AU2021106840A4 - Utilization of Lactobacillus rhamnosus ARJD granules for anti-colon cancer and against different bowel disorders - Google Patents

Abstract

Utilization of Lactobacillus rhamnosus ARJD granules for anti-colon cancer andagainst different bowel disorders. This invention formulates a new granule formulation containing Lactobacillus rhamnosus ARJD isolated from Indian sheep milk with antimicrobial, antioxidant and anti-colon cancer activity. The granule is prepared using the maltodextrin and starch as excipients by spray dry technique. It is formulated as dry granule with long shelf life in both room temperature and refrigeration (more viability). It is non-toxic, easy to prepare and can be used as nutraceutical mediators against different bowel diseases. The normal fermentation technique is used for the growth of lactobacillus in the natural buffalo milk as natural media. The prepared granule is palatable which enhances its various probiotics properties by adhesion, gastrointestinal tract pH and bile salt tolerance. The prepared granules was found to be cost-effective and producedin less time by spray dry method. SIGNATORY Dr. Abhinadan R Patil Mr. Ravsaheb Patil Dr. Priyanka Patil Mr. Amit Patil Dr. Shivaji Pawar Mr. Amrut Patil Ms. Sohili Patil Ms. Pallavi Patil Ms. Sujata Patil

Description

EDITORIAL NOTE 2021106840

There are 14 pages of description.

Title Utilization of Lactobacillus rhamnosus ARJD granules for anti-colon cancer and against different bowel disorders

FIELD OF INVENTION

[0001] This invention relates to the field of neutraceutical and medical sciences more particularly development of granules from new isolated strain of Lactobacillus rhamnosus preventing and curing colon cancer; acting as nutraceutical mediators.

[0002] Here the granules of new isolated strain of Lactobacillus rhamnosus ARJD isolated from Indian sheep milk were prepared from maltodextrin and starch by spary dry technique. The isolated strain exhibited probiotic properties like adhesion, gastrointestinal tract pH and bile salt tolerance. The prepared granules was found to be non-toxic, cost-effective and produced in less time by spray dry method.

PRIOR ART AND PROBLEM TO BE SOLVED

[0003] Cancer is considered as the disorder of the cell in which the natural death (apoptotic) pathway of the cell cycle is lost resulting in uncontrolled growth. The World health organization (WHO) has declared cancer as the second leading cause of death universally with a mortality rate of 9.5 million people's in only 2018. The report further says that 1 in 6 deaths reported globally is due to cancer, in which 70% of deaths are from low- and middle-income countries. The main reason behind all these death are the 5 leading dietary and behavioral risks which include obesity, low intake of fruit and vegetable, sluggish physical activity, alcohol, and tobacco consumption. Colorectal is the leading third most cancer in which nearly 8, 62, 000 deaths were reported in 2018. To cope up with this problem, few new synthetic medicines are discovered after several years of clinical trials and many are discarded at the last phase of trials due to host-drug incompatibility issues. The treatments of colorectal cancer are mostly given by the systematic route rather than the local action at the site of the disorder. This results in the systematic toxicity and adverse effects on vital organs. So there is a need of the drug that can be delivered by per-oral route with its local action, minimizing the systemic toxicity controlling morbidity and preventing the mortality.

[0004] Use of synthetic drugs are coming with many side effects and not fall under patient comfort zone. Thus there is a need of therapeutic mediators that can be given easily to patients with nearly zero side effects and act as nutraceutical agent. Recent studies in relation to probiotics have proven that these microbes as living non-pathogenic microbiota after the administration in appropriate doses acts as antioxidant mediators preventing and curing many diseases and disorders. This microbiota shows different pharmacological and physiological activity that plays a vital role in the human immune system. Many of the chemicals released by the Lactobacillus strains acts like SOD (superoxide dismutase) which role-plays as antioxidant mediators. These microbes also chelate various metal ions during the digestion process. Lactobacillus casei proved to chelate Fe2+ or Cu2+, due to its antioxidant nature.

[0005] The Lactobacillus (LAB's) genera from the probiotics category are found in the milk of different animals. These microbes are used since a long period as a functional food to treat many gastrointestinal related disorders. LAB's is the most acceptable form of probiotics, obtained mostly from milk and milk products. These microbes act as a nutraceutical agent conferring health benefits. The LAB's such as Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus plantarum, etc., are being used by many dairy industries as the active starter cultures for many preparations including milk products and beverages.

