Lots of cancer chemotherapeutics which are currently in usage, affect the normal
cells of patients strongly. They have hepatotoxic, nephrotoxic, cardiotoxic,
etc., side effects on normal cells and become serious medical problems. This
induced scientists to search for new medicaments from various sources, for instance,
from medicinal plants. There is a growing trend for herbal drugs because of
low toxicity and high medical effectiveness of plant extracts. The anticancer
features of plants have been recognized for centuries: millions of plant products
exist possessing such properties (Desai et al.,
2008). Recently, 52 and 36 plants with tumoricidal character have been presented
in the reviews of Kaur et al. (2011) and Nirmala
et al. (2011), respectively.
Medicine was an inseparable part of ancient Armenian culture and its roots
come from deep in the past. Armenia has a rich history in the field of natural
medicine, particularly, in phytotherapy. Since the V century BC, some medical
plants were disseminated in Europe from here (Vardanian,
1982). Herbal medicine developed thanks to medieval Armenian scholars Mkhitar
Heratsi, Amirdovlat Amasiatsi, Grigor Magistros and many others (Torosian,
2006). Several dietary plants widespread in Armenia are used in folk medicine.
For example, melilot (Melilotus officialis), various parts of sea buckthorn
(Hippophae rhamnoides L.), walnut (Juglans regia L.), grape (Vitis
vinifera L.), sorrel (Rumex L.) and rose (Rosa damascene),
etc., show different pharmacological effects and are used in traditional and
current medicine for treatment of various health disorders. However, at present,
little information is available about their anticancer properties.
Earlier the ability of aqueous extracts of 27 plants of Armenian Highland origin
to inhibit the purified preparations of dipeptidyl peptidase IV and adenosine
deaminase-the enzymes, activities of which are elevated at diabetes mellitus,
were tested in vitro (Mardanyan et al., 2011).
The obtained results have shown that such plants, as: Blackberry, melilot, oregano,
St. Johns wort, sea-buckthorn leaves, etc., can be used in combination
with the usual antidiabetic drugs to enhance their effectiveness at treatment
of diabetes mellitus.
In the present study, the cytotoxicity of the extracts of leaves of grape,
sea buckthorn and sorrel, rose petals, pellicles of walnut kernels and aboveground
parts of melilot toward cancer cells (mice Ehrlich ascites carcinoma, EAC) was
scanned. This study was compared with their influence on the viability of normal
cells (leukocytes of mice peripheral blood, MBL). The ethanol extracts of petals
of rose, leaves of grape and sorrel were subjected to fractionation by gel-filtration.
The extracts and the obtained fractions were characterized by chemical analysis,
thin layer chromatography, UV-VIS absorbance and antioxidant activities. The
described in the present article results signify the potential of studied plants
as sources of anticancer therapeutic agents. The findings also validate their
use in traditional medicine.
MATERIALS AND METHODS
Reagents and equipments: 2, 2-diphenyl-1-picrylhydrazyl (DPPH); 2, 2
azobis-(3-ethylbenzothiozoline-6-sulphonic acid) (ABTS); Cell cultivation RPMI-1640
medium and additions were purchased from Sigma Ltd, USA; G-25 and LH-20 Sephadex-from
Pharmacia Biotech, Upsala, Sweden. Other chemicals were of the highest purity.
UV-Vis absorption spectra were recorded on Specord M-40 (Germany) spectrophotometer.
For monitoring the cell growth and for DNA comet assay the light and fluorescence
microscopes model BH- 2RFCA, Olympus, with a digital camera, model c35AD-4,
Olympus were used.
Plant material: The leaves of grape (Vitis vinifera), sorrel
(Rumex crispus) and sea buckthorn (Hippophae rhamnoides), rose
(Rosa damascena), melilot (Melilotus officinalis) and the walnut
(Juglans regia) were collected from the flanks of Aragats Mountain and
in Ararat valley of Armenia. A voucher specimen has been deposited at the herbarium
of the Botanical Department of Yerevan State University (Dr. Narine Zaqaryan).
