RSC Advances

RSC Advances
In silico docking, in vivo UHPLC-MS/MS analysis and

platelet enhancing potential of Carica papaya leaf juice and
its fraction in rodents
Journal: RSC Advances
Manuscript ID RA-ART-11-2017-012748
Article Type: Paper
Date Submitted by the Author: 24-Nov-2017
Complete List of Authors: Anjum, Varisha ; Jamia Hamdard, New Delhi, Department of
Pharmacognosy and phytochemistry
Singh, Anjali; Jamia Hamdard, New Delhi, Department of Pharmaceutical
medicine
Arora, Poonam; Jamia Hamdard Faculty of Pharmacy, Pharmacognosy and
Phytochemistry
Parveen, Rabea; Jamia Hamdard, New Delhi, Department of Pharmaceutics
Ahmad, Sayeed; Jamia Hamdard, New Delhi, Department of
Pharmacognosy and phytochemistry;
Subject area & keyword: Bioanalytical < Analytical

Page 1 of 28 RSC Advances
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RSC Advances Page 2 of 28
In silico docking, in vivo UHPLC-MS/MS analysis and platelet enhancing

potential of Carica papaya leaf juice and its fraction in rodents
Varisha Anjuma, Anjali Singhb, Poonam Aroraa, Rabea Parveenc, Sayeed Ahmada*
a
Bioactive Natural Product Laboratory, Department of Pharmacognosy & Phytochemistry, School of
Pharmaceutical Education and Research, Jamia Hamdard, New Delhi 110062, India.
b
Department of Pharmaceutical Medicine, School of Pharmaceutical Education and Research, Jamia Hamdard,
New Delhi 110062, India
c
Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi
110062, India
*Corresponding Author
Dr Sayeed Ahmad
Bioactive Natural Product Laboratory

School of Pharmaceutical Education and Research
Jamia Hamdard, New Delhi-110062, India
Email ID: sahmad_jh@yahoo.co.in

