Antimalarial activity of phytol derivatives: in vitro and in vivo study

Article abstract of DOI:10.1007/s00044-017-2132-2

Severe malaria is one of the leading causes of mortality among children and pregnant woman globally. Resistance development against the frontline antimalarial drugs has created an alarming situation, which requires intensive drug discovery to develop effective, affordable, and accessible antimalarial agents possessing novel modes of action. As a part of our drug discovery program for antimalarial agents from plants, phytol a very commonly occurring diterpene alcohol in the plant was investigated for its antimalarial potential. In vitro antiplasmodial activity against the chloroquine-sensitive Plasmodium falciparum NF54 by measuring the parasite specific lactate dehydrogenase (pfLDH), showed moderate activity (IC₅₀ 211.5 ± 0.93 μM). Further, phytol was chemically converted into thirteen derivatives, which were evaluated for their antiplasmodial potential. All the derivatives were moderate active, but among all the derivatives palmitoyl (PhY-3; IC₅₀ 12.13 ± 0.31 μM) has found most active without any cytotoxic effect on macrophage cells. PhY-3 was further validated in an in vivo system for its efficacy and safety profile in mice. Oral administration of PhY-3 showed significant reduction of parasitemia and increased mean survival time. It also attributed significant increase in blood glucose and hemoglobin level, when compared with vehicle-treated P. berghei infected mice without any toxic effect on normal mice at a higher dose. These findings confirm suitability of the phytol derivatives as new chemical entities for further investigation towards the management of malaria.

Full text of DOI:10.1007/s00044-017-2132-2

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-017-2132-2
ORIGINAL RESEARCH
Antimalarial activity of phytol derivatives: in vitro and in vivo study
Archana Saxena1 Harish C. Upadhyay2,4 Harveer S. Cheema1,3 Santosh K. Srivastava2,3 Mahendra P. Darokar1,3
Dnyaneshwar U. Bawankule1,3
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Received: 21 June 2017 / Accepted: 30 December 2017
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Severe malaria is one of the leading causes of mortality among children and pregnant woman globally. Resistance development
against the frontline antimalarial drugs has created an alarming situation, which requires intensive drug discovery to develop
effective, affordable, and accessible antimalarial agents possessing novel modes of action. As a part of our drug discovery
program for antimalarial agents from plants, phytol a very commonly occurring diterpene alcohol in the plant was investigated
for its antimalarial potential. In vitro antiplasmodial activity against the chloroquine-sensitive Plasmodium falciparum NF54 by
measuring the parasite specific lactate dehydrogenase (pfLDH), showed moderate activity (IC50 211.5 0.93 µM). Further,
phytol was chemically converted into thirteen derivatives, which were evaluated for their antiplasmodial potential. All the
derivatives were moderate active, but among all the derivatives palmitoyl (PhY-3; IC50 12.13 0.31 µM) has found most active
without any cytotoxic effect on macrophage cells. PhY-3 was further validated in an in vivo system for its efficacy and safety
profile in mice. Oral administration of PhY-3 showed significant reduction of parasitemia and increased mean survival time. It
also attributed significant increase in blood glucose and hemoglobin level, when compared with vehicle-treated P. berghei
infected mice without any toxic effect on normal mice at a higher dose. These findings confirm suitability of the phytol
derivatives as new chemical entities for further investigation towards the management of malaria.
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Keywords Phytol In vitro In vivo Plasmodium Malaria Mice
Introduction
approximately 350–500 million clinical cases every year
with a corresponding mortality rate of 2–3 million deaths
each year, including young children under the age of five
and pregnant women (WHO 2012). Malaria is also an
enormous burden to the socioeconomic progress of people
in endemic areas, where most people suffer from poverty,
malnutrition and infectious diseases (Snow et al. 2005). So
far, no preventative vaccination against malaria exists, and
its control depends heavily upon antimalarial drugs that kill
parasites inside the human body (Snounou et al. 2005). The
World Health Organization (WHO) currently promotes
artemisinin-based combination therapy (ACT) as the first-
line treatment for management of falciparum malaria and to
reduce the risk of resistance. Although the standard anti-
malarial drugs are available, the burden of this disease is
getting worse, mainly due to the increasing resistance of
Plasmodium falciparum against the widely available anti-
malarial drugs, including emerging resistance to the arte-
misinin component of artemisinin-based combination
therapies (Ariey et al. 2014). Therefore, there is an urgent
need to discover new, highly effective antimalarial drug
candidates with new mechanisms of action to overcome the
Malaria is a major public-health problem associated with
high morbidity and mortality in many countries affecting
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2132-2) contains supplementary
material, which is available to authorized users.
* Santosh K. Srivastava
skscimap@gmail.com
* Dnyaneshwar U. Bawankule
du.bawankule@cimap.res.in
1
Molecular Bioprospection Department, CSIR-Central Institute of
Medicinal and Aromatic Plants, Lucknow 226015, India
2
Medicinal Chemistry Department, CSIR-Central Institute of
Medicinal and Aromatic Plants, Lucknow 226015, India
3
Academy of Scientific and Innovative Research (AcSIR),
Anusandhan Bhawan, New Delhi 110 001, India
4
Present address: Applied Science Department, Rajkiya
Engineering College, Sonbhadra, UP 231206, India

Medicinal Chemistry Research
problem of the rapid emergence of drug resistance and to
achieve long-term clinical efficacy (Silva et al. 2011).
