Biofield Energy Healing Based Herbomineral Formulation: An Emerging Frontier in Cosmetic Medicine

Journal: American Journal of Biomedical and Life Sciences

  • 298
  • 43
Biofield Energy Healing Based Herbomineral Formulation

Abstract:

The application of the herbomineral formulations in general skin health are increasing day-by-day due to the
excellent outcomes without any adverse effects. This study was designed to evaluate the influence of The Trivedi Effect®-
Consciousness Energy Healing Treatment on an herbomineral formulation and cell medium against various skin health
parameters. The test formulation consists of minerals (zinc chloride, sodium selenate, and sodium molybdate) and L-ascorbic
acid along with herbal extracts, Centella asiatica, and tetrahydrocurcumin (THC). The test formulation and DMEM media were
divided into two equal parts, one was treated with a Biofield Treatment (BT) by Dezi Ann Koster and denoted as treated, while
the other part was coded as the untreated (UT) groups. MTT assay results showed that test formulation was safe and nontoxic
with more than 89% cell viability in the tested cell lines. BrdU assay showed an improved cell proliferation by 2.82% in the
UT-DMEM + BT-Test formulation group compared with the untreated group. The level of collagen was significantly increased
by 32.42%, 33.64%, and 29.13% at 2.5, 1.25 and 0.625 μg/mL, respectively in the UT-DMEM + BT-Test formulation,
while34.17%, 26.73%, and 17.56% increased at 2.5, 1.25 and 0.625 μg/mL, respectively in BT-DMEM + BT-Test formulation
group compared with the untreated group. Elastin level was increased by 408.6% at concentration of 0.625 μg/mL in UTDMEM
+ BT-Test formulation group compared with untreated group. However, hyaluronic acid (HA) level was increased by
31.88%, 15.52%, and 58.29% at 2.5, 1.25, and 0.625 μg/mL, respectively in the BT-DMEM + BT-Test formulation group
compared with untreated group. Besides, melanin synthesis was significantly inhibited by 16.09% and 18.93% in the UTDMEM
+ BT-Test formulation and BT-DMEM + UT-Test formulation groups, respectively at 0.125 μg/mL compared with the
untreated group. Anti-wrinkling activity in HFF-1 cells showed an improved cell viability by 5.49% and 11.26% at 1.25 and
0.625 μg/mL, respectively in BT-DMEM + BT-Test formulation group compared with the untreated group. Wound healing
scratch assay results showed a significant healing rate by 5% and 10% in HFF-1 and HaCaT cells lines, respectively with high
cellular migration of fibroblast and keratinocytes. Overall, it can be concluded that the Biofield Energy Healing (The Trivedi
Effect®) based test formulation and cell medium could be helpful against various skin disorders and can be used in psoriasis,
seborrheic dermatitis, skin cancer, rashes from bacterial or fungal infections as anti-wrinkling, skin-whitening, anti-ageing, and
rejuvenating agent.

Keywords:

Consciousness Energy Healing Treatment, Biofield Energy Treatment, Extracellular Matrix, Hyaluronic Acid,
Scratch Assay, Tetrahydrocurcumin, HFF-1 and HaCaT Cells Lines

1. Introduction

The statistics from the World Health Organization (WHO) state that there is a high level of belief worldwide
and global movement towards the use of alternative medicinal approaches such as energy medicine, herbal based products, etc. as a way to adopt natural way of treatment. People instead of using synthetic drugs, prefer
natural and organic foods, herbal medicines, and naturopathic treatments for healthy life [1]. The use of
organic foods and farming instead of fertilizers based vegetable products and its demand have been increased. In
personal care system, use of herbal based products or cosmetics are highly preferred due to the lower incidence of
adverse effect compared with the synthetic compounds [2-4]. The increased demand of herbal based cosmetics is due
to high prevalence of human health hazards and side effects that lead to various diseases [5]. Many invasive skin
treatment measures such as gene therapy, chemical peels, and several devices such as laser energy, injectable, etc. are used for skin health and rejuvenation [6, 7], however many photo-aging products, antioxidant agents (i.e. vitamin B3, C, and E) are also available in the market for skin health. However, the new cosmetic market is driven towards herbal drugs, herbal cosmetics, nutraceuticals, and natural dyes.However, herbomineral products are the best way adopted in order to utilize the modern as well as alternative aspect, which includes the vital minerals required to maintain quality of life. The present research work deals with the novel cosmetic product, an herbomineral formulation prepared in order to improve overall skin health using various skin health parameters against cells lines such as human foreskin fibroblast (HFF-1), human keratinocytes (HaCaT) and mouse melanoma cell lines (B16-F10).

