Haemostatic effects of latex from Croton sparsiflorus Morang, in vitro, in vivo, in silico approaches

Accepted Manuscript Title: Haemostatic effects of latex from Croton sparsiflorus Morang, in vitro, in vivo, in silico approaches Authors: M.C. Kamaraj, S.Mohan Raj, D.Palani selvam, S. Subashchandrabose, A. Kalaiselvan PII: DOI: Reference:

S1476-9271(17)30334-1 https://doi.org/10.1016/j.compbiolchem.2018.03.025 CBAC 6830

To appear in:

Computational Biology and Chemistry

Received date: Revised date: Accepted date:

10-5-2017 23-3-2018 25-3-2018

Please cite this article as: Kamaraj, M.C., Raj, S.Mohan, selvam, D.Palani, Subashchandrabose, S., Kalaiselvan, A., Haemostatic effects of latex from Croton sparsiflorus Morang, in vitro, in vivo, in silico approaches.Computational Biology and Chemistry https://doi.org/10.1016/j.compbiolchem.2018.03.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Haemostatic effects of latex from Croton sparsiflorus Morang, in vitro, in vivo, in silico approaches

M. C. Kamaraja, S. Mohan Rajb* D. Palani selvama, S. Subashchandrabose c* A. Kalaiselvand aDepartment

of Biotechnology, PRIST University, Thanjavur-613403, Tamil Nadu, India for Research and Development, PRIST University, Thanjavur-613403, Tamil Nadu, cCentre for Functionalized Magnetic Material (FunMagMa), Immanuel Kant Baltic Federal University, Kaliningrad, Russia-236041 d Dept of Biochemistry and Heamatology, Thyrocare Technologies Ltd, Navi Mumbai, Maharastra, India.

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Crud sample extracted from Traditional plant Molecular structure of the target sample was found In vitro and in vivo blood clotting activity studied Explained the mechanism of the blood clotting activity Docking and DFT computational studies were carried out

ABSTRACT

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Highlights

The present investigations are phytochemical screening of Latex aqueous (Laq) extract of C. sparsiflorus and study its role in homeostasis. It is being traditionally used over fresh cuts to stop bleeding immediately. To know the contents of extract, the quantitative phytochemical analysis were performed it showed the contents such as saponins (15.2%), alkaloids (7.61%), phenols (0.62%), tannins (1.1%), and flavonoids (0.224%). The in vitro

and in vivo blood clotting mechanism was observed in Wister albino rats to understand the blood clotting activity. The in vitro cytotoxicity assay was performed by 3T3L1 cell lines evaluated by Laq extract of C. sparsiflorus to determine the toxic effects of the extract. The gas chromatographic and liquid chromatographic mass spectra (GCMS and LCMS) were observed there were three components observed namely, 1) methyl-hexafuranoside, 2) cumarandione, and 3) crotonosine, in addition to that the NMR (1H and 13C) elemental

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analysis, FT-IR (4000-400cm-1) and UV-Vis (800-200nm) spectra were also recorded in aqueous solution. The molecular docking studies performed, in which the blood clotting

factors have a potential interaction with crotonosine. This in-silico study demonstrates the

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interactions of active components of C. sparsiflorus with blood clotting factors.

Furthermore, since the crotonosine compound has more blood clotting factor the molecular structure was treated with density functional theory calculation (DFT) to

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understand the optimized geometry, vibrational behavior and electronic excitation states.

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1. Introduction

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*Corresponding Author: [email protected]

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Keywords: Croton sparsiflorus; Blood clotting; FT-IR; DFT; docking studies;

The genus Croton (Euphorbiaceae) comprises above 1300 species growing as

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herbs, shrubs and trees in tropic and subtropical regions of both hemispheres (Salatino, et al., 2007). Its various species are reported to possess diverse medicinal properties. One of

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its species is Croton sparsiflorus Morong (Syn. C. bonplandianum), commonly known as “Ban Tulsi” which is a woody shrub growing in road sides of Asia and South America. The leaf extract of this plant was reported to control high blood pressure (Yadav and Das,

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2013). It is also being traditionally applied over fresh cuts to stop bleeding immediately, thus enhances the wound healing process. However, there is no scientific evidence on its blood clotting properties and/or wound healing processes to-date. Understanding the science behind the association and/or reactions of latex on secondary hemostasis will be much useful as it can be used for medical emergency diseases, like hemophilia, and to

treat fresh cuts or sealing wounds in humans. Disorders of coagulation can lead to an increased risk of bleeding (hemorrhage). In patients with advanced liver disease, bleeding and thrombosis are dangerous complications (Satish et al., 2012). World Health Organization (WHO) reported that about 80% peoples are used traditional medicine for the completion of their day to day needs regarding primary health care (Shivaprasad et al., 2011). Accumulated evidences indicate that the latex bearing plants used in the treatment

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of various diseases such as asthma, diabetes, dysentery, malaria and skin problems (Badgujar, 2011). Plant latex is the milky juice, found in long branching tubes known as

latex tubes. This juice is white, yellow or pinkish in colour. It is a viscous fluid and