Artemisinin, a sesquiterpene lactone and its derivatives
are being used as antimalarial agents, which encouraged us
to investigate antimalarial activity in other terpenoids. As
part of our antimalarial drug discovery programs, we
recently reported significant antimalarial activity in penta-
cyclic triterpenoids glycyrrhetinic acid (Kalani et al. 2013)
and ursolic acid (Kalani et al. 2015). This encouraged us to
investigate antimalarial activity in other terpenoids. A lit-
erature search revealed that phytol was another such acyclic
terpenoid, occurring as major constituent in several Indian
medicinal plants. Recent reports have shown that phytol
possesses antimalarial activity (da Silva et al. 2004; Grace
et al. 2012) prompted us to carry out chemical transfor-
mation of phytol into several derivatives, evaluation of their
antimalarial activity and study their structure–activity rela-
tionship (SAR). As most of the time, this strategy has
provided exciting results with high bioactivity and low
toxicity (Upadhyay et al. 2014).
General procedure for synthesizing phytol (PhY)
derivatives 1–10
Phytol (592 mg, 2 mmol) was dissolved in dry pyridine, and
the respective acyl/aryl chloride (3 mmol) was added. After
adding a catalytic amount of 4-dimethylaminopyridine
(DMAP), the reaction mixture was stirred at 30–35 °C for
14 h. After completion of the reaction, crushed ice was
added, and the reaction mixture was extracted with
chloroform (3 × 25 mL). The combined chloroform extract
was washed with 6% aqueous HCl solution to remove the
pyridine. Finally, the combined CHCl3 extract was washed
with distilled water and dried over anhydrous Na2SO4, and
the solvent was removed under vacuum. The resulting crude
products were separately purified by column chromato-
graphy (silica gel, 60–120 mesh, 24 g, column diameter
2 × 30 cm) to afford the respective derivatives, 1–10, in high
purity (>95%).
PhY-11(1-bromo-3, 7, 11, 15-tetramethyl-2-hexadecene)
PBr3 (3 mmol) was added to solution of phytol (2 mmol) in
dry benzene. The reaction mixture was left at 4 °C for
30 min, followed by stirring at room temperature (30–35 °C)
for 3 h. After completion, the solvent was removed by
evaporation, and the residue was extracted with chloroform.
The combined organic extracts were dried and evaporated to
give crude products, which on purification by column
chromatography on silica gel, afforded pure product 11
(PhY-11) as a viscous dirty-white liquid in 86% yield
(619.5 mg) (purity >95%).
Materials and methods
General experimental procedure
Chemistry
All the chemicals and reagents including phytol (procured
as cis–trans mixture) were obtained from Sigma-Aldrich
Pvt. Ltd., India and were used without further purification.
The solvents used in chromatographic separations were
purchased from Merck India Pvt. Ltd. The 300 MHz nuclear
magnetic resonance (NMR) (Avance, Bruker, Switzerland)
was used to record 1H and 13C NMR with tetramethylsilane
(TMS) as internal standard. Hyphenated LC-PDA-MS
(Prominence LC and mass MS-2010EV, Shimadzu,
Japan) was used for mass spectra. Infrared (IR) spectra were
recorded in CCl4 using Spectrum BX FTIR spectrometer
(Perkin Elmer, USA). The progress of the reaction was
monitored by thin layer chromatography on silica gel
60F254 (Merck, Germany) readymade aluminum sheets,
first examined under ultraviolet (UV) illumination at 254
and 365 nm and then sprayed with vanillin-sulfuric acid
(1:5, w/v) solution in ethanol followed by heating at 95 °C
for 5 min. Chromatographic purifications were performed
on silica gel (60–120 Mesh) procured from Merck (India).
PhY-12(3, 7, 11, 15-tetramethyl-2-hexadecenal)
Pyridinium chlorochromate (3 mmol) was added to a solu-
tion of phytol (2 mmol) in dry CH2 Cl2. The reaction mix-
ture was reflux for 6 h, and then the solvent was removed by
evaporation. The residue was extracted with chloroform in
excess water. The combined organic extracts were dried and
evaporated to give crude products which, after purification
by column chromatography on silica gel, afforded pure
product 12 (PhY-12) as a viscous colorless liquid in 74%
yield (435.5 mg) (purity >95%).
3, 7, 11, 15-Tetramethyl-2'-phenyloctadec-1(1'), 2(3)-diene-
2'-one (13)
BF3-Et2O (5 mmol) was gradually added to a stirred solution
of 12 (2 mmol) and acetophenone (2 mmol). The reaction
mixture was reflux for 1 h and then left at room temperature
overnight (14 h). After completion of the reaction, the
mixture was diluted with diethyl ether (50 mL), followed by
washing with distilled water (5 × 100 mL) to discharge the
Semi-synthesis of phytol derivatives
Phytol was chemically modified into thirteen derivatives.
