LC-MS, GC-MS, and NMR Spectroscopy Based Evaluation of the Energy of Consciousness Healing Treated Withania somnifera (Ashwagandha) Root Extract

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Journal of Drug Design and Medicinal Chemistry
2017; 3(2): 18-26
http://www.sciencepublishinggroup.com/j/jddmc
doi: 10.11648/j.jddmc.20170302.11
ISSN: 2472-355X (Print); ISSN: 2472-3576 (Online)
LC-MS, GC-MS, and NMR Spectroscopy Based Evaluation
of the Energy of Consciousness Healing Treated Withania
somnifera (Ashwagandha) Root Extract
Mahendra Kumar Trivedi
1
, Alice Branton
1
, Dahryn Trivedi
1
, Gopal Nayak
1
,
Ariadne Esmene Afaganis
1
, Barbara Marie Bader
1
, Brian A. Weekes
1
, Daphne Luisa Dumas
1
,
Denise Marie Fiedler
1
, Dennille Mellesia Smith
1
, Desi Pano
1
, Donna Felice Galla
1
,
Donna Maria Alija
1
, Elaine Barbara Mullins
1
, Elaine M. Scorza
1
, Ellia O'Donnell
1
,
Fabio Massimo Paciucci
1
, Frances Goodman Warlick
1
, Haddon Norman Salt
1
, Inthirani Arul
1
,
Jacqueline Y. Andrews
1
, James Jay McLeran
1
, James Stephen Burnett
1
, Jean Caroline White
1
,
Parthasarathi Panda
2
, Kalyan Kumar Sethi
2
, Snehasis Jana
2, *
1
Trivedi Global, Inc., Henderson, Nevada, USA
2
Trivedi Science Research Laboratory Pvt. Ltd.,
Bhopal, Madhya Pradesh, India
Email address:
publication@trivedieffect.com (S. Jana)
*
Corresponding author
To cite this article:
Mahendra Kumar Trivedi, Alice Branton, Dahryn Trivedi, Gopal Nayak, Ariadne Esmene Afaganis, Barbara Marie Bader, Brian A. Weekes,
Daphne Luisa Dumas, Denise Marie Fiedler, Dennille Mellesia Smith, Desi Pano, Donna Felice Galla, Donna Maria Alija, Elaine Barbara
Mullins, Elaine M. Scorza, Ellia O'Donnell, Fabio Massimo Paciucci, Frances Goodman Warlick, Haddon Norman Salt, Inthirani Arul,
Jacqueline Y. Andrews, James Jay McLeran, James Stephen Burnett, Jean Caroline White, Parthasarathi Panda, Kalyan Kumar Sethi,
Snehasis Jana. LC-MS, GC-MS, and NMR Spectroscopy Based Evaluation of the Energy of Consciousness Healing Treated Withania
somnifera (Ashwagandha) Root Extract. Journal of Drug Design and Medicinal Chemistry. Vol. 3, No. 2, 2017, pp. 18-26.
