Fl ELD OF THE I NVENTI ON The present invention is directed to a method of producing anticoagulant composition including selective active fraction of Leucas indica (AFLI) and synergistic preparations (AAP) involving selective active fraction of Leucas indica (AFLI) in combination with -sitosterol, and also to compositions /preparations involving such manufacture. The extract and/or active fraction (AFLI) of Leucas indica of said active anticoagulant preparation (AAP) of the present advancement involves selective extraction method and formulations based thereon for a surprising activity of prevention of blood coagulation and hyperfibrinogenem ia related disorders. The synergistic anticoagulant composition comprising said active anticoagulant preparation (AAP) involving said AFLI of Leucas indica of the present advancement is found to have unexpected and significant synergistic activity for inhibition of thrombin and blood coagulation FXa showing fibrinogenolytic and fibrinolytic activity in combination with β-sitosterol.

BACKGROUND ART

Anticoagulants are the drugs, remain the primary strategy for the prevention and treatment of thromboembolic disorders. The inappropriate formation of blood clots, also known as thrombosis, in a blood vessel is a major public health problem. Blood also must be anticoagulated during certain surgical procedures, such as open heart surgery and hip replacement surgery, and during certain medical procedures, such as coronary angioplasty, to prevent the formation of blood clots.

Heparin, a widely used to anticoagulant drug prevents clotting by accelerating the ability of a naturally occurring blood protein, antithrombin, to inhibit a variety of active enzymes in the blood clotting system. Thus heparin itself is not believed to have antithrombin activity, but acts by intensifying the ability of the body's natural antithrombin to inhibit activated coagulation factors. Low-molecular- weight heparins (LMWHs) were developed during the 1970s [Chaoy et al., 1980]. Today, unfractionated heparin and LMWHs are widely used in clinics for the prevention and treatment of acute thromboembolic events. Chemically, heparin is a proteoglycan formed by binding several heteroglycan chains to the -OH groups of serine residues of an apoprotin. However, an overdose of heparin reduces rather than increases the activity of antithrombin, thus resulting in the risk of thrombosis [Davoran and Aster, 2006; Richard et al., 2008]. The major disadvantage of heparin is significant bleeding risk, allergic reactions and rebound thrombosis. It has multiple biological targets, and its effectiveness depends on the levels of antithrombin in the patient, with low levels of antithrombin limiting its effectiveness.

In addition to heparin, warfarin, and dicumarol, fraxiparine, enoxaparin are also used as anticoagulant drugs [Harter et al., 2015]. Warfarin mimics vitamin K, a coenzyme involved in the synthesis of proteins involved in blood coagulation, resulting in inactivation of coagulant factors which is a time taking process. Therefore, warfarin is not prescribed in acute conditions.

The known anticoagulants have several limitations. The oral anticoagulant warfarin has a narrow therapeutic index, hemorrhage, variations in its anticoagulant effect, numerous food and drug interactions and the need for regular monitoring [Ageno et al., 2012; Miyares and Davis 2012]. Hemorrhage is the most significant adverse effect associated with warfarin [Ageno et al., 2012].

There is a need, therefore, to explore for anticoagulant compositions/ actives having antithrombin and/or FXa inhibition activity without any adverse side effects. There also continues to be an unmet need to provide oral anticoagulant which significantly reduces such side effects and are safer to administer.

OBJECTS OF THE I NVENTI ON

The primary object of the present invention is thus directed to provide for advancement in manner of manufacture of selective active fraction from leaves of Leucas indica such as to have selective anticoagulant activity and also favouring producing anticoagulant composition including said selective active fraction of extract of leaves of L. indica and active anticoagulant preparation (AAP) thereof of a natural renewable material as an extract and/or active fraction and also a method of manufacturing of such compostions/preprations.

It is another object of the present invention to provide for synergistic anticoagulant composition comprising said active anticoagulant preparation (AAP) involving extract and/or active fraction of a renewable material with antithrombin and FXa inhibiting properties.

Another object of the present invention is to provide a process of preparation of a synergistic anticoagulant composition which can function effectively at normal physiological conditions, for example at pH 7.4 and at 37 °C.

Yet another object of the invention is to provide for advancement related to direct inhibitor of thrombin.

Another object of the present invention is to provide for advancement related to indirect inhibitor of thrombin.

Another object of the invention is to provide a FXa inhibitor.

Yet another object of the present invention is to provide for said active anticoagulant preparation (AAP) comprising extract and/or active fraction (AFLI) of natural renewable material including from plant material Leucas indica showing anticoagulant activity that is devoid of any haemolytic activity, and proteolytic activity against blood proteins such as albumin and globulin.

Yet another object of the present invention is to provide for said synergistic anticoagulant composition comprising said active anticoagulant preparation (AAP) of Leucas indica that reveals synergistic anticoagulant activity.

SUMMARY OF THE I NVENTI ON

Thus according to the basic aspect of the present invention there is provided a process for producing anticoagulant composition from leaves of L. indica including selective active fraction of extract of leaves of L. indica comprising i. subjecting dried leaves of L. indica to solvent extraction ii. homogenizing the L. indica extract followed by separation of the solids by centrifugation to get the supernatant iii. drying of the supernatant to obtain a dried crude L. indica extract iv. fractionating said dried crude L. indica extract in ion-exchange resins such as to reach to a selective active fraction therefrom the extract (AFLI) comprising - sitosterol, methyl palmitate and octamethylcyclotetrasiloxane along with a major 35 kDa protein possessing protease activity and having anticoagulant and fibrin(ogen)olytic activity without any haemolytic and proteolytic activity against blood proteins.