To perform the blind pharmacological antimalarial screen-
ing to avoid the biases, phytol was coded as PhY.

Medicinal Chemistry Research
BF3-Et2O complex. The combined organic extract was dried
over anhydrous Na2SO4 and evaporated to give crude pro-
ducts which, after purification by column chromatography
on silica gel, afforded pure product 13 as a reddish viscous
liquid in 62% yield (491 mg) (purity >95 %).
Purpose of Control and Supervision of Experimental Ani-
mals (CPCSEA), New Delhi, Government of India
(Registration No: 400/01/AB/CPCSEA).
In vitro cytotoxicity study
In vitro antiplasmodial activity
The most active derivative of phytol in in vitro antimalarial
screening was further taken up for its cytotoxicity profile in
normal mammalian cells. In brief, the macrophage cells
were isolated from the peritoneal cavities of mice (8-week-
old female BALB/C mice) after an intra-peritoneal injection
of 1.0 mL of 1% peptone (BD Biosciences, USA) 3 days
before harvesting. Mice were euthanized by cervical dis-
location under ether anesthesia and peritoneal macrophages
were obtained by intra-peritoneal (i.p.) injection of phos-
phate buffer saline (PBS), pH-7.4. Membrane debris was
removed by filtering the cell suspensions through sterile
gauze. Macrophage cells were treated with PhY-3 (10,
30, and 100 µg/mL) for 24 h. The MTT (stock solution
5 mg/ml) was added to a final concentration of 0.5 mg/mL
and cells were incubated for an additional 4 h. The medium
was removed and formazon precipitate was solubilized in
100 µL dimethyl sulfoxide (DMSO) for 10 min and absor-
bance was measured at 570 nm (molecular device, USA).
The amount of color produced is directly proportional to the
number of viable cells. Cell cytotoxicity was calculated as
the percentage of MTT absorption as follows: Percentage
(%) of survival = (mean experimental absorbance/mean
control absorbance × 100).
Chloroquine-sensitive P. falciparum (NF-54) was cultivated
in human B+ red blood cells using RPMI-1640 medium
supplemented with 25 mM HEPES, 0.2% NaHCO3, 370 µM
hypoxanthine, 40 µM gentamycin, 0.25 µM fungizone, and
0.5% albumax at 37 °C (Butler and Buss 2006). Culture was
maintained in a standard gas mixture consisting of 1% O2,
5% CO2, 94% N2, and culture medium was changed after
every 24 h. The culture was routinely monitored through
Giemsa staining of thin smears. The culture was synchro-
nized by 5% sorbitol treatment to obtain ring-stage parasites
(Lambros and Vanderberg 1979). Antiplasmodial activity
of phytol and its derivatives was estimated spectro-
photometrically in control and drug-treated cultures by
measuring the parasite specific lactate dehydrogenase
activity (Trager and Jensen 1976). Briefly, a synchronous
ring stage culture with 1.2% parasitemia and 2% hematocrit
was incubated in 96-well microtiter plate in different con-
centrations of phytol and its derivatives (µM) at 37 °C for
72 h. Chloroquine was used as positive control. After
incubation, plates were subjected to three 20-min freeze-
thaw cycles to release the cell content, then cultures were
mixed carefully and aliquots of 20 µL were transferred to
another microtiter plate containing 100 µL of Malstat
reagent (0.125% Triton X-100, 130mM L-lactic acid, 30
mMTris buffer and 0.62 µM 3-acetylpyridine adenine
dinucleotide) and 25 µL of NBT-PES (1.9 µM nitro blue
tetrazolium and 0.24 µM phenazine ethosulphate) solution
per well. The plate was incubated in the dark for 30 min and
absorbance was measured at 650 nm using a microplate
reader (BMG Labteck Pvt. Ltd, Germany). The absorbance
values of test compounds and standard were converted to
percentage inhibition using absorbance of control wells.
Antiplasmodial activity of the test compounds were
expressed as IC50 (Mean SEM) of the three separate
experiments performed in triplicate. IC50 responses were
compared with drug-free controls.
In vivo study
Experimental animals
BALB/C female mice obtained from the animal house of
the institute were used for the antimalarial and toxicity
studies. The animals were housed in plastic polypropylene
cages under standard animal house conditions with a 12 h
light/dark cycle and temperature of 25 2°C. The animals
had ad libitum access to drinking water and food.
In vivo antimalarial activity
The in vivo antimalarial activity of in vitro potentially
active palmitoyl derivatives of phytol (PhY-3) was carried
out on Plasmodium berghei infected mice, based on percent
parasitemia, hemoglobin, survival kinetics, and mean sur-
vival time. The schizontocidal activity of PhY-3 was eval-
uated using the method described by Knight and Peters
(Makler et al. 1993). Briefly, in the experiments; the
animals were infected intraperitoneally with inoculum of
1 × 106 Plasmodium berghei K-173 infected erythrocytes.
