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Contemporary Clinical Trials Communications 17 (2020) 100499
Available online 27 November 2019
2451-8654/© 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Research paper
Clinical evaluation of Emblica Ofcinalis Gatertn (Amla) in healthy human
subjects: Health benets and safety results from a randomized,
double-blind, crossover placebo-controlled study
Mahendra Parkash Kapoor
a
,
*
, Koji Suzuki
b
,
c
, Timm Derek
d
, Makoto Ozeki
a
, Tsutomu Okubo
a
a
Taiyo Kagaku Co. Ltd., Nutrition Division, 1-3 Takaramachi, Yokkaichi, Mie, 510-0844, Japan
b
Department of Molecular Pathobiology, Mie University, 2-174 Edobashi, Tsu City, Mie, 514 8507, Japan
c
Suzuka University of Medical Sciences, Suzuka City, Mie, 510-0221, Japan
d
Taiyo International Inc., Minneapolis, Minnesota, 55416, USA
ARTICLE INFO
Keywords:
Amla (Emblica Ofcinalis Gaertn)
Clinical trial
Vascular functions
Hematology
Lipid prole
ABSTRACT
The preventive efcacies and safety of Emblica Ofcinalis Gatertn (Amla), a most important and extensively
studied plant in the traditional Indian Ayurvedic system of medicine, are presented. Eligible healthy adult
subjects (n ¼15) were randomized to receive either amla or placebo (500 mg per day) during an 18-week study.
The efcacy parameters evaluated were the vascular function, blood hematology, oxidative and inammatory
biomarkers, glucose and lipid proles, urinalysis, and liver hepatotoxicity. The amla intake showed signicant
improvements in the primary efcacy parameter of blood uidity. There were also improvements in the sec-
ondary endpoints including lowering of von Willebrand factor (vWF), reduced 8-hydroxy-20-deoxyguanosine (8-
OHdG) as well as thrombin (TM) biomarkers of oxidative stress along with a signicant improvement in HDL-
cholesterol and lowering the LDL-cholesterol levels. No substantial changes were observed in liver hepatotox-
icity, urinalysis, and hematology after consumption of amla compared to baseline or placebo. In addition, no
adverse events, changes safety parameters or tolerance issues were observed after consumption of amla. In
conclusion, amla supplementation showed acceptable palatability, improved endothelial functions and reduced
oxidative stress.
1. Introduction
The metabolic syndrome is dened as having two of the following:
hyperglycemia, hyperlipidemia, high blood pressure, low HDL-
cholesterol, or abdominal obesity and has become an epidemic during
the last few decades [1]. In recent years there has been a growing in-
terest and awareness of the role of functional dietary supplements to
help with the prevention of lifestyle-related diseases [2,3]. Emblica
Ofcinalis Gatertn, commonly called amla, has traditionally been used
for different medicinal purposes including: rheumatic pains, gonorrhea,
asthma, hemorrhage, jaundice, dyspepsia, nausea, constipation, diar-
rhea, eye disease, brain health, intestinal ailments, diabetes mellitus,
coronary heart diseases, and various cancers [4,5]. Modern science has
shown amla to have hypoglycemic, anti-inammatory, anti--
hyperglycemic, anti-hyperlipidemic, and antioxidant properties in ani-
mal and human studies [6–8]. These properties may be due to the amla
fruit containing high levels of vitamin C, tannins, polyphenols, bers,
minerals, proteins, and amino acids [4,9]. Standardized amla formula-
tion usually contains the complex tannins and ellagitannins such as
corilagin, geraniin, chebulagic acid, and elaeocarpusin, etc., which are
the most active components and have high antioxidant activity [9].
Apart from the useful antioxidant activity, they have anti-thrombosis
properties to promote vascular health by improve blood uidity,
anti-coagulant, and antiplatelet activity, which can cause a warming
sensation. Amla also supports natural immunity and digestive functions.
It is not completely understood, which amla components are responsible
for each activity and they may be mediated through multiple different
mechanisms [3,10–12]. The combined anti-inammatory, anti-th-
rombosis, anti-coagulant, and anti-platelet activities of amla make it
attractive target for the prevention of a variety of vascular disorders [13,
14].
Further, there is limited evidence to support the longevity-promoting
* Corresponding author.
E-mail address: mkapoor@taiyokagaku.co.jp (M.P. Kapoor).
Contents lists available at ScienceDirect
Contemporary Clinical Trials Communications
journal homepage: http://www.elsevier.com/locate/conctc
https://doi.org/10.1016/j.conctc.2019.100499
Received 31 July 2019; Received in revised form 6 November 2019; Accepted 21 November 2019
Contemporary Clinical Trials Communications 17 (2020) 100499
2
Table 1
Anthropometric physiological and vascular parameters of subjects of amla supplementation clinical trial.
