The present invention generally relates to a herbal composition. Specifically, the present invention relates to a polyherbal alcohol free composition comprising of Glycyrrhiza glabra root extract, Ocimum sanctum leaf extract, Elettaria cardamomum fruit extract, Mentha spicata (Spearmint) oil and Tween 80 and method of preparation thereof. The polyherbal alcohol free composition of the present invention possesses excellent antimicrobial properties and useful for oral cavity.
BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. [0003] Oral health has a great impact on the general quality of life, with poor oral hygiene causing varied systemic diseases and chronic complications. More than 700 bacterial species have been detected in the oral cavity (Aas et al., 2005). Some of these bacteria have been implicated in oral diseases such as caries and periodontitis. Conventional treatments for maintaining oral care are mainly based on mechanical techniques and oral hygiene procedures (Salzer et al., 2020). Although mechanical techniques are considered quite effective, achieving an adequate level of oral hygiene requires patients' motivation, skill and cooperation. Rinsing with chemical agents is an effective alternative oral hygiene technique, particularly for patients with limitations in the use of mechanical approaches and in the initial phase of oral disorders. Chlorhexidine has been presented as the most efficacious rinsing agent for maintaining overall oral health (Solderer et al., 2019; Cai et al., 2020). It has broad spectrum antiseptic and antimicrobial effects, mainly on gram positive bacteria, but also on gram negative organisms (Solderer et al., 2019). The most common reported side effects are notably staining of teeth and other oral surfaces, an increase in supragingival calculus formation and less

commonly, an alteration in taste perception (Cai et al., 2020; Chiewchalermsri et al., 2020).
[0004] Interest in herbal medicines has risen rapidly with improvements in analysis and quality control, in addition to advances in clinical research demonstrating side effects from chemical agents and the value of herbal medicine in treating and preventing disease. The major strength of the herbs used in dentistry is that no side effects have been reported following their use (Kumar et al., 2013). Furthermore, herbal based oral care products do not contain alcohol and/or sugar, unlike most chemical products (Moghadam et al., 2020). Microorganisms responsible for bad breath and halitosis, feed on these ingredients, and release by products that further exaggerate halitosis. Avoiding these ingredients is one step towards better oral hygiene.
[0005] Oral spray formulation contain therapeutically active phyto ingredients dissolved or suspended in solutions or mixtures of excipients (e.g., preservatives, emulsifiers, sweetener agents) in non-pressurized dispensers that deliver a spray containing a metered dose of the active ingredient. The dose can be metered by the spray pump or could have been pre-metered during formulation testing. A oral spray unit can be designed for unit dosing or can discharge up to several hundred metered sprays of formulation containing the herbal extracts. Oral sprays are applied to the oral cavity for local and/or systemic effects.
[0006] Glycyrrhiza glabra (commonly called as Liquorice) root is a medicinal herb which has been an integral part of Ayurveda and Chinese medicine for centuries. Liquorice is a sweet, moist, soothing herb that is native to Mediterranean countries and Asia (Sidhu et al., 2020). Due to its sweet taste, liquorice has been used worldwide as a sweetener and a flavouring agent in food and medicine production and is listed in the USA by the Food and Drug Administration (FDA) as generally recognized as safe (GRAS). Ayurvedic texts like Charaka and Sushruta have mentioned the role of liquorice in Dantaroga (diseases of teeth) and Dantmamsaroga (diseases of gums). Recently the benefits of liquorice in oral diseases have been of great interest. Pharmacological

