DESC:FIELD OF INVENTION

[001] The present invention relates to a herbal composition for treating throat related infections and a method for preparation thereof. Particularly, the present invention relates to a herbal composition exhibiting potential antiviral, anti-inflammatory, antibacterial, and antimicrobial action that effectively relieves sore throat, tonsillitis and other throat related infections.

BACKGROUND OF THE INVENTION
[002] There has been an increase in respiratory tract infections due to the presence of abundant pathogens around us. Some of the most common respiratory tract infections include cold, bronchitis, sinusitis, laryngitis, etc. Such infections are caused by a number of harmful virus and bacteria. The major symptoms of respiratory tract infections include sore throat, cough, sneezing, headache, etc.

[003] There are various conventional methods which intend to provide relief from sore throat and other throat related infections. Some conventional methods include the use of antibiotics. However, excessive consumption makes body resistant to antibiotics. Moreover, antibiotics are associated with a number of side effects such as diarrhea, abdominal pain and so on. Other conventional methods include the consumption of lozenges. However, lozenges are effective for only a short period of time. Some other conventional methods include readymade mouth wash for use as gargling solution.

[004] However, such mouthwashes are astringent in nature. Moreover, such mouth wash may contain some harmful compounds such as Chlorhexidine which may cause allergy and worsen the infected area. Yet other conventional methods include spray with numbing compounds. However, such sprays are difficult to use and may result in unwanted numbing of tongue and surrounding areas.

[005] Therefore, keeping in view the problems associated with the state of the art there is a need for a natural, safe and efficient means for providing relief from sore throat, tonsillitis and other throat related infections.
OBJECTIVES OF THE INVENTION
[006] The primary objective of the present invention is to provide a herbal composition for treating throat related infections and a method for preparation thereof.

[007] Another objective of the present invention is to enhance immune response of the body by providing a herbal composition exhibiting medicinal properties.

[008] Another objective of the present invention is to provide antiviral, anti-inflammatory and antibacterial composition for providing effective relief from sore throat, tonsillitis, and other throat- related infections.

[009] Yet another objective of the present invention is to provide a herbal composition comprising a number of essential oils ensuring persisting antimicrobial action and less side effects.

[0010] Yet another objective of the present invention is to provide a herbal mouth rise for gargling to treat sore throat, tonsillitis and other throat related infections.

[0011] Other objects and advantages of the present invention will become apparent from the following description taken in connection with the accompanying drawings, wherein, by way of illustration and example, the aspects of the present invention are disclosed.

BRIEF DESCRIPTION OF DRAWINGS
[0012] The present invention will be better understood after reading the following detailed description of the presently preferred aspects thereof with reference to the appended drawings, in which the features, other aspects and advantages of certain exemplary embodiments of the invention will be more apparent from the accompanying drawing in which:

[0013] Figure 1 illustrates graphical representation depicting percentage reduction in antimicrobial activity of several pathogens by time kill assay;

[0014] Figure 2 illustrates graphical representation depicting anti-inflammatory activity by inhibition of inflammatory cytokine (IL-6) in Immune cells;

[0015] Figure 3 illustrates graphical representation depicting anti-allergic property by inhibition of histamine in immune cells;

[0016] Figure 4 illustrates graphical representation depicting analgesic property by inhibition of COX-2 activity; and

[0017] Figure 5 illustrates graphical representation depicting mucolytic property by inhibition of egg viscosity.

SUMMARY OF THE INVENTION
[0018] The present invention relates to a herbal composition for treating throat related infections and a method for preparation thereof. The herbal composition comprises of one or more essential oils, one or more herbal extracts; at least one solubilizing agent, a salt; one or more additives, and water. The essential oils are present in a range of 1% to 12% weight by volume of the total composition. The herbal extracts are present in a range of 1% to 20% weight by volume of the total composition. The solubilizing agents are present in a range of 1% to 16% weight by volume of the total composition. The salt is present in the range of 8% to 12% weight by volume of the total composition. The additives are present in a range of 11% to 15% weight by volume of the total composition. Water is present in a range of 25% to 40% by weight of the total composition. The composition of the present invention is prepared using a microemulsion technique. The composition of the present invention possesses potential antiviral, anti-inflammatory, antibacterial, and antimicrobial action that effectively relieves sore throat and other throat related infections.