The test formulation consisted of minerals (zinc chloride,
sodium selenate, and sodium molybdate), L-ascorbic acid,
tetrahydrocurcumin (THC), and an herbal extract of Centella
asiatica (commonly known as Jal Brahmi). Minerals such as
zinc, copper, and selenium are very commonly used in
cosmetic products and have wide range of application in
cosmetology. These minerals have been reported with strong
antimicrobial, antioxidant, and free radical scavenging effects
with improved skin extra cellular component synthesis [8, 9].
In addition, L-ascorbic acid (Vitamin C) along with other
vitamins (such as Vit-A and E) play an important role in skin
health, anti-wrinkling, and significant wound healing action
[10]. THC is one of the major active metabolite of curcumin
[11], which has strong antioxidant property and role in skin
health [12, 13]. C. asiatica is well known medicinal herb
used for skin ageing, and used as one of the natural weapons
in various skin health strategies. Moreover, C. asiatica has
been reported for wound healing action along and has been
used in many skin care products [14].

Biofield science and healing is emerging in the frontier in
medicine and its increased use as alternative integrative
approaches have been reported in order to promote wellness
by uncovering the root cause of diseases with universal
solutions [15]. Data suggest that most of the U. S. population
has been focused towards the use of natural products as a
complementary and alternate medicine (CAM) [16]. The
National Center for Complementary and Alternative
Medicine (NCCAM), now demarcated Biofield Therapies as
one form of the five complementary medicine domain.
Biofield is the name given to the low electromagnetic field
that permeates and surrounds living organisms [16]. Biofield
Healing has been accepted world-wide with significant
clinical outcomes including wound healing [17-19]. Biofield
Energy Treatment (The Trivedi Effect®) has been reported
with noteworthy results in nonliving materials and living
organisms. The Trivedi Effect® has been reported with
momentous results in the field of microbiology [20-22],
agriculture science [23-25], livestock [26], and materials
science [27-29]. On this basis, a novel Biofield Energy
Healing based herbomineral formulation has been designed
for skin care treatment. The study was conducted for the
evaluation of skin health parameters of test formulation using
HFF-1, HaCaT, and B16-F10 cell lines.

2. Materials and Methods

2.1. Chemicals and Reagents

The test formulation components consisted of zinc
chloride, which was purchased from TCI, Japan, sodium
selenate from Alfa-Aesar, USA, while sodium molybdate
from Sigma-Aldrich. Tetrahydrocurcumin and Centella
asiatica extract were procured from Novel Nutrients Pvt.
Ltd., India and Sanat Products Ltd., India respectively. Lascorbic
acid as a positive control was purchased from Alfa-
Aesar, while kojic acid and 3-(4, 5-diamethyl-2-thiazolyl) 2,
5 diphenyl-2 H-tetrazolium) (MTT) were purchased from
Sigma Chemical Co. (St. Louis, MO). Epidermal growth
factor (EGF) was procured from Gibco, ThermoFisher, USA.
ELISA kits for the estimation of extracellular matrix
components were procured from CUSABIO and CusAb Co.
Pvt. Ltd., USA. Fetal bovine serum (FBS) and DMEM were
purchased from Gibco, USA. Antibiotics solution (Penicillin-
Streptomycin) were procured from HiMedia, India, while
Direct Red 80 and EDTA were purchased from Sigma, USA.
All the other chemicals used in this experiment were
analytical grade procured from local vendors.

2.2. Cell Culture

HFF-1 (human foreskin fibroblast) cells were procured
from American Type Culture Collection (ATCC), USA,
originated from normal human skin fibroblast cells. B16-F10
(mouse melanoma) and HaCaT (human keratinocytes) cells
were procured from National Centre for Cell Science
(NCCS), Pune, India. HFF-1, HaCaT, 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) at the concentrations ranges from 10
μM to 1000 μM, while kojic acid (for melanin)
concentrations ranges from 1 mM to 10 mM. FBS (0.5%)
was used in cell proliferation assay in BrdU assay, while
EGF 10 μM used in non-cytotoxic concentration in MTT
assay.

2.3. Experimental Design

The experimental groups consisted of cells in normal
control group, 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 Biofield Energy Treated and
untreated test formulation/DMEM. It consists of four major
treatment groups on specified cells with UT-DMEM + UTTest
formulation, UT-DMEM + Biofield Energy Treated test
formulation (BT-Test formulation), BT-DMEM + UT-Test
formulation, and BT-DMEM + BT-Test formulation.

