FIELD OF INVENTION:
[001] The present invention relates to the field of antibacterial and antifungal formulation. The present invention in particular relates to a an antibacterial and antifungal formulation using grape seed extract and pomegranate peel extract and its method of preparation.
DESCRIPTION OF THE RELATED ART:
[002] Grape seed is a waste by-product of wine or juice making and a good source of functional compounds, such as polyphenols, which have broad potential applications as antioxidants. For example, grape seed proanthocyanidins were found to possess cardioprotective and nephroprotective abilities and can directly scavenge reactive oxygen species (ROS), including hydroxyl and peroxyl radicals.
[003] Also Constitutes of pomegranate have been studied for their antiviral and antifungal effects. For example, U.S. Pat. No. 5,840,308 (61) describes an antiviral and antifungal composition comprising a mixture of a ferrous salt and an extract of a plant including, inter alia, pomegranate rind. U.S. Pat. No. 5,411,733 (32) describes an antiviral agent containing a crude drug from, inter alia, the root bark and fruit peel of pomegranate.
[004] Reference may be made to the following:
[005] Patent No. US10542758 relates to the methanol extract of grape seed nanoparticles is prepared from grape seeds washed in distilled water and oven-dried at 60° C. for 12 hours. The seeds are milled or ground to a powder and sieved to a maximum size of 0.355 mm. The powder is added to concentrated HCl and stirred at 3000 rpm at 30° C. for one hour, and then distilled water is added with stirring for an additional 2 hours. The mixture is filtered, and the marc is dried to recover grape seed nanoparticles. The nanoparticles are added to methanol at the rate of 100 mg/ml, left in a shaker for 24 hours at room temperature, centrifuged, filtered, and the resulting extract (the supernatant) is recovered. Agar well diffusion testing showed that the nanoparticle extract exhibited greater antibacterial activity than a methanol extract of grape seeds alone, and testing showed greater antioxidant levels in the nanoparticle extract as well whereas in present invention 200 mg of Pomegranate peel powder was mixed with 1000ml of ethanol. The mixture was kept in a magnetic stirrer maintained at 50oC for 8hrs and stirred at 200 rpm.
[006] Patent No. US9061049 has found the topical, over the counter, all natural, organic, antifungal, antibacterial cream that eliminates and prevents the growth of the rash-causing fungus whereas in the present study the antifungal and antibacterial effect of combination of grape seed extract and pomegranate peel extract are used. This was then used against Staphylococcus aureus and Candida albicans that were obtained after isolation of same from the intaoral swab of patients with Periimplantitis.
[007] Patent No. US6375993 has provided, an antioxidative composition comprising an extract from pomegranate. A method of reducing lipid peroxidation, aggregation or retention, HDL oxidation in a sample and a method of alleviating atherosclerosis in a patient whereas the present study deals with the antibacterial and antifungal property of pomegranate peel extract against Staphylococcus aureus and Candida albicans (organisms causing periimplantitis in dental implants).
[008] Publication No. US5840308 relates to antiviral and antifungal compositions comprising a mixture of a ferrous salt and a plant extract of pomegranate rind, Viburnum plicatum leaves or flowers, tea leaves, or maple leaves in an aqueous solution are disclosed. The compositions are mainly used to prevent the growth of, or kill, viruses or fungi on surfaces. The compositions do not substantially affect bacterial viability as measured by colony forming ability whereas in present study combination of ethanolic extract of grape seed and pomegranate peel was used to prevent the growth of Staphylococcus aureus and Candida albicans (obtained from patients with Periimplantitis).The combination showed significant antibacterial and antifungal effect.