[0006] Many researchers use milk of different animals and fruit juices as natural media to enhance the growth and survival of these bacteria. The organism isolated from the any other sources may show growth and viability in some other natural or synthetic media. Newly discovered probiotics showing curing action against the diseases should by-passing these stress conditions and showing the tolerance against it are real contributors in showing the desired pharmacological activities. The various properties of Lactobacillus i.e. its survival in the gastrointestinal tract is very important aspects. Various stress boosters are present in the gut region of the host that interact with the microbial flora. These stress conditions include the effects of different enzyme and enzymatic system. The major other factors affecting the growth and survival of the LAB are the bile salt secretion and the effects of the cellular immunity system inside the gut of the host.

[0007] The most concerning part of direct use of the probiotics is its short shelf life; as the formulations deteriorate by a changes in temperature during storage conditions. Many studies revealed that cfu (colony forming unit) per gram value above 108 counts are found to show the health benefits. But it is found that during the storage condition the cfu level falls down below the threshold, resulting in zero pharmacological activities. Thus, there is need of proper and optimised drying techniques to maintain the cfu/g of LAB, making suitable for long-term storage

[0008] To resolve the above stated problem, Lactobacillus rhamnosus is isolated from sheep milk as a source; designed in a dry granule formulation for enhancing the antibacterial, antioxidant and anticancer properties. The dry granules of probiotics are not showing any adverse effect on gastrointestinal tract in a concentration of 108 counts/gm cfu count. Due to good gastrointestinal mucosal binding and probiotics properties it is conferring good health benefits as nutraceutical agent and act as anticoloncancer agent.

THE OBJECTIVES OF THE INVENTION:

[0009] Gastrointestinal tract is the red carpet area for the many microbes living in it showing dysbiosis and symbiosis. The chemical base injury or alternation in the flora result in the disturbance giving invitation to many bowel disorder if not treated in time led to dreadful diseases including cancer. This problem can be prevented by inoculation of the probiotics with desired pharmacological activities.

[0010] It has already been proposed where treatments of colorectal cancer are mostly given by the systematic route rather than the local action at the site of the disorder. This results in the systematic toxicity and adverse effects on vital organs. So there is a need of the drug that can be delivered by per-oral route with its local action, minimizing the systemic toxicity controlling morbidity and preventing the mortality.

[0011] The principal objective of the invention is the use of strain of Lactobacillus rhamnosus to prevent and cure colon cancer; acting as nutraceutical mediators. Here the granules of the isolated strain of Lactobacillus rhamnosus ARJD isolated from Indian sheep milk prepared from maltodextrin and starch by spary dry technique. The isolated strain exhibited probiotic properties like adhesion, gastrointestinal tract pH and bile salt tolerance.

[0012] Another objective of the invention is to screen some additional probiotic properties. These properties such as cell adhesion, auto-aggregation, and hydrophobicity are found vital, to fight against pathogens. Cell adhesion of LAB is studied by many researchers using the fish intestine mucus model. Physiochemical abilities of cell surface are found vital in cell adhesion to the intestinal mucosa by means of hydrophobicity. This will keep the microbial flora intact and conferring additional nutraceutical benefits like health gut working.

[0013] The further objective of the invention is to use granules of lactobacillus as not only nutraceutical agent against different bowel disorder but also to treat and prevent the colon cancer at the early stage.

SUMMARY OF THE INVENTION

[0014] Probiotics are the basic source of the functional food used as 'yogurt, dahi' but come with the short shelf life. Functional foods are used as the alternative source of healing component not morphological but by the physiological pathways. These are used as the therapeutical agent along with the diet to reduce the risks of various diseases as a prophylactic agent. The most common diseases that are treated by neutraceutical agents are gastrointestinal diseases and buccal cavity disorders. The pharmacological activities that are observed in case of probiotics are mostly by antioxidant mode of actions without the liver damage and carcinogenesis. The antioxidant mechanisms of probiotics could be due to metal ion chelation, enzyme inhibition and free radical scavenging mechanism. The synthetic drugs that are of the chemical origin or synthesized in the lab precipitate toxicity after long use.