2.4. Energy of Consciousness Treatment Strategies

The test formulation and DMEM were divided into two
parts. One part of the test samples were treated with the
Biofield Energy by a renowned Biofield Energy Healer (also
known as The Trivedi Effect®) and coded as the Biofield
Energy Treated formulation, while the second part of the test
samples did not receive any sort of treatment and was defined
as the untreated test samples. Dezi Ann Koster, a renowned
Biofield Energy Healer, provided the Biofield Energy
Treatment remotely for 5 minutes through the Healer’s
unique Energy Transmission process remotely to the test
formulation under laboratory conditions. The Biofield
Energy Healer was located in the USA, while the test
samples were located in the research laboratory of Dabur
Research Foundation near New Delhi, India. The Biofield
Energy Healer, Dezi Ann Koster, in this study never visited
the laboratory, nor had any contact with the test formulation
and the medium. Further, the control samples were treated
with a “sham” healer for comparative purpose. 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
study.

2.5. Determination of Non-cytotoxic Concentrations

The cell proliferation in cell lines such as HFF-1, HaCaT,
and B16-F10 were performed by MTT assay. The cells
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 cells were incubated overnight
under specific growth conditions that were allowed the cell
recovery and exponential growth, which were subjected to
serum stripping or starvation. The cells were subsequently
treated to the Biofield Energy Treated and untreated groups
of the test formulation/DMEM at a range of concentrations
(0.008 to 10 μg/mL) and ascorbic acid (10 and 50 μM)
followed by incubation from 24 to 72 hours in CO2 incubator
at 37°C, 5% CO2 and 95% humidity. Further, serum free
MTT media (20 μL of 5 mg/mL) was added to each well
followed by incubation for 3 h at 37°C. The supernatant was
aspirated and 150 μL of DMSO was added to each well to
dissolve the formazan crystals. Thereafter, the absorbance of
each well was recorded at 540 nm using Synergy HT micro
plate reader, BioTek, USA. The concentrations that exhibited
percentage cytotoxicity of less than 30% was considered as
non-cytotoxic [30].

2.6. Effect of Test Formulation on Human Foreskin
Fibroblast Cell Proliferation Using 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 then incubated overnight under growth
conditions so as to allow cell recovery and exponential
growth. After overnight incubation, the above cells were
subjected to serum starvation. Following serum starvation,
the cells were treated with non-cytotoxic concentrations of
test formulation in different defined experimental groups and
positive control. After 24 to 72 hours of incubation with the
test substance and positive control, the plates were taken out
and BrdU (5-bromo-2′-deoxyuridine) estimated using Cell
Proliferation ELISA, BrdU estimation kit (ROCHE –
11647229001) as per manufacturer’s instructions.

2.7. Estimation of Extracellular Matrix Component
Synthesis

Synthesis of extracellular matrix components (i.e.
collagen, elastin and hyaluronic acid) in HFF-1 cell line
was estimated for determining the potential of test
formulation to improve skin strength, overall elastin, 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 then
incubated overnight under specified growth conditions
followed by cells to serum stripping. Further, the cells were
treated with the test formulation at different experimental
combination groups with DMEM group viz. vehicle control
(DMSO, 0.05%), and positive control (ascorbic acid, at 10
μM). 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 of elastin and hyaluronic acid levels.
However, the corresponding cell layers were processed for
the estimation of collagen levels using Direct Sirius red dye
binding assay. 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- Skin Depigmentation
Effect

B16-F10 cells were used for the estimation of melanin
synthesis, cells were counted using hemocytometer and
plated in 90 mm culture dish at the density corresponding to
2 X 106/6 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-melanocytestimulating
hormone (α-MSH) for a time point ranging from 4 to 24 hours for stimulation of intracellular melanin
synthesis. Further, the cells were incubated with α-MSH, and
then treated with concentration at 0.625, 1.25 and 2.5 μg/mL
of test formulation with DMEM for a time period from 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-wrinkling Effects of 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 formulation.
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 formulation for 2 to 24 hours. After treatment, the cells
were subjected to 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 formula equation (1)

% Cell viability =
(X * 100)/R (1)

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

2.10. Wound-Healing Scratch Assay

HFF-1 and HaCaT cell lines 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 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 scratches that represent
wounds were created in the near confluent monolayer of cells
by gently scrap with the sterile 200 μL micropipette tip. The
cells were rinsed with serum free DMEM and treated with
test formulation. The scratched area was monitored for a time
period ranging from 0 to 48 hours for closure of wound area.
The representative photomicrographs of cells in different
groups were done at 16 hours for quantitative assessment of
cellular migration using digital camera, which was connected
to the inverted microscope. All the observations were
calculated and compared with the positive and vehicle
control [34].

