2016) 11:30
DOI 10.1186/s13020-016-0102-0
Chinese Medicine
Open Access
RESEARCH
Antioxidant and physiological effects
of Si‑Wu‑Tang on skin and liver: a randomized,
double‑blind, placebo‑controlled clinical trial
Hui‑Fang Chiu1, Ying‑Hua Wu2, You‑Cheng Shen3, Shing‑Jung Wang4, Kamesh Venkatakrishnan2
and Chin‑Kun Wang2*
Abstract
Background: Si-Wu-Tang (SWT) is used to treat various gynecological disorders in Chinese medicine. This study
investigated the antioxidant and physiological effects of SWT on the skin and liver in healthy adults.
Methods: This randomized, crossover, double-blind, placebo-controlled clinical trial was conducted at Chung Shan
Medical University Hospital in December 2008. Participants with uncontrolled diabetes, heart disease, liver disease,
kidney disease, cancer, and pregnancy were excluded. Sixty healthy volunteers taking no medications were recruited
from the community based on the results of their medical history questionnaires and biochemical analyses to confirm
their health status. The participants were assigned to two groups: one group drank 125 mL of placebo (n = 30) and
the other drank SWT (n = 30) for six continuous days per month for 6 months. The placebo and SWT were then
switched between the groups after a 1-month washout period. Anthropometric measurements (body weight, body
fat, and body mass index) were performed and fasting blood samples were drawn for various biochemical assays at
1, 3, 6, 10 and 13 months. Abdominal ultrasound and skin examinations were performed at 1, 6 and 13 months. The
skin examinations involved assessment of skin roughness, sebum content, hydration, surface water loss, erythema,
melanin index, and elasticity on the face (sunlight-exposed sites: middle of ear and nose) and inner arm (sunlightunexposed sites: center of wrist and elbow joint).
Results: Administration of SWT significantly increased the antioxidant index (P = 0.001) and antioxidant enzymes
activities (P = 0.001) from baseline to month 6. SWT also suppressed the concentration of serum lipids (triglycerides,
P = 0.01; high-density lipoprotein cholesterol, P = 0.23; low-density lipoprotein cholesterol, P = 0.48) and hepatic
marker enzymes (glutamic pyruvic transaminase, P = 0.76; glutamic oxaloacetic transaminase, P = 0.65) when com‑
pared with the placebo group. Abdominal ultrasound in the SWT group revealed a positive impact of SWT on mild
fatty liver, gallstones, and mild splenomegaly. Moreover, SWT intake concomitantly elevated erythema (P = 0.011) and
markedly lowered skin surface water loss (P = 0.016), sebum content (P = 0.021), and wrinkles (P = 0.024).
Conclusions: Oral administration of SWT for 6 months improved the antioxidant level and positively regulated the
lipid profile, liver function, and skin integrity and texture.
Background
The therapeutic essence of Chinese medicine (CM) can
be attributed to multiple elements rather than just one
in the product used [1]. Si-Wu-Tang (SWT) is a CM
*Correspondence: wck@csmu.edu.tw
2
School of Nutrition, Chung Shan Medical University, Taichung, Taiwan
Full list of author information is available at the end of the article
formula comprising four different medicinal herbs:
Radix Paeoniae Alba (bai shao yao), Rhizoma Ligusticum
Chuanxiong (chuan xiong), Radix Angelica Sinensis (dang
gui), and Radix Rehmanniae Preparata (shu di huang)
[2]. SWT is widely used for the treatment of gynecological disorders such as menstrual discomfort, climacteric
syndrome, dysmenorrheic and other estrogen-related
diseases [3], cutaneous diseases [4], and menstrual
© 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Chiu et al. Chin Med (2016) 11:30
discomfort [5]. Several preclinical (in vitro) studies of
SWT have demonstrated antioxidative, anti-inflammatory, antiaging, antibacterial, and antipruritic activities
[6, 7].
The major bioactive components in these four herbs
include phenolics, phthalides, alkaloids, terpene glycosides, and iridoid glycosides. Some major constituents
are gallic acid, paeoniflorin, senkyunolide A, ferulic
acid, Z-ligustilide, butylphthalide, sodium ferulate, and
catalpol [3]. Among these compounds, ferulic acid, paeoniflorin, and Z-ligustilide in SWT exhibited antioxidative, antimutagenic, anti-inflammatory, vasodilative, and
antiallergic effects [8–10]. Sodium ferulate in A. sinensis
and Lignsticum chuangxiong of SWT has lowered the
oxidation process and lipid levels in various animal models [11, 12]. Additionally, SWT is rich in organic acids,
amino acids, polysaccharides, vitamins, and minerals
such as magnesium, calcium, phosphorus, and iron [13].