[0015] The probiotics especially Lactobacillus genera are extensively explored for a long time as part of the diet. The origin of these microbes is mostly from the milk and its derived products. Since birth, these microbes work together as part of the digestive system as mutualism mode of adaption in the gastrointestinal tract. The probiotics fight the dysbiosis conditions with the pathogenic strains in the lumen of the intestine. In extreme condition, the inoculation of the Lactobacillus is found effective in suppressing and destroying the disease-causing microbes i.e. pathogens. The probiotics include many different bacterial strains, each of these species differs in nature and activity. Most of the times strains of the same species are unique in characteristics such as adherence, immunological activity and other biological actions on the host. The major mechanism involved is the lowering of the pH in the colonic region, inhibiting the adhesion of the pathogenic strains to the intestinal lumen. Auto-aggregation and co-aggregation are the other two mechanisms that are inhibiting the colonization of pathogens. The hydrophobicity of the host-probiotics interaction also prevents various diarrheal diseases. Currently, probiotic research aims in the isolation of new strains and investigation of microbiota and microbiome characteristics in each part of the gastrointestinal tract from the small intestine to the colonic region. The basic focus of research includes the host-microbe interactions within the intestine, microbe-pathogen interactions within the microbiota and the microbe-drug interactions in diseased condition. The discovery of the new strains of Lactobacillus from the new natural source from the heavy crowd of the microbiota is a tricky and time-consuming process. The mechanism of action shown by the lactobacillus genera and it's sub-strains vary in its pharmacological activity. By the genotypic phylogeny study, all of Lactobacillus strains may fall under the same ancestral category but varies in the biochemical and physiological activities. This indicates that newly discovered strain of Lactobacillus may come with new pharmacological activity against different diseases. The whole genomic sequencing of newly discovered culture highlights the potential of the strain as a functional food in treating many dreadful disorders of the intestine. The positive interaction seen in all the above cases is mediated by means of the antagonistic activity of these probiotics.

[0016] Currently, in the market, there is a need for such nutraceutical products which may provide health benefits. Mostly the world is suffering from dreadful diseases like cancer. There are various types of cancer cases, in which colon cancer ranks third in India. The prophylaxis measures available for cancerous diseases are nearly zero as the etiology of the disease is unknown. However, the probiotics are proven useful in treating the diseases of the gastrointestinal tract such as diarrhoea and minor infection or manifestation by the pathogenic strains. Improper bowel habit is found responsible for causing the disease called adenomatous carcinoma a hybrid type of colorectal cancer. This gives the major clue that the Lactobacillus may be used as the major source to heal any kind of the disease including colonic cancer. The drawbacks of using the Lactobacillus as functional food is its short shelf-life. Further, it gets contaminated if kept in a liquid formulations mode. So the marketing of this product is not only a risk factor to the manufacturer but also costly due to its preservation parameters such as refrigeration. Few of the products are nowadays available as dry powder format. They are coming with use of the coat of synthetic materials which is increasing the cost of the product. The use of natural drying material is the solution to overcome this cost in production but are rigid and require multiple time optimisation of the formulation. If the product gets developed the shelf life is the second issue or hurdle in large scale manufacturing to satisfy the needs of such a large population of our country. Here new strain of lactobacillus was discovered, grown in natural milk as media and converted to a dry granule form for long viability for exhibiting antibacterial, antioxidant and anticancer activity.

A DETAILED DESCRIPTION OF THE INVENTION

[0017] While the present invention is described herein by way of example, using various embodiments and illustrative drawings, those skilled in the art will recognize that the invention is neither intended to be limited to the embodiment of drawing or drawings described nor designed to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated with specific figures, for ease of illustration, and such omissions do not limit the embodiment outlined in any way. The drawings and detailed description of it are not intended to restrict the invention to the form disclosed, but on the contrary, the invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. The headings are used for organizational purposes only and are not meant to limit the scope of the description or the claims. As used throughout this specification, the word "may" be used in a permissive sense, rather than the mandatory sense.

[0018] Further, the words "an" or "a" mean "at least one" and the word "plurality" means one or more unless otherwise mentioned. Furthermore, the terminology and phraseology used herein are solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents and any additional subject matter not recited, and is not supposed to exclude any other additives, components, integers or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents acts, materials, devices, articles, and the like are included in the specification solely to provide a context for the present invention.

[0019] In this disclosure, whenever an element or a group of elements is preceded with the transitional phrase "comprising", it is also understood that it contemplates the same element or group of elements with transitional phrases "consisting essentially of, "consisting", "selected from the group comprising", "including", or "is" preceding the recitation of the element or group of elements and vice versa.