According to another aspect the present invention the above process for manufacture of synergistic active anticoagulant preparation (AAP) involving said selective active fraction (AFLI) comprising mixing of said selective active fraction (AFLI) exhibit anticoagulant and fibrin(ogen)olytic activity without any haemolytic and proteolytic activity against blood proteins with -sitosterol to produce a synergistic active anticoagulant preparation (AAP).

More preferably in said process an infusion of the L. indica leaves is prepared using double distilled water and said step of solvent extraction is carried out by extracting with double distilled water.

According to another preferred aspect of the present invention there is provided for said process wherein leaves are shade dried at room temperature (23 °C ± 2 °C).

Preferably in said process the pH of the double distilled water is maintained from 7.3 to 7.5.

More preferably, in said process the extraction process is carried out at 4 °C to room temperature ( 23 °C ± 2 °C) for a period of from 4 to 6 hours. According to another preferred aspect of the present invention there is provided for said process wherein the extract is filtered followed by centrifugation from 8000-12000 rpm for 10-15 min at temperature range for centrifugation is from 4 °C to room temperature (~ 23 °C ± 2 °C). Preferably in said process the supernatent is desiccated dried at room temperature or freeze dried the liquid portion and storing it at low temperature (8-12 °C).

More preferably in said process, the crude dried extract is dissolved in suitable buffer having buffer pH range of 7.0 to 8.0 preferably 20 iM potassium phosphate, 50 mM Tris-HCI buffer.

Preferably in said process, the crude dried extract is fractionated through ion exchange chromatography (cation exchange followed by anion exchange and vice versa) and the unbound fraction comprises the active fraction of L. indica (AFLI).

According to another aspect of the present invention there is provided an anticoagulant composition comprising selective active fraction of extract of leaves of L. indica (AFLI) comprising -sitosterol, methyl palmitate and octamethylcyclotetrasiloxane along with a major 35 kDa protein possessing protease activity and having anticoagulant and fibrin (ogen) olyt ic activity without any haemolytic and proteolytic activity against blood proteins. Preferably, in said anticoagulant composition said selective fraction having anticoagulant activity is selectively anyone or more of: a. 0.5 to 5.0 μς/ιτιΙ of said active fraction (AFLI) of Leucas indica shows anticoagulant effect;

b. 1.0 to 10 μg/ml of said active fraction (AFLI) of Leucas indica effects prothrombin time (PT) ;

c. 0.25 to 3.0 μg/ml of said active fraction (AFLI) of Leucas indica inhibits fibrinogen clotting time of thrombin;

d. atleast 1.0 μg/ml of said active fraction (AFLI) of Leucas indica inhibits amidolytic activity of thrombin; e. atleast 1.0 μg/ml of said active fraction (AFLI) of Leucas indica inhibits amidolytic activity of FXa;

f. atleast 1.0 μς/ιτιΙ of said active fraction of AFLI shows fibrin (ogen) olyt ic activity;

g. 0.1 to 1.0 μς/ιηΙ of said active fraction of AFLI shows defibrinogenating activity.

Preferably said anticoagulant composition is provided as powder, granule, tablet, chewing tablet, capsule or beverage type having effective amount of from 0.25 to 10.0 μg/ml.

According to yet another aspect of the present invention there is provided a synergistic anticoagulant preparation (AAP) including active fraction of L. indica (AFLI) comprising

(a) said active fraction of L. indica (AFLI) having -sitosterol, methyl palmitate and octamethylcyclotetrasiloxane as the three key ingredients of AFLI along with a major 35 kDa protein possessing protease activity and having anticoagulant and fibrin(ogen)olytic activity without any haemolytic and proteolytic activity against blood proteins, and

(b) -sitosterol. Preferably, in said synergistic anticoagulant preparation (AAP) the AFLI: sitosterol is in the range of 1:1 to 10:1 preferably in 5: 1 ratio (w/w).

More preferably, there is disclosed a synergistic anticoagulant preparation (AAP) having significantly special anticoagulant activity is selected from anyone or more of: a. 0.5 to 5.0 μg/ml of said active anticoagulant preparation (AAP) shows anticoagulant effect;

b. 1.0 to 10 μg/ml of said active anticoagulant preparation (AAP) effects prothrombin time (PT); c. 0.25 to 3.0 μς/ιηΙ of said active anticoagulant preparation (AAP) inhibits fibrinogen clotting time of thrombin;

d. atleast 1.0 μg/ml of said active anticoagulant preparation (AAP) inhibits amidolytic activity of thrombin;

e. atleast 1.0 μg/ml of said active anticoagulant preparation (AAP) inhibits amidolytic activity of FXa;

f. atleast 1.0 μg/ml of said active anticoagulant preparation (AAP) shows fibrin(ogen)olytic activity;

g. 0.1 to 1.0 μg/ml of said active anticoagulant preparation (AAP) shows defibrinogenating activity.

According to yet another preferred aspect of the present invention said synergistic anticoagulant preparation is provided as powder, granule, tablet, chewing tablet, capsule or beverage type having effective amount of from 0.25 to 10.0 μg/ml.

