2.1.1 with the help of a grinder. Papaya

2.1.1 Plant material:

Fresh and middle stage age carica
papaya leaves were purchased from a nursery in Delhi, India. Leaves were
thoroughly washed with running tap water and then shade dried. Leaves were then
finely powdered with the help of a grinder. Papaya leaves powder was then
weighed and mixed in autoclaved Milli Q water  to make papaya leaf aqueous extract (PLE). The
extract was further filtered and stored at 4oC   for further experiments.

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2.1.2 Cells :

THP-1 cell line, maintained in RPMI (Sigma) was used
as an infection model of DENV and the C6/36 cell line, maintained in MEM
(Sigma) was used to propagate DENV. RPMI was supplemented with 10 % Fetal
bovine serum (FBS) (Sigma), 100 U penicillin (Sigma), and 100 µg streptomycin
(Sigma), per ml at 370C in a 5% CO2 atmosphere in an
incubator (Sanyo, Japan).

 

2.1.3 Animals :

Male Sprague Dawley rats weighing about 180-200 gm and age
between 8-9 weeks were used for cyclophosphamide induced thrombocytopenia study.
Two experimental trials were done using 18 animal in first study and 30 in
second. First experiment was a pilot study to standardize the cyclophosphamide
dose, its duration and minimum effective dose of papaya. Second experiment
provided the result presented here. Animals were housed in standard conditions
of temperature and light in the animal house of DIPAS,DRDO. Ethical clearance
was taken from the animal ethical review committee of  DIPAS, DRDO Delhi. The animals were given
standard rat foods and allowed to drink water. Proper cleaning measures were
taken regularly.

 

2.2  Development of virus infection model

 

DENV serotype 2 New Guinea C strain was
propagated in C6/36 cell line and virus stock 
so obtained was used to infect THP-1 cell line as per protocol already
published in our paper (Sharma et al. 2016) .THP-1 cells were infected with
DENV as per protocol already published in our paper (Sharma et. al. 2016).
After viral infection cells were harvested, and the cell-free supernatant was
stored at -80oC until assayed for cytokine profiling.

 

2.3 LC-MS analysis

The PLE extract was
dried under a gentle stream of nitrogen and reconstituted in methanol. The
compounds present in the extract were analyzed by LC-MS/MS. The UPLC system was
coupled to a hybrid quadrupole, orthogonal time-of-flight (Q-TOF) tandem mass
spectrometer (SYNAPT G2 HDMS, Waters, Manchester, U.K.) equipped with ESI. The
chromatographic separation was performed on an Acquity UPLC BEH C18 Column (3.0
mm × 150 mm, 1.7 ?m, waters, Ireland) at a temperature of 40°C. The mobile
phases consisted of eluent A (0.1% formic acid in water, v/v) and eluent B
(0.1% formic acid in acetonitrile, v/v). These eluents were delivered at a flow
rate of 0.4 mL/min with a linear gradient program as follows: 20–80% B from 0
to 15 min, 80–95% B from 15.0 to 15.5 min, held at 95% B from 15.5 to 18.0 min,
95-20% B from 18.0 to 19.0 min and held at 25% B from 19.0 to 20.0 min. The
operating parameters were as follows: capillary voltage of 3 kV (ESI+), sample
cone voltage of 35 V, extraction cone voltage of 4 V, source temperature of
100°C, desolvation temperature of 300°C, cone gas flow of 50 L/h and
desolvation gas flow of 800 L/h. In MSE mode, the trap
collision energy for the low-energy function was set at 5 eV, while the ramp
trap collision energy for the high-energy function was set at 20–50 eV. Argon
was used as the collision gas for collision-induced dissociation (CID) in MSE mode.
To ensure mass accuracy and reproducibility, the mass spectrometer was
calibrated over a range of 50–1500 Da using a solution of sodium formate.

