Immunomodulatory antiviral NANO herbal composition and method to prepare the same

Abstract

Disclosed herein is an advanced synergistic nano herbal composition having significant immunomodulatory and antiviral effects in human subjects, said formulation comprising the specific combination of HAVEs of Citrus limon, Cymbopogon citratus, Origanum vulgare, Thymus serpyllum, Tinospora cordifolia, Withania somnifera, Zea mays, Meyna laxiflora, Blumea lacera, Boswellia serrata, Cinnamomum verum, Mentha pipperata, Mentha arvensis, Nigella sativa, Cocos nucifera, Fagonia arabica, Azadirachta indica, Eucalyptus globulus, Citrus sinensis, Coriandrum sativum, Saccharum officinarum, Syzygium aromaticum, Dolichos biflorus. Resulting composition is sonicated to obtain the final advanced synergistic nano herbal composition having average particle size 133.2nm.

Description

[0001] BEFORE THE INTERNATIONAL BUREAU OF THE WORLD INTELLECTUAL PROPERTY ORGANIZATION

[0002] NON-PROVISIONAL APPLICATION FOR PATENT PCT Rule 19.1(a)(iii) *** Non-Provisional Specification ***

[0003] “Immunomodulatory antiviral nano herbal composition and method to prepare the same”

[0004] Field of the invention

[0005] This invention belongs to the field of phytomedicine involving the use of plants or plant extracts for medicinal purposes. More particularly, the disclosures hereof are directed to a novel approach of modern herbalism based on scientific research and formulation principles to obtain a synergistic herbal composition having validated immunomodulatory properties for broad-spectrum antiviral and prophylactic applications.

[0006] Definitions and interpretations

[0007] Before undertaking the detailed description of the invention below, it may be advantageous to set forth definitions of certain words or phrases used throughout this patent document: the terms “include” and “comprise,” as well as derivatives thereof, mean inclusion without limitation; the term “or” is inclusive, meaning and / or; the phrases “associated with” and “associated therewith,” as well as derivatives thereof, may mean to include, be included within, interconnect, with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have, have a property of, or the like; and certain definitions are set forth for this document, as follows: -

[0008] (a) "Immunomodulatory" refers to the ability to modify or regulate the immune system's activity.

[0009] (b) “RSV” refers to respiratory syncytial virus.

[0010] (c) “HRV” refers to human rhinovirus.

[0011] (d) “HSV” refers to herpes simplex virus.

[0012] (e) "NHF" means a nano herbal formulation, and refers to a herbal product that incorporates nanotechnology, involving the manipulating materials at the nanoscale, which can offer unique properties and advantages compared to traditional formulations.

[0013] (f) “HAVE” means and refers to hydro alcoholic volatile extract.

[0014] (g) “Sieve No. 22 / 40” refers to a specific type of sieve used in particle size analysis.

[0015] The numbers 22 and 40 refer to the mesh sizes (number of openings per inch) of the sieve. In this case, the sieve has a number 22 mesh size on the top and a number 40 mesh size on the bottom.

[0016] (h) “Q.S” refers to Quantity Sufficient, which is that quantity of an ingredient which is enough to achieve the desired total volume or quantity of the final product. The quantity to be added isn't explicitly specified but is left to the discretion of the person preparing the formulation, ensuring that the final volume or quantity is as intended.

[0017] (i) “Bitter” refers to a class of compounds present in herbs which are essential component of various tonics.

[0018] Background of the invention

[0019] Herbal medicine is one of the oldest forms of healthcare known to humanity and is practiced in various cultures around the world. Herbal medicine encompasses a wide range of practices ranging from traditional folk remedies to the approach of this invention of amalgamating the traditional knowledge of medicinal herbs to modern medicine to empower health-care and allow individuals to actively pursue their wellbeing.

[0020] Traditional healers, herbalists, and indigenous peoples have accumulated vast knowledge about the therapeutic properties of plants and their uses for treating various ailments. However, this vast knowledge is often incomplete, scattered or merely based on anecdotal data. Irrespectively, herbal medicine encompasses a vast array of practices and remedies used by cultures worldwide wherein different cultures have developed their own unique systems of herbal medicine based on local plants, indigenous knowledge, and historical traditions. Some examples of traditional herbalism are-

[0021] (a) Ayurveda: An ancient healing system from India that focuses on achieving balance among the body, mind, and spirit, and is a vast compendium of knowledge on thousands of plants for their therapeutic properties. Key plants I herbs prescribed include turmeric, ashwagandha, holy basil, triphala, and neem. Characteristically, herbal formulations in this branch are tailored to an individual's constitution and specific health needs.

[0022] (b) Traditional Chinese Medicine: Dating back thousands of years, this branch emphasizes the concept of balance and harmony within the body and uses herbs to restore health by addressing imbalances in Qi (life energy) and Yin-Yang. Key plants I herbs prescribed include ginseng, astragalus, licorice root, ginger, and ginkgo biloba. Characteristically, formulations are prescribed on individual patterns of disharmony recognized by practitioners in this domain.

[0023] (c) Traditional Western Herbalism: This encompasses herbal healing practices from Europe and North America. It draws on indigenous knowledge as well as Greco- Roman, Celtic, and other historical herbal traditions. Key plants I herbs prescribed include echinacea, chamomile, elderberry, valerian, and St. John's wort. Characteristically, formulations conform to personalized formulas or single herbs are recommended based on specific health concerns.

[0024] (d) Native American Herbalism: This relates to native American tribal traditions of herbal medicine, using plants native to their regions for healing purposes. Key plants I herbs prescribed include sage, cedar, tobacco, echinacea, and sweetgrass, their use being mentioned for a wide range of purposes, including spiritual healing, ceremony, and physical ailments.

[0025] (e) Native traditional medicine: This relates to native African tribal traditions of herbal medicine, encompassing diverse healing practices across the continent, incorporating herbal remedies, spiritual rituals, and cultural beliefs. Key plants I herbs prescribed include African ginger, bitter kola, moringa, rooibos, and hoodia, which may be administered as infusions, decoctions, or poultices, depending on the specific tradition.

[0026] (f) Islamic medicine: This combines elements of traditional Arabic, Persian, and Greco-Roman medicine with Islamic principles and teachings, combined with dietary and lifestyle recommendations. Key plants I herbs prescribed include black seed (Nigella sativa), saffron, myrrh, frankincense, and fenugreek for various ailments.

[0027] Each of the above-mentioned traditional branches of herbal medicine has distinctive unique philosophies, diagnostic methods, and therapeutic approaches, but they all share a common emphasis on using plants for healing and promoting well-being. The applicants named herein identify principally with this approach, with progression along with principles of modern medicine based on evidence-based approach to diagnosis, treatment, and prevention of diseases, as well as its emphasis on preventive care to reduce the risk of disease and promote overall health and wellness.

