DESC:PRIORITY CLAIM
[0001] This application claims priority from the provisional application numbered 201941052810 filed with Indian Patent Office, Chennai on 19th December 2019 entitled “Phytochemicals with potent antimicrobial activity and process of extraction thereof” the entirety of which is expressly incorporated herein by reference
Preamble to the description
[0002] The following specification describes the invention and the manner in which is to be performed:
Description of the invention
Technical field of the invention
[0003] The present invention relates to isolation of a mixture of anacardic acids from plant sources and the process of extraction thereof and demonstration of their in vitro and in vivo antimicrobial activity. More particularly, the invention discloses a unique cold solvent extraction process of phytochemicals from cashew nut shells and demonstration of their potent in vitro and in vivo antibacterial activity against Gram positive bacteria that cause serious infections in humans and farm animals.
Background of the invention
[0004] Anacardium occidentale is a tropical tree that produces cashew seed and cashew apple. The fruit contains a single seed or a nut. The seed is surrounded by a double shell that contains phenolic resin. The shell of the cashew nut contains a soft honeycomb structure containing a dark reddish-brown viscous liquid (pericarp fluid) of the cashew nut. The viscous liquid is called the cashew nut shell liquid.
[0005] The Cashew Nut Shell Liquid (CNSL) is generally obtained from a solvent extraction or cold press from cashew nut shells. The composition and percentage content of the extract may vary based on the method of extraction used. The liquid is usually rich in cardol cardanol, anacardic acids and few other phenols.
[0006] One of the important component of CNSL is anacardic acid, which is isolated from cashew shells that are the most easily accessible source. Several other sources such as peels of Mangifera indica and to a lesser extent in Pelargonium contain anacardic acid mixtures. Anacardic acid is the general name given to a family of four different 6-alkyl salicylic acids having varying degrees of unsaturation in the 15-carbon alkyl chain.
[0007] CNSL has a wide range of applications in several industries, specifically in the polymer industry. Its applications range from resin, rubber, binders, adhesives, cashew cements, foundry chemicals, surfactants, paints and varnishes. Further, CNSL has multiple uses as, antioxidants, fungicides and other biomaterials.
[0008] Various studies have reported the in vitro antibacterial activity Minimum Inhibitory Concentration (MIC) of the CNSL only. However there are no reports demonstrating the in vitro killing kinetics and in vivo antibacterial efficacy of CNSL derived products in animal models of infectious diseases.
[0009] The traditional or conventional extraction methods employed to extract CNSL from the shell are based on thermal-mechanical method, which involves steam processing followed by quick roasting at high temperature or traditional solvent extraction methods. Such extraction process usually results in loss of the active ingredients during processing. Herein, an extraction process at ambient temperature and solvent removal process without exposing the extracts to extreme conditions of temperature and pressure, thereby ensuring the best recovery of active components of the CNSL is disclosed.
[0010] The US Patent Application “US2431127A” titled “Process of treating cashew nut shell liquid” discloses a process to easily separate the acid constituent from cashew nut shell liquid or hydrogenated cashew nut shell liquid. The process provides an appropriate separation of the acid from the toxic phenolic constituents of the liquid compared to other separation processes. The process comprises reacting the acid in the cashew nut shell liquid with alkaline earth metal ion to obtain an alkaline earth metal salt and separating the salt from toxic phenolic constituents, followed by regenerating the organic acid from the separated alkaline earth metal salt.
[0011] The Patent Application “WO2001062110A2” titled “Process for shelling fruits with shells” discloses a method of treating nuts with shells containing a non-aqueous liquid. The method comprises the steps of firstly removing at least 50% of the non-aqueous liquid from the shells of the nuts by subjecting to heat treatment which involves roasting the shells, followed by cold treatment to embrittle the shells. This method relates especially to cashew nuts and to the shelling of such nuts. Alternatively, the nuts may be subjected to stream treatment to remove cashew nut shell liquid (CNSL) and partially replace the CNSL with water.
[0012] The US Patent Application “US8338638B2” titled “Antimicrobial derivatives of anacardic acid and process for preparing the same” discloses derivatives of anacardic acid having antimicrobial properties and method for preparing the anacardic acid derivatives. The activity of various anacardic acid derivatives were assayed in vitro for antimicrobial activity ,
Minimum Inhibitory Concentration (MIC), against Staphylococcus aureus, methicillin resistant Staphylococcus aureus and Enterococcus fecalis.
[0013] The US Patent Application “US8377485B2” titled “Antimicrobial composition and use thereof” discloses compositions with broad antimicrobial activity against bacteria, fungi, and protozoans. The compositions and methods are used for prevention and treatment of pathogenic processes in people and animals by any mode of administration such as oral, topical, or parenteral. Additionally, the composition is used as a performance improver in animals, infection controller in fermentations, and preserver of grains, seeds, and food for animals and people.
[0014] The publication entitled “Antibacterial action of anacardic acids against Methicillin Resistant Staphylococcus aureus (MRSA)” by Isao Kubo, Ken-ichi Nihei and Kazuo Tsujimoto, discloses the structural and antibacterial activity (MIC) relationship of 6-alk(en)ylsalicylic acids, also known as anacardic acids and are investigated against Gram positive bacteria, specifically on Staphylococcus aureus ATCC33591 (MRSA) strain. The unsaturation in the alkyl side chain is not essential in eliciting activity but is associated with increasing the activity. The antibacterial activity (MIC) of methicillin against MRSA strains was significantly enhanced in combination with C12:0-anacardic acid, and the fractional inhibitory concentration index for this combination was calculated as 0.281.
[0015] Although, in vitro antimicrobial activity (MIC) is reported in the listed prior arts the Minimum Bactericidal Concentration (MBC), killing kinetics and in vivo antibacterial efficacy of CSNL derived molecules have not been reported. Moreover, extraction process reported in the prior-arts, does not focus on enriching the bio-active components that are responsible for its efficacy. Therefore, there is a clear need for a scalable and robust extraction process which prevents the loss of bio-active components in extracts.
Summary of the invention
[0016] The invention discloses a process of extraction of phytochemical active ingredient from cashew nut shells and demonstration of their in vitro and in vivo antimicrobial activity against various Gram positive bacterial pathogens. The process comprises of a unique cold extraction process followed by processing the extract to separate the antimicrobial active ingredient, which is mixture of anacardic acids. The invention also demonstrates the potent in vivo efficacy of the extracted compounds against various pathogens in animal models of infection.
[0017] The extraction process comprises crushing of approximately 1000g-3000 kgs of the cashew nut shells using hammer pulveriser. The crushed cashew nut shells are soaked in a large tank containing organic solvents, preferably solvents in the ratio between 1:3 to 1:12 for a duration of about 24-48 hours. The solvent mixture is separated from the residual cashew nut shells by decanting or filtration process using dume or bag filter. The separated contents are distilled at a temperature range of 400-500C under vacuum (around 400mm) with the help of a vacuum pump, using a chilled condenser to ensure maximum recovery of the solvents and the recovered solvent is recycled at least thrice to extract CNSL efficiently from the shells. The obtained crude residual CNSL oil is treated with 1%-5% activated charcoal and subjected to filtration using sparkler filters and the filtered solvent comprising the extract is subjected to evaporation, under vacuum and the residual material is dried to obtain a crude CNSL extract referred as VYX0007. The extracted CNSL is dissolved in a protic polar solvent preferably an alcoholic solvent such as ethanol or methanol at a concentration ratio between 1:3 to 1:12. The powdered metal hydroxide such as sodium hydroxide or potassium hydroxide or calcium hydroxide is added in portions over a period of 30-60 min to the solution of CNSL in protic polar solvent, in the concentration range between 1:0.15 to 1:0.5. The reaction mixture is continuously stirred at temperature range between 400C to 550C for a duration between 2 hr to 18 hrs and as a result salt formation is observed. Then the reaction mixture is allowed to cool to obtain the precipitated salt, which is filtered and washed using suitable solvents to obtain a free flowing solid. The mother liquor is used in the isolation of cardol and cardanol that are tested for its biochemical profile. The precipitated salt is vacuum dried for small scale trials or spin-drying or Vacuum Tray-Drying (VTD) technique is employed for larger capacities to obtain salt mixture of anacardic acids as VYX0008. VYX0008 is dissolved in water and is treated with 1.2:2.5(w/v) ratio of hydrochloric acid by maintaining the reaction pH between 1-2, stirred at room temperature for a duration between 2 h to 6 h. After which a solvent such as dichloromethane or ethyl acetate is added and stirred to ensure efficient extraction of the organic product. The organic layer is separated and washed once with water, separate the aqueous layer from the organics. The organic layer is evaporated under reduced pressure to yield an extract enriched with the mixture of anacardic acids as an oil as VYX0009. The mixture of anacardic acids (VYX0009) or the CNSL extract (VYX0007) is dissolved in a protic solvent preferably in alcoholic solvent in the ratio 1:10 v/v. At step (117), the solution from the above step, is subjected to hydrogenation under hydrogen pressure of around 4-5 kg, in presence of a 5% - 10% palladium catalyst at room temperature for a of 24 h to yield completely saturated CNSL extract, which is purified to obtain anacardic acid as a single component. The obtained pure anacardic acid is referred as VYX0002.