Liver cells (hepatocytes) metabolize toxins such as
alcohol and drugs into their inactive metabolites. Hepatocytes are extremely prone to peroxide (free radical) formation because of their higher metabolic rate. These free
radicals are effectively eliminated by antioxidants, especially glutathione peroxidase (GPx). GPx plays a major
role in the reductive detoxification of peroxides in hepatocytes [14]. A decreased antioxidant level may lead to
oxidative stress and eventual hepatic dysfunction. Several
reports have proven that hepatic dysfunction might contribute to various dermal disorders such as dermatitis,
eczema, psoriasis, acne, and boils or rashes secondary to
decreased detoxification (excessive toxin buildup in skin)
and elevated inflammation and oxidative stress [15, 16].
Hence, this study was designed to investigate the antioxidant and physiological effects of SWT on skin and liver
in healthy adults.
Methods
Chemicals
Folin–Ciocalteu phenol reagent, sodium hydroxide,
sodium nitrite, hydrochloric acid, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), hydrogen
peroxide, sodium dihydrogen phosphate, and trichloroacetic acid were purchased from Sigma (St. Louis, MO,
USA).
SWT and placebo
Commercially available SWT and placebo as an oral suspension (glass vial) were provided by Standard Foods
Corporation (Taipei, Taiwan). Each vial contained
125 mL of SWT (Radix P. Alba, Rhizoma L. Chuanxiong, Radix A. Sinensis, and Radix R. Preparata with cinnamon twig, rose hip extract, sucrose, vitamin C, malic
acid, citric acid, and gelatin) or placebo (simulated SWT
Page 2 of 10
flavor with herb-rinsed water and sucrose solution). Both
sample vials were indistinguishably packaged with a similar color, flavor, size, and shape.
Participants
This randomized, crossover, double-blind, placebocontrolled clinical trial was conducted in Chung Shan
Medical University Hospital. The trial was carried out in
accordance with the declaration of Helsinki and subsequent revisions and approved by the Institutional Review
Board (IRB) of Chung Shan Medical University Hospital, Taichung, Taiwan (CSMUHCS08008) and registered
with clinicaltrials.gov (NCT02634242) (Additional file 1).
Written informed consent was received from all participants before enrollment (Additional file 2). Taking into
account an expected dropout rate of approximately 25 %
and the crossover design, the required sample size (95 %
CI, α = 0.05) was 60 participants (30 participants in each
group) [17]. All participants (aged 20–80 years) were
enrolled in the present experiment via advertisements
displayed in public places.
The participants were requested to avoid using medications or supplements during the intervention. They
could withdraw from the study at any time if desired.
The exclusion criteria were a history of smoking, alcoholism, pregnancy or lactation, chronic diseases, and
hepatic or renal dysfunction. A physical examination
was performed in the 60 voluntary participants at the
beginning of the study, and they were segregated into
two groups of 30 participants in each (five male and 25
female): those who drank 125 mL of placebo and those
who drank SWT for 6 days per month for 6 months. The
placebo and SWT were switched between the two groups
after a 1-month washout period. Anthropometric analyses of body weight, body fat, and body mass index were
performed at 1, 3, 6, 10 and 13 months. The SWT and
placebo vials were labeled with the participant number
by an electrical randomization method [17]. The average
percentage intake of SWT or placebo was 88.47 % at the
end of the experiment based on the participants’ records.
During the crossover study, three and two female participants in the placebo and SWT groups, respectively, were
excluded from the study because of an unwillingness to
cooperate. Thus, the present study was performed with
27 and 28 participants in the placebo and SWT group,
respectively (n = 55).
Blood sampling
At baseline (months 1 and 7), the middle time point
(months 3 and 10), and the end of each intervention
period (months 6 and 13), fasting blood samples were
collected in an EDTA-coated vacuum tube, and plasma
was separated by centrifugation (Supercentrifuge 1K15;
Chiu et al. Chin Med (2016) 11:30
Sigma) at 1500×g. Blood samples were isolated after separating the intermediate film, and the settled portion was
washed with isotonic saline and centrifuged at 1500×g to
obtain the erythrocytes for assaying antioxidant enzymes.
All samples were stored at −80 °C until analyses.
Abdominal ultrasonic examination was performed at
month 1, 6 and 13 at Chung Shan Medical University
Hospital, Taichung, Taiwan with the help of an experienced gastroenterology physician (Dr. Chen Ziyan).
Various oxidative indexes
The total antioxidant capacity (TEAC) [18, 19], total thiobarbituric acid reactive substances (TBARS) [20], and
glutathione content [21] in plasma were determined by
previously reported methods.
Antioxidative enzymes
Superoxide dismutase (SOD) activity was determined
with the Ransod SD 125 kit (Randox Labs, Crumlin, UK),
GPx and glutathione reductase (GR) activity were determined with the Ransel RS 504 kit (Randox Labs, Crumlin,
UK), and catalase (CAT) activity was measured by Aebi
[22]. The red blood cell protein content was determined
based on the biuret reaction of the BCA kit from Thermo
Fisher Scientific (MA, USA).