Parameters Placebo intake (mean �sem) Amla intake (mean �sem) ANCOVA (Among
Groups)
Before (W1, W2) or
(W10, W11)
2 Weeks (W3, W4)
or (W12, W13)
4 Weeks (W5, W6)
or (W14, W15)
Post (3 Weeks)
(W7,W9,W9) or
(W16,W17,W18)
Before (W1, W2) or
(W10, W11)
2 Weeks (W3, W4) or
(W12, W13)
4 Weeks (W5, W6)
or (W14, W15)
Post (3 Weeks)
(W7,W9,W9) or
(W16,W17,W18)
Weight (kg) 70.6 �2.97 70.4 �3.03 70.4 �3.01 70.9 �2.87 70.6 �3.04 70.8 �2.98 70.4 �3.02 71.1 �3.00 2W: P ¼0.42
4W: P ¼0.89
Post: P ¼0.51
P ¼0.31 P ¼0.48 P ¼0.44 P ¼0.43 P ¼0.70 P ¼0.17
BMI (kg/m
2
) 25.1 �0.63 25.1 �0.67 25.0 �0.63 25.2 �0.66 25.0 �0.64 25.3 �0.64 25.2 �0.62 25.3 �0.66 2W: P ¼0.43
4W: P ¼0.21
Post: P ¼0.59
P ¼0.23 P ¼0.47 P ¼0.36 P ¼0.47 P ¼0.24 P ¼0.18
Body Fat (%) 30.5 �1.99 30.3 �1.93 30.2 �2.03 30.5 �1.97 30.2 �1.97 30.5 �2.04 30.9 �1.94 30.6 �1.97 2W: P ¼0.29
4W: P ¼0.038*
Post: P ¼0.39
P ¼0.52 P ¼0.60 P ¼1.0 P ¼0.22 P ¼0.007* P ¼0.18
SBP (mmHg) 136.6 �8.02 148.4 �6.62 143.9 �5.82 148.4 �6.76 135.6 �8.53 134.11 �8.05 142.2 �5.23 147.9 �6.17 2W: P ¼0.30
4W: P ¼0.78
Post: P ¼0.84
P ¼0.26 P ¼0.50 P ¼0.20 P ¼0.42 P ¼0.53 P ¼0.29
DBP (mmHg) 86.0 �5.03 93.0 �4.91 91.6 �4.35 91.9 �4.19 83.9 �4.85 84.7 �3.95 89.9 �4.26 94.4 �3.26 2W: P ¼0.57
4W: P ¼1.0
Post:P ¼0.10
P ¼0.39 P ¼0.50 P ¼0.43 P ¼0.43 P ¼0.45 P ¼0.15
Pulse Rate (Beats/
min)
68.9 �2.11 64.6 �2.10 65.7 �2.29 65.3 �2.29 72.7 �3.11 70.7 �3.94 65.0 �2.29 66.8 �2.86 2W: P ¼0.64
4W: P ¼1.0
Post: P ¼0.69
P ¼0.27 P ¼0.43 P ¼0.39 P ¼0.61 P ¼0.18 P ¼0.33
Blood Fluidity (
μ
L/
sec)
2.77 �0.09 2.74 �0.07 2.43 �0.25 2.65 �0.10 2.63 �0.06 2.89 �0.09 2.89 �0.08 2.86 �0.19 2W: P ¼0.012*
4W: P ¼0.024*
Post:P ¼0.036*
P ¼0.81 P ¼0.27 P ¼0.41 P ¼0.018* P ¼0.039* P ¼0.069
Vascular Age
(Waveform index)
0.04 �0.15 0.24 �0.09 0.21 �0.16 0.11 �0.13 0.04 �0.11 0.15 �0.14 0.12 �0.09 0.24 �0.12 2W: P ¼0.10
4W: P ¼0.82
Post:P ¼0.041*
P ¼0.33 P ¼0.46 P ¼0.70 P ¼0.08 P ¼0.56 P ¼0.29
M.P. Kapoor et al.
Contemporary Clinical Trials Communications 17 (2020) 100499
3
effects of Emblica Ofcinalis, but preliminary evidence suggests potent
antioxidant activity that may explain these effects. In brain cells amla
has high antioxidant activity [15]. Renal injury occurs when reactive
oxygen species exceed the antioxidant reserve of renal tissue. Amla
contains high concentrations of antioxidants ascorbic acid, gallic acid
and phenolic compounds [16] suggesting that antioxidant activity of
amla may be benecial for the prevention of age-related renal disease
and improvement of urinalysis parameters [7,17]. Also, amla extracts
are reported to have the ability to modulate basal oxidative markers and
enhance endogenous antioxidant defenses in a hepatocyte cell line
(HepG2) [18]. Overall, amla has promise to help multiple systems due to
its antioxidant activity.
The present randomized, placebo-controlled, double-blind crossover
study sought to determine the preventive effect of amla on vascular
functions, blood hematology, hemorheology, glucose and lipid proles,
oxidative stress and inammatory biomarkers. Additionally, this study
aimed to examine the safety prole of amla including urinalysis and
hepatic risk parameters in healthy Japanese volunteers. An improve-
ment in blood uidity as the primary objective, and reduction of von
Willebrand factor (vWF), 8-hydroxy-20-deoxyguanosine (8-OHdG), and
thrombin (TM) biomarkers of oxidative stress were secondary parame-
ters along with a signicant improvement in HDL-cholesterol and
lowering the LDL-cholesterol levels were hypothesized.
2. Materials and method
2.1. Ethics statement
The study protocol was reviewed and approved by the Medical
Ethical Committee of Mie University, Japan and registered (H-AO-
00012006) at the hospital clinical trial registry network. Before partic-
ipation, all participants gave written informed consent to the protocol
approved by the Ofce of Research Promotion and Integrity of Mie
University. Study-related procedures were carried out by the approved
guidelines and ethical standards established in the Helsinki Declaration.
2.2. Subjects and criteria for their selection
Twenty four volunteers (n ¼24) aged between 36 and 67 years were
recruited to participate in the study and underwent screening mea-
surements to determine eligibility for participation in the full protocol.
Fifteen subjects (n ¼15; M7, F8) were recruited after screening based on
triglycerides levels, cholesterol levels, and the blood uidity in whole
human blood estimated using a microchannel array ow analyzer (MC-
FAN; [19]. The following inclusion and exclusion criteria were used for
the subjects to participate in the study. Healthy subjects with somewhat
elevated blood triglycerides levels (>130 mg/mL to <250 mg/mL),
lower HDL cholesterol level (<54 mg/mL), and average values of blood
uidity (2.6–2.8
μ
L/s) were recruited for the study. Subject were
excluded if (i) have an allergy and/or allergic disease including atopy,
(ii) symptoms of cerebrovascular disorder, (iii) a history of myocardial
infarction, (iv) atrial brillation and severe arrhythmia, (v) high renal
Fig. 1. (a) Schematic presentation of the study protocol and procedures, and (b) Illustration of procedural details for evaluation of amla efcacies in randomized,
double-blind, crossover placebo-controlled trial in healthy human subjects.
M.P. Kapoor et al.