According to another aspect of the present invention there is provided a method of inhibition of blood coagulation, hyperfibrinogenemia related disorders, inhibition of thrombin and blood coagulation FXa showing fibrinogenolytic and fibrinolytic activity, the method comprising administering an effective amount of a said synergistic anticoagulant preparation (AAP) comprising (a) said active fraction of L. indica (AFLI) having -sitosterol, methyl palmitate and octamethylcyclotetrasiloxane as the three key ingredients of AFLI along with a major 35 kDa protein possessing protease activity and having anticoagulant and fibrin(ogen)olytic activity without any haemolytic and proteolytic activity against blood proteins and (b) sitosterol. DETAI LED DESCRI PTI ON OF THE I NVENTI ON

As discussed hereinbefore the basic aspect of the advancement is directed to method of producing anticoagulant composition including selective extraction of active fraction Lecuas indica (AFLI) as an extract and/or active fraction and an anticoagulant preparation (AAP) of Leucas indica obtained thereof in combination with -sitosterol comprising the steps of providing selective solvent for extraction and chromatographic techniques for obtaining said actives with anyone or more of the following surprisingly special characteristics of inhibition of blood coagulation and hyperfibrinogenemia related disorders, inhibition of thrombin and blood coagulation FXa showing fibrinogenolytic and fibrinolytic activity devoid of any haemolytic activity and proteolytic activity against blood proteins such as albumin and globulin. According to another aspect of the present advancement there is provided for active anticoagulant preparation (AAP) involving an extract and/or active fraction of Leucas indica (AFLI) with surprisingly special inhibitory activity against thrombin and FXa, the major enzymes of the blood clotting system, to be useful as an anticoagulant for clinical and laboratory applications.

According to another aspect of the present advancement there is provided for active anticoagulant preparation (AAP) involving an extract and/or active fraction of Leucas indica (AFLI) with defibrinogenating activity, to be useful for hyperfibrinogenemia related disorders.

According to yet another aspect of the present advancement there is provided a synergistic anticoagulant composition comprising said active anticoagulant preparation (AAP) of Leucas indica in combination with -sitosterol including asignificantly special selective amounts preferably in 5:1 ratio (w/w).

The above synergistic anticoagulant composition involving said active anticoagulant preparation (AAP) of Leucas indica is found to have surprisingly special and unexpected activities including: anticoagulant activity of at least about 80 Units/mg wherein one unit of anticoagulant activity is defined as 1 s increase in clotting time of the control PPP (platelet poor plasma) in presence of the active, which is found to be much enhanced as compared to the individual anticoagulant activities of either each of the components of AFLI of Leucas indica or - sitosterol taken together.

The nature of the present invention is discussed hereunder in greater detail in relation to the following non-limiting exemplary illustrations, figures and tables:

BRI EF DESCRI PTI ON OF THE Fl GURES:

Fig. 1a: illustrates dose-dependent in vitro anticoagulant activity of AFLI and AAP against human PPP. All values are means ± S.D. of twelve independent experiments. The Ca- Clotting time of PPP under identical experimental conditions (control) was found to be 91.2 s. Significance of difference with respect to control (without AFLI and AAP) ^* p < 0.01 ;

Fig. 1b: illustrates time-dependent anticoagulant activity by AFLI and AAP (5 μg/ml) against human PPP. AFLI and AAP (5.0 μg/ml) was pre-incubated with 300 μΙ of PPP from 3 to 15 min at 37 °C, pH 7.4 before addition of CaCI ₂ to initiate the clot formation. The Ca- Clotting time of PPP under identical experimental conditions (control) was found to be 91.2 s. Values are mean ± S.D. of twelve determinations. Significance of difference with respect to control (without AFLI and AAP) ^* p < 0.01 ;

Fig. 2a: Illustrates the in vitro effect of AFLI on APTT and PT time. Effect AFLI (1- 10 μς/ιτιΙ) on APTT and PT of PPP isolated from human blood. Values are mean ± S.D. of twelve determinations. Significance of difference with respect to control (without AFLI) ^* p< 0.05; Fig.2b: Illustrates the in vitro effect of AAP on APTT and PT time. Effect AAP (1 - 10 μς/ιτιΙ) on APTT and PT of PPP isolated from human blood. Values are mean ± S.D. of twelve determinations. Significance of difference with respect to control (without AAP) ^* p < 0.05;

Fig. 3a: illustrates inhibition of fibrinogen clotting activity of thrombin by different doses of AFLI and AAP (in terms of fibrinogen clotting assay) at 37 °C, pH 7.4. The fibrinogen clotting time of thrombin under identical experimental conditions (control) was found to be 39.9 ± 1.3 s. The values are mean of triplicate determinations. Significance of difference with respect to control (without AFLI and AAP) ^* p < 0.05;

Fig. 3b: illustrates time-dependent inhibition of fibrinogen clotting time of thrombin by AFLI and AAP (1.0 μg/ml) at 37 °C, pH 7.4. The fibrinogen clotting time of thrombin under identical conditions (control) was found to be 39.9 ± 1.3 s. The values are mean of triplicate determinations. Significance of difference with respect to control (without AFLI and AAP) ^* p < 0.05;

Fig. 4a: illustrates effect of AAP (1.0 μg/ml) on amidolytic activity of FXa (0.13 mM) against its chromogenic substrate F3301 (0.2 mM). The values are mean of triplicate determinations;

Fig. 4b: illustrates effect of AFLI (1.0 μg/ml) on amidolytic activity of thrombin (36.6 nM) against its chromogenic substrate T1637 (0.2 mM). The values are mean of triplicate determinations;

Fig 5a: illustrates control thrombin (incubated with buffer) and AAP-treated thrombin were separated on 15% SDS-PAGE under reduced conditions. Lane 1, Molecular weight markers; lane 2, control thrombin (without AAP treatment); lanes 3, 4, 5, and 6, thrombin-treated with 0.25 mg/ml of AAP at 10, 15, 30 and 60 minutes of incubation respectively, at 37 °C, pH 7.4. Fig. 5b: illustrates quantification of thrombin degradation by AAP at different incubation times (10 - 60 min). Gels were analyzed by Image J program. Data were expressed as percentage (%) of thrombin degradation. Thrombin control incubated at 37 °C during 60 min represents 0% degradation. Values are mean ± SD of triplicate determinations. Significance of difference with respect to control (without AAP) * p < 0.01

Fig. 5c: illustrates inhibition of prothrombin activation property of FXa by AAP. After reduction with -mercaptoethanol, degradation products were separated by 12.5% SDS-PAGE. Lane 1, protein molecular markers; lane 2, 12 μg PTH; lane 3, PTH (12 g) incubated with FXa (0.1 g) for 30 min at 37 °C, pH 7.4; lane 4, [FXa (0.1 g) pre-incubated with AAP (1.0 g) for 15 min] + PTH (12 g); lane 5, PTH + AAP.