[0028] On another note, while modern medicine has made significant advancements in diagnosing and treating diseases, it also has its limitations and challenges, including rising healthcare costs, access disparities, over-reliance on pharmaceuticals, and concerns about medicalization and overdiagnosis. Hence, the applicants named herein strongly propound that a recourse to traditional branches of herbal medicine along with principles of modern medicine holds the promise needful to augment ongoing research and efforts to improve healthcare delivery, address healthcare disparities, and integrate complementary and alternative approaches to promote holistic patient care.

[0029] From a basal understanding of the human body’s biochemistry, the immune system is a complex network of cells, tissues, and organs that defends the body against harmful pathogens, such as bacteria, viruses, and parasites, as well as abnormal cells, like cancer cells - however, an overactive or underactive immune system can lead to various health problems too. Also, it is essential to use them judiciously and under medical supervision, as improperly modulating the immune system can lead to adverse effects or complications. Additionally, individual responses to immunomodulatory therapies can vary, so treatment plans should be tailored to each patient's specific needs and health condition(s). Therefore, for the purposes and applications mentioned above, immunomodulatory activities of herbal ingredients and their ability to enhance the immune system's antiviral response assume prime importance.

[0030] While herbal medicine has been used for centuries and continues to be popular worldwide, it also is inundated with several potential issues and limitations, including-

[0031] (a) Limited scientific evidence: While some herbs have been studied extensively and have demonstrated therapeutic effects, many herbal remedies lack sufficient scientific evidence to support their efficacy and safety. The lack of standardized research protocols, inconsistent study results, and methodological challenges make it difficult to draw definitive conclusions about the effectiveness of herbal medicines.

[0032] (b) Variability in Quality and Potency: Herbal products vary widely in quality, potency, and composition. Factors such as plant species, growing conditions, harvesting methods, processing techniques, and storage can influence the concentration of active ingredients in herbal remedies. Poor quality control and contamination issues may result in variability in the effectiveness and safety of herbal products.

[0033] (c) Safety Concerns: While herbal remedies are often perceived as natural and safe, they can still pose risks, especially when used inappropriately or in high doses. Some herbs may interact with medications, exacerbate underlying health conditions, or cause adverse reactions such as allergic reactions, gastrointestinal upset, liver toxicity, or hormonal imbalances. Certain herbs may also be contraindicated during pregnancy, breastfeeding, or in specific populations.

[0034] (d) Misleading Marketing and Misinformation: The herbal supplement and drug industry is largely unregulated in many countries, leading to concerns about misleading marketing practices, false claims, and inadequate labeling of herbal products. Consumers may be misled by exaggerated claims, unsupported health claims, or pseudoscientific information promoted by manufacturers, sellers, or online sources.

[0035] (e) Herb-Drug Interactions: Herbal remedies can interact with prescription medications, over-the-counter drugs, or other supplements, potentially altering their effectiveness, absorption, metabolism, or elimination from the body. Some herbs may inhibit or induce drug-metabolizing enzymes in the liver, leading to unexpected side effects or therapeutic failures.

[0036] (f) Cultural and Ethical Concerns: The commercialization of traditional herbal knowledge and practices raises ethical concerns about cultural appropriation, intellectual property rights, and fair compensation for indigenous communities and traditional healers. The overharvesting of wild medicinal plants and habitat destruction may also threaten biodiversity and the sustainability of herbal resources.

[0037] (g) Lack of Regulation and Standardization: Herbal supplements and formulations are often regulated as dietary supplements rather than drugs in many countries, resulting in less stringent oversight by regulatory agencies. Quality control standards, manufacturing practices, labeling requirements, and safety regulations may vary widely among herbal products, leading to concerns about product consistency, purity, and authenticity.

[0038] (h) Delayed Diagnosis and Treatment: Relying solely on herbal remedies for serious or life-threatening medical conditions may delay appropriate diagnosis and treatment by healthcare professionals. While herbal medicine may complement conventional medical care for certain conditions, it should not be used as a substitute for evidence-based medical treatments, especially in emergencies or severe illnesses.

[0039] There is hence a pressing need for scientific research and validation of the diverse array of active ingredients offered by herbs and furthermore their combinatorial effects in order to realize a holistic approach to health and wellness, considering the interconnectedness of the body, mind, and spirit being accounted for in traditional medicine, especially Ayurveda.

[0040] Description of related art

[0041] Several herbs have been traditionally used and studied for their potential immunomodulatory and antiviral properties. Examples include-

[0042] (a) Echinacea purpurea - Antiviral effect particularly against respiratory viruses like the common cold and flu - this effect being attributed to the presence of alkamides, caffeic acid derivatives, polysaccharides, and glycoproteins, Nicotiflorin and rutin being dominant flavonoids in the same.

[0043] (b) Sambucus nigra - Also known commonly as Elderberry, this has been used traditionally used to fight colds and flu, with ingredients that may inhibit the replication of certain viruses, including influenza viruses.

[0044] (c) Glycyrrhiza glabra - Also known commonly as Licorice root, is known for antiviral effects against respiratory viruses. Glycyrrhizin, a compound found in licorice root, has been shown to inhibit the replication of certain viruses.

[0045] (d) Zingiber officinale - Also known commonly as ginger, this has antimicrobial properties and may help support immune function, antiviral effects against respiratory viruses, including RSV and HRV.

[0046] (e) Allium sativum - Also known commonly as garlic, an ingredient Allicin, has been shown to have antiviral activity against a variety of viruses, including influenza viruses, rhinovirus, and HSV.

[0047] (f) Andrographis paniculata - Referred in both Ayurvedic and traditional Chinese medicine, its ingredient, andrographolide, is determined to have antiviral effects against respiratory viruses, including influenza viruses and RSV.

[0048] (g) Astragalus membranaceus - Referred in Chinese medicine to support the immune system, studies have shown that it’s extract may have antiviral effects against certain viruses, including influenza viruses and HSV.

[0049] Therefore, while the above-mentioned and other herbs may offer potential benefits, especially for immunomodulation and antiviral properties, they have not been able to replace conventional medical treatments due to the core issues mentioned in the preceding section of this document.

[0050] Technical issues on hand

[0051] Advent of this invention owes its genesis to the following persisting technical issues- (a) Most herbal extracts mentioned in traditional medicine are in crude format, which prevents their active ingredients from rightful presentation for the therapeutic effect intended.

[0052] (b) Being in crude and non-optimized format, herbal ingredients take more time to act on the immunological response and thus the therapeutic effect intended.

[0053] (c) Desirable bioavailability, efficacy, targeted delivery of active herbal compounds, minimizing off-target effects is not possible with crude extracts.