SOURCE OF BIOLOGICAL MATERIAL USED IN THE INVENTION
[0019] The essential oils used in the herbal composition including such as, but not limited to, Lemon oil, Rosemary oil, Ginger oil, Peppermint oil, Cinnamon oil, Basil oil, Clove oil, or a combination thereof are procured from vendors. The herbal extracts used in present invention including such as, but not limited to, Kalmegh extract, Licorice extract, Ginseng extract, Tea extract, or a combination thereof are also procured from vendors. The test strains used in the present invention for ensuring validity of the tests for total aerobic microbial count including Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Candida albicans are procured from American Type Culture collection (ATCC). The ATCC number of the test strains includes ATCC 6538 for Staphylococcus aureus, ATCC 6633 for Bacillus subtilis, ATCC 8739 for Escherichia coli, and ATCC 2091 and ATCC 10231 for Candida albicans. The test strains used for determining microbial contamination including Salmonella abony was procured from National Collection of Type Cultures with NCTC number – NCTC6017 and Pseudomonas aeruginosa is procured from American Type Culture Collection (ATCC) with ATCC number – ATCC9027. The Wistar rat for evaluating acute inhalation toxicity of the composition was procured from Genotox Bio Services Pvt. Ltd (CPCSEA registration No.: 1242/PO/RcBiBt/S/08/CPCSEA).

DETAILED DESCRIPTION OF THE INVENTION
[0020] The following description describes various features and functions of the disclosed system. The illustrative aspects described herein are not meant to be limiting. It may be readily understood that certain aspects of the disclosed system can be arranged and combined in a wide variety of different configurations, all of which have not been contemplated herein.

[0021] Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness.

[0022] Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.

[0023] The terms and words used in the following description are not limited to the bibliographical meanings, but, are merely used to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustrative purpose only and not for the purpose of limiting the invention.

[0024] It is to be understood that the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0025] It should be emphasized that the term “comprises/comprising” when used in this specification is taken to specify the presence of stated features, steps or components but does not preclude the presence or addition of one or more other features, steps, components or groups thereof. The equations used in the specification are only for computation purpose.

[0026] Accordingly, the present invention provides a herbal composition for treating throat related infections and a method for preparation thereof. Particularly, the present invention relates to a herbal composition exhibiting potential antiviral, anti-inflammatory, antibacterial, and antimicrobial action that effectively relieves sore throat, tonsillitis and other throat related infections.

[0027] In an embodiment, the herbal composition of the present invention comprises of one or more essential oils, one or more herbal extracts; at least one solubilizing agent; a salt; one or more additives, and water.