 

2.5 Cell
viability assay

 

Cytotoxicity of
papaya in THP-1 cells was determined by MTT assay. THP-1 cells were treated
with PLE at different concentrations of 50, 75, 100, 200 and 300 µg/ml  for 24 h, 48 h and 72 h. The cytotoxicity of
PLE  in THP-1 cells was determined by
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) (Sigma
–Aldrich, USA) dye . 10 µl of MTT stock was added to  the cells and further incubated for 4 h at 370C.
MTT, a yellow tetrazole was reduced to purple formazan crystals by NADPH
dependent oxidoreductase enzyme present in viable THP-1 cells. The crystals
were dissolved by addition of DMSO and the optical density was measured  at 570 nm using a spectrophotometer (Biotek
Instruments, USA)

 

2.6
Determination
of antiviral activity
of PLE

 

Mock infected
and virus infected THP-1 cells were treated with or without PLE at 100 µg/ml
and 200 µg/ml dose, incubated for 48 h and whole cell lysate was prepared.Protein
was estimated by Bradford method and 40 µg protein of each sample was loaded
on  10% 
sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to
polyvinylidene difluoride (PVDF) membranes. Binding of   non specific antibodies was blocked by 3 %
BSA in TBS buffer (0.1 M Tris – HCl, pH 7.4 ,0.9% NaCl), followed by washing
with TBST20 (0.1% Tween-20 in TBS) 
and incubated in primary antibody,
rabbit anti dengue envelop
protein (Cat. No. PA5-32246, Thermoscientific USA) and NS1 protein (Cat No.
SAB2700022 SIGMA). PVDF membrane was again washed thrice with TBST20 and
incubated with goat anti rabbit immunoglobulin (IgG) biotinylated (Cat. No.
B8895 SIGMA)  and streptavidin peroxidase
(Cat.No. S2438 SIGMA). The proteins were detected by chemiluminescence .

 

2.7 Intracellular
staining of DENV

DENV infected
THP-1 cells at a concentration of 2 × 106 cells/ml were treated with
PLE 100 and 200µg/ml  and incubated for
48 h. Cells were harvested and then washed twice with 0.01M PBS, fixed and
permeabilised with cytofix/ cytoperm buffer (BD Biosciences, USA) for 20 min
followed by incubation with anti-DENV FITC –conjugated monoclonal antibodies
(Biorbyt, UK) at a 1:200 dilution for 60 min. Unbound antibodies were removed
by washing it again with PBS. Cells were finally suspended in 0.5 ml PBS and
analyzed using a flow cytometer (BD FACS Calibur) with Cell Quest Pro software.

 

2.8 Determination of IFN-?  by ELISA

Virus infected THP-1 cells were
treated with PLE and incubated for 48 h in IRPMI supplemented with 2% FBS.
Supernatant was collected and kept at -800C. IFN-?   was determined by human IFN- ? ELISA kit
(Cat No. 201-12-0077 Sunred biological technology China)

 

 

 

2.9 Haemolytic
activity

 

Seven ml of
fresh venous blood was collected from healthy human volunteers (n=3) in 15 ml

falcon tubes
containing heparin, washed three times with sterile saline solution (0.9% W/v

NaCl, pyrogen
free)  by centrifugation at 1500 rpm for
five min. The pellet was re-suspended in

0.5% saline
solution. A volume of 0.5ml of the cell suspension was mixed with 0.5ml of

different
concentrations of PLE (40 mg/ml, 20 mg/ml, 10 mg/ml, 5mg/ml,2.5 mg/ml) in
saline solution. The mixtures were incubated at 37°C for 30 min and centrifuged
at 2000 rpm for 10 min. Free haemoglobin content in the supernatants was
measured spectrophotometrically at 540 nm. Saline and distilled water were used
as minimal and maximal haemolytic controls. The haemolysis percent was
calculated as:

% Haemolysis =
{(O.D.test-O.D.contol min)/ (O.D. control max-O.D. control min)}/*100

 

 

2.10 Anti-haemolytic
activity

 

