Skin Protective Activity of Consciousness Energy Healing Treatment Based Herbomineral Formulation

Journal: Journal of Food and Nutrition Sciences

  • 136
  • 22
Skin Protective Activity of Consciousness Energy Healing Treatment Based Herbomineral Formulation

Abstract:

The current study was attempted to evaluate the impact of the Consciousness Energy Healing (The Trivedi
Effect®) Treatment based herbomineral test formulation and cell medium (DMEM) against skin health. The test formulation
and DMEM were divided into two parts. One of each part was received the Consciousness Energy Healing Treatment by
Dennille Mellesia Smith and was termed as the Biofield Energy Treated samples, while the other parts were denoted as the
untreated test items. MTT showed >78% viable cells, indicating that the test formulation was safe and nontoxic in all the
tested concentrations in three cell lines. The percent cell proliferation by BrdU assay was significantly increased by
238.30%, 192.06%, and 43.96% in the UT-DMEM + BT-Test formulation, BT-DMEM + UT-Test formulation, and BTDMEM
+ BT-Test formulation groups, respectively at 17.5 μg/mL with respect to the UT-DMEM + UT-Test formulation
group. The level of collagen was significantly increased by 55.55%, 32.65%, and 52.48% in the UT-DMEM + BT-Test
formulation, BT-DMEM + UT-Test formulation and BT-DMEM + BT-Test formulation groups, respectively at 1.25 μg/mL
compared to the untreated group. Elastin was significantly (p≤0.001) increased by 6.30%, 105.04%, and 29.41% in the UTDMEM
+ BT-Test formulation, BT-DMEM + UT-Test formulation, and BT-DMEM + BT-Test formulation groups,
respectively at 10 μg/mL compared to the untreated group. Hyaluronic acid was increased by 4.78%, 29.71%, and 58.29%
in the UT-DMEM + BT-Test formulation, BT-DMEM + UT-Test formulation, and BT-DMEM + BT-Test formulation
groups, respectively at 0.63 μg/mL compared to the UT-DMEM + UT-Test Formulation group. The level of melanin was
reduced by 14.64% and 18.25% in the UT-DMEM + BT-Test formulation and BT-DMEM + UT-Test formulation,
respectively at 0.13 μg/mL compared to the untreated group. Skin protection against UV-B data displayed that cell
proliferation was increased by 17.88%, 20.14%, and 9.89% in the BT-DMEM + BT-Test formulation at 0.63, 1.25, and 2.5
μg/mL, respectively compared to the untreated group. Wound healing activity exhibited significant wound closure and cell
migration in all the tested groups compared to the untreated group. Overall, result suggests that the Biofield Energy Treated
DMEM and test formulation exhibited better responses compared to the untreated medium and test formulation. Therefore,
the Biofield Energy Treated herbomineral formulation could be useful for the development of an effective cosmetic product
for the prevention and treatment of several skin problems such as erythema, contact dermatitis, skin aging, wrinkles and/or
change in the skin color, etc.

Keywords:
The Trivedi Effect®, Consciousness Energy Healing, Skin Protection, HFF-1, B16-F10, HaCaT, Scratch Assay, Extracellular Matrix

INTRODUCTION:

Skin is continuously exposed to pro-oxidant
environmental stresses from various sources like air
pollutants, ultraviolet (UV) light, chemical oxidants,
microorganisms, and ozone. Reactive oxygen species
(ROS) are considered as the main factor that causes several
skin disorders such as skin cancer and photoaging. In recent
years, particular antioxidants have gained considerable
attention as a means for neutralizing various ROS [1]. The
minerals and plant extracts play a vital role in skin repair,
growth, and development. Important minerals such as zinc
play a critical role in overall human physiology. It is an
essential cofactor of various metalloenzymes and it protects
the skin from UV irradiation and has been used for wound
healing and to reduce inflammation. Deficiency and
abnormal metabolism of zinc causes a hereditary disorder
like acrodermatitis enteropathica in infants along with skin
lesions. [2-4]. Several scientific evidences suggest that
selenium plays an important role in protecting skin from the
harmful effects of UV-B. It is an essential trace element is
found in many foods including meat, fish, eggs, dairy
products, and grains. In humans, low selenium status is
associated with increased the risk of developing skin cancer
[5, 6]. Zinc, and selenium are involved in the destruction of
free radicals through cascading enzyme systems. Apart
from zinc and selenium, molybdenum is involved in many
biochemical processes of life such as respiration, DNA and
RNA reproduction, maintenance of cell membrane integrity,
and sequestration of free radicals [7]. Vitamin C is an
essential constituent for the production of collagen and a
potent antioxidant that can help rejuvenate aged and
photodamaged skin [8, 9]. Sugiyama et al. [10]
demonstrated that tetrahydrocurcumin (THC) also exhibited
strong anti-oxidant and anti-cancer activity. However, it
was also reported that THC has less effective as
chemopreventive agent in mouse skin than curcumin [11].
The extract of Centella asiatica is effective for the treatment
of small wounds, hypertrophic wounds, burns, psoriasis and
scleroderma through promoting the proliferation of
fibroblast. It increases the synthesis of collagen,
intracellular fibronectin, and the tensile strength of newly
formed skin as well as inhibiting the inflammatory phase of
hypertrophic scars and keloids [12]. Owing to the
importance of minerals and vitamins, a new proprietary
herbomineral formulation was formulated consisting of
essential minerals (zinc chloride, sodium selenate, and
sodium molybdate), vitamin (L-ascorbic acid),
tetrahydrocurcumin (THC), and herbal extract (Centella
asiatica). Each ingredient already has been proven for its
potential activity on skin health as various medicine as well
as cosmeceuticals. Exposure to UV radiation and
environmental pollutants can accelerate the skin aging by
degrading collagen and triggering oxidative stress in the
skin.