[0028] The essential oils are concentrated liquid extracts derived from different parts of the plants. In an exemplary embodiment, the essential oils are selected from a group consisting of, such as, but not limited to, Lemon oil, Rosemary oil, Ginger oil, Peppermint oil, Cinnamon oil, Basil oil, Clove oil, or a combination thereof. The essential oils are derived from different parts of herbs such as, but not limited to, Lemon oil extracted from peel of Citrus limon, Rosemary oil extracted from flowers of Rosmarinus officinalis, Ginger oil extracted from roots of Zingiber officinale, Peppermint oil extracted from leaves, flowers and stems of Mentha piperita L., Cinnamon oil extracted from leaves of Cinnamomum zeylanicum, Basil oil extracted from leaves from Ocimum basilicum and Clove oil extracted from stem of Eugenia caryophyllus. The essential oils used in the present invention are present in liquid form such as, but not limited to, emulsion, solvent, solution, oil and so on. The essential oils are present in a range of 1% to 12% by weight of the total herbal composition. In an exemplary embodiment, each essential oil is present in a range of 1% to 2% by weight of the total herbal composition. The essential oils contain a number of chemical compounds known as phytoactive compounds, which possesses antimicrobial, antiviral and anti-inflammatory properties. In an exemplary embodiment, the phytoactive compound present in each essential oils include such as, but not limited to, Citral in Lemon oil, Cineol in Rosemary oil , Zingiberene in Ginger oil, L-menthol in Peppermint oil, Aldehyde in Cinnamon oil, Chavicol in Basil oil, Eugenol in Clove oil. In another exemplary embodiment, the amount of phytoactive compound present in the essential oils are a range of 4% to 95% by total weight of the essential oils. The essential oils possess a wide range of medicinal properties as discussed below:
a. Lemon oil: Lemon oil possesses antioxidant and antimicrobial properties that aid in treating sore throat. It also aids in preventing other throat related infections. Lemon oil contains large amounts of vitamin-C which enhances immunity. The acidic nature of lemon juice aids reduce inflammation of throat tissues and creates an unfavorable environment for the harmful bacteria and viruses.
b. Rosemary oil: Rosemary oil exhibits antiviral and antimicrobial properties which aid in treating sore throat. The soothing aroma of rosemary oil helps in treating sore throat, cold and cough. The potential antimicrobial property of Rosemary oil also provides protection from harmful microbes.
c. Ginger oil: Ginger oil exhibits anti-inflammatory, antimicrobial, and antioxidant properties. The anti-inflammatory property provides relief from sore throat. The antimicrobial properties of the oil aids in combating viral and bacterial infections, causing painful throat. The antioxidants present in ginger exhibit therapeutic action against a number of diseases.
d. Peppermint oil: Peppermint oil exhibits anti-inflammatory, antimicrobial, and antioxidant properties. The antimicrobial property of the oil fights against various harmful pathogens entering the body through mouth. The antibacterial property of the peppermint oil aids fighting off harmful bacteria. Peppermint also contains an organic compound known as menthol, which is added to a number medicines used in healing sore throat. Peppermint oil also prevents bad breath.
e. Cinnamon oil: Cinnamon oil exhibits anti-inflammatory, antimicrobial and antifungal properties. The anti-inflammatory property reduces inflammation of sore throat. The antimicrobial property of the oil destroys the growth of microorganisms such as bacteria, virus, and fungi. The antifungal property of Cinnamon oil makes it favorable for providing relief against painful throat and other throat related infections. Cinnamon oil stimulates the immune system, fights free radicals, and provides protection against germs.
f. Basil oil: Basil oil exhibits antibacterial and antiviral property which provides protection against harmful pathogen causing throat related infections. The anti-inflammatory property of the
basil oil aids in treating swelling of the throat tissues.
g. Clove oil: Clove oil exhibits antiviral, antifungal, antimicrobial and anti-inflammatory properties. Such properties aid in killing germs, relieving respiratory conditions such as sore throat, cough, cold and sinus. The antioxidant property of clove oil acts as a scavenger against free radicals capable of causing various diseases. Clove oil also provides cooling effect on throat and nasal
cavity. Clove oil also prevents bad breath.

[0029] The herbal extract is a solution of a number of herbs dissolved in a solvent. The process of herbal extraction involves separation of a mixture containing a number of secondary metabolites present in the herbs using a solvent. Such extraction process results in separation of soluble secondary metabolites present in the herbs and insoluble cellular portion. The resulting secondary metabolites are bio-active chemical components produced by plants. Such metabolites may be classified into various categories such as, but not limited to, terpenoids, alkaloids, phenolic compounds and so on. The secondary metabolites present in the herbal extracts exhibits antibacterial, antiviral and anti-inflammatory properties, thereby imparting medicinal properties to the herbal extracts. In an exemplary embodiment, the herbal extracts are selected from a group, consisting of, such as, but not limited to, Kalmegh extract, Licorice extract, Ginseng extract, Tea extract, or a combination thereof. In another exemplary embodiment, the herbal extracts are present in solid form including such as, but not limited to, powder, pellet, crystal and capsule and so on. The herbal extracts are present in a range of 1% to 20% by weight of the total herbal composition. In an exemplary embodiment, each herbal extract is present in a range of 1% to 5% by total weight of the herbal extracts. The herbal extracts used in the present invention exhibits antibacterial, antiviral and anti-inflammatory properties imparted by the secondary metabolites. In yet another exemplary embodiment, the secondary metabolites present in the herbal extracts may include, such as, but not limited to, andrographolide in Kalmegh extract; Glycerrhizic acid in Licorice extract, Ginsenoside in Ginseng extract and polyphenols in Tea extract. In another exemplary embodiment, the secondary metabolites present in the herbal extracts are inrange of 10% to 95% by total weight of the herbal extracts. The herbal extracts possess healing potential and possess antioxidant, anti-inflammatory, antimicrobial and antiseptic properties as discussed below:

a. Kalmegh extract- Kalmegh helps in providing relief from tonsilitis. Kalmegh exhibits anti-inflammatory, antimicrobial and immunomodulatory properties. Kalmegh prevents swelling
of tonsils. It also aids in treating sore throat, painful swallowing, cough and swollen tonsil associated with tonsillitis and other throat related infections Kalmegh extract also prevents decay of tooth.
b. Licorice extract- Licorice aids in treating sore throat and cough. Licorice exhibits antioxidant, antimicrobial, and anti-inflammatory properties. Licorice reduces
inflammation of throat and provide relief from other throat infections.
c. Ginseng extract- Ginseng extract possesses antioxidant and anti-inflammatory properties. It aids in treating sore throat and dry cough.
d. Tea extract – The tea extract exhibits a prophylactic effect. The tea extracts possess antioxidant, anti-inflammatory and anti-bacterial properties and helps in combating harmful
pathogens causing sore throat.
[0030] The herbal composition also comprises of at least one solubilizing agent that aids in effective solubilization of the essential oils in water. In an exemplary embodiment, the solubilizing agent is selected from a group consisting of, such as, but not limited to, PEG 400, Propylene glycol, Poloxomer 188 and Polysorbate 80, or a combination thereof. The solubilizing agents are present in a range of 1% to 16% by weight of the total herbal composition. In an exemplary embodiment, each solubilizing agent is present in different amount such as propylene glycol is present in a range of 6% to 7% by total weight of the solubilizing agent, PEG 400 is present in a range of 1% to 5% by total weight of the solubilizing agent; poloxomer 188 is present in range of 0% to 1% by total weight of the solubilizing agent; and polysorbate 80 is present in a range of 0% to 1% by total weight of the solubilizing agent.

[0031] The salt used in preparation of the herbal composition aids in lowering the pH of the saliva, thereby inhibiting the growth of harmful pathogens, which may cause irritation and pain in throat. In an exemplary embodiment, the salt used in the present invention may include such as, but not limited to, Sodium bicarbonate. Sodium bicarbonate is a potential anti-irritant and aids in reducing irritation in the throat. The salt used in the present invention is present in the range of 8% to12% by weight of the total herbal composition.

[0032] The additives used in the present invention may be selected from a group consisting of such as, but not limited to, sweetener, or solubilizer, or a combination thereof. The sweetener used in the present invention may include such as, but not limited to, Sorbitol. The solubilizer used in the present invention may include, such as, but not limited, to, PEG 400. The additives are present in a range of 11% to 30% by weight of the total herbal composition. In an exemplary embodiment, the sweetener is present in a range of 10-20% by total weight of the additives. In another exemplary embodiment, the solubilizer is present in a range of 1-5% by total weight of the additives.

[0033] Water is present in a range of 25% to 40% by weight of the total herbal composition.

[0034] The herbal composition prepared in the present invention exhibits a pH in the range of 6.5 to 9.5.