Contemporary Clinical Trials Communications 17 (2020) 100499
4
dysfunction (serum creatinine >4.0 mg/dL), (vi) advanced liver func-
tion disorder, (vii) diabetes mellitus, (viii) anemia (hemoglobin <7
mg/dL), (ix) pregnant or lactating women, (x) consumption of medica-
tions or supplements affecting blood uidity, (xi) other exclusions,
judged by study medical practitioner during examination. Clinical
characteristics of the subjects at baseline are presented in Table 1.
2.3. Study design and sample collection protocol
The study was a randomized, placebo-controlled, double-blind,
crossover study (Fig. 1a), and a schematic illustration of the study prole
is presented in Fig. 1b. Randomization was conducted using StatsDirect
system by a researcher who was not involved in taking measurements
and analysis (1:1; block randomization with random block size between
3 and 6). Subjects were randomly assigned into the amla or placebo
group after two weeks (W1, W2) of controlled diet. No signicant dif-
ferences in the study parameters (age, height, body weight, body mass
index (BMI), total fat mass, etc.) were observed between the treatment
and placebo groups at baseline. The treatment group (T; n ¼8) received
amla capsules after each meal, while the placebo group (P; n ¼7)
received identical placebo capsules. After four weeks on the respective
treatment (W3 to W6) a three-week washout period (W7 to W9) was
implemented before crossover the other treatment. Two subjects (M1,
F1) from decided to withdraw from the study due to personal reasons.
After crossover, both groups consumed a controlled diet for two weeks
(W10, W11) and then groups received the other treatment for four
weeks (W12 to W15). At the end of the supplementation period, a post-
intake period for three weeks (W16 to W18) was completed.
All subjects were asked to refrain from smoking, drinking alcohol or
caffeine-containing beverages and excess consumption of coffee and tea
(>2 cups/day) because caffeine may have a synergic effect with amla
and the ability to modulate study parameters. All subjects were asked to
abstain from any strenuous physical activity during the study. Subjects
came to the laboratory in the morning after an overnight fast at the
beginning of W1 where physical measurements (body weight, height,
body fat, BMI, etc.), as well as fasting blood and urine samples, were
conducted to determine baseline values. Before blood sample collection,
a medical practitioner examined the subjects for the state of breathing as
well as the presence or absence of epigastric/abdominal pains, diarrhea/
constipation, vomiting, nausea, anorexia, and other subjective symp-
toms. Blood samples were drawn by a registered nurse in a seated po-
sition from an antecubital vein with anticoagulation by heparin solution
after resting in a chair for at least 5 min.
Similarly, the fasted blood and urine samples were collected after at
the end of W4, W6 and W9. Likewise blood and urine samples were
collected at the beginning of W10 and then, at the end of W13, W15 and
W18. Blood samples were snap-frozen in liquid nitrogen and stored at
80 �C until analysis.
2.4. Test supplementation and dosages
The commercial proprietary formulation of amla (SunAmla-PD1;
Taiyo Kagaku Co. Ltd., Japan) as a dry powder was investigated in this
study. It is prepared as a water extract of amla fruit pulp that has been
hydrolyzed with pectinase followed by centrifugation with the super-
natant spray dried. Study capsules were 250 mg hard gelatin capsules
containing amla formulation (125 mg) and dextrin (125 mg), while
placebo capsules were only dextrin (250 mg). Both supplements were
identical in size and appearance. They are considered safe and well-
tolerated at the reported dose. Subjects received (4 capsules/day) dur-
ing both the treatment and placebo period. The total dose of amla was
500 mg per day (ellagitannins 1.2–1.5%; ellagic acid 0.1–0.2%; gallic
acid, 1.5–2.0%; total polyphenols, 10–14%). The major active compo-
nents of ellagitannins are corilagin, geraniin, elaeocarpusin, and che-
bulagic acid. Subjects were instructed to ingest two capsules every day
immediately after breakfast and dinner during the four-week
supplementation periods (W3 to W6) and (W12 to W15). Subjected were
asked to comply with their supplementation and maintain a prescribed
lifestyle during the study. A mandatory daily log of meal timing,
including approximate calorie intake, supplementation intake, and total
working, resting and sleeping hours were completed during the study.
2.5. Measured study parameters
The measured items were body temperature, pulse rate, blood
pressure, vascular function, blood uidity, hematological parameters,
and biochemical parameters. Axillary body temperature was measured
using a mercury thermometer of high precision. Automatic sphygmo-
manometer (Terumo; ES-P2000A) was used to measure blood pressure
and pulse rate after resting at the sitting position at a stable position and
height. Both systolic and diastolic blood pressures, body temperature,
and pulse rate were measured three times consecutively, and the mean
of the three measurements was recorded. Vascular age was measured
using the acceleration pulse wave measurement system (Arnett PDU-M
100; Yumedica Co. Ltd. Osaka, Japan), and was conducted before the
blood sample collection. The acceleration pulse waveform data as
waveform index-I was measured twice, and the mean value of the
waveform was used to estimate the blood vessel age. The measurement
of blood uidity as a primary efcacy parameter was done immediately
after blood samples were drawn. Blood was collected in a separate tube
for the measurement of blood passage time for the calculation of blood
uidity.
Hematological test parameters along with the specic tests of blood
coagulation including Red blood cell count (RBC), White blood cell
count (WBC), Hematocrit (Ht), Hemoglobin (Hb), Blood uidity,
Platelet counts (Plt), Platelet aggregation ability (PAA), Prothrombin
time (PT), Activated partial thromboplastin time (APTT), Mean
corpuscular hemoglobin (MCH), Mean corpuscular volume (MCV),
Mean corpuscular hemoglobin concentration (MCHC) were measured by
using an automatic cell counter (Beckman Coulter Co. Ltd.).