Fig.6: illustrates fluorescence spectra showing (a) thrombin (36.6 nM), (b) interaction of AFLI (1.0 μg/m\) with thrombin (36.6 nM), (c) interaction of AFLI (2.0 μg/ml) with thrombin (36.6 nM), (d) interaction of AFLI (3.0 μg/ml) with thrombin (36.6 nM), (e) interaction of AFLI (4.0 μg/ml) with thrombin (36.6 nM) and (f) AFLI (1.0 μg/ml).

Fig. 7a: illustrates kinetics of fibrinogenolytic activity of AAP. After reduction with -mercaptoethanol, degradation products were separated by 12.5% SDS-PAGE. Lane 1, control human fibrinogen (0.25% w/v in 20 mM K-phosphate buffer, 150 mM NaCI, pH 7.4); lanes 2-5, human fibrinogen degradation by AAP (100 μg/ml) at 30, 60, 90 and 120 minutes of incubation, respectively, at 37 °C, pH 7.4.

Fig. 7b: illustrates kinetics of fibrinolytic activity of AAP. After reduction with - mercaptoethanol, degradation products were separated by 12.5% SDS-PAGE. Lane 1, control human fibrin; lanes 2 - 4, human fibrin degradation by 100 μg/ml AAP at 60, 90 and 120 minutes of incubation, respectively, at 37 °C, pH 7.4. Fig. 8: illustrates dose-dependent in vitro defibrinogenating activity of AAP and AFLI at 60 minutes of incubation with human PPP, respectively, at 37 °C, pH 7.4. The values are mean ± S.D of twelve determinations. EXAMPLE 1 : Method of preparation

As discussed hereinbefore the present invention is based on an active anticoagulant preparation (AAP) and anticoagulant composition including extract/ active fraction of Leucas indica (AFLI) as the active and provides a method of extraction of said selective active fraction from said plant material involving a selective extraction/isolation procedure as discussed hereunder:

For the purposes of the exemplary illustrations Leaves of L. indica were collected from 20-cm-tall herb, from the areas surrounding the Sivasagar district, Assam (26.9844° N, 94.6314° E). The identity of this plant, collected in December 2013, was confirmed by Botanical survey of India (BSI), Shillong with the accession number 37604. L. indica herb are used in India folk medicine as antipyretic, antihelmintic, antibacterial and for the treatment of various ailments for example nasal congestion, sinusitis, and poisonous bites and nowhere in the traditional knowledge literature is it known to have any relationship in influencing the hemostatic system of a subject.

100 gm of leaves of Leucas indica was shade dried at room temperature. It was then crushed and powdered using mortar and pestle or commercial grinder. The extraction was carried out by stirring the shade dried crushed leaves in 250 ml double distilled water (pH 7.3 to 7.5 adjusted with 0.01 N NaOH) for 4 - 6 hours. The temperature range for extraction may vary from 4 °C to room temperature. After extraction, the extract was filtered through muslin cloth or through a whatman no. 1 filter paper (0.45 μιη) and centrifuged at 8,000 to 12,000 rpm for 10 to 15 min at 4 °C to room temperature. The supernatant was separated, desiccated dried or freeze-dried to obtain the crude dried leaves extract. The crude dried extract was then dissolved in 20 mM potassium phosphate buffer having pH in the range of 7.0 to 8.0 (50 iM Tris-HCI buffer or 20 oiM HEPES buffer and their like can also be used). After dissolving in suitable buffer, the crude dried leaves extract was fractionated through ion exchange chromatography (cation exchange followed by anion exchange and vice versa). The unbound fraction of the ion exchange chromatography of the present process showed significantly superior anticoagulant and antithrombin activity than the crude aqueous leaves extract of L. indica and comprises the active fraction (AFLI) of the present invention.

The GC-MS analysis of the AFLI indicates -sitosterol, methyl palmitate and octamethylcyclotetrasiloxane are the three key ingredients of AFLI. SDS-PAGE analysis of the AFLI further shows the presence of a prominent protein band of 35 kDa and LC-MS/MS analysis of the tryptic digested protein reveals a new protein not reported in the plant protein databases, which AFLI containing all actives could only be extracted by following the above selective methodology.