[0018] The extracts obtained are characterized by Proton Nuclear Magnetic Resonance (1H NMR), the Carbon-13 Nuclear Magnetic Resonance (13C NMR) and Mass Spectrometry. VYX0002 isolated by the unique cold solvent extraction process of phytochemicals from cashew nut shells is processed into a specific formulation. In order to improve the solubility of VYX0002, it is formulated into a salt form.
[0019] VYX0002 exhibited antibacterial activity against bacterial strains Propionibacterium acnes (MTCC1951), Streptococcus pneumoniae (TCH8431 HM145), Clostridium difficile (NR-13427), Staphylococcus epidermidis (ATCC35984), Clostridium perfringens (WAL-14572 HM-310), Staphylococcus hominis (SK119 HM-119), Enterococcus faecalis (ATCC29212), Staphylococcus aureus (ATCC25923), Methicillin resistant Staphylococcus aureus (USA-300 ATCC43300), Vancomycin Resistant Staphylococcus aureus [VRSA] ([BEI]NR-46410, [BEI]NR-46411, [BEI]NR-46412, [BEI]NR-45858, [BEI]NR-46422). VYX0002 showed pharmacologically active concentrations in mice following single intravenous dose ( 5mg/kg) and single oral dose (50 mg/kg) with an oral bioavailability of 13%. In in vitro ADME studies, VYX0002 showed high protein binding (>90%) in mouse and human plasma; low permeability in Caco2 cells, moderate to low metabolic stability in mouse and human liver microsomes.
[0020] VYX0002 exhibited antibacterial effect in animal models of ear infection, skin infection, bacteremia, neutropenic septicaemia. Further, the invention demonstrates formulation/s of CNSL as very potent antimicrobial activity against various bacterial pathogens, indicating that CNSL extracts have the potential to treat infections caused by several Gram positive negative bacteria.
Brief description of the drawings
[0021] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.
[0022] Figure 1 illustrates the process of extraction of the phytochemicals from the cashew nut shells.
[0023] Figure 2a illustrates the Proton Nuclear Magnetic Resonance (1H NMR) of VYX0002.
[0024] Figure 2b illustrates the Carbon-13 Nuclear Magnetic Resonance (13C NMR) of VYX0002.
[0025] Figure 3a illustrates the Mass Spectrometry of VYX0002 in positive ion mode.
[0026] Figure 3b illustrates the Mass Spectrometry of VYX0002 in negative ion mode.
[0027] Figure 4a illustrates the structural formula of the phytochemicals present in VYX0007 obtained from the shells.
[0028] Figure 4b illustrates the mixture of anacardic acids with variable R1 group and their percentage concentration in VYX0009.
[0029] Figure 5a illustrates the HPLC report of blank run.
[0030] Figure 5b illustrates the HPLC report of the VYX0007.
[0031] Figure 5c illustrates the HPLC report of VYX0008.
[0032] Figure 5d illustrates the HPLC report of VYX0009.
[0033] Figure 5e illustrates the HPLC report of VYX0002.
[0034] Figure 5f illustrates the 1H NMR(DMSO-d6) of VYX0014.
[0035] Figure 5g illustrates the illustrates the MS report of VYX0014.
[0036] Figure 5h illustrates the HPLC profile of VYX0014 at 280 and 254nm (HPLC conditions is same as the earlier experiments).
[0037] Figure 5i illustrates the 1H NMR(DMSO-d6) of VYX0015.
[0038] Figure 5j illustrates the illustrates the MS report of VYX0015.
[0039] Figure 5k illustrates the HPLC profile of VYX0015 at 280 and 254nm (HPLC conditions is same as the earlier experiments).
[0040] Figure 6a illustrates the GC report of blank run.
[0041] Figure 6b illustrates the GC report of VYX0009.
[0042] FIGURE 7 illustrates the bidirectional permeability of VYX0002 in Caco-2 cells.
[0043] FIGURE 8 illustrates the in vitro plasma protein binding of VYX0002.
[0044] FIGURE 9 illustrates the in vitro metabolic stability of VYX0002 in mouse and human liver microsomes.
[0045] FIGURE 10 illustrates the oral and intravenous pharmacokinetics of VYX0002 in mouse.
[0046] Figure 11a tabulates the MIC and MBC of test samples against Gram positive bacteria.
[0047] Figure 11b tabulates the MIC and MBC of test samples against Gram positive bacteria.
[0048] Figure 12 illustrates the time kill kinetics of different concentrations of VYX0002 against various bacterial strains.
[0049] FIGURE 13 illustrates the effect of VYX0002 against Propionibacterium acnes (MTCC1951) in a mouse model of ear infection.
[0050] Figure 14 demonstrates the in vivo antibacterial efficacy of VYX0002 against MRSA (USA 300) in a murine model of bacteremia.
[0051] Figure 15 demonstrates the in vivo antibacterial efficacy of VYX0002 against Propionibacterium acnes (MTCC 1951) induced skin infection in mouse model.
[0052] Figure 16 demonstrates the dose dependent response of intravenous VYX0002 against MRSA (USA300) induced neutropenic septicemia in mouse model.
[0053] FIGURE 17a illustrates the efficacy of VYX0002 against MRSA [USA300] in mouse model of neutropenic septicemia.
[0054] FIGURE 17b illustrates the efficacy of VYX0002 against MRSA [43300] in mouse model of neutropenic septicemia.
[0055] FIGURE 17c illustrates the efficacy of VYX0002 against VRSA [NR-46410] in mouse model of neutropenic septicemia.
[0056] FIGURE 18 illustrates the dose response efficacy of single sub-cutaneous administration of VYX0002 on MRSA [USA300] in a neutropenic septicemia mouse model.
[0057] FIGURE 19 illustrates the efficacy of VYX002 against Clostridium difficile (ATCC43255) in Hamster infection model.
[0058] FIGURE 20 illustrates the efficacy of VYX008 against Clostridium perfringens (WAL-14572 HM-310) induced necrotic enteritis in Broilers.
Detailed description of the invention
[0059] Reference will now be made in detail to the description of the present subject matter, one or more examples of which are shown in figures. Each example is provided to explain the subject matter and not a limitation. Various changes and modifications obvious to one skilled in the art to which the invention pertains are deemed to be within the spirit, scope and contemplation of the invention.
[0060] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.
[0061] The term "in vivo" studies refers to studies in living organisms (animal models)
[0062] The term "in vitro" refers to tests or studies performed in microtiter plates, culture dish or a test tube (outside living organism)
[0063] The term "Centrifugation" is a separation technique, uses spinning technique to separate particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed.
[0064] The term "Thin layer Chromatography (TLC)" is a chromatography technique used to separate mixtures using a thin uniform layer of silica gel or alumina coated or a suitable adsorbent on to a piece of glass, metal or rigid plastic surface.
[0065] The term "Minimum Inhibitory Concentration (MIC)" refers to the lowest concentration of antimicrobial agent, required to inhibit growth of a microorganism.
[0066] The term "Minimum Bactericidal Concentration (MBC)" refers to the lowest concentration of an antimicrobial agent required to kill 99% of microorganism.
[0067] The term "Gram positive bacteria" refers to bacteria which stain purple in the Gram staining technique.
[0068] The term “Liquid Chromatography Mass Spectrometry (LC-MS)” means an analytical chemistry technique that combines the separation capabilities of liquid chromatography with the mass detection capabilities of mass spectrometry, as the context requires.
[0069] The term "HPLC" refers to High Performance Liquid Chromatography, a separation technique in analytical chemistry used to separate and quantify a test sample or a mixture, by a suitable detection technique, by injecting the test sample, through a pre-packed column of a suitable solid adsorbent and pumping a liquid (mobile phase) mixture through the column and detector to observe the elution/separation of components of the test sample.
[0070] The term "GC" refers to Gas Chromatography which is a chromatography technique used in analytical chemistry for separating and analyzing compounds that can be vaporized without decomposition. The technique is mainly used for analyzing purity or separating different components from the sample.
[0071] The term "ANOVA" refers to Analysis of Variance, a collection of statistical models and their associated estimation procedures used to analyze the differences among group means in a sample.