2.11. Statistical Analysis

Each experiment was carried out in three independent
assays and was represented as mean values with standard
error of mean (SEM). Student’s t-test was used to compare
two groups to judge the statistical significance. For multiple
group comparison, one-way analysis of variance (ANOVA)
was used followed by post-hoc analysis using Dunnett’s test.
Statistically significant values were set at the level of p≤0.05.

3. Results and Discussion

3.1. MTT Assay- Non-cytotoxic Effect of the Test
Formulation

The cytotoxic effects of test formulation was tested on all
the three cell lines i.e. HFF-1, HaCaT and B16-F10. The
results were compared with respect to ascorbic acid (10 μM)
and EGF (10 ng/mL) at defined concentrations for the
estimation of percentage cell viability. The results of
percentage cell viability in all the tested cell lines showed the
cell viability range of 73% to 118% in different test
formulation groups with DMEM, while for ascorbic acid it
was found more than 89% (Figure 1). These data suggest that
the test formulation was found safe at all the tested
concentrations range up to maximum of 40 μg/mL against
the tested cell lines.

Paper-14-4-1

Figure 1. Effect of the test formulation on HaCaT, HFF-1, and B16-F10 cell
lines for cell viability after 72 hours using the MTT assays. EGF-10:
Epidermal growth factor (10 μM).

3.2. Effect of the Biofield Energy Treated Test Formulation
on Human Foreskin Fibroblast Cell Proliferation
(BrdU Method)

The results of human fibroblast cell proliferation using
BrdU method at different combinations of the Biofield
Energy Treated test formulation with DMEM on percentage
cellular proliferation of HFF-1 cells after 48 hours of
incubation is represented in Figure 2. In the presence of FBS,
the percentage cell proliferation was significantly increased
to 250%. The study results showed FBS (0.5%) group have
significantly improved cell proliferation by 150% and 133%,
compared with the normal and vehicle control groups,
respectively. Besides, the Biofield Energy Treated group,
UT-DMEM + BT-Test formulation showed an increased
cellular proliferation at the concentration of 17.5 μg/mL by
2.82% compared with the UT-DMEM + UT-Test formulation
group. However, the test formulation also showed a
significantly improved cell proliferation at all the tested
concentrations viz. 2.13, 8.75, and 17.5 μg/mL compared
with the normal and vehicle control group. This suggests that
the Biofield Energy Treatment results in increased cellular
proliferation in HFF-1 cells at 17.15 μg/mL using BrdU
assay.

Paper-14-5-1

Figure 2. Effect of the Biofield Energy Treated test formulation with DMEM on cellular proliferation in HFF-1 cells after 48 hours. VC: Vehicle control; FBS:
Fetal bovine serum; UT: Untreated; BT: Biofield Treated.

3.3. Analysis of Extracellular Matrix Component Synthesis

The extra cellular matrix components (ECM) were
assessed in order to identify the skin strength, hydration level
and overall elasticity using the combination of the Biofield
Energy Treated and untreated test formulation/DMEM in
HFF-1 cell line. The analysis outcomes of ECM are
presented as collagen, elastin, and hyaluronic acid levels.

3.3.1. Collagen Analysis

The effect of the Biofield Energy Treated test formulation
on the collagen level showed a significant increase in the
collagen content at various experimental test formulation
concentrations on HFF-1 cell line. Collagen is the most
abundant protein and major skin components for skin health,
structure and most abundant fibrous protein present in ECM.
Experimental results of collagen with respect to ascorbic acid
and Biofield Energy Treated test formulation/DMEM groups
are presented in Figure 3. Ascorbic acid (10 μM) showed a
significant increase collagen content by 55%, while the
Biofield Energy Treated test formulation at selected
concentration range from 0.625 to 2.5 μg/mL reported with
the significant increased percentage of collagen amount.