[0054] Prior art therefore, does not list a single effective solution embracing all considerations mentioned hereinabove, thus preserving an acute necessity- to-invent for the present inventor / s who, as result of focused research, has come up with novel solutions for resolving all needs once and for all. Work of the applicant / s hereof, specifically directed against the technical problems recited hereinabove and currently part of the public domain including earlier filed patent applications, is neither expressly nor impliedly admitted as prior art against the present disclosures.

[0055] A better understanding of the objects, advantages, features, properties and relationships of the present invention will be obtained from the following detailed description which sets forth an illustrative yet-preferred embodiment.

[0056] Objectives of the present invention

[0057] The present invention is identified in addressing at least all major deficiencies of art discussed in the foregoing section by effectively addressing the objectives stated under, of which:

[0058] It is a primary objective to provide a synergistic herbal composition which exhibits significant immunomodulatory activity.

[0059] It is another objective further to the aforesaid objective(s) that the composition so propounded exhibits broad-spectrum prophylactic antiviral activity.

[0060] It is another objective further to the aforesaid objective(s) that the composition so propounded is affordable, accessible, and constitutes a culturally-appropriate healthcare solution.

[0061] The manner in which the above objectives are achieved, together with other objects and advantages which will become subsequently apparent, reside in the detailed description set forth below in reference to the accompanying drawings and furthermore specifically outlined in the independent claims. Other advantageous embodiments of the invention are specified in the dependent claims. Brief description of drawings

[0062] The present invention is explained herein under with reference to the following drawings, in which:

[0063] FIGURE 1 is a graph illustrating the particle size distribution of Formulation No. 7, as prepared by one protocol, according to the present invention.

[0064] FIGURE 2 is a graph illustrating the particle size distribution of Formulation No. 7, as prepared by another protocol, according to the present invention.

[0065] FIGURE 3 (A to E) showcase the results of the docking analysis of SARS-CoV-2 MProbinding with nobiletin, in which (A) hydrophobicity surface 3D representation (B) 3D representation of tricin with SARS-CoV-2 MProinteraction (C) Interactions of tricin through H-bond in a pocket site of SARS-CoV-2 MPro(D) Interactions through hydrophobic bond in a pocket site of SARS-CoV-2 MPro(E) 2D representation in active site of SARS-CoV-2 MPro.

[0066] FIGURE 4 (A to E) showcase the results of the docking analysis of SARS-CoV-2 MProbinding with tangeretin, in which (A) hydrophobicity surface 3D representation (B) 3D representation of tricin with SARS-CoV-2 MProinteraction (C) Interactions of tricin through H-bond in a pocket site of SARS-CoV-2 MPro(D) Interactions through hydrophobic bond in a pocket site of SARS-CoV-2 MPro(E) 2D representation in active site of SARS-CoV-2 MPro.

[0067] FIGURE 5 (A to E) showcase the results of the docking analysis of SARS-CoV-2 MProbinding with sideroxylonal-C, in which (A) hydrophobicity surface 3D representation (B) 3D representation of tricin with SARS-CoV-2 MProinteraction (C) Interactions of tricin through H-bond in a pocket site of SARS-CoV-2 MPro(D) Interactions through hydrophobic bond in a pocket site of SARS-CoV-2 MPro(E) 2D representation in active site of SARS-CoV-2 MPro.

[0068] FIGURE 6 (A to E) showcase the results of the docking analysis of SARS-CoV-2 MProbinding with coriandron, in which (A) hydrophobicity surface 3D representation (B) 3D representation of tricin with SARS-CoV-2 MProinteraction (C) Interactions of tricin through H-bond in a pocket site of SARS-CoV-2 MPro(D) Interactions through hydrophobic bond in a pocket site of SARS-CoV-2 MPro(E) 2D representation in active site of SARS-CoV-2 MPro.

[0069] FIGURE 7 (A to E) showcase the results of the docking analysis of SARS-CoV-2 MProbinding with ombuin, in which (A) hydrophobicity surface 3D representation (B) 3D representation of tricin with SARS-CoV-2 MProinteraction (C) Interactions of tricin through H-bond in a pocket site of SARS-CoV-2 MPro(D) Interactions through hydrophobic bond in a pocket site of SARS-CoV-2 MPro(E) 2D representation in active site of SARS-CoV-2 MPro.

[0070] FIGURE 8 (A to E) showcase the results of the docking analysis of SARS-CoV-2 MProbinding with tamarixetin, in which (A) hydrophobicity surface 3D representation (B) 3D representation of tricin with SARS-CoV-2 MProinteraction (C) Interactions of tricin through H-bond in a pocket site of SARS-CoV-2 MPro(D) Interactions through hydrophobic bond in a pocket site of SARS-CoV-2 MPro(E) 2D representation in active site of SARS-CoV-2 MPro.

[0071] FIGURE 9 (A to E) showcase the results of the docking analysis of SARS-CoV-2 MProbinding with 6-Deacetylnimbin, in which (A) hydrophobicity surface 3D representation (B) 3D representation of tricin with SARS-CoV-2 MProinteraction (C) Interactions of tricin through H-bond in a pocket site of SARS-CoV-2 MPro(D) Interactions through hydrophobic bond in a pocket site of SARS-CoV-2 MPro(E) 2D representation in active site of SARS-CoV-2 MPro.

[0072] FIGURE 10 (A to E) showcase the results of the docking analysis of SARS-CoV-2 MProbinding with Nimbolide, in which (A) hydrophobicity surface 3D representation (B) 3D representation of tricin with SARS-CoV-2 MProinteraction (C) Interactions of tricin through H-bond in a pocket site of SARS-CoV-2 MPro(D) Interactions through hydrophobic bond in a pocket site of SARS-CoV-2 MPro(E) 2D representation in active site of SARS-CoV-2 MPro.

[0073] FIGURE 1 1 (A to E) showcase the results of the docking analysis of SARS-CoV-2 MProbinding with tricin, in which (A) hydrophobicity surface 3D representation (B) 3D representation of tricin with SARS-CoV-2 MProinteraction (C) Interactions of tricin through H-bond in a pocket site of SARS-CoV-2 MPro(D) Interactions through hydrophobic bond in a pocket site of SARS-CoV-2 MPro(E) 2D representation in active site of SARS-CoV-2 MPro.

[0074] FIGURE 12 (A and B) are graphs showcasing the change in body weight for male and female rats when administered with Formulation No. 7 of the present invention.

[0075] FIGURE 13 (A and B) are graphs showcasing the change in biochemical parameters for male and female rats when administered with Formulation No. 7 of the present invention.

[0076] FIGURE 14 (A and B) are photographs showcasing results of renal and Liver histopathological examination in male and female rats, when administered with Formulation No. 7 of the present invention.