[0072] The invention discloses isolation of phytochemicals with potent antimicrobial activity, and the process of extraction and enrichment of the active component from Cashew Nut Shells (CNS). More particularly, the invention relates to the process of extraction of the Cashew Nut Shells Liquid (CNSL), which enriches the active phenolic components present in the CNSL. Further, the invention demonstrates formulation/s of CNSL and very potent antimicrobial activity of CNSL against various bacterial pathogens, indicating that components of CNSL extracts have the potential to be an active ingredient of medicinal products to treat infections caused by several Gram positive bacteria.
[0073] The cashew nut obtained from the cashew tree (Anacardium occidentale) used in the invention has a geographical source of origin from Indian and African sub-continent.
[0074] The phytochemicals of the CNSL also comprises cardol and cardanols, which have applications in polymer and automobile industry. One of the interesting component of the CNSL is anacardic acid. The phenolic contents such as cardanols and cardols in the CNSL are high, when CNS are subjected to extraction process involving high temperatures, there is an increase in the concentration of cardanol, cardol and methylcardols, with loss of anacardic acid mixtures. The extraction processes involving heat converts the anacardic acids in CNSL to cardol and cardanols by thermal decarboxylation. Cardanol, cardol and methyl cardol are used in polyurethane based polymers, surfactants, epoxy resins, foundry chemicals and other chemical and automobile industry.
[0075] The invention discloses a process of extraction of anacardic acid/s from cashew nut shells, the process comprises a unique cold solvent extraction of cashew nut shells and processing of the CNSL extract to separate the anacardic acid by chromatography, salt formation, crystallization or an acid-base work-up technique. The product of extraction, after processing is subjected to reduction to obtain the purest form of anacardic acid.
[0076] Figure 1 illustrates the process of extraction of the phytochemicals from the cashew nut shells. The extraction process (100) of the present invention comprises a step (101) of crushing of approximately 1000g-3000kg, specifically 800kg per batch, of the cashew nut shells using hammer pulveriser. At step (102), the crushed cashew nut shells are soaked in a large tank containing mixture of organic solvents, preferably in the ratio between 1:3 to 1:12 (weight of crushed shells: volume of solvent) for a duration of about 24-48 hours. At step (103), the solvent mixture is separated from the residual cashew nut shells. The separation of the solvent is achieved either by decanting for smaller volumes or filtration process for larger volumes. The filtration of solvent is achieved using dome or bag filter. At step (104) the separated contents are distilled at a temperature range of 400-500C under vacuum (around 400mm) with the help of a vacuum pump, using a chilled condenser to ensure maximum recovery of the solvents. At step (105), the recovered solvents are recycled for repetition of steps (102)-(104) at least thrice to extract CNSL efficiently from the shells. At step (106), the obtained crude residual CNSL oil obtained in step (105) is treated with 1%-5% activated charcoal and subjected to filtration using sparkler filters. At step (107), the filtered product comprising the extract is subjected to evaporation, under vacuum and the residual material is dried to obtain a crude CNSL extract which is referred as VYX0007. The yield achieved for VYX0007 is 25% to 40%. At step (108), the extracted CNSL extract is dissolved in a protic polar solvent, preferably an alcoholic solvent such as ethanol or methanol at a concentration ratio between 1:3 to 1:12 (w/v of CNSL: Solvent). At step (109), powdered metal hydroxide such as sodium hydroxide or potassium hydroxide or calcium hydroxide is added in portions over a period of 30-60 min to the solution of CNSL in protic polar solvent, in the concentration range between 1:0.15 to 1:0.55 (v/w of CNSL: metal hydroxide). At step (110), the reaction mixture is continuously stirred at temperature range between 400C to 550C for a duration between 2 hr to 18 hrs. Salt formation is observed slowly upon stirring. At step (111), the reaction mixture is allowed to cool to obtain the precipitated salt. At step (112), the precipitated salt is filtered and washed using solvents such as methanol, ethyl acetate, toluene or 2 propanol or a solvent mixtures in the ratio ranging between 1:3 to 1:6 (w/v of CNSL: solvent) to obtain a free flowing solid. At step (113), the precipitated salt is vacuum dried for small scale trials or spin-drying or VTD technique is employed for larger capacities to obtain salt mixture of anacardic acids. The dried salt mixture of anacardic acids is referred to as VYX0008. At step (114), about 1: 5 (w/v) ratio of salt mixture of anacardic acid is dissolved in water and is treated with 1.2:2.5 ratio of mineral acid preferably 6N hydrochloric acid, concentrated hydrochloric acid. The reaction pH is maintaining between 1-2, continuously stirred at room temperature for a duration between 2 h to 6 h. The organic solvent such as ethyl acetate or dichloromethane is added and stirred well to extract out the mixture of anacardic acids. Similarly, multiple extractions are done to ensure that the aqueous layer has no components of anacardic acid. The combined organic layers are washed once with water. The organic layer is separated and evaporated under reduced pressure to yield an extract enriched with the mixture of anacardic acids as an oil. The oil comprising mixture of anacardic acids is referred to as VYX0009. At step (115), the mixture of anacardic acids (VYX0009) or the CNSL extract (VYX0007) is dissolved in a protic solvent preferably in alcoholic solvent in the ratio 1:10 v/v. At step (116), the solution from the above step, is subjected to hydrogenation under hydrogen pressure of around 4-5 kg, in presence of a 5% to 10% palladium catalyst at room temperature for a of 24 h to yield completely saturated CNSL extract, which is purified to get pure anacardic acid as a single component. The obtained pure anacardic acid is referred as VYX0002.
[0077] The cashew nut shells of 3 mm to 6 mm thickness are sourced from the Indian sub-continent or from African continent. The shells are crushed manually or using a mechanical device such as a hammer pulveriser and soaked in a suitable organic solvent. The solvents are selected from a group comprising methanol, 2-propanol, ethanol, ethyl acetate, ether, acetone, methyl vinyl ketone, methyl isobutyl ketone (MIBK), cyclohexane, hexanes or heptane. Most preferably cyclohexane or hexanes and a suitable alcohol are used for the extraction. Additionally, to ensure best recovery of the solvents, post soaking and filtration, distillation is performed under vacuum and the condenser is maintained at sub-zero temperature. The recovered solvent was recycled for repetition of step (102) to (104) at least thrice to ensure extraction of CNSL efficiently from the shells.
[0078] Further, the crude residual CNSL as described in step (106) is subjected to a clean-up process, which partially removes dark color in the extract by a simple clarity filtration over hyflo or celite bed using activated charcoal for large volumes of CNSL. In case of lab trials column chromatography is preferred, with silica gel as the stationary phase and the mixture of organic solvents such as cyclohexane or hexane and ethyl acetate, acetone or MIBK as the mobile phase. The clean-up process yields a yellowish-brown viscous liquid, which is the CNSL extract (VYX0007).
[0079] The batch size for cold solvent extraction to get VYX0007 is achieved by extraction of approximately 3750kg shells to get 1050kg of VYX0007, which is further processed as per the requirements.
[0080] As described in FIG 1, VYX0007 is dissolved in polar solvents selected from a group comprising acetone, 2-propanol, methyl ethyl ketone, methanol, ethanol or MIBK in the range between 1:3 or 1:12 (w/v of CNSL : solvent). Further, purification is achieved by salt formation using a suitable metal hydroxides, selected from a group comprising of lithium, sodium, potassium, magnesium, calcium or barium and added in the range between 1:0.15 or 1:0.55 (v/w of CNSL: metal hydroxide), preferably sodium or potassium or calcium metal hydroxides are chosen. The precipitated salt is filtered and washed with solvent such as methanol, ethyl acetate, toluene or 2 propanol mixture, in the ratio ranging between 1:3 to 1:6 (w/v of CNSL:solvent). For smaller scale of 1g to 100g of CNSs, standard lab filtration equipment such as Buchner funnel, filter flasks etc. are used. For larger volume, filtration is performed using a nutsche filter or centrifuged to separate the solvent from the precipitated salt. The solid obtained is vacuum dried for smaller scale batches or dried using spin dryer to expel off any traces of solvent for larger scale batches. The solids thus obtained comprises salt mixtures of anacardic acids referred as VYX0008 obtained from the VYX0007 and possess potent antimicrobial activity.