Serum lipids and hepatic markers
The serum concentrations of total cholesterol (TC),
triglycerides (TG), and high-density lipoprotein cholesterol (HDL-c) were measured by commercially available lipid profile kits (CHODPAP for TC, GPO-PAP
for TG, and a precipitation method for HDL-c; Roche
Diagnostics, Mannheim, Germany) on a Hitachi 747
autoanalyzer. Low-density lipoprotein cholesterol
(LDL-c) was calculated by the Friedewald equation.
The plasma glutamic oxaloacetic transaminase (GOT)
and glutamic pyruvic transaminase (GPT) concentrations were measured by AppliedBio assay kits (Hercules, CA, USA).
Skin examination
A multifunctional skin detector (MPA 580; Courage and
Khazaka Electronic GmbH, Cologne, Germany) and skin
roughness analyzer (VD 300; Courage and Khazaka Electronic GmbH) were used to detect the biophysical characteristics of skin in the face and arm regions. No skin
care products were applied to the measured sites for at
least 24 h prior to the measurements. The skin sebum
content, hydration, surface water loss, erythema, melanin
index, and elasticity were measured with the following
respective probes: Sebumeter, Corneometer, TEWAmeter, Mexameter, and Cutometer (Courage and Khazaka
Electronic GmbH).
Page 3 of 10
The Sebumeter SM 815 utilizes the difference in light
intensity via a plastic strip to indicate the quantity of
absorbed sebum. The sebum level is expressed as μg/cm2
[23]. Skin hydration was measured by Corneometer CM
825, which uses the high dielectric constant of water
to evaluate the water-related changes in the electrical
capacitance of the skin. It displays hydration measurements in system-specific arbitrary units [24]. A melanin
index is calculated by the Mexameter MX 18 from the
strength of the absorbed and reflected light at 660 and
880 nm, respectively. Erythema is processed similarly at
568 and 660 nm, respectively [25]. The measurement of
cutaneous water loss by TEWAmeter TM 300 was analyzed based on the diffusion in an open chamber and is
expressed as g/m2/h [26]. The Cutometer MPA 580 pulls
the targeted skin into the probe with controlled vacuum
pressure [27]. Skin wrinkles were measured using a Visioscan VC 98 (Courage and Khazaka Electronic GmbH).
In brief, two points were measured: one at the middle of
the nose and right ear, and the other at the middle of the
wrist and inner elbow joint of right hand. All participants
were instructed to wash and clean their face and arm, and
then rested calmly for 30 min in a room kept at 20 ± 2 °C
and a relative humidity of 50 ± 2 % before testing.
Statistical analysis
The results are expressed as a mean ± standard deviation.
The paired t test was employed to compare differences
within the same group (baseline vs. end of treatment),
and Student’s t test was used to compare differences
between the experimental (SWT) and control (placebo)
groups. All variables were analyzed via one-way analysis
of variance with the post doc least-significant differences
test. A P < 0.05 was considered statistically significant. The Statistical Package for the Social Sciences (SPSS) version 17.0 (Chicago, IL, USA) was used for analysis. Results and discussion A crossover design was chosen for the present investigation because of its numerous advantages such as decreased confounding covariants (because the same participants ingested both SWT and placebo) as well as statistically effective during chronic condition [28]. Based on our literature survey, a 1-month wash-out period was adopted for the present study to suppress the carry-over effect. A flowchart of the present study is illustrated in Fig. 1. The anthropometric parameters in the placebo and SWT groups are presented in Table 1. At baseline, the body weight, body fat, and body mass index were within the normal range in both the placebo and SWT groups. There were no significant differences in any of the anthropometric parameters between the placebo and SWT groups at the end of the intervention. Chiu et al. Chin Med (2016) 11:30 Page 4 of 10 Fig. 1 Flow chart of present study Table 1 The anthropometric parameters and SWT treated healthy subjects Group in placebo Body fat (%) BMI (kg/m2) 57.46 ± 9.43a a 29.23 ± 5.82a a 22.29 ± 3.18a 57.74 ± 9.80a a 29.47 ± 5.91a a 22.39 ± 3.28a 57.70 ± 9.28a 29.32 ± 6.09a 22.32 ± 3.46a Weight (kg) Baseline Placebo SWT 3rd month Placebo SWT 6th month Placebo SWT 57.45 ± 9.58 57.70 ± 9.48 57.41 ± 9.67 a 29.27 ± 5.77 29.15 ± 5.70 a 29.25 ± 5.77 22.35 ± 3.19a 22.44 ± 3.21a 22.07 ± 3.43a Values were expressed as mean ± SD (n = 55). Data within the same column of each group sharing different