[0077] FIGURE 15 is a graph showcasing results of the Ex vivo intestinal absorption everted sac method undertaken with the Formulation No. 7 of the present invention. The above drawings are illustrative of particular examples of the present invention but are not intended to limit the scope thereof. Though numbering has been introduced to demarcate reference to specific components in relation to such references being made in different sections of this specification, all components are not shown or numbered in each drawing to avoid obscuring the invention proposed.

[0078] Attention of the reader is now requested to the detailed description to follow which narrates a preferred embodiment of the present invention and such other ways in which principles of the invention may be employed without parting from the essence of the invention claimed herein.

[0079] Statement / Summary of the invention

[0080] This invention is directed to an advanced synergistic nano herbal composition having significant immunomodulatory and antiviral effects in human subjects, said formulation comprising the specific combination of HAVEs of Citrus limon, Cymbopogon citratus, Origanum vulgare, Thymus serpyllum, Tinospora cordifolia, Withania somnifera, Zea mays, Meyna laxiflora, Blumea lacera, Boswellia serrata, Cinnamomum verum, Mentha pipperata, Mentha arvensis, Nigella sativa, Cocos nucifera, Fagonia arabica, Azadirachta indica, Eucalyptus globulus, Citrus sinensis, Coriandrum sativum, Saccharum officinarum, Syzygium aromaticum, Dolichos biflorus. Resulting composition is sonicated to obtain the final advanced synergistic nano herbal composition having average particle size 133.2nm.

[0081] Detailed description

[0082] Principally, general purpose of the present invention is to assess disabilities and shortcomings inherent to known systems comprising state of the art and develop new systems incorporating all available advantages of known art and none of its disadvantages. Accordingly, the disclosures herein are directed towards

[0083] Approach: The present invention follows a foundational approach by progression marked as per the following steps-

[0084] (a) Screening of herbs for needful therapeutic effects

[0085] (b) Validation of traditional knowledge

[0086] (c) Identification of active compounds in shortlisted herbs

[0087] (d) Understanding mechanisms of action of the identified active compounds (e) Establishing nano-formulations of herbal ingredients. Principles of nanotechnology are incorporated herein for enhancing bioavailability, allowing targeted delivery with minimal off-target interactions and effects, improving efficacy, and enabling targeted delivery of active herbal compounds.

[0088] (f) Optimization of nano-formulations and dosages made using the studied active compounds

[0089] (g) Investigating the safety profile of formulations and dosages devised.

[0090] (h) Empowering individuals to adopt herbal medicine with evidence-based information that enables making of informed decisions about their healthcare and integrate herbal medicine into even their self-care practices safely and effectively.

[0091] Reference is now made to the preferred embodiment and other alternative embodiments via which the present invention is intended to be implemented. The content is illustrative and not restricting in any form or interpretation.

[0092] Method of preparation:

[0093] The advanced synergistic nano herbal composition having significant immunomodulatory and antiviral effects in human subjects is prepared by formulation of a HAVE. The two-stage process for preparation of the HAVE comprises the following sequence of steps-

[0094] Stage 1 :

[0095] (a) Fresh or dried plant material, free from any contaminants, is carefully collected in weighed quantities.

[0096] (b) Weighed quantities of the plant material were pulverized using suitable common art methods to obtain moderately coarse powder having average particle size passing through sieve No. 22 / 40.

[0097] (c) The pulverized material was then transferred to the distillation unit followed by the addition of water (1 to 3 times the amount of plant material) and left for 24 to 48 hours for soaking.

[0098] (d) Subsequently, distillation (50-80°C) was carried out over a period of 3-8 hours, with the distillate being collected in tubes

[0099] (e) the plant material was dried using an oven at a temperature range of 50-80°C until completely dry.

[0100] Stage 2:

[0101] (a) The dried materials were then extracted using reflux I Soxhlet extraction methods employing water, alcohol between concentrations of 60-95%, and / or mixtures thereof; repeating these steps up to 3-8 times for durations ranging from 3-6 hours each time.

[0102] (b) The extracts were filtered and combined before undergoing drying under reduced pressure conditions (100-235 mBar) with temperatures maintained within the range of (50-75°C).

[0103] (c) The extracts were dried till the moisture content was achieved in the range of 7.5 to 15%.

[0104] Formulation Development (Composition 1-7):

[0105] (a) Respective distillate and HAVE extract were mixed in ratio ranging from (1 :100 to 1 :1000).

[0106] (b) Different HAVE mixtures were prepared as per the formulation composition tables (Table 1 to 7 below) with addition of excipients and vehicle Q.S.

[0107] (c) To prepare the formulation prepared HAVE were mixed in specific quantity with mixing at speeds between 150 to 200 RPM for duration from 30 to 60 minutes to obtain the final formulations of the immunomodulatory antiviral nano herbal composition hereof.

[0108] Table 1 (Formulation No. 1 )

[0109]

[0110] Table 2 (Formulation No. 2)

[0111] Table 3 (Formulation No. 3)

[0112] Table 4 (Formulation No. 4)

[0113] Table 5 (Formulation No. 5)

[0114]

[0115] Table 6 (Formulation No. 6)

[0116]

[0117] Table 7 (Formulation No. 7)

[0118] Approach for formulation: 23 plants were initially selected for their antioxidant and immunomodulatory properties. From these plants, various formulations, F-1 (4 plants), F-2 (9 plants), F-3 (7 plants), F-4 (7 plants) were prepared. Antioxidant and antiinflammatory potential of these formulations were scientifically validated, and thereafter F-1 and F-2 combined to form F-5, F-3 and F-4 combined to form F-6 and further screening was performed using antioxidant and anti-inflammatory assays. On the basis of results of F-5 and F-6 , F-2 was combined with the F-6 to formulate F-7 which was superior to previous formulations which was evident from the in vitro results, this the formulation F-7 shown in Table 7 above was determined to be optimal for production of the composition of this invention.

[0119] Preparation of nano formulations: Solubility and Compatibility of the HAVE were screened in various solvents to ensure optimal performance. Thereafter, the formulations No. 1 to 7 described above were processed further, via two alternative protocols, for nano-sizing, as under

[0120] Protocol A:

[0121] (a) HAVE was admixed with water as % concentration in Formulation composition tables above, to form a solution.

[0122] (b) The solution was sonicated for 25-30 min in a bath sonicator followed by homogenization for 5-10 min at 9000 to 10000 RPM to obtain a nano-sized formulation. Size distribution of resultant nano-sized formulation for Formulation No. 7 as obtained by Protocol A is shown in the accompanying FIGURE 1 .

[0123] Protocol B:

[0124] (a) Have extracts were then meticulously combined with excipients such as (Polyethylene glycol, Lecithin, TPGS 1000, Brij). Mixing was carried out by vortexing for 5 minutes.