The National Center for Complementary and Integrative
Health (NCCIH), allows the use of Complementary and
Alternative Medicine (CAM) therapies like Biofield Energy
as an alternative treatment in the healthcare sector. About
36% of US citizens regularly use some form of CAM [13],
in their day-to-day life. Researchers reported that a shortlived
electrical action potential exists in the mammalian
cells such as neurons, muscles, and endocrine. When the
cells are present in the central nervous system of human
body that communicate with each other by means of
electrical signals that propagate along the nerve impulses
[14]. Therefore, it was hypothesized that the Biofield can
exist around the human body and evidence was found using
electromyography, electrocardiography and
electroencephalogram [15]. Thus, a Biofield Energy
Healing Practitioner has the ability to harness the energy
from the environment and can transmit it into any object
(living organism or non-living material) around the globe.
The object(s) always receive the energy and respond in a
useful way that is called “Biofield Energy Treatment”. This
process is known as “Biofield Energy Healing”. Biofield
Energy Healing has been approved as an alternative method
that has an impact on various properties of living organisms
in a cost-effective manner [16, 17]. The Trivedi Effect® –
unique Biofield Energy Treatment has been known to alter
the response in a wide-spectrum field in living and nonliving
systems viz. materials science [18-20], agriculture
[21, 22], microbiology [23-25] biotechnology [26, 27].
Based on the excellent outcome of the Biofield Energy
Treatment, authors designed this study to investigate the
impact of the Biofield Energy Healing based DMEM and
test formulation on various skin health parameters using
three cell lines such as human foreskin fibroblast (HFF-1),
human keratinocytes (HaCaT), and mouse melanoma (B16-
F10) cells.

2. Materials and Methods

2.1. Chemicals and Reagents

L-ascorbic acid was purchased from Alfa-Aesar, while
kojic acid was purchased from Sigma, USA. Epidermal
growth factor (EGF) was procured from Gibco,
ThermoFisher, USA. ELISA kits were procured from
CUSABIO and CusAb Co. Pvt. Ltd., USA. Zinc chloride
purchased from TCI, Japan, sodium selenate from Alfa-
Aesar, USA, while sodium molybdate from Sigma-
Aldrich, USA. Tetrahydrocurcumin and Centella asiatica
extract were procured from Novel Nutrients Pvt. Ltd.,
India and Sanat Products Ltd., India, respectively. Fetal
bovine serum (FBS) and Dulbecco’s Modified Eagle’s
Medium (DMEM) were purchased from Gibco, USA.
Antibiotics solution (penicillin-streptomycin) was
procured from Himedia, India, while 3-(4, 5-diamethyl-2-
thiazolyl)-2, 5-diphenyl-2H-tetrazolium) (MTT), Direct
Red 80 and ethylene diamine tetra acetic acid (EDTA)
were purchased from Sigma, USA. All the other chemicals
used in this experiment were analytical grade procured
from India.