Other blood biochemical parameters including: GOT (AST), GPT
(ALT), and γ–GTP (Randox kits), total protein (Biuret method), albumin
(Bromocresyl green method), albumin/globulin (A/G ratio), blood
glucose (Hexokinase glucose-6-pyruvate dehydrogenase method), tri-
glycerides (Glycerin-1-phosphate-oxidase method), total cholesterol
(Enzyme method), HDL-cholesterol (Homogeneous method), LDL-
cholesterol (Friedewald equation), HbA1c (Chromatographic method),
ALP (Spectroscopic method), uric acid (Enzymatic spectrophotometry
method), total bilirubin (Microassay method), urea nitrogen (BUN) and
creatinine (Enzyme method), Na, K, Ca, P (Flame photometry) and Cl
(titration method) were measured. In addition, the secondary efcacy
parameters thrombin receptor (TM), von Willebrand factor (vWF), 8-
hydroxy-20-deoxyguanosine (8-OHdG), thiobarbituric acid reactive
substances (TBARS), adiponectin (ELISA kit) were also measured.
Urinalysis, a physical and chemical examination of the urine, was
performed by collecting 5 mL of midstream urine by the subjects in a
sample tube. A dipstick (test strips based on necessary wet micro-
chemistry reactions) was used to detect the pH, specic gravity, urobi-
linogen, sugar, a ketone body, and occult blood. Qualitative and semi-
quantitative results are expressed as either adverse or varying degrees
of positive, indicating different amounts present.
2.6. Blood hemorheology assay using MC-FAN
Blood uidity in whole blood collected from subjects was estimated
using a microchannel array ow analyzer (MC-FAN; WBA-Neo, Tokyo,
Japan). These results were employed for the initial screening and
recruiting the subjects for this study. Briey, the microchannel passage
time for the 100
μ
L physiological saline as control was initially
measured, followed by 100
μ
L heparinized blood obtained from the
subjects. The revised values of the blood passage measure for the blood
uidity estimation was corrected and expressed as a function of the
M.P. Kapoor et al.
Contemporary Clinical Trials Communications 17 (2020) 100499
5
Table 2
Changes in hematological and anticoagulant activity parameters in amla and placebo intake groups throughout the trial duration.
Parameters
Placebo intake (mean �sem) Amla intake (mean �sem) ANCOVA (Among
Groups)
Before (W1, W2) or
(W10, W11)
2 Weeks (W3, W4) or
(W12, W13)
4 Weeks (W5, W6) or
(W14, W15)
Post (3 Weeks)
(W7,W9,W9) or
(W16,W17,W18)
Before (W1, W2) or
(W10, W11)
2 Weeks (W3, W4) or
(W12, W13)
4 Weeks (W5, W6) or
(W14, W15)
Post (3 Weeks)
(W7,W9,W9) or
(W16,W17,W18)
WBC (/
μ
L) 5860 �419 5929 �480 5840 �383 5946 �419 5961 �383 5724 �452 6005 �471 6239 �530 2W: P ¼0.52
4W: P ¼0.92
Post: P ¼0.67
P ¼0.86 P ¼0.92 P ¼0.81 P ¼0.11 P ¼0.87 P ¼0.43
RBC (x10
4
/
μ
L) 484.7 �13.6 486.6 �12.8 480.5 �12.0 481.5 �13.8 481.3 �13.3 478.5 �12.5 475.5 �12.1 486.1 �12.8 2W: P ¼0.38
4W: P ¼0.78
Post: P ¼0.24
P ¼0.59 P ¼0.25 P ¼0.35 P ¼0.61 P ¼0.31 P ¼0.42
Hemoglobin (g/dL) 14.6 �0.42 14.2 �0.39 14.1 �0.37 14.5 �0.42 14.7 �0.41 14.5 �0.38 14.3 �0.38 14.4 �0.41 2W: P ¼0.56
4W: P ¼0.79
Post: P ¼0.23
P ¼0.05* P ¼0.015* P ¼0.80 P ¼0.04* P ¼0.002* P ¼0.004*
Hematocrit (%) 44.5 �1.21 44.5 �1.51 44.1 �1.09 44.1 �1.29 44.1 �1.21 44.0 �1.17 43.7 �1.12 44.6 �1.20 2W: P ¼0.92
4W: P ¼1.0
Post: P ¼0.16
P ¼0.86 P ¼0.20 P ¼0.18 P ¼0.84 P ¼0.42 P ¼0.39
Platelets Count
(x10
4
/
μ
L)
28.4 �2.46 28.5 �2.41 28.1 �2.17 29.2 �2.55 28.9 �2.64 28.1 �2.45 28.3 �2.23 28.7 �2.53 2W: P ¼0.48
4W: P ¼0.82
Post: P ¼0.46
P ¼0.92 P ¼0.57 P ¼0.37 P ¼0.18 P ¼0.47 P ¼0.82
PT (Sec) 11.6 �0.47 10.9 �0.17 11.9 �1.30 10.47 �0.12 11.6 �0.33 10.9 �0.23 10.3 �0.22 10.38 �0.29 2W: P ¼0.11
4W: P ¼0.01*
Post: P ¼0.013*
P ¼0.13 P ¼0.87 P ¼0.07 P ¼0.025* P ¼0.007* P ¼0.008*
APTT (Sec) 31.7 �1.13 32.6 �1.44 35.8 �2.28 32.3 �1.71 32.4 �1.58 32.3 �1.56 33.5 �1.75 31.9 �1.56 2W: P ¼0.73
4W: P ¼0.60
Post: P ¼0.81
P ¼0.67 P ¼0.15 P ¼0.81 P ¼0.59 P ¼0.64 P ¼0.83
Platelet
Aggregation (%)
96.7 �2.50 100 �0 100 �0 93.7 �5.04 86.5 �4.81 99.7 �0.28 94.0 �4.49 95.1 �2.51 2W: P ¼0.30
4W: P ¼0.20
Post: P ¼0.28
P ¼0.095 P ¼0.23 P ¼0.33 P ¼0.027* P ¼0.20 P ¼0.16
MCV () 91.7 �0.98 92.0 �0.99 91.9 �0.97 91.7 �0.90 91.9 �1.07 91.5 �0.92 91.8 �0.90 91.7 �0.98 2W: P ¼0.04*
4W: P ¼0.15
Post: P ¼0.52
P ¼0.16 P ¼0.38 P ¼1.0 P ¼0.05* P ¼0.52 P ¼0.37
MCH (pg/cell) 30.4 �0.39 29.7 �0.32 29.7 �0.35 29.9 �0.27 30.3 �0.27 29.8 �0.31 29.7 �0.32 29.9 �0.36 2W: P ¼0.57
4W: P ¼0.48
Post: P ¼0.89
P �0.001* P �0.001* P ¼0.003* P �0.001* P �0.001* P ¼0.017*
MCHC (g/dL) 33.1 �0.21 32.5 �0.12 32.4 �0.16 32.6 �0.18 33.1 �0.15 32.3 �0.20 32.3 �0.15 32.6 �0.20 2W: P ¼0.08
4W: P ¼0.03*
Post: P ¼0.28
P �0.001* P �0.001* P ¼0.005* P �0.001* P �0.001* P ¼0.03*
M.P. Kapoor et al.