[0125] (b) Sonication was carried out using a Probe Sonicator for 25-30 minutes with Amplitude 20-45 kHz and temperature 25-45°C followed by homogenization for 5- 10 min at 10000 to 15000 RPM to obtain a nano-sized formulation

[0126] Size distribution of resultant nano-sized formulation for Formulation No. 7 as obtained by Protocol B is shown in the accompanying FIGURE 2. Protocol B was adopted for nano formulation preparation since uniform particle size (Average particle size 133.2nm) was obtained using this process. It shall be understood by the reader that the formulation proposed herein is amenable to be formulated alternatively as one selected from among teas, tinctures, capsules, powders, and extracts, by using common art principles of drug formulation.

[0127] Synergistic Action: A commonly used approach to assess the impact of combining plant extracts is through the fractional index. This method helps determine whether a combined therapy exhibits synergy, addition, or antagonism. The inhibitory concentration is determined by measuring IC values. The cumulative fractional inhibitory concentration index (£FIC) involves adding up individual FIC values for each extract. Interpretation table below is referred to for FIC

[0128] Formulations 1 to 7 was compounded from different HAVE components. Subsequently these formulations were screened for Antioxidant activity and Antiinflammatory action.

[0129] Synergies of these formulations were identified using the Fractional inhibitory concentration index as below.

[0130] Formulation 7 showed better FIC when compared to formulation 1 to 6 which is in accordance to interpretation table for FIC. Similar results were obtained on carrying out the anti-inflammatory activity. Hence Formulation 7 was determined as the optimum for further experimental studies.

[0131] EXPERIMENTAL VALIDATION

[0132] The final formulations of the immunomodulatory antiviral nano herbal composition hereof obtained as per the foregoing narrative were screened using in vitro tests.

[0133] Characterization studies for quality of the final formulation:

[0134] (a) Constituent content: Final formulations as reached in accordance with the aforesaid narrative was characterized for Quality standards parameters on the basis of quantification of secondary metabolites such as Total phenol, Alkaloids, Glycosides, Bitter, Tannins, Terpenoids using the following methods:

[0135] Gravimetry: The sample (3.0gm) was mixed with 50mL of methanol in a 100mL round bottom flask and refluxed for one hour. The mixture was then cooled, filtered through Whatman no.1 filter paper, and the residue in the flask was subjected to two more cycles of refluxing with 50mL of methanol, followed by cooling and filtering using the same filter paper each time. The combined extracts were evaporated to remove methanol completely, and the residue was dissolved in distilled water (25mL). After transferring it to a separating funnel through cotton, the cotton was washed with warm water (10 mL) before rinsing the beaker with ethyl acetate and transferring it to another separating funnel through cotton. After shaking well for five minutes, the ethyl acetate layer was removed into another separating funnel before repeating this extraction process four times over using 25mL of ethyl acetate each time on aqueous layer. Finally, all these layers were combined after which they were transferred into a tare dish where they were evaporated on a water bath before being dried at 105°C constantly in an oven up until there wasn't any change detected in weight anymore.

[0136] Spectrophotometry: The total phenolic content in extracts was determined using the Folin-Ciocalteu procedure. Samples (2mL, triplicates) were mixed with 1 .0 mL of Folin-Ciocalteu’s reagent and 0.8 mL of sodium carbonate (7.5%) in test tubes and allowed to stand for 30 min before measuring the absorption at 765 nm using a UV-visible Spectrophotometer. The total phenolic content was expressed as gallic acid equivalents per gram dry material. Accordingly, the determined by the aforesaid methods is shown in Table 9 below.

[0137] Table 9

[0138] (b) Assessment of Anti-oxidant Potential of Prepared compositions 1-7: The free radical scavenging activity was measured in terms of hydrogen donating or radical scavenging ability using the stable radical DPPH. 200 pM solution of DPPH in methanol was prepared and 50pl of this solution was added to 150 pl of different test compounds at different concentrations (6.25 -200 pl / ml). After 30 minutes, the absorbance was measured at 517 nm

[0139] Absorbance of control - Absorbance of test

[0140] % Scavenging = X 100

[0141] Absorbance of control

[0142] Results of this study are presented in Table 10 below.

[0143] Table 10

[0144] (c) Screening for Anti-inflammatory activity: Different concentrations of extracts and extract combinations ranging from 1 to 20 mg / mL were mixed with 5 mL of a 1 .5 mg / mL BSA solution and incubated at 37°C for a period of 15-20 minutes. Following this, the reaction mixtures were heated for exactly three minutes at a temperature of 80°C. Instead of sample buffer, a Control was used in this experiment. The tubes were then allowed to cool down to room temperature before measuring the absorbance at a wavelength of 660 nm using a spectrophotometer.

[0145] % inhibition was calculated using the formula:

[0146] Absorbance of control - Absorbance of test

[0147] % inhibition = - - - - - - - - - X 100

[0148] Absorbance of control

[0149] Results are shown in Table 1 1 below.

[0150] Table 11 Individual extracts were screened, and composition mixes were created to evaluate their anti-inflammatory action. Upon analysis, it was discovered that composition 7 displayed a notable synergistic effect when compared with the % inhibition at identical dose levels. Composition 7 was found to be most potent and repetitive trials were carried out to ensure the reproducibility of results after which it was further screened for its immunomodulatory and anti-inflammatory activity in non-clinical and clinical studies.

[0151] (d) Antimicrobial activity: Antibacterial activity of aqueous and solvent extracts was determined by agar well diffusion method. Inoculum containing 106CFU / ml of each bacterial culture to be tested was spread on nutrient agar plates. Subsequently, wells of 6 mm diameter were punched into the agar medium and filled with 20pl prepared formulation (as such and diluted with sterile saline) and allowed to diffuse at room temperature for 30 min. The plates were then incubated in the upright position at 37° for 24h. Wells containing the same volume of saline, extraction solvent and distilled water served as negative controls while standard antibiotic discs as positive control. After incubation, the diameters of the growth inhibition zones were measured in mm. Three replicates were carried out for each extract against each of the test organism. Results are shown in Table 12 below.

[0152] Table 12

[0153] (e) In silico screening of selected ingredients from formulation No. 7: Screening carried with the help of crystallographic structure of COVID-19 Mpro as ligand, Structure of CoV-2 was derived from protein data base RCSB PDB, using software Autodock (version 4.2.6.). Open Babel GUI 3.0 software was used for ligand to convert .sdf file in to .pdb format. Optimization of receptor was carried out by elimination of excess water molecules and heteroatoms. Addition of hydrogen atoms, kollman charges grid parameter was set as coordinates of X, Y and Z which were arranged as per active sites with grid box. Docking was processed by using genetic algorithm (GA) with addition of 1750000 generation by using Autodock tools, Auto dock vina and MGL tools. After interaction of molecules 10 best docking hits were selected depending upon average binding energy. Docking site and position were analysed by PyMol 2.3. In silico studies were carried out for only those ingredients showing interaction with Mpro (Protein target of SARS-Cov). Two-dimensional map was generated by using Discovery Studio Visualizer 2020 to understand receptor-ligand interaction. Results are listed in Table 13 below.