2.2. Cell Culture

HFF-1 (human fibroblast) cells were procured from
American Type Culture Collection (ATCC), USA,
originated from normal human skin fibroblast cells. B16-
F10 (mouse melanoma) cells were procured from National
Centre for Cell Science (NCCS), Pune. HFF-1, and B16-
F10 cell lines were maintained in the growth medium,
DMEM supplemented with 15% FBS, with added
antibiotics penicillin (100 U/mL) and streptomycin (100
μg/mL). The growth condition of cell lines were 37°C, 5%
CO2, and 95% humidity. L-ascorbic acid (for ECM, UV-B
protection, and wound healing assay) in concentrations
ranges from 10 μM to 1000 μM, while kojic acid (for
melanin synthesis) concentrations ranges from 1 mM to 10
mM, FBS (0.5%) was used in cell proliferation (BrdU)
assay, while EGF 10 μM was used in MTT assay.

2.3. Experimental Design

The experimental groups consisted of cells in normal
control, vehicle control group (0.05% DMSO), positive
control group (L-ascorbic acid/kojic acid/EGF/FBS) and
experimental tested groups. Experimental groups included
the combination of the Biofield Energy Treated and untreated
test formulation/DMEM. It consisted of four major treatment
groups on specified cells with UT-DMEM + UT-Test
formulation, UT-DMEM + BT-Test formulation, BT-DMEM
+ UT-Test formulation, and BT-DMEM + BT-Test
formulation.

2.4. Consciousness Energy Healing Treatment Strategies

The test formulation and DMEM were divided into two
parts. One of each part of the test formulation was treated with
the Biofield Energy by renowned Biofield Energy Healer (also
known as The Trivedi Effect®) and coded as the Biofield
Energy Treated samples, while the second part of the test
formulation and DMEM did not receive any sort of treatment
and was defined as the untreated test samples. This Biofield
Energy Healing Treatment was provided by Dennille Mellesia
Smith, who participated in this study and performed the
Biofield Energy Treatment remotely for ~5 minutes. Biofield
Energy Healer was remotely located in the USA, while the test
samples were located in the research laboratory of Dabur
Research Foundation, near New Delhi, India. This Biofield
Energy Treatment was provided for 5 minutes through the
Healer’s unique Energy Transmission process remotely to the
test samples under laboratory conditions. The Biofield Energy
Healer, Dennille Mellesia Smith, in this study never visited the
laboratory in person, nor had any contact with the test
formulation and DMEM. Further, the control groups were
treated with a 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
experimental study.

2.5. Determination of Non-cytotoxic Concentration

The cell viability was performed by MTT assay in HFF-1
(human fibroblast), HaCaT (human keratinocytes), and B16-
F10 (mouse melanoma) cells. The cells were counted and
plated in 96 well plates at the density corresponding to 5 X
103 to 10 X 103 cells/well/180 μL 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 formulation
and DMEM/positive controls. The untreated cells were
served as baseline control. The cells in the above plate(s)
were incubated for a time point ranging from 24 to 72 hours
in CO2 incubator at 37°C, 5% CO2 and 95% humidity.
Following incubation, the plates were taken out and 20 μL of
5 mg/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 150 μL of DMSO was added
to each well to dissolve formazan crystals. The absorbance of
each well was read at 540 nm using Synergy HT micro plate
reader, BioTek, USA. The concentrations exhibiting %
cytotoxicity of < 30 % was considered as non-cytotoxic [28,
29]. The percentage cell viability at each tested
concentrations of the test substance were calculated using the
following Equation 1:

% Cell viability = (X ∗ 100)/R (1)

Where, X represent the absorbance of the cells
corresponding to positive control and test groups and R
represent the absorbance of the cells corresponding to the
baseline (control cells) group.

2.6. Effect of the Test Item on Fibroblast Proliferation by
5-bromo-2′-deoxyuridine (BrdU) Method

HFF-1 cells were counted using hemocytometer and plated
in 96 well plate at the density corresponding to 1 X 103 to 5
X 103 cells/well in DMEM supplemented with 15% FBS.
The cells/plates were incubated overnight under growth
conditions so as to allow cell recovery and exponential
growth. Following overnight incubation, the above cells were
subjected to serum starvation. Following serum starvation,
the cells were treated with non-cytotoxic concentrations of
test substance and positive control. Following 24 to 72 hours
of incubation with the test substance and positive control, the
plates were taken out and 5-bromo-2′-deoxyuridine (BrdU)
estimation using cell proliferation ELISA, BrdU estimation
kit (ROCHE – 11647229001) as per manufacturer’s
instructions.