LC-MS/MS (Peptide mass fingerprinting) analysis of major protein present in AFLI:

The 35 kDa SDS-PAGE protein band was cut into small pieces and after reduction and alkylation, it was trypsin digested overnight at 37 °C [Mukherjee et al., 2014]. The tryptic peptides were reconstituted in 15 μΙ of 2% acetonitrile with 0.1% formic acid and 1 μΙ sample was injected to C ₁₈ column. Digested peptides were subjected to 110 minute RPLC gradient, followed by acquisition of the data on LTQ-Orbitrap-MS. Generated data was searched for the identity on the MASCOT 2.4 search engine using Swiss-Prot, and TrEMB, databases from NCBI. A minimum of two high confidence peptides was used as a prerequisite to identify the protein. Furthermore, matching peptides and proteins showing 1 Olg P value > 23 and 20, respectively were used as filtration parameters. The tryptic peptide sequences of 35 kDa protein were further subjected to a BLASTp search in the NCBI database of non-redundant protein sequence (nr), Swissprot protein sequences (Swissprot), and protein databank proteins (pdb) against Lamiaceae family protein database (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The 35 kDa protein, a major constituent of AFLI did not show significant similarity with any plant protein deposited in protein database suggesting it to be a new protein isolated from a plant source. However, one of the MS-MS derived tryptic fragment of 35 kDa protein viz. I ITHPNFNGNTLDNDI MLI K demonstrated conserved domain of trypsin-like superfamily suggesting this protein may be a previously uncharacterized protein from plant source. Biochemical characterization revealed protease activity contained in this active fraction which may be correlated to this 35 kDa protein. The other tryptic sequences of this 35 kDa protein viz. LGEHNI DVLEGNEQFI NAAK, VATVSLPR, LSSPATLNSR, APVLSDSSCK did not show significant hit to Lamiaceae family of proteins deposited in databases.

In the present invention, while preparation of dried leaves aqueous extract of L. indica was tried at two different conditions for comparison shade dried at room temperature or sun dried for 14 days; and while the pH of the water was adjusted to pH 7.4 by adding 0.01 N NaOH and other pH ranges were tried; and while the preparation of 100% methanol leaves extract of L. indica was also carried out using shade dried leaves that was homogenized in a blender during 5- 10 min and the extraction was carried out by stirring the crushed leaves in 100% methanol, 100% ethanol, 50%, 25%, 12.5 % ethanol in water for 4 - 6 hours at 4 °C-room temperature and the leaves extract was prepared; and while 20 mM potassium phosphate buffer leaves extract of L. indica and further while preparation of 50 mM Tris-CI buffer leaves extract of L. indica was prepared and various pH ranges were tried for elution with buffer, it was surprisingly found that the shade dried aqueous leaves extract when extracted in double distilled water having pH 7.4, centrifuged to obtain a supernatant that was further dried and when taken in buffer to be further fractionated using anion and cation exchange resins using buffer as eluant in the pH range of 7-8 showed surprisingly enhanced anticoagulant and fibrinogenolytic activities over other comparative extracts of sun dried and fresh leaves extract as discussed above. It was also found by way of the present invention that the preferred critical process parameters due to which the AFLI extraction from L. indica was possible with the desired actives with said enhanced anticoagulant and fibrinogenolytic activities are the following

a. Solvent for extraction: Water, pH 7.4

b. Temperature of extraction: Room temperature (23 ± 2°C)

c. Optimum time for extraction: 4 hours

d. Centrifugation: 10,000 rpm for 10 min

e. Selective chromatographic combination of (anion and cation exchange) resins involving buffer as eluant having pH in the range of f. Flow rate: 1 -5 ml/min

g. pH of the equilibration buffer: 7.4

Different chromatographic processes such as gel filtration chromatography, RP- HPLC chromatographic processes such as gel filtration, RP-HPLC and removal of particles from 3KDa cut off membrane, which were tried could not provide for the desired AFLI with actives giving the desired activities.

The viability of the process for the generation of AFLI from L. indica involving actives with surprisingly enhanced anticoagulant and fibrinogenolytic activities thus resides on the above discussed process parameters.

Importantly, surprising and selective indications of the AFLI, active anticoagulant preparation (AAP) of Leucas indicia as an extract and/or active fraction, obtained by way of the present advancement was confirmed as none of the ingredients of AFLI demonstrate better anticoagulant and antithrombin activities as compared to the activity shown by AFLI.

Also, the AFLI obtained by way of the present advancement was found to further reveal significant and unexpected efficacy in combination with -sitosterol preferably in 5:1 ratio (w/w) providing AAP that showed synergistic anticoagulant activity. The AAP thus exhibits significantly enhanced anticoagulant and antithrombin activity as compared to the crude plant extract or AFLI. The active fraction (AFLI) and active anticoagulant preparation (AAP) according to the present invention were subjected to various pharmacological studies.

Table 1. A summary of fibrin (ogen)olytic, anticoagulant and thrombin inhibition (in terms of fibrinogen clotting time of thrombin) activities of the crude aqueous leave extract of L. indica, active fraction (AFLI), is set forth below. Values are mean ± S.D. of triplicate determinations.

ND: Not detected, NA: Not applicable ^a One unit (U) of fibrinolytic activity is defined as 1.0 μg of tyrosine liberated per m in per m I of sample

b One unit (U) of fibrinogenolytic activity is defined as 1.0 μg of tyrosine liberated per min per ml of sample

c One unit of anticoagulant activity is defined as 1 s increase in clotting time of the control PPP in presence of sample

d One unit of inhibition of fibrinogen clotting time of thrombin is defined as 1 s increase in clotting time of the control fibrinogen in presence of sample. Control (thrombin and fibrinogen) showed a fibrinogen clotting time of 39.9 ± 1.3 s (mean ± S.D., n=3)

^* p value < 0.01. Significance of difference with respect to crude aqueous leaves extract,

^{* *} p value < 0.01. Significance of difference with respect to the control (thrombin and fibrinogen). Control showed a fibrinogen clotting time of 39.9 ± 1.3 s (mean ± S.D., n= 3)

Preparation of active anticoagulant preparation (AAP) :

The AFLI and 3- sitosterol was mixed in the ratio of 1:1, 5:1 and 10:1 (w/w). The anticoagulant and thrombin inhibition (in terms of fibrinogen clotting time of thrombin) activity was assessed by taking 1.0 μg of each formulation according to the standard protocol as described in examples 20.1 and 20.3, respectively. It was observed that the anticoagulant activity was significantly enhanced when AFLI was mixed with 3- sitosterol. Saturation in anticoagulant activity was observed after the ratio of 5:1 (AFLI: 3- sitosterol) and this comprises the active anticoagulant preparation (AAP) of the present invention.