effects of liquorice (Pastorino et al., 2018; Sidhu et al., 2020). Clinical trials have been conducted worldwide to evaluate the effects of liquorice and its metabolites in preventing and treating various oral diseases such as dental caries, periodontal diseases, candidiasis, aphthous ulcers and debilitating diseases like oral cancer. Moreover, liquorice has also been studied as a root canal medicament which can prevent failed root canal therapies and lead to higher success rate of the treatment (Sidhu et al., 2020).
[0007] The genus Ocimum includes aromatic herbs yielding essential oils, and has tropical distribution with nearly two-third of the 160 species reported from West Africa and the remaining one- third from Asia and America (Singh et al., 2018). India is represented by nine species of Ocimum, among which Ocimum sanctum L. (also known as holy basil) is one of them. Renowned as a general tonic and vitalizer, "The Elixir of Life", O. sanctum has been traditionally employed in hundreds of different formulations for the treatment of a wide range of disorders, including those of the mouth and throat, lungs, heart, blood, liver, kidney, and the digestive, metabolic, reproductive and nervous systems (Pattanayak et al., 2010). It has been a component of oral health, dental products, and has been used in the fragrance and food industry. Preclincal and clinical investigations have validated the antimicrobial effects of basil against bacterial, fungal and viral strains (Gupta et al., 2008).
[0008] Elettaria cardamomum (popularised as cardamom), often referred to as queen of spices because of its very pleasant aroma and taste, has a history as old as human race. It is especially common in the Indian subcontinent and is known for its delicious aroma, aphrodisiac properties and also as a common folk remedy to treat stomach aches (Ashokkumar et al., 2019). Both in Middle East and India, it is used for flavoring hot beverages such as tea and coffee as well as sweets. But the most common use of cardamom in India has been as an excellent breath freshener. Besides, flavouring agent it possess antimicrobial effects against oral pathogenic organisms like Streptococcus mutans and Candida albicans (Abdullah etal., 2017).

[0009] In the recent times the use of herbal based products for oral care is on the rise due to the spread in the awareness of the effect of complementary and alternative medicine. It is also due to the much stronger belief that the alternative therapy is with less side effects. They are also popular in dentistry due to their antimicrobial, and antioxidant properties (Byeon et al., 2018). There is a dynamic and rapid accumulation of clinical trials showing that herbal exacts and essential oils can probably be an effective treatment to maintain oral hygiene including microbial infections. There are numerous natural ayurvedic plants which have a great significance in dentistry (Byeon et al., 2018). Previous literature suggest that herbal/natural product for management of dental ailments has shown favorable results where some of these natural plants have been used for preparation of oral care products such as green tea, turmeric, neem, cranberry, Aloe vera, pot marigold, and triphala, and studies conducted have proved to be effective in reducing plaque accumulation and gingival inflammation (Taheri et al., 2011). [0010] Majority of oral health issues are due to bacterial and fungal infections. The most prevalent oral conditions are dental caries and periodontal diseases, and they are largely preventable. Dental caries, an irreversible chronic disease initiated by microbes, causes the dissolution of tooth mineral (primarily hydroxyapatite, CalO (P04)6(0H)2) by acids derived from bacterial fermentation of sucrose and other dietary carbohydrates. Periodontal disease is an inflammatory response of the gingiva and surrounding connective tissue to the bacterial or plaque accumulations on the teeth (Colombo et al., 2015). Bacteria in the plaque touching the tissue elaborate various compounds, such as H2S, NH3, amines, endotoxins, enzymes (such as collagenases) and antigens, all of which penetrate the gingiva and elicit an inflammatory response (Colombo et al., 2015; Arshad et al., 2020). In the absence of effective oral hygiene, dental plaque can accumulate to levels that are no longer compatible with health and microbial homeostasis breaks down, thereby predisposing sites to disease. Moreover, poor oral hygiene is now considered as a one of the major risk factors for COVID-19 complications, especially in patients with diabetes, hypertension or cardiovascular disease (Sampson et al., 2020). Therefore, it recommended improving oral hygiene during

