Preclinical and Clinical Studies to Evaluate the Effect of Carica papaya Leaf Extract on Platelets

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RESEARCH ARTICLE
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Preclinical and Clinical Studies to Evaluate the Effect of Carica
papaya Leaf Extract on Platelets
Deepak K. Semwal1,*, Randhir S. Dahiya2, Naveen Joshi3, Ruchi B. Semwal4,
Sonali Aswal5, Ankit Kumar5, Ashutosh Chauhan6 and Abhimanyu Kumar7
1Department of Phytochemistry, Faculty of Biomedical Sciences, Uttarakhand Ayurved University,
Harrawala, Dehradun-248001, India; 2Department of Pharmacology, Maharishi Markandeshwar
College of Pharmacy, MM University, Mullana, Ambala-133207, Haryana, India; 3Department of
Swasthavritta and Yoga, Faculty of Ayurveda, Uttarakhand Ayurved University, Harrawala, Deh-
radun-248001, India; 4Department of Chemistry, Pt. Lalit Mohan Sharma Government Postgradu-
ate College, Rishikesh- 249201, Uttarakhand; 5Research and Development Centre, Faculty of Bio-
medical Sciences, Uttarakhand Ayurved University, Harrawala, Dehradun-248001, India;
6Department of Biotechnology, Faculty of Biomedical Sciences, Uttarakhand Ayurved University,
Harrawala, Dehradun-248001, India; 7Vice Chancellor, Uttarakhand Ayurved University, Harra-
wala, Dehradun-248001, India
Abstract: Background and Objectives: Carica papaya leaves have been used in traditional
medicine in Asian countries mainly in treating dengue. Various earlier studies proved their
role in increasing platelet count in dengue patients. A precise mechanism of their action re-
mains unknown. The present study aimed to evaluate the effect of C. papaya leaf extract on
platelets and unravelling its possible mode of action.
Materials and Methods: The methanol (MEA) and acetone (ACA) extracts were studied for
antiplatelet activity and bleeding time in rats whereas the decoction of leaves powder was
clinically used in patients with low platelet count.
Results: The results showed that MEA, ACA and positive control (heparin) attenuated the
platelets aggregation by 68%, 56% and 72%, respectively. On the other hand, MEA and
ACA at 400 mg/kg significantly increased bleeding time in rats by 3 and 4 seconds, respec-
tively, while the standard (aspirin) at 100 mg/kg increased the bleeding time by 7 seconds in
comparison to the control. The clinical studies revealed that decoction of the leaf (1 g thrice
a day) significantly increased the platelet count in the patients.
Conclusion: The study concludes that the papaya leaf has potential to increase platelet count
via attenuating their aggregation.
A R T I C L E H I S T O R Y
Received: August 20, 2018
Revised: November 03, 2018
Accepted: January 08, 2019
DOI:
10.2174/2215083805666190124162640
Keywords: Aedes aegypti, Carica papaya, Aspirin, Dengue, Platelet aggregation, Thrombocytopenia.
*Address correspondence to this author at the Department of
Phytochemistry, Faculty of Biomedical Sciences, Uttarak-
hand Ayurved University, Harrawala, Dehradun-248001,
India; Tel: +91-987-676-1502;
E-mail: dr_dks.1983@yahoo.co.in
1. INTRODUCTION
Dengue is a deadly disease caused by 4 distinct,
but closely related serotypes of the virus called
DEN-1, -2, -3 and -4, mainly transmitted by fe-

298 Current Traditional Medicine, 2018, Vol. 4, No. 4 Semwal et al.
male Aedes aegypti mosquito. Delay in its treat-
ment may cause severe dengue including severe
mucosal bleeding [1]. According to the WHO
(2009) report, it has different clinical presentations
with unpredictable clinical evolution and outcome.