Contemporary Clinical Trials Communications 17 (2020) 100499
6
actual whole blood passage time over physiological saline solution
passage time of 12 s at a pressure of 20 cm of water.
Further, platelet aggregation ability based on thrombus formation in
blood treated with activating agent thrombin was determined by screen
ltration pressure (SFC) detected using an aggregometer by measuring
the decrease in ow rate as a function of agent concentration according
to the manual of the manufacturer as described in Ref. [19]. Briey, 200
μ
L of blood collected from subjects was mixed with 22
μ
L of thrombin at
the concentration of 5U/mL of thrombin. The platelet aggregation
ability was measured and expressed as % of inhibition against thrombin
agonists.
2.7. Estimation of anticoagulant activity
Anticoagulant activities of the amla or placebo supplementation
were determined by prothrombin time (PT) and activated partial
Fig. 2. Illustration of oxidative and inam-
matory biomarkers assay (A) 8-hydroxy-2
0-
deoxyguanosine; 8-OHdG, (B) von Wille-
brand factor; vWF, (C) Thrombin; TM and
(D) Thiobarbituric acid reactive substances;
TBARS during the amla supplementation
duration. The result depicts the comparison
between the groups (a to b denote signi-
cance of p-value p <0.05; a to ab denote
nearly signicance of p-value p <0.07;
measured by one-way ANOVA), and among
the groups (* denote signicance of p-values
p <0.05; **denote signicance of p-values
p <0.01; measured by ANCOVA).
Table 3
Assessment of characteristic oxidative and inammatory biomarkers and their statistical comparison to review the potential of amla preventive effects among healthy
human subjects.
Parameters
Placebo intake (mean �sem) Amla intake (mean �sem) ANCOVA
(Among
Groups)
Before
(W1, W2) or
(W10, W11)
2 Weeks
(W3, W4) or
(W12, W13)
4 Weeks
(W5, W6) or
(W14, W15)
Post
(3 Weeks)
(W7,W9,W9) or
(W16,W17,
W18)
Before
(W1, W2) or
(W10, W11)
2 Weeks
(W3, W4) or
(W12, W13)
4 Weeks
(W5, W6) or
(W14, W15)
Post
(3 Weeks)
(W7,W9,W9) or
(W16,W17,
W18)
TM (FU/mL) 1.98 �0.22 1.86 �0.21 2.10 �0.21 1.96 �0.26 2.05 �0.23 1.80 �0.27 1.64 �0.15 2.02 �0.27 2W: P ¼0.67
4W: P ¼0.16
Post: P ¼0.86
P ¼0.68 P ¼0.76 P ¼1.0 P ¼0.11 P ¼0.12 P ¼0.92
vWF (mU/mL) 9.05 �0.71 8.54 �0.51 8.73 �0.60 8.02 �0.56 10.2 �0.94 8.52 �082 8.22 �0.41 8.09 �0.48 2W: P ¼0.27
4W: P ¼0.58
Post: P ¼0.18
P ¼0.56 P ¼0.75 P ¼0.28 P ¼0.011* P ¼0.045* P ¼0.056*
TBARS (
μ
M) 4.56 �1.11 4.96 �0.93 5.54 �0.73 4.55 �0.51 5.41 �1.33 3.18 �0.43 3.72 �0.67 4.99 �0.95 2W: P ¼0.19
4W: P ¼0.46
Post: P ¼1.0
P ¼0.80 P ¼0.54 P ¼1.0 P ¼0.14 P ¼0.34 P ¼0.80
8-OHdG
(ng/mL)
0.25 �0.02 0.24 �0.03 0.24 �0.01 0.19 �0.02 0.27 �0.02 0.23 �0.03 0.21 �0.01 0.22 �0.02 2W: P ¼0.22
4W: P ¼0.002*
Post: P ¼
0.003*
P ¼0.77 P ¼0.60 P ¼0.06 P ¼0.033* P ¼0.036* P ¼0.031*
Adiponectin
(ng/mL)
1.33 �0.19 1.47 �0.21 1.50 �0.21 1.51 �0.23 1.41 �0.23 1.32 �0.17 1.47 �0.19 1.40 �0.17 2W: P ¼0.47
4W: P ¼0.24
Post: P ¼0.11
P ¼0.69 P ¼0.63 P ¼0.61 P ¼0.86 P ¼0.83 P ¼1.0
M.P. Kapoor et al.
Contemporary Clinical Trials Communications 17 (2020) 100499
7
thromboplastin time (APTT) assays. The assays were performed with 50
μ
L blood serum collected from the subjects added with 100
μ
L of PT or
50
μ
L of APTT and 50
μ
L of CaCl
2
to measure coagulation time (sec) by a
CA50 coagulometer (SYSMEX, Kobe, Japan) according to the manu-
facturer instructions.