[0020] Before explaining at least one embodiment of the invention in detail, it is to be understood that the present invention is not limited in its application to the details outlined in the following description or exemplified by the examples. The invention is capable of other embodiments or of being practised or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for description and should not be regarded as limiting.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Besides, the descriptions, materials, methods, and examples are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0022] The present invention is described herein by way of example, using various embodiments and illustrative drawings, those skilled in the art will recognize that the invention is neither intended to be limited to the embodiment of drawing or drawings described nor designed to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated with specific figures, for ease of illustration, and such omissions do not limit the embodiment outlined in any way. The drawings and detailed description of it are not intended to restrict the invention to the form disclosed, but on the contrary, the invention covers all modifications, equivalents, and alternatives falling within the spirit and scope of the present invention as defined by the appended claims. The headings are used for organizational purposes only and are not meant to limit the scope of the description or the claims. As used throughout this specification, the worn "may" be used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning, must).

[0023] Further, the words "an" or "a" mean "at least one" and the word "plurality" means one or more unless otherwise mentioned. Furthermore, the terminology and phraseology used herein is solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents and any additional subject matter not recited, and is not supposed to exclude any other additives, components, integers or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents acts, materials, devices, articles and the like are included in the specification solely to provide a context for the present invention.

[0024] In this disclosure, whenever an element or a group of elements is preceded with the transitional phrase "comprising", it is also understood that it contemplates the same element or group of elements with transitional phrases "consisting essentially of, "consisting", "selected from the group comprising", "including", or "is" preceding the recitation of the element or group of elements and vice versa.[0018] Before explaining at least one embodiment of the invention in detail, it is to be understood that the present invention is not limited in its application to the details outlined in the following description or exemplified by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for description and should not be regarded as limiting.

[0025] Unless otherwise defined, all technical and scientific terms used here in have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Besides, the descriptions, materials, methods, and examples are illustrative only and not intended to be limiting. Methods and materials similar or equivalent to those described herein are used in the practice or testing of the present invention.

[0026] In the present embodiment a new sub-strains of LAB is isolated from sheep milk. The physiological and biochemical investigations is the clue in the identification of the Lactobacillus at the lab level. The strains was confirmed as Lactobacillus rhamnosus by whole genomic sequencing. The isolate is found with additional properties of cholesterol assimilation, lysozyme survival, haemolytic and DNAse activity. This shows that the isolate is with the potential of sustaining the physiological stress condition of the human gut.

[0027] After isolation, the most important part is the flourishing of LAB in natural growth media. Mostly the existing marketed probiotics are grown in artificial growth media which is not only costly but may precipitate toxicity after prolonged use in the host. On the contrary, our microbe is showing active growth and flourishing in milk as natural media. Our results show that the growth of all LAB is optimum in the milk of buffalo as compared to the milk of sheep, cow, and goat.

[0028] For proper utilisation of the LAB as an end product, it is needed to be converted into a dosage form with good viability and shelf life. The shelf life of LAB can be enhanced by granule or powder formulations. In addition, very few probiotics formulations are available in the market which comes in granule or powder formulations by use of pharmaceutical excipients. Currently, the marketed probiotics formulations come in liquid dosage forms with short shelf life and involve refrigeration's. The use of pharmaceutical excipients to enhance the shelf life of LAB by granule formulations is a tricky process which involves proper optimizations for ease of manufacturing and end-user satisfactions, else it causes heavy product losses. The cost of the products may increase if the final products as formulations are not properly optimised. In the case of our product, both spray dry and lyophilization techniques are employed. The study shows that the use of maltodextrin in combinations with starch enhances shelf life as compared to use of lactose in combination. Spray dried products are screened for a period of 12 months at two different extreme conditions i.e. at 4 °C and at 37 °C. Combinations of maltodextrin and starch in ratio 3:1 shows greater viability as compared to other pharmaceutical excipients in combinations.

[0029] These encapsulated formulation keep the LAB viable, protecting the probiotics antioxidant and its antagonistic activities. Further, our LAB is screened for antioxidant and supportive antagonistic activities by various assays. The antioxidant activities determined by the scavenging of hydroxyl radicals, DPPH and other assays show that the LAB as lysate with significantly greater scavenging activity than an intact cell.

