Author’s Accepted Manuscript

Haemostatic property of new Cystein protease(s)

f r o m Sesbania grandiflora: It's action on
fibrinogens

C.S Madhu, K.S Balaji, J Shankar

www.elsevier.com/locate/bab

PII: S1878-8181(17)30214-1
DOI: http://dx.doi.org/10.1016/j.bcab.2017.08.008
Reference: BCAB603
To appear in: Biocatalysis and Agricultural Biotechnology
Received date: 6 April 2017
Revised date: 29 June 2017
Accepted date: 13 August 2017
Cite this article as: C.S Madhu, K.S Balaji and J Shankar, Haemostatic property
of new Cystein protease(s) from Sesbania grandiflora: It's action on fibrinogens,
Biocatalysis and Agricultural Biotechnology,
http://dx.doi.org/10.1016/j.bcab.2017.08.008
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Haemostatic property of new Cystein protease(s) from Sesbania grandiflora: It’s action on
fibrinogen.
C.S Madhu1, K.S Balaji2, J Shankar2*
1
Research scholar, Yuvaraja’s College, Department of Biochemistry, Yuvaraja’s College,
University of Mysore, Mysore, Karnataka-570-005.
2
Research scholar, Post Graduate Department of Biotechnology, Teresian College, Affiliated to
University of Mysore, Mysore, Karnataka-570-011.
*
Corresponding author, Assistant Professor, Post Graduate Department of Biotechnology,
Teresian College, Affiliated to University of Mysore, Mysore, Karnataka-570-011.
madhucs89@gmail.com

Abstract:
This study aims to explore possible involvement of Sesbania grandiflora leaves extract
on haemostatic pathway. The crude dialyzed fraction (SgCDF) hydrolyses casein and exhibiting
a protease activity (19.7±1.5 U/Hr) at 100ug/ml. Zymography (casein) studies further reveal the
presence of two high molecular weight protease(s). Presence of possible protease(s) was
confirmed by inhibition of Proteolytic activity by mercury chloride (HgCl2) suggesting the
presence of Cysteine protease. The stability of protease activity against various parameters viz.,
temperature, pH, salt (NaCl) has been evaluated. These protease(s) exhibiting a promising
Fibrinogenolytic activity by hydrolyzing Aα and Bβ subunit of fibrinogen at 25ug and 50ug
concentration, whereas SgCDF fails to degrade the γ-subunit at the same concentration. The
protease shows a promising recalcification time up to 80±2% against Trypsin. These findings
suggest the possible role of proteolytic fraction of Sesbania grandiflora in haemostatic and
wound healing process.
Keywords: Hemostasis, procoagulant, Cystein protease, Fibrinogen, etc.
1. Introduction:

Blood clotting/coagulation is a complex and dynamic process involving a tightly

regulating events at cellular level, which restores/regain the tissue integrity (Colette et al., 2004,
Walker et al., 1985). During blood clotting process series of zymogens’ (proenzymes) activation
has been taking place, invivo (Monroe et al., 2010). Upon activation of these proteases which
acts on, fibrinogen a heterotrimeric protein (Aα, Bβ & γ) hydrolyzed into fibrin mesh which
stops the bleeding on fresh cuts/ injury region. During pathophysiological condition/ infections,
host fails to response and in most of the cases results in the delayed in healing of chronic wounds
(Singer et al., 1999). It is debatable, that uses of conventional drugs such as antibiotics, synthetic
molecules and anti-inflammatory drugs are used in treating wounds were associated with
undesirable side effects including pain, tissue damage and restoration, skin discoloration,
complications of histocompatibility and more importantly cost of the product (Dorsett-Martin et
al., 2008). To overcome/minimize the complications and accelerate the healing process, a drug
that can enhance healing mechanism, when applied tropically, should prove ideal.
Medicinal plants have been used from our ancient times as a complementary and
alternative medicine (CAM)/ traditional medicine for various ailments. Recent years CAM has
been challenging part of the pharmacology fields, to understand the cellular and molecular events
taking place (Wachtel-Galor et al., 2011). In this regard, herbal based medicines are liable, cost
effective and have an immense potential to treat wounds and provide suitable environment to
promote physiological healing process in the short time with minimal pain and infection
complications (Mackay et al., 2003, Purna et al., 2000). Use of crude extracts from different
medicinal plant parts to enhance the blood clot at the injured area and also promote wound
healing has been practice in India and other parts world (Corider et al., 2012). Folklore
practitioners and tribal people were used freshly prepared plant crushed juices to treat various
types of wounds including burns, cuts, ulcers, boil, warts and crack; this is due to the presence of
various health sustaining bioactive constituents (Korpenwar et al., 2012, Thakur et al., 2011).
Wound healing activity of plants were attributed partly from secondary metabolites and mostly
to protease enzyme found in it (Upadhyay et al., 2011). This virtue of knowledge has been
scientifically examined for several years. The earlier reports show that protease from different
plant parts such as Eumilin (cystein), Ficin (Cystein), Papain (Cystein), Hirtin (Serine), LGP
(Serine), Crinumin (Serine), etc are shows the significant towards this healing process
(Shivaprasad et al., 2016). However, the detail mechanisms of action, underlying the wound
healing property of plant proteases are unclear.