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colloidal in nature. Known ingredients of latex are proteins, alkaloids, tannins, terpens, starch, sugars, oils, resins, gums and enzymes (Pandey, 2001). The pharmacological

application of plant latices as hemostasis and wound healing purpose involve their role on

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haemostasis and fibrinolysis. Hemostasis and fibrinolysis both are two separate process

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maintained by the action of specific proteins. Therefore, the aim of the present study is to investigate the phytochemical screening, in vitro and in vivo blood clotting activity and

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cytotoxicity assay, phytoanalytical study such as GCMS, LCMS, NMR, FTIR, and UV

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spectra were recorded to characterization of the compounds. Further the isolated compounds are used for docking studies to understand the interactions of blood clotting

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proteins. The plant compound Crotonosine, the major alkaloid of Croton linearis (Haynes and stuart 1964). Crotonosine C17H17NO3, the key compound of the series contains one

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methoxyl, one phenolic group, a secondary nitrogen atom, two double bonds and a

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carbonyl group in a cross-conjugated dienone.

2. Experimental

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2.1 Collection and Preparation of plant Sample Croton sparsiflorus latex was collected from the waste lands in and around

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papanasam, Thanjavur district, Tamil Nadu, India, where it was found naturally. The plant was identified by Dr. S. John Britto, Director, Rabinat Herbarium and Centre for Molecular systematic, St. Joseph’s College, Trichy, and Tamil Nadu, India. The latex of C. sparsiflorus was obtained as exudates by and plucking of fresh leaves from actively growing plants. The latex was collected into sterile, plastic containers by pressing and squeezing in between fingers. The apex of the leaves was collected to release as much as

possible latex into the containers. The collections of latex were made in the mornings. After collection, the latex was centrifuged with 3000 rpm for 15 minutes to remove any solid particles present in it. The latex was transferred into containers and closed tightly and stored at 4°C until the time of use. This latex was diluted 1:1 ratio with distilled water and mixed well and used for further analysis. 2.2 Phytochemical screening of aqueous extracts of Croton sparsiflorus

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The Laq extract of C. sparsiflorus was characterized for its phytochemical

constituents. The presence of alkaloids, phenols, tannins, saponins, flavonoids,

carbohydrates, steroids, terpenoids, and glycosides were determined according to the

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methods described by (Trease and Evans 1989). Alkaloids, phenols, saponins, flavonoids and tannins were estimated by (Harborn, et al., 1973). 2.3 In-vitro and in- vivo blood clotting activity

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The In vitro and in vivo blood clotting activity of Laq extract of C. sparsiflorus

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were analyzed using the standard procedure suggested by Lee and White method (Lee and White, 1986). The blood sample was then subjected to morphological evaluation using

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Light microscope.

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2.4. In-vitro cytotoxicity assay

In vitro cytotoxicity assay was performed by adipocytes cell line 3T3-L1. Cell

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lines were cultured in DMEM media supplemented with 10% inactivated Fetal Bovin Serum (FBS), penicillin (100 IU/ml), streptomycin (100 mg/ml) and amphotericin B (5

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mg/ml) in a humidified atmosphere of 5% CO2 at 37ºC until confluent. The cells were dissociated with TPVG solution (0.2% trypsin, 0.02% EDTA, 0.05% glucose in PBS).

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The stock cultures were grown in 25cm2 culture flasks and all experiments were carried

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out in 96 microtitre plates.

2.5Identification of bioactive compounds of Laq of C. sparsiflorus

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To identify the bioactive compounds present in Laq extract of C. sparsiflorus, the

sample was subjected to GCMS analysis using GCMS (Model GCMS-QP2010, Shimatzu) equipped with a flame ionization detector, a fused silica capillary column (VF-5ms, 30.0 m x 0.25mm and a film thickness 0.25 µm). Helium was used as the carrier gas at a constant flow rate of 1.52 ml/min and 2 µl of sample was injected. The column temperature was programmed to 70°C with increasing temperature of 10°C/min to

300°C/min. The mass spectra were obtained through ionization energy of 70 Ev in the EI mode. Total GC-MS running time was 30 min. The organic compounds were identified by comparison of mass spectra with the inbuilt libraries. 2.6 Liquid-Chromatography Mass Spectrometry (LC-MS) Analysis In addition to GC-MS profile of Laq extract of C. sparsiflorus, it was further subjected to LC-MS (LC-MS xevo TQD system, waters India Pvt. Ltd). To confirm the

activity. 2.7 Nuclear Magnetic Resonance (NMR Spectrum) Analysis

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molecular weight and identity of the lead components responsible for blood clotting

NMR and

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The compounds identified by GC-MS and LC-MS, further characterized by 1H C NMR spectra to understand the element present in the extract and it was

recorded using DMSO as solvent on BRUKER 300 MHz ultra shield spectrometer with

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2.8 Fourier Transform Infrared Spectrum (FT-IR)

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AVANCE II console.

Functional groups present in bioactive Laq extract of C. sparsiflorus, was studied

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by FT-IR spectrum in KBr medium in the range of 400-4000 cm-1 using Perkin Elmer

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RX1 infrared scanner.