-sitosterol are known to be used as blood cholesterol-lowering agents and there is no public domain knowledge on the use of -sitosterol as an anticoagulant. Table 2: Anticoagulant and antithrombin (in terms of fibrinogen clotting time of thrombin) activities of the AFLI mixed with β-sitosterol. Values are mean ± S.D. of triplicate determinations.

a One unit of anticoagulant activity is defined as 1 s increase in clotting time of the control PPP in presence of sample

b One unit of inhibition of fibrinogen clotting time of thrombin is defined as 1 s increase in clotting time of the control fibrinogen in presence of sample. Control (thrombin and fibrinogen) showed a fibrinogen clotting time of 39.9 ± 1.3 s (mean ± S.D., n=3)

^* p value < 0.05. Significance of difference with respect to AFLI ^{* *} p value < 0.01. Significance of difference with respect to AFLI

Other ratios of AFLI and - sitosterol 1:5, 1:8, 2:5 combined upto 1.0 μg were tried that did not reveal any enhancement of synergy reached otherwise by the ratios provided of 1:1-10:1 of AFLI : - Sitosterol provided under Table 2.

Example 2: In vitro anticoagulant assay of AFLI and AAP, Dose- and time- dependent human platelet-poor plasma clotting assay by Ca- clotting time method

To study the in vitro anticoagulant activity of AFLI and AAP, blood was collected from 12 healthy volunteers both male and female who were not under any medication (All the experiments were performed as approved by the Ethical Committees of Tezpur University, Tezpur). The blood was withdrawn using phlebotomy by expert technician and collected in sterile tubes containing 3.8% tri-sodium citrate. The platelet poor plasma (PPP) was prepared by centrifuging the blood twice at 4300 rpm for 20 min at 4 °C [Doley and Mukherjee, 2003]. Different concentrations of AFLI and AAP (0.5 -10.0 μg) (in a total volume of 20 μΙ) were pre-incubated with 300 μΙ of PPP for 3 min at 37 °C, and clotting was initiated by adding 40 μΙ of 250 mM CaCI ₂ . For control, instead of AFLI and AAP, the same volume of 1X PBS was used. One unit of anticoagulant activity has been defined as 1 s increase in clotting time of the control PPP in presence of AFLI [Mukherjee et al., 2000].

AFLI as well as AAP dose-dependently prolonged the Ca ²⁺ clotting time of platelet poor plasma (PPP); however, after a dose of 5.0 μg/ml of AFLI and AAP saturation in anticoagulant activity was observed (Fig. 1a). In another assay the AFLI and AAP (5.0 μg/ml) was pre-incubated with 300 μΙ platelet poor plasma (PPP) from 3 to 15 min and the recalcification clotting time of PPP was assessed against appropriate controls; the optimum anticoagulant activity was observed at 10 - 15 min of pre- incubation of PPP with AFLI and AAP ( Fig. 1 b). Example 3: Effect of AFLI and AAP on PT and APTT

Activated partial thromboplastin time (APTT) and prothrombin time (PT) of human PPP were measured using commercial kits (Tulip diagnostics, Mumbai) and according to the instructions of the manufacturer.

Both AFLI and AAP (1-10 μg/ml) had no effects on APTT of human plasma; however, AFLI and AAP (1- 10 μς/ιτιΙ) treatment of human PPP resulted in a significant (p < 0.05) enhancement of PTwith respect to control (Fig. 2a and 2b). This result indicates that AFLI and AAP affects the extrinsic coagulation system to enhance the clotting time suggesting that AFLI and AAP exerts its anticoagulant effect by inhibiting thrombin and FXa.

Example 4: Antithrombin effect of AFLI and AAP

Different concentrations of AFLI and AAP (0.25 - 3.0 μg/ml) were pre-incubated with thrombin (3 μΙ, 10 NIH U/ml in 20 mM potassium phosphate buffer, pH 7.4) for 30 min at 37 °C. A control was also set up in which thrombin was incubated with PBS under identical conditions. The reaction was started by adding 40.0 I of 0.25% (w/v) human plasma fibrinogen (in 20 mM potassium phosphate buffer, pH 7.4), and the time of fibrin clot formation, if any, was monitored with visual inspection [Mukherjee et al.2012].

In a dose-dependent manner, AFLI and AAP prolonged the fibrinogen clotting time of thrombin (Fig. 3a). In another assay the thrombin (1.0 μg/ml) was pre- incubated with AFLI and AAP (1.0 μg/ml) from 5 to 30 min and the fibrinogen clotting time of thrombin was assessed against appropriate controls; the optimum inhibition being observed at 15 min of pre-incubation of thrombin with AFLI and AAP (Fig.3b)

Example 5: Amidolytic activity assay

Amidolytic activity of thrombin and FXa towards their chromogenic substrate (0.2 mM) T1637 and F3301, respectively, was assayed as described earlier [Saikia et al., 2011; Mukherjee and Mackessy, 2013]. The release of p-nitroaniline was monitored for 15 min at intervals of 1 min at 405 nm in a plate reader (Multiskan GO, Thermo Scientific, USA). For every experiment, a control was run in parallel. The activity of thrombin or FXa towards their substrates was considered 100% activity and thus other values were compared with this.