In silico docking, in vivo UHPLC-MS/MS analysis and platelet enhancing

potential of Carica papaya leaf juice and its fraction in rodents
Varisha Anjuma, Anjali Singhb, Poonam Aroraa, Rabea Parveenc, Sayeed Ahmada*
a
Bioactive Natural Product Laboratory, Department of Pharmacognosy & Phytochemistry, School of
Pharmaceutical Education and Research, Jamia Hamdard, New Delhi 110062, India.
b
Department of Pharmaceutical Medicine, School of Pharmaceutical Education and Research, Jamia Hamdard,
New Delhi 110062, India
c
Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi
110062, India
Carica papaya leaf juice (CPLJ) presents itself as promising platelet enhancing remedy in dengue fever. Present study was aimed to
evaluate the potential role of fresh CPLJ and its fractions on platelet count (PC) and coagulation factors after oral administration in murine
model followed by ultra high-performance liquid chromatography tandem mass spectroscopy (UHPLC-MS/MS) for pattern recognition and
pharmacokinetics study. Docking study was done on CPLJ constituents to reveal the effect of the bioactive butanol fraction (BBF) on
hematology and coagulation parameters. In acute toxicity study, none of the groups exhibited alteration in behaviour or reduction in food
and water intake. The PC (p ≤ 0.01) and total leukocytes count (TLC; p ≤ 0.05) were significantly increased in the BBF treated when
compared to that of control group on day 7. However, there was a significant reduction in the activated partial thromboplastin time (aPTT;
p ≤ 0.01) response. The results of docking studies of selected compounds in the C. papaya showed binding energy between -3.40 to -8.00
Kcal/mol, where myricetin and kaempferol showed the highest docking scores. The docking analysis suggested that the myricetin and
kaempferol might act as anti-thrombocytopenic agents. It can thus be concluded that the BBF can mediate the release of platelets from
megakaryocytes and stimulate the antithrombocytopenic effect, providing evidence for the treatment of dengue fever related
thrombocytopenia.
1. Introduction
Dengue is an acute viral infection with potential fatal complications. Dengue viruses (DENV) belong to family
Flaviviridae and has four serotypes referred to as DENV-1, DENV-2, DENV-3 and DENV-4. It is mainly transmitted
by Aedes aegypti mosquito and A. albopictus.1 All four serotypes can cause full spectrum of disease from a
subclinical infection to a mild self-limiting disease. The diagnosis of dengue infection is made clinically on the basis
of fever, myalgia and skin rash during an epidemic and confirmed by laboratory investigations.2
Thrombocytopenia is the hallmark of dengue patients, which usually develops after the initial acute febrile phase,
followed by critical period of 24-48h duration and lastly by recovery phase. Deaths due to dengue are usually a
consequence of patients developing complications like dengue hemorrhagic fever and dengue shock
syndrome,2,3 with a mortality rate of 10-20%. It mainly occurs due to progression of thrombocytopenia by
reduced platelet production in the bone marrow (BM) or excessive peripheral destruction of platelets and
development of increased vascular permeability and plasma leakage.4
Carica papaya L. (papaya) is native to Central America and now cultivated throughout the world. The
therapeutic effects of papaya leaves are presumed to be due to presence of several active components such as
papain, chymopapain, cystatin, L-tocopherol, ascorbic acid, flavonoids, cyanogenic glucosides and glucosinolates.5
Previous animal studies suggest that C. papaya leaf extracts have potential therapeutic effect on disease
processes causing destabilization of biological membranes, as they inhibit hemolysis in vitro.6 They may cause
increased platelet and red blood cell counts.7 Inspite of these small-scale studies, the fact remains that the
dengue is mostly a self-limiting disease with spontaneous increase in platelets during recovery phase. Keeping
these observations in consideration, it seems interesting to investigate the effect of C. papaya leaf juice (CPLJ) and
their bioactivity guided and enriched fractions on platelet count in thrombocytopenic mice followed by evaluation
of enhancing activity using pharmacokinetic and in silico docking studies.
2. Results and discussion
The % yield (w/w) for freeze dried powder (FDP), bioactive hexane fraction (BHF), bioactive dichloromethane
fraction (BDF), bioactive butanol fraction (BBF), bioactive aqueous fraction (BAF), carotenoid enriched fraction
(CEF), alkaloid enriched fraction (AEF) and glycoside enriched fraction (GEF) were found to be 4.84%, 0.33%,
0.28%, 0.86%, 3.42%, 0.26%, 0.21% and 1.51%, respectively. The FDP was used for the quality control analysis,
whereas all fractions were used to select the most active fraction. Further, the selected fraction was quantified
and validated by UHPLC-MS/MS for two flavonoids and four phenolics in rat plasma.
2.1. Standardization of FDP
The standardization of FDP for various primary and secondary metabolites showed the presence of carbohydrates
(17.80% w/w), glycosides (0.33% w/w), total alkaloids (03.25% w/w), total saponins (02.02% w/w), total
flavonoids (0.97% w/w), total phenolic (0.31% w/w) and tannins content (0.43% w/w).
2.2. Acute toxicity studies of FDP
No treatment related toxic symptom or mortality was observed after oral administration of the tested plant
extract at a dose of 300, 1000 and 2000 mg/Kg. The general behaviour of the FDP-treated animals and control
group was observed first for short period (4h) followed by long period (72h), did not display any drug related
changes in behaviour, breathing, skin effects, water consumption, impairment in food intake and temperature.
Therefore, the FDP seems to be safe at a dose level of 2000 mg/Kg, and the LD50 was considered to be >2000
mg/Kg. However, there were signs of sedation, lethargy and drowsiness after the administration of CPLJ and
fractions, when compared to control group. The parameters observed for acute toxicity study after the
administration of FDP compared with the normal group (Table S1).
In acute toxicity study, there was no mortality upto a maximum dose of 2000 mg/Kg body weight of Balb/c
after per oral administration. The changes in body weight have been used as an indicator of adverse effect of
CPLJ.8 Since, no remarkable changes were observed, hence it can be inferred that CPLJ is non-toxic at the doses
administered. Data analysis of animals’ blood parameters can be translated for risk evaluation in humans, since
the changes in hematological system have a higher predictive value for human toxicity.
2.3. Preliminary platelet enhancing potential of FDP and fractions
Amongst various bioactive fractions, treatment of animals with BBF and CEF significantly enhanced the production
of platelet count (PC; p<0.05) compared to untreated animals. The increase in cell count was also observed for
CPLJ, FDP and other fractions-treated animals but results were not as significant as BBF and CEF (Table 1). The BBF
treatment also showed significant increase in total leukocyte count (TLC) and activated partial thromboplastin
time (aPTT; p<0.05), when compared to animals treated with FDP, 3.43 g/Kg fresh leaves equivalent CPLJ and
other fractions (Table 1).
There was a transient increase in PC and TLC, which might be due to the increased absorption of iron in
presence of vitamin C. The result indicates that CPLJ is neither toxic to the circulating red cells, nor does it
interfere with their production. The fractions were also screened for clotting time and bleeding time that were
found significant (p<0.01) in BBF and CEF, when compared with normal control animals on day 0 and day 7,
respectively (Table 1).
The papaya leaf is a rich source of α-tocopherol, ascorbic acid, flavonoids, phenolics, cynogenetic glucosides
and glucosinolates5,9 and has been used since ages in dengue fever.7 Solubility of flavonoids and their glycosides
depend upon the polarity in solvent. In our study, moderately polar solvent fraction i.e., butanol, yielded maximum %
content of phenolic and flavonoids. Maximum pharmacological activity by BBF and CEF during bioactivity guided
screening may be due to presence of moderately polar flavonoid and their glycosides in C. papaya leaves.10
The multiple mechanisms mediated by many active principles in CPLJ may be responsible for increasing the
blood cell counts. The compounds stimulate and/or improve the megakaryocytes to produce sufficient number of
platelets to maintain a suitable platelet count in mammals, in particular during chemotherapy.11 Biologically active
compounds present in leaf are proteolytic enzymes, for example papain and chymopapain. As pro-platelet
formation is regulated by α-caspase (protease) activation, the protein digestion by these enzymes may increase
platelet count.12 The flavonols and flavonoids have shown anabolic effect,13 which may be responsible for
stimulant effect on blood cell production.
The inhibited heat-induced and hypotonicity-induced by CPL extracts possess membrane-stabilizing
properties and protect blood cells against stress-induced destruction and possibly prevent platelet lysis. This
effect could be due to the presence of flavonoids and other phenolic compounds in the papaya leaf.6 The aPTT
reflect the activities of multiple clotting factors, and it has been shown that, a significant decrease in any one
factor must occur before the PT or aPTT, is significantly prolonged.14 In particular, decreased factor VII activity is
supported by previous studies that describe a slight decrease of factor VII activity in fresh frozen plasma.15 This
decrease of factor VII activity also explains the small but significant decrease in the activity of factor X that was
reported in dogs, when samples were stored for 48h14 and in humans that occurred between baseline testing and
analysis after thawing the plasma samples.15 As factor X activity affects both prothrombin time and aPTT results,
marked loss of factor X activity may explain the statistically significant prolongation of both hemostatic tests
reported in the present study.
2.4. Quantitative estimation of BBF by UHPLC-MS/MS in rat plasma
Quantitative analysis was performed on a Waters Acquity UHPLC where optimum values for compound
dependent parameters like cone voltage (V) and collision energy (eV) were set at 28 and 24 for para-coumaric
acid; 30 and 18 for vanillic acid; 34 and 20 for caffeic acid; 32 and 16 for trans-ferulic acid; 60 and 36 for
kaempferol and 46 and 24 for myricetin, respectively. Unit mass resolution was employed and the dwell time was
set at 0.025 s. The detection of the ions was performed in the selected resonance monitoring (SRM) mode, by
monitoring the transition pairs (precursor to product ion) of m/z 162.94→92.915 for para-coumaric acid; m/z
166.904→151.96 for vanillic acid; m/z 178.94→106.897 for caffeic acid; m/z 192.94→133.954 for trans-ferulic
acid; m/z 284.913→92.914 for kaempferol and m/z 316.94→150.961 for myricetin. Mass Lynx software version
4.1 was used to control all parameters of UHPLC and MS.
2.4.1. Method validation. In order to achieve better peak shapes and a shorter run time for simultaneous
analysis of the six compounds, optimization of the mobile phase was conducted by using methanol, acetonitrile,
water and 0.1% formic acid as candidate solvents. Acetonitrile was found to improve the stability and resolution
of kaempferol and para-coumaric acid. Using 0.1% formic acid as the mobile phase additives to acetonitrile could
improve the peak shape of the analytes. Under the developed chromatographic condition, all analytes were
eluted out within four min. It was reported that cross-talk may exist to affect the accuracy of quantification, when
molecules are fragmentized within the ion source giving rise to multiple sources for the precursor ion selected.16
Specifically myricetin and kaempferol were easily fractured to produce corresponding polyphenol. No cross-talk
was observed between the flavonoids and polyphenols. Under the pre-condition of baseline separation, the
linearity of calibration curves, precision, accuracy, stability of the method were not significantly affected. The
developed method was reliable and suitable for the quantitative analysis of the six analytes.
2.4.2. Specificity and selectivity. The SRM chromatograms of analytes are shown in Fig. 1. The retention times
of vanillic acid, para-coumaric acid, caffeic acid, trans-ferulic acid, myricetin and kaempferol were 0.20, 0.20, 0.20,
0.21, 0.21 and 0.24 min, respectively. No significant interferences from endogenous components were observed
in samples. All the peaks of the analytes and samples were detected with excellent resolution and exhibited good
peak shape.
2.4.3. Relative sensitivity. The calibration curves for the six analytes were found to be linear in the
concentration range tested (Table 2). The regression coefficients (r) were higher than 0.990. The lower limit of
quantification (LLOQs), whose accuracy and precision met the acceptance criteria mentioned in the guidelines.
The LLOQ can be accurately quantified within a 20% bias of the nominal concentration and with a precision not
exceeding 20% in plasma. The negative ionization model was selected for the detection of six analytes. Due to the
presence of -OH, -COO, the negative ion ionization model had the higher efficiency for ionization and could offer
better sensitivity for six analytes. By optimizing the mass spectrometric parameters, the maximum abundance of
the parent ions and product ions for all analytes was obtained.
2.4.4. Linearity and sensitivity. The calibration curves and LLOQ of the six analytes are summarized in Table 2.
The correlation coefficient (r) for every calibration curve was higher than or close to 0.990.
2.4.5. Precision. The precision was obtained from multiple sampling of the homogenous sample under the
similar conditions. The exactness of the proposed method was obtained by repeatability and intermediate
precision. The inter-day, intra-day and intermediate precisions were determined and reported in terms of percent
relative standard deviation (%RSD) (Table 3).
2.4.6. Stability studies. The stability of the analytes in rat plasma under different storage conditions was
evaluated. The results shown in Table S2 indicate that the six analytes were stable in plasma samples through
three freeze-thaw cycles. The analytes were also found to be stable for at least 4h at room temperature, for 24h
in the autosampler after sample preparation and for long term stability.
2.4.7. Extraction recoveries, matrix effect and process efficiency. The mean extraction recoveries of the
investigated analytes at three different concentration levels in plasma were found to be 84.37 ± 1.91, 85.33 ±
3.65, 86.38 ± 2.64, 88.56 ± 2.06, 89.50 ± 0.14 and 90.48 ± 1.14% for vanillic acid, myricetin, para-coumaric acid,
caffeic acid, trans-ferulic acid and kaempferol, respectively. The recoveries of the low quality control (LQC),
medium quality control (MQC), and high quality control (HQCs) showed acceptable consistency (%RSD < 4%),
which indicates that the protein precipitation method was acceptable.
The liquid-liquid extraction (LLE) full form technique using ethyl acetate, or n-butanol as an extracting
solvent exhibited a low recovery and a high variability. Moreover, the procedure was complicated. In contrast, the
solvent-induced PP was found to be effective for the removal of proteins from rat plasma due to the low
probability of losses during the sample preparation. The results showed good sensitivity, acceptable recovery,
clear supernatants, and good separation between the interfering peaks, when methanol was used as the plasma
protein precipitation (PP) reagent. The acceptable limit of recovery for single laboratory validation provided a
mean recovery of 90-108% of analyte concentration at 0.1%.17
The matrix effect ranged from 76.43% to 92.40% for different QC levels with %RSD of less than 3.00%, which
demonstrates that the matrix effect on the ionization of the analytes is negligible. The process efficiency was
found in limits (ranged from 41.26% to 77.79% for different QC levels) with %RSD of less than 0.83% showing that
the method was acceptable.
2.5. Application in pharmacokinetic studies
Pharmacokinetic parameters of the myricetin, kaempferol, trans-ferulic acid, para-coumaric acid, caffeic acid and
vanillic acid are shown in Table 4. The plasma concentration-response curve of these six metabolites in rat is
shown in Fig. 2 demonstrating that phenolics was rapidly absorbed and then slowly decreased. The maximum
peak plasma concentrations (Cmax) of para-coumaric acid, caffeic acid, kaempferol, myricetin, trans-ferulic acid,
and vanillic acid were 07.92, 09.05, 10.48, 14.05, 25.35, and 32.56 µg/mL, respectively, with time of maximum
concentration (Tmax) of 4h for all these metabolites (Table 4). The concentration versus time profiles of all these
six metabolites were biphasic, with a rapid distribution phase and a slower terminal elimination phase lasting up
to 72h after administration.
The validated method was applied to a pharmacokinetic study in rats received oral BBF. The non-
compartmental pharmacokinetic analysis of the concentration-time data was performed using Kinetica 5.1
(Thermo Scientific™). The Cmax and Tmax were obtained directly from the plasma concentration-time plots. The
elimination rate constants (Ke) were determined through linear regression analysis of the logarithmic
transformation of the last four data points of the curve.18 The half-life (T1/2) and area under the concentration–
time curve (AUC0→t) were calculated by Kinetica software. The AUC0→t of caffeic acid, para-coumaric acid,
kaempferol, myricetin, trans-ferulic acid, and vanillic acid in BBF were 25.51, 28.24, 48.50, 87.05, 164.50 and
284.56 µg.h/mL, respectively. Although the administered doses of caffeic acid, para-coumaric acid, trans-ferulic
acid and kaempferol were rather lower than those of the myricetin and vanillic acid, the AUC0→t of vanillic acid
284.56 µg.h/mL was higher than those of caffeic acid, para-coumaric acid, trans-ferulic acid, which demonstrates
that the bioavailability of the hydroxycinnamic acid derivative (trans-ferulic acid, vanillic acid, caffeic acid, etc) was
much lower than that of the dihydroxybenzoic acid derivative (kaempferol, etc).
The T1/2 of caffeic acid was higher in BBF, which indicates that the dihydroxybenzoic acid derivative is metabolized
significantly higher than the dihydroxycinnamic acid derivatives in vivo. In addition, the elimination rate of ferulic
acid was the fastest,19 and the AUC0→t and Cmax values obtained for trans-ferulic acid and vanillic acid were
significantly higher than those obtained from the other flavonoids. This finding demonstrates that the phenolics
were more easily absorbed into the blood and more quickly metabolized compared with the flavonoids.
2.6. Comparative metabolomics analysis of BBF in plasma after its oral administration by UHPLC-MS
The present study deals with the targeted and untargeted metabolic profiling of plant extract and fate of
metabolites present in extract after its oral administration. By comparing chromatograms (peak pattern) of BBF as
well as blood plasma (Fig. 3), the patterns of metabolites were screened out to know about as well as fate of
metabolites. Mass spectrum of every metabolites separated through UHPLC-MS were tentatively identified by
comparing m/z values and literature survey along with verifying with publicly available database. A total of 89
metabolites were screened by UHPLC-MS chromatograms of BBF and plasma showing the number of metabolites
separated by UHPLC-MS. The UHPLC-MS data shows that BBF contained 60 metabolites. Among which, 25
metabolites were absorbed after 4h and another 18 metabolites were absorbed after 12h of oral administration.
Four metabolite of BBF were not absorbed in systemic circulation.
From mass spectrum data, metabolites were tentatively identified on the basis of their base and molecular
ion peak as well as literature survey. The number of metabolites obtained from BBF and blood plasma are shown
in Table 5. There were changes in pattern of metabolites in BBF and in plasma. Dissimilarity in metabolites pattern
between different time intervals of blood sample leading to a conclusion that some metabolites expressions were
being changed after oral administration of BBF. Similarly, changes in BBF and plasma showed that all metabolites
are not being absorbed or some of them being changed their expression in biological environment.
2.7. Docking and interaction analysis

