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

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American
Journal
of
Biomedical
and
Life
2017; 5(2): 16-25
http://www.sciencepublishinggroup.com/j/ajbls
doi: 10.11648/j.ajbls.20170502.11
ISSN: 2330-8818 (Print); ISSN: 2330-880X (Online)
Evaluation of the Energy of Consciousness Healing Treated
Withania Somnifera (Ashwagandha) Root Extract Using
LC-MS, GC-MS, and NMR Spectroscopy
Mahendra Kumar Trivedi
1
, Alice Branton
1
, Dahryn Trivedi
1
, Gopal Nayak
1
, Alan Joseph Balmer
1
,
Dimitrius Anagnos
1
, Janice Patricia Kinney
1
, Joni Marie Holling
1
, Joy Angevin Balmer
1
,
Lauree Ann Duprey-Reed
1
, Vaibhav Rajan Parulkar
1
, Parthasarathi Panda
2
, Kalyan Kumar Sethi
2
,
Snehasis Jana
2, *
1
Trivedi Global, Inc., Henderson, USA
2
Trivedi Science Research Laboratory Pvt. Ltd., Bhopal, India
Email address:
publication@trivedieffect.com (S. Jana)
*
Corresponding author
To cite this article:
Mahendra Kumar Trivedi, Alice Branton, Dahryn Trivedi, Gopal Nayak, Alan Joseph Balmer, Dimitrius Anagnos, Janice Patricia Kinney,
Joni Marie Holling, Joy Angevin Balmer, Lauree Ann Duprey-Reed, Vaibhav Rajan Parulkar, Parthasarathi Panda, Kalyan Kumar Sethi,
Snehasis Jana. Evaluation of the Energy of Consciousness Healing Treated Withania somnifera (Ashwagandha) Root Extract Using LC-MS,
GC-MS, and NMR Spectroscopy American Journal of Biomedical and Life Sciences. Vol. 5, No. 2, 2017, pp. 16-25.
doi: 10.11648/j.ajbls.20170502.11
Received: February 27, 2017; Accepted: March 8, 2017; Published: March 31, 2017
Abstract:
Withania somnifera (Ashwagandha) root extract is very useful herbal medicine since from ancient time. The
current study designed to investigate the impact of The Trivedi Effect
®
- Energy of Consciousness Healing Treatment on the
structural properties of the ashwagandha root hydroalcoholic 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 Energy of Consciousness Healing Treatment remotely by seven 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 both the control and treated samples, whereas the peak area % at respective retention time was significantly altered. The
peak area% of the treated sample at R
t
of 5.3, 5.5, 6.4, 6.5, 6.8, 6.9, 7.1, 7.3, 7.8, 7.9, 8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.1, and 10.1
minutes were significantly reduced by 0.57% to 38.10% compared to the control sample. In addition, the peak area% of the
treated sample at R
t
of 5.7, 6.7, and 8.1 minutes were significantly increased by 16.00%, 244.44% and 19.62%, respectively
compared with the control sample. A total of 21 withanolides such as withanoside IV, coagulin Q, viscosa lactone B,
withanolide A, withaferin A, withanone, withanolide D, ixocarpalactone A and withanolide sulfoxide, ixocarpalactone A,
withanolide sulfoxide, withanolide B, etc. were proposed with their structure from the molecular mass at m/z 783, 569, 621,
489, 473, 767, 471, 505, 992, and 455 at retention times of 6.4, 6.5, 6.8, 7.1, 7.9, 8.1, 8.4, 9.1, and 10.1 minutes with the help
of GC-MS and NMR data of both the control and Biofield Energy Treated samples. There were significant changes observed in
the peak intensity values at the same retention time in the Biofield Energy Treated sample in the range of -75.32% to 108.51%
compared with the control sample. These findings suggest that The Trivedi Effect
®
- Energy of Consciousness 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 inflammatory diseases,
immunological disorders, stress, arthritis, cancer, diabetes, sexual disorders, aging and other chronic infections.