[009] Publication No. US8734867 relates to a method and a composition for producing and using a plant-based biocidal solution. The plant-based biocidal solution contains a bioactive material and a plant-based substance formed from the cellular material of a plant. The plant-based substance is capable of binding to the bioactive material. In some embodiments, the bioactive material is hydrogen peroxide. The hydrogen peroxide can be added exogenously or generated endogenously. In accordance with further embodiments, the plant-based biocidal solution can be applied to a target, thereby impairing the target. In some embodiments, the target can be a pathogen. In accordance with another embodiment, the plant-based substance of the plant-based biocidal solution can form a microscopic cluster, a complex, or an aggregate for providing sufficient bioactive material to overcome the defense mechanism of the target whereas in present study combination of ethanolic extract of grape seed and pomegranate peel was used .The antibacterial and antifungal property can be contributed to the presence of substantial amount of phenolic compounds , including flavonoids (anthocyanins, catechins and other complex flavonoids) and hydrolyzable tannins (punicalin, pedunculagin, punicalagin, gallic and ellagic acid). The antibacterial phenolic contents present in grape seed are partially hydrophobic, they are considered to interact with the bacterial cell wall and lipopolysaccharide interfaces by decreasing the membrane stability. This can be taken in accordance with antibacterial and antifungal property of grape seed extract.
[010] Reference may be made to an article entitled “Investigation of different interactions between Staphylococcus aureus phages and pomegranate peel, grape seed, and black cumin extracts” by Emine K. Tayyarcan, Esra Acar Soykut, Ozay Mentes Yilmaz, Ismail H. Boyaci, Maha Khaaladi, Sami Fattouch; Journal of Food Safety; 09 July 2019. The present study was focused on antibacterial and antifungal property of combination of ethanolic extract of pomegranate peel and grape seed extract. This extract combination was used in three different concentrations 1.25mg/ml, 25mg/ml, 125mg/ml. This combination showed significant effect against Staphylococcus aureus and Candida albicans (Isolated from intraoral swab obtained from patients with Periimplantitis surrounding dental implants).This combination can be made commercially available in form of gel, paste etc to be used in the treatment of Periimplantitis.
[011] Reference may be made to an “Bactericidal effect of grape seed extract on methicillin-resistant Staphylococcus aureus (MRSA)” by Aamar Al-Habib, Esmaeil Al-Saleh, Abdel-Majeed Safer, Mohammad Afzal; The Journal of Toxicological Sciences Volume 35, Issue 3; 2010. In the present study commercially available grape seed extract were obtained and it was dried. 200 mg of grape seed powder was mixed with 1000ml of ethanol. The mixture was kept in a magnetic stirrer maintained at 50oC for 8hrs and stirred at 200 rpm. Following this, the extract was filtered through Whatmann No. 1 filter paper and filtrate was evaporated to dryness in water bath. The ethanolic extract was weighed and reconstituted in ethanol to give final concentration of 1.25mg/mL, 25mg/ml and 125mg/ml. Same procedure was repeated for pomegranate peel extract. 25microlitre of each of the extracts in three concentrations were combinated in 1:1ratio to prepare solutions of combined extract in exact concentrations of 1.25mg/ml, 25mg/ml, 125mg/ml. This combination is then used to check for antibacterial and antifungal effect against Staphylococcus aureus and Candida albicans(isolated from patient’s swab) respectively using well diffusion method.
[012] Reference may be made to the article entitled “Antioxidant and antibacterial potential of pomegranate peel extracts” by Shalini Malviya, Arvind, Alok Jha, Navam Hettiarachchy; J Food Sci Technol. ; 51(12): 4132–4137; 2013 Feb 22. In the present study ethanolic extract of pomegranate peel was used. This was prepared by obtaining commercially available pomegranate fruits and their peel was dried. 200 mg of Pomegranate peel powder was mixed with 1000ml of ethanol. The mixture was kept in a magnetic stirrer maintained at 50oC for 8hrs and stirred at 200 rpm. Following this, the extract was filtered through Whatmann No. 1 filter paper and filtrate was evaporated to dryness in water bath. The ethanolic extract was weighed and reconstituted in ethanol to give final concentration of 1.25mg/mL, 25mg/ml and 125mg/ml. This was combined with grape seed extract (prepared in similar manner).Pomegranate was not used alone rather combination of pomegranate peel and grape seed extract was used in concentration of1.25mg/mL ,25mg/ml and 125mg/ml. to check the antibacterial and antifungal effect against Staphylococcus aureus and Candida albicans.
