Potential Estrogenic Effects of Biofield Energy Treatment Using Human Endometrial Adenocarcinoma Cell Line
Alice Branton1 and Snehasis Jana2*
1Trivedi Global, Inc., USA
2Trivedi Science Research Laboratory Pvt. Ltd., India
Submission: November 15, 2018; Published: January 22, 2019
*Corresponding author: Snehasis Jana, Trivedi Science Research Laboratory Pvt. Ltd., Bhopal, India
How to cite this article: Alice Branton, Snehasis Jana. Potential Estrogenic Effects of Biofield Energy Treatment Using Human Endometrial Adenocarcinoma Cell Line. Glob J
Reprod Med. 2019; 6(3): 555690. DOI:10.19080/GJORM.2019.06.555690.
The present study was aimed to investigate the potential role of Consciousness Energy Healing (The Trivedi Effect®) based DMEM medium on the level of alkaline phosphatase enzyme (ALP) in Ishikawa cells. The test item (DMEM medium) was divided into two parts, one part received Biofield Energy Treatment by a renowned Biofield Energy Healer, Alice Branton and was categorized as the Biofield Energy Treated DMEM group, while another part did not receive any sort of treatment and coded as the untreated DMEM group. The cell viability using MTT assay showed a significantly improved cell viability upto 98% in the test item group, which suggested a safe and nontoxic profile of the test item. Estrogenic potential using estimation of ALP level showed a significantly increased by 30.8% in the Biofield Energy Treated group as compared with the untreated DMEM group. The Biofield Energy Treated DMEM showed a significant role in the growth of Ishikawa cells along with an improved ALP level that can be utilized significantly in the promotion and maintenance of estrogen level. It is concluded that Biofield Energy Healing Treatment showed a significant improved ALP level, which can be used in different menstrual and estrogenic disorders like hypophosphatasia, osteoporosis, malnutrition, hypothyroidism, magnesium deficiency, heart surgery, chronic myelogenous leukemia, enteritis in children, pernicious anemia, bacterial infection and intrauterine infection is a leading cause of pelvic inflammatory disease, subfertility, infertility, endometritis, early pregnancy loss, fetal defects, and preterm birth.
Abbrevations: CAM: Complementary and Alternative Medicine; ER: Estrogen Receptor; NCCAM: National Center for Complementary and Alternative Medicine; ALP: Alkaline phosphatase; DMEM: Dulbecco’s Modified Eagle’s Medium; FBS: Fetal Bovine Serum
Ishikawa cell line is a well‐differentiated human endometrial adenocarcinoma cell line, which was established to study the estrogenic potential due to the presence of estrogen and progesterone receptors (i.e., ERα and PR) . Ishikawa cell line is derived from human endometrium that plays a significant role as a fertility-determining factor [2,3]. Hence, Ishikawa cell line was selected as a test system for this study. Continues basic research area in this field using this cell line like reproductive biology and molecular science, reported its vital role to compare its action using various parameters such as alkaline phosphate (ALP), a zinc-containing metalloenzymes. Human endometrial cell lines are the best characterized cell lines that are easy to cultivate for estrogenic potential. ALP is more abundant in liver, bone, and a small amount in placenta, which were denoted as ALP-1, ALP-2, and ALP-3, respectively.
Maintained the level of ALP is very important for conception as it significantly regulates the estrogen level and endometrium growth [4-6]. Various menstrual disorders take place in the presence of low level of ALP during implantation and conception. A decreased ALP level may be due to zinc deficiency, hypothyroidism, vitamin C deficiency, folic acid deficiency, excess vitamin D intake, low phosphorus levels, celiac disease, malnutrition with low protein assimilation, insufficient parathyroid gland function, pernicious anemia, vitamin B6 insufficiency, and also with the frequent use of synthetic contraceptive, which Results and Discussion in the loss of endocrine functions via estrogen receptor (ER) . Thus, for identification of estrogenic potential, Ishikawa cell line was selected as a test system for this study in order to find the effect of the Biofield Energy Treated DMEM media for ALP as a biomarker.