Docking studies were carried out in order to find out the enhancing activity of the compounds present in C.
papaya BBF on AD 4.2 using PyMol visualizer. We got binding interaction of ligands with amino acid residues of
PHE 97, ARG 95, GLU 64, SER 65, TYR 24, GLU 23, PRO 691, CYS 690, THR 221 etc., involved in docking. Out of the
six components, kaempferol and myricetin had the highest docking score. Fig. 4 and 5 show the molecular docking
of thrombopoietin, α2 adrenergic receptor, integrin αIIbβ3 and integrin α2β1 with two ligands. Table 6 shows the
ligand used, the docking score and interacting residues formed by the compounds. Medicinal plants appear to be
rich source of secondary metabolites, widely used in traditional medicine to combat and cure various ailments. In
this study, we determined and compared the docking scores of six compounds presented in the CPLJ with
different platelet receptor using an automated docking model. Platelet receptors represent the forefront of
recent research and major advances have been made in understanding their molecular functions and their
downstream signaling pathways.20
Initial tethering and firm adhesion of platelets to the exposed sub-endothelium is mediated by glycoprotein
(GP) Ib/IX/V, GP Ia/IIa complex and collagen receptors (integrin α2β1 and αIIbβ3), in the platelet surface.21
Interactions between these elements are largely influenced by flow and trigger signaling events that reinforce
adhesion and promote platelet activation. Specific interactions of these agonists with their G-protein coupled
receptors generate inside-out signaling leading to conformational activation of integrin αIIbβ3, increasing their
ligand affinity. Binding of αIIbβ3 to its ligands, mainly fibrinogen supports processes such as clot retraction and
platelet aggregation. Stabilization of thrombi is supported by the late wave of signaling events promoted by close
contact between aggregated platelets. The best known contact-dependent signaling is outside-in signaling
through αIIbβ3,22 whereas thrombopoietin (TPO), the most potent cytokine plays a central role in the survival and
proliferation of haematopoietic stem cells23 that stimulates megakaryocytes to increase in cell size and ploidy, and
to form proplatelet processes that then fragment into single platelets. The platelet adhesion at the site of vessel
damage involves interactions of collagen with GPVI and integrin α2β1 for thrombus formation.
3. Experimental
3.1. Chemicals and solvents
Formic acid, acetonitrile and methanol were purchased from J.T. Baker (Baker Mallinckrodt, Mexico). Carboxy
methyl cellulose used was of AR grade and procured from Sigma Eldrich, St. Louis, MO. The standards namely
myricetin (98%), trans-ferulic acid (99%), caffeic acid (98%) para-coumaric acid (98%), kaempferol (98%) and
vanillic acid (98%) were purchased from Chromadex (Life Technology, Bangalore, India). Solvents used for
chromatography were of HPLC and LCMS grade and distilled water was obtained by a Milli-Q plus water
purification system (Millipore Corp., Bedford, MA). The marketed preparation Edoxin (Cyclophosphamide
injection; 1 g; Baxter, United Kingdom) was procured from Local Pharmacy. Docking analysis was carried out using
molecular graphics laboratory (MGL) tools, AutoDock (AD) 4.2.6 and EduPyMOL-v1.7.4.5 software’s.
3.2. Preparation of plant material
The tender leaves of C. papaya were collected from Jamia Hamdard Campus in the month of March 2015 and
authenticated from NISCAIR by taxonomist Dr. H.B.Singh (NISCAIR/RHMD/Consult/-2012-13/2158/164).
Previously weighed, 300 g of cleaned and chopped leaves were crushed with addition of small amount of distilled
water using a grinder for one min after removal of woody parts. The mixture volume was made upto 3500 mL
with distilled water and kept for 24h with occasional shaking. Then, it was filtered through muslin cloth and marc
left was washed with distilled water. The filtrate and the washing were pooled and final volume was made upto
4000 mL. The obtained juice was divided into four portions to be used for CPLJ (500 mL), FDP (1000 mL),
bioactivity guided fractions (1500 mL) and metabolite enriched fractions (1000 mL). The % yields were calculated
after drying and stored at -20°C until analysis. Freeze drying prevents enzymatic degradation of phytoconstituents
present in the fresh plant material.10
For bioactivity guided polarity based fractionations, accurately measured quantity of CPLJ (1500 mL) was
fractionated using n-hexane, dichloromethane and n-butanol thrice to separate total metabolites in four polarity
based fractions. The remaining of juice was used as such as bioactive aqueous fraction. All the fractions thus
obtained were evaporated to dryness in rotavapor (BUCHI Labortechnik AG, Switzerland) to get BHF, BDF, BBF,
and BAF. For extraction of alkaloid and glycoside rich fractions, the defatted CPLJ (500 mL) was basified using
ammonia (pH > 10.5). The solution was fractionated twice with chloroform. The chloroform layer was collected
and evaporated to dryness to obtain AEF. To the remaining layer, hydrochloric acid (HCl) was added drop wise to
make pH neutral and further extracted with ethyl acetate and evaporated to dryness to get GEF. For carotenoid
isolation, whole experiment was performed in dark. Ice cold acetone having 0.1% butylated hydroxy toluene
(BHT) (500 mL) was mixed in fresh leaf juice (500 mL) and left over night in freezer. The filtrate was washed thrice
with acetone (1.0 L) after filtering with whatman filter paper and the filtrate was concentrated in rotavapor (250
mL). To the concentrate, 10% sodium chloride (NaCl) solution (250 mL) was added and further extracted with
diethyl ether (400 mL) thrice. The collected ethereal layer was dried over anhydrous sodium sulphate (Na2SO4)
and then evaporated to dryness in a rotavapor. The BHT (0.1%) in petroleum ether and 10% methanolic
potassium hydroxide (KOH; 50 mL each) was added to the residue and kept overnight in dark. The resultant
solution was partitioned thrice with distilled water. The organic phase was collected and passed over anhydrous
Na2SO4 and then concentrated using rotavapor (30°C). The obtained residue was CEF. The extractive values of all
the fractions were calculated and stored for further analysis.
3.3. Standardization of FDP
The total carbohydrate, alkaloid, glycoside, tannin and saponin content in FDP were determined24 and the total
flavonoid and phenolic contents were determined by the method of Bedir et al.25
3.4. Acute toxicity studies of CPLJ
The oral acute toxicity study of FDP was evaluated according to the Organization for Economic Co-operation and
Development (OECD) guidelines 42326 on Balb/c mice (25-30 g),8 where the limit test dose of 2000 mg/Kg was
used. All the animals were kept at overnight fasting before every experiment with free access to water ad libitum.
The animals were divided into four groups, each comprising five animals. The 1st group served as a normal
control, while 2nd, 3rd and 4th were considered as tested groups receiving orally FDP (dissolved in normal saline)
at a dose of 300 mg/Kg, 1000 mg/Kg and 2000 mg/Kg, respectively. Before administration, the human dose (10-20
g) was converted to dose to be administered in mice by using the formula given by Reagan-Shaw.27 The animals
were observed for any toxic effect for first 4h after the treatment period. Further, the animals were investigated
for a period of three days for any toxic effect. The behavioural changes and other parameters such as body
weight, urination, food intake, water intake, respiration, convulsion, tremor, temperature, constipations, changes
in eye and skin color, etc were recorded.
3.5. Platelet enhancing potential of CPLJ
3.5.1. Animals and dosing schedule. Female Balb/c mice, weighing 25-30 g, were procured from Central Animal
House Facility, Jamia Hamdard, New Delhi. The experimental protocol was approved by Institutional Animal Ethics
Committee (IAEC) (registration number 173/GO/RE/S/2000/CPCSEA/1169). The animals were kept in
polypropylene cages under standard laboratory conditions (25 ± 2°C and 55 ± 5°C RH; photoperiod of 12h) and
had a free access to commercial pellet diet and tap water ad libitum. The experiment was conducted as per the
AOAC guidelines for Care and Use of Animals. The animals were divided in ten groups of six each. Group I served
as normal control and received normal saline solution (0.2 mL p.o). Group II and III received CPLJ and FDP,
respectively. Group IV, V, VI and VII received BHF, BDF, BBF and BAF, respectively. Group VIII, IX and X received
CEF, AEF and GEF, respectively. All the groups received dose of fractions equivalent to 3.42 gm/Kg b.wt. of fresh
leaves p.o., except Group I. The two experimental trials were conducted using Balb/c mice. The first trial was the
acute toxicity studies, where the toxicity of the dose was considered and the second trial (screening of all groups)
a pilot study, where platelet function analysis was considered.
3.5.2. Platelet function analysis. Blood samples were analyzed using autoanalyzer (XP-100, Sysmex
Corporation, Japan) for the following hematological parameters: platelet count (PC), total leukocyte count (TLC),
bleeding time and clotting time28 on day 0 and day 7th of the experiment for selection of most active fraction. The
aPTT was estimated from citrated plasma using standard kit erba actime made especially for CA 50 Coagulation
Analyzer, Transasia (Sysmex Corporation Kobe, Japan) on day 7.
3.6. Quantitative estimation of BBF by UHPLC-MS/MS in rat plasma
The sample solution for BBF (1.0 µg/mL) was prepared in LCMS grade methanol and filtered using 0.2 µM
polytetrafluoroethylene membrane filter (PTFE, Merck). The initial stock solutions of 250 µg/mL of each standard
(kaempferol, myricetin, trans-ferulic acid, para-coumaric acid, caffeic acid and vanillic acid) were prepared
separately, which were mixed in equal volumes to get mixed standard (50 µg/mL). Further, a series of working
solutions were prepared by diluting the mixed standard solution with LCMS grade methanol at appropriate ratios
to yield concentrations in the range of 1-100 ng/mL. For method validation, three concentrations of the mixed
standard solution at low quality control (LQC), medium quality control (MQC) and higher quality control (HQC)
(15, 50, 90 ng/mL), respectively, were used to prepare the quality control (QC) plasma samples to assess the
accuracy and precision. The prepared samples and standards were stored at 4°C until analysis.
The PP method was used for extraction of markers from plasma. Briefly, to the plasma samples (0.25 mL),
methanol (0.75 mL) was added and centrifuged to 10,000 rpm for 10 min to precipitate proteins. From the
supernatant, metabolites were extracted by LLE with different solvents (n-butanol, ethyl acetate and methanol).
Further, the hoarded organic layer was parched over nitrogen (N2) gas and re-constituted in methanol, and stored
at -20°C until analysis. Final extracting solvent was chosen on the basis of their percentage recovery. The
extraction recovery analysis was conducted with spiked mixed standard in bio-samples at three QC levels and
calculated by comparing the peak area of bio-samples in spiked plasma with those found by direct injection of
standard solutions at the same concentration.29
3.6.1. UHPLC-MS/MS conditions and optimization Quantitative analysis was performed on a Waters Acquity
UHPLC H-Class-Xevo TQD system (MA, USA) equipped with electrospray ionization operated in the negative
ionization mode. The chromatographic separation of analytes was carried out on Acquity UHPLC HSS C18 (50 × 2.1
mm, 1.8 m) column using a gradient mobile phase consisting of 0.1% formic acid (A) and acetonitrile (B) at a flow
rate of 0.4 mL/min, under following conditions: A:B (90:10, initial min; 90:10, 1 min; 10:90, 3 min; 90:10, 5 min;
90:10, 6 min). The column and the pressure were maintained at 40°C and 15000 psi, respectively. The source
dependent parameters namely cone gas flow, desolvation gas flow, capillary voltage, source temperature and