a COVID-19 infection in order to reduce the bacterial load in the mouth and the risk of a bacterial superinfection. Hence, using an oral care product with strong antimicrobial effects seems to be a viable strategy for maintaining oral health. In the present study, the prepared optimized mouth spray showed strong inhibitory effects against E. coli, S. aureus and C. albicans and these effects were statistically similar with chlorhexidine gluconate. Also, the prepared optimized formulation exhibited strong antioxidant effects. Glycyrrhiza glabra root, Ocimum sanctum leaf and Elettaria cardamomum fruit are reported to contain diverse armoury of chemical compounds including flavonoids, tannins, terpenoids, saponins etc that are documented to have wide array of biological effects including antioxidant and anti-inflammatory activities (Pattanayak et al., 2010; Pastorino et al., 2018; Ashokkumar et al., 2019; Sidhu et al., 2020). [0011] There is, therefore, a need to develop a novel polyherbal alcohol free oral spray that is safe, economical and having antimicrobial effects.
OBJECTS OF THE INVENTION
[0012] An object of the present invention is to provide a polyherbal oral spray
formulation.
[0013] Another object of the present invention is to provide a polyherbal
alcohol free composition useful for oral cavity.
[0014] Another object of the present invention is to provide a polyherbal
alcohol free composition having excellent antimicrobial activity useful for oral
cavity.
SUMMARY OF THE INVENTION
[0015] The present invention relates to a polyherbal alcohol free composition comprising of Glycyrrhiza glabra root extract, Ocimum sanctum leaf extract, Elettaria cardamomum fruit extract, Mentha spicata (Spearmint) oil and Tween 80 and method of preparation thereof. The polyherbal alcohol free composition of the present invention possesses excellent antimicrobial properties and useful for oral cavity.

[0016] In one aspect, the present invention relates to a polyherbal alcohol free composition comprising of:
(a) Glycyrrhiza glabra root extract;
(b) Ocimum sanctum leaf extract;
(c) Elettaria cardamomum fruit extract;
(d) Mentha spicata (Spearmint) oil; and
(e) Tween 80.
[0017] In another aspect of the present invention, the composition further
comprises optionally Stevioside.
[0018] In another aspect of the present invention, the composition further
comprises water in sufficient quantity.
[0019] In another aspect, the present invention relates to a polyherbal alcohol
free composition comprising of:
(a) Glycyrrhiza glabra root extract in an amount ranges from 0.2% to 1.5 % w/v of the composition;
(b) Ocimum sanctum leaf extract in an amount ranges from 0.15% to 1.25 % w/v of the composition;
(c) Elettaria cardamomum fruit extract in an amount ranges from 0.1%) to 1%> w/v of the composition;
(d) Mentha spicata (Spearmint) oil in an amount ranges from 0.1% to 1%> w/v of the composition; and
(e) Tween 80 in an amount ranges from 0.25% to 1% w/v of the composition.
[0020] In another aspect, the present invention relates to a polyherbal alcohol free composition comprising of:
(a) Glycyrrhiza glabra root extract in an amount ranges from 0.2% to 1.5 % w/v of the composition;
(b) Ocimum sanctum leaf extract in an amount ranges from 0.15% to 1.25 % w/v of the composition;
(c) Elettaria cardamomum fruit extract in an amount ranges from 0.1%) to 1%) w/v of the composition;

(d) Mentha spicata (Spearmint) oil in an amount ranges from 0.1% to 1%> w/v of the composition;
(e) Tween 80 in an amount ranges from 0.25% to 1% w/v of the composition;
(f) optionally stevioside in an amount ranges from 0.01% to 0.1% w/v of the composition; and
(g) water q.s.
[0021] In yet another aspect of the present invention, wherein pH of the
composition is in the range of 6 to 6.2.
[0022] In yet another aspect of the present invention, wherein the composition
is in the form of spray or solution.
[0023] In another aspect, the present invention relates to an oral spray poly
herbal alcohol free formulation comprising a combination of:
(a) Glycyrrhiza glabra root extract;
(b) Ocimum sanctum leaf extract;
(c) Elettaria cardamomum fruit extract;
(d) Mentha spicata (Spearmint) oil;
(e) Tween 80;
(f) optionally stevioside; and
(g) water q.s.
[0024] In another aspect, the present invention relates to an oral spray poly herbal alcohol free formulation comprising a combination of:
(a) Glycyrrhiza glabra root extract;
(b) Ocimum sanctum leaf extract;
(c) Elettaria cardamomum fruit extract;
(d) Mentha spicata (Spearmint) oil;
(e) Tween 80;
(f) optionally stevioside; and
(g) water q.s.
[0025] In another aspect, the present invention relates to an oral spray poly herbal alcohol free formulation comprising a combination of:

(a) Glycyrrhiza glabra root extract in an amount ranges from 0.2% to 1.5 % w/v of the composition;
(b) Ocimum sanctum leaf extract in an amount ranges from 0.15% to 1.25 % w/v of the composition;
(c) Elettaria cardamomum fruit extract in an amount ranges from 0.1%) to 1%> w/v of the composition;
(d) Mentha spicata (Spearmint) oil in an amount ranges from 0.1% to 1%> w/v of the composition;
(e) Tween 80 in an amount ranges from 0.25% to 1% w/v of the composition;
(f) optionally stevioside in an amount ranges from 0.01% to 0.1% w/v of the composition; and
(g) water q.s
[0026] In yet another aspect, the present invention relates to a method of preparing an oral spray a polyherbal alcohol free composition comprising the steps of:
(a) mixing the ingredients with water to obtain a solution; mays seed fine powder;
(b) filtering the solution to obtain a filtrate; and
(c) packing the filtrate in spray bottle to obtain the oral spray polyherbal alcohol free composition.
[0027] In another aspect, the composition of the present invention possesses
excellent antimicrobial activity.
[0028] In another aspect, the composition of the present invention is useful for
oral cavity.
[0029] Various objects, features, aspects and advantages of the inventive
subject matter will become more apparent from the following detailed description
of preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 depicts the zone of inhibition (diameter) against test organisms, A: Formulation of the present invention and B: Standard Control. [0031] Figure 2 shows the in-vitro antimicrobial swab test.
DETAILED DESCRIPTION OF THE INVENTION
[0032] The following is a detailed description of embodiments of the
disclosure. The embodiments are in such detail as to clearly communicate the
disclosure. However, the amount of detail offered is not intended to limit the
anticipated variations of embodiments; on the contrary, the intention is to cover all
modifications, equivalents, and alternatives falling within the spirit and scope of
the present disclosure as defined by the appended claims.
[0033] Unless the context requires otherwise, throughout the specification
which follow, the word "comprise" and variations thereof, such as, "comprises"
and "comprising" are to be construed in an open, inclusive sense that is as
"including, but not limited to."
[0034] Reference throughout this specification to "one embodiment" or "an
embodiment" means that a particular feature, structure or characteristic described
in connection with the embodiment is included in at least one embodiment. Thus,
the appearances of the phrases "in one embodiment" or "in an embodiment" in
various places throughout this specification are not necessarily all referring to the
same embodiment. Furthermore, the particular features, structures, or
characteristics may be combined in any suitable manner in one or more
embodiments.
[0035] As used in this specification and the appended claims, the singular
forms "a," "an," and "the" include plural referents unless the content clearly
dictates otherwise. It should also be noted that the term "or" is generally
employed in its sense including "and/or" unless the content clearly dictates
otherwise.
[0036] In some embodiments, the numbers expressing quantities of
ingredients, properties such as concentration, reaction conditions, and so forth,

used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
[0037] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. [0038] All processes described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. [0039] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments. [0040] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered

to include other remaining combinations of A, B, C, or D, even if not explicitly
disclosed.
[0041] All publications herein are incorporated by reference to the same
extent as if each individual publication or patent application were specifically and
individually indicated to be incorporated by reference. Where a definition or use
of a term in an incorporated reference is inconsistent or contrary to the definition
of that term provided herein, the definition of that term provided herein applies
and the definition of that term in the reference does not apply.
[0042] Groupings of alternative elements or embodiments of the invention
disclosed herein are not to be construed as limitations. Each group member can be
referred to and claimed individually or in any combination with other members of
the group or other elements found herein. One or more members of a group can be
included in, or deleted from, a group for reasons of convenience and/or
patentability. When any such inclusion or deletion occurs, the specification is
herein deemed to contain the group as modified thus fulfilling the written
description that follows, and the embodiments described herein, is provided by
way of illustration of an example, or examples, of particular embodiments of the
principles and aspects of the present disclosure. These examples are provided for
the purposes of explanation, and not of limitation, of those principles and of the
disclosure.
[0043] It should also be appreciated that the present invention can be
implemented in numerous ways, including as a system, a method or a device. In
this specification, these implementations, or any other form that the invention may
take, may be referred to as processes. In general, the order of the steps of the
disclosed processes may be altered within the scope of the invention.
[0044] Various terms as used herein are shown below. To the extent a term
used in a claim is not defined below, it should be given the broadest definition
persons in the pertinent art have given that term as reflected in printed
publications and issued patents at the time of filing.
[0045] In a general embodiment, the present invention relates to a polyherbal
alcohol free composition comprising of Glycyrrhiza glabra root extract, Ocimum