[0030] The zone of inhibition studies was carried out for LAB against all tested Gram positive and Gram-negative microorganism. The maximum zone of inhibition is observed as the whole organism as compared to lysate and supernatant. The disc diffusion method is easy, important and most widely method for antibacterial activity study. In the disc diffusion method, the first target organism suspension spread on the surface of nutrient agar medium and then the paper disc of antibacterial agents or tested samples deposited on the surface of sterile medium plate. Further, the tested sample discs diffused in medium by plates kept in refrigerator for 10 min. After the plates are incubated at 37 0 C for 24 h, the zone of inhibition (ZOI) around each disc are determined by using the scale in millimeter.

[0031] In agar well diffusion process, the wells (6 mm in diameter) are prepared by applying sterile cork borer and the target organism suspension is spread over the nutrient agar medium. The tested liquid sample of volume 7 0 -8 0 1 u is aseptically dispensed into each well and plates are kept at 4 C for 10 min to facilitate the diffusion of sample in the agar medium. After plates are incubated at 37 0 C for 24 h and the ZOI is measured around each well.

[0032] The tests showed that that the antibacterial activity of LAB is due to its adhesion to mucosa and probiotics activities Adherence and auto-aggregation abilities of LAB formulations of spray dry and lyophilization granules were tested in the Red snapper fish intestinal mucus. L. rhamnosus shows higher auto-aggregation and adhesion properties as compared to L. acidophilus which is used as standard.

[0033] Cell line studies are carried out using LAB, as the drug or nutraceutical products. The formulations or drug decreasing the viability of cancer cell lines is considered as an ideal candidature to heal the cancer disease. The viability study conducted by SRB and MTT assay on HCT 115 cell line show a decrease in percentage cell viability in case of the L. rhamnosus for 50 pg/ml dose as compared to other LAB under trails. Thus, it revealed that L. rhamnosus as an effective antiproliferative agent against HCT 115 cell line after 48 h. The apoptotic proteins are investigated by the up-regulation and down-regulation of the Bax, Bcl2 and beta-actin along with its expression ratio. L. rhamnosus shows the apoptotic activity by up-regulation of Bax and down-regulation of Bcl2 gene.

[0034] Chemical-induced oxidative stress studies is carried out using L. rhamnosus on Wistar rat's anti-colon cancer model. The study reflects that DMH (Dimethyl hydrazine) does not affect the physiological metabolism of L. plantarum treated group and the values found are the same as that of the vehicle control group. The L. rhamnosus group of animal model shows a significant increase in body weights after 6 weeks period which are the same as the vehicle control group, not observed in case of the synthetic drug group. The decrease in Hb level results in anemia and is found to be greatly affecting the pathophysiological conditions of a cancer patient. The present study shows that L. rhamnosus not only increased the Hb level but maintained neutrophils and lymphocyte count nearly the same as a vehicle control group. In liver and spleen indices studies DMH drastically affected the colonic regions with minor effects on liver cells. This indicates that L. rhamnosus prevents the splenomegaly conditions. No any sign of hepatomegaly is observed in L. rhamnosus treated group indicating normal liver functioning. Thus, in-vivo studies revealed that L. rhamnosus not only reduces the precancerous colonic lesions but also diminishes the chemically induced oxidative stress conditions, which is not observed by treatment with the synthetic drug observed b histopathological investigation. Thus, L. rhamnosus formulations may be considered as ideal neutraceutical product to heal adenomatous chemical induced carcinoma at the lowest cost and may act as a natural origin functional food.

[0035] While there has been illustrated and described embodiments of the present invention, those of ordinary skill in the art, to be understood that various changes may be made to these embodiments without departing from the principles and spirit of the present invention, modifications, substitutions, and modifications, the scope of the invention being indicated by the appended claims and their equivalents.

FIGURE DESCRIPTION

[0036] the accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate an exemplary embodiment and, together with the description, explain the disclosed embodiment. In the figures, the left and rightmost digit(s) of a reference number identify the figure in which the reference number first appears. The same numbers are used throughout the figures to reference features and components. Some embodiments of the system and methods of an embodiment of the present subject matter are now described, by way of example only, and concerning the accompanying figures, in which:

[0037] Figure 1 shows the schematic diagram of isolation of LAB, culturing in milk of milking animal and probiotics studies

[0038] Figure 2 shows the Schematic representation of the agar disc diffusion antibacterialactivity

[0039] Figure 3 illustrates the Schematic representation of the agar well diffusion method for antibacterial activity.

[0040] Figure 4 illustrates the Schematic representation of the antioxidant and anticancer activity.