[013] Reference may be made to the article entitled “A study on antibacterial effect of grape seed extracts in common clinical and drug resistant isolates” by Mohanakrishnan Kandasamy, Sowmya Nasimuddin, Jeevan Malayan, Nithyalakshmi J, Sumathi Gnanadesikan, Mayuri Chandrasekar; International Journal of Clinical Trials, Vol 3, No 3; 2016. In the present study grape seed was not used alone rather it was used in a combined extract form with pomegranate peel extract. This combination was used in concentration of 1.25mg/mL,25mg/ml and 125mg/ml. The mean zone of inhibition showing antibacterial property ranged from 9.63mm-14.80mm and the mean zone of inbition showing antifungal property ranged from 9.07mm-13.93mm. Maximum zone of inbition was seen by 125mg/ml of extract combination. So by increasing the concentration both the antifungal and antibacterial effects can be increased.
[014] Reference may be made to an article entitled “Antibacterial activity of a grape seed extract and its fractions against Campylobacter spp.” by Jose Manuel Silván, Elisa Mingo, Maria Hidalgo, Sonia de Pascual-Teresa, Alfonso V.Carrascosa, Adolfo J.Martinez-Rodriguez; Food Control Volume 29, Issue 1, Pages 25-31; 23 January 2012. In the present study grape seed was not used alone rather it was used in a combined extract form with pomegranate peel extract. This combination was used in concentration of 1.25mg/ml,25mg/ml and 125mg/ml. The combination had both antibacterial and antifungal that can be because of the presence of substantial amount of phenolic compounds, including flavonoids (anthocyanins, catechins and other complex flavonoids) and hydrolyzable tannins (punicalin, pedunculagin, punicalagin, gallic and ellagic acid). The antibacterial phenolic contents present in grape seed are partially hydrophobic, they are considered to interact with the bacterial cell wall and lipopolysaccharide interfaces by decreasing the membrane stability.
[015] Reference may be made to an article entitled “The pomegranate: effects on bacteria and viruses that influence human health” by Amy B. Howell ; Doris H. D'Souza ; Evid Based Complement Alternat Med.; 606212.; 2013. In the present study the antibacterial and antifungal property of extract is considered and to enhance the effect; an ethanolic combination of grape seed extract and pomegranate peel extract was used. This combination was used exclusively on the Staphylococcus aureus and Candida albicans that was obtained from patients who had periimplantitis surrounding dental implants.
[016] Reference may be made to an article entitled “Antibacterial and antifungal activities of punica granatum peel extracts against oral pathogens” by Sh. Abdollahzadeh, RY. Mashouf, H. Mortazavi, MH. Moghaddam, N. Roozbahani, and M. Vahedi; J Dent (Tehran). 2011 Winter; 8(1): 1–6; 2011 Mar 31. In the present study ethanolic extract of pomegranate peel was used in combination with ethanolic extract of grape seed. The combination showed both antibacterial and antifungal effect against Staphylococcus aureus and Candida albicans respectively.
[017] Reference may be made to an article entitled “Studies on antibacterial and antifungal activity of pomegranate (Punica granatum L.)” by Saad Sabbar Dahham, Mir Naiman Ali, Hajera Tabassum and Mazharuddin Khan; American-Eurasian J. Agric. & Environ. Sci., 9 (3): 273-281, 2010. The present study ethanolic extract of pomegranate peel was used in combination with ethanolic extract of grape seed. This combination was used exclusively on the Staphylococcus aureus and Candida albicans that was obtained from patients who had periimplantitis surrounding dental implants.
[018] Various forms of bacteria, including both Gram-negative and Gram-positive bacteria, have been implicated in food poisoning and other forms of bacterial contamination. While antibacterial agents are known, various strains of bacteria have developed resistance to some conventional antibacterial agents, and there is always a perennial effort to find ways of increasing the strength and effectiveness of antibacterial agents.
[019] In order to overcome above listed prior art, the present invention aims to provide an antibacterial and antifungal formulation using grape seed extract and pomegranate peel extract and its method of preparation.
OBJECTS OF THE INVENTION:
[020] The principal object of the present invention is to provide an antibacterial and antifungal formulation using grape seed extract and pomegranate peel extract and its method of preparation.