A continuous decrease in the platelets (thrombo-
cytes) caused thrombocytopenia, a stage when the
platelet count reached below 100×103/µL (normal
range 150-450×103/µL). In this condition, the
bleeding starts outside or inside the body that may
result in death [2]. As dengue is a viral disease,
there is no specific medicine or treatment available
except to treat only its symptoms in which complete
rest and adequate fluid intake are highly important
[3]. A specific and reliable treatment for dengue is a
need of the hour. Hence, scientists from the world
over are looking for a solution against this deadly
viral disease.
Carica papaya L. (Caricaceae), popularly
known as Papaya, grows widely in tropical and
subtropical regions, and is now cultivated world-
wide mainly for its edible fruits. Other parts of the
plant such as leaf, seed and latex are used in tradi-
tional medicine for a variety of health benefits [4].
The juice of the leaf of the plant is used as a tradi-
tional remedy for dengue fever in South Asian
countries [5-7]. Phytochemical reports revealed that
the leaves of the plant contain flavonoids, saponins,
cardiac glycosides, anthraquinones and alkaloids in
which carpaine, pseudocarpaine, dehydrocarpaine I
& II, kaempferol and quercetin are some individual
bioactive compounds [8]. Besides, papain, chymo-
papain, myricetin, caffeic acid, trans-ferulic acid,
manghaslin, clitorin, rutin, nicotiflorin, chitinase,
glutaminyl cyclase, cysteine endopeptidases of
class-II & III, benzyl isothiocyanate and linalool are
other secondary metabolites isolated from different
parts of C. papaya [4,9,10]. Many recent studies in
different experimental models confirmed its an-
tiplasmodial [11], anti-thrombocytopenic [10], anti-
inflammatory, immunomodulatory [12], nephropro-
tective [13], antiulcerogenic [14], antidiabetic [15],
antioxidant [16], and anticancer [17] activities. Alt-
hough previous studies proved the role of papaya
leaf extract in platelet count, no certain mode of its
action is yet available. Herein, we attempted in
vitro, in vivo and clinical experiments all together
to understand the actual mechanism of papaya leaf
extract on platelet count.
2. MATERIALS AND METHODS
2.1. Animals and Reagents
Wistar albino rats of either sex weighing 150-
200 g were used in the present study. All the animal
experiments were conducted at the Maharishi Mar-
kandeshwar College of Pharmacy, Ambala (Animal
Ethical Approval No. 585/05/A/CPCSEA) with
proper care of animals as per the guidelines of
Committee for the Purpose of Control and Super-
vision on Experiments on Animals (CPCSEA).
The animals were allowed commercial rat diet and
water ad lib throughout the experimental period.
The clinical studies were conducted at the Utta-
rakhand Ayurved University hospital (Human Eth-
ical Approval No. 3233/UAU/2017-18) under the
supervision of expert clinicians by obtaining writ-
ten consent from the patients. The clinical samples
were examined at the hospital pathology and a
separate register was maintained for the patients
who participated in the study. The chemicals used
for the extraction, phytochemical studies and other
experiments were purchased from Sigma Aldrich,
Germany and all these were of analytical grade.
2.2. Plant Material
Mature leaves (excluding the leaves turning
yellow) of C. papaya were collected from the
nearby area of Uttarakhand Ayurved University,
Dehradun. The plant material was authenticated
from Dravyaguna department of the University
and a specimen of the plant (Voucher No. UAU-
152) has been deposited in the laboratory for fu-
ture record.
2.3. Extraction
The fresh leaves (5 kg) were dried in a hot air
oven with a maintained temperature at 40 °C for
48 hrs. For preliminary phytochemical analysis,
the dried leaves were crushed to form a powder
(0.5 kg) and extracted separately with water,
methanol, acetone, chloroform and hexane using 5
g powder in 100 mL solvent each for 24 hrs at
room temperature. The extraction process was re-
peated three times for 24 hrs each to obtain water
extract (WE = 13.64%), methanol extract (ME =
11.93%), acetone extract (AE = 6.55%), chloro-
form extract (CE = 8.17%) and hexane extract (HE
= 4.85%). After drying, the respective extracts
were weighed and percentage yields were deter-
mined separately. The similar procedure was fol-

Preclinical and Clinical Studies Current Traditional Medicine, 2018, Vol. 4, No. 4 299
lowed to obtain the desired extracts for in vitro and
in vivo studies. The solvents were evaporated to
dryness in a rotary evaporator (Equitron Medica
Pvt. Ltd., India) with the maintained temperature
at 40°C, and the resulting extracts were stored in
screw-capped vials at 4°C temperature until fur-
ther used.