2.8. Data processing and statistical analysis
All data values are expressed as means �sem unless otherwise
stated. Data analysis was performed by one-way analysis of variance
(ANOVA) with study duration followed by Tukey’s post hoc test, as well
as using analysis of covariance (ANCOVA) to access the pre-post dif-
ferences between both amla and placebo supplementations. All differ-
ences were considered signicant at p �0.05. At the beginning of the
study, a sample size of at least 12 subjects was estimated to be required
to detect relevant physiological changes in study parameters between
groups, at a 5% signicance level with 90% power, assuming the drop
out of 10%. Further, a post hoc power calculation was conducted to
conrm the statistical power was adequate. An independent two-tailed
Student’s t-test was performed to verify the statistical signicance.
3. Results
In accordance with the established criteria of subject selection based
on blood rheology, we enrolled subjects with average values of blood
uidity (range 2.6–2.8
μ
L/s), because of the tendency for the blood
uidity to increase after test supplementations. The subjects who
completed the study trial (n ¼13; M6, F7) had a mean age of 51.9 �2.8
years.
3.1. Physiological and vascular parameters
Most of the physiological parameters changes with the time duration.
Weight and BMI were in the normal range, and no signicant differences
were found. Also, no signicant difference was observed for SBP, DBP,
and pulse rate over the study. On the other hand, the blood uidity was
higher after the amla treatment compared to baseline. Signicance
differences by ANCOVA were observed in blood uidity after two weeks
(P ¼0.012), four weeks (P ¼0.024), and even after withdrawal (P ¼
0.036) of the amla treatment. Vascular age measured from the waveform
index-I was only found to signicant after the withdrawal of the amla
intake (P ¼0.041) compared to placebo (Table 1).
3.2. Hematological parameters and anticoagulant activity
The hematocrit, red blood cell, and white blood cell counts did not
show signicant changes during the placebo or amla period. The he-
moglobin showed signicant changes within both placebo and amla
period compared to baseline, and also remained signicant after the
withdrawal of amla intake (P ¼0.004). However, the hemoglobin levels
were not signicantly different between the amla and placebo periods.
No signicant difference was noticed in platelets counts for both placebo
and amla intakes during the trial period, whereas compared to baseline
the MCH and MCHC showed very signicant changes within both pla-
cebo and amla intake duration (P <0.001), and even after the with-
drawal of the intake of both treatments (P <0.05). Whereas, MCV
Fig. 3. (a) Metabolic pathways of amla: Urolithins are the major tentative metabolites of ellagitannins of amla formulation associated with different health benets,
and (b) Amla inhibits the vWF stimulated thrombin (TM) generation by the reduction in vWF secretion.
M.P. Kapoor et al.
Contemporary Clinical Trials Communications 17 (2020) 100499
8
showed the signicance difference (P ¼0.04) between both placebo and
amla treatments at two weeks of supplementation (see Table 2).
The platelet aggregation showed signicant stepwise improvement
within the amla intake (P ¼0.027) after two weeks of supplementation,
but no difference were observed between placebo and amla intake for
platelet aggregation. Anti-coagulant activity parameter PT showed a
statistically signicant reduction (P <0.01) for the amla intake
throughout the trial and also after the post-treatment period. Further, a
signicant difference between the placebo and amla treatment groups
after four weeks of supplementation (P ¼0.01) and also after the post
treatment period (P ¼0.013) was observed. The anticoagulant activity
parameter APTT showed no signicant changes within and between the
placebo and amla treatment groups.
3.3. Blood biochemical parameters and biomarkers
Fasting blood glucose levels signicantly changed after four weeks
(P ¼0.03) and tended to after the withdrawal (P ¼0.06) of amla intake.
Both the fasting glucose and triglyceride levels remained in the normal
range during the amla and placebo intake periods. No signicant dif-
ference in HbA1c, triglycerides, and total cholesterol levels were
observed during both the placebo and amla periods. HDL cholesterol
levels were signicantly higher after two weeks (P ¼0.03) and remained
higher than baseline values when comparing to the placebo or amla
intakes. The LDL cholesterol levels decreased but did not reach statis-
tical signicance (P ¼0.09) after two weeks of amla supplementation
compared to placebo. The Ca, Na, K, P levels in the blood signicantly
changed with amla, while chloride levels signicantly changed in both
placebo and amla intake periods (see Table ST1; supplementary
information).
Of secondary efcacy parameters, the concentration of 8-hydroxy-20-
deoxyguanosine (8-OHdG) was signicantly decreased compared to
baseline during the amla intake period and even after the withdrawal (P
<0.05) of amla supplementation. Further, compared to placebo, a sig-
nicant difference (P <0.01) was also observed after four weeks and
even after post-intake of amla (Fig. 2a). Also, the von Willebrand factor
(vWF) was signicantly lowered (P <0.05) during the amla intake
period (Fig. 2b). While a non-signicant decrease in the thrombin (TM)
receptor concentration and thiobarbituric acid reactive substances
(TBARS) was observed (Fig. 2c and d) and remained in the normal range
during the amla intake period. No signicant changes were observed in
adiponectin (see Table 3).
3.4. Evaluation of hepatic risk parameters
The test results of the hepatic factors, which are helpful to determine
the liver functions are illustrated in Table ST2 (see supplementary in-
formation). During the amla and placebo period no difference in the
levels of AST (GOT). The ALT (GPT) level was lower after two weeks (P
¼0.02) of amla intake and remained at lower than baseline levels. Also,
a lower ALP and γ-GPT levels were noticed during the trial periods, but
were not statistically decreased. All critical hepatic risk parameters
remained in the normal range throughout for both amla and placebo
periods. Total bilirubin signicantly decreased after two weeks of amla
intake (P ¼0.04) compared to baseline, while no signicant differences
were found in total protein, albumin, albumin/globulin ratio, creatinine,
uric acid, and urea-nitrogen levels assays.