sanctum leaf extract, Elettaria cardamomum fruit extract, Mentha spicata
(Spearmint) oil and Tween 80 and method of preparation thereof. The polyherbal
alcohol free composition of the present invention possesses excellent
antimicrobial properties and useful for oral cavity.
[0046] The term "about" as used herein refers to a defined range of the value
by plus or minus 10 %. For example, about 2 % means 1.8 % to 2.2 %, about 5 %
means 4.5 % to 5.5 %, about 10 % means 9 % to 11 % and about 40 % means 36
% to 44 %.
[0047] As used herein, "Tween 80" is a polysorbate 80 surfactant which helps
in stabilizing.
[0048] In an embodiment, the present invention relates to a polyherbal alcohol
free composition comprising of:
(a) Glycyrrhiza glabra root extract;
(b) Ocimum sanctum leaf extract;
(c) Elettaria cardamomum fruit extract;
(d) Mentha spicata (Spearmint) oil;
(e) Tween 80;
(f) optionally Stevioside and
(g) water q.s.
[0049] In another embodiment, the present invention relates to an oral spray polyherbal alcohol free composition comprising of:
(a) Glycyrrhiza glabra root extract;
(b) Ocimum sanctum leaf extract;
(c) Elettaria cardamomum fruit extract;
(d) Mentha spicata (Spearmint) oil;
(e) Tween 80;
(f) optionally Stevioside and
(g) water q.s.
[0050] In another embodiment of the present invention, the Glycyrrhiza glabra root extract is present in an amount ranges from about 0.2% to about 1.5 % w/v of the composition.

[0051] In another embodiment of the present invention, the Ocimum sanctum
leaf extract is present in an amount ranges from about 0.15% to about 1.25 % w/v
of the composition.
[0052] In another embodiment of the present invention, the Elettaria
cardamomum fruit extract is present in an amount ranges from about 0.1% to
about P/o w/v of the composition.
[0053] In another embodiment of the present invention, the Mentha spicata
(Spearmint) oil is present in an amount ranges from about 0.1% to about 1% w/v
of the composition.
[0054] In another embodiment of the present invention, the Tween 80 is
present in an amount ranges from about 0.25% to about 1% w/v of the
composition.
[0055] According to the present invention, the oral/mouth spray was brown in
colour and has pleasant taste and odour. In an embodiment, pH of the mouth spray
of the present invention is in the range of 6.0 to 6.2. Preferably, pH is 6.1.
[0056] The present invention provides a simple and cost-effective method for
preparation of an oral/mouth spray polyherbal alcohol free composition.
[0057] In yet another embodiment of the present invention, the polyherbal
alcohol free composition is prepared in the form of spray, liquid, or cream. The
person having ordinary skill in the art would easily prepare the various forms of
the polyherbal alcohol free composition using the common general knowledge
and routine experimentation based on the composition of the present invention.
[0058] The polyherbal alcohol free composition of the present invention
possesses excellent antimicrobial properties.
[0059] In an embodiment, the polyherbal alcohol free composition of the
present invention is useful in the treatment of oral cavity and mouth related issues.
[0060] While the foregoing describes various embodiments of the disclosure,
other and further embodiments of the disclosure may be devised without departing
from the basic scope thereof. The scope of the invention is determined by the
claims that follow. The invention is not limited to the described embodiments,
versions or examples, which are included to enable a person having ordinary skill

in the art to make and use the invention when combined with information and knowledge available to the person having ordinary skill in the art. EXAMPLES
[0061] The present invention is further explained in the form of following examples. However, it is to be understood that the following examples are merely illustrative and are not to be taken as limitations upon the scope of the invention. [0062] Procurement of ingredients: Standardized Glycyrrhizin glabra root aqueous extract, Ocimum sanctum leaf aqueous extract, Elettaria cardamomum fruit aqueous extract, stevioside, Mentha spicata (Spearmint) oil and menthol were given by Hindustan Mint & Agro Products, Pvt. Ltd., Chandausi, Uttar Pradesh, India as generous gift samples [0063] Example 1 [0064] Ingredients