2.9 UV spectrophotometer analysis of Laq extract of C. sparsiflorus

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The Laq extract of C. sparsiflorus was dissolved in de-ionized water. Its optical behavior was examined by UV spectrophotometer (systronics).

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3. Computational details

The quantum chemical calculations of pytochemical structure of extracts of

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C.sparsiflorus have been performed using the B3LYP level of theory supplemented with the standard 6-311 G(d,p) basis set, using the Gaussian 03 program. The entire

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calculations were performed at DFT levels on a personal computer using Gaussian 03W (Schlegel, 1982) program package, invoking gradient geometry optimization (Vayner,

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2000).

3.1 Molecular Docking A computational tool offers the advantage of delivering new drug candidates more quickly and at a lower cost. The present work by computational approach used for the following software manipulation of drugs using molecular docking server online web service for

calculation of drug likenes. The identified compounds from Laq extract of C. sparsiflorus was used to interact with blood clotting proteins retrieved from PDB. 3.2 Protein Data Bank (PDB) The PDB is the particular, international files for information about the 3D structure of bio macromolecules and their complexes as determined by X-ray crystallography, NMR spectroscopy and includes more than a few Nobel Prize winning structure. Blood clotting

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protein was downloaded from protein data bank with the specific resolution and the PDB id is 1AHW and 1dan. 3.3 Preparation of protein

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A characteristic PDB structure file consists of heavy atoms and may include a cocrystallized ligand, ions and cofactors, metal and water molecules. Some structures are multimeric and may need to be reduced to a single unit. Schrodinger has accumulated a

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set of tools to prepare proteins in a form that is suitable for modelling calculations. The

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tools are come together in the Protein Preparation Wizard under Maestro. 3.4 Preparation of ligand

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The Schrödinger ligand preparation was constructed using the fragment library of Maestro

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9.2 and the compounds were arranged by using the LigPrep 2.4 (LigPrep, 2011), which can produce a number of structures from each input structure through various ionization

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states, tautomers, stereochemistries and ring conformations to eliminate molecules using various condition including molecular weight or specified numbers and types of

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functional groups present with correct chiralities for each successfully processes input structure. GLIDE (Grid-based Ligand Docking with Energetics) is a ligand binding

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program put together by Schrodinger that searches for favourable interactions among one or more ligand and receptor molecule, generally a protein. It provides a complete solution

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for ligand-receptor docking. The mixture of site and direction of a ligand relative to the receptor, along with its conformation in flexible docking is referred to as a ligand pose.

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The ligand poses that Glide generates pass through a sequence of hierarchical filters that calculate the ligand interaction with the receptor. In conclusion, the minimized poses are re-scored to build the Score or Glide score is the sum total of various figures cause for each ligand during the docking process. The OPLS-2005 force field was used for optimization, which produces the low-energy conformer of the ligand (Hayes, et al., 2004).

3.5 ADMET predictions by QikProp 3.2 QikProp is performed on minimized structures to calculate the ADMET properties for evaluate the 3D structure of the predicted ligand was analyzed to assess their various physio-chemical properties responsible for compose them potential drug like candidates. Validate the molecules are biologically active without any toxic functional groups. Their absorption, distribution, metabolism, excretion and toxicity (ADME) properties were

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calculated Insilco using QikProp component of Schrodinger (QikProp, 2012). The best Glide Score is obtained as the most negative value and the most active ligands in terms of

G Score are enlisted in descending order. In the ligand preparation, the structure of

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crotonosine was developed by using (Jorgensen and Duffy, 2000). Some of the computed

ADMET descriptors are mentioned along with their recommended ranges for 95% of known drugs.

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3.6 Binding site analysis

maestro to classify the active sites on the grid.

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3.7 GLIDE / Ligand Docking

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The major binding site analysis was carried out using the Sitemap tool from Schrodinger’s

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Grid generated output file was uploaded as an input for Ligand docking against protein

4. Results and Discussion

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prepared targets in GLIDE mode was adopted. Flexible docking mode was selected.

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4.1. Qualitative analysis of Laq of C. sparsiflorus The qualitative analysis for the presence of major groups of chemical compounds suggest

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that Laq extract of C. sparsiflorus has saponins, followed by alkaloids, phenols, flavonoids, tannins, terpenoids, steroids, glycosides and carbohydrates are present (Table

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1).

4.2. Quantitative phytochemical analysis of Laq extract of C. sparsiflorus

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The Quantitative phytochemical analysis of Laq extract revealed that the extract has more saponins (15.2%), followed by alkaloids (7.61%), phenols (0.62%), tannins (1.1%), and flavonoids at 0.224% is shown in the (Table 2). 4.3. In vitro and in vivo blood clotting analysis of Laq extract of C. sparsiflorus The in-vitro clotting time for control blood sample was observed to be 2.0±5 min, while it was 8±10 sec when equal amount of Laq (100µl) was added onto the blood sample (Table

3). The bleeding time accounted on animal was 5±1 min, while it was only 5±1 sec upon adding Laq extract of C. sparsiflorus. In vitro test sample demonstrated that coagulation factors I, II, V, VII, VIII, IX, X, XI and XIII was not affected by the addition of crude and Laq extract of C. sparsiflorus to fresh blood, plasma and serum samples. These study indicates that the crude and Laq extract induced network formation depends upon interactions between plant extract and