2.7. Estimation of Extracellular Matrix (ECM)

Synthesis of extracellular matrices component (i.e. collagen,
elastin, and hyaluronic acid) in HFF-1 was estimated for
determining the potential of the Biofield Energy Treated test
formulation and DMEM to improve the skin strength, elasticity,
and hydration level. HFF-1 cells were counted using hemocytometer and plated in 48 well plate at the density
corresponding to 10 X 103 cells/well in DMEM supplemented
with 15% FBS. The cells were incubated overnight under
specified growth conditions followed by cells to serum stripping.
Further, the cells were treated with different groups viz. vehicle
control (DMSO-0.05%), positive control (L-ascorbic acid, at 10
μM concentration), and the test items at different concentrations.
Further, 72 hours of incubation with the test items and positive
control, the supernatants from all the cell plates were taken out
and collected in pre labeled centrifuge tubes for the estimation
elastin and hyaluronic acid levels. However, the corresponding
cell layers were processed for estimation of collagen levels using
Direct Sirius red dye binding assay [30]. Elastin and hyaluronic
acid were estimated using ELISA kits from Cusabio Biotech Co.
Ltd., Human Elastin ELN Elisa kit 96T and Human hyaluronic
acid, Elisa kit 96T, respectively [31].

2.8. Estimation of Melanin Synthesis

B16-F10 cells were used for melanin synthesis estimation,
cells were counted using hemocytometer and plated in 90
mm culture dish at the density corresponding to 2 X 106 per
6 mL in culture plates. Further, the cells were incubated
overnight under specified growth conditions and allowed for
cell recovery and exponential growth. After incubation, the
cells were treated with α-melanocyte-stimulating hormone
(α-MSH) for a time point ranging from 4 to 24 hours for the
stimulation of intracellular melanin synthesis. Further, the
cells were incubated with α-MSH and then treated with the
test formulation with DMEM at different concentrations for
48 to 96 hours. After incubation, intracellular melanin was
extracted in NaOH and the absorbance was recorded at 405
nm. The level of melanin was extrapolated using standard
curve obtained from purified melanin [32].

2.9. Anti-wrinkle Effects of the Test Formulation on HFF-1
Cells against UV-B Induced Stress

UV-B induced stress was evaluated in HFF-1 cells and cell
viability was estimated in the presence of test items. The
cells were counted using hemocytometer and plated in 96
well plate at the density corresponding to 5 X 103 to 10 X 103
cells/well in DMEM supplemented with 15% FBS
cells/plates, which were incubated overnight under growth
conditions to allow cell recovery and exponential growth.
The cells were treated with non-cytotoxic concentrations of
test items for 2 to 24 hours. After treatment with test items,
the cells were subjected to the lethal dose of UV-B
irradiation (200 mJ/cm2) that can lead to approximately 50%
cytotoxicity (302 nm, CL-1000 M, UVP, USA) [33]. The
percent cell viability was assessed using following Equation
2:

% Cell viability = (X ∗ 100)/R (2)

Where,
X represents the absorbance of cells corresponding to
positive control and test group,
R represents the absorbance of cells corresponding to the
baseline (control cells) group.

2.10. Wound Healing Activity by Scratch Assay

HFF-1 and HaCaT cells were counted using
hemocytometer and plated in 12 well plates at the densities
0.08 X 106/well/mL of cell growth medium. The cells/plates
were incubated overnight under growth conditions and
allowed cell recovery and exponential growth. After
overnight incubation, the cells were subjected to the serum
starvation in DMEM for 24 hours. Mechanical scratch that
represents wound was created in the near confluent
monolayer of cells by gently scraping with sterile 200 μL
micropipette tip. The cells were then rinsed with serum free
DMEM and treated with the test formulation. The scratched
area was then monitored for a time period ranging from 0 to
48 hours for closure of wound area. The photomicrographs
(x10) were done at the selected time point (at 16 hours) of
migrated cells using digital camera. It represented the
fibroblast distance covered and subsequent scratch closure
[34].

2.11. Statistical Analysis

Each experiment was carried out in three independent
assays and the values were represented as mean values with
standard error of mean (SEM). 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.

3. Results and Discussion

3.1. Cell Viability by MTT Assay

MTT assay was used for the assessment of the viable
cells in three different cells like HFF-1, HaCaT, and B16-
F10 cells and the results are shown in Figure 1A to 1C. The
result exhibited about >78% viable cells in the tested
concentrations ranges from 0.63 to 10 μg/mL in the HFF-1
cells (Figure 1A), which indicated that the test formulation
was safe and nontoxic. The selected concentrations were
used for the estimation of collagen, elastin, and hyaluronic
acid. Furthermore, the cell viability in HaCaT cells
exhibited >95%. The concentrations of the test formulation
from 5 to 40 μg/mL were used for the evaluation of wound
healing activity by scratch assay (Figure 1B). The
percentage of viable cells in the B16-F10 cells revealed that
the test formulation was non-cytotoxic (i.e. percentage cell
viability value >98%) and to be safe. The tested
concentrations were used further for the measurement of
melanin level at the concentrations ranging from 10 to 40
μg/mL (Figure 1C).