Following the infection, the animals were orally
Ethics statement
In vitro cytotoxicity study on isolated peritoneal macro-
phages cells from healthy mice and In vivo animals
experimentation on mice was performed as per the approved
protocol by the Institutional Animal Ethics Committee
(IAEC) of CSIR-Central Institute of Medicinal and Aro-
matic Plants, Lucknow followed by the Committee for the

Medicinal Chemistry Research
administered with PhY-3 at doses 3, 10 and 30 and PhY-3
(3 mg/kg) in combination with chloroquine at semi curative
dose 2.5 mg/kg body weight for a period of 4 days after
suspending them in 0.7% carboxymethyl cellulose (CMC)
along with 0.1% ethyl acetate. However, chloroquine alone
at 2.5 mg/kg dose was orally administered, while oral dose
of 0.7% CMC was given to the vehicle group. On day 4, tail
blood was used to make thin blood slides and parasitemia
was counted by microscopic examination of Giemsa stained
blood smears. Parasitemia was determined by counting
RBCs comprising of both the parasitized RBCs and the
normal RBCs. Parasitemia was counted on 4th, 6th, 8th, and
onwards up to 28th day. Percentage of parasitemia was
counted based on infected erythrocytes calculated per 100
erythrocytes. Throughout the test, the general condition of
the animals in terms of behavior and clinical signs were also
evaluated and the survival of the recovered mice was
observed until day 28. Percent suppression was calculated
as 100[(A−B)/A], where A is the mean percent parasitemia
of the mice taken as vehicle control and B is the mean
parasitemia in the test group. In addition, mortality in the
mice was followed up to 28 days post-infection to evaluate
the percent survival and mean survival time.
pain and signs of illness were conducted daily during the
period of 7 days. On day 7, blood was collected from the
retro-orbital plexus of the animals for hematological and
biochemical parameters.
Statistical analysis
Results were presented as the mean SEM and analyzed
using Graph Pad Prism 4. Student’s “t” test was performed
to compare the results of vehicle and treatments. P < 0.05
was considered statistically significant.
Results
Phytol and its semi-synthetic derivatives
Phytol was procured from Sigma-Aldrich Pvt. Ltd., India.
Since phytol is a diterpene primary alcohol, it was decided
to prepare its simple acyl, benzoyl, and other related deri-
vatives. The rationale for preparing these acyl and benzoyl
ester derivatives was that we prepared the similar acyl and
benzoyl ester derivatives for our pentacyclic triterpenoid
antimalarial leads glycyrrhetinic acid (Kalani et al. 2013)
and ursolic acid (Kalani et al. 2015) and some of them
showed significant antimalarial activity. For this purpose, a
total of thirteen derivatives were synthesized for evaluation
of antiplasmodial activity and SAR study. Spectral data of
phytol derivatives has been depicted as supplementary data
(Supplementary Fig.1).
Estimation of hemoglobin and glucose from blood
from malaria-infected mice
Hemoglobin was estimated by standard drabkin’s cyan-
methemoglobin method (Knight and Peters 1980). In brief,
2 µL of whole blood was added into 500 µL of drabkin’s
solution (Monozyme Ltd., India) and kept at room tem-
perature for 5 min. Optical density of test was measured
against drabkin’s solution at 540 nm against blank using
CMG as standard through spectrophotometer (SpectraMax,
Mol Devices, USA). Blood glucose was tested using a
portable glucometer (Ascensia, Bayer). In brief, blood was
obtained by pricking the tail of mice on glucometer strips to
analyze the blood glucose level using glucometer (Balasu-
bramaniam and Malathi 1992). Hemoglobin and blood
glucose level were estimated on the day when mortality
started in vehicle treated infected mice.
In vitro antiplasmodial activity
The phytol derivatives (1–13) were evaluated for their
in vitro antiplasmodial activity against P. falciparum NF-54
and the results are presented in Table 1. The results showed
that although the parent compound phytol was marginally
active (IC50 211.5 0.93 µM), but the PhY-3 was most
active (IC50 12.13 0.31 µM) followed by PhY-13 (IC50
21.2 0.29 µM) and PhY-8 (IC50 18.8 0.29 µM). Four
concentrations (1, 10, 50, and 100 µM) of the test com-
pounds and standard drug were used for the In vitro
screening. Chloroquine was used as standard antiplasmodial
drug, which exhibited IC50 (0.042 0.006 µM).