S.No Ingredient Amount
(% w/v)
1 Glycyrrhiza glabra root extract Antimicrobial and sweetening agent 1.0
2 Ocimum sanctum leaf extract Antimicrobial agent 0.75
3 Elettaria cardamomum fruit extract Flavouring agent 0.50
4 Stevioside Natural agent 0.02
5 Mentha spicata (Spearmint) oil Flavouring agent 0.17
6 Tween 80 Surfactant 0.5
7 Water q.s. 10 ml
[0065] Each ingredient was carefully weighed and mixed together in 10 ml water. The prepared formulation was filtered through Whatman filter paper and packed in spray bottle for further use.

[0066] Example 2-5: The following formulation was prepared by following the method of example 1.

Ex.
No Glycyrrhiza glabra
(%w/v) Ocimum sanctum
(%w/v) Elattaria cardamomum
(%w/v) Spearmint
(%v/v) Tween 80
2 0.50 0.38 0.25 0.17 0.5
3 0.33 0.25 0.17 0.17 0.5
4 0.25 0.19 0.13 0.17 0.5
5 0.20 0.15 0.10 0.17 0.5
Preparation of microbial culture
[0067] A loop full of lyophilized bacterial [S. aureus, (MTCC-1430), E. Coli (MTCC-1687)] and fungal [C. Albican (MTCC-227)] strains were inoculated, separately, in nutrient broth and sabouraud dextrose broth, respectively and incubated at 37 °C for 48-72h. The strains were later sub-cultured in respective sterilised media and 24h old culture was employed for further analysis (Rodriguez-Tudela et al., 2001; Yasuyuki et al., 2010; Ewnetu et al., 2013). Cell count by turbidometric method
[0068] Active bacterial and fungal cultures (24h old) were analysed spectrophotometrically at 600 nm and 530 nm respectively, taking nutrient media as blank. The absorbance value of the culture was adjusted to achieve final culture concentration equivalent to 0.5 McFarland standards. The sample was diluted with broth till absorbance values reaches the detectable limits and the total cell count in sample was calculated by multiplying number of colonies with the dilution employed (Rodriguez-Tudela et al., 2001; Ewnetu et al., 2013). Evaluation of minimum inhibitory concentration (MIC)
[0069] 100 uL of prepared cell suspensions (S. aureus, E. coli and C. albicans strain; OD600&530 = 1.0) was added separately to the test tubes containing 5 mL of prepared different formulations. The test tubes were incubated at 37 °C for 5-7 h. Thereafter, 50 uL of this suspension was spread over the petriplates containing sterilized respective agar media and incubated at 37 °C for 24-48h. The petriplates

were then analysed for cell count and the formulation with minimum
concentration which exhibited no growth was taken as MIC value.
Physical evaluation of the formulation
[0070] Colour, odour and consistency
[0071] The formulation was physically examined for colour, odour and
consistency.
[0072] pH : The pH of formulation was measured using digital pH meter.
Test for microbial growth in formulation
[0073] 50 uL of the mouth spray of the present invention was spread over the
plates containing sterilised solidified agar media with the help of sterilised
spreader. Simultaneously positive and negative control media was prepared
containing microbial strains and blank media respectively. The plates were
incubated at 37°C for 24-48h. After the incubation period plates were taken out
and checked for microbial growth and were compared with the control (Malik et
al., 2020).
Determination of antioxidant activity of the formulation
[0074] The antioxidant activity of the formulation of the present invention
was determined using DPPH assay. One ml of DPPH (0.1 mM in methanol) was
mixed with 1 ml of the formulation and the mixture was allowed to incubate in
dark for 30 minutes. A control containing 1 ml DPPH and 1 ml methanol was also
prepared and incubated for 30 minutes in dark. After incubation the absorbance of
mixture was measured at 415 nm using UV-Vis spectrophotometer and percentage
of DPPH inhibition was calculated by (Singh et al., 2016; Kaur et al., 2019; Pruthi
etal., 2021).
% inhibition = [(Abs of control - Abs of sample)/ Abs of control] x 100 Determination of zone of inhibition of the formulation
[0075] Agar diffusion method was employed to evaluate the antimicrobial potential of the formulation of the present invention against bacterial and fungal strains and compared with that of equivalent concentration of marketed preparation of chlorhexidine gluconate (Hexidine®). Bacterial strain was grown in nutrient agar media while sabouraud dextrose agar media was employed for the