desolvation temperature at 50 L/h, 900 L/h, 3.0 kV, 150°C and 450°C, respectively were maintained for the
analytes. Detection of analytes was performed using a triple-quadrupole mass spectrometer (XEVO-TQD #
QCA896; Fisher Scientific, San Jose, CA, USA) with an electrospray ionization source in negative ion mode, using
SRM. The spray voltage, capillary and vaporizer temperatures were set to 3.0 kV, 350°C and 300°C, respectively.
Mass Lynx software version 4.1 was used to control all parameters of UHPLC and MS.
3.6.2. Validation of developed UHPLC-MS/MS method Different parameters such as system suitability,
linearity, accuracy and precision, stability testing, extraction recoveries, matrix effect and process efficiency of
method were evaluated for the developed method.30 The validation was performed in six replicates. The BBF was
prepared and analyzed to investigate the potential interference from endogenous compounds. The comparison
study was performed on chromatograms of sample and sample spiked with mixed standards. The calibration
curve consisted of seven concentration levels, and the sample with each concentration level was prepared as
described above, in six replicates. The concentration of each QC samples was calculated using calibration curve
through least squares linear regression analysis. The sensitivity was determined by limit of detection (LOD) and
LLOQ using the calibration curve method, according to the ICH recommendations.31 The LLOQ can be accurately
quantified within a 20% bias of the nominal concentration and with a precision not exceeding 20% in plasma.
The precision and accuracy were assessed by analyzing the QC samples (LQC, MQC and HQC). The intra-day
precision and accuracy were evaluated by six replicate samples on the same day. The inter-day precision and
accuracy were evaluated by six replicate samples on three consecutive days, where precision and accuracy were
expressed as %RSD and percent relative error (%RE), respectively. The stability was analyzed for mixed analytes
stored at -20°C for 120 days with those of freshly prepared solutions at the same concentration. The stability of
mixed analytes was evaluated for post extraction, freeze-thaw cycle, short term and long term stability tests.32
The samples were considered stable, if the deviation from the nominal concentration was within ±15.0%. The
extraction recoveries (ER) and the effect of matrix were examined by comparing the mean peak areas of analytes
between sample sets. The matrix effect (ME) and percent process efficiency was also evaluated simultaneously.32
3.7. Comparative metabolomic analysis of BBF as such and in plasma
The study was based on the targeted and untargeted metabolites present in BBF. The high-throughput profiling of
metabolite in blood was carried out by UHPLC-MS. The system was operated under the Empower software
(Waters, USA). Data acquisition was done in positive modes. Chromatography was performed using 0.1% formic
acid (A) and acetonitrile (B) as the mobile phase on monolithic capillary silica based C18 column (ACQUITY UHPLC
(R) BEH C18, 1.7 μm, 2.1 × 100 mm), with the pre-column split ratio 1:5 min at ambient temperature. The
separation was achieved by stepwise gradients from 10% A to 100% A for 16 min. The flow rate of the nebulizer
gas and cone gas was set at 10 μL/min and 50 L/h, respectively and the source temperature (100°C) was fixed. The
capillary and cone voltages were set to 3.0 and 40 kV, respectively. For collision, argon was employed at a
pressure of 5.3 × 10-5 Torr. The accurate mass and composition for the precursor ions and for the fragment ions
were calculated using the Mass Lynx V 4.1 software incorporated in the instrument.
Separated metabolites present in BBF were tentatively identified based on their m/z ratio and on literature.
Analyses of markers in plasma as well as in BBF were carried out as per the previous literature. Data obtained
from metabolomics analysis, was correlated with before and after treatment through orally administered BBF.
This correlation study was based on metabolites present in plasma before and after administration of BBF and in
its original form. A metabolite based comparison in extract and in plasma of animal has led to the determination
of fate of metabolite in blood.33 The comparative metabolite analysis was carried out to find the change in the
pattern of metabolites at different time intervals. The samples of plasma collected at 2, 4, 8, 12 and 24h were
prepared as described above and used in UHPLC-MS analysis for metabolomic study.
3.8. In silico analysis of polyphenols present in BBF
Initial step in the in silico drug designing procedure is the identification and selection of the appropriate drug
target or receptor. To study the nature of interaction, binding modes and selectivity of platelet receptors with
quantified secondary metabolites in BBF, docking was carried out with Autodock 4.2. The crystal structure of
target enzymes namely thrombopoietin (2ZKH), α2 adrenergic receptor (1HO9), integrin αIIbβ3 (2N9Y) and integrin
α2β1 (1AOX) were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) protein data
bank. The ligands namely trans-ferulic acid, para-coumaric acid, vanillic acid, kaempferol, myricetin and caffeic
acid were downloaded as 3D structure SDF file from PubChem34 and optimized using Ligands Input in the AD 4.2.
The preparation of the target enzyme with the AutoDock Tools (ADT) involved addition of all polar hydrogen
atoms and merging of its nonpolar hydrogen atoms along with the removal of water molecule and other
heteroatoms to the target enzyme, which is a necessary step for the computation of partial atomic charges. For
the ligands, Gasteiger charges were added and all the rotatable bonds were set to be rotatable. The protein-
ligand docking was done using the Lamarckian Genetic Algorithm (LGA) method. Three-dimensional affinity grids
(126 × 126 × 126 Å) were considered for each of the following atom types: HD, C, A, N, OA, and SA, representing
all probable atom types in the target enzyme. The ADT provide various methods to analyze the results of docking
simulations such as, conformational similarity, visualizing the binding site and its energy and other parameters like
inter-molecular energy and inhibition constant.35 The optimized ligand molecules were docked with refined
platelet receptors using AutoDock 4.2. The docking results were analyzed using PyMOL molecular graphics
visualization tool. An extended PDB format, termed as PDBQT file was used for coordinate files, which includes
atomic partial charges. The ADT was used for generating PDBQT files from traditional PDB files.36 The docking of
the ligands into the active site of platelet receptor proteins was carried out using AD 4.2.37 After completion of
docking searches, the best conformation was chosen from the most populated cluster with the minimum binding
energy. The interaction of docked protein-ligand complex conformations, including hydrogen bond and other
interactions were analyzed using PyMOL Molecular Graphics Visualizer 1.7.4.5.
All the data were evaluated statistically with SPSS version 22 software. The platelet count was represented
as mean ± SEM and compared with one way ANOVA among groups. For post hoc analysis Dunnett test was
performed. P ≤ 0.05 was considered significant.
Conclusions
The findings of the present study strongly suggest that there could be some active compounds in BBF that
enhances hemopoiesis and thrombopoiesis in animals. Chemical analysis of C. papaya leaves showed the
presence of guanine, 1-(O-beta-D-glucopyranosyl)-(1,3R,27R)-octaconsanetriol,myricetin, kaempferol, trans-
ferulic acid, caffeic acid, vanillic acid, para coumaric acid, N-arachidonoyl-L-alanine and unidentified constituents.
The pK analysis proved good bioavailability of vanillic acid, myricetin, trans-ferulic acid and kaempferol. Based on
our observations of in silico analysis, the flavonoids myricetin and kaempferol in BBF proved to have good binding
with integrin α2β1, integrin αIIbβ3, thrombopoietin, α2 adrenergic receptors and might exert thrombocyte
enhancing property.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This research was financially supported by Department of Science and Technology, Ministry of Science and
Technology, Government of India, New Delhi. Authors are also thankful to Dr. A.K.Tiwari, Incharge, Central Animal
House Facility for his cooperation in animal studies.
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Figure Captions
Fig. 1: Representative extracts ion SRM chromatogram of vanillic acid, para-coumaric acid, trans-ferulic acid, caffeic acid,
myricetin and kaempferol.
Fig. 2: Plasma concentration response curve of BBF.
Fig. 3: LCMS chromatogram showing mass of (A) BBF and (B) Plasma at 4 h.
Fig. 4: Docked pose of kaempferol with different receptors where a: α2 adrenergic receptor, b: integrin α2β1, c: integrin
αIIbβ3 and d: thrombopoietin.
Fig. 5: Docked pose of myricetin with different receptor where a: α2 adrenergic receptor, b: integrin α2β1, c: integrin αIIbβ3
and d: thrombopoietin.
TABLES
Table S1 General appearance and behavioural observations of acute toxicity study for control and treated groups
Parameters observed Control group 300 mg/Kg 1000 mg/Kg 2000 mg/Kg
Digestion Not observed Not observed Not observed Not observed
Body weight Normal Not change Not change Not change
Temperature Normal Normal Normal Normal
Food intake Normal Normal Normal Normal
Urination Normal No effect No effect No effect
Rate of respiration Normal No effect No effect No effect
Change in skin No effect No effect No effect No effect
Drowsiness Not present Not present Present Present
Sedation No effect No effect Observed Observed
Eye color No effect No effect No effect No effect
Diarrhoea Not present Not present Not present Not present
General physique Normal Normal Lethargy Lethargy
Coma Not present Not present Not present Not present
Death Alive Alive Alive Alive
Table S2 Stability of quality control samples (n = 3)
Stability tests Time Analytes Theoretical Conc (Mean ± Found Conc (Mean ± SD, Precision Accuracy
period SD, ng/mL) ng/mL) (%RSD) (%RE)
Myricetin 109445.40 ± 2716.99 109632.50 ± 2556.68 2.33 -0.17
Kaempferol 103846.20 ± 2304.75 103941.30 ± 2577.74 2.48 0.09
Stock solution
Trans-ferulic acid 137612.60 ± 2528.25 137791.70 ± 2893.62 2.10 -0.13
stability 120 days
Para-coumaric acid 101098.80 ± 3126.13 101526.00 ± 2355.40 2.32 4.22
(-30ºC)
Caffeic acid 83308.48 ± 1404.73 83645.53 ± 2526.09 3.02 4.04
Vanillic acid 93508.64 ± 3054.26 93666.32 ± 2397.85 2.56 0.16
BLOOD PLASMA
15 16.79 ± 1.24 7.35 11.93
Myricetin 50 53.42 ± 1.33 2.48 6.84
90 93.83 ± 2.62 2.79 4.25
15 15.64 ± 0.15 0.95 4.26
Kaempferol 50 53.22 ± 2.07 3.88 6.44
90 93.97 ± 2.74 2.91 4.41
15 16.43 ± 0.55 3.34 9.53
Freeze/ Trans-ferulic acid 50 50.91 ± 1.98 3.89 1.82
Three 90 94.57 ± 2.57 2.71 5.07
Thaw stability
Cycle
(-20°C to 25°C) 15 16.24 ± 0.65 4.00 8.26
Para-coumaric acid 50 54.76 ± 4.75 8.67 9.52
90 94.06 ± 1.92 2.04 4.51
15 17.01 ± 0.63 3.70 13.40
Caffeic acid 50 54.39 ± 3.40 6.25 8.78
90 94.20 ± 3.95 4.19 4.66
15 16.40 ± 0.93 5.67 9.33
Vanillic acid 50 53.26 ± 4.44 8.33 6.52
90 94.79 ± 3.12 3.29 5.32
15 16.36 ± 1.56 9.53 9.06
Myricetin 50 51.77 ± 3.24 6.25 3.54
90 93.78 ± 2.76 2.94 4.20
15 14.63 ± 0.67 4.57 -2.46
Kaempferol 50 51.95 ± 1.91 3.67 3.90
90 93.32 ± 2.23 2.38 3.68
15 15.91 ± 1.39 8.73 6.06
Trans-ferulic acid 50 51.47 ± 4.00 7.77 2.94
Short term
Four 90 94.48 ± 3.69 3.90 4.97
stability (in ice
hours 15 15.42 ± 1.33 8.62 2.80
water bath)
90 93.26 ± 2.75 2.94 3.62
15 15.16 ± 1.04 6.86 1.06
Caffeic acid 50 51.43 ± 2.05 3.98 2.86
90 91.47 ± 0.82 0.89 1.63
15 15.34 ± 1.16 7.56 2.26
Vanillic acid 50 51.60 ± 2.54 4.92 3.20
90 95.42 ± 3.27 3.42 6.02
15 16.07 ± 1.45 8.83 7.13
Myricetin 50 49.43 ± 1.12 2.24 -1.14
Post extraction 90 94.98 ± 1.40 1.47 5.53
stability (in 23 15 14.87 ± 0.78 5.24 -0.86
autosampler, hours Kaempferol 50 52.21 ± 2.34 4.48 11.05
15°C) 90 94.29 ± 2.10 2.22 4.76
15 14.72 ± 1.12 7.60 -1.86
Trans-ferulic acid
50 51.41 ± 2.47 4.80 2.82
90 94.41 ± 1.61 1.70 4.90