Acute toxicity study
Acute oral toxicity of PhY-3 was done on female BALB/c
mice. Ten mice were taken and divided into two groups of
five mice in each group. Animals of group 1 were con-
sidered as control and treated with 0.7% CMC; 10 mL/kg
body weight while animals of group 2 were taken as
experimental wherein the animals were treated with PhY-3;
300 mg/kg body weight as a single oral dose. Surviving
animals were weighed and visual observations for mortality,
behavioral pattern, changes in physical appearance, injury,
Structure–activity relationship (SAR) study
The careful analysis of the results (Table 1) revealed that
antiplasmodial activity of phytol got decreased in its acetyl
derivative (PhY-1). The antiplasmodial activity got further
decreased in its trans-crotonyl derivative (PhY-5), which
consisted of a four carbon chain with olefinic bond, but got
significantly increased in its pivaloyl derivative (PhY-4),

Medicinal Chemistry Research
Table 1 Anti-plasmodial activity of phytol and its derivatives against chloroquine sensitive Plasmodium falciparum NF-54 by measuring the
parasite specific lactate dehydrogenase (pfLDH)
Codes
Structure
IC50 (µM) (mean SEM)
211.5 0.93
Phytol (PhY)
OR
R = H
PhY-1
PhY-2
PhY-3
PhY-4
PhY-5
PhY-6
R = –COCH3
263.9 0.43
53.9 0.23
12.13 0.31
77.7 0.61
>250
R = –CO–CH2 CH2 (CH2)7 CH2 CH3
R = –CO–CH2 CH2(CH2)10 CH2 CH2 CH3
R = –CO–C (CH3)3
R = –CO–CH=CH CH3
>250
O
R=
C
PhY-7
PhY-8
213.3 0.34
OCH3
O
C
R=
18.8 0.29
OCH3
OCH3
OCH3
O
R= C
PhY-9
171.2 0.26
Cl
Cl
O
C
R=
PhY-10
PhY-11
177.8 0.34
O
R=
C CH=CH
>250
Br

Medicinal Chemistry Research
Table 1 (continued)
Codes
Structure
IC50 (µM) (mean SEM)
109.3 0.93
PhY-12
O
H
PhY-13
21.2 0.29
O
CQ
0.042 0.006
IC50 concentration that inhibits 50% growth of parasites, n = 3
which consisted of five carbon branched chain. This
increase in antiplasmodial activity with increase in carbon
chain length was further observed in lauroyl (PhY-2)
and palmitoyl (PhY-3) derivatives having twelve and
sixteen carbon chains respectively. On comparing the
antiplasmodial activities of lauroyl (PhY-2, IC50 53.9
0.23 µM) and palmitoyl (PhY-3, IC50 12.13 0.31 µM)
derivatives, it was observed that PhY-3 was four times more
active than PhY-2, which may be considered due to four
carbon increase in the chain length of PhY-3 than of PhY-2.
From the above SAR study, it may be concluded that acy-
lation of the hydroxyl group in phytol by long chain
(lipophilic) groups significantly increases antiplasmodial
activity. We postulate that many of these ester derivatives
will be working as prodrugs as these ester derivatives (eg.
PhY-3) might have better cell penetration than the phytol
and would be getting hydrolyzed back to phytol inside the
cells. At the same time, it can’t be ruled out that the activity
is not due to phytol. Similarly, SAR could also be deduced
among the acyl derivatives of phytol bearing substituted
benzoyl groups. The antiplasmodial activity got reduced in
the benzoyl (PhY-6, IC50 > 250 µM), which got slightly
improved in m-anisoyl (PhY-7, IC50 213.3 0.34 µM)
derivative. Further, it increased slightly in the dichloro
substituted derivative (PhY-9; IC50 171.2 0.26 µM), but it
was drastically increased in the trimethoxy substituted deri-
vative (PhY-8; IC50 18.8 0.29 µM). On comparing the
antiplasmodial activities of monomethoxy (PhY-7, IC50 213.3
0.34 µM) and tri-methoxy (PhY-8, IC50 18.8 0.29 µM)
benzoyl derivatives, a ten-fold increase in the activity was
observed due to the increase of two more methoxy
groups in PhY-9. Hence, it may be concluded that,
substitution of hydroxyl group in phytol with benzoyl group
however reduced the antiplasmodial activity, but substitu-
tion with tri-methoxy benzoyl derivative significantly
increase the activity.
Finally, the other two phytol derivatives were also
evaluated for their antiplasmodial activity. In comparison
to phytol, a two-fold enhancement in the activity of
phytol (PhY-12, IC50 109.3 0.93 µM) was observed. The
enhancement in activity may be due to the oxidation of
aldehyde into the corresponding carboxylic acid inside the
cells. The final benzoyl derivative PhY-13 showed fourfold
increase (IC50 21.2 0.29 µM) in its activity, which may be
accounted for its unhydrolysable keto group.
Cytotoxicity study
The in vitro cytotoxicity of PhY-3, a most active anti-
malarial was carried out in the peritoneal macrophage cells
isolated from the mice using 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium (MTT) assay. No significant
change in percent (%) dead cell population was observed
(P < 0.05) at 10, 30 and 100 µg/mL concentration of the
treatment when compared with un-treated cells, confirming
that PhY-3 is non-toxic (Supplementary Fig. 2).