As an alternative way of treatment, Complementary and
Alternative Medicine (CAM) therapies are emerging as one of
the best and safe way to treat against acute and chronic diseases
. Among CAM, Biofield Energy Healing Treatment (The Trivedi
Effect®) one of the best approach that has provided a scientific
groundwork in the past years by many renowned healers in
order to understand the complex homeodynamic regulation of
living systems . National Institute of Health (NIH) and National
Center for Complementary and Alternative Medicine (NCCAM)
recommend and included various Energy Healing therapies
such as natural products, deep breathing, yoga, Tai Chi, Qi Gong,
chiropractic/osteopathic manipulation, meditation, massage,
special diets, homeopathy, progressive relaxation, guided imagery,
acupressure, acupuncture, relaxation techniques, hypnotherapy,
healing touch, movement therapy, pilates, rolfing structural
integration, mindfulness, Ayurvedic medicine, traditional Chinese
herbs and medicines, naturopathy, essential oils, aromatherapy,
Reiki, cranial sacral therapy and applied prayer under CAM
category that has been accepted by the most of the U.S. population
with several advantages .
The Trivedi Effect®- Consciousness Energy Healing Treatment
contains a putative bioenergy, which is channeled by a renowned
practitioner from a distance. Biofield Energy Healing as a CAM
showed a significant result in biological studies . The Trivedi
Effect®- Consciousness Energy Healing Treatment has been
reported with significant revolution in the physicochemical
properties of metals, chemicals, ceramics and polymers [12-14],
improved agricultural crop yield, productivity, and quality [15,16],
transformed antimicrobial characteristics [17-19], biotechnology
[20,21], improved bioavailability [22-24], skin health [25,26],
nutraceuticals [27,28], cancer research [29,30], bone health [31-
33], human health and wellness.
In pursue with the outstanding Results and Discussion of Biofield Energy
Healing Treatment outcome, authors in this study evaluates the
impact of the Biofield Energy Treatment (The Trivedi Effect®) on
DMEM as a test sample for estrogenic potential with respect to
ALP parameter using standard in vitro assay in Ishikawa cells.
Naringenin was purchased from Sigma, India. Fetal bovine
serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM)
were purchased from Life Technology, USA. Antibiotics solution
(penicillin-streptomycin) was procured from HiMedia, India,
while 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium)
(MTT), Direct Red 80, and ethylenediaminetetraacetic acid (EDTA)
were purchased from Sigma, USA. All the other chemicals used in
this experiment were analytical grade procured from India.
Ishikawa cell line (human endometrial adenocarcinoma)
from human endometrial tissue was used as test system in the
present study. Ishikawa cell line was maintained in DMEM growth
medium for routine culture supplemented with 10% FBS. Growth
conditions were maintained at 37 °C, 5% CO2, and 95% humidity
and subcultured by trypsinisation followed by splitting the cell
suspension into fresh flasks and supplementing with fresh cell
growth medium. Before the start of the experiment, the growth
medium of near-confluent cells was replaced with fresh phenolfree
DMEM, supplemented with 10% charcoal-dextran stripped
FBS (CD-FBS) and 1% penicillin-streptomycin for 3 days .
The experimental groups consisted of group 1 (G-I) the
untreated DMEM. Group 2 (G-II) consisted of positive control at
non-cytotoxic concentrations. Further, group 3 (G-III) included the
Biofield Treated DMEM.
DMEM as the test item was divided into two parts, one part
was treated with the Biofield Energy by a renowned Biofield
Energy Healer (The Trivedi Effect®) and coded as the Biofield
Energy Treated DMEM group, and the other part did not receive
any sort of treatment and denoted as the untreated DMEM group.