The dried in shade plant material was grinded and the 10% (w/v) extracts were
prepared in bi-distilled water (30 min in boiling water bath) and in 70% ethanol
(72 h at ambient temperature). The extracts were filtered through a sterile
cheese cloth, dried at 37°C to constant weight and stored at -18°C until
Fractionation of plant extracts: The appropriate amount of the dried
extract, usually, 1-3 mg, was dissolved in 2 mL of 70% ethanol and subjected
to gel-filtration on LH-20 Sephadex containing column (2x40 cm), equilibrated
with 20% ethanol. The fractions were eluted with increasing concentration of
ethanol up to 70%. The fractions containing the mixture of different compounds
were subjected to gel-filtration on G-25 Sephadex column, equilibrated with
20% ethanol and eluted with an appropriate concentration of ethanol. The received
fractions were dried to constant weight by evaporation.
Identification of chemical constituents of plant extracts: The constituents
of the extracts and the fractions were characterized by qualitative chemical
analysis (Grinkewich and Safronich, 1983). The appearance
of characteristic colors in the following specific reactions were used: Bright
yellow-for flavonoids (Fl) in basic medium (5% ethanol solution of AlCl3
was also used); dark violet-for phenol glycosides (PhG) in the presence
of FeSO4 crystals; black-green-for tannins at reaction with 1% iron
ammonium alums; pink-red-for anthracene derivatives (AD)-in the presence of
5% ethanol solution of sodium hydroxide; bright red-cherry-for coumarins (Cm)
at adding of Paulis reagent after boiling in basic medium. Then, for the
detection of phloroglucides (Phlg)-Paulis reagent; alkaloids-the Dragendorffs
reagent; carbohydrates-the Benedicts test and cardio glycosides-reactions
of Legal and Keller-Kiliani were used. Saponins presence was checked by the
formation of a frothing after boiling and vigorous mixing.
Thin-layer chromatography: The constituents of the extracts and fractions,
dissolved in different concentrations of ethanol (depending on the compound
type), were characterized by TLC analysis on silica gel sheets (glass support,
Fluke), in the appropriate solvent system: For PhG-EtOAc/CH2O2/H2O
(3/2/1), revealing by 1% solution of FeCl3 in ethanol; for AD-EtOAc/MeOH/H2O
(100/17/13), revealing by 5% solution of KOH in methanol; for Fl-EtOAc/CH2O2/H2O
(8/3/1), revealing by 1% solution of AlCl3 in ethanol; for Cm-EtOAc/MeOH/H2O
(3/1/1), revealing by 10% H2SO4 followed by heating until
the appearance of spots.
Antioxidant assays: The antioxidant activities of the extracts and fractions
were estimated by two methods: DPPH and ABTS radicals scavenging effects. Each
sample was dissolved in DMSO at concentration 10 mg mL-1 and diluted
in 70% ethanol to 1 mg mL-1. Then, definite concentrations in 70%
ethanol were prepared right before duplicate analysis for antioxidant capacity:
||The DPPH assay was done according to the method of Brand-Williams
et al. (1995). Briefly: The methanol solution of DPPH, with absorbance
of about 1.05 (±0.02) at 517 nm was prepared daily. A 1 mL aliquot
of this solution was mixed with 100 μL of a sample at appropriate concentration.
The solution in the test tube was shaken well and incubated in the dark
for 30 min at room temperature. Then the absorbance at 517 nm was measured
||The ABTS Radical scavenging activity was assayed according to Re
et al. (1999): 7 mM ABTS solution reacted with 2.45 mM potassium
persulfate, kept overnight in the dark and diluted with 0.05 M phosphate
buffer, pH 7.4 to get an absorbance of about 0.800 (±0.02) at 743
nm. A 1.0 mL of this solution was mixed with 100 μL of a sample at
appropriate concentration and in 6 min the absorbance at 743 nm was measured
Along with the analysis of samples, ascorbic acid solution was used as a standard.
At both assays, the control mixtures contained 70% ethanol instead of the test
solution and the percentage scavenging activity of the plant sample (and of
the standard, ascorbic acid) solution was estimated using the following equation:
Scavenging effect (%) = [(control absorbance-sample absorbance)/(control
The scavenging activity of a plant sample was expressed as the concentration
of ascorbic acid with the activity, equal to the activity of sample. IC50
value was determined as the concentration of a plant preparation in μg
mL-1 with ability to scavenge 50% of free radicals.