3.5. Urinalysis parameters
Urinalysis of the most common chemicals before and after the pla-
cebo and amla intake periods were collected. The pH range was slightly
acidic (5.5–6.1) and the specic gravity range of urine was always less
than 1.02 during the trial periods, but no signicant changes were
noted. Quantitative assay of urobilinogen showed normal levels for all
subjects (n ¼13) throughout the study. Quantitative protein and sugar
levels were also normal except somewhat doubt (no immediate concern)
was registered for one subject during the post-supplementation periods
of placebo (protein, n ¼1) and amla (sugar, n ¼1), respectively. The
placebo group had nine subjects with normal occult blood in urine and
four subjects with some borderline doubt but in healthy condition (n ¼
9, A1-judgment; n ¼4, A2-judgment) at baseline and remained un-
changed during the placebo period. The amla intake group registered
twelve subjects without abnormalities and one with no immediate
concerns at baseline, however, eight subjects without abnormalities and
ve subjects with no immediate concerns were reported at the end of
amla treatment possibly due to crossover nature of the study trial (see
Table ST3; supplementary information).
3.6. Adverse events and examination opinions
No signicant conditions or adverse events were found in the
physician examination implemented on the rst day of both study pe-
riods through the completion of the study. Some minor conditions were
reported mainly during the placebo intake. One subject reported diar-
rhea feelings during the placebo intake of the trial. While, one subject
reported for the loose bowel movements after two weeks, and other
reported constipation after four weeks of placebo supplementation.
Also, one subject reported cold after 2–4 weeks of placebo intake
duration of the trial. One subject reported edema before the start of amla
intake; however, no other conditions were reported during the amla
intake period (see Table ST4; supplementary information).
Furthermore, no anomalies were observed in the subjective symptom
surveys or daily activity diaries wherein the subjects recorded any
concerning information during the study, such as unusual circumstances
or the side effects. There was 100% compliance with the placebo and
amla intakes during the trial for all subjects who completed this study.
Also, the diet and meal intake were not noteworthy, and there were no
subjects with eating and lifestyle habits that deserve special mention.
4. Discussion
Vegetables and fruits form essential elements of a healthy diet;
however, ellagitannins and ellagic acid are the polyphenol often
underestimated in our diets [20,21]. Interest in ellagitannins is still
growing [22]; therefore, it is important to identify safer and more
cost-effective strategies for managing common health issues. The pre-
sent study was designed to evaluate the safety and efcacy of ellagi-
tannins (hydrolyzable to ellagic acid and gallic acid) rich amla (500
mg/day) compared to a placebo in healthy humans. Risk factors such as
hypertension, dyslipidemia, and atherosclerosis are emerging as critical
components associated with the pathophysiology of accelerated
impaired endothelial functions [2,12,13]. The oxidative stress in the
vascular endothelium cell is primarily associated with thrombosis, while
increased platelet aggregation is yet another signicant risk factor for
cardiovascular diseases [23–25]. The high blood lipid proles and
platelet aggregation in blood vessels can lead to hypertension. Inhibition
of platelet aggregation by oral supplementation of amla is attributable
primarily to the abundance of low molecular weight (<1000 Da)
hydrolysable ellagitannins (i.e. chebulagic acid, pedunculagin, geraniin,
corilagin, elaeocarpusin, etc.), gallic acid, ellagic acid, and their me-
tabolites which are illustrated in Fig. 3a. Hydrolysis of ellagitannins
yields galloyl-glucose residues, which are eventually transformed into
gallic acid, pyrogallol and resorcinol [26,27], along with HHDP moiety,
which undergoes lactonization and spontaneous rearrangement to stable
ellagic acid. Due to very poor bioavailability of ellagitannins, they are
found relatively low concentrations in the gastrointestinal (GI) tract
including feces; therefore the ellagitannins have not been reported in the
human systematic circulation system or urine. Urolithins are the pri-
mary metabolites of ellagitannins of amla in human plasma and urine
mostly present as a conjugated form with glucuronic acid or sulfate.
Urolithin-A metabolite was recently reported to possess several
M.P. Kapoor et al.
Contemporary Clinical Trials Communications 17 (2020) 100499
9
biological activities such as anti-inammatory effects [27]. Thus, ella-
gitannins of amla are associated with health benets that are mediated
by the bioconversion of ellagitannins by gut microbiota.
Earlier studies demonstrated the benecial antioxidant activity and
cytoprotective effect of amla formulations on dyslipidemia and lipid
peroxidation [23,28]. Amla has an anti-thrombotic and anti-oxidative
effect. It may also help control vascular injuries in humans due to hy-
perglycemia that has been positively associated with oxidative stress,
which is one of the principal mechanisms involved in the damage of
endothelial function. In this study, amla supplementation showed a
benecial effect on endothelial function by modulating thrombin and
endothelial vWF, along with a signicant improvement in biomarkers of
oxidative stress, including TBARS and 8-OHdG. The vWF is a critical
adhesive protein that captures platelets from circulation, which usually
changes to an elongated form that provides an adhesive surface for
platelets via binding to glycoprotein [29]. Oral supplementation of amla
inhibits the vWF stimulated thrombin generation by the reduction of
vWF secretion in the circulatory system and, thus prevent the vWF
mediated adhesion of platelets to brin (Fig. 3b), which is a prerequisite
for the thrombin to expose anionic phospholipids at the plasma mem-
brane of platelets to facilitate the adhesion. The vWF appears to be an
essential factor in the generation of thrombin and subsequent conver-
sion of plasma protein brinogen into brin [30]. Therefore, amla
intake reduces the thrombin formation in vWF decient platelet-rich
plasma (Fig. 3b and c). The oral administration of amla reduced
TBARS levels in plasma, suggesting amla consumption could also
ameliorate oxidative stress due to aging-related mitochondrion
dysfunction. A signicant decrease in the cellular oxidative stress
biomarker 8-OHdG excretion, an oxidized nucleoside of DNA, represents
a decrease in oxidative DNA damage as a result of amla supplementation
compared to the placebo. The overall ability to reduce oxidation induced
DNA damage and repair of DNA lesions is believed to depend on the time
phase relationship. However, an inuence of metabolic rate (oxygen
consumption) on the 8-OHdG excretion cannot be excluded due to its
standard dual function in base excision repair (BER; the cellular mech-
anism that repairs damaged DNA) as well as in the oxidation of DNA
[31]. Further, it is also postulated that metabolites of ellagitannins and
polyphenols of amla could be radially adsorbed by cell tissues during
regular supplementation, and thus enhancing antioxidant protection
against oxidation-induced DNA damage and mutation.