This Biofield Energy Healing Treatment was provided by Alice
Branton remotely for ~5 minutes through the Healer’s unique
Energy Transmission process to the test sample under laboratory
conditions. Biofield Energy Healer was located in the USA, while
the test items were located in the research laboratory of Dabur
Research Foundation, New Delhi, India. Biofield Energy healer
in this study never visited the laboratory in person, nor had any
contact with the test item (DMEM medium). Further, the control
group was treated with “sham” healer for comparative purposes.
The “sham” healer did not have any knowledge about the Biofield
Energy Treatment. After that, the Biofield Energy Treated and
untreated samples were kept in similar sealed conditions for
The cell viability was performed by MTT assay in human
endometrial adenocarcinoma cell line (Ishikawa). The cells were
counted and plated in 96-well plates at the density corresponding
to 5 X 103 to 10 X 103 cells/well/180μL of cell growth medium. The
above cells were incubated overnight under growth conditions
and allowed the cell recovery and exponential growth, which were
subjected to serum stripping or starvation. The cells were treated
with the test items (DMEM) and positive control. The cells in the
above plate(s) were incubated for a time point ranging from 24 to
72 hours in a CO2 incubator at 37 °C, 5% CO2, and 95% humidity.
Following incubation, the plates were taken out and 20μL of
5mg/mL of MTT solution were added to all the wells followed by
additional incubation for 3 hours at 37 °C. The supernatant was
aspirated and 150μL of DMSO was added to each well to dissolve
formazan crystals. The absorbance of each well was read at
540nm using Synergy HT microplate reader, BioTek, USA . The
percentage cytotoxicity at each tested concentrations of the test
substance were calculated using the following equation (1):
X = Absorbance of treated cells;
R = Absorbance of untreated cells
The percentage cell viability corresponding to each treatment
was obtained using the following equation (2):
The concentrations exhibiting ≥70% cell viability was
considered as non-cytotoxic.
The cells were counted and plated in 96-well plates at the
density corresponding to 5 X 103 cells/well/180μL phenol-free
DMEM+ 10% CD-FBS. The above cells were incubated overnight
under growth conditions for 48 hours in a CO2 incubator at
37°C, 5% CO2, and 95% humidity to allow the cell recovery and
exponential growth. The above cells were incubated with the
test samples or positive control for 6 days. Re-addition of the test
sample or positive control was done on day 3. After incubation
with the test samples, the ALP enzyme activity was determined
by monitoring the hydrolysis of p-nitrophenyl phosphate to
p-nitrophenol (pNPP). The cells were washed with 1X PBS and
lysed by freeze-thaw method i.e., incubation at -80°C for 20
minutes followed by incubation at 37 °C for 10 minutes. Lysates
were prepared in 0.1% triton-X. 50μL of substrate solution i.e.,
10mM of pNPP in 1M diethanolamine and 0.24mM magnesium
chloride (MgCl2) solution, pH 10.4 was added to all the wells
containing 50μL of lysates followed by incubation for 1 hour at 37
°C. The absorbance of the above solution was recorded at 405nm
using Synergy HT microplate reader. The percentage increase in
ALP enzyme activity with respect to the untreated DMEM group
was calculated using equation (3):
X = Absorbance of cells corresponding to positive control and
R = Absorbance of cells corresponding to untreated group
All the values were represented as Mean ± SEM (standard
error of mean) of three independent experiments. The statistical
analysis was performed using SigmaPlot statistical software
(v11.0). For two groups comparison student’s t-test was used.
For multiple group comparison, one-way analysis of variance
(ANOVA) was used followed by post-hoc analysis by Dunnett’s
test. Statistically significant values were set at the level of p≤0.05.
The Biofield Energy Treated and untreated test samples were
tested for cell viability using MTT assay in Ishikawa cells. The
outcomes in terms of percentage cell viability are represented in
Figure 1. The MTT data showed that the test samples were found
to have significant cell viability after Biofield Energy Treatment
by 98%, while in the naringenin (positive control) group the
cell viability was 75% to 96%. Thus, the experimental MTT data
suggested that the Biofield Energy Treated DMEM was found to be
safe in the Ishikawa cells as compared with the untreated DMEM.