Primary cell culture of mice ehrlich ascites carcinoma (EAC) cells:
The line of EAC cells was supported by re-inoculation into the peritoneal cavity
of laboratory mice (Harutyunyan, 2010). On the 7th day
after infection, the ascitic fluid, containing EAC cells, was taken from animals
under aseptic conditions. Cells were washed twice with the sterile Phosphate
Buffered Saline (PBS), pH 7.2 and suspended in the RPMI-1640 medium, containing
10% fetal bovine serum and penicillin (50 IU mL-1). The concentration
of cells were adjusted to 0.5x106 cells mL-1 and cultivated
during 5 days in the humidified chamber with 5% CO2 at 37°C.
Primary cell culture of mouse peripheral blood leucocytes: The blood
was taken from mouse by cardiac puncture with sterile heparinized (20-50 units
mL-1) syringe. After centrifugation of whole blood at 380xg during
10 min, the rich of leucocytes fraction was collected from buffy coat. Leucocytes
were washed three times in PBS and suspended in RPMI-1640 medium containing
fetal bovine serum (15%), penicillin (50 IU mL-1) and phytohemagglutinin-M
(200 μg mL-1) (Miller and Enders, 1968).
The final concentration and the cultivation conditions of leucocytes were as
for the EAC cells.
Viability of cells: To investigate the cytotoxicity of plant extracts
and their constituents toward EAC and MBL cells, a definite amount of plant
sample was dissolved in DMSO and added to the cultivation medium up to the appropriate
concentration. The amount of DMSO in the medium did not exceed 1%. Special test
ensured the absence of affectivity of this DMSO concentration. The concentration
of cells was measured daily by light microscopy using the counting chamber.
The amount of viable cells was determined by trypan blue exclusion test as percentage
of the total amount of cells (Strober, 2001). DNA Comet
assay by Ostling and Johanson (1984) was used for genotoxisity
qualifying, as it was done previously (Movsisyan et al.,
Statistical analyses: The obtained data were analyzed using the statistical
software InStat, version 3 for Windows (GraphPad Software, Inc., San Diego,
CA, USA). All results are the averages of three independent analyses. Data are
presented as Mean±S.E.
Screening of plant extract influence on the growth of cultured EAC cells:
The EAC cells were incubated in the medium containing water or ethanol extract
of either Grape Leaves, (GL), Sorrel Leaves (SL), Sea Buckthorn Leaves (SBL),
Rose Petals (RP), above ground part of Melilot (M) and Pellicles of Walnut Kernels
(WKP). The effects of the water extracts of these plants were not clearly pronounced.
In Fig. 1, the 3 day effects of 1% ethanol extracts of the
studied six plants on the EAC cells growth are shown. Here, the amount
of alive cells in the presence of the extract is expressed as percentage of
the amount in control sample. One can see that in 2-3 days, the studied extracts
inhibited the growth of EAC cells by 50-100 percent compared with that in the
||Inhibition of growth of ehrlich ascites carcinoma cells by
1% ethanol extracts of six edible plants. The amount of alive cells in the
presence of plant extract is expressed as percentage of the amount in the
control sample: (0) Control, without plant extract, (1) Leaves of grape,
(2) Pellicles of walnut kernels, (3) Petals of rose, (4) Leaves of sorrel,
(5) Melilot and (6) Leaves of sea buckthorn
|| Chemical composition of plant extractsa
|aComparative quantity evaluated by quality analysis
in the identical aliquots of the extracts: +++>++>+, bNo-not
Chemical composition of plants: The qualitative chemical analysis of
the water and ethanol extracts of GL, SL, SBL, RP, M and WKP revealed the existence
in them of the compounds, presented in Table 1. From these
data we can see that in WKP the most of the analyzed chemicals were obtained:
Tannins, phenol glycosides, cardio glycosides, phloroglucides, alkaloids but
not coumarins, the flavonoids were found nearly in all of the studied extracts.
They are the main chemical compounds in GL. RP were rich in phenol glycosides
and phloroglucides, but anthracene derivatives were found only in SL. Saponins
were not found in any of the studied plants.
Fractionation of extracts: A more detailed investigation was continued
with ethanol extracts of GL, SL and RP, regularly used in Armenian cuisine.