Amidolytic activity of FXa (Fig. 4a) and thrombin (Fig. 4b) was significantly reduced in presence of 1.0 μg/ml AAP.

Example 6: Assay of thrombin and FXa inhibition using SDS-PAGE analyses

15 μg of thrombin was incubated with 5.0 μg of AAP from 10 to 60 min at 37 °C in total volume of 20 μΙ of 20 mM sodium phosphate buffer at a pH 7.4. The AAP- treated and control thrombin (incubated with 1X PBS, pH 7.4) were analyzed by 15% SDS-PAGE under reducing conditions [Laemmli et al, Nature 227, 680-685, 1970]. For FXa inhibition assay, AAP (1.0 μg) was pre-incubated with FXa (0.1 g) in total volume of 20 μΙ of 20 mM sodium phosphate buffer at a pH 7.4 at 37 °C for 30 min. Thereafter, 12 g of prothrombin (PTH) in a total volume of 3 I was added and the reaction mixture was incubated at 37 °C for 4 h. A control was run in parallel where instead of AAP, 1X PBS was added. The prothrombin degradation products were analyzed by 12.5% SDS-PAGE under reducing conditions [Thakur et al., 2015]. The ImageJ (version 1.47) was used to calculate the percent degradation of thrombin by AAP. The SDS-PAGE bands intensity of control thrombin was considered as 100% (base value) and percent decrease in intensities of thrombin bands after treatment with AAP was compared with that to determine the percent of thrombin degradation by AAP.

To elucidate the optimum time of action of AAP, digested fragments of thrombin were analyzed using SDS-PAGE [Majumdar et al., 2015]. The thrombin degradation pattern demonstrated that AAP time dependently degrade the thrombin band; the optimum inhibition being observed at 10 min of preincubation of thrombin with AAP (Figs. 5 a, b). The optimum pre-incubation time for fibrinogen clotting time of thrombin was observed at 15 min (Fig. 7 b) which correlates that optimum time of action of AAP to degrade thrombin is 10 min. AAP also inhibited the prothrombin activating property of FXa (Fig. 5 c) suggesting FXa inhibition by this active anticoagulant preparation (AAP) of L. indica leave extract is one of its mechanisms to exert anticoagulant action. Example 7: Interaction studies of AFLI with thrombin by spectrofluorometric analysis

Thrombin (36.6 nM, 10 NIH U/ml) in 1X PBS, pH 7.4 was incubated with different concentrations (1.0 - 4.0 ig/m\) of AFLI for 60 s at room temperature. The fluorescence intensity was monitored by exciting the reaction mixture at 280 nm and the emission spectrum was recorded in the range of 300 nm to 425 nm using a fluorescence spectrometer (LS55, Perkin Elmer) [Dutta et al., 2015]. All the binding experiments were done in triplicate to ensure their reproducibility.

A steady decrease in the fluorescence intensity of thrombin was monitored in the presence of AFLI, suggesting the quenching of the tryptophan residues of thrombin was due to its binding with AFLI (Fig. 6). This indicates binding of AFLI with thrombin to form an enzyme-substrate complex [thrombin-AFLI] before its degradation to exert the anticoagulant activity by AFLI.

Example 8: Fibrin(ogeno) lytic activity assay

Effect of AAP on fibrinogen/fibrin was evaluated by 12.5% SDS-PAGE using a Tris-Glycine system. Briefly, 40.0 μΙ of 0.25% (w/v) human fibrinogen or 0.6% (w/v) human fibrin (dissolved in 20 mM K-phosphate buffer, 150 mM NaCI , pH 7.4) was incubated with AAP (5.0 μg in 20 mM K-phosphate buffer, pH 7.4) in a total reaction volume of 50 μΙ for different time interval (15 to 120 min) 37 °C. After incubation, the reaction was centrifuged at 10000 rpm for 10 minutes and the supernatant was subjected to 12.5% SDS-PAGE. The gels were stained with 0.25% Coomassie blue R250 (Sigma-Aldrich, USA) to visualize the protein bands. To elucidate the mode of action of AAP, digested fragments of fibrin/fibrinogen were analyzed using 12.5% SDS-PAGE [Majumdar et al., 2015]. The fibrin/fibrinogen degradation pattern demonstrated that AAP preferentially degraded the A - chain of fibrin and fibrinogen and slow degradation of B -chain of fibrinogen (Fig. 7a); however, the -chain of fibrinogen/fibrin was not degraded within 2 h of incubation (Fig. 7b) suggesting the active fraction (AAP) contains a fibrin(ogen)olytic protease. Example 9 : In vitro hemolytic activity assay

The hemolytic activity of AAP was determined using the method as described by of Doley et al., [2004]. Briefly, 5 ml of blood was collected from healthy human volunteers in sterile tubes containing 0.5 ml of 3.8 % tri-sodium citrate (anticoagulant), centrifuged at 4300 rpm for 15 min. The platelet poor plasma (PPP) was discarded, and the erythrocytes pellet was re-suspended and washed thrice with PBS (pH 7.4), and diluted to 5% (v/v) in PBS (pH 7.4). To 2 ml of 5% erythrocytes suspension graded concentrations of AAP (0.25, 0.5, 1.0, 2.5, 5 g/ml) were added, gently inverted and incubated at 37 °C for 90 min. The tubes incubated with 1% Triton X-100 are used as positive control and incubated with 1XPBS (20 iM K-phosphate, 150 mM NaCI, pH 7.4) was used as negative control under identical experimental conditions. After incubation, all the tubes were centrifuged at 10,000 rpm for 10 min to pellet down the erythrocytes. The absorbance of the supernatant was measured at 540 nm for released hemoglobin using UV-Vis spectrophotometer. The percentage of hemolytic index (%) was calculated by using the following formulae:

samples " f.eqauveccRio!:

% hemolysis = — ^:

ΏΕ> positive- tpfiiro:: -.QD .negative ccMi!-ai

The biocompatibility of AAP was carried out by performing hemolytic assay and the results obtained are shown in Table 1. The AAP, at a concentration of 5.0ig/m\ demonstrated very marginal (3.2%) hemolysis of mammalian erythrocytes (Table 1 ) .