Sesbania grandiflora (Agathi), belongs to the family fabaceae. The various parts of the
plant have been used to treat different ailments such as fever, cough, diabetes, inflammation and
cancer. The freshly prepared leaves juices were directly applied to treat all kinds of sprains and
bruises. The leaves and flowers of the plant having good astringent property, which helps to
promote contracting body tissue and blood vessel (Dale et al., 1987, Orwa et al., 2009). Earlier
studies show that, Sesbania grandiflora exhibits a strong antioxidant and anticancer (Ramesh et
al., 2010 & Longanayaki et al., 2012). The protein fraction isolated from leaves and flower
shows the antioxidant (Zarena et al., 2014), anticancer (Laldhas et al., 2010), wound healing
(sheikh et al., 2011) and α–glucosidase inhibitory (Boonmee et al., 2007) activity. It is believed
that raw juices from medicinal plant leaves can able to stop the bleeding in injured area. On the
basis of this, we first time report the presence of possible Cystein proteases from Sesbania
grandiflora leaves involved in the wound healing process. The dialyzed fraction (SgCDF)
exhibits a promising fibrinogenolytic and procoagulant activity. Further studies required to check
the possible role and molecular mechanism of protease(s), which is involved in the blood
coagulation cascade.
2. Materials and Methods:
2.1. Sample preparation:
Sesbania grandiflora leaves were collected from local garden area, Mysore and authenticated
by Dr. Sharvani K.A, Assistant professor, Department of Botany, Yuvraja’s College, University
of Mysore, Mysuru. Herbarium was prepared and deposited at Department of Botany, Yuvraja’s
College, University of Mysore, Mysuru, Mysore (Accession number: YCM(UOM)0266). The
leaves were cleaned by using double distilled water and air dried under shade and finely course
powdered. Leaf powder (5gm) was weigh and mixed with 100mL distilled water in a rotary
shaker at 120 rpm for overnight at room temperature. Centrifuge the mixture at 10,000 g at 40C
for 15 min and collected supernatant was subjected to ammonium sulphate (80%) precipitation
for to overnight at 40C. The pellet (protein) was collected by centrifugation and extensively
dialysis (molecular cutoff 3.0 KDa) against distilled water 24 hours and lyophilized to
concentrate the protein. Further separation of low and high molecular weight proteins was
carried out by using Amicon Ultra centrifugation filters (Merk Millipore, Billericia, MA, USA)
with 50K cut-off used. The aliquots of 0.5mL dialysis sample was transferred to
ultracentrifugation filters and centrifuged at 14,000 x g for 15min. The resulting filter solution
was collected and named as Sesbania grandiflora crude dialysis fraction (SgCDF) and used for
the further studies.
 2.2. Protein Estimation:

The protein content was determined using Bradford’s reagent using Bovine Serum Albumin
as standard (Bradford et al., 1976).

2.3. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE):

One dimensional (1D) Electrophoresis was performed according to the method described
earlier (Laemmli et al., 1970). Ten percent gels SDS-PAGE (10%) contains, was carried out for
the SgCDF. Upon reaching of the bands to the bottom of the gel, carefully removed from the
system and stained with commassie brilliant blue R-250, followed by distained with methanol,
acetic acid & water(3:1:6).