Keywords:
Ashwagandha, Energy of Consciousness Healing Treatment, Biofield Energy Healers, The Trivedi Effect
®
,
LC-MS, Withanolides, GC-MS, NMR
17 Mahendra Kumar Trivedi et al.: Evaluation of the Energy of Consciousness Healing Treated Withania somnifera
(Ashwagandha) Root Extract Using LC-MS, GC-MS, and NMR Spectroscopy
1. Introduction
Herbal medicines have been getting importance
worldwide for the prevention and of the various diseases
because of their impressive therapeutic effects and fewer
side effects as compared to the modern medicines [1]. The
roots of Withania somnifera (L.) Dunal (Family-
Solanaceae) is an ancient Rasayana 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 nervous and sexual disorders,
infectious diseases, diabetes, 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, starch, reducing sugars,
peroxidases, glycosides, dilcitol, withanicil, benzyl
alcohol, 2-phenyl ethanol, benzoic acid phenyl acetic acid,
3, 4, 5-trihydroxy cinnamic acid, etc. [7-9]. Isolated
withanolides from W. somnifera possess various
pharmacological activities include antioxidant, anticancer,
immunomodulating, neuroprotective, hepatoprotective,
anti-inflammatory, antiarthritic, antimicrobial,
hypoglycaemic, 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 nutraceuticals [31, 32], pharmaceuticals [33,
34], organic compounds [35-37], 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 root extract.
2. Materials and Methods
2.1. Chemicals and Reagents
Withania somnifera (Ashwagandha) root hydroalcoholic
extract was procured from Sanat Product Ltd, India (Batch
no: 063012). The HPLC grade acetonitrile and Milli Q
water were purchased 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 was one of the components of
American Journal of Biomedical and Life Sciences 2017; 5(2): 16-25 18
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
®
- Energy of Consciousness
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 seven renowned Biofield Energy Healers who
participated in this study and performed the Biofield Energy
Treatment remotely. Six Biofield Energy Healers were
remotely located in the U. S. A. and one of which was
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 solvent’s 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 liquid 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 21 peaks
at different retention times and their LC data are presented in
the Table 1. There were no change in the retention time of the
chromatographic peaks in the Biofield Energy Treated sample
compared with the control sample indicated that, the polarity
of the phytoconstituents in ashwagandha root extract remained
unaltered.
There was a significant decrease in the peak area% of the
Biofield Energy Treated sample in the range of 0.57% to
38.10% at R
t
of 5.3, 5.5, 6.4, 6.5, 6.8, 6.9, 7.1, 7.3, 7.8, 7.9,
8.0, 8.2, 8.4, 8.6, 8.8, 9.0, 9.1, and 10.1 minutes compared with
the control sample. Consequently, the peak area% of the
Biofield Energy Treated sample at R
t
of 5.7, 6.7, and 8.1
minutes were significantly increased by 16.00%, 244.44% and
19.62%, respectively compared to the control sample. The
peak area% provides the relative amounts of components in
the chromatogram, when all components respond in the
detector and are eluted [43, 44]. Here, the liquid
chromatographic conditions for both 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. So, the provided peak area% are revealed the relative
amounts of the phytoconstituents of W. somnifera root extract.
19 Mahendra Kumar Trivedi et al.: Evaluation of the Energy of Consciousness Healing Treated Withania somnifera
(Ashwagandha) Root Extract Using LC-MS, GC-MS, and NMR Spectroscopy
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 Biofield Energy Treated % Change
*
1 5.3 1.3 1.20 -7.69
2 5.5 5.59 3.46 -38.10
3 5.7 3.00 3.48 16.00
4 6.4 3.68 3.13 -14.95
5 6.5 8.01 7.25 -9.49
6 6.7 0.18 0.62 244.44
7 6.8 1.6 1.36 -15.00
8 6.9 0.64 0.49 -23.44
9 7.1 10.06 8.97 -10.83
10 7.3 1.31 0.98 -25.19
11 7.8 2.12 2.00 -5.66
12 7.9 2.95 2.91 -1.36
13 8.0 0.54 0.34 -37.04
14 8.1 27.11 32.43 19.62
15 8.2 4.45 3.57 -19.78
16 8.4 8.79 8.74 -0.57
17 8.6 0.45 0.34 -24.44
18 8.8 1.2 1.00 -16.67
19 9.0 0.33 0.21 -36.36
20 9.1 2.66 2.41 -9.40
21 10.1 3.03 2.92 -3.63
*
denotes the percentage change in the peak area (%) of the Biofield Energy Treated sample with respect to the control sample.
The Table 1 revealed that Biofield Energy Healing
Treatment might have the significant effect on the relative
amount 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 alter due to the Biofield Energy Healing
Treatments [28-35].
9 chromatographic peaks out of the 21 peaks, only at the
R
t
of 6.4, 6.5, 6.8, 7.1, 7.9, 8.1, 8.4, 9.1, and 10.1 minutes
having higher peak area% than other R
t
responded to the
mass spectrometric analysis and afforded the respective ESI-
MS spectra. A total of 21 withanolides as shown in the
Figure 2 were proposed along with GC-MS and NMR data
(Figure 3 and 4). Compounds (Figure 2) were proposed 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 crude extract
according to the approach described in our recent literature
[43].