2.4. Phytochemical Analysis
Previously used methods for phytochemical
screening was adopted to determine the presence
of major classes of secondary metabolites in vari-
ous extracts of the papaya leaves [18, 19]. Chemi-
cal tests adopted for preliminary phytochemical
analysis of the extract are shown in Table 1.
2.5. Acute Toxicity
The toxicity was measured as per the standard
protocol in accordance with the Organization for
Economic Cooperation and Development guide-
line 423 adopted on December 17, 2001 [20].
Briefly, a single dose of the extracts at 2 g/kg per
oral was given to rats and thereafter, observations
were made once daily for a period of 14 days and
the animals were weighed every 4 days. The ob-
servation focused on mortality, behaviour change,
skin, eyes, fur, and somatomotor activity. Atten-
tion was directed to observations of tremors, con-
vulsions, salivation, diarrhoea, lethargy, sleep and
coma.
2.6. Antiplatelet Activity
Platelet-rich plasma (PRP) from rats was pre-
pared by centrifugation (1000 rpm for 5min) of
blood collected from normal rats. Acid citrate dex-
trose (1.5 mL) was used as an anticoagulant for
every 8.5 mL of blood and cuvettes were incubat-
ed at 37°C for 5 min. The aggregation was initiat-
ed by adding 20 µL of adenosine diphosphate
(ADP) to 1 mL of PRP. The aggregation was rec-
orded for 5 min at 600 nm. The effect of different
concentrations (50-250 µg/mL) of Carica papaya
Table 1. Chemical tests adopted for the phytochemical screening of Carica papaya leaf extracts.
Secondary Metabolites
Chemical Test (Color/Appearance)
Alkaloids
Mayer’s test (cream precipitate) and Dragendorff’s test (orange precipitate)
Anthraquinones
Borntrager’s test including modified Borntrager’s test (pink to red)
Cardiac glycosides
Keller–Kiliani test (formation of reddish brown ring)
Flavonoids
Ferric chloride test (red-brown) and Shinoda’s test (pinkish-red)
Iridoids
Trim–Hill test (coloured appearance)
Lignins
Labat test (olive green colour) and Lignin test (formation of red colour)
Resins
Turbidity test (coloured appearance with turbidity)
Saponins
Foam test (white foam) and sodium bicarbonate test (formation of froth)
Steroids
Salkowski test (reddish-brown) and Liebermann–Burchard test (blue-green)
Tannins
Gelatin test (white precipitate) and lead acetate test (reddish brown bulky precipitate)
Terpenoids
Salkowski test (reddish-brown) and Liebermann–Burchard test (blue-green)
Phenolics
Ferric chloride test (intense colour) and Ellagic acid test (brown muddy precipitate)
Proteins/ amino acids
Million’s test (white precipitate terns to red on heating); Ninhydrin test (blue colour)
Fatty acids
Greasy spot test (oil stains)
Quinones
Potassium hydroxide solution (range of red to blue colour)
Volatiles
Copper sulphate test (clear solution); Sudan red test (red colour precipitate)
Carbohydrates
Molisch’s test (violate ring formation); Fehling’s test (first yellow then brick red precipitate)
Coumarins
Sodium hydroxide solution (evolution of yellow fluorescence)