[021] Another object of the present invention is to provide an antibacterial and antifungal formulation which has improved efficacy.
SUMMARY OF THE INVENTION
[022] The present invention relates to an antibacterial and antifungal formulation which has improved efficacy and its method of preparation. The grape seed extract and pomegranate peel extract combined in the ratio of 1:1 in 1.25mg/ml, 25mg/ml, 125mg/ml concentrations on growth of Staphylococcus aureus and Candida albicans.
[023] Combination of pomegranate seed extract and grape seed extract has synergistic effect. This effect can be attributed to the fact that almost 26–30% of total fruit weight is comprised of the peel of pomegranate. Pomegranate peels are characterized by an interior network of membranes and is characterized by substantial amount of phenolic compounds, including flavonoids (anthocyanins, catechins and other complex flavonoids) and hydrolyzable tannins (punicalin, pedunculagin, punicalagin, gallic and ellagic acid). This accounts for both antibacterial and antifungal properties of Punica granatum peel. Similarly phenolic contents present in grape seed are partially hydrophobic, they are considered to interact with the bacterial cell wall and lipopolysaccharide interfaces by decreasing the membrane stability. This leads to the antibacterial and antifungal effect of grape seed extract.
DETAILED DESCRIPTION OF THE INVENTION:
[024] The present invention relates to an antibacterial and antifungal formulation which has improved efficacy and its method of preparation. The grape seed extract and pomegranate peel extract combined in the ratio of 1:1 in 1.25mg/ml, 25mg/ml, 125mg/ml concentrations on growth of Staphylococcus aureus and Candida albicans. The method includes following steps:
[025] 200 mg of Pomegranate peel powder was mixed with 1000ml of ethanol. The mixture was kept in a magnetic stirrer maintained at 50oC for 8hrs and stirred at 200 rpm. Following this, the extract was filtered through Whatmann No. 1 filter paper and filtrate was evaporated to dryness in water bath. The ethanolic extract was weighed and reconstituted in ethanol to give final concentration of 1.25mg/mL, 25mg/ml and 125mg/ml. Same procedure was repeated for grape seed extract. 25microlitre of each of the extracts in three concentrations were combinated in 1:1ratio to prepare solutions of combined extract in exact concentrations of 1.25mg/ml, 25mg/ml, 125mg/ml This combination of extract is then used to check the antibacterial and antifungal effect using well-diffusion method onStaphylococcus aureus and Candida albicans ( obtained from patients with Periimplantitis)respectively.
[026] ANTIBACTERIAL EFFECT OF EXTRACT SOLUTION ON S.auerus.
[027] ISOLATION OF S.auerus
[028] Swab was plated on Blood agar medium. Culture plate was incubated for 24 hours at 370C. Coagulase test was done to isolate S.auerusfrom culture. Inoculum suspension was prepared from isolated organism.
[029] PREPARATION OF INOCULUM SUSPENSION
[030] S.aureuswas grown on Mannitol salt broth and incubated in aerobic chamber for 24hr at 35°C.Microorganism was transferred to distilled water and microbial suspension was prepared. Turbidity of cell suspensions was adjusted to 1-2x 10 5 cells/ml.
[031] WELL DIFFUSION METHOD FOR EVALUATING ANTIBACTERIAL EFFICACY-
[032] Dilution of prepared ethanolic extract solution (combination of pomegranate peel extract and grape seed extract in ratio 1:1) was performed to get exact concentrations of 1.25 mg/ml, 25mg/ml and 125 mg/ml. The obtained inoculum containing S.aureuswas spread on Soyabean casein digest agar plates. Four wells measuring 5mm in depth were made on agar plates. Extract solutions of different concentrations were added in three wells. Erythromycin was added in fourth well as positive control.
[033] This culture plate was then incubated at 350C for 24-48 hrs. The plate was observed for zones of inhibition around the wells. Zones of inhibition were measured in mm.