2.4. Evaluation of Protease activity and its nature and effect of pH, temperature,
salt (NaCl):

The Protease activity of SgCDF was carried out according to the method described earlier
(Murata et al., 1963) by using casein as substrate. Briefly, different doses (20-100ug/ml) of
SgCDF were incubated with casein (2%) in 0.2 M Tris-HCl buffer, pH 8.5, in a final volume of
1ml at 37 0C for 2.5 hr. By adding 0.44 M trichloroacetic acid (TCA), reaction was stopped and
allowed it to stand for 30 min, followed by centrifugation at 1500 g for 15 min. The supernatant
(1.5ml) was mixed with 0.4 M sodium carbonate (2.5ml) and 1:2 FC reagent (Folin–Ciocalteu)
(0.5ml). The absorbance was read spectrophotometrically at 660 nm. Activity was expressed as
units/h. One unit of enzyme activity was defined as the amount of enzyme required to increase
the absorbance by 0.01 at 660nm/h.
2.5. Determination of effect of temperature, pH, Salt stability and nature of
Protease enzyme:
The effect of temperature, pH and salt on protease (SgCDF) activity was determined by
incubating the sample for 30 minutes with different temperature (200-1000C), various buffers
with wide range of pH(2-12 pH) and salt concentration(0.5%-3%) respectively. After incubating
the samples, protease activity was determined as described earlier (Murata et al., 1963).
Similarly, by using specific protease inhibitors (5mM) was incubated with SgCDF (100ug) for
30 minutes at 370C and the activity was determined as described earlier (HgCl2:Cystein protease,
PMSF: Serine protease), EDTA: Metallo protease, Pepstatin A: Aspartic protease).
 2.6. Substrate-Gel assay:
Protease activity was further confirmed by substrate gel assay described earlier (Satish et al.,
2012). Briefly, SDS-PAGE (10%) containing casein (2%) was used for the study. The different
concentration of SgCDF (25ug, 50ug & 100ug) incubated with sample loading buffer in the
absence of 2-mercaptaethanol, without subjected to heat treatment. Later, the electrophoresis gel
was washed with Triton X-100 (2.5% in 10mM Tris-HCl buffer, pH 7.5) and followed by
distilled water wash (4X) until the foam was removed. Then the gel was transferred to incubating
buffer (50mM Tris-Hcl, 50mM CaCl2, 0.15mM NaCl) for 18hrs, at 370C. After incubation, gel
was stained with Commassie brilliant blue R-250 and then distained by using distaining solution.
The white clear bands indicate the presence of protease enzyme.
2.7. Coagulant activity:
Plasma re-calcification time was determined according to the method described earlier
(Condrea et al., 1983). Platelet poor plasma was prepared by centrifuge (2500 rpm, 15min) the
blood collected from non-smoke healthy donor in a tube containing anti-coagulant (0.11M tri-
sodium citrate) in the ratio of 9:1. The collected supernatant was considered as platelet poor
plasma (PPP). Platelet poor plasma (300µl) was pre incubated with different concentrations of
the SgCDF (20, 40, 60, 80, 100ug/ml) for 1 min at 370C. By adding 25µl of CaCl2 (0.25M) to the
reaction mixture clot formation was initiated and time taken for visible clot was recorded from
the time of adding CaCl2. Tris-Hcl buffer was used as a control. Clotting efficiency was
calculated is as follows:

2.8. Fibrinogenolytic activity:

Fibrinogenolytic activity of SgCDF was carried out according to the method described earlier
(Lee et al., 2006). Briefly, human fibrinogen (50ug) was pre-incubated with SgCDF (50ug &
100ug) in 10mM Tris-Hcl buffer pH 7.4 for 30min at 370C. Add sample buffer (4% β-
mercaptoethanol, 1M urea and 4% SDS and subjected to SDS-PAGE (10%) to reaction mixture
for terminating the reaction. The hydrolyzed fibrinogen pattern was observed by using
Commassie Brilliant Blue R-250 staining followed by distaining.
 2.9. Statistical analysis: All statistical analysis was performed by using GraphPad
prism 5.01. Data of 3 sets were expressed as the mean ± SEM and the analysis was
performed with ONE-WAY analysis of variance (ANOVA). Results were considered
statistically significant at **P<0.05, ***P<0.0001.