90 Dennille Mellesia Smith et al.: Skin Protective Activity of Consciousness Energy Healing Treatment Based Herbomineral Formulation

Paper-13-5-1

Figure 1. Evaluation of the Cell viability by MTT assay of the test formulation in three different cells. (A) HFF-1 cells after 72 hours of treatment; (B) HaCaT
cells after 48 hours of treatment; and (C) B16-F10 cells after 48 hours of treatment. LA: L-Ascorbic acid; EGF: Epidermal growth factor.

3.2. Cell Proliferation by BrdU Assay

The cell proliferation analyzed by bromodeoxyuridine
(BrdU) assay is shown in Figure 2. The cell proliferation was
100% and 250.4% in the vehicle control (VC) and positive
control (FBS-0.5 μg/mL) groups, respectively. Further, the
cell proliferation was significantly increased by 149.18% and
118.86% in the UT-DMEM + BT-Test formulation and BTDMEM
+ UT-Test formulation groups, respectively at 8.75
μg/mL compared to the UT-DMEM + UT-Test formulation
group. Moreover, the cell proliferation was enhanced by
238.30%, 192.06%, and 43.96% in the UT-DMEM + BTTest
formulation, BT-DMEM + UT-Test formulation, and
BT-DMEM + BT-Test formulation groups, respectively at
17.5 μg/mL with respect to the UT-DMEM + UT-Test

Paper-13-5-2

Figure 2. Effect of the test formulation on cellular proliferation by BrdU assay after 48 hours of treatment. VC: Vehicle control; FBS: Fetal bovine serum
(μg/mL); UT: Untreated; BT: Biofield Treated.

3.3. Impact of the Test Formulation on Synthesis of
Extracellular Matrix (ECM) Components in Human
Foreskin Fibroblast (HFF-1)

3.3.1. Collagen

Effect of the test formulation and DMEM on collagen level
in HFF-1 cells is shown in Figure 3. The level of collagen
was 93.14 ± 1.37 and 129.42 ± 8.50 μg/mL in the vehicle
control (VC) and positive control groups, respectively. The
level of collagen was significantly increased by 8.58% and
15.18% in the BT-DMEM + UT-Test formulation and BTDMEM
+ BT-Test formulation groups, respectively at 0.63
μg/mL compared to the UT-DMEM + UT-Test formulation
group. Additionally, collagen data showed 55.55%, 32.65%,
and 52.48% elevation in the UT-DMEM + BT-Test formulation group. At 35 μg/mL, the cell proliferation was
significantly elevated by 95.79%, 76.05%, and 45.97% in the
UT-DMEM + BT-Test formulation, BT-DMEM + UT-Test
formulation, and BT-DMEM + BT-Test formulation groups,
respectively compared to the UT-DMEM + UT-Test
formulation group. Cell proliferation is vital for cellular
homoeostasis and maintenance of an organism. The BrdU
assay was used for the evaluation of three major objectives
such as for measuring the rate of DNA replication, analysis
of metabolic activity and recognitions of cell surface antigen
activity [35]. Overall, the cell proliferation in the Biofield
Energy Treated test formulation and DMEM groups were
remarkably improved. formulation, BT-DMEM + UT-Test formulation and BTDMEM
+ BT-Test formulation groups, respectively at 1.25
μg/mL compared to the UT-DMEM + UT-Test formulation
group. Moreover, the expression of collagen was enhanced
significantly (p≤0.05) by 64.59%, 38.57%, and 57.41% in the
UT-DMEM + BT-Test formulation, BT-DMEM + UT-Test
formulation and BT-DMEM + BT-Test formulation groups,
respectively at 2.5 μg/mL compared to the UT-DMEM +
UT-Test formulation group. Several stimuli such as local
tissue ischemia, necrotic tissue, repeated trauma, etc. causes a
chronic wounds in the inflammatory phase. In chronic
wounds, there was an elevation of matrix metalloproteinases
(MMPs) enzymes that degraded the both viable as well as
non-viable collagen [36]. Collagen is an important
component responsible for wound healing and due to damage of collagen the repair process also delayed [37]. Therefore,
the control of collagen metabolism might be useful for a
variety of therapeutic and cosmetic applications. Overall, the
level of collagen synthesis was improved significantly in the
Biofield Energy Treated test formulation and DMEM group,
which might be due to The Trivedi Effect® – Energy of
Consciousness Healing Treatment.