American Journal of Biomedical and Life Sciences 2017; 5(2): 16-25 20
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
sample
Biofield Energy
Treated sample
%
Change
a
6.4 Withanoside IV (1) 783 [M + H]
+
9.64e5 2.01e6
108.51
Withanoside VI (2) 800 [M + NH
4
]
+
1.46e7 9.31e6 -36.23
621 [M + H – β-glucose]
+
1.30e6 1.42e6 9.23
6.5 2,3-Dihydrowitha-none-3β-O-sulfate (3) 569 [M + H]
+
3.77e6 2.29e6 -39.26
314 8.26e6 3.36e6 -59.32
6.8 Coagulin Q (4), 621 [M + H]
+
1.57e6 2.40e6 52.87
physagulin D (5) 471 4.70e6 1.16e6 -75.32
650 4.34e6 1.36e6 -68.66
7.1 Viscosa lactone B (6) 489 [M + H]
+
1.68e7 3.06e7 82.14
27-hydroxy withanolide A (7) 506 [M + NH
4
]
+
2.69e6 4.15e6 54.28
(20S,22R)-,6α-epoxy-4β,5β,27-trihydroxy-1-oxowitha-24-enolide (8)
(20S,22R)-4β,5β,6α,27-tetrahydroxy-1-oxo-with-2,24-dienolide (9)
7.9 Dihydrowithanolide D (10) 473 [M + H]
+
7.67e6 6.90e6 -10.04
490 [M + NH
4
]
+
1.02e6 9.07e5 -11.08
Withanoside V (11) 767 [M + H]
+
1.16e6 10.00e5 -13.79
784 [M + NH
4
]
+
1.08e7 8.37e6 -22.50
8.1 Withanolide A (12) 471 [M + H]
+
5.98e7 7.12e7 19.06
Withaferin A (13) 488 [M + NH
4
]
+
2.23e7 2.33e7 4.48
Withanone (14) 941 [2M + H]
+
8.04e6 8.46e6 5.22
Withanolide D (15) 958 [2M + NH
4
]
+
1.46e7 1.58e7 8.22
8.4 Ixocarpalactone A (16) 505 [M + H]
+
7.08e6 4.86e6 -31.36
488 [M – H
2
O + 2H]
+
2.44e7 2.57e7 5.33
471 [M – 2H
2
O + 3H]
+
5.22e6 4.98e6 -4.60
9.1 Withanolide sulfoxide (17) 992 [M + H]
+
1.25e6 9.83e5 -21.36
975 [M – H
2
O + 2H]
+
3.15e6 3.31e6 5.08
10.1 Withanolide B (18) 455 [M + H]
+
2.81e7 1.91e7 -32.03
Withanolide G (19) 472 [M + NH
4
]
+
3.03e7 2.09e7 -31.02
Withasomidienone (20) 496 6.45e6 6.41e6 -0.62
Withacoagin (21)
*As m/z 788 [M + H]
+
;
a
denotes the percentage change of the Biofield Energy Treated sample with respect to the control sample.
At R
t
of 6.4 minutes, withanoside IV (1) or withanoside
VI (2) (Figure 2) were proposed from the 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 ESI-MS spectra of the
control and Biofield Energy Treated sample. 2,3-
Dihydrowitha-none-3β-O-sulfate (3) at R
t
of 6.5 minutes
exhibited the molecular ion peak at m/z 569 [M + H]
+
(calcd
for C
28
H
41
O
10
S, 569) (Figure 2). Similarly, at R
t
of 6.8
minutes coagulin Q (4) or physagulin D (5) exhibited the
molecular ion peak at m/z 621 [M + H]
+
(calcd for
C
34
H
53
O
10
, 621) in the ESI-MS spectra of the control and
Biofield Energy Treated sample (Figure 2).
The LC-MS, GC-MS (Figure 3) and NMR (Figure 4)
spectral analysis and following the literature [43] confirmed
the presence of viscosa lactone B (6) or 27-hydroxy
withanolide A (7) or (20S,22R)-3α,6α-epoxy-4β,5β,27-
trihydroxy-1-oxowitha-24-enolide (8) or (20S,22R)-
4β,5β,6α,27-tetrahydroxy-1-oxo-with-2,24-dienolide (9)
(Figure 2) in the control and Biofield Energy Treated samples
at R
t
of 7.1 minutes and m/z 489 [M + H]
+
. Consequently,
dihydrowithanolide D (10) and withanoside V (11) displayed
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 the control sample at
the R
t
of 7.9 minutes (Figure 2) [43].
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