3.0. Results & Discussion:

Proteases are acts as a “molecular knives” which specifically cleaves peptide bond
present between the aminoacids. Hemostasis involves two important proteolytic system,
fibrinolysis and coagulation (Lijnen et al., 2002). Series of reaction has been carried out during
blood coagulation or clotting, that involves activation of fibrin from fibrinogen by the action of
thrombin. Formation of fibrin mesh involves the activation of series of zymogen’s activation by
protease(s) that leads to blood coagulation. This causes the platelet aggregation and forms a
stable haemostatic plug which prevents the blood loss (Berkner et al., 2001). More importantly,
regulation of the proteases is known to be an important factor in the wound healing mechanism.
The innate mechanism of action of wound healing rely on the regulation of proteases levels at
wound site. When this fails to activation at wounded site, external application of proteases
become a must. Number of proteases from different sources are involved in hemostasis have
been identified and characterized. Earlier reports conducted against these molecules could
enhance the identification and development of novel molecules which has been used to treat the
disorders related with coagulation cascade (Costa et al., 2010).
Present investigation mainly concentrates upon wound care carried out using plant
extracts and their proteases present in it. Also we provide the methodological approach towards
studying the wound healing property of the proteases. In this study, Protease sample was
prepared via extract with double distilled water followed by 80% ammonium sulphate
precipitation, centrifugation and subsequent dialysis to remove ammonium sulphate salt and
other phenolic metabolites. The crude protein lysate was subjected reducing gel resolved into
multiple bands corresponds to variety of proteins present in the sample (supplementary data1).
SgCDF exhibited protease activity by hydrolyzing casein as substrate. The activity was steadily
increasing with increasing concentration. SgCDF (20-100ug) protein lysate gave a mean activity
of 4.52± 1.5U/hr to 19.7± 1.5U/hr against trypsin as standard, exhibited higher protease activity
(Fig1A). Further, protease activity was confirmed by substrate gel assay by incorporating 2%
casein into 10% SDS-PAGE. The hydrolyzing pattern of SgCDF was resolved into two distinct
bands with higher molecular weight as shown (Fig1B). The similar results have been reported
earlier from curcuma species (Shivlingu et al., 2016). Stability and nature of protease present in
the sample has been evaluated viz different parameters. Incubating the sample at different
temperature, pH & salt stability has been determined. The optimum activity of the SgCDF
against temperature (Fig2A), pH (Fig2B) & salt (NaCl) (Fig2C) was found to be 450C±5, 7.0±
0.5 & 3%. This results exhibit a protease in SgCDF can function at normal physiological
condition(s). Nature of protease has been determined by incubating the various inhibitors with
SgCDF, indicates that the SgCDF containing cystein protease (Fig2D) was inhibited by mercury
chloride (HgCl2) which is a general inhibitor of cystein protease. Other protease inhibitors such
as PMSF, EDTA & Pepstatin had no effect on the protease activity of SgCDF.
A plasma glycoprotein human fibrinogen (factor X) has only tyrosyl and glutamyl N-
terminal residues, with a molecular weight 340-kDa, made up of three subunits Aα (63.5kDa),
Bβ (56.0 kDa) and γ (47.0 kDa), whereas fibrin having glycyl N-terminal residues instead of
glutamyl residue (Laskowski et al., 1956). Fibrinogenolytic activity of SgCDF shows the
possible involvement of protease(s) on haemostatic mechanism. Fibrinogenolytic activity was
determined by incubating the samples (25ug & 50ug) with human fibrinogen (50ug) and
hydrolyzed pattern was analyzed by 10% SDS-PAGE (Fig3B). Protease present in the SgCDF
preferentially hydrolyses Aα & Bβ subunits with the incubation time of 30min, whereas γ chain
shows the resistant against protease action. The fibrinogenolytic activity of SgCDF resembles to
other crude protease reported earlier from Cucumis sativus (Nafeesa et al., 2015) & Atrocararpus
heterophyllus (Siritapetawee et al., BBA 2012). These enzymes preferentially hydrolyzes the
different subunits in Aα>Bβ>γ manner. SgCDF exhibits thrombin (serine protease) like activity,
which cleaves Aα & Bβ chains of fibrinogen at the N-terminal disulfide knot and form fibrin
mesh by releasing fibrinopepetide A and/or B (Komori et al., 1985). This is due to the absence
of carbohydrates in Aα chain, whereas in contrast to γ chain contains carbohydrate (N-glycosyl)
moiety at the end of fibrinogen E-domain (Zauner et al., 2012). Most of the proteases(s) from
plant sources lack the ability to hydrolyse the γ-subunit of the fibrinogen (Imamura et al., 2001,
Richter et al., 2002). Earlier studies say that cystein proteases from Calotropis gigantean,
Pergularia extensa and Asclepias curassavica fails to degrade the chain of the fibrinogen
(Rajesh et al., 2005, Shivaprasad et al., 2009, Shivaprasad et al., 2009). Supporting to the
fibrinogenolytic activity, we also evaluate the clot inducing ability by SgCDF, the plasma
recalcification time of platelet poor plasma was estimated (Fig3A). Formation of clot was
determined in a dose dependent manner and shows up to 78.58±1.5% clotting efficiency,
whereas trypsin shows 23.87±1.5% clotting efficiency. These data strongly suggests that the
SgCDF is a therapeutically important and effectively control the bleeding upon injuries and
could be the responsible for the procoagulant effect of SgCDF.
4.0. Conclusion
Till date there is no report on Proteolytic enzyme from Sesbania grandiflora aqueous
leaves extract and current study revealed the presence of a protease, which belongs to the
Cysteine Protease family. This study helps to identifying the new Cystein protease(s) from
Sesbania grandiflora providing scientific validation for its use in traditional medicinal plants
leaves in maintaining hemostasis. The SgCDF exhibit a promising Proteolytic activity (Cystien)
activity and relatively stable at optimum condition, with effective fibrinogenolytic and pro-
coagulant activity. Further studies require to purifying and characterizing the interfering protease
present in the SgCDF, behind the coagulation pathway.
Acknowledgement:

Authors acknowledge to Teresian Research foundation for the support towards this
research work. This research did not receive any specific grant from funding agencies in the
public, commercial, or not-for-profit sectors.

Conflict of interest:

Authors declare that they have no conflict of interest.

References:

Berkner, K.L., 2001. Blood Clotting: General Pathway. In:eLS. John Wiley & Sons, Ltd,
Chichester.
Boonmee, A., Reynolds, C.D., Sangvanich, P., 2007. Aplha-Glucosidase inhibitor
proteins from Sesbania grandiflora flowers. Planta Med. 73(11), 1197-1201.
Bradford, M.M., 1976. A Rapid and sensitive method for the quantification of microgram
quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 7(72),
248-254.
Colette, N.M., Kelly, G.J.M., Williams, C.M., 2004. Olive oil and hemostasis. Grasas y
Aceites. 55, 52–65.
 Condrea, E., Yang, C.C., Rosenberg, P., 1983. Anticoagulant activity and plasma
phosphatidyl serine hydrolysis by snake venom phospholipases A2. Thromb Haemost.
49, 151.
Corider, W., Steenkmp, V., 2012. Herbal remedies affecting coagulation: a review.
Pharma Biol. 50, 443-452.
Costa, J.O., Fonseca, K.C., Garrote-Filho, M.S., Cunha, C.C., De Freitas. M.V., Silva,
H.S., Araujo, R.B., Penha-Silva, N., Oliveira, F., 2010. Structural and Functional
Comparison of Proteolytic Enzymes from Plant Latex and Snake Venoms. Biochimie. 92,
1760-1765.

Dale, O.E., Peter P. Rotar., 1987. Sesbania in Agriculture. Westview Press (Verlag),
978-0-8133-7312-6 (ISBN).

Dorsett-Martin, M.A., Person, B., Wysocki, A., Lineaweaver, W., 2008. Wounds, 20(11), 292-
298. Doi: 10.1007/978-1-59745-285-4_65.
Imamura, T., Banbula, A., Pereira, P.J., Travis, J., Potempa, J., 2001. Activation of human
prothrombin by arginine-specific cysteine proteinases (Gingipains R) from
Porphyromonas gingivalis. The Journal of Biological Chemistry 276(22), 18984–18991.
Doi: 10.1074/jbc.M006760200.
Komori, Y., Sugihara, H., Tu, A.T., 1985. Specificity of hemorrhagic proteinase from
Crotalus atrox (Western diamond back rattle snake) venom. Biochimica et Biophysica
Acta. 829, 127–130. Doi: 10.1016/0167-4838(85)90076-7.
Korpenwar et al., 2012. Ethnomedicinal plants used by the tribal’s in cure of wounds in
Buldhana District (MS) India. International journal of Recent Trends in Science and
technology. 3(2), 49-53.

Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature. 227, 680–685.
Laladhas, K.P., Cheriyan, V.T., Puliappadamba, V.T., Bava, S.V., Unnithan, R.G.,
Vijayammal, P.L., Anto, R.J., 2010. A novel protein fraction from Sesbania grandiflora
shows potential anticancer and chemo preventive efficacy, in vitro and in vivo. J Cell
Mol Med. 14(3), 636-646. Doi: 10.1111/j.1582-4934.2008.00648.x

Laskowski, M., Thomas, J.R., Donnelly, H., Barbara, A., Tijn, V., Harold, A, S., 1956. The
proteolytic action of thrombin on fibrinogen. J. Biol. Chem. 222,815-821.

Lijnen, H.R., 2002. Matrix metalloproteinase’s and cellular fibrinolytic activity.

Biochemistry (Moscow) 67, 92–98. Doi: 10.1023/A:1013908332232.
Lee J. S., Baik, H. S., Park, S. S. 2006. Purification and characterization of two novel fibrinolytic
proteases from mushroom, Fomitella fraxinea. J Microbiol Biotechnol. 16, 264–271.
 Longanayaki, N., Suganya, N., Manian., 2012. Evaluation of edible flowers of agathi
(Sesbania L. Fabaceae) for invivo anti-inflammatory and analgesic, and invitro
antioxidant potential. Food Sci Biotechnol, 21 (2), 509-517.
Mackay, D., Miller, A.L., 2003. Nutritional support for wound healing. Altern Med Rev.
8(4) 359-377.
Monroe, D.M.III., Hoffman, M., Roberts, H.R., 2010. Williams Hematology (8th Eds.)
Molecular biology and Biochemistry of the coagulation factors and pathways of
hemostasis. New York NY: McGraw-Hill Professional Publishing. 614–616.
Murata, J., Satake, M., Suzuki, T., 1963. Studies on snake venom. XII. Distribution of
proteinase activities among Japanese and Formson snake venoms. J. Biochem. 53, 431–
443 (Tokyo).
Nafeesa, Z., Shivalingu, B.R., Vivek, H.K., Priya, B.S., Swamy, S.N., 2015. Exploring a
new serine protease from Cucumis sativus L. Appl Biochem Biotechnol. 175: 2787-2794.

Orwa, C., Mutua, A., Kindt, R., Jamnadass, R., Anthony, S., 2009. Agroforestree
Database: a tree reference and selection guide version 4.0. World Agroforestry Centre,
Kenya.

Purna, S.K., Babu, M., 2000. Collagen based dressings-a review. Burns, 26(1), 54-62.
Doi: 10.1016/S0305-4179(99)00103-5.

Ramesh, T., Sureka, C., Bhuvana, S., Begum, V.H., 2010. Sesbania grandiflora
diminishes oxidative stress and ameliorates antioxidant capacity in liver and kidney of
rats exposed to cigarette smoke. J Physiol Pharmacol. 61, 4, 467-476.
Richter, G., Hans, P.S., Friedrich, D., Peter, L., (2002). Activation and inactivation of human
factor X by proteases derived from Ficus carica. British Journal of Haematology, 119(4),
1042–1051.
Satish, A., Sairam, S., Ahmed, F., Urooj, A., 2012. Moringa oleifera Lam.: protease
activity against blood coagulation cascade. Pharmacognosy Res. 4(1), 44–49.
Sheikh, A.A., Sayyed, Z., Siddiqui, A.R., Pratapwar, A.S., Sheikh, S.S., 2011. Wound healing
activity of Sesbania grandiflora Linn flower ethanolic extract using excision and incision
wound model in wistar rats. Int J of Pharm Tech Research. 3(2):895-898.
Shivaprasad, H.V., Rajesh, R., Brigitte, M.F., Felix, J.F., Vishwanath, B.S., 2009.
‘Pergularain e I’ – a plant cysteine protease with thrombin like activity from Pergularia
extensa latex. Thromb Res, 125, e1000-e105.