Paper-13-6-1

Figure 3. Effect of the test formulation on collagen synthesis in human foreskin fibroblast cells (HFF-1). VC: Vehicle control; LA: L-Ascorbic acid; UT:
Untreated; BT: Biofield Treated. *p≤0.05 vs UT-DMEM + UT-Test formulation using one-way ANOVA (post-hoc Dunnett’s test).

3.3.2. Elastin

The effect of the test formulation and DMEM on elastin
level in the human foreskin fibroblast cells (HFF-1) is shown
in Figure 4. The level of elastin in the vehicle control (VC)
and positive control groups was observed as 6.06 ± 0.00 and
7.27 ± 0.15 pg/mL, respectively. The level of elastin was
significantly (p≤0.001) increased by 19.99% in the BTDMEM
+ UT-Test formulation group at 2.5 μg/mL compared
to the UT-DMEM + UT-Test formulation group. Moreover,
at 5 μg/mL the level of elastin was significantly (p≤0.001)
elevated by 72.54% and 27.56% in the BT-DMEM + UTTest
formulation and BT-DMEM + BT-Test formulation
groups, respectively compared to the UT-DMEM + UT-Test
formulation group. Further, at 10 μg/mL the expression of
elastin was also significantly (p≤0.001) increased by 6.30%,
105.04%, and 29.41% in the UT-DMEM + BT-Test
formulation, BT-DMEM + UT-Test formulation, and BTDMEM
+ BT-Test formulation groups, respectively
compared to the UT-DMEM + UT-Test formulation group.
Elastin is the important component of the skin that maintain
the mechanical and cell interactive properties. It induce a
wide-range of cellular activities such as cell migration and
proliferation, matrix synthesis, and protease production [38].
Elastin enhanced the process of wound healing due to its
inherent properties. Cutaneous ageing is the result of two
biological processes, which may occur simultaneously as
termed as intrinsic ageing and extrinsic ageing. The intrinsic
aged skin is due to dryness and lack of elastin than youthful
skin [39]. Altogether, the level of elastin synthesis was
improved significantly in the Biofield Energy Treated test
formulation and DMEM group, which might be due to The
Trivedi Effect® – Energy of Consciousness Healing
Treatment.

Paper-13-6-2

Figure 4. Effect of the test formulation and DMEM on elastin formation in human foreskin fibroblast cells (HFF-1). VC: Vehicle control; LA: L-Ascorbic acid;
UT: Untreated; BT: Biofield Treated. ***p≤0.001 vs UT-DMEM + UT-Test formulation using one-way ANOVA (post-hoc Dunnett’s test).

3.3.3. Hyaluronic Acid (HA)

The effect of the test herbomineral formulation and
DMEM for the expression of HA in HFF-1 cells is shown in
Figure 5. The results of HA synthesis in the presence of Lascorbic
acid (10 μM), showed significant increase in HA
content by 26.37% compared with the vehicle control (VC)
group (9.67 ± 0.08 ng/mL). The level of HA was increased
significantly (p≤0.001) by 4.78%, 29.71%, and 58.29% in the
UT-DMEM + BT-Test formulation, BT-DMEM + UT-Test
formulation, and BT-DMEM + BT-Test formulation groups,
respectively at 0.63 μg/mL compared to the UT-DMEM +
UT-Test formulation group. Further, at 1.25 μg/mL the HA
level was significantly (p≤0.001) increased by 15.54% in the
BT-DMEM + BT-Test formulation group compared to the
UT-DMEM + UT-Test formulation group. Additionally, the
level of HA was significantly increased by 31.91% in the
BT-DMEM + BT-Test formulation group with respect to the
UT-DMEM + UT-Test formulation group at 2.5 μg/mL. The
overall data suggested that the Biofield Energy Treated test
formulation and DMEM have the significant capacity to
increase the level of hyaluronic acid.

Paper-13-7-1

Figure 5. Effect of the test formulation on the expression of hyaluronic acid in human foreskin fibroblast cells (HFF-1). VC: Vehicle control; LA: L-Ascorbic
acid; UT: Untreated; BT: Biofield Treated. ***p≤0.001 vs UT-DMEM + UT-Test formulation using one- way ANOVA (post-hoc Dunnett’s test).