In vivo study
Antimalarial activity in mice
The in vivo antimalarial model was employed for this study
to validate the in vitro antiplasmodial potential of the most
active PhY-3. Our results indicated that the intra-peritoneal

Medicinal Chemistry Research
Table 2 Anti-malarial activity
of PhY-3 against P. berghei in
Balb/c female infected mice
Groups
Dose (mg/kg)
Parasitaemia (%)
Suppression (%)
Mean survival time (Days)
Normal
–
NA
NA
NA
Vehicle control
PhY-3
–
34.05 2.62
29.70 0.85
20.34 2.24
15.36 0.79
8.21 1.32
7.28 0.88
0.00
10.17
11.00
13.33
16.83
18.17
21
3
12.77 2.51
40.26 6.59
54.89 2.32
75.88 3.48
78.62 2.60
PhY-3
10
PhY-3
30
CQ + PhY-3
CQ
2.5 + 3
2.5
Data are expressed as mean SEM. n = 6
NA not applicable, CQ chloroquine
*P < 0.05; vehicle vs. treatment (ANOVA; Tukey’s test)
Fig. 2 Effect of PhY-3 on percent survival in P. berghei-infected mice.
n = 6
Fig. 1 Effect of PhY-3 on percent parasitemia in P. berghei-induced
malaria in mice. Data are expressed as mean SEM; *P < 0.05;
vehicle vs. treatment (ANOVA; Tukey’s test), n = 6
CQ were capable to restore the blood hemoglobin and
glucose level towards normal (Fig. 3a, b).
injection of P. berghei infected RBCs induced the peak of
parasitemia on day 12 of post infection. Oral administration
of PhY-3 at 3, 10, and 30 mg/kg exhibited significant
(P < 0.05) dose-dependent inhibition of parasitemia as com-
pared to vehicle-treated infected control mice. The repre-
sentative data of percentage of parasitemia and percentage of
suppression of parasitemia are depicted in Table 2. Mortality
rate improvement was observed in treated groups when
compared to vehicle-treated infected control mice. The mean
survival time of vehicle-treated control mice was 10.17 days
whereas, PhY-3 (3, 10 and 30 mg/kg) treated groups were
13.33, 16.83, and 18.17 days, respectively and chloroquine
(2.5 mg/kg) and combination of PhY-3 (3 mg/kg) plus
chloroquine (2.5 mg/kg) was 21 and 18.17 days, respec-
tively. The result of percent parasitemia and percent survival
of mice are depicted in Figs. 1 and 2, respectively.
Acute oral toxicity study in mice
The acute oral toxicity study of the most potent PhY-3
showed that the administration of a single oral dose of
300 mg/kg body weight did not produce any toxic effects on
the general behavior or appearance of mice as compared to
the control group and all the mice survived during the whole
experimental period. Similarly, no significant changes were
recorded in body weight, serum biochemical (SGOT,
SGPT, creatinine, cholesterol, and triglycerides,) as well as
hematological parameters (RBCs and WBCs) of the treated
group when compared to the control group (Table 3).
Discussion
Effect of PhY-3 on hemoglobin and blood glucose
A safe and effective treatment is essential to achieve sig-
nificant and sustained reductions in malaria-related mor-
bidity and mortality. The widely used antimalarial drugs in
the market are plant-based quinine and artemisinin deriva-
tives. The complexity of the Plasmodium falciparum
The level of hemoglobin and glucose in blood were sig-
nificantly decreased in vehicle-treated infected mice when
compared with normal mice. The treatments with PhY-3 and

Medicinal Chemistry Research
Fig. 3 Effect of PhY-3 on
hemoglobin and blood glucose
level post infection of P.
berghei-induced malaria in
mice. a Blood glucose b
hemoglobin. Data are expressed
as mean SEM; *P < 0.05;
vehicle vs. treatment; # vehicle
vs. normal; n = 3
Table 3 Effect of PhY-3 on
acute oral toxicity at 300 mg/kg
as a single oral dose in Balb/c
female mice
Parameters
Basal value
Control
7th day
PhY-3
Control
PhY-3
Body weight (g)
28.5 0.31
24.15 3.36
19.57 1.33
1.62 0.36
101.09 3.04
118.52 7.69
10.52 0.29
6.12 0.27
7.82 0.37
27.84 0.34
25.27 2.78
23.52 1.92
1.69 0.13
107.63 4.87
116.55 7.00
11.19 0.75
6.19 0.46
7.79 0.56
28.2 0.47
22.64 1.98
20.53 2.04
1.51 0.17
104.22 3.45
117.05 5.68
11.17 0.68
6.47 0.35
7.44 0.34
28.06 0.67
23.31 2.73
21.98 2.12
1.58 0.20
111.07 6.53
121.10 1.87
10.93 0.78
6.23 0.29
7.5 0.40
SGOT (U/L)
SGPT (U/L)
Creatinine(mg/dL)
Cholesterol (mg/dL)
Triglycerides (mg/dL)
Haemoglobin (gm/dL)
RBC (million/mm3)
WBC (thousands/mm3)
Data are expressed as mean SEM, n = 4
parasite leads to develop the progressive resistance to these
frequently used drugs. World Health Organization (WHO)
has recommended the use of combinations of drugs to
replace the monotherapy in the treatment of malaria. Hence,
as a part of our drug discovery program for antimalarial
agents from plants; phytol, a very commonly occurring
diterpene alcohol in the plant was investigated for anti-
plasmodial potential against the chloroquine-sensitive
Plasmodium falciparum NF54 by measuring the parasite
specific lactate dehydrogenase (pfLDH) activity.
Identification of potential drugs targeting malaria parasites
are aimed at the blood stages of the parasites (Singhal et al.