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blood proteins, such as fibrinogen. These plant extract might affect fibrinogen and other proteins by agglutination of these molecules. Macroscopic appearance of protein network

formation before and after adding crude and Laq extract of C. sparsiflorus to human

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plasma, serum and blood. The basic mechanism of action for crude and Laq extract shows to be the formation of encapsulated protein network that provides crucial points for erythrocyte aggregation. Rather than affecting an individual clotting factor. This protein

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mesh affects the entire physiological hemostatic process that controls the bleeding. In

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vitro cytotoxicity assay were also performed by 3T3L1 cell lines evaluated by Laq extract of C. sparsiflorus to determine the toxic effects of the extract. The cytotoxicity assay

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revealed that there was no toxic effect on 3T3L1 cell lines.

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4.4. Morphological evaluation

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resulted in the formation of a protein network that serves as the basis for the aggregation of red blood cells (Fig 1). Microscopic observations showed that contact between the

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blood with crude and Laq extract result in the formation of a substance that is in the form of concentric filaments at the magnification of 100 X.

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4.5. Assessment of cytotoxicity assay of C. sparsiflorus Cytotoxicity assays are performed to predict potential toxicity, using cultured cells which

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may be normal or transformed cells. The ability of a plant extract to inhibit cellular growth and viability can also be ascertained as an indication of its toxicity. Assessment

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parameters for cytotoxic effects include inhibition of cell proliferation, cell viability markers (metabolic and membrane), morphologic and intracellular differentiation markers (O’Brien and Haskings, 2006). In the present study, the Laq extract of C. sparsiflorus was subjected to determine the antiproliferation property. The 3T3-L1 preadipocytes were seeded in the 96 wells at the density of 1X105 / well. After 24 hours, different concentration of plant extract was supplemented and read the optical density at 450nm

after 24 and 48 hours incubation. The results showed that the plant extract treatment influenced its toxic effect on the 3T3-L1 cells viability (Table 4 and Table 5). Up to 0.1% at 24 hours and 0.025% of plant extract at 48 hours did not possess any cytotoxic effects. So, these concentrations were safe, and it can be used for the further experiment. But, cytotoxicity effects of this plant extract may be varied between the cells. The % of live cells was reduced as compared with control cells when increased concentration of

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plant extract. The LD 50 values for 3T3-L1 cells at 24 and 48 hours were 0.50% and 0.03% respectively.

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4.6. GC-MS analysis of Laq extract of C. sparsiflorus

The GC-MS spectrum of Laq of C. sparsiflorus showed three major peaks at retention times (RT) 15.512, 26.260, and 26.635 min in GC. The GC-MS spectrum and peak fragmentation are shown in (Fig 2). GC-MS plays a key role in the analysis of known and

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unknown components of the plant origin. GC-MS ionizes compound and measures their

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mass numbers. The use of mass spectrometry (MS) is most cases coupled to an

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appropriate separation technique as gas chromatography. GC-MS analysis of phyto constituents in plants gives a clear picture of the pharmaceutical value of the plant. There

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are very less reports on the analysis of phytochemicals in plants. The result of the GC-MS analysis led to the identification of number of compounds peak from GC fractions of the

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Laq extract of C. sparsiflorus (Table 6)

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4.7. LC-MS analysis of Laq extract of C. sparsiflorus The chromatographic analysis of Laq extract of C. sparsiflorus allowed to identifying three

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major compounds (Fig 3). Shows the LCMS-UV profile at 220 nm and illustrates the LCMS (ESI) profile in positive mode of Laq extract of C. sparsiflorus. The chromatogram

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shows the presence of one main peak 1 and two other peaks 2 and 3 respectively. 4.8. The 1H NMR and 13C NMR Spectral studies of Laq extract of C. sparsiflorus The proton magnetic resonance spectrum of aqueous extract of C. sparsiflorus was

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recorded in DMSO. The spectrum is given in (Fig. 4) and the resonance signals are shown in Table 7. The integration of the spectrum indicates the numbers of protons as 17. Resonance doublet at δ 2.4 ppm is due to –CH2- protons. The aromatic proton appear as multiplets in the range δ 7.0-6.7 and 6.3-6.1 ppm and the aromatic protons of tri substituted benzene appears in the range δ 7.0-6.7 ppm. The amine NH proton appears at δ 5.8 ppm. The signal due to methane proton appears at 4.7 ppm (triplet). The methoxy

proton appears in the range of 2.9 (doublet). Thus the 1H NMR spectrum reveals the presence of aromatic, amine NH and methylene CH2 groups in the compound. The intensity ratio obtained for the signals correlates well with the total number of protons under each chemically equivalent and magnetically active nucleus. The data presented in (Table 8). The spectrum shows absorption of carbonyl carbon at δ186ppm. The chemical shifts of aromatic carbons appear at δ150-149 and 128 - 126 ppm.