3.4. Effect of the Test Formulation on Skin Depigmentation

The effect of the test formulation and DMEM on alphamelanocyte-
stimulating hormone (α-MSH) stimulated
melanin synthesis in B16-F10 cells is shown in Figure 6. The
level of melanin was significantly decreased by 75.92% in
the kojic acid (KA) group (5.96 ± 0.2 μg/mL) compared to
the α-MSH group (24.75 ± 0.22 μg/mL). The cellular content
of melanin was reduced by 12.49%, 6.74%, and 8.79% in the
UT-DMEM + BT-Test formulation, BT-DMEM + UT-Test
formulation, and BT-DMEM + BT-Test formulation groups,
respectively at 0.06 μg/mL compared to the UT-DMEM +
UT-Test formulation group. Besides, the level of melanin
synthesis was significantly (p≤0.001) inhibited by 14.64%
and 18.25% in the UT-DMEM + BT-Test formulation and
BT-DMEM + UT-Test formulation groups, respectively at
0.13 μg/mL compared to the UT-DMEM + UT-Test
formulation group. Thus, it can be concluded that the
Biofield Energy Treated test formulation and DMEM inhibits
the melanin production significantly in the B16-F10 cells.
This improvement could be beneficial for the development of
a cosmeceuticals for hyperpigmentation and different types
of skin conditions.

Paper-13-7-2

Figure 6. Effect of the test formulation on alpha-MSH stimulated melanin in B16-F10 cells. α-MSH: Alpha-melanocyte-stimulating hormone, KA: Kojic acid
(mM); UT: Untreated; BT: Biofield Treated. ***p≤0.001 vs UT-DMEM + UT-Test formulation using one-way ANOVA (post-hoc Dunnett’s test).

3.5. Anti-wrinkle Effects of the Test Formulation on HFF-1
Cells against UV-B Induced Stress

The effect of the test formulation with DMEM after
pretreatment with UV-B challenge in HFF-1 cells is
represented in Figure 7. The cell viability was identified
using hemocytometer. The cells were subjected to lethal dose
of UV-B irradiation (200 mJ/cm2) showed 26.73% cell
viability. The cell viability was 100% and 27.78% in the
normal control (NC) and vehicle control (VC) groups
respectively. The cell viability was increased by 55.11% in
the positive control (L-ascorbic acid) group compared to the
VC group. After UV-B induce stress condition the level of
cell viability was significantly increased by 17.88%, 20.14%,
and 9.89% in the BT-DMEM + BT-Test formulation at 0.63,
1.25, and 2.50 μg/mL, respectively compared to the UTDMEM
+ UT-Test formulation group. Besides, the rest of the
concentrations did not show any alteration with respect to the
UT-DMEM + UT-Test formulation group. Several factors are
responsible for skin wrinkles such as aging, genetics, and
environmental factors such as ultraviolet radiation, smoking
and due to deficiency of estrogen [40, 41]. Aging is one of
the most important factor responsible for skin wrinkles. In
humans, due to aging the skin becomes thin and decrease
elasticity, collagen, etc. [42, 43]. The results suggested that
both the Biofield Energy Treated test formulation and
DMEM could be significantly used for skin protective effect
with anti-wrinkling potential.
Figure

Paper-13-8-1

Figure 7. Percentage restoration of the cell viability in HFF-1 cells after 20 hours of pretreatment before UV-B challenge. NC: Normal control; VC: Vehicle
control LA: L-Ascorbic acid; UT: Untreated; BT: Biofield Treated.

3.6. Wound Healing Activity by Scratch Assay

The wound healing activity by scratch assay of the test
formulation and DMEM was performed for the measurement
of cell migration in HFF-1 and HaCaT cells. The
representative photomicrographs are presented in Figure 8.
The cell coverage area was increased by 10.5%, 8.8%, and
7.0% in the UT-DMEM + BT-Test formulation, BT-DMEM +
UT-Test formulation, and BT-DMEM + BT-Test formulation
groups, respectively at 0.63 μg/mL in HFF-1 cells compared to
the UT-DMEM + UT-Test formulation group. Additionally,
the cell coverage area was increased by 1.8% (at 2.5 μg/mL)
and 8.8% (1.25%) in the BT-DMEM + UT-Test formulation
and BT-DMEM + BT-Test formulation groups, respectively in
HFF-1 cells compared to the UT-DMEM + UT-Test
formulation group (Figure 8A). Moreover, the cell coverage
area was increased by 8.3% at 2.5 μg/mL in the UT-DMEM +
BT-Test formulation, BT-DMEM + UT-Test formulation, and
BT-DMEM + BT-Test formulation groups in HaCaT cells
compared to the UT-DMEM + UT-Test formulation group.
Furthermore, the cell coverage area was increased by 2.8%,
2.8%, and 1.4% in the UT-DMEM + BT-Test formulation,
BT-DMEM + UT-Test formulation, and BT-DMEM + BTTest
formulation groups, respectively in HaCaT cells
compared to the UT-DMEM + UT-Test formulation group
(Figure 8B). In vitro scratch assay is a well-established method
for the estimation of cell migration, cell-matrix, and cell-to-cell
interactions during wound healing [44]. The wound healing
results indicated that the test formulation and DMEM showed
significant wound closure activity.