superscript letters were significantly different (p < 0.05) BMI body mass index Antioxidants interact with free radicals and stabilize by donating their electrons or protons. If untreated, these free radicals can cause various chronic diseases [29]. Table 2 shows the plasma oxidative indexes including, TEAC, glutathione (GSH) concentration, and TBARS in the placebo- and SWT-treated participants. The TEAC reflects the relative ability of hydrogen or electron-donating antioxidants to scavenge the 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical cation compared with that of Trolox. Treatment with SWT for 6 months substantially increased the levels of TEAC (0.35–0.59 µM; P = 0.001) and GSH (14.19–14.35 µM; P = 0.88), whereas the TBARS level (1.40–0.68 µM; P = 0.001) was lower than that in the baseline period; additionally, notable changes were found between the SWT and placebo groups. SWT comprises four medicinal plants with numerous active components including gallic acid, paeoniflorin, ferulic acid, and Z-ligustilide, which contain numerous OH groups that scavenge free radicals through electron delocalization [6]. Liu et al. [30] reported that polysaccharide from A. sinensis could directly scavenge oxygen-derived free radicals in colonic tissues of rats with colitis. The antioxidant defense system consists of endogenous antioxidants SOD, CAT, GPx, and GR. SOD catalyzes the dismutation of superoxide into hydrogen peroxide and water molecules. After the decomposition of hydrogen peroxide by GPx at the expense of GSH, the GR-catalyzed Chiu et al. Chin Med (2016) 11:30 Page 5 of 10 Table 2 Various plasma oxidative indexes in placebo and SWT treated healthy subjects Group TEAC (µM/mL) p value* TBARS (μM/L) p value* GSH (μM/L) p value* 0.34 ± 0.07a 0.82 1.43 ± 0.47a 0.87 14.18 ± 2.30b 0.67 0.34 ± 0.04a 0.001 1.42 ± 0.43a 0.001 14.19 ± 2.79a 0.83 0.35 ± 0.03a 0.001 1.45 ± 0.32a 0.001 14.16 ± 2.85a 0.88 Baseline Placebo SWT 3rd month Placebo SWT 6th month Placebo SWT b a 0.35 ± 0.07 14.19 ± 2.48b 1.40 ± 0.44 b b 0.40 ± 0.07 14.31 ± 2.72a 0.83 ± 0.28 a c 0.59 ± 0.06 14.35 ± 2.94b 0.68 ± 0.23 Values were expressed as mean ± SD (n = 55). Data within the same column of each group bearing different superscript letters were significantly different (p < 0.05) * Student’s t test was used to assess statistical significance between placebo and Si Wu Tang (SWT) regeneration of GSH from its oxidized form can sustain the GSH-dependent oxy-radical scavenging activity [31]. The antioxidant enzymes evaluated in this study (SOD, CAT, GPx, and GR in the erythrocytes of the placeboand SWT-treated participants) are epitomized in Table 3. No notable changes were found from baseline to month 6 in the placebo-treated group. However, treatment with 125 mL of SWT showed a marked escalation in the levels of SOD (1478.12–1812.47 IU/g Hb; P = 0.001), CAT (565.82–803.32 IU/g Hb; P = 0.001), GPx (51.03– 52.59 IU/g Hb; P = 0.46), and GR (10.51–11.93 IU/g Hb; P = 0.02) between baseline and the end of the treatment (month 6). Meanwhile, significant changes were also noted between the SWT and placebo groups at month 6. Nuclear factor erythroid 2-related factor 2 (Nrf2) by Z-ligustilide was involved in the antioxidant activity in the SWT group. Activation of Nrf2 triggered the downstream antioxidant genes (SOD, CAT) and thereby enhanced antioxidant enzyme levels [32]. Additionally, the polyphenolics and numerous phytochemical constituents present in SWT might have elevated the levels of antioxidant enzymes by their free radical quenching ability [7]. Hou et al. [33] reported that treatment with L. chuanxiong and A. sinensis showed a better protection against hydrogen peroxide-induced endothelial damage by suppressing reactive oxygen species production. Furthermore, trace elements (calcium, magnesium, and selenium) in SWT act as cofactors to antioxidant enzymes [34]. Lipids are metabolized and regulated by hepatocytes. Any changes in the hepatic lipid status could directly reflect the serum lipid status. Although they are not major hepatic markers, lipids are still effective markers with which to determine whether SWT impacts physiological changes in hepatic tissue by estimating the lipid profile in serum. Table 4 shows the lipid profiles in the serum of the placebo and SWT groups. Supplementation with SWT for 6 months resulted in a significant decline in the levels of TG (102.12–85.89 mg/dL; P = 0.01) with a slight decrement in the level of LDL-c (113.05– 112.84 mg/dL; P = 0.48), whereas the level of HDL-c was concomitantly impro ... Purchase answer to see full attachment