doi: 10.11648/j.jddmc.20170302.11
Received: March 14, 2017; Accepted: March 27, 2017; Published: April 18, 2017
Abstract: Withania somnifera root extract which contains withanolides as major active constituent and it is used
traditionally for the prevention and treatment of several diseases. The objective of the current study was to investigate the
impact of The Trivedi Effect
®
- Consciousness Energy Healing Treatment on the structural properties of the ashwagandha root
extract using LC-MS, GC-MS, and NMR spectroscopy. Ashwagandha root extract was divided into two parts one part was
control (without treatment), while other part was treated with the Consciousness Energy Healing Treatment remotely by twenty
renowned Biofield Energy Healers and defined as the Biofield Energy Treated sample. The LC-MS analysis revealed that the
retention time of the phytoconstituents remained same in the control and Biofield Energy Treated samples, whereas the peak
area% i.e. the relative amount of the phytoconstituents at respective retention time was significantly altered. The peak area% at
R
t
of 5.6, 6.8, 6.9, 7.2, 7.9, 8.4, 8.5, 8.6, and 9.2 minutes of the treated sample were increased significantly in the range of
1.46% to 253.06% compared to the control sample. In the contrary, the peak area% of the treated sample at R
t
of 6.4 and 8.2
minutes were significantly decreased by 12.72% and 17.35%, respectively with respect to the control sample. A total of 16
withanolides such as withanoside IV, coagulin Q, viscosa lactone B, dihydrowithanolide D, withanolide A, withaferin A,
withanone, withanolide D, ixocarpalactone A, withanolide sulfoxide, withanolide B, etc. were proposed with their structure
from the molecular mass at m/z 783, 621, 489, 473, 767, 471, 505, and 992 at retention times of 6.4, 6.8, 7.2, 7.9, 8.2, 8.4, and
9.1 minutes, respectively with the help of LC-MS, GC-MS and NMR data of both the control and Biofield Energy Treated
samples. The mass peak intensities of the Biofield Energy Treated sample were significantly changed in the range of -81.36%
to 1720.90% compared with the control sample at the same retention time. These findings suggest that The Trivedi Effect
®
-
Consciousness Energy Healing Treatment could be beneficial for altering the concentration of the phytoconstituents in the
ashwagandha root extract by modifying their intrinsic physicochemical properties, which might be helpful to improve the
bioavailability of active constituents of W. somnifera extract that might provide better therapeutic response against
19 Mahendra Kumar Trivedi et al.: LC-MS, GC-MS, and NMR Spectroscopy Based Evaluation of the Energy of
Consciousness Healing Treated Withania somnifera (Ashwagandha) Root Extract
inflammatory diseases, immunological disorders, stress, arthritis, cancer, diabetes, sexual disorders, aging, and other chronic
infections.
Keywords: Ashwagandha, Biofield Energy Healers, The Trivedi Effect
®
, Consciousness Energy Healing Treatment,
Biofield Energy Healing Treatment, LC-MS, Withanolides, GC-MS
1. Introduction
Herbal medicines have been getting reputation worldwide
for the prevention and treatment of the various diseases
because of their impressive therapeutic effects and fewer side
effects [1]. The roots of Withania somnifera is an ancient
herb and is popularly known as ‘Ashwagandha’ or winter
cherry or ‘Indian ginseng’ [2, 3]. W. somnifera is mostly used
in the herbal drugs and nutraceuticals for the prevention and
treatment of various diseases include sexual and nervous
disorders, diabetes, infectious diseases, cancer, ulcer,
immunological disorders, stress, arthritis, etc. As a tonic, it is
useful to arrest the aging process, rejuvenate the body and
boost the defense system against infectious disorders as well
as to promote the longevity [2-6]. The major active
phytoconstituents of W. somnifera root extract are highly
oxygenated withanolides. Besides withanolides,
ashwagandha root contains alkaloids, numerous
sitoindosides, withanamides, reducing sugars, starch,
peroxidases, glycosides, dilcitol, withanicil, benzyl alcohol,
2-phenyl ethanol, 3,4,5-trihydroxy cinnamic acid, benzoic
acid, phenyl acetic acid, etc. [7-9]. Isolated withanolides
from W. somnifera possess various pharmacological activities
include antioxidant, anticancer, neuroprotective,
immunomodulating, hepatoprotective, anti-inflammatory,
antiarthritic, hypoglycaemic, antimicrobial, etc. [10-12].
Therefore, ashwagandha root extract was considered as one
of the components in a novel proprietary herbomineral
formulation, and can be used for the prevention and treatment
of various human disorders.
A unique vital force preserved by every living organisms
which is usually believed to create the source of life is co-
related with the soul, spirit and mind and is also recognized
as prana by the Hindus, qi or chi by the Chinese, and ki by
the Japanese from the ancient-time. Now-a-days, this
hypothetical vital force is considered as the Bioenergetics
Field. This energy field is infinite, paradimensional and
dynamic electromagnetic field surrounding the human body.