Paper-13-8-2

Figure 8. Effect of the test formulation and DMEM on wound closure and
cell migration after 16 hours of treatment. Representative photomicrograph
(X10) of the test formulation shown in A. HFF-1 and B. HaCaT cells. UT:
Untreated; BT: Biofield Treated.

4. Conclusions

The cell viability using MTT assay exhibited more than
78% cells were viable, indicating that the Biofield Energy
Healing based herbomineral test formulation was safe and
nontoxic in all the tested concentrations. The percent cell
proliferation using BrdU was significantly increased by
238.30%, 192.06%, and 43.96% in the UT-DMEM + BT-Test
formulation, BT-DMEM + UT-Test formulation, and BTDMEM
+ BT-Test formulation groups, respectively at 17.5
μg/mL compared to the UT-DMEM + UT-Test formulation
group. The level of collagen was significantly increased by
55.55%, 32.65%, and 52.48% in the UT-DMEM + BT-Test
formulation, BT-DMEM + UT-Test formulation and BTDMEM
+ BT-Test formulation groups, respectively at 1.25
μg/mL compared to the UT-DMEM + UT-Test Formulation
group. Elastin was significantly (p≤0.001) increased by 6.30%,
105.04%, and 29.41% at 10 μg/mL in the UT-DMEM + BTTest
formulation, BT-DMEM + UT-Test formulation, and BTDMEM
+ BT-Test formulation groups, respectively compared
to the UT-DMEM + UT-Test formulation group. Hyaluronic
acid was increased significantly by 4.78%, 29.71%, and
58.29% in the UT-DMEM + BT-Test formulation, BT-DMEM
+ UT-Test formulation, and BT-DMEM + BT-Test
formulation groups, respectively at 0.63 μg/mL compared to
the UT-DMEM + UT-Test Formulation group. Melanin level
was significantly (p≤0.001) reduced by 14.64% and 18.25% in
the UT-DMEM + BT-Test formulation and BT-DMEM + UTTest
formulation groups, respectively at 0.13 μg/mL with
respect to the UT-DMEM + UT-Test formulation group. Antiwrinkling
effect using UV-B induced stress in HFF-1 cells
showed that the Biofield Energy Treated test formulation was
increased significantly by 17.88%, 20.14%, and 9.89% at 0.63,
1.25, and 2.5 μg/mL, respectively in the BT-DMEM + BTTest
formulation group compared to the UT-DMEM + UTTest
formulation group. Wound healing results displayed a
significant effect of the test formulation and DMEM on wound
closure and cell migration in all the tested groups in both HFF-
1 and HaCaT cells compared to the untreated groups. Overall,
the Consciousness Energy Healing Treated test formulation
(The Trivedi Effect®) and DMEM have shown significant
protective effects on various skin health parameters such as
wrinkling, aging, skin whitening, and wound healing.
Therefore, the Biofield Energy Healing based herbomineral
test formulation would be suitable for the development of
herbal cosmetics, and it would be useful for the management
of wounds and various skin related disorders viz. abscess,
pimple, cellulitis, impetigo, scabies, syringoma,
photosensitivity, urticaria, hives, warts, abscess, callus, acne,
chickenpox, eczema, rosacea, seborrheic dermatitis, athlete’s
foot, psoriasis, erythema, contact dermatitis, cutis
rhomboidalis nuchae, skin aging, wrinkles and/or change in
skin color etc.

Abbreviations

HaCaT: Human keratinocytes, HFF-1: Human fibroblast
cell line, B16-F10: Mouse melanoma cell line, THC:
Tetrahydrocurcumin, ECM: Extracellular matrix, EGF:
Epidermal growth factor, α-MSH: Alpha-melanocytestimulating
hormone, ANOVA: One-way analysis of
variance, HA: Hyaluronic acid, UT: Untreated, BT: Biofield
Treated, FBS: Fetal bovine serum, BrdU:
Bromodeoxyuridine, ROS: Reactive oxygen species, CAM:
Complementary and alternative medicine, DMEM:
Dulbecco’s modified eagle’s medium, ATCC: American type
culture collection, NCCS: National centre for cell science,
UV: Ultra-violet.

Acknowledgements

Authors are grateful to Dabur Research Foundation,
Trivedi Global, Inc., Trivedi Science, Trivedi Testimonials,
and Trivedi Master Wellness for their support throughout the
work.

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