However, among the test formulation groups, UT-DMEM
+ BT-Test formulation group reported with significant
increase in the collagen level at 2.5, 1.25 and 0.625 μg/mL
by 32.42%, 33.64%, and 29.13%, respectively compared to
the UT-DMEM + UT-Test formulation group. Similarly, in
the BT-DMEM + UT-Test formulation group at
concentration 2.5, 1.25 and 0.625 μg/mL showed an
increased collagen level by 27.14%, 18.20%, and 14.46%,
respectively. However, in BT-DMEM + BT-Test formulation
group, at 2.5, 1.25 and 0.625 μg/mL the collagen level was
increased by 34.17%, 26.73%, and 17.56%, respectively
compared with the UT-DMEM + UT-Test formulation group.
Experimental data showed that after the Biofield Energy
Healing Treatment, a significant increase in the collagen
level was observed at all the tested concentrations. It can be
suggested that collagen synthesis was improved after
Biofield Energy Healing based test formulation with respect
to the respective untreated groups. Hence, it can be assumed
that procollagen peptides and cross-linking (aldol reaction)
among various tropocollagen molecules was increased after
Biofield Treatment, which results in improvement of the
collagen fibrils. Fibrils are required to provide strength and
structure to the skin [35, 36]. The study data suggest that the
Biofield Energy (The Trivedi Effect®) treated test
formulation, showed a remarkable increase in the collagen
level that might be useful for application in skin health,
strength and structure, and wound healing.

Paper-14-6-1

Figure 3. Concentration-dependent effects of test formulation on human dermal fibroblast (HFF-1) cell line for collagen level. ***p≤0.001 and *p≤0.05
statistical comparison with respect to untreated DMEM and untreated test formulation using one way ANOVA (Dunnett’s test). VC: Vehicle control; LA-10: LAscorbic
acid at 10 μM concentration; UT: Untreated; BT: Biofield Treated.

3.3.2. Assessment of Elastin

The effect of the Biofield Energy Healing on the test
formulation was evaluated for change in the elastin level.
Elastin helps to retain shape in body tissue and very elastic
tissue of the body. Elastin is one of the important part of the
ECM, it forms tight junction with collagen fibrils, which
helps to maintain the cellular integrity [37]. The current data
(Figure 4.) exhibited a significant enhancement in the elastin
synthesis in the Biofield Energy Healing based test
formulation in HFF-1 cell line. Ascorbic acid (50 μM) group
showed a significant increased elastin content by 55%
compared with the normal control group. However, among
other tested groups, UT-DMEM + BT-Test formulation
group displayed highly significant increased elastin level by
408.6% at concentrations 0.625 μg/mL compared with UTDMEM
+ UT-Test formulation group. Other experimental
groups were reported with altered level of elastin level after
treatment with the Biofield Energy Treated Test formulation
compared with the untreated groups. This suggests the
importance of the Biofield Energy Healing based Test
formulation as an anti-ageing formulation, as fibroblast and
elastin are responsible for ageing and health. Data suggests a
significant increased elastin level, which represented
enhanced elasticity and strength of the skin with activated
dermal metabolism. Therefore, the Biofield Energy Healing
based Test formulation and DMEM can be significantly used
as an alternative approach in order to improve the elastin
level that helps to improve cell growth, survival,
differentiation and morphogenesis.

Paper-14-6-2

Figure 4. Concentration-dependent effect of the Biofield Energy Treated test formulation on human dermal fibroblast (HFF-1) cell line for elastin level.
***p≤0.001 statistical comparison with respect to untreated DMEM and untreated test formulation using one way ANOVA (Dunnett’s test). VC: Vehicle control;
LA-50: L-Ascorbic acid at 50 μM concentration UT: Untreated; BT: Biofield Treated.

3.3.3. Analysis of Hyaluronic Acid

Hyaluronic acid (HA) is a natural glycosaminoglycan
(polysaccharides) and regarded as one of the major
connective tissue. Besides, HA is distributed extensively all
over the connective, neural, and epithelial tissue and it can be
derived from multiple resources, foods, supplements, HA
powders, etc. It helps to retain skin moisture, secures
moisture, creates fullness, and regulates the skin water
balance. Skin care cosmetics found many products such as
hyaluronic acid creams, serums, injectable, and hyaluronic
acid supplements. Lower level of HA results in loss of skin
elasticity and expose the signs of aging, while it plays a
critical role in skin health due to its high and unique ability to
hold the moisture. These all parameters defined HA as “smart
nutrient” due to its ability to adjust moisture with high
absorption rate. The level of hyaluronic acid after the
Biofield Energy Treatment in test formulation was evaluated
in the HFF-1 cell line (Figure 5). The results of ascorbic acid
showed a significant increase in the hyaluronic acid content
by 183.6%. However, untreated test formulation/DMEM
group showed a significant change in HA levels in all the
groups with respect to normal control group. After Biofield
Energy Treatment, UT-DMEM + BT-Test formulation group,
at 0.625 μg/mL showed an increase in HA level by 4.76%. In
addition, BT-DMEM + UT-Test formulation group presented
significant increased HA level by 29.71% at 0.625 μg/mL
with respect to the UT-DMEM + UT-Test formulation group.
Further, in BT-DMEM + BT-Test formulation group at 2.5,
1.25, and 0.625 μg/mL HA levels were significantly
increased by 31.88%, 15.52%, and 58.29%, respectively
compared with the UT-DMEM + UT-Test formulation group.
Overall, results showed a significantly improved HA level
using the Biofield Energy Healing based test formulation
would be a new approach in cosmetology. HA based skin car
product are widely available in the market for skin health
[38]. However, the Biofield Energy Healing (The Trivedi
Effect®) based test formulation and DMEM could be used as
better alternative to improve the content of hyaluronic acid
along with various skin disorders.