15 15.59 ± 0.45 2.88 3.93
90 94.29 ± 2.28 2.41 4.76
15 14.61 ± 0.42 2.87 -2.60
Caffeic acid 50 53.02 ± 1.48 2.79 6.04
90 93.16 ± 2.88 3.09 3.51
15 16.08 ± 0.36 2.23 7.20
Vanillic acid 50 52.58 ± 3.54 6.73 5.16
90 93.86 ± 1.78 1.89 4.28
15 16.44 ± 1.11 6.75 9.60
Myricetin 50 53.82 ± 4.48 8.32 7.64
90 94.57 ± 0.81 0.85 5.07
15 15.82 ± 0.55 3.47 5.46
Kaempferol 50 51.65 ± 2.04 3.94 3.30
90 94.98 ± 1.80 1.89 5.53
15 15.03 ± 0.67 4.45 0.20
Trans-ferulic acid 50 52.99 ± 4.65 8.77 5.98
Long term 90 94.87 ± 1.96 2.06 5.41
55 days
stability 15 15.39 ± 1.14 7.40 2.60
90 94.64 ± 2.34 2.47 5.15
15 15.38 ± 0.54 3.51 2.53
Caffeic acid 50 52.15 ± 0.68 1.30 4.30
90 92.98 ± 1.99 2.14 3.31
15 15.83 ± 0.64 4.04 5.53
Vanillic acid 50 52.02 ± 2.88 5.53 4.04
90 95.38 ± 2.96 3.10 5.97
Conc- concentration; SD- standard deviation; RE-relative error
Table 1 The effect of PC, TLC count, aPTT, bleeding time and clotting time in different fractions
5
Groups PC × 10 lac/cumm TLC /cumm aPTT in sec Bleeding time in sec Clotting time in sec
(Mean ± SEM) (Mean ± SEM) (Mean ± SEM) (Mean ± SEM) (Mean ± SEM)
Day 0 Day 7 Day 0 Day 7 Day 0 Day 7 Day 0 Day 7 Day 0 Day 7
Group I 08.95 ± 0.55 08.80 ± 0.54 7066.66 ± 154.20 7033.33 ± 166.67 27.83 ± 0.94 27.66 ± 0.88 274.16 ± 23.62 269.83 ± 22.19 178.66 ± 05.60 177.33 ± 06.13
ns ns ns ns ns ns ns ns
Group II 09.95 ± 0.28 10.37 ± 0.48 5416.66 ± 215.12** 6250.00 ± 176.54 25.33 ± 0.80 19.00 ± 1.00** 285.50 ± 17.73 270.00 ± 18.04 205.33 ± 07.78 192.16 ± 04.92
Group III 09.19 ± 0.67 11.43 ± 0.36* 5100.00 ± 159.16** 6100.00 ± 123.83 26.50 ± 0.84 23.66 ± 1.54 288.00 ± 15.47 272.50 ± 12.27 181.16 ± 11.43 171.16 ± 09.61
ns ns ns ns ns ns
Group IV 09.87 ± 0.39 08.31 ± 0.29 5166.66 ± 249.89** 5516.66 ± 402.84** 19.83 ± 0.87** 21.16 ± 0.87** 282.50 ± 12.61 280.50 ± 12.18 171.33 ± 04.61 173.66 ± 05.20
ns ns ns ns ns ns ns
Group V 09.71 ± 0.34 11.38 ± 0.72* 4866.66 ± 420.85** 6250.00 ± 449.26 24.83 ± 1.81 23.33 ± 1.72* 268.00 ± 08.08 256.83 ± 06.94 190.00 ± 10.44 184.83 ± 07.47
ns ns ns ns ns
Group VI 10.18 ± 0.27 13.97 ± 0.17** 6016.66 ± 313.49* 7283.33 ± 192.21 32.66 ± 0.80** 22.50 ± 0.88* 276.83 ± 18.64 251.33 ± 18.50 167.83 ± 05.17 146.00 ± 04.00*
ns ns ns ns ns ns
Group VII 10.20 ± 0.39 11.66 ± 0.83** 4616.66 ± 181.51** 5350.00 ± 309.57** 20.00 ± 0.73** 27.33 ± 1.28 281.50 ± 14.76 278.66 ± 13.42 177.83 ± 04.08 176.83 ± 05.51
ns ns ns ns ns ns ns
Group VIII 09.81 ± 0.23 11.78 ± 0.45** 5816.66 ± 273.76** 6066.66 ± 477.96 25.94 ± 1.11 19.66 ± 0.80** 267.33 ± 16.28 248.33 ± 17.78 173.33 ± 08.35 164.50 ± 07.72
ns ns ns ns ns ns
Group IX 09.70 ± 0.34 09.42 ± 0.91 4733.33 ± 098.88** 5750.00 ± 408.86* 21.16 ± 0.65** 23.16 ± 0.60* 280.83 ± 17.06 267.33 ± 17.00 190.50 ± 03.96 184.66 ± 03.45
Group X 10.03 ± 0.50 10.76 ± 0.48 5566.66 ± 206.02** 6583.33 ± 151.47 20.00 ± 0.89** 24.50 ± 0.84 272.33 ± 20.11 253.83 ± 18.29 189.83 ± 04.52 185.83 ± 03.03
Note: Effect of leaf juice, bioactive guided fractions and metabolite enriched fractions on PC, TLC, aPTT, bleeding time and clotting time in mice. Values shown are mean ± S.E.M. (n=6);
** *
p<0.05 is considered extremely significant compared to normal; p<0.01 is considered signification. (PC- platelet count; TLC-total leukocyte count; aPTT- activated partial thromboplastin
time; SEM- standard error mean; ns- non significant). Where Group1: group received normal saline solution; Group II: received C. papaya leaf juice (CPLJ); Group III: freeze dried powder
(FDP); Group IV: bioactive hexane fraction (BHF); Group V: bioactive dichloromethane fraction (BDF); Group VI: bioactive butanol fraction (BBF); Group VII: bioactive aqueous fraction (BAF);
Group VIII: carotenoid enriched fraction (CEF); Group IX: alkaloid enriched fraction (AEF) and Group X: glycoside enriched fraction (GEF).
Table 2 Linearity data of chromatographic UHPLC-MS/MS method for mixed standards (n = 6)
Biomarkers Linearity (ng/mL) Equation Regression ± SD LOD (ng/mL) LLOQ (ng/mL)