2011) phytol, a parent molecule showed moderate activity
(IC50 211.5 0.93 µM). On the basis of SAR strategy which
generally provides exciting results with high bioactivity and
low toxicity (Upadhyay et al. 2014). We, further, phytol
was chemically converted into thirteen derivatives, which
were also evaluated for their antiplasmodial potential.
To test the effect of phytol and its derivatives on malaria
parasites, we performed parasite specific lactate

Medicinal Chemistry Research
dehydrogenase (pfLDH) assay in the chloroquine-sensitive
P. falciparum NF54. All the derivatives were active, but
the PhY-3 (IC50 12.13 0.31 µM) followed by PhY-8
(IC50 18.8 0.29 µM) and PhY-13 (21.2 0.29 µM) were
significantly active against the P. falciparum. The P. falci-
parum lactate dehydrogenase enzyme (PfLDH) has been
considered as a potential molecular target for antimalarials
due to this parasite’s dependence on glycolysis for energy
production. Because the LDH enzymes found in P. vivax,
P. malariae, and P. ovale exhibit around 90% identity to
PfLDH, it would be desirable to have new anti-pLDH
drugs, particularly ones that are effective against P. falci-
parum, the most virulent species of human malaria (Penna-
Coutinho et al. 2011). Among all this most active derivative
was PhY-3. To confirm the safety profile, in vitro cyto-
toxicity of the most active PhY-3 in primary macrophages
isolated from mice revealed that it was non-toxic even at the
higher concentration. To substantiate the physiological
function of PhY-3, we further evaluated it’s therapeutic
efficacy and safety profile in mice. In vivo efficacy study in
infected mice indicated that the oral treatment with PhY-3
(3, 10, and 30 mg/kg), showed antimalarial activity by
reducing the parasitemia in a dose-dependent manner when
compared to vehicle treated infected control mice. Further,
treatment was able to improve the mean survival time of
P. berghei infected mice. There are several reports on the
antimalarial activity of plant-derived molecules and its
derivatives against the blood stages of the parasites by
reducing the parasitemia and improve the mean survival
time (Mohanty et al. 2015; Saxena et al. 2016). Hyperpar-
asitemia, hypoglycemia, cerebral malaria, severe anemia,
respiratory distress are the clinical manifestations of severe
malaria (Perkins et al. 2011) and it has been correlated with
mortality in many parts of the world especially in children
and pregnant women (Schantz-Dunn and Nour 2009).
Infection and destruction of red blood cells is central to the
malaria-causing plasmodium parasite’s reproduction and
survival. Since red blood cells consist of hemoglobin, an
oxygen transport protein, their destruction causes severe
anemia (Boeuf et al. 2012). Malaria parasites also depen-
dent on glucose as a nutrient source. As Plasmodium has no
capacity to store energy in the form of glycogen they rely
entirely on an exogenous supply of glucose and the para-
sites use a large portion of this energy both to break apart
hemoglobin proteins within the cells (Ogetii et al. 2010).
The oral treatment of PhY-3 to the P. berghei infected mice
also attributed the improvement of blood glucose and
hemoglobin level when compared with vehicle-treated
infected mice. PhY-3 treatment kills the malaria parasite,
so loss of blood glucose and hemoglobin due to malaria
infection in mice was improved in PhY-3 treated groups.
In this study, we have attempted to find out any syner-
gistic interaction standard antimalarial drug chloroquine and
PhY-3. Chloroquine (CQ) is a cost effective antimalarial
drug with a relatively good safety profile. However, CQ is
no longer used alone to treat patients with P. falciparum due
to the emergence and spread of CQ-resistant (Aguiar et al.
2014). The results on combination study of CQ and PhY-3
showed no synergism. In vitro cytotoxicity in primary
macrophage cells and in vivo acute oral toxicity study in
mice revealed that the PhY-3 was non-toxic even at the
higher concentration. There are several reports concluding
that plant-derived molecules are safe at higher concentration
(Mohanty et al. 2015; Saxena et al. 2016).
Conclusion
Phytol and its derivatives showing antiplasmodial activity.
The phytol derivative PhY-3 possesses significant anti-
malarial potential in both in vitro and in vivo bioassay and
also non toxic even at the higher dose to the mice.
Acknowledgements We acknowledge the Council of Scientific and
Industrial Research (CSIR), New Delhi, India for financial support
through XII FYP networking projects BSC-0203. We are thankful to
Director, CSIR-Central Institute of Medicinal and Aromatic Plants,
Lucknow, India for rendering essential research facilities and support.