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The substituted aromatic carbon can be distinguished from other carbons by its decreased

peak height. It lacks a proton and hence suffers from longer relaxation time with a

diminished Nuclear Overhauser Effect (NOE). The peak at δ153ppm may be assigned to

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the substituted carbon in the aromatic ring attached with O-CH3. The methine carbon which is bonded to NH exhibits a signal at δ 80 ppm. The methylene carbon of pyridine para carbon resonates at δ22 and 47 ppm. From the above assignments the

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C NMR

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signals of the compound are in good agreement with the 1H NMR data and also agree with

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the computed values (Table 9).Then the result shows a good sign of crotonosine structural confirmation and leads us to further investigation.

molecular

name

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cyclopent[ij]isoquinolin]-4-one,

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Spiro[2,5-cyclohexadiene-1,7'(1'H)-

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The

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4.9. Molecular geometries

2',3',8',8'a-tetrahydro-5'-hydroxy-6'-methoxy.

The

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common name of this compound is Crotonosine. This molecule is extracted by plant source, aqueous extract of C. sparsiflorus. In this compound the functional groups such as

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carbonyl, methoxyl, and hydroxyl derivatives are presented. The molecular formula of the compound is C17H17NO3, and its molecular weight is about 283.1208. The geometric

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parameter was calculated by B3LYP with 6-311G(d,p) as the basis set is listed in Table S1 (Supporting). To the best of our knowledge, experimental data on the geometric

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structure of crotonosine is not available in the literature so far. The optimized molecular

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structure of crotonosine is shown in (Fig. 5).

4.10. Fourier Transform Infrared Spectroscopy Analysis (FT-IR) The functional groups of phytocompounds were detected by using FT-IR spectroscopy analyses are shown in (Fig. 6) and the data are presented in (Table. S2). The FT-IR analysis of Laq of C. sparsiflorus gave a broad band at 3401 cm-1 is assigned to the νO-H stretching vibration. The medium band at 2976 cm-1 is attributed to aromatic νC-H

stretching vibrations. The absorption in the region of 2360 cm-1 is due to the νC-H of aliphatic group. The band due to νC=O appears at 1640 cm-1 and aromatic amine band due to the δN-H (in- plane bending) vibrations appears at 1528 cm-1. The medium band at 1049 cm-1 is assignable to νC-N-C. The presence of absorption bands in the region 879678 cm-1 is due to out-of-plane bending vibrations of C-H bonds of aromatic ring. The strong absorption band observed at 678 cm-1 is due to mono substituted aromatic ring. In

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general primary amines show two νN-H stretching bands resulting from symmetrical and asymmetrical νN-H stretching and secondary amines show only one absorption band for

amine group is revealed from the IR spectrum of the extract.

4.11. Molecular Docking

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νN-H. Thus, the presence of mono substituted aromatic ring and secondary aromatic

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The 3D structure of Tissue factor and labile factor were optimized to achieve minimal

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potential energy using Schrodinger. The minimization values are summarized in the table. Docking simulation of 10 runs of plant compound crotonosine was performed for a set of

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catalytic active site of tissue factor and labile factor. The best docked conformation was

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selected based on lowest docking energy and binding free energy among ten different poses generated for each drug molecule. The docked conformations to analyze the binding

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mode of each factor docking score, glide g score, glide EVDW, glide ECOUL and glide energy are summarized in (Table 10). We are further use the potential energy, van der

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Waals interaction, electrostatic energy, dihedral energy, stretch energy and bend energy of tissue factor and stable factor are summarized in (Table 11). We also validated our

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docking results with the experimentally measured metabolic rate of blood clotting factors. The molecular property used for absorption, distribution, metabolism, and excretion

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(ADME) are fundamental for drug design. The ADME properties in the drug discovery process are able to reduce the costs of drug development and decrease the disintegration

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rate of drug candidates. The bioactive compound of crotonosine was within the acceptable range (results shown in the Table 12). Docking simulation of crotonosine into Tissue factor (PDB id: 1AHW) resulted in formation of three hydrogen bond interaction with ASP 165, ASP 167 and VAL 167 respectively. The hydrogen bond distance of 2.82Å, 2.30Å, 2.57 Å (Fig. 7) and it was observed that side chain NH group of ASP 167 act as hydrogen bond donor. The amino acid residues pro40, pro171, trp41, phe170, val167 are

involved in hydrophobic interactions with an active site. The other amino acid residues thr169, thr164, hid168, ser168, ser164 and ser165 are the polar bond interaction. The Glide score and docking score of the complex were -5.881 and -5.47 kcal/mol respectively. The docking results of crotonosine with stable factor (PDB id: 1dan) showed that only one hydrogen bond interaction. This hydrogen bond was formed between the side chain NH groups of GLU26 with a bond distance of 2.14 Å (Fig. 8). The amino acids

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cys27, pro23, cys22, val157, leu137, and trp207 are involved in hydrophobic interaction with an active site. In side chain asn159 act as the polar bond interaction. The Glide score and docking score of the complex were -3.578 and -3.518 kcal/mol respectively.