Myricetin 05-100 Y= 13.51x + 011.79 0.997 ± 0.002 01.76 05.33
Kaempferol 05-100 Y= 35.96x – 152.37 0.992 ± 0.001 02.31 07.00
Trans-ferulic acid 05-100 Y= 54.40x – 169.84 0.995 ± 0.002 01.79 05.45
Para-coumaric acid 05-100 Y= 48.01x – 232.32 0.990 ± 0.001 01.80 05.46
Caffeic acid 05-100 Y= 34.87x – 061.08 0.997 ± 0.002 01.62 04.93
Vanillic acid 05-100 Y= 49.25x – 134.19 0.995 ± 0.003 02.07 06.28
LOD- limit of detection; LLOQ- lower limit of quantification; SD- standard deviation
Table 3 Intra- and inter-day accuracy and precision of quality control samples
Analytes Theoretical Intra-day (n = 6) Inter-day (n = 6 × 3)

Conc (ng/mL) Found Conc (Mean Precision Accuracy Found Conc (Mean ± Precision Accuracy
± SD, ng/mL) (%RSD) (%RE) SD, ng/mL) (%RSD) (%RE)
15 15.96 ± 1.23 7.70 6.40 16.64 ± 1.22 7.33 10.93
Myricetin 50 53.13 ± 1.67 3.14 6.26 52.85 ± 1.50 2.83 5.70
90 92.22 ± 0.95 1.03 2.46 92.64 ± 2.66 2.87 2.93
15 15.49 ± 0.38 2.45 3.26 15.71 ± 0.95 6.04 4.73
Kaempferol 50 52.36 ± 2.58 4.92 4.72 52.75 ± 4.48 8.49 5.50
90 94.03 ± 1.63 1.73 4.47 92.44 ± 1.67 1.80 2.71
15 15.77 ± 0.17 1.07 5.13 16.10 ± 0.04 0.24 7.33
Trans-ferulic
50 51.65 ± 2.71 5.24 3.30 51.78 ± 3.61 6.97 3.56
acid
90 94.69 ± 2.23 2.35 5.21 94.27 ± 2.21 2.34 4.74
15 14.68 ± 0.80 5.51 -2.13 15.56 ± 0.56 3.59 3.73
Para-coumaric
50 52.90 ± 1.69 3.19 5.80 51.92 ± 1.95 3.75 3.84
acid
90 94.16 ± 1.98 2.10 4.62 94.83 ± 3.17 3.34 5.36
15 15.70 ± 0.46 2.92 4.66 16.31 ± 0.20 1.22 8.73
Caffeic acid 50 53.30 ± 1.76 3.30 6.60 52.96 ± 1.67 3.15 5.92
90 92.99 ± 2.07 2.57 3.32 93.61 ± 3.42 3.65 4.01
15 16.05 ± 0.68 4.23 7.00 16.69 ± 0.33 1.97 11.26
Vanillic acid 50 51.98 ± 3.02 5.80 3.96 51.58 ± 2.84 5.50 3.16
90 93.59 ± 2.41 2.57 3.98 93.67 ± 2.76 2.76 4.07
Conc- concentration; RSD- relative standard deviation; SD- standard deviation; RE-relative error
Table 4 Pharmacokinetic parameters of metabolites in Wistar rats upon oral administration of BBF (n= 5)
-1
Analytes Cmax (µg/mL) Tmax (h) Ke (h ) T1/2 (h) AUC0-t (µg.h/mL) AUC0-∞ (µg.h/mL) AUMC0-t (µg.h/mL) AUMC0-∞ (µg.h/mL) MRT (h)
Myricetin 14.05 4 0.074 09.28 87.05 87.47 830.61 866.07 9.541
Kaempferol 10.48 4 0.070 09.78 48.50 48.81 427.51 454.24 8.813
Trans-ferulic acid 25.35 4 0.080 08.61 164.50 165.01 1497.69 1540.72 9.104
Para-coumaric acid 07.92 4 0.061 10.97 28.24 28.49 234.17 255.58 8.289
Caffeic acid 09.05 4 0.063 11.22 25.51 23.66 176.57 189.08 6.919
Vanillic acid 32.56 4 0.076 09.09 284.56 285.53 2738.92 2821.60 9.625
0-t 0-t
Cmax- maximum concentration; Tmax- time at maximum concentration; AUC - Area under curve 0 to time t; Ke- elimination rate constant; T1/2- half-life; AUMC - ; area under metabolic
curve 0 to t; MRT- mean residual time.
Table 5 Peak pattern of metabolites in plasma and as such after its oral administration of BBF separated through UHPLC-MS in plasma
S.No Rt m/z Metabolite profile in extract & blood Tentative identification Formula Source ID
BBF 2h 4h 8h 12 h 24 h
1. 0.98 151.1304 + - + - - - Guanine C5H5N5O CID 764
2. 173.2079 + - + - + - N-Acetyl-L-Leucine C8H15NO3 KO000243
3. 195.1987 + - + - + - Glucosaminic acid C6H13NO6 KO000903
4. 209.1876 + - + - + - 2,6,10-trimethyl-undecanoic acid C14H28O2 45765
5. 225.2367 + - + - - - 6-Benzylaminopurine C12H11N5 KO000336
6. 241.2843 + - - - - - Tetrabutylammonium C16H36N 85164
7. 4.11 135.1317 + - + - - - Adenine C5H5N5 KNA00567
8. 183.1345 + - + - - - 4-hydroxy-undecanoic acid C11H22O3 74541
9. 209.2621 + - + - - - Nicotinoylcholine C11H17N2O2+ PB000343
10. 223.2813 + - + - - - 5-Aminonaphthalene-2-sulfonic acid C10H9NO3S SM834151
11. 257.3055 + - + - - - 9-methoxy-2,2-dimethyl-6-hydro-2H-pyrano [5,6-c] quinolin-5-one C15H15NO3 BML80656
12. 313.3142 + - - - - - 6-methylheptadecane C18H38 95536
13. 343.4228 + - - - - - Hydroxybutorphanol C21H29NO3 CO000226
14. 360.4658 + - + - - - 2',3'-O-p-anisylidenecytidine C17H19N3O6 SD 15060
15. 426.5662 + - + - - - 10,12,14-nonacosatriynoic acid C29H46O2 SD 19071
16. 451.6133 + - + - + - Cyanidin-3-O-(2"-xylosyl-6"-(6"-caffeoyl-glucosyl)-galactoside) C41H45O23 86122
17. 487.6037 + - + - + - Icosafluorononane C9F20 SD 19892
18. 495.6943 + - + - + - 2,4,6-triphenylthiopyrylium-p-toluenesulfonate C30H24O3S2 SD 24403
19. 8.14 160.9341 + - + + - - Vanillic acid C8H8O4 CID 8468
20. 162.9281 - - - + - - Methyl-seleno-pyruvate C4H6O3Se 72665
21. 175.0717 + - + - - - Arthonioic acid C29H36O9 44454
22. 197.9215 - - + + - - 4,6-dinitro-o-cresol C7H6N2O5 EQ312051
23. 201.9209 + - + + - - Trans-ferulic acid C10H10O4 CID 45039253
24. 246.2634 + - + - + - n-Pentadecylamine C15H33N 34540
25. 249.1173 - - + + + - (S)-3-Methylthiohexyl acetate C9H18O2S 88451
26. 301.2404 + - + - + - 1-(O-beta-D-glucopyranosyl)-(1,3R,27R)-octaconsanetriol C34H68O8 46586
27. 311.3663 + - - - + - S-benzyl-N-(tert-butoxycarbonyl)-L-cysteine C15H21NO4S SD 19458
28. 325.3976 + - - - + - 3-Hydroxy-3-(4-methylpent-3-en-1-yl)glutaryl-CoA C32H52N7O20P3S 66238
29. 341.3945 + - - - - - Fluoromethacin C19H16FNO4 WA001375
30. 396.4106 + - - - - - 5,5'-dithiobis (2-nitrobenzoic acid) C14H8N2O8S2 SD 460
31. 433.4461 + - - - - - Palmityl myristate C30H60O2 97025
32. 476.5156 - - - + - - (E)-1,2-bis (o-tolyl)vinylene bis (o-toluate) C32H28O4 SD 26980
33. 500.5295 - - - + + - O-bis(beta-(phenylthio) phenethyl) benzene C34H30S2 SD 11576
34. 504.5452 - - - + + - 5-cholesten-3beta-yl phenylacetate C35H52O2 SD 5989