ICMR-Senior Research Fellowship to Ms. Archana Saxena is duly
acknowledged.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
References
Aguiar AC, Pereira DB, Amaral NS, De Marco L, Krettli AU (2014)
Plasmodium vivax and Plasmodium falciparum ex vivo suscept-
ibility to antimalarials and gene characterization in Rondonia,
West Amazon, Brazil. Malar J 13:73
Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim
N, Kim S, Duru V, Bouchier C et al. (2014) A molecular marker
of artemisinin-resistant Plasmodium falciparum malaria. Nature
505:50–55
Balasubramaniam P, Malathi A (1992) Comparative study of hemo-
globin estimated by Drabkin′s and Sahli′s methods. J Postgrad
Med 38:8–9
Boeuf PS, Loizon S, Awandare GA, Tetteh JK, Addae MM, Adjei GO,
Goka B, Kurtzhals JA, Puijalon O, Hviid L, Akanmori BD, Behr
C (2012) Insights into deregulated TNF-α and IL-10 production
in malaria: implications for understanding severe malarial anae-
mia. Malar J 11:253
Butler MS, Buss AD (2006) Natural products—the future scaffolds for
novel antibiotics? Biochem Pharmacol 71:919–929
da Silva TBC, Alves VL, Mendonça LVH, Conserva LM, da Rocha
EMM, Andrade EHA, Lemos RPL (2004) Chemical constituents
and preliminary antimalarial activity of humiria balsamifera.
Pharma Biol 42:94–99
Grace MH, Lategan C, Graziose R, Smith PJ, Raskin I, Lila MA
(2012) Antiplasmodial activity of the ethnobotanical plant Cassia
fistula. Nat Prod Commun 7:1263–1266

Medicinal Chemistry Research
Kalani K, Agarwal J, Alam S, Khan F, Pal A, Srivastava SK (2013) In
silico and in vivo anti-malarial studies of 18β glycyrrhetinic acid
from Glycyrrhiza glabra. PLoS One 8:e74761
Kalani K, Cheema HS, Tripathi H, Khan F, Daroker MP, Srivastava
SK (2015) QSAR-guided semi-synthesis and in vitro validation
of antiplasmodial activity in ursolic acid derivatives. RSC Adv
5:32133–32143
Saxena A, Yadav D, Mohanty S, Cheema HS, Gupta MM, Darokar
MP, Bawankule DU (2016) Diarylheptanoids rich fraction of
Alnus nepalensis attenuates malaria pathogenesis: in-vitro and in-
vivo study. Phytother Res 30:940–948
Schantz-Dunn J, Nour NM (2009) Malaria and pregnancy: a global
health perspective. Rev Obstet Gynecol 2:186–192
Silva JR, Ramos Ade S, Machado M, de Moura DF, Neto Z, Canto-
Cavalheiro MM, Figueiredo P, do Rosario VE, Amaral AC,
Lopes D (2011) A review of antimalarial plants used in traditional
medicine in communities in Portuguese-speaking countries:
Brazil, Mozambique, Cape Verde, Guinea-Bissau, Sao Tome and
Principe and Angola. Mem Inst Oswaldo Cruz 1:142–158
Singhal J, Nagaprashantha L, Vatsyayan R, Awasthi S, Singhal S
(2011) RLIP76, a glutathione-conjugate transporter, plays a major
role in the pathogenesis of metabolic syndrome. PLoS One 6:
e24688
Knight DJ, Peters
W (1980) The antimalarial activity of N-
benzyloxydihydrotriazines. The activity of clociguanil (BRL
50216) against rodent malaria, and studies on its mode of action.
Ann Trop Med Parasitol 74:393–404
Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium
falciparum erythrocytic stages in culture. Parasitol Res
65:418–420
Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins B,
Hinrichs DJ (1993) Parasite lactate dehydrogenase as an assay for
Plasmodium falciparum drug sensitivity. Am J Trop Med Hyg
48:739–741
Mohanty S, Maurya AK, Jyotshna, Saxena A, Shanker K, Pal A,
Bawankule DU (2015) Antimalarial and safety evaluation of
Pluchea lanceolata (DC.) Oliv. & Hiern: in-vitro and in-vivo
study. Curr Pharm Biotechnol 16:544–552
Snounou G, Gruner AC, Muller-Graf CD, Mazier D, Renia L (2005)
The Plasmodium sporozoite survives RTS, S vaccination. Trends
Parasitol 21:456–461
Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI (2005) The
global distribution of clinical episodes of Plasmodium falciparum
malaria. Nature 434:214–217
Ogetii GN, Akech S, Jemutai J, Boga M, Kivaya E, Fegan G, Maitland
K (2010) Hypoglycaemia in severe malaria, clinical associations
and relationship to quinine dosage. BMC Infect Dis 10:334
Penna-Coutinho J, Cortopassi WA, Oliveira AA, França TC, Krettli
AU (2011) Antimalarial activity of potential inhibitors of Plas-
modium falciparum lactate dehydrogenase enzyme selected by
docking studies. PLoS One 6:e21237
Perkins DJ, Were T, Davenport GC, Kempaiah P, Hittner JB,
Ong’echa JM (2011) Severe malarial anemia: innate immunity
and pathogenesis. Int J Biol Sci 7:1427–1442
Trager W, Jensen JB (1976) Human malaria parasites in continuous
culture. Science 193:673–675
Upadhyay HC, Dwivedi Gaurav R, Roy S, Sharma A, Darokar MP,
Srivastava SK (2014) Novel phytol derivatives as drug resistance
reversal agents. Chem Med Chem 9:1860–1868
WHO (2012) World Malaria Report. Geneva. http://www.who.int/ma
laria/publications/world_malaria_report_2012/wmr2012_summa
ry_en.pdf?ua=1