300 Current Traditional Medicine, 2018, Vol. 4, No. 4 Semwal et al.
leaf extracts (methanol and acetone extracts) was
assessed by incubation with PRP at 37°C for 5 min
before the addition of ADP. Commercial heparin
(Sigma-Aldrich/Merck) was used as reference
standard. The maximal aggregation was recorded
as per Alban and coworkers [21]. The aggregation
is expressed as % inhibition (X) calculated by us-
ing the following equation:
X (%) = (A-B)/A×100
Where, A=maximal aggregation of the control,
and B=maximal aggregation of drug-treated PRP
2.7. Evaluation of Bleeding Time
Bleeding time in rats was evaluated using an
adapted method described by Dejana and cowork-
ers [22]. All samples and vehicle were given orally
60 min prior to experiment. The incision was made
2 mm from the tip of anaesthetised rat tail and the
blood was soaked on a filter paper, which was
monitored at an interval of 2s till the bleeding
stopped. The time elapsed from the tail tip incision
to the stoppage of bleeding was recorded as the
bleeding time. Aspirin (Sigma-Aldrich/Merck) at
100 mg/kg was used as a standard.
2.8. Clinical Studies
To assess the efficacy of the extract on platelet
count in human patients, a decoction of 1 g of the
papaya leaf was given orally thrice a day. The de-
coction was prepared as per Ayurvedic standards
[23]. Briefly, 1 g of papaya leaf powder was mixed
with 100 mL of water and boiled in an open metal-
lic pot until the volume of water remained one
fourth. The resulted decoction was filtered and left
at room temperature for some time before being
consumed. The platelets were counted before the
treatment and after 24, 48 and 72 hrs after treat-
ment with a follow-up of 15 days up to 3 months.
Fifty patients were selected for this study who
came to the out-patient department (OPD) for the
treatment of acute viral fever. All the patients se-
lected for this study were clearly explained about
the treatment and a written consent was obtained
from all of them. The patients having platelet
count below 100×103/µL, diagnosed cases of den-
gue, patients taking platelet inhibiting drugs like
aspirin and inherited platelet disorder were ex-
cluded from the study. The average values of
platelet count were calculated for the data of be-
fore treatment and after treatment.
2.9. Statistical Analysis
Statistical analysis was carried out using
GraphPad Prism 6. Values were expressed as mean
± SEM and one-way analysis of variance (ANO-
VA) was used for statistical analysis. ANOVA was
followed by Tukey’s as post hoc multiple compar-
ison test. The results were considered significant if
p ≤ 0.05.
3. RESULTS
3.1. Phytochemical Screening
The preliminary phytochemical screening stud-
ies revealed that the plant contains alkaloids, fla-
vonoids, saponins and tannins as main secondary
metabolites. The phytochemicals present in the
different extracts of Carica papaya leaves are giv-
en in Table 2. Herein, based on the diversity of
secondary metabolites, only methanol and acetone
extracts were used for further in vitro and in vivo
studies.
3.2. Acute toxicity
The toxicity study revealed that the leaf extract
of papaya was found safe for the animals up to 2
g/kg body weight. It did not show any toxic sign in-
cluding mortality, lethargy, sleep, coma, diarrhoea,
behaviour change and somatomotor activity.
3.3. Antiplatelet Activity
The platelet aggregation study revealed that at
higher concentration i.e. 500 µg/mL, both metha-
nol and acetone extracts showed significant activi-
ty (p<0.005) by inhibitory percentage of 68% and
56%, respectively, which was found comparable to
that of heparin, a standard which showed activity
by 72% (Table 3). The inhibitory effect of extracts
at the concentration up to 200 µg/mL was found
non-significant.
3.4. Bleeding Time in Rats
The results (Fig. 1) for a bleeding time showed
that the methanol extract is more effective than
that of acetone extract at a lower dose. However,
at higher doses, both the extracts showed almost
similar results. The results for ME at 100, 200 and
400 mg/kg were found significant when compared
with the control whereas, for AE, the results with
200 and 400 mg/kg were found significant. The
positive control, aspirin was found most significant

Preclinical and Clinical Studies Current Traditional Medicine, 2018, Vol. 4, No. 4 301
Table 2. Secondary metabolites present in different extracts of Carica papaya leaf.