Human RBCs were diluted in
PBS buffer to obtain a 4 % suspension. The plant extracts were prepared in PBS
buffer at five concentrations: 40, 20, 10, 5, and 2.5 mg mL-1. To 2
mL of RBC suspension we added 1 mL of plant extract (in the above
concentrations) and enough PBS to reach the final volume of 5 mL. After 5 min
of incubation at room temperature, 0.5 mL of 0.3 % H2O2 was added to induce
oxidative degradation of membrane lipids and the mixture was shaken at 37°C for
240 min. The samples were then centrifuged at 1500 g for 10 min and the
resulting supernatant was removed and used to evaluate their haemolytic
activity using a spectrophotometer (UV– Visible EZ201, Perkin Elmer, Norwalk,
CA, USA) at the absorbance wavelength of 540 nm. RBC lysis in the presence of H2O2
and absence of a plant extract was considered as 100 % haemolytic activity.
Haemolysis in the presence of extracts was calculated relative to this control haemolysis.
Haemolysis inhibition was calculated as follows:

%
antihaemolysis=(Ao?A1)/Ao×100

where Ao was the absorbance
of control (H2O2+RBC, without extract) and A1 the
absorbance in the of the extract or vitamin C as the reference
antioxidant  used in the same concentrations
as the extracts (02.5-40 mg mL-1).
Each set of experiments was performed in triplicate and the inhibitory activity
expressed as percentage.

 

 

2.11 Establishment of
Thrombocytopenia in the Rat Model

 

Animals
were divided in five groups of six animals each: Group one was control, in
which rats were given saline orally for fourteen days, second was cyclophosphamide
treated group, in which rats were given cyclophosphamide (50 mg/kg)  subcutaneously for two consecutive days , third
was papaya only treated group, in which rats were given papaya dose (200mg/kg)
or 40mg/ml  orally through gavage from
day one to day six , fourth group was cyclophosphamide treated and papaya dose
was given prophylactically from day one for six days and in  fifth group 
rats were cyclophosphamide treated and papaya dose was given
therapeutically i.e. after establishment of thrombocytopenia on day seven, for
six days.

Blood collection

Blood was collected from each of the rats by drawing  from retroorbital plexus of eye on the 1st,
4th, 7th 11th and 14th day of study. Analysis
of haematological parameters of rat blood was done by Haematological analyzer.

 

2.12Histopathological analysis

The
histopathologic analysis was performed by light microscopy. Liver, spleen and
kidney tissue sections were fixed in 10% buffered formalin. After fixation, the
sample were washed with running water and processed to obtain 5 ?m thick
paraffin sections. All sections were stained with hematoxilin and eosin (HE).

 

2.13 Estimation of TPO and IL-6 in
Rat plasma of thrombocytopenic rats by ELISA

Blood plasma from thrombocytopenic
rats was collected at different time point i.e. first day, fourth day, seventh
day, eleventh day and fourteenth day. It was stored at -800C for
cytokine analysis.TPO and IL-6 level was determined by ELISA (My Biosource, MBS262061
and e Bioscience) as per manufacturer’s protocol.

 

2.14 Statistical
analysis

Data were analyzed by using a
commercially available statistics software package (SPSS  for Windows, version 14.0, Chicago,
USA).One-way analysis of variance (ANOVA) test was performed. Results were
presented as means ± standard error 
(S.E.M),  p values 0.001) . Similarly
it was found to significantly increase in prophylactic group to 435.39 ± 61.7
pg/ml and  therapeutic group also to 465.81
± 70.07 on Day 14.TPO level was found to increase significantly in papaya only
treated group on day-11 and 14  (147.53±76.21 pg/ml  and 218.52 ± 11.7 pg/ml)  respectively 
in comparison to cyclophosphamide 
treated group (5.77 ± 2.9 pg/ml and 15.72 ± 3.7 pg/ml respectively)
(Fig. 15 and 16)

 

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