Although there is minimal human evidence on amla in the scientic
literature, it appears to be very promising as it could lower blood glucose
in both healthy people and diabetics [6]. In animal research, amla ap-
pears to be able to reduce triglycerides, improve the cholesterol prole,
and benet cardiovascular health. Most of these actions are attributed to
its antioxidant properties, which are partially derived from a reasonably
high vitamin C content in addition to the ellagitannins. Amla is bene-
cial in preventing age-related kidney diseases when administration
resulted in signicantly decreased levels of lipids [32]. Benets appear
to extend to humans, and the present study demonstrated that four
weeks of oral supplementation of standardized amla effectively alters
the lipid levels by decreasing LDL cholesterol, total triglycerides, and
total cholesterol levels along with a concomitant increase of HDL
cholesterol, whereas placebo did not have any considerable effect on any
of the study parameters. The results were comparable to studies reported
previously [32]. Lipid-lowering properties and endothelial function
management activity of amla may be attributed to the polyphenol
content and ellagitannins of the formulation. It is also postulated that
the ellagitannins present in amla delay the oxidation of vitamin C, while
the synergic effect of pectin has also been reported to decrease the
cholesterol levels in humans [12] along with the avonoid components
of amla are mainly responsible for a potent hypolipidemic effect.
Although several mechanisms [5,33] such as interference with choles-
terol absorption, an increase in lecithin-cholesterol acyltransferase ac-
tivity, and inhibition of HMG-CoA reductase activity are reported in
literature for the alteration in the lipid proles upon supplementation of
amla formulations; however conclusive pieces of evidence are not yet
clear [8]. Further, the decreasing trend of total cholesterol could also be
a determinant of the increased blood uidity, since erythrocyte mem-
brane structure that usually decreases the membrane uidity in the su-
percial layer with any signicant alteration of membrane cholesterol
and TBARS.
Further, the white blood cell counts, a surrogate marker of inam-
mation, which usually correlates positively with platelet counts showed
no evidence on their possible association with aforementioned risk
factors indicate the possibility of a shared mechanism that drives a
detrimental effect of amla intake on the improvement in the blood
rheology [25]. Therefore, it may be suggested that hematocrit and WBC
could be the determinants of the blood uidity a primary efcacy
parameter of this study. Moreover, the albumin is also a vital protein
regarding blood uidity as it could have a negative correlation with
blood passage, but the measured values of albumin were at normal levels
in the present study indicates the possibility of the relevance of a weak
plasma lipid concentration for blood uidity. Thus, a signicant increase
in blood uidity with amla compared to placebo could be attributed to
soft and balanced oxygen transport to the cells. Also, vascular age
derived from the waveform index can reect vascular resistance, as the
pathogenesis of small size vessels may affect the vascular age. Since,
hemorheology is estimated using MC-FAN which is an in vitro assay that
uses articial blood vessels (lumen measures <10 mm; assumed to
correspond to small-sized capillary vessels), and signicant post-trial
changes observed between amla and placebo intake suggests the
improvement in vascular resistance and reduced impairment of hem-
orheology with increased blood uidity possibly due to sympathetic
activation on vascular functions. The prothrombin time was signi-
cantly lowered upon amla intake compared to placebo without signi-
cant alteration in APTT (almost no interfering with coagulation complex
assembly on phospholipid surface). Also, levels of both PT and APTT
remained within the normal range, suggested that amla on limits the
platelet aggregation due to reduced thrombin, and help accelerate the
anticoagulant activity, which could result from a moderate increase in
blood uidity. No considerable differences were noticed in any blood
test item MCV, MCH, and MCHC between placebo and amla groups as
changes were small and within the normal range, suggesting that
observed changes were not medically relevant. Also, no signicant dif-
ferences were noted in urinalysis ndings between placebo and amla
groups. Also, the blood tests related to liver and renal/kidney functions
(blood urea nitrogen, AST, ALT, γ-GPT, creatinine, uric acid, phosphate,
calcium, sodium, potassium, and chloride) were standard in all subjects.
Although a few adverse events were noted during the trial, they were
reported during the placebo and were common conditions likely unre-
lated to study participation. No symptoms considered to be a side effect
were observed during the amla treatment period. These results indicate
the tolerance and safety of a daily 500 mg dose of amla in healthy
humans. The ndings above indicate that amla and its components
(ellagitannins, polyphenols and their metabolites) could be recognized
as a potent antioxidant with the determinant function of vascular ho-
meostasis, regulating several physiologic properties, including vascular
permeability as well as antithrombotic properties that could prevent the
arterial thrombosis.
Despite the positive ndings of this study, there were few limitations.
The rst is MC-FAN can detect somewhat lower platelet aggregation
related hemorheological data due to its in vitro characteristics that use
articial blood vessels and absence of an inuence of vascular factors
such as endothelial cells or smooth muscle, which must be considered
when interpreting the results. Another limitation that this is a relatively
small study of only fteen subjects and despite a strong study design, a
larger study is necessary to conrm the health benets of amla.
5. Conclusions
In this clinical study, we conrm that the proprietary amla
M.P. Kapoor et al.
Contemporary Clinical Trials Communications 17 (2020) 100499
10
formulation showed a signicant improvement in endothelial function
as well as a reduction in biomarkers of oxidative stress. Also, the results
suggest that amla intake may increase plasma antioxidant potential and
decrease oxidative stress, which can help promote oxidative homeo-
stasis. All of these benets are possible without inuencing hepatic or
renal function, or diabetic indices in healthy humans. Lastly, the results
from this human clinical study conclusively established that amla has an
acceptable sensory and safety prole while providing enormous poten-
tial for the management of a healthy lifestyle.
Acknowledgments
The authors thank all study subjects for their voluntary participation.
All authors acknowledge and thank their respective Institutes and Uni-
versities. The authors have no conicts of interest to declare.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.conctc.2019.100499.
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