This is also known as The Biofield Energy. It can easily flow
between the human and environment that leads to the
continuous movement or matter of energy [13, 14]. Thus, the
human has the capability to harness energy from the earth,
the “Universal Energy Field” and transmit it to any living or
nonliving object(s) around the globe. The objects always
receive the energy and respond in a useful way. This process
is known as Biofield Energy Healing Treatment [15-17].
Biofield (Putative Energy Fields) based Energy Therapies
have been practiced worldwide in different health disease
profiles [18]. The National Center of Complementary and
Integrative Health (NCCIH) has been recognized and
accepted Biofield Energy Healing as a Complementary and
Alternative Medicine (CAM) health care approach in
addition to other therapies, medicines and practices 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 (as is common in all
religions, like Christianity, Hinduism, Buddhism and
Judaism) [19]. The Biofield Energy Treatment (The Trivedi
Effect
®
) has been extensively studied with significant
outcomes in many scientific fields such as cancer research
[20]; altered antimicrobial sensitivity of pathogenic microbes
in biotechnology [21, 22], genetics [23, 24], microbiology
[25-27], changing the structure of the atom in relation to the
various metals, ceramics, polymers and chemicals materials
science [28-30], altered physical and chemical properties of
organic compounds [31-33], nutraceuticals [34, 35],
pharmaceuticals [36, 37], and improved overall growth and
yield of plants in agricultural science [38, 39].
Modern sophisticated techniques such as high-
performance liquid chromatography (HPLC) with photodiode
array and evaporative light scattering detection, ultra-
performance liquid chromatography (UPLC) electrospray
ionization (ESI) normally hyphenated with mass
spectrometry, gas chromatography (GC), nuclear magnetic
resonance (NMR) are very useful for the metabolite profiling
and identification of the crude herbal extract [8, 40-42]. The
LC-MS/MS, GC-MS and NMR analysis of W. somnifera
hydroalcoholic root extract revealed the presence of several
known withanolides including withaferin A, withanolide D,
withanoside IV or VI, withanolide sulfoxide, etc. along with
two new withanolides i.e. dihydrowithanolide D and
ixocarpalactone A [43]. For this reason, LC-MS/MS, GC-
MS, and NMR analysis were conducted in this study for the
profiling and structure elucidation of the phytoconstituents of
the Biofield Energy Treated (The Trivedi Effect
®
) W.
somnifera hydroalcoholic root extract.
2. Materials and Methods
2.1. Chemicals and Reagents
Withania somnifera (Ashwagandha) root hydroalcoholic
extract was procured from Sanat Product Ltd., India. The
HPLC grade acetonitrile and Milli Q water were purchased
Journal of Drug Design and Medicinal Chemistry 2017; 3(2): 18-26 20
from Merck and Millipore. All other chemicals used in the
experiment were of analytical grade available in India.
2.2. Energy of Consciousness Healing Treatment Strategies
Ashwagandha root extract powder was one of the
components of the new proprietary herbomineral
formulation, developed by our research team and it was used
per se as the test compound for the current study. The test
compound was divided into two parts, one part of the test
compound was treated with The Trivedi Effect
®
-
Consciousness Energy Healing Treatment (Biofield Energy
Treatment) by renowned Biofield Energy Healers and
defined as Biofield Energy Treated sample. The second part
of the test compound did not receive any sort of treatment
and defined as untreated or control ashwagandha root extract
sample. This Biofield Energy Treatment was provided by the
group of twenty renowned Biofield Energy Healers who
participated in this study and performed the Biofield Energy
Treatment remotely. Eighteen Biofield Energy Healers were
remotely located in the U.S.A. and two of which were
remotely located in Canada, while the test compound was
located in the research laboratory of GVK Biosciences Pvt.