Paper-14-7

Figure 5. Synthesis of extracellular matrix component, hyaluronic acid by the Biofield Energy Treated Test formulation in human dermal fibroblasts (HFF-1)
cell lines. ***p≤0.001 statistical comparison with respect to untreated DMEM and untreated test formulation using one way ANOVA (Dunnett’s test). VC:
Vehicle control; LA-50: L-Ascorbic acid at 50 μM concentration; UT: Untreated; BT: Biofield Treated.

3.4. Estimation of Melanin Synthesis Inhibition

Melanin is a brown pigment that results in different shades
and colors of human skin. However, amount of melanin in
the skin defines the skin pigmentation. Depigmentation
results in various disorders, while skin coloration also
depends upon the genetic makeup. Moreover, sun ultraviolet
radiations (UV-A and UV-B) initiated the process of
melanogenesis in the melanocytes results in skin darkening
[39]. The Biofield Energy based test formulation were
cultured in DMEM supplemented media for melanogenesis
on mouse melanoma (B16-F10) cells containing several
concentrations along with positive control, kojic acid (10
μM) for 48 to 96 hours. The results of percentage decrease in
α-MSH induced melanin synthesis in all the experimental
groups are presented in Figure 6. Kojic acid, a skin whitening
compound was used as positive control showed a significant
decrease in the level of melanin synthesis by 188.2%
compared with stimulated in presence of alpha melanocyte
stimulating hormone group. In addition, the Biofield Energy
based test formulation exhibited a significant decrease in the
melanin synthesis by 8.24% and 16.09% in the UT-DMEM +
BT-Test formulation group at 0.0625 and 0.125 μg/mL
respectively, compared with the UT-DMEM + UT-Test
formulation group. Moreover, at 0.125 μg/mL, BT-DMEM +
UT-Test formulation group showed a significant decreased
melanin synthesis by 18.93% compared with the UT-DMEM
+ UT-Test formulation group. However, at 0.0625 μg/mL in
the BT-DMEM + BT-Test formulation group, melanin
synthesis was decreased by 3.58% compared with the UTDMEM
+ UT-Test formulation group in B16-F10 melanoma
cell line. The Biofield Energy Treated Test formulation
significantly decreased the level of melanin synthesis. The
results suggest that the Biofield Energy Treated test
formulation might alter and inhibit the tyrosinase enzymes
activity, which is mainly responsible for melanin synthesis [40]. Skin whitening action of most of the cosmetic
formulation are based on melanin inhibition, however the
chemicals have several side effects. However, the test
formulation is the combination of minerals, Centella asiatica
extract and THC, which were reported to have significant
effect against skin infection, inflammatory dermatoses, with
strong antioxidant action [41, 42]. Therefore, it can be
concluded that the Biofield Energy Healing based test
formulation and DMEM would be useful approach to
decrease the melanogenesis process and would be a novel
approach for skin-related disorders with significant skin
whitening action.

Paper-14-8-1

Figure 6. Inhibitory effect of the Biofield Energy Treated test formulation on melanogenesis (skin whitening potential) in mouse melanoma (B16-F10) cell line.
***p≤0.001 statistical comparison with respect to untreated DMEM and untreated test formulation using one way ANOVA (Dunnett’s test). α-MSH: Alpha
melanocyte stimulating hormone, UT: Untreated; BT: Biofield Treated.