[0035] In an embodiment, the present invention also provides a method for preparing herbal composition for treating throat related infections. Microemulsion technique is employed for the preparation of the herbal composition of the present invention. The Microemulsion is an optically transparent colloidal system involving the dispersion of tiny drops of one liquid, which is immiscible in another. The method involves the following steps:
(a) adding salt in water contained in apparatus (A) followed by dissolving the salt thoroughly in the water;
(b) heating the solution obtained in step (a) at a temperature ranging between 60-70?;
(c) adding herbal extracts to the solution obtained in step (b);
(d) stirring the solution prepared in step (c);
(e) filtering the solution obtained in step (d);
(f) adding solubilizing agent and essential oils in an apparatus (B);
(g) stirring the solution obtained in step (f);
(h) adding additives and water to the solution obtained in step (e);
(i) mixing the solution obtained in step (h) and the solution obtained in step (g), forming another solution; and
(j) homogenizing the solution obtained in step (i), forming a herbal composition;

[0036] In an exemplary embodiment, the salt added in step (a) may include such as, but not limited to, Sodium bicarbonate. The salt used in the present invention is present in the range of 8% to12% by weight of the total herbal composition.

[0037] In an exemplary embodiment, the herbal extracts added in step (c) may be selected from a group, consisting of, such as, but not limited to, Kalmegh extract, Licorice extract, Ginseng extract, Tea extract, or a combination thereof. The herbal extracts are present in a range of 1% to 20% by weight of the total herbal composition. In an exemplary embodiment, each herbal extract is present in a range of 1% to 5% by total weight of the herbal extracts.

[0038] In an exemplary embodiment, the solubilizing agent added in step (f) may be selected from a group consisting of, such as, but not limited to, PEG 400, Propylene glycol, Poloxomer 188 and Polysorbate 80, or a combination thereof. The solubilizing agents are present in a range of 1% to 16% by weight of the total herbal composition. In an exemplary embodiment, each solubilizing agent is present in different amount such as propylene glycol is present in a range of 6% to 7% by total weight of the solubilizing agent, PEG 400 is present in a range of 1% to 5% by total weight of the solubilizing agent; poloxomer 188 is present in range of 0% to 1% by total weight of the solubilizing agent; and polysorbate 80 is present in a range of 0% to 1% by total weight of the solubilizing agent.

[0039] In an exemplary embodiment, the additives added in step (h) may be selected from a group consisting of, such as, but not limited to, sweetener, or solubilizer, or a combination thereof. The sweetener used in the present invention may include such as, but not limited to, Sorbitol. In an exemplary embodiment, the sweetener is present in a range of 10-20% by total weight of the additives. The solubilizer used in the present invention may include, such as, but not limited, to, PEG 400. In another exemplary embodiment, the solubilizer is present in a range of 1-5% by total weight of the additives. The additives are present in a range of 11% to 30% by weight of the total herbal composition.

[0040] Water added in step (h) may be present in a range of 25% to 40% by weight of the total herbal composition.

[0041] In a preferred embodiment, the herbal composition prepared in the present invention may be used as a mouth a mouth rinse for the purpose of gargling to treat sore throat, tonsillitis and other throat related infections.

[0042] In an embodiment, the herbal composition prepared in the present invention may be used in the following forms:
• cream, gel, and rub;
• inhalant;
• directly as drops;
• gargling solution prepared by adding few drops of the herbal composition in water; and
• a mixture prepared by adding few drops of the herbal composition in water for the purpose of steam inhalation.

[0043] In an exemplary embodiment, Table 1 shows quantity of each compound and the amount of phytoactive compound present in different compounds.

Sr. No. Components Quantity (%) Percentage of Phytoactive compound present
in different compounds
1. Sodium
bicarbonate 8-12% -
2. Tea extract 1-5% Polyphenol 85-95%
3. Kalmegh
extract 1-5% -
4. Licorice extract 1-5% Glycerrhizic acid –
10-20%
5. Lemon oil 1-2% Citral 4-8%
6. Rosemary oil 1-2% Cineol 35-45%
7. Ginger oil 1-2% Zingiberene – 30-
40%
8. Peppermint oil 1-2% L-menthol 35-45%
9. Ginseng extract 1-5% Ginsenoside – 10-
20%
10. Cinnamon oil 1-2% Aldehyde – 75-85%
11. Basil oil 1-2% Chavicol 70-80%
12. Clove oil 1-2% Eugenol 80-90%
13. Propylene
glycol 6-7% -
14. PEG 400 (solubilizing agent) 1-5% -
15. Poloxomer 0-1% -
16. Polysorbate 80 0-1%
17. PEG 400 (additive) 1-5%
17. Sorbitol 10-20% -
18. Water 25-40% -
Table 1
[0044] The following illustrates the experimental data of the present invention and should not be construed to limit the scope of the present invention.