Ltd., Hyderabad, India. This Biofield Energy Treatment was
provided for 5 minutes through Healers Unique Energy
Transmission process remotely to the test compound under
the laboratory conditions. None of the Biofield Energy
Healers in this study visited the laboratory in person, nor had
any contact with the compounds. Similarly, the control
compound was subjected to “sham” healers for 5 minutes,
under the same laboratory conditions. The sham healer did
not have any knowledge about the Biofield Energy Healing
Treatment. After that, the Biofield Energy Treated and
untreated samples were kept in similar sealed conditions and
characterized thoroughly by LC-MS, GC-MS and NMR
spectroscopy.
2.3. Characterization
2.3.1. Liquid Chromatography Mass Spectrometry (LC-MS)
The LC-MS analysis of the test samples were conducted
by following the almost same method as mentioned in the
recent literature [43] using The Waters
®
ACQUITY UPLC,
Milford, MA, USA equipped with a binary pump (The
Waters
®
BSM HPLC pump), autosampler, column heater and
a photo-diode array (PDA) detector. A Triple Quad (Waters
Quattro Premier XE, USA) mass spectrometer equipped with
an electrospray ionization (ESI) source was used for the mass
spectrometric analysis. The control and Biofield Energy
Treated extract powders were dissolved in dimethylsulfoxide
to afford a 1 mg/mL stock solution. An aliquot of 2 µL of the
stock solution was used for LC-MS analysis with a total run
time of 25 min. Mass spectra were recorded in the positive
ionization mode and with the full scan (m/z 50-1400).
Percent change in peak area (%), P was calculated using
following equation (1):
%changeinpeakarea% =




× 100 (1)
Where, P
Control
and P
Treated
are the peak area (%) of the
control and Biofield Energy Treated samples, respectively.
2.3.2. Gas Chromatography-Mass Spectrometry (GC-MS)
Analysis
GC-MS analysis of the test samples were analyzed by
following the same procedure as mentioned in the recent
scientific literature [43] with the help of Agilent 7890B with
5977A Mass selective detector, USA equipped with a
Quadrupole detector with pre-filter and flame ionization
detector (FID). The control and Biofield Energy Treated
extract powders were dissolved in dimethylsulfoxide to
afford a 1 mg/mL stock solution. An aliquot of 1.0 µL of the
stock solution was injected with a total run time of 44.0 min.
The identification of analyte was performed using the
retention time with a comparison of the mass spectra of the
identified substances with references.
2.3.3. Nuclear Magnetic Resonance (NMR) Analysis
1
H NMR and
13
C NMR analysis of the test samples extract
powders were performed on
a 400 MHZ VARIAN FT-NMR
spectrometer and 100.00 MHz on a VARIAN FT-NMR
spectrometer, respectively using the same procedure as
mentioned in the recent literature [43].
1
H NMR
multiplicities were labelled as singlet (s), doublet (d), doublet
of doublet (dd), triplet (t), quartet (q), multiplet (m), broad
(br), apparent (app). Chemical shifts (δ) were in parts per
million (ppm) relative to the solvents residual proton
chemical shift (CD
3
OD, δ = 3.31, 4.80 ppm) and solvent’s
residual carbon chemical shift (CD
3
OD, δ = 49.15 ppm).
3. Results and Discussion
The LC chromatograms and their chromatographic data of
the control and Biofield Energy Treated samples of W.
somnifera root extract are presented in the Figure 1 and Table
1, respectively. The liquid chromatograms of the control and
Biofield Energy Treated samples (Figure 1) showed several
peaks at different retention times. Among the obtained
chromatographic peaks, only 11 peaks showing almost same
retention time and higher peak area in the control and
Biofield Energy Treated sample (Table 1). A change in the
retention time along with the disappearance and appearance
of some peaks was observed in the Biofield Energy Treated
sample compared the control sample. Thus, the polarity of
the phytoconstituents in the Biofield Energy Treated
ashwagandha root extract was altered compared with the
control sample.