These three extracts were fractionated by gel-filtration on columns with LH-20
and G-25 Sephadex equilibrated with 20% ethanol solution and eluted by increasing
concentration of ethanol. These procedures resulted in receiving of several
fractions, among them-coumarin, phenol glycoside and anthracene derivative fractions
from the extract of SL; phloroglucide, 2 coumarin and 2 phenol glycoside fractions
from the extract of GL, 2 coumarin and three phenol glycoside fractions from
||Thin-layer chromatography pictures of some plant preparations.
(a) The extract of sorrel leaves (1) and isolated from it anthracene derivative
(2) in EtOAc/MeOH/H2O (100/17/13) solvent system. (b) The extract
of sorrel leaves (1), isolated from it fraction of flavonoids (2) and quercetin
as a control (3) in EtOAc/CH2O2/H2O (8/3/1)
solvent system. (c) The phenol glycoside fractions from extracts of rose
petals (1), grape leaves (2) and sorrel leaves (3) in EtOAc/CH2O2/H2O
(3/2/1) solvent system. (d) The coumarin fractions from extracts of rose
petals (1), sorrel leaves (2) and grape leaves (3) in EtOAc/MeOH/H2O
(3/1/1) solvent system
Thin layer chromatography: In Fig. 2a and b,
the Thin Layer Chromatography (TLC) pictures of sorrel extract and of isolated
from it AD and Fl fractions are presented. The absence of tails and of residues
in the initial points of the traces 2 evidenced the purity of isolated AD and
Fl fractions. In Fig. 2c, the TLC pictures of PhG fractions
from GL, RP and SL (traces 1, 2 and 3, respectively) are presented. The characteristic
colors of spots prove the results of chemical analysis (Table
1). In Fig. 2d, the TLC picture of Cms from extracts of
RP, SL and GL (traces 1, 2 and 3, respectively) are shown. The red-brown color
of low-mobility spots manifests that these fractions contain a significant amount
of sugar. The glycoside group contamination in these fractions along with Cms
was proved also by the qualitative analysis for carbohydrides, as well as by
appearance of specific lines in the region of chemical shifts between 3 and
4 ppm in NMR spectra (Homans et al., 1987) (not
shown). These fractions were further subjected to phase separation in the H2O/EtOAc
system which resulted in obtaining glycoside and coumarin fractions in the water
and the ethyl acetate phases, respectively. The following investigation demonstrated
the cytotoxicity of both of the separated fractions toward EAC cells.
Optical absorbance of isolated constituents: In Fig. 3,
the spectra of optical absorbance in UV-VIS region of fractions, isolated from
plant extracts upon gel-filtration procedures, are presented. In general, the
characters of the registered spectra reaffirm the results of chemical and TLC
identifications. Two coumarin fractions received both from RP and GL differed
by the wavelengths of absorption peaks. The spectra of Cm from SL and Cm1 from
RP are similar, with absorption peaks at 270 nm (Fig. 3a,
curve 1). The absorption peak of Cm2 from RP is at 315 nm (Fig.
3a, curve 2). In spectra of Cm1 and Cm2 from GL the absorption peaks are
shifted to lower wavelength (Fig. 3b, curves 1 and 2). Based
on the publication of Kim et al. (2003), we
can conclude, that only Cm2 from RP with λmax >300 nm is in monomer
form, all the other Cms which λmax <300 nm, with high probability are
in the dimeric form.
||Optical absorbance spectra of fractions, isolated from plant
extracts by gel-filtration. (a) Coumarin from sorrel leaves, coumarin1 from
rose petals (1) and coumarin2 from rose petals (2), (b) Coumarin1 (1) and
coumarin2 (2) from grape leaves, (c) Anthracene derivative from sorrel leaves,
(d) Phloroglucide from grape leaves, (e) Phenol glycosides and (f) Flavonoids.
The last two compounds existed in all of the studied extracts (Table
The spectrum of anthracene derivative from SL (Fig. 3c)
allows suggesting that it has a structure of emodin.
Antioxidant properties: In Table 2, the antioxidant
activities of RP, SL and GL extracts and their constituents at concentration
1 mg mL-1, evaluated by DPPH and ABTS methods, are shown. The activities
of plant preparatons determined by the both methods are equal to the activity
of ascorbic acid at concentration intervals: 1-3.3 μg mL-1 for
Cms; 45-100 μg mL-1 for Phlg of GL; 20-200 μg mL-1
for PhGs. The AD from SL scavenged only ABTS radicals with the activity corresponding
to the activity of ascorbic acid at concentration 14 μg mL-1.