35. 528.5615 - - - + + - Onjisaponin F C75H112O36 71674
36. 578.5791 - - - + + - 1-bromo-1,1,3,3-tetrabutyl-3-hydroxydistannoxane C16H37BrO2Sn2 SD 33933
37. 588.6111 - - - + + - N,N'-ethylenedioctadecanamide C38H76N2O2 SD 5845
38. 589.6378 - - - + + - 2,2'-(methylimino)-bis-(N,N-dioctylacetamide) C37H75N3O2 SD 53468
39. 619.5999 - - - + - - Cholesteryl ester C44H78O2 41721
40. 297.3079 - - - - + - Dihydrophytol C20H42O 102900
41. 313.3425 + - - - + - 1-desoxy-1-(p-nitroanilino)-D-glucofuranuronamide C12H15N3O7 SD 32635
42. 394.2766 + - - - + - N-Arachidonoyl-L-Alanine C23H37NO3 64920
43. 476.5236 + - - - + - 4-acetyl-3-hydroxy-o-phenylene bis (p-toluene sulfonate) C22H20O8S2 SD 28111
44. 529.5684 - - - - + - Dimethyl dialkyl ammonium chloride C35H74N 88278
45. 602.6238 - - - - + - Trans-1,2-bis (tributylstannyl) ethylene C26H56SN2 SD 50703
46. 10.76 147.0658 + - - - - - 1-hydroxy-2-pentanone C5H10O2 96265
47. 175.0766 + - - - - - Methylhildgardtol A C22H24O4 47375
48. 255.4091 + - - - - - 4-ethylthiomethyl-6-morpholino-1 3,5-triazin-2-amine C10H17N5OS SID 26426
49. 311.3663 + - - - - - N,N-bis (phenylsulfonyl) methylamine C13H13NO4S2 SD 31186
50. 325.4042 + - - - + - N-ethylbis (phenylsulfonyl) amine C14H15NO4S2 SD 31187
51. 353.4261 + - - - - - N-[(9-fluorenyl) methoxycarbonyl]-L-isoleucine C21H23NO4 SD 11747
52. 160.9341 - - + - + - Dimethyl trisulfide C2H6S3 66929
53. 197.9163 - - + - + - 4,7-dichloroquinoline C9H5Cl2N SD 6268
54. 293.3383 - - + - + - 1-(2-oxo-3-oxazolidinyl) anthraquinone C17H11NO4 SD 33732
55. 339.3857 - - + - + - Naphthyl-2-oxomethyl-succinyl-CoA C36H46N7O20P3S 69813
56. 383.3394 - - - - + - 9-Pentacosyne C25H48 97955
57. 397.3647 - - - - + - 9,10-Hexacosadiene C26H50 97924
58. 12.30 175.0815 + - - - - - Tetrahydroalstonine C21H24N2O3 64328
59. 227.3504 + - - - - - 4-chloro-3-nitrocinnamic acid C9H6ClNO4 SD 10620
60. 311.3728 + - - - - - S-benzyl-N-(tert-butoxycarbonyl)-L-cysteine C15H21NO4S SD 19458
61. 325.4042 + - - - - - N-ethylbis (phenylsulfonyl) amine C14H15NO4S2 SD 31187
62. 339.4196 + - + - - - N-[(9-fluorenyl) methoxycarbonyl]-L-valine C20H21NO4 SD 11740
63. 395.4947 + - - - - - Trinonylamine C27H57N SD 41243
64. 134.9735 - - + - - - Methyl 2-propenyl selenide C4H8Se 87247
65. 162.9281 + - + - - - Para-coumaric acid C9H8O3 CID 637542
66. 249.1115 - - + - - + 2Z-Dodecenedioic acid C12H20O4 74918
67. 270.9399 - - + - - - 2,5-Dichloro-4-oxohex-2-enedioate C6H4Cl2O5 69492
68. 399.3469 - - + - - - 8-Hydroxy-6-docosanone C22H44O2 90920
69. 465.5471 - - + - + - N-(3alpha,7alpha,12alpha-trihydroxy-24-oxo-5 beta-cholan-24-yl) glycine C26H43NO6 SD15420
70. 485.5104 - - + - + - 1,7-Pentatriacontadien-11-ol C35H68O 95467
71. 502.8776 - - + - + - 1-O-acetyl-2,3,5-tri-o-benzoyl-beta-D-ribofuranose C28H24O9 SD13683