Experimental Data

1. General Parameters
[0045] The general parameters of the herbal composition prepared in the present invention were evaluated. Such characteristics include color, odor, pH and viscosity of the composition. The herbal composition was perceived to be in dark brown colorand exhibits a characteristic odor.

2. pH (as per API, Part I, Vol. VI Appendix 3, 3.1.3)
[0046] The pH of the herbal composition prepared in the present invention was evaluated using pH meter. The pH meter was calibrated by using buffer solution before use. The pH of the herbal composition was determined in triplicate and an average of the triplicate values was evaluated. The pH of the composition was evaluated to be 8.85.

3. Viscosity ( API, Part-I, Vol. VI, Appendix -3, 3.4)
[0047] The viscosity of the herbal composition prepared in the present invention was evaluated. The liquid test sample was filled in a U-tube viscometer in accordance with the expected viscosity so that the fluid level stands within 0.2 mm of the filling mark of the viscometer when the capillary was vertical and a specified temperature was attained by the liquid sample. The liquid sample was pipetted/blown to a specified weight of the viscometer and the time taken for the meniscus to pass the two specified marks was determined. The kinematic viscosity (in centistokes) was calculated from the following equation:
Kinematic viscosity = kt
wherein,
k = constant of the viscometer tube determined by observing liquids of known kinematic viscosity;
and
t = time for meniscus to pass through the two specified marks (seconds).
The viscosity of the test sample was evaluated to be 10.3 cps.

4. Microbial Contamination (as per API, Part-I, Vol.-IX (Extracts); Appendix-3, 3.2)
[0048] The microbial contamination of the herbal composition prepared in the present invention was evaluated. The number of viable aerobic microorganisms present in the herbal composition and the presence of specific microbial species in the herbal composition (if any) was determined by performing following tests:

[0049] Preliminary Testing: Prior to performing preliminary tests, the diluted test sample to be examined was inoculated with separate viable cultures of Escherichia coli, Salmonella abony, Pseudomonas aeruginosa and Staphylococcus aureus, in order to ensure that the test samples prevent the inhibition of multiplication under the test conditions of microorganisms that may be present in the composition. The inoculation was performed by adding 1 ml of 24 hours broth culture containing at least 1000 microorganisms to the first dilution (in buffer solution with pH 7.2 using Fluid Soyabean-Casein Digest Medium or Fluid Lactose Medium) of the test sample and following the test procedure. In case the organisms fail to grow in the relevant medium then the volume of diluent was increased with the quantity of test solution remaining the same or a sufficient amount of a required inactivating agent in the diluents was incorporated to permit growth of the organisms in the medium. In case inhibitory substances were present in the samples, 0.5% of soya lecithin and 4% of polysorbate were added to the culture medium. Alternatively, the same procedure may be repeated using Fluid Casein Digest Soya Lecithin-Polysorbate 20 medium for demonstrating neutralization of preservatives or other antimicrobial agents in the test sample. In case the inhibitory substances are contained in the test sample and the test sample was soluble then the membrane filtration method was employed.

[0050] The failure to isolate the inoculated microorganism even after the incorporation of a desired inactivating agents and/or significant increase in the volume of diluent and/or in case the test sample was not suitable for applying the membrane filtration method, may be due to the bactericidal activity of the test sample. Such failure may indicate a probability that the test sample may not be contaminated with the given species of microorganisms. The monitoring of the sample was resumed to establish a spectrum of inhibition and bactericidal activity of the sample.