[0081] VYX0008 is synthesized by dissolving 1 kg of VYX0007 in polar solvents such as methanol, ethanol or MIBK in the range between 1:3 to 1:12 (w/v of CNSL : solvent), precisely 1: 6(w/v) when used with methanol and stirred at 400-500C for 30 mins. Finely powdered Calcium hydroxide (1.15eq) is added portion wise in small lots over a period of 30-45mins. The contents are stirred at this temperature for 4-6h during which the contents form a clear solution and allowed to cool to room temperature, the salt of anacardic acid crystallizes out. However, cardol and cardanol that does not form any salts are separated out and retained in the solution. The salt thus formed is filtered using a filtration assembly, which is further washed with 2-2.5l methanol and solid dried using Vacuum Tray Drier (VTD) at 400C for 6h. The weight of pure salt obtained by this process is 800g. The solids thus obtained comprises calcium salt mixtures of anacardic acids (VYX0008).
[0082] The salt mixtures of anacardic acids can be directly used for efficacy studies or can be neutralized to get back the active CNSL extract. VYX0008 is converted to free acid form by treatment with a suitable acid. Around 1: 5 (w/v) ratio of VYX0008 is taken in water and treated with a suitable acid, preferably 6N hydrochloric acid or concentrated hydrochloric acid or equivalent (1.25-unit volumes) The contents are stirred at room temperature, while maintaining the reaction pH between 1 to 2 for a duration between 2 hr to 6 hr. The organic solvent such ethyl acetate, dichloromethane(3-5 volumes) are added for extraction of the free acids. The aqueous layer is separated and extracted thrice to ensure effective extraction. The organic layer is separated, and washed once with water, followed by combining the organic layers and further evaporated under reduced pressure to obtain extract enriched with mixture of anacardic acids as an oil (VYX0009).
[0083] The mixture of anacardic acid is subjected to reduction which converts the unsaturated sidechain of the anacardic acid (R1= C15H31-2n) to get pure anacardic acid where n=0, thus yielding a side chain of R1=C15H31, which is found to be at a concentration of only 3-6% in the CNSL extracts obtained at step (107). The extract as described in step (115) is dissolved in a suitable protic polar solvent such as ethanol, methanol, 2-propanol or butanol or a mixture of these solvents in a suitable proportion, preferably in a ratio of 1:10 ( v/v of anacardic acids mixture to solvent/s) and subjected to catalytic hydrogenation. The catalysts used in the process can be palladium in the form of Pd-C (5%-10% palladium loading), or platinum in the form of PtO2 or nickel in the form of Raney-Nickel. About 300 g of VYX0007 or VYX0009 is dissolved in methanol or ethanol and purged with nitrogen. 10% (w/w) of the Pd-C catalyst (50% moist) with 5%-10% palladium loading is added. The contents are stirred under hydrogen pressure between 5 kg to 8 kg for a time duration between 24 h to 36 h at room temperature. The reaction is monitored by HPLC for the presence of single peak corresponding to anacardic acid. Upon completion of the reaction, the contents are filtered over a celite bed and washed with methanol or ethanol. The solvent washings are combined and dried over sodium sulfate to remove the solvent under reduced pressure to yield the desired product with a purity greater than 95%. The obtained product is further crystallized using a mixture of protic polar solvent and water to obtain pure product of greater than 98% by HPLC, which comprises pure anacardic acid as the single component (VYX0002). The scale-up of this product was performed on a 300g scale under similar conditions to obtain pure VYX0002 in quantitative yields. VYX0002 is analysed by Proton Nuclear Magnetic Resonance (1H NMR), Carbon-13 nuclear magnetic resonance (13C NMR) and Mass Spectrometry (MS). Further, moisture content is determined by standard analytical methods. The melting point of VYX0002 at ambient temperature and pressure is found to be in the range of 910C-930C.
[0084] The mother liquor obtained from step (113) is utilized to isolate cardol and cardanol. An aliquot of mother liquor 14g is dissolved in dichloromethane, adsorbed on silica gel, and chromatographed on a silica gel column using hexane and ethyl acetate as eluant. The fractions are collected and the non-polar spot is isolated and found to be a mixture of (4g) cardanols and the next polar spot is found to be mixture of (4.5) cardols. These mixtures of cardanols and cardols are subjected to hydrogenation in the presence of 10% palladium catalyst using ethanol as solvent resulted in completely saturated 1.2g of VYX0014 (saturated cardanol) and 2g of VYX0015 (saturated cardol). These two compounds are characterized by 1H NMR and Mass Spectrometry.
[0085] Figure 2a illustrates the Proton Nuclear Magnetic Resonance (1H NMR) of VYX0002. The test identified the hydrogen atoms in the sample.
[0086] Figure 2b illustrates the Carbon-13 nuclear magnetic resonance (13C NMR) of VYX0002. The test identified the carbon atoms in the sample.
[0087] Figure 3a illustrates the Mass Spectrometry of VYX0002 in positive ion mode.
[0088] Figure 3b illustrates the Mass Spectrometry of VYX0002 in negative ion mode.
[0089] Figure 4a illustrates the structural formula of the phytochemicals present in VYX0007 obtained from the shells. The CNSL extract obtained at step (107) relating to VYX0007 as described in FIG 1, comprises cardol, cardanol, methyl cardanol and mixture of anacardic acids.
[0090] Figure 4b illustrates the mixture of anacardic acids with variable R1 group and their percentage concentration in VYX0009. The variable group R1= C15H31-2n, where n is the no. of C=C in the chain. If n=0, R1=C15H31 which is found to around 3-4% in the VYX0009. If n=1, R1=C15H29 which is found to around 34-36% in the CNSL extract. If n=2, R1=C15H27 which is found to around 21-22% in the CNSL extract. If n=3, R1=C15H25 which is found to around 40-41% in the CNSL extract.
[0091] The extracts obtained at different steps during the extraction process are checked for the integrity using various analytical methods like TLC, HPLC and GC.
[0092] A HPLC system of Agilent 1290 series with a column composition of Shodex-4E C18 (250mm x 4.6mm x 5µm) is used for the analysis. The extract obtained at different steps as described in the FIG 1 are analyzed by HPLC with a mobile phase composition in the ration of 95% acetonitrile: 2.5% water: 2.5% acetic acid. The test samples comprise a blank containing only the mobile phase solvent, sample 1 comprises VYX0007, sample 2 comprises VYX0008, sample 3 comprises VYX0009, sample 4 comprises VYX0002. Approximately 1 mg of each sample is transferred to an Eppendorf tube and mixed with 1 ml of the mobile phase. The samples are dissolved to obtain a final concentration of 1.0 mg/mL for each sample. The resulting sample solution is diluted 10 folds with the mobile phase. 20 µl of each sample is injected with a flow rate set to 1.5 ml/min and a run time of about 25 mins. The samples are analyzed at 280 nm wavelength.
[0093] Figure 5a illustrates the HPLC report of blank run. The chromatogram illustrates the sample blank run comprising the mobile phase used in the sample preparation.
[0094] Figure 5b illustrates the HPLC report of VYX0007. The peaks in the chromatogram illustrates the presence of cardol, methyl cardanol, cardanol and mixture of anacardic acids in sample 1.
[0095] Figure 5c illustrates the HPLC report of acids VYX0008. The peaks in the chromatogram illustrates the salt mixtures of anacardic acids devoid of cardol, cardanol, methyl cardanol in sample 2.
[0096] Figure 5d illustrates the HPLC report of VYX0009. The peaks in the chromatogram illustrates the presence of active phytochemicals in sample 3.
[0097] Figure 5e illustrates the HPLC report of VYX0002. The single peak in the chromatogram illustrates the presence of the anacardic acid as the single component in sample 4.
[0098] Figure 5f illustrates the 1H NMR(DMSO-d6) of VYX0014.
[0099] Figure 5g illustrates the MS analysis of VYX0014.
[00100] Figure 5h illustrates the HPLC profile of VYX0014. The HPLC profile of VYX0014 is analyzed at 280 and 254nm.
[00101] Figure 5i illustrates the 1H NMR(DMSO-d6) of VYX0015.
[00102] Figure 5j illustrates the MS analysis of VYX0015.
[00103] Figure 5k illustrates the HPLC profile of VYX0015. The HPLC profile of VYX0015 is analyzed at 280 and 254nm.
[00104] Figure 6a illustrates the GC reports of blank run. The chromatogram relates to the solvent blank run, injected prior to the sample run to check for impurities in the solvent used for sample preparation. Dimethyl sulfoxide (DMSO) is used as the solvent for the sample preparation.
[00105] Figure 6b illustrates the GC reports of VYX0009. The sample VYX0009 at a concentration of about 1 mg was dissolved in 1 ml of DMSO. Approximately 1 µl of the sample was injected with a run time of 40 mins to determine the purity of the sample.