[034] ANTIFUNGAL EFFECT OF EXTRACT SOLUTION ON C.albicans
[035] ISOLATION OF C.ALBICANS
[036] Swab was plated on Blood agar medium. Culture plate was incubated for 24 hrs at 370C on SDA. Coagulase test was done to isolate C.albicans from culture. Inoculum suspension was prepared from isolated organism.
[037] PREPARATION OF INOCULUM SUSPENSION-
[038] C.albicanswas grown on SDA and incubated for 24hr at 35°C.Microorganism was transferred to distilled water and microbial suspension was prepared. Turbidity of cell suspensions was adjusted to 1-2x 10 5 cells/ml.
[039] WELL DIFFUSION METHOD FOR EVALUATING ANTIFUNGAL EFFICACY-
[040] Dilution of prepared ethanolic extract solution (combination of pomegranate peel extract and grape seed extract in ratio 1:1) was performed to get exact concentrations of 1.25 mg/ml, 25mg/ml and 125 mg/ml.). The obtained inoculum containing C.albicans was spread on Potato dextrose agar plates. Four wells measuring 5mm in depth were made on agar plates. Extract solutions of different concentrations were added in three wells. Itraconazole was added in fourth well as positive control.
[041] This culture plate was then incubated at 350C for 24-48 hrs. The plate was observed for zones of inhibition around the wells. Zones of inhibition were measured in mm.
[042] The obtained inoculum containing S.aureus was spread on Soyabean casein digest agar plates. Four wells measuring 5mm in depth were made on agar plates. Extract solutions of different concentrations were added in three wells. Erythromycin was added in fourth well as positive control.
[043] This culture plate was then incubated at 350C for 24-48 hrs. The plate was observed for zones of inhibition around the wells. Zones of inhibition were measured in mm.
[044] Combination of pomegranate seed extract and grape seed extract has synergistic effect. This effect can be attributed to the fact that almost 26–30% of total fruit weight is comprised of the peel of pomegranate. Pomegranate peels are characterized by an interior network of membranes and is characterized by substantial amount of phenolic compounds, including flavonoids (anthocyanins, catechins and other complex flavonoids) and hydrolyzable tannins (punicalin, pedunculagin, punicalagin, gallic and ellagic acid). This accounts for both antibacterial and antifungal properties of Punica granatum peel. Similarly phenolic contents present in grape seed are partially hydrophobic, they are considered to interact with the bacterial cell wall and lipopolysaccharide interfaces by decreasing the membrane stability. This leads to the antibacterial and antifungal effect of grape seed extract.
[045] Both grape seed extract and pomegranate peel extract show antibacterial and antifungal effect individually so on combination of these two there is additive effect shown by the contents of both especially by the phenolic content. Therefore, the combination of both show more antibacterial and antifungal effect even at lower concentrations.
[046] This has an in-vivo and an in-vitro component. In-vivo includes swab collection from selected patients with periimplantitis. The in-vitro analysis was performed by checking the antibacterial and antifungal property of combination of pomegranate peel extract and grape seed extract (1:1) on Staphylococcus .aureus and Candida.albicans (organisms causing periimplantitis). A total number of 15 samples. Patients with implant prosthesis and periimplantitis for more than six months were selected . Intraoral examination of selected patients showed inflammation, bleeding on probing, pocket formation and bone loss around the implant site. Immunocompromised patients, patients with failed implants , and with full mouth implants prosthesis were excluded from the analysis.
[047] COLLECTION OF SAMPLE (SWAB COLLECTION)-
[048] A total patient with Periimplantitis was made to sit on a dental chair. Personal protective equipment (PPE) were used. The area near the culture plate was sterilized with heat using Bunsen burner and then scrubbed with surgical spirit .Swab pouch was opened and inserted into the side of gingiva affected with Periimplantitis and rubbed in circular motion . It was placed into vacutainer that contained sterile normal saline. Vacutainer was wiped using a disposable paper towel. Sample was protected from breakage/ leakage during transportation by wrapping the collection tube in a paper towel. Vacutainer was wrapped and transported in a plastic leak proof packaging container to the laboratory.
