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 < 0.05 were regarded as statistically significant.       3 Results                                                                    3.1 Virus infection model established in THP-1 cell line Virus infection model was established in THP-1 cell line as per protocol published in our previous paper.   3.2 LC-MS analysis Papaya leaf aquous extract was prepared and on LCMS analysis followed by integrated library research, carpaine was found to be major constituent of molecular mass 479.384 in the PLE extract.(Fig. 1)   3.3  Cell viability assay  The cytotoxicity of  PLE  in  THP-1 cells was tested by MTT assay. Treatment of  cells with  PLE at concentration of 50, 75,100 and 200 µg/ml does not result in any cytotoxicity  in THP-1 cells even upto 72 h of incubation. Lower doses of PLE upto 75 µg/ml, in fact causes slight cell proliferation. Cell viability at 300 µg/ml slightly decreased to 94.22 ± 0.57% at 72 h of incubation in THP-1 cells (Fig. 2A-B). Lower doses of PLE 100 and 200 µg/ml respectively were chosen to determine their antiviral activity.   3.4 Determination of antiviral activity of PLE  Effect of PLE on DENV infection was determined by evaluating envelope protein expression of dengue by western blotting. DENV infected and PLE (100 µg/ml)  treated THP-1 cells showed a marked decrease of more than four fold in envelope protein expression, in comparison to the DENV infected cells (Fig. 3A  and 3B). PLE (200 µg/ml) dose completely inhibited envelope protein expression. Similarly, NS1 protein expression was also significantly decreased by more than four fold by 100 µg/ml PLE treatment in DENV infected cells. (Fig. 4A and 4B ) indicating the antiviral activity of PLE.   3.5 Determination of  Intracellular viral load Intracellular viral load in DENV infected and PLE treated THP-1 cells was determined by FACS analysis. PLE at 100 µg/ml decreases mean fluorescent intensity (MFI) of FITC labelled DENV-2 specific antibody decreased by 1.5 fold from 76.6 ±10.5 to 49.15 ± 9.3  and 200 µg/ml dose  decrease to 47.25 ± 6.7 (Fig.5A and 5B) in PLE treated THP-1 cells in comparison to virus infected cells, hence  further confirming the antiviral activity of PLE against DENV.   3.6 Determination of IFN-?  by ELISA Changes in the expression of IFN-? cytokines during DENV infection and PLE treatment were investigated by ELISA. The expression of IFN-? was found to be almost 1.6 fold higher in DENV-infected THP-1-cells treated with PLE (200 µg/ml) than in the control, increasing significantly from 213.57 ± 28.98  pg/ml to 343.341 ± 12.35 pg/ml.(Fig. 6)   3.7 Haemolytic activity This assay was performed to determine haemolytic activity of PLE at different concentration in human blood. Distilled water was used as positive control and 0.5% saline as negative control. PLE extract does not exhibit haemolytic activity  at any of the dose (40 mg/ml, 20 mg/ml, 10 mg/ml, 5 mg/ml and 2.5 mg/ml) in comparison to the saline treated cells.(Fig. 7)   3.8 Anti-hemolytic activity Reactive oxygen species are a major cause of oxidative damage and hemolysis of the RBCs. We determine the effect of PLE on hydrogen peroxide (H2O2) induced oxidation of RBCs by anti-hemolytic assay.PLE was found to exhibit significantly high anti-hemolytic activity in comparison to negative control (H2O2) at different concentrations of PLE (40 mg/ml, 20 mg/ml, 10 mg/ml, 5 mg/ml and 2.5 mg/ml). PLE  shows significantly high anti-hemolytic activity even more than ascorbic acid (positive control) for 40 mg/ml,20 mg/ml and 10 mg/ml dose.(p<0.001).(Fig.8)   3.9  Platelete augmentation activity of PLE Platelet augmentation effect of PLE was determined in two phases of experiments. In the first phase, dose of cyclophosphamide and PLE was standardized so as to ensure survival of the rats. Cyclophosphamide dose was given subcutaneously (s/c) at 50 mg/kg for two consecutive days and PLE extract was administered orally at 200 mg/kg through gavage. Average platelet counts of Control,Cyclo, papaya only, prophy and thera  groups were 595.33±15.9 ×103/?L, 623.833±         55.6×103/?L, 572± 25.61×103/?L, 559.333± 31.3 ×103/?L and  576.333± 24.09 ×103/?L respectively on the first day of the experiment. Platelet counts within the cyclo and therapeutic group started to fall after Day 3 (239.5 ± 40.63×103/?L in cyclo and  252.33 ± 20.36 × 103      /?L in thera) and remarkable thrombocytopenia developed after 7 days (200.5 ±31.26 × 103/?L    and  ± 172.66 ± 14.1 × 103/?L in thera).PLE was given from day one in prophylactic group, interestingly PLE does not let platelet count to fall in this group (415 ± 13.2 ×103/µl) on day 4 and (466.83± 41.07×103/?L) on day 7. In therapeutic group PLE was given from day 7 after cyclophosphamide treatment and platelet counts observed to increase on day 11 ( 389 ± 47.3 × 103/?L) and reach a normal level at Day 14 (578.33 ± 57×103/µl). (Fig. 9)   3.10  Histopathological analysis of Rat liver, spleen and kidney The histology of liver, spleen and kidney presented normal morphology in control, PLE only, prophylactic and therapeutic group rats. Hepatocytes in control rats showed normal cell morphology  with well-preserved cytoplasm and nucleus on the other hand cyclophosphamide treated rats showed altered staining characteristic of nucleii. The liver morphology of rats treated with PLE only, prophylactic treated group, therapeutic group was similar to the control group. Histopathology of spleen shows that both cyclophosphamide treatment and PLE treatment in does not cause any significant change in splenocyte morphology. Renal tubules in control rats shows normal cell structure lined by columnar epithelium while cyclophosphamide treated cells showed renal tubules having edematous changes in epithelial cell lining.PLE only, prophylactic and therapeutic treated rats shows normal renal tubules.(Figure 12, 13 and 14)   3.11 Estimation of  IL-6 and TPO in Rat plasma of thrombocytopenic rats by ELISA IL-6 cytokine is reported to stimulate thrombopoiesis through thrombopoietin. We evaluated the expression of IL-6 cytokine and TPO in plasma of cyclophosphamide induced thrombocytopenia rat model. It was found that  IL-6 level increases significantly in papaya treated group in comparison to cyclophosphamide group from 262.83 ± 14.04 pg/ml to 523.17 ±  25.7 pg/ml on day 14 (p>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)