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, M.A., Morita, T., (eds), Toxins and Hemostasis. Dordrecht, Heidelberg,
London, New York: Springer+Business Media: 591-603.
Shivalingu, B.R., Vivek, H.K., Nafeesa, Z., Priya, B.S., Swamy, S.N., 2016. Comparative
analysis of procoagulant and fibrinogenolytic activity of crude protease fractions of
turmeric species. J Ethnopharmocol. 172, 261-264.

Singer A.J., Clark, R.A., 1999. Cutaneous wound healing. N Engl J Med, 2, 341(10).
738-746. Doi: 10.1056/NEJM199909023411006.

Siritapetawee, J., Thumanu, K., Sojikul, P., Thammasirirak, S., 2012. A novel serine
protease with human fibrinogenolytic activities from Atrocarpous heteophyllus latex.
Biochim Biophys Acta. 1824(7), 907-912.

Thakur, R., Jain, N., Pathak, R., Sandhu, S.S., 2011. Practices in wound healing studies
of plants. Evid Based Complement Alternat Med. Vol (2011), Article ID-438056. Doi:
10.1155/2011/438056.

Upahyay, N.K., Kumar, R., Siddiqui, M.S., Gupta, A., 2011. Mechanism of wound
healing activity of Hippophae rhamnoides L. leaf extract in experimental burns. Evid
Based Complement Alternat Med. Vol(2011), Article ID-659705. Doi: 10.1093/ecam/nep
189.

Walker, I.D., Davidson, J.F., Thomson, J.M., 1985. Blood coagulation and hemostasis,
Churchill Livingstone, Edinburgh, 208–263.

Wachtel-Galor, S., Benzie, I.F.F., 2011. Herbal Medicine: An Introduction to Its History,
Usage, Regulation, Current Trends, and Research Needs. In: Benzie, I.F.F., Wachtel-
Galor, S., (eds). In: Herbal Medicine: Biomolecular and Clinical Aspects. Chapter I, 2 nd
Edn. Boca Raton (FL): CRC Press/ Taylor & Francis.

Zauner, G., Hoffmann, M., Rapp, E., Koeleman, C.A.M., Dragan, I., Deelder, A.M.,
Wuhrer, M., Hensbergen, P.J., 2012. Glycoproteomic analysis of human fibrinogen
reveals novel regions of O-glycosylation. J Proteome Res, 11, 5804–5814.
List of Figure captions:

Fig1A: Protease activity of SgCDF crude fraction was detrmined in dose dependent manner.
Values represent mean ±SEM (n=3), ***P<0.0001. Fig1B: Casein zymogram by using 10% SDS
PAGE, L1: 10ug; L2: 25ug; L3: 50ug *L=lane
Fig2A: Evaluation of stability of protease against Temperature (0C). SgCDF (100µg) was
incubated at different temperature (200C-1000C) and protease activity was determined. For each
experiment, n=3, mean±SEM; p<0.05. Fig 2B: Evaluation of stability of protease against pH.
SgCDF (100ug) was incubated at different pH (2-12pH) and protease activity was determined.
For each experiment, n=3, mean±SEM; p<0.05. 2C: Evaluation of stability of protease against
different salt concentration. SgCDF (100ug) was incubated at different salt concentration (NaCl:
0.5%-3%) and protease activity was determined. For each experiment, n=3, mean±SEM; p<0.05.
2D: Determination of nature of protease. SgCDF (100ug) was incubated at different protease
inhibitors at 5mM concentration and protease activity was determined. For each experiment,
n=3, mean±SEM; p<0.05.
Fig 3A: Recalcification time (procoagulant effect) of SgCDF (filled square) was evaluated in
dose dependent manner (10ug-100ug) against Trypsin (filled circle). Values were expressed in
Mean ± SEM, n=3, ***p<0.0001. 3B: Fibrinogenolytic activity was determined by using 10%
SDS-PAGE human fibrinogen degradation by SgCDF. LaneC: Control (50ug Fibrinogen),
Lane2: (Fibrinogen+25ug SgCDF), Lane3: (Fibrinogen+50ug SgCDF). 3B: Fibrinogenolytic
activity was determined by using 10% SDS-PAGE human fibrinogen degradation by SgCDF.
LaneC: Control (50ug Fibrinogen), Lane2: (Fibrinogen+25ug SgCDF), Lane3:
(Fibrinogen+50ug SgCDF).