Table 1. Assessment of hemolytic activity, if any, of active anticoagulant preparation (AAP). The hemolytic activity of AAP (0-5.0 ug/ml) was tested against 0.5% (V/V) washed human erythrocytes suspension. Data represent mean ± S.D of triplicate experiments.

Samples % hemolysis

Triton X-100 Positive control

PBS Negative control AAP( g/ml)

0.25 0.5

0.5 1.4

1.0 2.1

2.0 2.3

5.0 3.2

Example 10: In vitro defibrinogenating activity assay

The blood was withdrawn from 12 healthy volunteers using phlebotomy by expert technician and collected in sterile tubes containing 3.8% tri-sodium citrate. The platelet poor plasma (PPP) was prepared by centrifuging the blood twice at 4300 rpm for 20 min at 4 °C [Doley and Mukherjee, 2003]. Different concentrations of AFLI and AAP (0.1 , 0.5 and 1.0 μg/ml) were pre-incubated with 300 μΙ of PPP for 60 minutes at 37 °C, and clotting was initiated by adding 3 μΙ of 10 NIH U/ml of thrombin. For control, instead of AFLI and AAP, the same volume of 1X PBS was used. The fibrinogen content of plasma was determined by using a Ca-thrombin reagent as described by Burmester et al (1970) and modified by Mukherjee and Mackessy (2003).

AFLI and AAP shows dose-dependent in vitro defibrinogenation of human PPP (Fig.8).

The above in vitro experiments as illustrated in examples 2, 3, 4, 5, 8 and 10 substantially demonstrated the following:

i) 0.5 to 5.0 μg/ml of said active anticoagulant preparation (AAP) and/or active fraction (AFLI) of Leucas indica shows anticoagulant effect.

ii) 1.0 to 10 μg/ml of said active anticoagulant preparation (AAP) and/or active fraction (AFLI) of Leucas indica effects prothrombin time (PT).

iii) 0.25 to 3.0 μg/ml of said active anticoagulant preparation (AAP) and/or active fraction (AFLI) of Leucas indica inhibits fibrinogen clotting time of thrombin.

iv) 1.0 μg/ml of said active anticoagulant preparation (AAP) and/or active fraction (AFLI) of Leucas indica inhibits amidolytic activity of thrombin v) 1.0 μς/ιηΙ of said active anticoagulant preparation (AAP) and/or active fraction (AFLI) of Leucas indica inhibits amidolytic activity of FXa vi) 1.0 ig/m\ of said active anticoagulant preparation (AAP) and/or AFLI shows fibrin(ogen)olytic activity.

vi i ) 0.1 to 1.0 μg/ml of said active anticoagulant preparation (AAP) and/or

AFLI shows defibrinogenating activity.

In an average healthy adult, the volume of blood is about one-eleventh of the body weight. The volume of blood in an average human adult, who is between 150 to 160 pounds, iss between 4.7 and 5.5 liters [Lan Na, 1998 Hall, 2015]. Therefore, based on above fact and in vitro test results, active anticoagulant preparation (AAP) and/or active fraction of L. indica (AFLI) from the dose ranging from 2.75 mg to 27.5 mg in 5.5 litres of blood would provide for desired anticoagulant effect in average healthy adult and will be useful to treat hyperfibrinogenemia related disorders, inhibition of thrombin and blood coagulation FXa.

It is thus possible for the present invention to provide for anticoagulant preparations and synergistic compositions comprising active anticoagulant preparation (AAP) involving extract and/or active fraction of Leucas indica (AFLI), and also provides a method of such manufacture. The extract and/or active fraction of said active anticoagulant preparation (AAP) of Leucas indica involves selective extraction method for a surprising activity of prevention of blood coagulation and hyperfibrinogenemia related disorders, which is surprising special and significant considering the activities of the individual components of said preparation in isolation. Advantageously further, the synergistic anticoagulant composition involving said active anticoagulant preparation (AAP) of Leucas indica of the present advancement is found to have unexpected synergistic activity for inhibition of thrombin and blood coagulation FXa showing fibrinogenolytic and fibrinolytic activity in combination with β-sitosterol. The advancement would thus provide for anticoagulant composition including selective active fraction of Leucas indica (AFLI) and much desired preparations and synergistic compositions active anticoagulant preparation (AAP) involving selective active fractions of a natural renewable material as an extract and/or active fraction manufactured from leaves of L. indica. Advantageously, the active anticoagulant preparation (AAP) of said natural renewable material involving an extract and/or active fraction (AFLI) would provide for synergistic anticoagulant composition which can function effectively at normal physiological conditions, for example at pH 7.4 and at 37 °C favouring advancement related to direct and indirect inhibitor of thrombin, providing for a FXa inhibitor. Importantly, also the active anticoagulant preparation (AAP) of a natural renewable material as an extract and/or active fraction from plant material Leucas indica would enable anticoagulant activity that is devoid of any haemolytic activity, and proteolytic activity against blood proteins such as albumin and globulin.