There was a significant increase in the peak area% of the
Biofield Energy Treated sample in the range of 1.46% to
253.06% at R
t
of 5.6, 6.8, 6.9, 7.2, 7.9, 8.4, 8.5, 8.6, and 9.2
minutes compared with the control sample (Table 1).
Consequently, the peak area% of the Biofield Energy Treated
sample at R
t
of 6.4 and 8.2 minutes were significantly
decreased by 12.72% and 17.35%, respectively with respect
to the control sample (Table 1). The peak area% provides the
relative amounts of components in the chromatogram, when
all components respond in the detector and are eluted [43,
21 Mahendra Kumar Trivedi et al.: LC-MS, GC-MS, and NMR Spectroscopy Based Evaluation of the Energy of
Consciousness Healing Treated Withania somnifera (Ashwagandha) Root Extract
44]. The liquid chromatographic conditions for the control
and Biofield Energy Treated samples were same. It is
assumed that all the components in both the samples were
equally responded in the detector. Thus, the obtained peak
area% are revealed the relative amounts of the
phytoconstituents present in the W. somnifera root extract.
The Table 1 revealed that Biofield Energy Healing Treatment
might have the significant effect on the relative
amount/concentration of the phytoconstituents. It is assumed
that the intrinsic physicochemical properties of ashwagandha
root extract such as morphology, particle size, shape, etc. of
the compounds that are related to the solubility of the
compounds might have altered due to the Biofield Energy
Healing Treatments [28-35].
Figure 1. Liquid chromatograms of the control and Biofield Energy Treated W. somnifera (Ashwagandha) root extract.
Table 1. Liquid chromatographic data of the control and Biofield Energy
Treated W. somnifera (Ashwagandha) root extract.
Sl.
No.
Retention time
(R
t
, min)
Peak area%
Control
sample
Biofield
Treated sample
% Change
*
1 5.6 1.36 1.69 24.26
2 6.4 2.83 2.47 -12.72
3 6.8 1.66 2.33 40.36
4 6.9 0.52 1.42 173.08
5 7.2 18.49 18.76 1.46
6 7.9 3.85 6.00 55.84
7 8.2 31.53 26.06 -17.35
8 8.4 9.31 10.38 11.49
9 8.5 2.45 8.65 253.06
10 8.6 1.18 1.58 33.90
11 9.2 4.98 7.00 40.56
*
denotes the percentage change in the peak area (%) of the Biofield Energy
Treated sample with respect to the control sample.
Among these 11 peaks, only 7 peaks at the R
t
of 6.4, 6.8,
7.2, 7.9, 8.2, 8.4, and 9.2 min having higher peak area% than
other R
t
responded to the mass spectrometric analysis and
afforded the respective ESI-MS spectra (Table 1). From the
ESI-MS spectra, a total of 16 withanolides shown in the
Figure 2 were proposed along with the help GC-MS and
NMR data (Figure 3 and 4, respectively). Compounds
proposed (Figure 2) from the mass of the molecular ion and
its fragmentation pattern at corresponding retention time
(Table 2) along with the GC-MS (Figure 3) and NMR data
(Figure 4) of the ashwagandha root extract according to the
approach described in our recent literature [43].
The withanoside IV (1) or withanoside VI (2) (Figure 2)
were proposed from the protonated molecular ion peak at m/z
783 [M + H]
+
(calcd for C
40
H
63
O
15
, 783) along with
ammonium adduct ion mass m/z 800 [M + NH
4
]
+
and
m/z 621
[M + H - β-glucose]
+
in the mass spectra of the control and
Biofield Energy Treated sample at R
t
of 6.4 minutes.
Similarly, at R
t
of 6.8 minutes coagulin Q (3) or physagulin
D (4), which exhibited the molecular ion peak at m/z 621 [M
+ H]
+
(calcd for C
34
H
53
O
10
, 621) in the mass spectra of the
control and Biofield Energy Treated sample (Figure 2) were
proposed.