[00106] VYX0002 isolated by the unique cold solvent extraction process of phytochemicals from cashew nut shells of the present invention is processed into a specific formulation. In order to enhance the solubility of extract VYX0002, VYX0002 is formulated into a salt form. Generally, the active ingredient is combined with other carriers or excipients to safely produce an acceptable drug product to comprise a formulation, which is utilized in various forms for suitable administration such as injection, capsule, ointment or as a power. However, the process of formulation shall not modify the pharmacological effects of the active ingredient. In order to enhance the water solubility of VYX0002 isolated by the process of the present invention, the active ingredient VYX0002 is formulated into a salt form to improve the aqueous solubility and stability of VYX0002.
[00107] According to an embodiment of the invention, a known weight of the pure VYX0002 is taken in a glass reactor to which an equi-molar equivalent of alkali metal hydroxide is added. The alkali metal hydroxide used is selected from lithium hydroxide, sodium hydroxide, cesium hydroxide or potassium hydroxide. The contents are allowed to stir at 400-500C for a duration of 3 hours. During this time, the contents forms a clear solution and the insoluble particles are filtered off and the contents are allowed to cool to room temperature during which the corresponding salt of VYX0002 is crystallized. The crystalline material is filtered and washed with water. The crystalline material is dried under vacuum at 350-400C to obtain the salt of VYX0002. The sodium salt of VYX0002 is referred as VYX0004.
[00108] VYX0002 isolated by the unique cold solvent extraction process of the present invention is analyzed for different in vitro and in vivo characteristics.
[00109] VYX0002 is analyzed for in vitro Absorption, Distribution, Metabolism and Excretion (ADME) to evaluate the pharmacokinetics through analysis of low permeability in hepatocyte cell line Caco-2 cells.
[00110] FIGURE 7 illustrates the bidirectional permeability of VYX0002 in Caco-2 cells. Caco-2 cells were grown in Dulbecco's Modified Eagle Medium comprising GlutaMAX I, 4,500 mg/L D-glucose and sodium pyruvate supplemented with 10% Fetal Bovine Serum (FBS) in the presence of antibiotics. For transport studies, cells were seeded onto polycarbonate Transwell filter membranes (Millipore) at a density of 60,000 cells/well. After 24 h of seeding, the medium is changed and cultured for another 21 days before transport experiments. For transport studies, donor solutions were prepared by diluting the stock solutions of test compounds in transport medium (HBSS buffer with 10mM HEPES, pH 7.4). The receiver solutions were the same HBSS buffer with 10mM HEPES, pH 7.4. The transport of test compounds (5 µM) was measured in duplicate in two directions [apical to basolateral (A?B) and basolateral to apical (B?A)]. The bioanalysis was performed by LC/MS/MS. The apparent Permeability coefficient [Papp] was estimated using the equation
Papp (cm/sec) = (Vr/C0) (1/S) (dC/dt)

Wherein, (Vr = volume of medium in the receiver chamber, C0 = PAR of the test drug in the receiver chamber, S = surface area of monolayer, dC/dt = drug PAR in the receiver chamber with time).
Area of 24-well = 0.7 cm2
Peak area ratio[PAR] = Analyte peak area/IS peak area
Efflux ratio [ER] = Papp, B ? A / Papp,A ? B
(where Papp, B ? A and Papp,A ? B represent the apparent permeability of test compound from the receiver to donor and donor to receiver side of the cellular monolayer, respectively).

Criteria: Papp value: < 1 x 10-6 cm/sec - low permeability; 2 to 10 x 10-6 cm/sec – medium permeability and >10 x 10-6 cm/sec - high permeability. ER <2.0 - not a substrate for efflux; ER>2.0 - substrate for efflux.
[00111] The results indicated that VYX0002 exhibited low permeability in Caco-2 cells.
[00112] VYX0002 is analyzed for Plasma Protein Binding (PPB).
[00113] FIGURE 8 illustrates the plasma protein binding of VYX0002. PPB was estimated using the Rapid Equilibrium Dialysis (RED) method. The dialysis membranes [HT Dialysis (12-14 kDa)] were soaked in Milli-Q water for 60 min and subsequently in 20% ethanol in Milli-Q water for 30 min. The membranes were rinsed well with Milli-Q water twice before loading onto the HT dialyzer. 150 µl of mouse or human plasma containing 3 µM final concentration of test compound/reference standard was placed into one half of the 96-well Micro-Equilibrium Dialysis Device [Reusable 96-well Micro-Equilibrium Dialysis Device HTD 96: HT Dialysis LLC-HTD 96b-1006] and 150 µl of blank sodium phosphate buffer (100 mM, pH = 7.4) was placed into the buffer half of the same well of the above plate, and equilibrated at 37 ± 5°C for 5 h, with constant rotation at 130 rpm on an orbital shaker. After equilibration, 10 µl of plasma sample from the plasma half of the well was pipetted to a vial/plate containing 200 µl of quenching solution and 50 µl of blank buffer was added to it. Similarly, 50 µL of buffer sample from the buffer half of the wells was pipetted to a vial/plate containing 200 µL of quenching solution and 10 µL of blank plasma was added to it. The samples were centrifuged at 14,000 rpm for 5 min, and the supernatants were transferred to LC-MS/MS vials or plate for analysis by LC-MS. For recovery experiments, 10 µL of plasma from plasma half and 50 µL from the buffer half of the same well were added to a vial\plate containing 200 µL of quenching solution, centrifuged at 14000 rpm and the supernatants were analysed by LC-MS/MS. The experiment was done in triplicates and bioanalysis was done by LC-MS/MS. The results indicated that VYX0002 showed high plasma protein binding in mouse and human plasma.
[00114] It is important to understand the in vitro metabolic stability of VYX0002. VYX0002 is analyzed for in vitro metabolic stability in mouse or human liver microsomes.
[00115] FIGURE 9 illustrates the in vitro metabolic stability of VYX0002 in mouse and human liver microsomes. Mouse or human liver microsomes were removed from -700C deep freezer and thawed on an ice bath. After thawing, 1 µl of test compound or control (1.1 mM) and 27.5 µL microsomes (20 mg/ml) were added to 971.5 µl of 66.7 mM potassium phosphate buffer (pH7.4) in propylene tubes and termed as Incubation mix. 180 µl of incubation mixture is transferred to 4 tubes labelled as T0, T5, T15 and T30. All the tubes were pre-incubated at 37 ± 10C for 5 min in a shaking water bath. Tubes of NADPH (10 mM) were similarly pre-incubated. After pre-incubation, 20 µL NADPH solutions (10 mM) were added to the T5, T15 and T30 tubes, and 20 µl of buffer was added to the T0 tube. At the end of the incubation period (0, 5, 15 and 30 min) of the respective tubes, an aliquot of 200 µl of quenching solution was added to each tube to stop the reaction. The samples were centrifuged at 1934 g for 20 min. The assay was done in duplicates and bioanalysis was done by LC-MS/MS. The peak area ratios of test compound or verapamil to IS were used for the calculations. The natural logarithm (Ln) of the mean peak area ratio was plotted against time to assess the first order nature of the reaction, and the slope (Ke, the 1st order elimination rate constant) was calculated from the linear portion of the curve using the equation
Ke = [-In (c2-c1)/t2-t1], where c2 is the Peak area ratio at time t2 and c1 is the peak area ratio at time t1.
[00116] The half-life (t1/2) and intrinsic microsomal clearance (CLint ) were calculated using the following equations:
Half Life (t1/2) (min) = 0.693/Ke
CLint (µl/min/mg) = [ln2/half-life (min) / [Microsomal protein (mg)]] * (Volume of Incubation (ml) *1000
[00117] The percent of the substrate metabolized (% Metabolized) was also calculated using the equation [1-(test area ratio/control area ratio)]*100.
[00118] The results indicated that VYX0002 showed high clearance in human liver microsomes and moderate clearance in mouse liver microsomes.
[00119] VYX0002 is analyzed for in vivo pharmacokinetics in mouse. The pharmacokinetics of VYX0002 is evaluated post a single oral dose of 50 mg/kg and a single intravenous dose of 5 mg/kg in male CD-1 mice aged 6-8 weeks.
[00120] FIGURE 10 illustrates the oral and intravenous pharmacokinetics in mouse. The pharmacokinetic study of VYX0002 was approved by the Institutional Animals Ethics Committee (IAEC) of the test facility. The study is performed in male CD-1 mice. Sterile water is used as a vehicle for oral and intravenous studies. Appropriate amounts of VYX0004 i.e. sodium salt of VYX0002 was weighed and dissolved in appropriate volumes of sterile water. Animals in the oral dosing group were fasted two hours prior to dosing and feed was given half-hour post dose, and water was provided ad libitum. Feed and water were given ad libitum for the intravenous dosing group. The blood samples were collected post dose and at different time points (3 animals per time point). Blood samples were collected in 1.5 ml Eppendorf tubes containing 0.005 ml of 10% K2EDTA, mixed gently and placed in ice before centrifugation. Blood was centrifuged at 10,000 p.m. for 2 minutes, plasma was harvested and stored at –80°C. Finally, the compound concentrations were quantified in plasma by LCMS/MS.