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4.12. UV Spectral Studies

Ultra-violet visible (UV-Vis.,) absorption spectrum was recorded for Crotonosine at the visible region between 200-800 nm. The recorded spectrum is shown in Fig 9. The

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recorded UV-Visible spectrum of Laq extract of C. sparsiflorus exhibits absorption band at 365 nm which can be assigned to n → π* transition of carbonyl group. The absorption

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band at 380 nm can be assigned to π → π* transition of carbonyl and aromatic groups.

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This gives an idea about the saturated compounds containing hetero atoms such as

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nitrogen, oxygen, etc, having non-bonding electrons in addition to σ electrons. The TDDFT calculated results of electronic excitation were presented in Table S3 (Supporting

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information).

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5. Summary and Conclusion

In phytochemical screening of Laq extract of C. sparsiflorus revealed the presence

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of alkaloids, phenols, saponins, flavonoids, steroids, terpenoids, glycosides and carbohydrates in plant extract. Especially, the alkaloids and saponins are greatly present in

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the extract. Bleeding can cause significant mortality and morbidity in any clinical setting. C. sparsiflorus is a traditional folkloric medicinal plant extract it is a novel effective

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haemostatic agent that has the therapeutic potential to be used in the management of haemorrhage. The aqueous extract of C. sparsiflorus was the appropriate extract promoting the highest hemostatic activity of both in vitro and in vivo. The in-vivo study, confirmed the significant ability of this plant extract to stop bleeding in rats. Mechanism of blood coagulation, when add the Laq extract shows the formation of network of proteins. It is basis for cell aggregation and this mechanism appears independently of

coagulation factors. This offers a very interesting therapeutic perspective because these plants extract could be used as local haemostatic with coagulation disorders such as haemophilia. The toxicological investigation of the Laq extracts of C. sparsiflorus was carried out in which no cytotoxicity was observed, hence there is no toxic in nature. In silico molecular docking studies revealed that the crotonosine exhibit good interactions with many blood clotting factors, and thus Crotonosine is the lead compound responsible 13

C), analysis confirms the

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for accelerating the haemostatic activity. NMR (1H and

elements of crotonosine compound, the FT-IR vibrational spectra showed the

characteristic functional groups of crotonosine, moreover the measured UV-Visible spectrum

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of C. sparsiflorus exhibit n → π* and π → π* transitions. The structure geometry, vibrational

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frequencies and electronic excited states were investigated and agree well with literatures.

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6. References 1. Badgujar, S.B., 2011. Proteolytic enzymes of some latex bearing plants belonging to Khandesh region of Maharashtra (Ph.D. thesis). North Maharashtra University, Jalgaon, Maharashtra State, India. 2. Vayner, E., Ball, D.W., 2000. Ab initio and density functional optimized structures, proton affinities, and heats of formation for aziridine, azetidine, pyrrolidine, and piperidine, J. Mol. Struct. (THEOCHEM) 496, 175-183. 3. Evans W.C., Trease and Evan’s., 1989, Textbook of pharmacognosy, 13th ed, Cambridge University press, London, p.546. 4. H.B. Schlegel, 1982. Optimization of equilibrium geometries and transition structures, J. Comput. Chem. 3, 214-218. 5. Harborn JB. Phytochemical methods: A guide to modern techniques of plant analysis. 1973. 6. Hayes,M.J.,Stein,M.,Weiser,J., 2004. Accurate calculation sof ligand binding free energies. J.Phys.Chem. A, 108, 3572–3580. 7. Jorgensen WL, Duffy EM., 2000. Prediction of drug solubility from Monte Carlo simulations. Bioorg Med Chem Lett 10,1155–1158.

8. Lee and White. Coagulation procedures 1968. Ortho Diagnostics, Raritan, New Jersy. 9. LigPrep, 2011. Version 2.5, Schrödinger, LLC, New York 10. Pandey, B.P., 2001. Plant Anatomy, 6th revised ed. S. Chand and Company Ltd., New Delhi, India. 11. L. J. Haynes, K. L. Stuart, D.H.R. Barton, and G. W. Kirby, Proc.Chem.Soc.,

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1964, 261. 12. QikProp, version 3.5, schrodinger, LLC, New York, NY, 2012. 13. Salatino, A., Maria, L., Salatino, F., Negri, G. J. Braz., 2007. Traditional uses, Chemistry and Pharmacology of Croton species (Euphorbiaceae), Chem. Soc. 18, 11-33. 14. Satish, A., Sairam, S., Ahmed, F., Urooj, A., 2012. Moringa oleifera Lam.: protease activity against blood coagulation cascade. Pharmacogn. Res. 4, 44–49. 15. Shivaprasad, H.V., Rajesh, R., Yariswamy, M., Vishwanath, B.S., 2011, Procoagulant properties of plant latex proteases. In: Kini, R.M., Clemetson, K.J., Markland, F.S., McLane, T., Morita, T. (Eds.), Toxins and Hemostasis. Springer, Netherlands, pp. 591–603. 16. Yadav SK and Das S. 2013. Phytochemical screening and antidiarrhoeal activity of aqueous extracts of Croton sparsiflorus Morong. Int J Pharma Res Rev. 2, 1216.