[0051] Table 2 illustrates experimental results of the trace element analysis of the herbal composition
S No Test Result Standard Limits
1 Total Aflatoxin Below Quantification Limit Not more than 5mcg/Kg
2 Aflatoxin B1 Below Quantification Limit 1-2mcg/Kg
3 Aflatoxin B2 Below Quantification Limit 1-2mcg/Kg
4 Aflatoxin G2 Below Quantification Limit 1-2mcg/Kg
5 Aflatoxin G1 Below Quantification Limit 1-2mcg/Kg
Table 2
Culture Media
[0052] Different culture mediums were prepared for evaluating microbial contamination or a dehydrated culture media was used such that the medium possess similar ingredients when reconstituted as directed by the manufacturer and/or yield media comparable to the media obtained from the formulae given below for preparing different culture mediums. Agar with moisture content within 15% was used for the preparation of the culture mediums, if required. Purified water was also used in the culture mediums, if required. The culture mediums were sterilized by heating the mediums in an autoclave at 15 psi and at 121? for 15 minutes, if required. The soluble solids were dissolved in water for preparing the mediums by the formulas (for the preparation of the culture mediums) provided below. A required amount of 0.1N hydrochloric acid or 0.1N sodium hydroxide solutions were added by using heat, if required, for yielding a desired pH in the medium before the use. The pH of the test sample was determined at temperature of 25? ± 2?. The procedure for preparing different culture mediums is described herein:
Baird-Parker Agar medium –

[0053] The Baird-Parker Agar medium was prepared by adding 10.0 g of Pancreatic digest of casein, 5.0 g of Beef extract, 1.0 g of Yeast extract, 5.0 g of Lithium chloride, 20.0 g of Agar, 12.0 g of Glycerin, and 10.0 g of Sodium pyruvate. Water was added to make the volume of the mixture up to 1000ml. The solution was heated to boiling for 1 minute along with stirring. The solution was then sterilized by heating the solution in an autoclave at a temperature in range of 45?- 50? followed by cooling the solution. 10 ml of a 1% weight by volume of sterile potassium tellurite solution was added to the solution and 50 ml of egg yolk emulsion was added to the solution. The egg yolk emulsion was prepared by disinfecting the surface of whole shell eggs and aseptically cracking the eggs followed by separating intact yolks into a sterile graduated cylinder. The prepared solution was thoroughly mixed and poured into plates. A required amount of sterile saline solution was added to the prepared solution to obtain a ratio of 3:7 of egg-yolk to saline solution. The solution was added to a sterile blender cup and then mixed at high speed for about 5 seconds. The pH of the solution was adjusted to 6.8 ± 0.2 after sterilization.