[0154] Table 13

[0155] FIGURES 3 to FIGURE 1 1 showcase the results of the docking analysis of SARS- CoV-2 MProbinding with sub-figures representing, for each compound above - (A) hydrophobicity surface 3D representation (B) 3D representation of tricin with SARS- CoV-2 MProinteraction (C) Interactions of tricin through H-bond in a pocket site of SARS-CoV-2 MPro(D) Interactions through hydrophobic bond in a pocket site of SARS-CoV-2 MPro(E) 2D representation in active site of SARS-CoV-2 MPro

[0156] (f) Acute toxicity of Formulation 7: Acute toxicity study was done in rats. Animals were administered with dose of formulation 300mg / kg and 2000mg / kg. The control group received water. Safety evaluation was carried out using parameters such as change in body weight, biochemical and haematological changes and vital organ histopathology.

[0157] An acute oral toxicity of Formulation No. 7 was been carried out using Wistar rats. Single oral administration was given to female and male rats up to a dose of 2000mg / kg. It does not show any mortality or signs of toxicity in animals. Change in body weight for male and female rats is shown in FIGURE 12 (A and B) respectively. Change in biochemical parameters for male and female rats is shown in FIGURE 13 (A and B) respectively. No Significant Change in Body Weight Data or Biochemical markers were observed, indicating no toxicity of Formulation No. 7. During the observation period of 14 days no adverse effect was seen in animals. Gross pathology observation of vital organs showed normal architecture and anatomy. During pharmacological screening of formulation No. 7, no signs of toxicity or mortality were observed in animals after repeated dosing.

[0158] Renal and Liver histopathological examination in male and female rats, as showcased in FIGURES 14 (A and B) and FIGURE 14 (C and D) respectively, established no evidence of toxicity. No Significant Change was observed in Liver function test.

[0159] (g) Delayed type hypersensitivity assay in SRBC immunized rat model for Formulation No. 7: Wistar rats were treated with test drug and vehicle as described in the experimental protocol. After blood collection, rats were challenged by injection of SRBC (0.5 x 109 cells / ml / 100 g) into the left hind foot pad. Isotonic saline was injected in right hind paw. Paw oedema was measured at 0 and 24h after SRBCs challenge using digital plethysmometer (Ugobasile, Italy). The pre- and post-challenge difference in the thickness of footpad was expressed in millimeter and specific paw swelling (D%) was calculated which will be taken as a measure of DTH 7. Effect on Antibody titer in Rats can be appreciated from the Table 14 below.

[0160] Table 14

[0161] Effect on Delayed Hypersensitivity in Rats can be appreciated from the Table 15.

[0162] Table 15

[0163] (h) Cyclophosphamide induced immunosuppression in mouse model study of Formulation No. 7: Mice were subjected to immunosuppression by hypodermic injection of CPA (70 mg / kg / day) on days 4, 8 and 12 to establish the immunosuppressive animal model, while mice in normal control group was administered with the same volume of sterile physiological saline solution. The animals were treated with test drugs for 14 days. On 14thday, the animals were anesthetized and blood was collected by retro-orbital plexus for platelet count, determination of IgG and TNF-a levels. Effect on IgG levels can be appreciated from the Table 16 below.

[0164] Table 16

[0165] (i) Carbon clearance: A carbon clearance test was used to determine the phagocytic index, on day 14, 2 hours after the last administration, mice weighed and then injected with Indian ink (0.1 mL / 10 g BW) via the tail vein. A 20 pL specimen of blood were collected from the retro-orbital plexus at 2 and 10 min immediately after ink injection. Blood samples mixed with 2 mL of 0.1 % Na2COs solution, and the absorbance measured at 650 nm. Afterwards, mice sacrificed by cervical dislocation. The spleen and liver were excised and immediately weighed. The rate of carbon clearance (K) and the phagocytic index (a) were calculated as follows-

[0166] Effect on formulation on Carbon Clearance can be readily realized from Table 17.

[0167] Table 17

[0168] To summarize, when Formulation no. 7 was evaluated for its immunomodulation action using rodent model, it showed reduction of haemagglutination by 76% in comparison to disease group and normalization of humoral immune response. It also showed decrease in inflammation and attenuates paw volume via delayed type hypersensitivity. Said Formulation no. 7 also showed normalization of Platelet count in comparison to disease control group when used in Cyclophosphamide induced immunocompromised rodent model. Infective agents virus / bacteria survive in body present in body require either activation of cytokine / chemokines for activation of immune action. Activation and increase in levels of IgG along with augmentation of phagocytic index and Carbon clearance. It also decreased the levels of TNF-a which is helpful in regulating inflammation.

[0169] Clinical trial of Formulation no. 7: A single blind randomized placebo-controlled phase I trial in healthy subjects aged between 18 to 65 years. Subjects received either formulation (10ml b.i.d.) or placebo for 30 days. Efficacy was evaluated by comparing the change in levels of immune cell (Lymphocyte, CD19+, CD3+, CD4+’ CD8+and NK cells). Also for safety evaluation changes in haematological and biochemical parameters were compared.

[0170] It was observed that HF-2 group showed increase in absolute count of other cells involved in immunity such as CD3+, CD19+(Helper T Cells), CD8+(Suppressor T cells) and NK cells (CD16+CD56). Observations of this study are as reflected in Table 18 below.

[0171] Table 18

[0172] In HF-2 group there was an increase (4.3%) in the mean absolute lymphocytes count though it was not significantly different (p-Value 0.207) on comparison of Day 30 with baseline. However, HF-2 showed significant difference in Mean change for Absolute count of lymphocytes in comparison to P-1 on Day 30 (P < 0.01 ).

[0173] Lymphocytes help the immune system fight disease (cancer) and infectious pathogens (viruses and bacteria), and they help the immune system for antigen recognition. HF-2 showed increase in the absolute count of CD19+(B-cells) with a value of 9.6% and this change was significantly different when compared with Baseline (Day 0) levels, whereas the change observed in other groups was not significantly different. CD19+ cells, also known as B cells, play a significant role in humoral immunity by producing antibodies that neutralize microorganisms and initiating signaling. After Completion of treatment the mean absolute count in P-1 showed a decrease by 4.5% in CD3+cell count. An increase of 6.4% was observed in HF- 2 when compared to baseline values. Although these changes were not statistically significant.