[00121] Pharmacokinetic analysis was performed using noncompartmental methods in WinNonlin. Pharmacokinetic parameters such as Clearance (CL), Volume of Distribution at steady state (Vss), Ke (elimination rate constant), terminal half-life (t1/2), AUC0-t (Area under the Concentration Time Curve up to last non-zero conc), AUC0-8 (Area under the Concentration Time Curve up to infinity) were estimated for intravenous delivery and Cmax (Peak concentration achieved in plasma), tmax (time at which Cmax is achieved), Ke(elimination rate constant), t1/2 (the terminal half-life), AUC0-t (Area under the Concentration Time Curve up to last non-zero conc), AUC0-8 (Area under the Concentration Time Curve up to infinity and Oral Bioavailability (F) for oral administration.
[00122] The results indicated that the a single intravenous dose of 5 mg/kg VYX0002 showed low systemic clearance, a low Volume of Distribution with a t1/2 of ~ 1 h in mice. The single oral dose of 50 mg/kg VYX0002 showed rapid absorption with tmax, Cmax, AUCinf, and t1/2 of 0.5 h, 4.9 µg/ml, 10.35 µg.h/ml and 2.8 h, respectively in mice. The oral bioavailability was 13% in mice and the pharmacologically active exposures achieved with intravenous 5 mg/kg VYX0002.
[00123] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner.
Example 1: MIC and MBC studies related to test samples VYX0007, VYX0008, VYX0009 and VYX0002 against Gram positive
[00124] The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of test samples against Gram positive and Gram negative bacteria are determined by the microdilution method using CLSI guidelines. The MIC and MBC assay is determined against bacterial strains Propionibacterium acnes (MTCC 1951), Streptococcus pneumoniae (TCH8431 HM145), Clostridium difficile (NR-13427), Staphylococcus epidermidis (ATCC35984), Clostridium perfringens (WAL-14572 HM-310), Staphylococcus hominis (SK119 HM-119), Enterococcus faecalis (ATCC29212), Staphylococcus aureus (ATCC25923), Methicillin resistant Staphylococcus aureus (ATCC USA-300 ATCC43300), Vancomycin Resistant Staphylococcus aureus [VRSA] ([BEI]NR-46410, [BEI]NR-46411, [BEI]NR-46412, [BEI]NR-45858, [BEI]NR-46422).
[00125] The MIC is defined as the lowest concentration of the test compound that prevents bacterial growth tested by lack of turbidity or the Optical Density (OD) at 600 nm wavelength measured in a Ultra-Violet (UV) spectrophotometer, relative to no growth control.
[00126] The MBC is defined as lowest concentration of test compound that reduces the viability of the initial bacterial inoculum by 99%. Ciprofloxacin is used as the positive control in both the MIC and MBC studies.
[00127] Figure 11a tabulates the MIC and MBC of test samples against Gram positive bacteria. The MIC is found to be <0.0039% against Gram positive bacteria such as Propionibacterium acnes (MTCC 1951), Streptococcus pneumoniae (TCH8431 HM145), Clostridium difficile (NR-13427), Staphylococcus epidermidis (ATCC35984), Clostridium perfringens (WAL-14572 HM-310), Staphylococcus hominis (SK119 HM-119), Enterococcus faecalis (ATCC29212), Staphylococcus aureus (ATCC25923), Methicillin resistant Staphylococcus aureus (ATCC USA-300 ATCC43300), Vancomycin Resistant Staphylococcus aureus [VRSA] ([BEI]NR-46410, [BEI]NR-46411, [BEI]NR-46412, [BEI]NR-45858, [BEI]NR-46422). The MIC of ciprofloxacin against the tested bacterial stains is consistent with literature reports. In conclusion, the test samples displayed potent MIC on all the Gram positive bacteria, with MIC ranging between 0.0019% and 0.0039%. The test samples exhibited potent bactericidal activity on all Gram positive bacteria (MBC=0.0039%) with bacterial numbers being below the limit of quantitation at 24 h. In conclusion, the test samples displayed potent MBC on all Gram positive bacteria suggesting that the samples have the capability to rapidly kill pathogenic bacteria.
[00128] Figure 11b tabulates the MIC and MBC of test samples against Gram positive bacteria. The MIC and MBC are analyzed against Enterococcus faecalis (ATCC29212), MRSA (USA-300), Staphylococcus epidermidis (ATCC35984) and Streptococcus pyogenes (ATCC12202).

Example 2: Study for determination of in vitro time-kill kinetics.
[00129] Time-kill assay in vitro is performed to determine the antimicrobial activity of VYX0002 against the bacterial strains Propionibacterium acnes (MTCC1951), Streptococcus pneumoniae (TCH8431 HM-145), Staphylococcus epidermidis (ATCC35984), Staphylococcus hominis (SK119 HM-119), Staphylococcus aureus (ATCC25923), Methicillin resistant Staphylococcus aureus (ATCC USA-300).
[00130] Figure 12 illustrates the in vitro time kill kinetics of VYX0002 against various bacterial strains. VYX0002 exhibited concentration dependent killing of the micro-organisms. VYX0002 resulted in rapid killing of Staphylococcus aureus (ATCC25923) and MRSA (ATCC USA-300). The bacterial load was reduced to Below Limit of quantitation within few minutes of addition of VYX0002 at a concentration between 128 ug/ml and 512 ug/ml. Similarly, the bacterial load of Staphylococcus epidermidis (ATCC35984) to BLOQ was reduced by 2h at 256 ug/ml and that of Propionibacterium acnes (MTCC1951) to BLOQ by 12h at 32 µg/ml. This is the first report of in vitro killing kinetics of VYX0002 on Gram positive bacteria. The rapid killing property of VYX0002 suggests its potential to be a unique antibiotic for the treatment of bacterial infections caused by Gram positive bacteria such as bacteremia, Bacterial Skin and Skin Structure Infections (BSSSI), Acute Bacterial Skin and Skin Structure Infections (ABSSSI), hospital Ventilator Acquired Pneumonia (VAP), osteomyelitis, skin acnes, malodor in arm pits, urinary tract infections and complicated Urinary Tract Infections (cUTI), endocarditis, and necrotic enteritis in poultry.
Example 3: Efficacy study of VYX0002 against Propionibacterium acnes (MTCC1951) in a mouse model of ear infection
[00131] VYX0002 is analyzed against Propionibacterium acnes (MTCC1951). The study is performed in a mouse model of ear infection. Female BALB/c mice were rendered neutropenic using cyclophosphamide, anesthetised and infected with approximately 20 µl of P. acnes culture (~109 CFU/ml) intra-dermally into the left ear of each mouse. At 8 h post infection, mice were treated with a vehicle and VYX0002, by topical application of 0.5%, 1% and 2% twice daily (q12h) for two days. Following treatment, ears are harvested, homogenised and plated for bacterial counts.
[00132] FIGURE 13 illustrates the effect of VYX0002 against Propionibacterium acnes (MTCC1951) in a mouse model of ear infection. VYX0002 at 1% and 2% (15 mg b.i.d) displayed significant dose dependent reduction of bacterial load on skin when compared to the vehicle (p<0.05) in the ear infection model. In conclusion, VYX0002 has the potential to be used to treat skin infections such as acne.
Example 4: Evaluation of in vivo antibacterial efficacy of VYX0002 against MRSA (USA300) in a murine model of bacteremia
[00133] The antibacterial efficacy of VYX0002 is evaluated against MRSA (USA300) in a murine model of bacteremia. Female CD-1 mice aged 6-8 weeks are neutropenised with cyclophosphamide and infected intra-peritoneally with 0.5 ml of Staphylococcus aureus MRSA (USA300) ~5 x 108 CFU/animal. Two hours post infection, groups of mice are treated intravenously with vehicle (3 doses, q1h), sub-cutaneously with vancomycin (110 mg/kg, q12h) and intravenously with VYX0002 (5 mg/kg, 3 doses, q1h). The blood samples are drawn at different time points and bacteria are enumerated. The clinical signs and mortality rates are recorded. Mean ± SD Log10CFU/ml blood is estimated at the indicated time points for each group. The significant differences between treatment group means are assessed using a one-way ANOVA, followed by a Dunnett’s multiple comparison test.