[049] PREPARATION OF EXTRACTS:
[050] Commercially available Pomegranate fruits were obtained and their peel was dried. 200 mg of Pomegranate peel powder was mixed with 1000ml of ethanol. The mixture was kept in a magnetic stirrer maintained at 50oC for 8 hrs and stirred at 200 rpm. Following this, the extract was filtered through Whatmann No. 1 filter paper and filtrate was evaporated to dryness in water bath. The ethanolic extract was weighed and reconstituted in ethanol to give final concentration of 1.25mg/mL, 25mg/ml and 125mg/ml. Same procedure was repeated for grape seed extract.
[051] ANTIBACTERIAL EFFECT OF EXTRACT SOLUTION ON S.auerus
[052] Isolation of S.auerus- Swab was plated on Blood agar medium. Culture plate was incubated for 24 hours at 370C. Coagulase test was done to isolate S.auerus from culture. Inoculum suspension was prepared from isolated organism.
[053] Preparation of inoculum suspension
[054] S.aureus was grown on Mannitol salt broth and incubated in aerobic chamber for 24hr at 35°C.Microorganism was transferred to distilled water and microbial suspension was prepared. Turbidity of cell suspensions was adjusted to 1-2x 10 5 cells/ml.
[055] Well diffusion method for evaluating antibacterial efficacy-
[056] Dilution of prepared ethanolic extract solution (combination of pomegranate peel extract and grape seed extract in ratio 1:1) was performed to get exact concentrations of 1.25 mg/ml, 25mg/ml and 125 mg/ml. The obtained inoculum containing S.aureus was spread on Soyabean casein digest agar plates. Four wells measuring 5mm in depth were made on agar plates .Extract solutions of different concentrations were added in three wells. Erythromycin was added in fourth well as positive control. This culture plate was then incubated at 350C for 24-48 hrs. The plate was observed for zones of inhibition around the wells. Zones of inhibition were measured in mm.
[057] ANTIFUNGAL EFFECT OF EXTRACT SOLUTION ON C.albicans.
[058] Isolation of C.albicans - Swab was plated on Blood agar medium. Culture plate was incubated for 24 hrs at 370C on SDA. Coagulase test was done to isolate C.albicans from culture. Inoculum suspension was prepared from isolated organism.
[059] Preparation of inoculum suspension- C.albicans was grown on SDA and incubated for 24hr at 35°C.Microorganism was transferred to distilled water and microbial suspension was prepared. Turbidity of cell suspensions was adjusted to 1-2x 10 5 cells/ml.
[060] Well diffusion method for evaluating antifungal efficacy-
[061] Dilution of prepared ethanolic extract solution (combination of pomegranate peel extract and grape seed extract in ratio 1:1) was performed to get exact concentrations of 1.25 mg/ml, 25mg/ml and 125 mg/ml.) . The obtained inoculum containing C.albicans was spread on Potato dextrose agar plates. Four wells measuring 5mm in depth were made on agar plates. Extract solutions of different concentrations were added in three wells. Itraconazole was added in fourth well as positive control. This culture plate was then incubated at 350C for 24-48 hrs. The plate was observed for zones of inhibition around the wells. Zones of inhibition were measured in mm

WE CLAIM

1. An antibacterial and antifungal formulation with improved efficacy comprises grape seed extract and pomegranate peel extract combined in the ratio of 1:1 in 1.25mg/ml, 25mg/ml, 125mg/ml concentrations on growth of Staphylococcus aureus and Candida albicans.
2. The antibacterial and antifungal formulation with improved efficacy, as claimed in claim 1, wherein the method of preparation includes following steps-
a) Peel of pomegranate fruits were dried and 200 mg of Pomegranate peel powder was mixed with 1000ml of ethanol.
b) The mixture was kept in a magnetic stirrer maintained at 50oC for 8 hrs and stirred at 200 rpm.
c) Following this, the extract was filtered through Whatmann No. 1 filter paper and filtrate was evaporated to dryness in water bath.
d) The ethanolic extract was weighed and reconstituted in ethanol to give final concentration of 1.25mg/mL, 25mg/ml and 125mg/ml.
e) Same procedure was repeated for grape seed extract.