[0174] CD3+ cells are a type of T lymphocyte that plays a crucial role in the immune system by recognizing and responding to foreign antigens. They activate T cells, differentiate T cells which help immune system in recognizing and control pathogens. In HF-2 group 57% participants showed increase in CD4+ Cell count in comparison to P-1 group (40%). After activation, CD4+ T cells differentiate into distinct effector subtypes, including T helper 1 (Th1 ), T helper 2 (Th2), T helper 17 (Th17), and regulatory T cells (Tregs), which play a major role in mediating immune response through the secretion of specific cytokines. The count of CD8+cells was also increased in 50 % participants of HF-2 group in comparison to P-1 (40%). CD8+ T cells recognize and kill infected cells by recognizing specific antigens presented on the surface of the infected cells. This process is important for controlling viral infections, such as HIV and hepatitis C, and for preventing the development of cancer.

[0175] HF-2 treatment showed increase of 2.7% in absolute cell count level for CD3 / CD (16+56), P-1 group showed decrease in count by 28.2% which was significantly lower in comparison to Day 0 levels.

[0176] CD3 / CD16+56 cells are best known for killing virally infected cells and detecting and controlling early signs of cancer. They are also involved in natural cytotoxicity against infected cells without prior immunization, antibody-dependent cellular cytotoxicity (ADCC) against antibody-coated target cells, and cytokine and chemokine production and secretion, which not only are important in innate immunity but also influence the subsequent adaptive immune response.

[0177] A randomized placebo controlled double blind clinical trial was conducted to assess the effectiveness and safety of Composition 7 in SARS-COVID-19 patients. Patients were administered either Composition 7 (10ml t.i.d.) or a placebo for 10 days alongside standard care. The evaluation of efficacy was based on the recovery rate (change in RT-PCR positive cases) among subjects. Treatment with Composition 7 led to a reduction in the duration of hospitalization and achieved a recovery rate of 100% within 7 days compared to the standard treatment group's rate of 63.33%.

[0178] The average LDH levels fell within the normal range for both groups, which is 81 to 234 U / L. At the beginning of the study, the average LDH was 213.60 ± 45.42 U / L in composition 7 group and slightly lower than that of the Placebo Group at 228.97 ±80.14 U / L. On Day 10, there was a statistically significant increase of LDH (9.56%) in the placebo group and a decrease (4.18%) in composition 7 group (p value=0.046).

[0179] Initially, CRP levels were similar between composition 7 group (9.80±9.50 mg / L) and placebo (9.38±18.17 mg / l), with no statistical significance; after treatment completion however, there was a striking reduction of CRP by 63% among participants using composition 7. A reduction of 41 % seen in those who used placebos from baseline CRP values (p value=0.03).

[0180] The average IL-6 levels were 8.64 ± 12.41 pg / ml at baseline, with composition 7 and placebo measuring 5.65 ± 5.51 pg / ml, showing no significant difference between them. The composition 7 treated group exhibited a substantial reduction of 54.72% on Day 10 (p value=0.009) compared to the baseline IL-6 levels, while the decline in the placebo group was only 26.48%, which was not statistically significant

[0181] No adverse effects were reported during the clinical evaluation of composition 7 during the complete clinical trial and all the biochemical parameters were within normal range during the complete study.

[0182] SOURCING OF BIOLOGICAL INGREDIENTS

[0183] All biological ingredients included in this invention have been sourced from India using sustainable harvesting practices while keeping a core outlook for their ethical sourcing, cultivation, and conservation of said resources which preserves biodiversity, protects endangered species and supports the livelihoods of local communities. Sourcing information as to the biological ingredients used in this invention is provided in the Table 19 below.

[0184]

[0185] Table 19

[0186] Source and Place for access for all ingredients above: Purchased from Local market at Pune, Maharashtra, India.

[0187] From the foregoing narration, the reader shall appreciate that the advanced nano herbal formulation of this invention establishes an innovative approach to harness the therapeutic potential of herbal medicine while addressing challenges related to bioavailability, stability, and targeted delivery with high safety, efficacy, and absence of any long-term adverse effects.

[0188] Industrial applicability

[0189] The present invention has been applied to practice by the applicants named herein, and, through rigorous scientific experimentation, proven to have the following salient advantages- a) Enhanced Bioavailability (Assessed by Ex vivo intestinal absorption everted sac method): Hen (Gallus gallus domesticus) small intestine obtained from local slaughter house was excised by cutting across 10 cm from the upper end of the duodenum and 3 cm above the upper end of the colon and stripping the mesentery manually. The intestine was carefully washed with the cold Krebs Ringer (K R) bicarbonate Buffer solution having pH 7.3 to 7.4. using a syringe equipped with blunt end and then gently everted over a glass rod of 3 mm diameter. The everted intestine was then slipped off the glass rod and placed in a dish containing the KR buffer comprising 10 mM glucose and bubbled at 37°C. Then it was cut into segments of 5-7 cm length. One end of intestinal segment was clamped and tied with a silk suture. Then sacs were filled with one ml of KR buffer at 37°C using a 1 ml syringe. The filled intestinal sac was then slipped off the needle carefully and the loose ligature at the proximal end was then tightened. Evervted sacs were incubated with formulation- 7 (without nanotization), nanotaized formulation- 7 and HAVE extracts maintained with constant aeration at 37°C. At 2 hours, sacs were removed from incubation buffer, washed thoroughly with buffer and blotted dry. The sacs were cut open. The serosal fluid was collected into eppendorf tubes. Samples were proceeded for HPTLC fingerprinting and quantification of marker, results of which are shown in the accompanying FIGURE 15. b) Increased Stability: Prepared formulation-7 along with normal formulation was subjected to Accelerated stability studies as per ICH Guidelines and was evaluated for the change in Antioxidant (ICso) capacity as marker for stability. Change in IC50 value using DPPH inhibition action can be seen in Table 20 below.

[0190] Table 20 c) Improved Solubility: The solubility study of HAVE extract and Formulation-7 were determined by the shake-flask method. 1 ml of extract and formulation quantity equivalent to active was separately added into to 50 ml of water and vortex to facilitate mixing. Mixtures were shaken for 48 h in a reciprocating water bath shaker maintained at room temperature. After 48 h, each tube was centrifuged at 5000 RPM for 10 min, and clear water phase collected separately and filtration through a 0.45-pm membrane filter. The filtrate was suitably extracted with toluene and the

[0191] Table 21

[0192] As will be realized further, the present invention is capable of various other embodiments and that its several components and related details are capable of various alterations, all without departing from the basic concept of the present invention. Accordingly, the foregoing description will be regarded as illustrative in nature and not as restrictive in any form whatsoever. Modifications and variations of the system and apparatus described herein will be obvious to those skilled in the art. Such modifications and variations are intended to come within ambit of the present invention, which is limited only by the appended claims.