Secondary Metabolites
Water
Methanol
Acetone
Chloroform
Hexane
Alkaloids
+
+
+
+
-
Anthraquinones
-
-
-
-
-
Cardiac glycosides
+
+
-
-
-
Flavonoids
+
+
+
-
-
Iridoids
-
+
+
-
-
Lignins
-
-
-
-
-
Resins
-
+
+
-
-
Saponins
+
-
-
-
-
Steroids
-
+
+
+
+
Tannins
+
+
+
-
-
Terpenoids
-
+
+
+
+
Phenolics
+
+
+
+
-
Proteins/ amino acids
+
-
-
-
-
Fatty acids
-
-
-
-
-
Quinones
+
-
-
-
-
Volatiles
+
+
-
-
+
Carbohydrates
+
+
-
-
-
Coumarins
+
+
-
+
-
Where, positive (+) stands for present and negative (-) stands for absent.
Fig. (1). Effect of Carica papaya leaf extract on bleeding time in rats. ME (methanol extract); AE (acetone extract);
Asterisk (*) denoted for significant values (p<0.005).

302 Current Traditional Medicine, 2018, Vol. 4, No. 4 Semwal et al.
Table 3. Effect of Carica papaya leaf extract on platelet aggregation.
Concentration
Percentage Inhibition per Minute (±SD)
Methanol Extract
Acetone Extract
Heparin
050 µg/mL
09.19±4.3
06.81±6.4
17.88±4.5
100 µg/mL
16.24±5.6
13.24±5.6
21.98±6.2*
200 µg/mL
23.52±3.0*
18.16±1.0
34.23±1.5*
300 µg/mL
34.56±3.5*
30.96±1.5*
48.87±1.0*
400 µg/mL
54.02±2.6*
48.66±5.7*
59.34±3.0*
500 µg/mL
68.12±2.3*
56.40±2.0*
72.65±2.5*
Where, asterisk (*) means significant (p<0.005) values.
Fig. (2). Effect of Carica papaya leaf powder decoction on platelet count in patients with acute viral fever. Asterisk
denoted for significant values (p<0.005).
which delayed the bleeding time up to 21 seconds
at 100 mg/kg.
3.5. Clinical Studies
The decoction of the papaya leaf powder was
found effective in increasing platelet count in pa-
tients with acute viral fever. The effect at 48 and
72 hrs was found significant when compared with
the platelet count of before treatment.
4. DISCUSSION
For the past few years, papaya (Carica papaya
L.) leaf has been a subject of research due to its
possible role in dengue associated with thrombo-
cytopenia by increasing platelet count in the pa-
tients. Various studies have been conducted, main-
ly the clinical trials to evaluate its role in dengue
treatment [24], a precise mechanism of action, as
well as responsible bioactive constituents, still re-
main unknown. A clinical study on patients with
dengue fever and dengue hemorrhagic fever also
found that papaya leaf juice significantly increased
the platelet count within 48 hrs of treatment. This
study also revealed that ALOX 12 and PTAFR
genes were highly expressed among the patients
treated with juice [5]. The leaf powder was also
found active in in vivo models which suggested
that the leaf powder at 15 mg/kg significantly in-
creased platelet count in mice within 12 hrs [25].
An in vitro study by Chinnappan et al. [26] re-
vealed that papaya leaf extract capsule of 500 mg
once daily can directly act on platelet, and also pos-
sesses a neutralizing effect on dengue viral-infected
plasma that may exert a protective role on platelets.
However, the study could not clarify the mode of
action of papaya leaf extract on the patients. Aziz et
al. [27] designed an in vitro study with papaya leaf
extracts to understand the mechanism of its
thrombopoietic cytokines (interleukin 6 and stem
cell factor) induction. This study suggested that
treatment potentially increased the concentration of

Preclinical and Clinical Studies Current Traditional Medicine, 2018, Vol. 4, No. 4 303
both interleukin 6 and stem cell factor by mesen-
chymal stem cells and haematopoietic cells.