growth of fungal strain. 50 uL of active inoculum (equivalent to 0.5 McFarland
standards) was then spread over the surface of the solidified media base with the
help of sterilized spreader. Wells were made in the solidified medium using sterile
borer and were filled with 100 uL the test material (Example 3, commercial
preparation and solvent). Sterilised water was used as a solvent control. The plates
were then incubated at 37°C for 24-48h. Anti-microbial activity of the test sample
was determined from the diameter of zone of inhibition (ZOI) and compared with
that of control and the standard (Dua et al., 2015; Malik et al., 2020).
Swab Test
[0076] Buccal swab test was performed to determine and compare the in-vivo
antimicrobial potentials of the formulation of the present invention with that of
commercial preparations. Nutrient agar media was opted as a medium for growth
analysis. For this, sterilised cotton swab was rubbed/swiped over the inner buccal
area (mouth, gums, tongue and teeth) separately before and after the application of
formulation (Example 3 and commercial preparation) and was then dipped in the
5ml of sterilised water for injection. 50ul of the above sample was taken and
spread over the sterilised solidified nutrient agar medium in petriplate using
spreader. The samples were later incubated at 37°C for 24h and observed for
growth and compared with that of negative control (only media) (Taraghi et al.,
2011).
Result
MIC value of herbal mouth spray
[0077] Results of the present study showed that commercially available 0.2%
chlorhexidine gluconate mouthwash and the herbal mouth spray of the present
invention were effective against test organisms. Formulation of Examples 1-5
showed antibacterial and antifungal efficacy. To calculate MIC, the formulations
were tested for its inhibitory effects against both test bacterial and fungal strains.
The results showed that minimum amount of extracts required to exhibit
antimicrobial effects were present in the formulation of example 3.
[0078] Physical evaluation of the mouth spray of the present invention

[0079] The colour, odour, taste and consistency of the mouth spray are shown in the table below. The pH of the formulation was found to be 6.1. The formulation was found to be free from microbes as it did not produce any microbial growth when inoculated in the agar medium. [0080] Physical evaluation of the mouth spray

Parameter Result
Color Brown
Odor Pleasant
Taste Sweet
Consistency Liquid
pH 6.1
Zone of inhibition
[0081] Agar diffusion assay revealed that the formulation of the present invention exhibited the antimicrobial effects, against varied bacterial and fungal strains, similar to that of commercial preparation as could be observed from the table given below and figure 1 depicting almost similar zone of inhibition. [0082] Zone of inhibition (diameter) against test organisms

Formulation E. coli S. aureus C. albicans
Formulation of Example 3 1.8 cm 1.7 cm 2.8 cm
0.2% Chlorhexidine gluconate (Hexidine®) 2.0 cm 1.8 cm 3.0 cm
DPPH scavenging activity of the mouth spray of the present invention
[0083] The mouth spray of the present invention showed demonstrated
appreciable antioxidant effects. It showed 92.74 % inhibition of DPPH.
[0084] Swab Test
[0085] Analysis of salivary samples from oral cavity, incubated 24h in
nutrient media, displayed the absence of any growth and the results were found to