3.5. Anti-wrinkling Effects of Test Formulation on UV-B
Induced Photoaging

HFF-1 cells are used to study the impact of the Biofield
Energy Healing based test formulation in cell viability
potential and stress was induced due to UV-B. Various skin
related diseases are reported due to exposure of UV-B
induced such as skin disorders, stress, free radical generation,
etc. This results in downregulation of the human skin
fibroblasts through various inflammatory responses, DNA
damage, wrinkles and skin-ageing [43]. Anti-wrinkling
effects of cell viability from UV-B rays is presented in Figure
7. The HFF-1 cells were subjected to the lethal dose of UV-B
irradiation (200 mJ/cm2) and percentage cell viability due to
UV-B was identified. The HFF-1 cells while exposure of
UV-B reported with high degree of cell death with
approximately 25.21% of cell viability. The cell viability in
vehicle control (DMSO) group was found as 20.51% due to
UV-B irradiation (200 mJ/cm2) (Figure 7). However,
ascorbic acid (50 μM) showed a significant increase in the
cell viability by 110.48%. In addition, the experimental
groups showed that all the tested groups in specified
concentrations reported with improve cell viability. Among
the tested groups, at 2.5 μg/mL in the UT-DMEM + BT-Test
formulation and BT-DMEM + UT-Test formulation groups
showed an increase cell viability by 5.37% and 3.15%,
respectively compared with the UT-DMEM + UT-Test
formulation group. Similarly, in BT-DMEM + BT-Test
formulation group at 1.25 and 0.625 μg/mL showed an
increase cell viability by 5.49% and 11.26%, respectively
compared with the UT-DMEM + UT-Test formulation group.
UV-B results in loss in cell viability and cause skin damage,
however results suggest improved cell viability by Biofield
Energy Healing based test formulation. Therefore, it can be
predictable that the Biofield Energy Treated test formulation
and DMEM would be a better alternative treatment for skin
protection and cell viability against UV-B radiations and can
be effectively used as anti-wrinkling action.

Paper-14-8-2

Figure 7. Anti-wrinkling potential and cytoprotective potential of the Biofield Energy Treated test formulation against UV-B induced stress in human dermal
fibroblasts (HFF-1) cell lines. % cell viability of HFF-1 cells after treatment in various groups. NC: Normal control; VC: Vehicle control; LA-50: L-Ascorbic
acid at 50 μM concentration, UT: Untreated; BT: Biofield Treated.

3.6. Wound-Healing Scratch Assay

Wound healing scratch assay was performed in HFF-1 and
HaCaT cells with different combinations of the Biofield
Energy Treated test formulation/DMEM. The representative
HFF-1 and HaCaT cells migration photographs in different
groups were monitored and shown in Figure 8. The effect of
the Biofield Energy Treated test formulation/DMEM
determined the cellular migration, and defines the cell-to-cell
and cell-to-matrix interactions during wound healing process
[45]. Higher rate of cell migration was reported at 16 hours in
the presence of positive control, EGF group compared with
vehicle control group (Figure 8). However, in UT-DMEM +
BT-Test formulation group at 10 μg/mL showed a significant
cellular migration rate in HFF-1 cells, while in HaCaT cells,
BT-DMEM + BT-Test formulation group showed an
effective healing and migration rate compared with other
tested groups. Overall, the scratched monolayer in both cells
lines showed a significant migration after treatment with the
Biofield Energy Treated test formulation/DMEM compared
with the untreated test formulation/DMEM group.
Experimental data suggest that the percent cell covered area
in the Biofield Energy Treated test formulation group was
significantly higher with improved healing rate compared
with the Biofield Energy Treated DMEM. HaCaT and HFF-1
cells showed upto 5% increase in wound coverage area in the
BT-DMEM + BT-Test formulation group, with respect to the
UT-DMEM + UT-Test formulation group. The percentage
cell coverage area due to the Biofield Treated test
formulation/DMEM in HFF-1 cells with respect to the
concentration of untreated test formulation in untreated
medium ranged from 2% to 10% in UT-DMEM + BT-Test
formulation group. Similarly, wound healing rates in HaCaT
cells were found with 1.5% to 8% in BT-DMEM + BT-Test
formulation group compared with the untreated group. The
results after treatment of wound healing scratch assay in EGF
group as shown in Figure 8 (b), normal control group in Figure 8
(a). Similarly, in the UT-DMEM + BT-Test formulation and BTDMEM
+ UT-Test formulation experimental group showed a
significant rate of cellular migration rate along with wound
closure as shown in Figure 8 (d, e, f), compared with the UTTest
formulation + UT-DMEM group (Figure 8 c).

Paper-14-9

Figure 8. Representative pictures of HFF-1 and HaCaT cell migration cells after induction of a scratch. All the pictures were taken immediately after the
scratch was induced (i.e. at 0 hours), after 16 hours in the presence of EGF and Biofield Energy Treated test formulation. Pictures are taken at 50 times
magnification. Images represents HFF-1 cells migration in presence of (a) baseline control media, (b) EGF, (c) UT-DMEM + UT-Test formulation, (d) UTDMEM
+ BT-Test formulation, (e) BT-DMEM + UT-Test formulation, and (f) BT-DMEM + BT-Test formulation.