[00134] Figure 14 demonstrates the in vivo antibacterial efficacy of VYX0002 against MRSA (USA300) in a murine model of bacteremia. VYX0002 showed rapid in vivo antibacterial effect following three intravenous doses of 5 mg/kg given every 1 h. The bacterial load in blood is BLOQ at 20 h post last dose. In contrast, vancomycin (110 mg/kg, SC, q12h) showed 1.5 Log10CFU/ml reduction of bacteria in blood. The mice treated with VYX0002 are apparently normal and showed 100% survival. In comparison, mice in the vehicle control group showed 0% survival and died within 24 h. Mice treated with vancomycin showed 0% survival at 48 h. VYX0002 is superior to vancomycin in curing bacteremia caused by MRSA in mice and has the potential to be used in the clinic for treating sepsis. Moreover, VYX0002 is found to be safe and tolerated following intravenous administrations of 5 mg/kg thrice daily.
Example 5: Evaluation of efficacy of VYX0002 against Propionibacterium acnes (MTCC 1951) Induced Mouse Skin Infection Model
[00135] The efficacy of topical VYX0002 formulation was analyzed against Propionibacterium acnes (MTCC 1951) induced skin infection in mouse model. Female BALB/c mice were rendered neutropenic with cyclophosphamide. Mice were anaesthetised by intraperitoneal (IP) administration of cocktail of 50 mg/kg ketamine and 10 mg/kg xylazine followed by injection of 50 µL P. acnes (MTCC 1951) culture (~109 CFU/ml) intra-dermally into dorsal area of the skin of each mouse. Eight hours post infection, clindamycin as positive control and the formulations of VYX0002 were applied topically on the infection site, twice daily for seven days. Following treatment, animals were terminated, skin i.e. site of infection was homogenized in 1 ml BHI broth containing 1% glucose and plated on BHIA plates for 3-4 days in an anaerobic atmosphere to enumerate the CFU’s. The mean ± SD of bacterial load skin (per gram) was estimated for each group. The significance of differences between the control and treatment groups were evaluated using an appropriate statistical test at 95% confidence level. VYX0002 can be formulated into gel form or other suitable formulation using lauryl glucoside, coco glucoside, glutamate VLT as thickener at different weight percentage of the ingredients.
[00136] Figure 15 demonstrates the in vivo antibacterial efficacy of VYX0002 against Propionibacterium acnes (MTCC 1951) induced skin infection in mouse model. The results indicated that VYX0002 exhibited anti-bacterial effect on Propionibacterium acnes (MTCC 1951) induced skin infection following topical administration for seven days when compared to the vehicle control (p<0.05).
Example 6: Dose Response Efficacy of VYX0002 on MRSA (USA300) following intravenous administrations in a neutropenic septicemia mouse model
[00137] The dose dependent response of intravenous VYX0002 was analyzed against MRSA (USA300) induced neutropenic septicemia in mouse model. Female CD-1 mice aged 6-8 weeks were neutropenised with cyclophosphamide and infected intra-peritoneally with 0.5 ml of MRSA (USA300) ~5 x 108 CFU/animal. Two hours post infection, the groups of mice were treated intravenously with vehicle, intravenously with 110 mg/kg single dose of vancomycin and intravenously with single doses of VYX0002 (25, 50 and 75 mg/kg) and repeated doses (three) of 5 mg/kg, every 5 min. VYX0002 was administered as a sodium salt (VYX0004) and the vehicle was sterile MilliQ water. Blood samples were drawn at different time points and bacteria were enumerated. The duration of the study was eight hours and the clinical signs along with percent mortality were recorded. Mean ± SD Log10CFU/ml blood was estimated at the indicated time points for each group. Significant differences between treatment group means were assessed using a one way ANOVA, followed by a Dunnett’s multiple comparison test.
[00138] Figure 16 demonstrates the dose dependent response of intravenous VYX0002 against MRSA (USA300) induced neutropenic septicemia in mouse model. The results indicated that VYX0002 showed dose dependent antibacterial effect on MRSA following single intravenous doses. VYX0002 exhibited antibacterial effect at 4 h and 6 h PI and at 6 h (5 mg/kg, IV, 3 doses, q 5min) when compared to the vehicle control (p<0.05). VYX0002 showed significantly dose dependent higher survival at 8 h, when compared to the vehicle control. The efficacy of VYX0002 at 75 mg/kg intravenous single dose was comparable to that of single dose 110 mg/kg, vancomycin.
Example 7: Efficacy of VYX0002 on MRSA [USA300, 43300] and VRSA [NR-46410] in a non-neutropenic septicemia mouse model
[00139] The efficacy of VYX0002 is analyzed against MRSA [USA300, 43300] and VRSA in mouse model of neutropenic septicemia. The groups of female CD-1 mice aged 6-8 weeks were infected intra-peritoneally with 0.5 ml of Staphylococcus aureus MRSA (USA300), MRSA (43300) and VRSA [NR-46410], all at ~5 x 108 CFU/animal. Two hours post infection, the groups of mice were treated intravenously with vehicle, intravenously with 110 mg/kg, single dose vancomycin and intravenously with repeated doses (three) of 5 mg/kg VYX0002, every 5 min. VYX0002 was administered as a sodium salt (VYX0004) and the vehicle was sterile MilliQ water. Blood samples were drawn at different time points and bacteria were enumerated. The duration of the study was eight hours and the clinical signs and percent mortality were recorded. Mean ± SD Log10CFU/ml blood was estimated at the indicated time points for each group. There was a significant differences between treatment group means and were assessed using a one way ANOVA, followed by a Dunnett’s multiple comparison test.
[00140] FIGURE 17a illustrates the efficacy of VYX0002 against MRSA [USA300] in mouse model of neutropenic septicemia. VYX0002 showed significant antibacterial activity on MRSA (USA300) at 4 and 6 h when compared to the 2h control. Mice dosed with VYX0002 showed 67% survival at 8h PI whereas controls showed 100% mortality.
[00141] FIGURE 17b illustrates the efficacy of VYX0002 against MRSA [43300] in mouse model of neutropenic septicemia. The results indicated that intravenous VYX0002 at 5mg/kg 3 doses q5min showed antibacterial activity on MRSA 43300 when compared to the vehicle control at all the time points.
[00142] FIGURE 17c illustrates the efficacy of VYX0002 against VRSA [NR-46410] in mouse model of neutropenic septicemia. The results indicated that intravenous VYX0002 at 5 mg/kg, 3 doses, q5min showed significant antibacterial activity on VRSA [NR-46410] at 4, 6 and 8h when compared to the 2h control. Intravenous single dose of vancomycin was not significantly active at any of the time points.
Example 8: Analysis of dose response efficacy of single sub-cutaneous administration of VYX0002 on MRSA [USA300] in a neutropenic septicemia mouse model
[00143] The dose response efficacy of single sub-cutaneous administration of VYX0002 on MRSA [USA300] is analyzed in mouse model of neutropenic septicemia. Female CD-1 mice aged 6-8 weeks were neutropenised with cyclophosphamide and infected intra-peritoneally with 0.5 ml of Staphylococcus aureus MRSA (USA300) ~5 x 108 CFU/animal. Two hours post infection, groups of mice were treated intravenously with vehicle, intravenously with 110 mg/kg, single dose vancomycin and subcutaneously with single doses of VYX0002 (25, 50 and 75 mg/kg). VYX0002 was administered as a sodium salt (VYX0004) and the vehicle was sterile MilliQ water. Blood samples were drawn at different time points and bacteria were enumerated. The duration of the study was eight hours and the clinical signs and percent mortality were recorded. Mean ± SD Log10CFU/ml blood was estimated at the indicated time points for each group. Significant differences between treatment group means were assessed using a one way ANOVA, followed by a Dunnett’s multiple comparison test.
[00144] FIGURE 18 illustrates the dose response efficacy of single sub-cutaneous administration of VYX0002 on MRSA [USA300] in a neutropenic septicemia mouse model. The results indicated that VYX0002 exhibited antibacterial activity on MRSA [USA300] when compared to the 2h control at 6h PI and 8 h PI when dosed SC at doses 75 mg/kg, 50 mg/kg and 25 mg/kg.
Example 9: Analysis of efficacy of VYX002 against Clostridium difficile (ATCC43255) in Hamster infection model
[00145] The efficacy of VYX002 against Clostridium difficile (ATCC43255) in Hamster infection model. Male Golden Syrian hamsters were pretreated 24 h prior to infection with a single subcutaneous injection of clindamycin at 10 mg/kg. On the day of infection (day 0), animals were inoculated by oral gavage with approximately 106 C. difficile (ATCC43255) vegetative cells per hamster. Saline, 20 mg/kg vancomycin and VYX0002 at doses of 30, 100, 300mg/kg was administered orally beginning 24 h after infection. Treatment continued once daily, for a total of 2 days. On Day 2, all surviving animals were euthanized by CO2 inhalation and used for sampling of cecal contents. These samples were tenfold serially diluted with phosphate-buffered saline (PBS) and subjected to enumeration of Clostridial spore counts.