be similar to that of negative control (Figure 2). However the samples taken before the formulation application exhibited the microbial colonization (Figure 2). Thus, the formulation of the present invention could be concluded to possess antimicrobial activity and effective against an array of microbial strains persistent in the oral cavity.
ADVANTAGES OF THE PRESENT INVENTION
[0086] The advantageous properties of the instant formulation of the present invention are as follows:
• provides a safe and effective practice of oral hygiene,
• eradicates harmful oral microbial load,
• removes bad breath,
• reduces chances of microbial infection in oral cavity,
• provides long lasting mouth freshness,
• easy to carry, easy to handle, easy to use,
• cost effectiveness , and
• useful in COVID-19, Flu, Microbial Diseases of the Mouth and Oral Cavity.
[0087] A skilled artisan will appreciate that the quantity and type of each
ingredient can be used in different combinations or singly. All such variations and
combinations would be falling within the scope of present disclosure
[0088] The foregoing examples are merely illustrative and are not to be taken
as limitations upon the scope of the invention. Various changes and modifications
to the disclosed embodiments will be apparent to those skilled in the art. Such
changes and modifications may be made without departing from the scope of the
invention.
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We Claim:
1. A polyherbal alcohol free composition comprising combination of:
(a) Glycyrrhiza glabra root extract;
(b) Ocimum sanctum leaf extract;
(c) Elettaria cardamomum fruit extract;
(d) Mentha spicata (Spearmint) oil; and
(e) Tween 80.

2. The polyherbal alcohol free composition as claimed in claim 1, wherein the composition further comprises optionally Stevioside.
3. The polyherbal alcohol free composition as claimed in claim 1 or 2, wherein the composition further comprises water in sufficient quantity.
4. The composition as claimed in claim 1, wherein the composition comprisies:

(a) Glycyrrhiza glabra root extract in an amount ranges from 0.2% to 1.5 % w/v of the composition;
(b) Ocimum sanctum leaf extract in an amount ranges from 0.15% to 1.25 % w/v of the composition;
(c) Elettaria cardamomum fruit extract in an amount ranges from 0.1%) to 1%> w/v of the composition;
(d) Mentha spicata (Spearmint) oil in an amount ranges from 0.1% to 1%> w/v of the composition; and
(e) Tween 80 in an amount ranges from 0.25% to 1% w/v of the composition.
5. The polyherbal alcohol free composition as claimed in claim 2, wherein the
composition comprises:
(h) Glycyrrhiza glabra root extract in an amount ranges from 0.2% to
1.5 % w/v of the composition;
(i) Ocimum sanctum leaf extract in an amount ranges from 0.15% to
1.25 % w/v of the composition;
(j) Elettaria cardamomum fruit extract in an amount ranges from
0.1%) to 1%) w/v of the composition;

(k) Mentha spicata (Spearmint) oil in an amount ranges from 0.1% to
1% w/v of the composition;
(1) Tween 80 in an amount ranges from 0.25% to 1% w/v of the
composition;
(m) optionally stevioside in an amount ranges from 0.01% to 0.1% w/v
of the composition; and
(n) water q.s.
6. The polyherbal alcohol free composition as claimed in any one of the claims 1 to 5, wherein the pH of the composition is in the range of 6 to 6.2.
7. The polyherbal alcohol free composition as claimed in any one of the claims 1 to 6, wherein the composition is in the form of spray or solution.
8. A mouth spray poly herbal alcohol free formulation comprising a combination of:
(h) Glycyrrhiza glabra root extract;
(i) Ocimum sanctum leaf extract;
(j) Elettaria cardamomum fruit extract;
(k) Mentha spicata (Spearmint) oil;
(1) Tween 80;
(m) optionally stevioside; and
(n) water q.s.
9. A mouth spray poly herbal alcohol free formulation comprising a combination
of:
(h) Glycyrrhiza glabra root extract in an amount ranges from 0.2% to
1.5 % w/v of the composition;
(i) Ocimum sanctum leaf extract in an amount ranges from 0.15% to
1.25 % w/v of the composition;
(j) Elettaria cardamomum fruit extract in an amount ranges from
0.1%) to 1%> w/v of the composition;
(k) Mentha spicata (Spearmint) oil in an amount ranges from 0.1% to
1%) w/v of the composition;

(1) Tween 80 in an amount ranges from 0.25% to 1% w/v of the
composition;
(m) optionally stevioside in an amount ranges from 0.01%> to 0.1%> w/v
of the composition; and
(n) water q.s.