IP T SC R

Fig. 1. Microscopic view of the human blood before and after contact with the crude

A

CC

EP

TE

D

M

A

N

U

and Laq extract of C. sparsiflorus

Fig 2. GC-MS chromatogram of aqueous extract of C. sparsiflorus

IP T SC R U N

A

CC

EP

TE

D

M

A

Fig. 3. LC-MS chromatogram of Laq extract of C.sparsiflorus

IP T SC R U N A M D TE EP CC A Fig.4. 1H NMR and 13C NMR spectrum of aqueous extract of C.sparsiflorus

D

TE

EP

CC

A

IP T

SC R

U

N

A

M

IP T SC R U N

A

CC

EP

TE

D

M

A

Fig. 5. Optimized structure of crotonosine

 

FT-IR



IP T

949

1182

1523

2441

A



3037



2938w

SC R

 

M

D

2.0

1689 s

EP

2.5

1232ms

IR/B3LYP/6-31G(d,p)

1.5

TE

IR Intensity

1.0

1488ms

0.5

1158w

N

0.0

CC

3.0

A

1601w

U



833w

IR Intensity



3.5 4000

3500

3000

2500

2000

1500

1000

-1

Wavenumber (cm )

Fig. 6. Combined FT-IR spectra of C.sparsifloru

500

IP T SC R U

A

CC

EP

TE

D

M

A

N

Fig. 7. UV recorded and simulated of aqueous extract of C.sparsiflorus

D

TE

EP

CC

A

IP T

SC R

U

N

A

M

IP T SC R

A

CC

EP

TE

D

M

A

N

U

Fig. 8. Docking interactions with Tissue factor

IP T SC R

A

CC

EP

TE

D

M

A

N

U

Fig. 9. Docking interactions with Stable factor

SC R

A

N

U

Qualitative and quantitative phytochemical analysis Phytoconstituents Present (+)/absent (-) Alkaloids + Steroids + Tannins + Phenols + Terpenoids + Glycosides Carbohydrates + saponins + Quinine + Cardiac glycosides + Coumarins + Flavonoids +

IP T

Table 1. Qualitative phytochemical screening Laq extract of C. sparsiflorus

M

Table 2 Quantitative phytochemical analysis of Laq extract of C. sparsiflorus

CC

EP

TE

D

Quantitative phytochemical analysis Phytoconstituents Percentage (%) Alkaloids 7.61±0.2 Phenols 0.62±0.08 Tannins 1.1±0.17 saponins 15.2±0.91 Flavonoids 0.224±0.004 Results expressed as mean ±SEM

A

Table 3 Blood coagulation properties of latex and Laq of Croton sparsiflorus Parameter Blood Blood T Plasma Plasma T Serum Serum T

Blood coagulation time (In sec.) Control Latex 120 10 60 8 660 22 96 23 720 26 120 21

Laq 13 10 20 19 32 23

Blood = normal blood sample, BloodT = blood with Thromboplastin, plasma = fluid obtained when anti coagulated blood has been centrifuged. plasmaT = plasma with Thromboplastin, serum = fluid obtained when coagulated blood has been centrifuged. SerumT = serum with Thromboplastin

Cont rol

0.01 25

0.02 5

0.05

0.1

1

1.5

2

2.5

3

A

1.194

1.166

D

1.13

E

1.156

Aver age STD

1.155 8 0.026 2 100

0.63 8 0.57 5 0.61 8 0.59 9 0.52 5 0.59 1 0.04 36 51.1 334

0.50 7 0.67 4 0.65 9 0.58 9 0.51 2 0.58 82 0.07 87 50.8 911

0.485

C

0.77 6 0.90 9 0.76 5 0.75 9 0.81 1 0.80 4 0.06 20 69.5 622

0.536

1.133

0.77 8 0.79

0.594

B

1.10 7 1.20 8 1.06 4 1.15 1 1.24 1 1.15 42 0.07 21 99.8 615

0.37 5 0.36 1 0.40 8 0.34 3 0.37 1 0.37 16 0.02 38 32.1 508

EP CC A

D

M

0.592 8 0.008 2 51.28 91

0.523

0.522

0.475

0.516

0.549

0.646

0.522

0.570 4 0.063 2 49.35 11

0.510 8 0.030 3 44.19 45

U

0.587

0.632

N

0.606

A

0.592

TE

In %

0.78 5 0.73 6 0.78 1 0.77 4 0.02 17 66.9 666

0.585

3.5

4

4.5

0.37 3 0.51 8 0.43 3 0.44 5 0.37 8 0.42 94 0.05 90 37.1 517

0.38 9 0.39 6 0.47 8 0.42 6 0.51

0.32 9 0.33 3 0.34 3 0.34 9 0.35 9 0.34 26 0.01 21 44.2 635

SC R

Raw data

IP T

Table. 4.Cytotoxicity studies of Laq extract of C. sparsiflorus on 3T3L1 cell line at 24h