[00146] FIGURE 19 illustrates the efficacy of VYX002 against Clostridium difficile (ATCC43255) in Hamster infection model. The results indicated that VYX0002 showed significant dose dependent anti-Clostridial effect when compared to the vehicle control (p<0.05) in the Hamster infection model.
Example 10: Effects of VYX008 formulation on Clostridium perfringens (WAL-14572 HM-310) induced Necrotic enteritis in Broilers.
[00147] VYX0008 formulation: VYX0008 is formulated into calcium anacardate for analyzing the antibacterial effect in poultry. VYX0008 was finely powdered and 5-95% VYX0008 is mixed well with a carrier such as soya or silica. The ratio of carrier to VYX0008 is in the range of 8:1 to 2:1 (carrier: salt). The carrier and the salt were thoroughly mixed using a mechanical blender and finally mixed as a feed additive in the final feed formulation. This formulation was used for entire trial.
[00148] FIGURE 20 illustrates the efficacy of VYX008 against Clostridium perfringens (WAL-14572 HM-310) induced necrotic enteritis in Broilers. A total of 1800 broiler chicks (COBB) were divided into three groups comprising a basal diet without additive (Normal Control group), a basal diet with standard antibiotic (which is Standard of care) and a basal diet with VYX0008. A fresh Clostridium perfringens (WAL-14572 HM-310) inoculum was prepared daily on d 18, 19, and 20. Thus, on three consecutive days (d 18, 19, and 20), a round-tipped animal feeding needle (15 G, 78 mm; Solomon Scientific, PA, USA) attached to a syringe was used to orally deliver 1 mL(1x103CFU/mL) of the freshly prepared Clostridium perfringens (WAL-14572 HM-310) inoculum into the crop of each chick. The parameters such as daily Feed in/out and weekly body weight were recorded for computation of FCR and daily weight gain. After 35 days, the broilers were weighed and slaughtered. 10 birds from each group were randomly selected for blood sampling and caecal contents were collected for assessment of natural defence activity and enumeration of colony forming units.
[00149] The results indicated that VYX0008 exhibited significant anti-Clostridial (C. Perfringens) effect when compared to normal control (p<0.05) in necrotic enteritis induced broiler chickens. FCR improved by 6.6% when compared to standard of care and 11% when compared to groups without antibiotic feed. The feeding trial showed a distinctly higher activity of defence cells under the influence of the phytogenic feed additive VYX0008. Thus, VYX008 not only improved performance in broilers but also contributes to a strong immune response reducing the infections and the stress. Phytogenic feed additive VYX0008 has a beneficial and significant effect on the Natural defence activity of broilers. In addition, VYX0008 demonstrated its ability to improve growth performance in terms of weight gain and FCR. VYX0008 therefore has the potential to contribute to a healthy and robust immune defence system as well as improve performance in the poultry birds. VYX0008 is effective alternatice to antibiotic growth promoter, which is used as a feed additive in poutry.
[00150] The invention discloses a cold extraction process of a plant product by preserving the active constituents of the extract and further using the plant product to demonstrate in vitro and in vivo antimicrobial effects in animal models of infection. The antimicrobial activity in vitro and in vivo against many Gram positive and Gram negative bacteria of the plant extracts clearly shows its potential for use in the treatment of infections in humans and farm animals.

,CLAIMS:We Claim,

1. A cold solvent extraction process of phytochemicals from Cashew Nut Shells (CNS), the process (100) comprising the steps of:
a. crushing, 1000g to 3000 kgs of the cashew nut shells using suitable mechanical device such as hammer pulveriser (101);
b. soaking the crushed cashew nut shells in a large solvent tank containing solvents such as acetone, methanol, ethyl acetate hexanes or cyclohexanes in 1:3-1:12 ratios of crushed sheel:solvent at room temperature for 24-48 hours (102);
c. separating the solvent mixture from the residual cashew nut shells by filtration technique such as decanting or filtration using a device such as dume or a bag filter (103);
d. distilling the separated solvents at a temperature in the range between below 500C under vacuum using a vacuum pump and a chilled condenser for maximum recovery of the solvents for repetition of steps (102)-(104), (104);
e. recycling the recovered solvent for repetition of steps (102) to (104) at least thrice to extract Cashew Nut Shell Liquid (CNSL) from CNS (105);
f. treating the crude residual CNSL oil obtained in step (105) with 1%-5% activated charcoal and subjecting to filtration using sparkler filters (106);
g. subjecting the filtered solvent comprising the extract to evaporation under vacuum and drying the residual material to obtain a crude CNSL extract referred as VYX0007 with a yield 25% to 40% (107);
h. the extracted CNSL is dissolved in a protic polar solvent preferably in an alcoholic solvent at a concentration in the ratio 1:3 and 1:12; and adding suitable metal hydroxide and heated below 550C for 2-18h to get salt VYX0008 (108);
i. adding powdered metal hydroxide such as sodium hydroxide, potassium hydroxide or calcium hydroxide in portions over a period of 30-60 min to the solution of CNSL in protic polar solvent, in the concentration in the ratio 1:0.15 to 1:0.55 (109);
j. stirring the reaction mixture at a temperature in the range between 700C for a duration between 2 hr to 18 hrs to allow salt formation (110);
k. cooling the reaction mixture to obtain the precipitated salt (111);
l. filtering the precipitated salt and washing with one or more solvents such as methanol, ethyl acetate, toluene or 2 propanol alone or in combination in the ratio ranging between 1:3 to 1:6 to obtain a free flowing solid (112);
m. vacuum drying or spin-drying the solids to obtain salt mixture of anacardic acids as VYX0008 (113);
n. treating the salt mixture of anacardic acid VYX0008 in with mineral acid preferably 6N hydrochloric acid, concentrated hydrochloric acid at pH in the range between 1-2, after 2-6 h of continued stirring, extracted with organic solvent and usual work-up yielded VYX0009 (114);
o. dissolving the mixture of anacardic acids (VYX0009) or the CNSL extract (VYX0007) in a protic solvent preferably in alcoholic solvent in the ratio 1:10 v/v (115); and
p. subjecting the solution to catalytic hydrogenation under hydrogen pressure of around 4-5 kg, in presence of a 5% to 10% palladium catalyst at room temperature for a duration of 24 h to yield completely saturated and pure VYX0002 (116).

2. The cold solvent extraction process as claimed in claim 1, wherein the recovered solvent is subjected to recycling for repetition of step (102) to (104) at least thrice to ensure extraction of CNSL efficiently from the cashew nut shells such that the cost of extraction is reduced.

3. The cold solvent extraction process as claimed in claim 1, wherein the process resulted in extraction of VYX0007 as crude form with a mixtures of phenolic compounds, VYX0008 as salt of anacardic acid mixtures, VYX0009 as oil comprising the mixture of anacardic acids and VYX0002 a single component which is anacardic acid in addition to saturated cardol as VYX0015 and saturated cardanol as VYX0014.

4. Anacardic acid VYX0002 isolated from a cold solvent extraction process is formulated into a sodium salt as VYX0004 to enhance its aqueous solubility and VYX0008 isolated from a cold solvent extraction process, is formulated as calcium salt of mixture of anacardic acids

5. VYX0002 as claimed in Claim 4, wherein VYX0002 exhibited one or more properties such as Absorption, Distribution, Metabolism and Excretion (ADME) and bidirectional permeability in hepatocyte cell line and pharmacokinetics after oral and intravenous administration in mouse .

6. VYX0002 as claimed in Claim 4, wherein VYX0002 exhibited plasma protein binding in mouse and human plasma and in vitro metabolic stability in mouse and human liver microsomes.

7. VYX0002 as claimed in Claim 4, wherein VYX0002 or the salt forms such as VYX0008 or VYX0009 or VYX0004, exhibited antibacterial activity against Propionibacterium acnes (MTCC1951) in a mouse model of ear infection and skin infection, MRSA (USA300) in a murine model of bacteremia and neutropenic septicemia, MRSA (USA300, 43300) and VRSA (NR-46410) in a mouse model of non-neutropenic septicaemia, Clostridium difficile (ATCC43255) in Hamster infection model.

8. VYX0007 as claimed in Claim 3, wherein VYX0007 formulated into calcium anacardate improved growth performance and enhanced immune response in Clostridium perfringens induced necrotic enteritis in Broiler chicken thus suggesting as alternative feed additive in poultry.