Table 2. Compounds proposed from ESI-MS spectra of the control and Biofield Energy Treated ashwagandha root extract.
R
t
(min) Identified compounds ESI-MS (m/z)
Peak Intensity
Control Biofield Energy Treated
% Change
*
6.4 Withanoside IV (1) 783 [M + H]
+
9.93e5 5.77e5
-41.89
Withanoside VI (2) 800 [M + NH
4
]
+
8.48e6 6.78e6 -20.05
621 [M + H - β-glucose]
+
1.22e6 6.98e5 -42.79
6.8 Coagulin Q (3), 621 [M + H]
+
1.40e6 8.76e5 -37.43
Physagulin D (4) 471 2.88e6 3.38e6 17.36
650 3.61e6 3.35e6 -7.20
314 2.68e5 4.88e6 1720.90
7.2 Viscosa lactone B (5) 489 [M + H]
+
2.40e7 2.09e7 -12.92
Journal of Drug Design and Medicinal Chemistry 2017; 3(2): 18-26 22
R
t
(min) Identified compounds ESI-MS (m/z)
Peak Intensity
Control Biofield Energy Treated
% Change
*
27-hydroxy withanolide A (6) 506 [M + NH
4
]
+
4.94e6 3.65e6 -26.11
(20S,22R)-,6α-epoxy-4β,5β,27-
trihydroxy-1-oxowitha-24-enolide (7)
(20S,22R)-4β,5β,6α,27-tetrahydroxy-1-oxo-
with-2,24-dienolide (8)
7.9 Dihydrowithanolide D (9) 473 [M + H]
+
9.38e6 1.79e7 90.83
490 [M + NH
4
]
+
N. F. 2.35e6 N. D.
Withanoside V (10) 767 [M + H]
+
N. F. 1.94e6 N. D
784 [M + NH
4
]
+
6.49e6 1.78e7 174.27
8.2 Withanolide A (11) 471 [M + H]
+
5.56e7 1.92e7 -65.47
Withaferin A (12) 488 [M + NH
4
]
+
1.69e7 3.15e6 -81.36
Withanone (13) 958 [2M + NH
4
]
+
8.36e6 1.99e6 -76.20
Withanolide D (14) -17.28
8.4 Ixocarpalactone A (15) 505 [M + H]
+
3.53e6 2.92e6 14.47
488 [M - H
2
O + 2H]
+
3.04e6 3.48e6 30.26
471 [M - 2 H
2
O + 3H]
+
9.98e5 1.30e6 -12.04
443 5.48e5 4.82e5 -37.38
9.2 Withanolide sulfoxide (16) 992 [M + H]
+
4.04e6 2.53e6 -29.54
976 [M - H
2
O + H]
+
1.08e7 7.61e6 174.27
N. F.- Not found, N. D. – Not determined,
*
denotes the percentage change of the Biofield Energy Treated sample with respect to the control sample.
The ESI-MS, GC-MS (Figure 3) and NMR (Figure 4)
spectral analysis, along with the literature [43] confirmed the
presence of viscosa lactone B (5) or 27-hydroxy withanolide
A (6) or (20S,22R)- 3α, 6α-epoxy-4β, 5β, 27-trihydroxy-1-
oxowitha-24-enolide (7) or (20S,22R)- 4β, 5β, 6α, 27-
tetrahydroxy-1-oxo-with-2,24-dienolide (8) (Figure 2) at m/z
489 [M + H]
+
in the control and Biofield Energy Treated
samples at R
t
of 7.2 minutes. Consequently, at R
t
of 7.9
minutes dihydrowithanolide D (9) or withanoside V (10) was
proposed with the molecular ion peak at m/z 473 [M + H]
+
(calcd for C
28
H
41
O
6
, 473) and 767 [M + H]
+
(calcd for
C
40
H
62
O
14
, 767), respectively in the ESI-MS spectra of both
the samples (Figure 2) [43].
Figure 2. Structure of proposed compounds 1-16.
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