0.43 98 0.05 26 38.1 043

I N U SC R

0.0125

0.025

A

1.943

2.255

1.433

B

2.021

2.039

1.361

C

2.152

2.41

D

2.416

1.942

E

2.339

Average

2.1742

0.05

0.1

1

1.5

2

2.5

3

3.5

4

4.5

0.608

1.115

0.525

0.586

0.437

0.632

0.934

0.479

0.355

0.34

0.709

0.649

0.766

0.528

0.536

0.585

0.395

0.539

0.391

0.344

1.238

1.317

0.613

0.651

0.549

0.515

0.572

0.397

0.539

0.391

0.329

1.711

0.601

0.562

0.656

0.543

0.699

0.564

0.42

0.551

0.37

0.32

2.35

1.47

0.686

0.552

0.72

0.629

0.503

0.576

0.378

0.59

0.38

0.324

2.1992

1.4426

0.7842

0.6982

0.6636

0.567

0.538

0.5858

0.5048

0.5396

0.3774

0.3314

M

Control

CC EP

TE

D

Raw data

A

Table. 5. Cytotoxicity studies of Laq extract of C. sparsiflorus on 3T3L1 cell line at 48h

0.2018

0.2013

0.1742

0.3015

0.2362

0.0909

0.0407

0.0973

0.0269

0.2403

0.0398

0.0152

0.0102

In %

100

101.1498

66.3508

36.0684

32.1129

30.5215

26.0785

24.7447

26.9432

23.2177

24.8183

17.3581

15.2423

A

STD

Table 6 Phytocomponents identified in the aqueous extracts of C.sparsiflorus by GC-MS S. No.

RT

Area%

Library/ID(C:\Database\NIST08.L)

Mol. F

Mol. W

15.512

9.16

Alpha-d-mannofuranoside

C7H14O6

194

2.

26.260

25.27

2,3-benzofurandione

C16H12O5

284

3.

26.635

65.57

Spiro[2,5-cyclohexadiene

C17H17NO3

IP T

1.

Chemical shift for protons Chemical shift in ppm

OH proton Aromatic proton NH Methine CH O-CH3 Methyl -CH2-

7.2 7.0 – 6.7 & 6.3 – 6.1 5.8 4.7(t) 2.9(d) 2.4(d)

D

M

A

N

U

Types of protons

SC R

Table 7 1H NMR resonance signals (δ ppm) for Laq extract of C.sparsiflorus

EP TE

Table 8. 13C NMR resonance signals (δppm) for aqueous extract of C.sparsiflorus

A

CC

Carbon environment C=O carbon Aromaric carbon attached with O-CH3 Aromatic ortho carbons Aromaric carbon attached with OH Aromatic meta carbons Aromatic carbon Aromatic para carbon Cyclic carbon next to NH group Pyridine meta carbon Quinone para carbon Pyridine ortho carbon Pyridine para carbon

Chemical shift (ppm) 186(s) 153(s) 150-149(d) 141(s) 128-126(m) 124-120(m) 110(s) 80(s) 73-67(m) 56(d) 47(d) 22(s)

283

Table 9. Elemental Analysis of Laq extract of C. sparsiflorus Calculated % 72.07 6.05 4.94 16.94

Observed % 72.89 5.71 9.96 11.44

Table 10. Docking interactions of crotonosine with blood clotting factors Docking score

GLIDE GLIDE Gscore EVDW

GLIDE ECOUL

SC R

PDB ID

GLIDE energy

1AHW

-5.47

-5.881

-24.024

-10.666

-34.69

1dan

-3.518

-3.578

-15.14

-5.055

-20.195

N

crotonosine

Target protein Tissue factor Stable factor

U

Compound name

IP T

Element Carbon Hydrogen Nitrogen Oxygen

EP TE

D

M

A

Table 11. Schrodinger values (Steepest Descent followed by conjugate Gradient method) crotonosine with tissue factor and stable factor. Energy levels with minimization max of 1000 steps (Kcal/mol) Molecules Potential Van der Electrostatic Dihedral Stretch Bend energy waals energy energy energy energy energy Tissue -3283.97 -11131.6 -6925.6 -6925.6 411.702 2037.09 factor Stable -1039.87 -2405.09 -1236.27 -1236.27 96.829 417.97 factor Table 12. The ADME properties of crotonosine using Qikprop tool. Properties Molecular weight H Bond Donor H bond Acceptor Dipole SASA CNS Volume QPlogPoct QPlogPw QplogPo/w QPlogS QPlogBB

A

CC

S. No 1 2 3 4 5 6 7 8 9 10 11 12

Crotonosine 283.326 2 5 8.07 497.164 1 883.09 16.122 10.214 1.311 -2.261 -0.151

Recommended values 130.0 – 725.0 0.0 – 6.0 2.0 – 20.0 1.0 – 12.5 300.0 – 1000.0 –2 to +2 500.0 – 2000.0 8.0 – 35.0 4.0 – 45.0 –2.0 – 6.5 –6.5 – 0.5 –3.0 – 1.2

QPlogKp IP(ev) EA(ev) HOA PSA Rule of three N &O

–8.0 – –1.0 7.9 – 10.5 –0.9 – 1.7 -1.5 – 1.5 >80% is high <25% is poor maximum is 3 2 – 15

-5.033 9.031 0.446 3 72.091 0 4

A

CC

EP TE

D

M

A

N

U

SC R

IP T

13 14 15 16 17 18 19