It can be noted that the values of antioxidant activities of compounds belonging
to the same chemical type are different. For instance, the different fractions
of PhG from RP, eluted from Sephadex columns by different concentrations of
ethanol, differ from each other up to 8 times.
|| Antioxidant activity of some plant preparationsa
|aScavenging activity of 1 mg mL-1 plant
preparation is expressed in equivalent of ascorbic acid (standard) in μg
mL-1 possessing this activity
It manifests that the obtained phenol glycosides possess different molecular
structures. The data in Table 2 evidence that the antioxidant
activities of the extracts of the three studied plants at concentration 1 mg
mL-1 are in the order: RP>GL>SL. Their IC50 values
were evaluated from the concentration dependencies of antioxidant activities
of the extracts. The data for DPPH and ABTS radicals scavenging were, respectively:
9.03±0.27 and 2.79±0.5 μg mL-1 for RP; 17.85±0.52
and 3.79±0.09 μg mL-1 for GL; and 115.94±1.09
and 14.38±0.14 μg mL-1 for SL. These values reaffirm
the order of reducing ability of the extracts.
Action of plant preparations on the viability of EAC and MBL cells:
In Fig. 4, the effects of ethanol extracts of SL, RP and GL
and of their constituents at concentration 200 μg mL-1 on the
viability of EAC cells and MBLs are shown. It is evident that the extracts of
the three plants inhibited the growth of cells differently. Unlike the extracts
of RP and GL, the extract of SL inhibited EAC cells more effectively, than MBLs.
In this figure, one can see that among coumarin fractions, Cm2 of RP inhibited
EAC cells most effectively. The inhibiting ability of Cm from SL and Cm1 from
RP on EAC cells amounted to 40-55%, while they practically had no influence
on MBLs. Since two Cms from GL manifested similar toxicity to both EAC and MBL
cells, we performed DNA-comet assay (Fig. 5) to research the
mechanism of killing the cells by Cm1 from GL. The obtained result evidenced
in favor of apoptotic mechanism.
PhG2 from GL and PhG3 from RP possessed some cytotoxicity toward EAC cells
(Fig. 4), but almost did not influence MBLs, while PhG from
SL, PhG1 from GL, PhG1 and PhG2 from RP possessed rather strong cytotoxicity
toward EAC cells and practically had no effect on MBLs. The anthracene derivative
fraction from the ethanol extract of SL also demonstrated a relatively mild
toxicity toward normal cells and strong toxicity to EAC cells.
||Influence of plant preparations on the viability of ehrlich
ascites carcinoma cells and mice blood leukocytes. The cells were cultivated
two days in the presence of 200 μg mL-1 of indicated plant
preparation. The amount of alive cells in each sample is expressed as percentage
of the amount in the control sample (a) Sorrel leaves, (b) Grape leaves
and (c) Rose patels
||DNA comet pictures of ehrlich ascites carcinoma cells after
24 h cultivation (a) In the absence and (b) In presence of coumarin from
The concentration dependencies of cytotoxicity of SL, GL and RP extracts toward
EAC cells and MBLs were studied. The results elucidated that the extracts of
these plants at concentration 4 μg mL-1 inhibited the growth
of EAC cells by 71, 73 and 79%, respectively, leaving the viability of MBLs
at 95-100% (not shown). This confirmed the high toxicity of the studied extracts
toward cancer cells and the low toxicity toward normal cells.
The IC50 values of the three plant extracts for inhibiting the growth
of MBLs, revealed from the concentration dependencies, were in the order: SL>GL>RP
(25±10 and 1.03±0.2 mg mL-1 and 73±3 μg
mL-1, respectively). This order is consistent with the decrease of
their antioxidant activities: RP>GL>SL (Table 2). IC50
values relating to the inhibition of growth of EAC cells for the extracts of
GL and RP (80±5 and 15±2 ng mL-1, respectively) were
of the same order; however, IC50 for the extract of SL (0.1±0.04
ng mL-1) was lower by three orders than that for GL.
|| IC50 values for inhibiting the growth of ehrlich
ascites carcinoma cells by plant fractions
In Table 3, the revealed from the concentration dependencies
IC50 values for inhibiting the growth of EAC cells by some fractions,
isolated from the three plant extracts, are presented.