[0193] Dated this 03rdDay of April 2024

[0194] Duly constituted attorney for the applicant,

[0195] Rohit Nitin Deshpande

[0196] Advocate [MAH / 4858 / 2012] & Patent Agent [IN / PA-1389]

[0197] Address for service: Inventillect Consultants, Office No. 307, Business Guild Condominium, Apex Colony, ILS Law College Road, Erandwane, Pune, Maharashtra, India - 411004 Phone: +91 -9422944630 Email: rd@inventillect.com

Claims

ClaimsWe claim,1 ] A synergistic nano herbal formulation exhibiting immunomodulatory and antiviral activities, comprising a combination of hydro alcoholic volatile extracts at relative proportion of 0.05 % to 2.5 % w / w of Citrus limon extract; 0.01 % to 3.5 % w / w of Cymbopogon citratus extract; 0.01 % to 3.5 % w / w of Origanum vulgare extract; 0.01 % to 3.5 % w / w of Thymus serpyllum extract; 0.02 % to 2.5 % w / w of Tinospora cordifolia extract; 0.01 % to 3.5 % w / w of Withania somnifera extract; 0.5 % to 3.5 % w / w of Zea mays extract; 0.01 % to 3.5 % w / w of Meyna laxi flora extract; 0.01 % to 3.5 % w / w of Blumea lacera extract; 0.1 % to 3.0 % w / w of Boswellia serrata extract; 0.01 % to 3.5 % w / w of Cinnamomum verum extract; 0.1 % to 2.5 % w / w of Mentha pipperata extract; 0.1 % to 2.5 % w / w of Mentha arvensis extract; 0.01 % to 3.5 % w / w of Nigella sativa extract; 0.1 % to 3.5 % w / w of Cocos nucifera extract; 0.01 % to 2.0 % w / w of Flacourtia jangomas extract; 0.01 % to 1 .5 % w / w of Fagonia arabica extract; 0.5 % to 3.0 % w / w of Azadirachta indica extract; 0.1 % to 3.0 % w / w of Eucalyptus globulus extract; 0.01 % to 3.0 % w / w of Citrus sinensis extract, 0.5 % to 3.5 % w / w of Coriandrum sativum extract; 0.1 % to 3.2 % w / w of Saccharum % w / w officinarum extract; 0.5 % to 2.8 % w / w of Syzygium aromaticum extract; 0.2 % to 2.5 % w / w of Dolichos biflorus extract being admixed with excipients selected among 0.01 to 5% w / w Polyethylene glycol, 0.01 % to 5% Lecithin, 0.01 % to 5%TPGS 1000, 0.01 % to 5% Brij and water as a vehicle, said composition being nanotized via sonication for 25 to 30 minutes in a bath sonicator at Amplitude ranging between 20 to 45kHz to thereby attain an average particle size 133.2nm which is the final synergistic nano herbal formulation exhibiting immunomodulatory and antiviral activities being ready for use.2] The synergistic nano herbal formulation exhibiting immunomodulatory and antiviral activities as claimed in claim 1 , being formulated alternatively as one selected from among teas, tinctures, capsules, powders, and extracts.3] A method to prepare the synergistic nano herbal composition claimed in claim 1 , comprising- a) Selecting plant materials, said materials being selected between fresh and dried variants; b) Preparing separately, a hydro alcoholic volatile extract of each plant material selected;c) Filtering and combining the hydro alcoholic volatile extracts produced under reduced pressure conditions ranging between 100 to 235 mBar and temperature ranging between 50° to 75°C to obtain their admixed composition; d) Drying the admixed composition at 80-1 10°C in a vacuum tray dryer er until a moisture content of 7.5 to 15%, to form a dried admixed composition; e) Nano-sizing the dried admixed composition, by a nano-sizing process, to thereby form the synergistic nano herbal composition being ready for use. The method to prepare the synergistic nano herbal composition as claimed in claim3, wherein the step of preparing a hydro alcoholic volatile extract of each plant material selected consists of- a) Pulverizing the plant material selected using a mill until moderately coarse powder having average particle size passing through sieve No. 22 / 40 is obtained; b) Soaking the powdered plant material over 24 to 48 hours in water admixed at 1 to 3 times the weight of said powdered plant material; c) Distilling the soaked powdered plant material at a temperature ranging between 50-80°C over a period of 3 to 8 hours to obtain a distillate for use in preparation of the synergistic nano herbal composition; d) Oven-drying the marc at a temperature ranging between 50 to 80°C until completely dry to obtain a dry residue; e) Subjecting the dry residue of step (d) to Soxhlet extraction process with concentration of water : alcohol ranging between 60-95% to obtain an extract; and f) Repeating step e) for up to 3 to 8 times for durations ranging from 3 to 6 hours for obtaining a hydro alcoholic volatile extract of said plant material. The method to prepare the synergistic nano herbal composition as claimed in claim3, wherein the nano-sizing process consists of-(a) Admixing the Hydro alcoholic volatile extracts with water;(b) Sonicating the admixture of step (a) for 25 to 30 minutes in a bath sonicator to result in a nano-sized admixture; and(c) Homogenizing the nano-sized admixture for 5-10 min at 9000 to 10000 RPM to thereby form the synergistic nano herbal composition being ready for use.6] The method to prepare the synergistic nano herbal composition as claimed in claim 3, wherein the nano-sizing process consists of-(a) Admixing the Hydro alcoholic volatile extracts with an excipient;(d) Sonicating the admixture of step (a) for 25 to 30 minutes in a bath sonicator at Amplitude ranging between 20 to 45kHz and temperature ranging between 25 to 45°C to result in a nano-sized admixture; and(b) Homogenizing the nano-sized admixture for 5-10 min at 10000 to 15000 RPM to thereby form the synergistic nano herbal composition being ready for use7] The method to prepare the synergistic nano herbal composition as claimed in claim 3, wherein the plant materials are chosen among Citrus limon, Cymbopogon citratus, Origanum vulgare, Thymus serpyllum, Tinospora cordifolia, Withania somnifera, Zea mays, Meyna laxiflora, Blumea lacera, Boswellia serrata, Cinnamomum verum, Mentha pipperata, Mentha arvensis, Nigella sativa, Cocos nucifera, Fagonia arabica, Azadirachta indica, Eucalyptus globulus, Citrus sinensis, Coriandrum sativum, Saccharum officinarum, Syzygium aromaticum, Dolichos biflorus.Dated this 03rdDay of April 2024Duly constituted attorney for the applicant,Rohit Nitin DeshpandeAdvocate [MAH / 4858 / 2012] & Patent Agent [IN / PA-1389]Address for service: Inventillect Consultants, Office No. 307, Business Guild Condominium, Apex Colony, ILS Law College Road, Erandwane, Pune, Maharashtra, India - 411004 Phone: +91 -9422944630 Email: rd@inventillect.com

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