Thus, DMEM was used to study the estrogenic potential (i.e., ALP
activity) of The Trivedi Effect®- Biofield Energy Healing in vitro
using human endometrial adenocarcinoma cell line (Ishikawa).
The level of ALP in terms of percentage change are presented
in Figure 2. Naringenin, positive control showed a significantly
increased the value of ALP by 43.75% and 200.89% (p≤0.001)
at 500 and 1000nM, respectively with respect to the untreated
DMEM group. The Biofield Energy Treated DMEM group showed a
significant increased the ALP level by 30.8% as compared with the
untreated DMEM group. Thus, the Biofield Energy Treated DMEM
showed a significant increment of ALP, which play a major role in
estrogen balance for conception. It might be highly significant in
case of infertility and helpful against various menstrual disorders.
The scientific literature reported that decreased ALP
level in placenta results in serious complications such as
amyloidosis, granulation tissue, gastrointestinal inflammation
such as inflammatory bowel disease, systemic infections, hypophosphatasia, postmenopausal women receiving estrogen
therapy that is due to the osteoporosis, severe anemia, heart
surgery, aplastic anemia, malnutrition, magnesium deficiency,
hypothyroidism, chronic myelogenous leukemia, children with
achondroplasia and cretinism, and pernicious anemia . Thus,
Biofield Energy Healing Treatment would significantly improved
the estrogenic potential and worked as an index of osteoblastic
differentiation as well as improved ALP enzyme activity .
Thus, in order to study the effect of Biofield Energy Treatment on
DMEM, ALP level was significantly improved in Ishikawa cell line.
It might be expected that Biofield Energy Treatment has altered
the osteoblastic differentiation, which is due to an increased ALP
The Trivedi Effect®- Consciousness Energy Healing Treatment
on DMEM was considered as a significant role to improve estrogenic
potential with respect to increased level of ALP in Ishikawa cells.
Cell viability data using MTT assay showed a significant improved
cell viability after Biofield Energy Healing Treatment with 98%
in the test sample group, while upto 96% in the positive control
group signifies the high safety profile of the test samples. The
level of ALP was significantly increased by 30.8% in the Biofield
Energy Treated DMEM group as compared with the untreated
DMEM group. Thus, The Trivedi Effect® on DMEM were found to
have a significant impact on ALP level, which results in a better
estrogenic potential and osteoblastic differentiation.
Therefore, with respect to the untreated DMEM, the Biofield
Energy Treated DMEM would be highly significant in growth and
viability of Ishikawa cells. Therefore, the Consciousness Energy
Healing based DMEM might be a suitable alternative media
for cell growth. It can be useful for the management of various
estrogenic and menstrual disorders viz. Dysmenorrhea with
painful cramps, Premenstrual Syndrome (PMS), Menorrhagia,
Oligomenorrhea, Amenorrhea, and Missed periods. Thus, Biofield
Energy Treatment would be useful to control the estrogen balance
and thus control overall hormonal balance, which can be useful
against stress, aging, osteoporosis, various bone diseases, cell
differentiation, could improve cell-to-cell communication, normal
cell growth, neurotransmission, cell cycling and proliferation,
skin health, immune and cardiovascular functions. Besides, it
controls various immune-related disease conditions such as
Aplastic Anemia, Pernicious Anemia, Hepatitis, Sjogren Syndrome,
Myasthenia Gravis, Parkinson’s Disease, Asthma, Atherosclerosis,
Graves’ Disease, Dermatomyositis, Dermatitis, Diabetes, Multiple
Sclerosis, Ulcerative Colitis, Alzheimer’s Disease, Irritable Bowel
Syndrome, Systemic Lupus Erythematosus, stress, etc. with a safe
therapeutic index to improve overall health and Quality of Life.
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