The results of investigations presented in this work manifested the toxicity
of ethanol extracts of rose petals, pellicles of walnut kernels, melilot, leaves
of sea buckthorn, sorrel and grape toward mice Ehrlich ascites carcinoma cells.
Taking into account that bioactivity-directed fractionation must be carried
out to identify compounds which can serve as candidates for preclinical testing,
several constituents of these extracts were isolated and studied for cytotoxicity
against both EAC cells and MBLs (Fig. 4). The observed higher
cytotoxic efficacy of crude extracts compared with the cytotoxicity of the isolated
components can be explained by synergy effect due to the probable presence of
substances which enhance the activity of the components responsible for the
researched toxicity (Gilbert and Alves, 2003).
The observed relatively mild toxicity of coumarins from three plants toward
both EAC cells and MBLs (Fig. 4), despite the known cytotoxicity
of such compounds (Luo et al., 2012; Riveiro
et al., 2010) was in accordance with their low antioxidant activity
(Table 2). Interestingly, among several coumarins, EAC cells
most effectively were inhibited by Cm2 from RP with the absorbance spectrum
(Fig. 3a, curve 2) characteristic for monomer coumarins (Kim
et al., 2003). Cm from SL and Cm1 from RP with the absorbance spectra,
characteristic for dimeric form (Fig. 3a, curve 1), provided
relatively low inhibition of EAC cells, having practically no effect on MBLs
(Fig. 4). The DNA-comet assay (Fig. 5) demonstrated
pronounced genotoxicity of Cm1 from GL, characterizing the general mechanism
of cytotoxicity of coumarins as preferentially apoptotic.
The therapeutic benefit of medical plants (particularly, the cytotoxicity and
anticancer activity), is often attributed to their antioxidant properties. However,
the obtained in our research cytotoxicity of six phenol glycosides from three
plants (Fig. 4) did not correspond to their various antioxidant
activities (Table 2). Contrary to high antioxidant activity,
PhG2 from GL and PhG3 from RP possessed low cytotoxicity toward EAC cells (Table
3) and had no effect on MBLs. Whereas PhG from SL, PhG1 from GL, PhG1 and
PhG2 from RP with relatively lower antioxidant activity possessed rather strong
cytotoxicity toward EAC cells (Table 2 and 3),
practically having no effect on the MBLs. This observation enhances the chemotherapeutic
significance of phenol glycoside fractions from all the three studied edible
plants: Rose petals, leaves of sorrel and grape. Phloroglucide fraction from
GL demonstrated similar peculiarity. We presume that these compounds can be
considered as candidates for preclinical testing of their anticancer properties.
The work, directed to the elucidation of the molecular structure of the perspective
compounds is underway.
The toxicity of the ethanol extracts from three plants toward mouse blood lymphocytes
(Fig. 4) was in keeping with their antioxidant features (Table
2). However, the toxicity to the Ehrlich ascites carcinoma cells of the
extract from sorrel leaves, despite its lower antioxidant activity, was higher
than the toxicity of the extracts both from rose petals and grape leaves with
higher antioxidant activities. Unlike this peculiarity of sorrel leaves, the
ability of ethanol extract of Rumex L. fruit to kill cancer cells, reported
by Wegiera et al. (2012), was in accordance
with the high antioxidant activity of methanol extract of ripe fruits of the
plant, observed by Maksimovic et al. (2011).
The anti-cancerous property of methanol extract of sorrel roots was also in
compliance with its free radical scavenging activity (Shiwani
et al., 2012).
Let us note that, like the extract of sorrel leaves, the anthracene derivative
from it also demonstrated relatively low toxicity toward normal cells and strong
toxicity to EAC cells. All these observations-the strong toxicity to EAC cells
of PhG, AD and the extract of SL as a whole, together with their low action
on normal cells, underlines the preference of sorrel leaves as an anticancer
In general, based on the described results of the implemented investigation,
one can conclude, that the extracts of rose petals, leaves of sorrel and grape,
as well as some of their constituents can be recommended as sources for searching
new anticancer medicaments.
This study was supported by a research Grant «11-1f105»
awarded in 2011 to Dr. A. Antonyan from the Committee of Science of Armenian
Ministry of Education and Science. We are grateful to Prof. Giulio Lupidi from
the University of Camerino, Italy, for performing the NMR study.