EDITORIAL NOTE 2021106840

There is 1 page of claims only.

Claims (5)

CLAIMS:

1. A method of formultating granule containing Lactobacillus rhamnosus ARJD for preventing and curing colon cancer; acting as nutracutical mediators consisting of; Lactobacillus rhamnosusARJD Maltodextrin and Starch.

2. The formulated granules as claimed in claim - 1, consist of Lactobacillus rhamnosus as probiotics, maltodextrin and starch as excipient used as lyoprotective or thermoprotective agent during the process of spray dry.

3. The formulated granules as claimed in claim - 1, uses the Lactobacillus rhamnosus as probiotics that is the optimal growth only in the milk of buffalo.

4. The formulated granules as claimed in claim - 1, is having long shelf life without losing viability upto 12 months if kept at 4 OC and for period of 6 months if kept at 37 0 (room temperature as per Indian climate)

5. The formulated granules as claimed in claim - 1, can be used against the normal bowel disorders due to probiotics properties and adhesion abilities.

SIGNATORY

Dr. Abhinadan R Patil

Mr. Ravsaheb Patil

Dr. Priyanka Patil

Mr. Amit Patil

Dr. Shivaji Pawar

Mr. Amrut Patil

Ms. Sohili Patil

Ms. Pallavi Patil

Ms. Sujata Patil

APPLICANT Name: Dr. Abhinandan R Patil, Mr. Ravsaheb Patil, Dr. Priyanka Patil, Mr. Amit Patil, 24 Aug 2021

Dr. Shivaji Pawar Mr. Amrut Patil, Ms. Sohili Patil, Ms. Pallavi Patil, Ms. Sujata Patil Page 1 of 4 2021106840

Figure 1: Schematic of discovery of LAB, culturing in milk of milking animal and probiotics studies SIGNATORY Dr. Abhinadan R Patil

Mr. Ravsaheb Patil

Dr. Priyanka Patil

Mr. Amit Patil

Dr. Shivaji Pawar

Mr. Amrut Patil

Ms. Sohili Patil

Ms. Pallavi Patil

Ms. Sujata Patil

APPLICANT Name: Dr. Abhinandan R Patil, Mr. Ravsaheb Patil, Dr. Priyanka Patil, Mr. Amit Patil, 24 Aug 2021

Dr. Shivaji Pawar Mr. Amrut Patil, Ms. Sohili Patil, Ms. Pallavi Patil, Ms. Sujata Patil Page 2 of 4 2021106840

Figure 2 shows the Schematic representation of the agar disc diffusion antibacterial activity SIGNATORY

Dr. Abhinadan R Patil

Mr. Ravsaheb Patil

Dr. Priyanka Patil

Mr. Amit Patil

Dr. Shivaji Pawar

Mr. Amrut Patil

Ms. Sohili Patil

Ms. Pallavi Patil

Ms. Sujata Patil

APPLICANT Name: Dr. Abhinandan R Patil, Mr. Ravsaheb Patil, Dr. Priyanka Patil, Mr. Amit Patil, 24 Aug 2021

Dr. Shivaji Pawar Mr. Amrut Patil, Ms. Sohili Patil, Ms. Pallavi Patil, Ms. Sujata Patil Page 3 of 4 2021106840

Figure 3 illustrates the Schematic representation of the agar well diffusion method for antibacterial activity. SIGNATORY

Dr. Abhinadan R Patil

Mr. Ravsaheb Patil

Dr. Priyanka Patil

Mr. Amit Patil

Dr. Shivaji Pawar

Mr. Amrut Patil

Ms. Sohili Patil

Ms. Pallavi Patil

Ms. Sujata Patil

APPLICANT Name: Dr. Abhinandan R Patil, Mr. Ravsaheb Patil, 24 Aug 2021

Dr. Priyanka Patil, Mr. Amit Patil, Dr. Shivaji Pawar Mr. Amrut Patil, Ms. Sohili Patil, Ms. Pallavi Patil, Ms. Sujata Patil Page 4 of 4 2021106840

Figure 4 illustrates the Schematic representation of the antioxidant and anticancer activity.

SIGNATORY

Dr. Abhinadan R Patil

Mr. Ravsaheb Patil

Dr. Priyanka Patil

Mr. Amit Patil

Dr. Shivaji Pawar

Mr. Amrut Patil

Ms. Sohili Patil

Ms. Pallavi Patil

Ms. Sujata Patil