72. 560.8798 - - + - + + D-myo-Inositol-1,3,4,5,6-pentaphosphate C6H17O21P5 44862
73. 653.6199 - - + - - - Hexacosanyl oleate C42H82O2 75409
74. 15.21 136.0741 + - - - - - Gamma-glutamyl-Lysine C11H22N4O4 86046
75. 249.3147 + - - - - - 5-(3-nitrobenzylidene)-2-thioxo-4-imidazolidinone C10H7N3O3S SD 26700
76. 255.4091 + - - - + - N,N-diethyldodecanamide C16H33NO SD 880
77. 265.3325 + - - - + - N-benzyloxycarbonyl-DL-norleucine C14H19NO4 SD 10884
78. 297.3460 + - + - + - N-(2,4-dinitrophenyl)-L-leucine C12H15N3O6 SD 7105
79. 311.3728 + - + - - - Myricetin C15H10O8 CID 5281672
80. 325.3976 + - - - - - 8-cyano-3,3-diphenyl-3,3a-dihydrocyclohepta-(b)-furan-2-one C22H15NO2 SD 30670
81. 339.4264 + - - - - - 6,7-dimethoxy-1-veratrylisoquinoline C20H21NO4 SD 33904
82. 409.5774 + - - - - - Heptafluorobutyric anhydride C8F14O3 SD 6072
83. 16.83 175.0815 + - - - - - 16-Hydroxytabersonine C21H24N2O3 64340
84. 261.1193 + - - - - - Nα-Acetyl-L-arginine C8H16N4O3 58241
85. 273.1483 + - - - - - 9-Acetoxyfukinanolide C17H24O4 90114
86. 289.1326 + - + - + - Kaempferol C15H10O6 CID 5280863
87. 305.1216 + - - - - - 4'-Hydroxy-5,7-dimethoxyflavan C17H18O4 47492
88. 311.3857 + - - - - - S-benzyl-N-(tert-butoxycarbonyl)-L-cysteine C15H21NO4S SD 19458
89. 325.4109 + - - - - - N-ethylbis (phenylsulfonyl) amine C14H15NO4S2 31187
Page 23 of 28 RSCnot
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PAPER
Table 6 The interaction energy analysis of six ligands with that of platelet receptors using PyMOL molecular graphics system 1.7.4.2
Protein name Ligands Modes Binding energy (Kcal/mol) Interacting residues

Kaempferol M-1 -8.0 TRP-47, ARG-95, PHE-97
Thrombopoietin
Myricetin M-1 -7.3 GLN-165, ASP-164, THR-163, SER-173
Kaempferol M-1 -4.9 GLU-30, ASN-25, SER-155, THR-290, SER-156
α 2 adrenergic receptor
Myricetin M-1 -4.7 ARG-31, THR-220, HIS-258, SER-153
Kaempferol M-1 -5.8 GLU-960, LEU-994,
Integrin αIIbβ3
Myricetin M-1 -6.1 ARG-962, PRO-691, CYS-690, GLU-961,
Kaempferol M-1 -6.7 ASN-154, SER-153, SER-155, THR-221
Integrin α2β1
Myricetin M-1 -6.7 SER-155, SER-153, THR-221, HIS-258
This journal is © The Royal Society of Chemistry 2017 RSC Adv., 2017, 00, 1-3 | 22
Please do not adjust margins

Representative extracts ion SRM chromatogram of vanillic acid, para-coumaric acid, trans-ferulic acid, caffeic
acid, myricetin and kaempferol.
49x60mm (300 x 300 DPI)

Plasma concentration response curve of BBF
254x190mm (96 x 96 DPI)

LCMS chromatogram showing mass of (A) BBF and (B) Plasma at 4 h
54x75mm (300 x 300 DPI)

Docked pose of kaempferol with different receptors where a: α2 adrenergic receptor, b: integrin α2β1, c:
integrin αIIbβ3 and d: thrombopoietin.
Docked pose of myricetin with different receptor where a: α2 adrenergic receptor, b: integrin α2β1, c:
integrin αIIbβ3 and d: thrombopoietin

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In silico docking, in vivo UHPLC-MS/MS analysis and

Journal: RSC Advances

Article Type: Paper

Date Submitted by the Author: 24-Nov-2017

Subject area & keyword: Bioanalytical < Analytical

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In silico docking, in vivo UHPLC-MS/MS analysis and platelet enhancing

Bioactive Natural Product Laboratory

Email ID: sahmad_jh@yahoo.co.in

In silico docking, in vivo UHPLC-MS/MS analysis and platelet enhancing

2. Results and discussion

2.1. Standardization of FDP

2.2. Acute toxicity studies of FDP

2.3. Preliminary platelet enhancing potential of FDP and fractions

2.4. Quantitative estimation of BBF by UHPLC-MS/MS in rat plasma

2.5. Application in pharmacokinetic studies

2.7. Docking and interaction analysis

3.2. Preparation of plant material

3.3. Standardization of FDP

3.4. Acute toxicity studies of CPLJ

3.5. Platelet enhancing potential of CPLJ

3.6. Quantitative estimation of BBF by UHPLC-MS/MS in rat plasma

3.7. Comparative metabolomic analysis of BBF as such and in plasma

3.8. In silico analysis of polyphenols present in BBF

Fig. 2: Plasma concentration response curve of BBF.

Table S2 Stability of quality control samples (n = 3)

90 94.41 ± 1.61 1.70 4.90

Table 2 Linearity data of chromatographic UHPLC-MS/MS method for mixed standards (n = 6)

Biomarkers Linearity (ng/mL) Equation Regression ± SD LOD (ng/mL) LLOQ (ng/mL)

Analytes Theoretical Intra-day (n = 6) Inter-day (n = 6 × 3)

34. 504.5452 - - - + + - 5-cholesten-3beta-yl phenylacetate C35H52O2 SD 5989

71. 502.8776 - - + - + - 1-O-acetyl-2,3,5-tri-o-benzoyl-beta-D-ribofuranose C28H24O9 SD13683

Protein name Ligands Modes Binding energy (Kcal/mol) Interacting residues

Please do not adjust margins

49x60mm (300 x 300 DPI)

Plasma concentration response curve of BBF

254x190mm (96 x 96 DPI)

LCMS chromatogram showing mass of (A) BBF and (B) Plasma at 4 h

54x75mm (300 x 300 DPI)

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