On the other hand, the responsible constituent
of papaya leaf for the anti-dengue activity is also
unknown. In the present study, the aggregation of
platelets was found to be attenuated in a dose-
dependent manner. The methanol extract was
found to be more effective than acetone extract
which suggested that the polar constituents includ-
ing glycosides of flavonoids and steroids might be
responsible for the antiplatelet activity.
Senthilvel et al. [28] studied quercetin, isolated
from papaya leaf against dengue virus and found
that the molecule showed strong binding energy
against the viral non-structural 2B and 3-protease
which is evident by the formation of six H-bonds
with amino acid residues at the binding site of the
receptor. The leaf juice (30 µL) was shown to have
a significant inhibition of hemolysis in vitro and
also a potential therapeutic effect on disease pro-
cesses causing destabilisation of biological mem-
branes [29].
The results of clinical studies in the present
work showed a significant increase in platelet
count in the patients of acute viral fever. The study
also found that the leaf powder in the form of a
decoction was safe as no side effects have been
observed during and after the treatment up to 3
months. The present study mainly highlights the
effect of papaya leaf extract on platelets using both
preclinical and clinical experiments to understand
the mechanism of action. The preclinical experi-
ments suggested its antiplatelet action while the
clinical study showed its potential in increasing
platelet count. In addition, the detailed phytochem-
ical profile of the extracts revealed that alkaloids,
cardiac glycosides, flavonoids, iridoids, steroids,
tannins and terpenoids are perhaps synergistically
responsible for the activity.
CONCLUSION
The papaya leaf extract significantly attenuated
the aggregation of platelets in rats when compared
to that of control. The bleeding time in rats was
significantly increased after treatment with papaya
leaf extract. The activity with crude extract was
found comparable to that of standards. On the ba-
sis of in vivo studies, it has been found that the
methanol extract is more effective than that of ace-
tone extract at 100 mg/kg. However, the methanol
extract, at higher doses i.e. 200 and 400, was
found comparatively lesser effective. The clinical
study suggested that the extract has potential in
increasing the platelet count in patients with acute
viral fever. The dual action i.e. increasing platelet
count and attenuating aggregation suggested that
the papaya leaf can be given to the dengue patients
with a high risk of blood clots, stroke, and heart
attack.
ETHICS APPROVAL AND CONSENT TO
PARTICIPATE
All the animal experiments were conducted at
the Maharishi Markandeshwar College of Pharma-
cy, Ambala (Animal Ethical Approval No. 585/05/
A/CPCSEA).
HUMAN AND ANIMAL RIGHTS
Both human & animals have been used for this
study. According to guideline set-up by UAU and
Helsinki Declaration (modified 1989), the follow-
ing guidelines were adhered to all the patients in-
volved in this study.
CONSENT FOR PUBLICATION
Written informed consent was obtained from all
the patients included in the study.
CONFLICT OF INTEREST
The authors declare no conflict of interest, fi-
nancial or otherwise.
ACKNOWLEDGEMENTS
This research was funded by National Medici-
nal Plants Board, Ministry of AYUSH, Govt. of
India, grant number Z.18017/187/CSS/R&D/UK-
01/2017-18-NMPB-IV A.
AUTHOR CONTRIBUTIONS
Conceptualization by Deepak Kumar Semwal
and Ruchi Badoni Semwal; Methodology by
Randhir Singh Dahiya and Ankit Kumar; Software/
Statistics by Ashutosh Chauhan; Validation by
Deepak Kumar Semwal and Randhir Singh Dahi-
ya; Formal Analysis by Randhir Singh Dahiya;
Clinical investigation by Naveen Joshi; Writing-
Original Draft Preparation by Ruchi Badoni
Semwal and Sonali Aswal; Writing-Review & Ed-
iting by Abhimanyu Kumar and Deepak Kumar
Semwal; Project Administration by Deepak Kumar

304 Current Traditional Medicine, 2018, Vol. 4, No. 4 Semwal et al.
Semwal; Funding Acquisition by Deepak Kumar
Semwal.
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