Therefore, it can be concluded that the Biofield Energy
Healing (The Trivedi Effect®) based test formulation has
the capacity to significantly improved cellular migration,
which can be used to improve wound healing activity.
However, enhanced collagen deposition due to The
Trivedi Effect®- Consciousness Energy Healing Treatment
results in improved strength, integrity and structure of the
skin. It might be suggested Biofield Energy Treatment
showed an improved wound healing with some effective
antioxidant mechanism.

4. Conclusions

Herbal based products have the potential to improve
various skin diseases. Currently, the world population
depends on alternative health care system for curing skin
related problems, although there is a movement towards
seeking alternative, natural approaches for treatment. The
Trivedi Effect®-Consciousness Energy Healing Treatment
based novel test formulation and DMEM showed a
significant outcomes, such as MTT assay displayed
significant cell viability in HFF-1, HaCaT and B16-F10
cells lines with more than 89% viable cells in the Biofield
Energy Healing based herbomineral test formulation.
Besides, BrdU assay exhibited significant high rate of
cellular proliferation in the UT-DMEM + BT-Test
formulation group. Collagen synthesis was improved by
32.42%, 27.14% and 34.17% in the UT-DMEM + BT-Test
formulation, BT-DMEM + UT-Test formulation, and BTDMEM
+ BT-Test formulation groups, respectively at 2.5
μg/mL compared with the UT-DMEM + UT-Test
formulation group. The elastin level was found to be
increased by 408.6% at concentrations 0.625 μg/mL in the
UT-DMEM + BT-Test formulation group compared with
the UT-DMEM + UT-Test formulation groups. However,
the amount of hyaluronic acid was increased by 29.71% and 58.29% in the BT-DMEM + UT-Test formulation and
BT-DMEM + BT-Test formulation groups, respectively at
concentration 0.625 μg/mL compared with the UT-DMEM
+ UT-Test formulation group. Melanin content was
inhibited by 16.09% and 18.93% in the UT-DMEM + BTTest
formulation and BT-DMEM + UT-Test formulation
group, respectively at 0.125 μg/mL compared with the
UT-DMEM + UT-Test formulation group in B16-F10
melanoma cell line. However, the Biofield Treated Test
formulation showed a significantly improved antiwrinkling
effects using UV-B induced stress in HFF-1
cells by 5.49% and 11.26% in the BT-DMEM + BT-Test
formulation group at 1.25 and 0.625 μg/mL, respectively
compared with the UT-DMEM + UT-Test formulation
group. Wound healing scratch assay results validated
significant migration of fibroblast and keratinocytes cells
with increase covered area up to 10% in the HFF-1 and
HaCaT cells after treatment with the Biofield Energy
based test formulation and DMEM. Overall, the study
results concluded that the Biofield Energy Healing based
test formulation and DMEM could be used to improve
various skin disorders and ageing.
Overall, the Biofield Energy Treated test formulation
can be used as a Complementary and Alternative Medicine
(CAM) with a safe therapeutic index for various skin
irregularities that are typically symptoms of a skin
disorders such as Eczema, diaper rash, chickenpox,
measles, warts, acne, hives, wrinkles, ringworm, Rosacea,
psoriasis, seborrheic dermatitis, skin cancer, rashes from
bacterial or fungal infections, rashes from allergic
reactions, raised bumps that are red or white, cracked skin,
discolored patches of skin, fleshy bumps, warts, or other
skin growths, changes in mole color or size, a loss of skin
pigment, scaly or rough skin, peeling skin, ulcers, open
sores or lesions, dry, excessive flushing. Further, the
Biofield Energy Healing based herbomineral test
formulation can also be used in the prevention of
temporary and permanent skin disorders, anti-aging,
improved overall health, and quality of life.

Abbreviations

THC: Tetrahydrocurcumin; ECM: Extracellular matrix;
EGF: Epidermal growth factor; DMEM: Dulbecco’s
Modified Eagle’s Medium; α-MSH: α-Melanocytestimulating
hormone; ANOVA: Analysis of variance; HA:
Hyaluronic acid; HFF-1: Human foreskin fibroblast cell
line, UV-B: Ultra violet B rays; CAM: Complementary
and alternative medicine; NCCAM: National Center for
Complementary and Alternative Medicine.

Acknowledgement

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

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