[0054] The Bismuth Sulphite Agar medium was prepared by forming two solutions. The first solution was prepared by adding 6.0 g of Beef extract, 10.0 g of Peptone, 24.0 g of Agar, 0.4 g of Ferric citrate, 10.0 mg of Brilliant green. Water was added to make the volume of the mixture up to 1000 ml. The solution was then mixed by heating the solution. The solution was sterilized by heating in an autoclave at a temperature of at 115? for 30 minutes. The second solution was prepared by adding 3.0 g of Ammonium Bismuth Citrate, 10.0 g of Sodium Sulphite, 5.0 g of Anhydrous Disodium Hydrogen Phosphate, and 5.0 g of Dextrose Monohydrate. Water was added to make the volume of the mixture up to 100 ml. The solution was thoroughly mixed and then boiled. The solution was allowed to cool at room temperature. 1 part of the second solution was added to 10 parts of first solution previously melted and cooled to a temperature of 55?. The solution was poured into plates. The Bismuth Sulphite Agar Medium was stored at a temperature in a range of 2? to 8? for 5 days before use.
Green Agar Medium
[0055] The Green Agar Medium was prepared by adding 10.0 g of Peptone, 3.0 g of Yeast extract, 10.0 g of Lactose, 10.0 g of Sucrose, 5.0 g of Sodium chloride, 80.0 g of Phenol red, 12.5 mg of Brilliant green, 12.0 g of Agar, and 5.0 g of Sodium chloride. Water was added to the make the volume of the mixture up to 1000 ml. The solution was mixed thoroughly and allowed to stand for 15 minutes. The solution was sterilized by heating in an autoclave at a temperature of at 115? for 30 minutes followed by mixing the solution. The solution was poured into plates.
Buffered Sodium Chloride- Peptone Solution pH 7.0
[0056] Buffered Sodium Chloride - Peptone Solution pH 7.0 was prepared by adding 3.56 g of Potassium dihydrogen phosphate, 7.23 g of Disodium hydrogen phosphate, 4.30 g of Sodium chloride, and 1.0 g of Peptone (meat or casein). Water was added to the make the volume of the mixture up to 1000 ml. 0.1 to 1.0% weight by volume of polysorbate 20 or polysorbate 80 was added to the solution. The solution was sterilized by heating in an autoclave at a temperature of 121? for 15 minutes.
Casein Soyabean Digest Agar Medium
[0057] Casein Soyabean Digest Agar Medium was prepared by adding 5.0 g Papaic digest of soyabean meal, 5.0 g of Sodium chloride, and 15.0 g of Agar. Water was added to make the volume of the mixture up to 1000 ml. The pH of the solution was adjusted to 7.3 ± 0.2 after sterilization.
Cetrimide Agar Medium
[0058] Cetrimide Agar Medium was prepared by adding 20.0 g of Pancreatic digest of gelatin, 1.4 g of Magnesium chloride, 10.0 g of Potassium sulphate, 0.3 g of Cetrimide, 13.6 g of Agar, and 10.0 g of Glycerin. Water was added to make the volume of the mixture up to 1000 ml. The solution was boiled for 1 minute along with shaking the solution. The pH of the solution was adjusted to 7.0-7.4 after sterilization.
Desoxycholate-Citrate Agar Medium
[0059] Desoxycholate-Citrate Agar Medium was prepared by adding 5.0 g of Beef extract, 5.0 g of Peptone, 10.0 g of Lactose, 8.5 g of Trisodium citrate, 5.4 g of Sodium thiosulphate, 1.0 g of Ferric citrate, 5.0 g of Sodium desoxycholate, 0.02 g of Neutral red, and 12.0 g of Agar. Water was added to make the volume of the mixture up to 1000 ml. The solution was mixed and allowed to stand for 15 minutes. The solution was gently boiled with continuous stirring until the solution was completely homogenized. The solution was allowed to cool at a temperature of 80?. The solution was then mixed and poured into plates. Precautions were taken to prevent overheating of Desoxycholate Citrate Agar while preparing the medium. Re-melting of the solution was also avoided. The surface of the plates were dried before use.
Fluid Casein Digest - Soya Lecithin - Polysorbate 20 Medium
[0060] Fluid Casein Digest - Soya Lecithin - Polysorbate 20 Medium was prepared by adding 20.0 g of Pancreatic digest of casein, 5.0 g of Soya lecithin, and 40.0 ml of Polysorbate 20. Water was added to make the volume of the mixture up to 1000 ml. The pancreatic digest of casein and soya lecithin was dissolved in water followed by heating the solution in a water-bath at a temperature in a range of 48? to 50? for about 30 minutes. Polysorbate 20 was added into the solution followed by mixing and pouring the solution into the plates.
Fluid Lactose Medium
[0061] Fluid Lactose Medium was prepared by adding 3.0 g of Beef extract, 5.0 g of Pancreatic digest of gelatin, and 5.0 g of Lactose. Water was added to make the volume of the mixture up to 1000 ml. The solution was then immediately cooled after sterilization. The pH of the solution was adjusted to 6.9 ± 0.2 after sterilization.
Lactose Broth Medium
[0062] Lactose Broth Medium was prepared by adding 3.0 g of Beef extract, 5.0 g of Pancreatic digest of gelatin, and 5.0 g of Lactose. Water was added to make the volume of the mixture up to 1000 ml. The pH of the solution was adjusted to 6.9 ± 0.2 after sterilization.
Levine Eosin - Methylene Blue Agar Medium
[0063] Levine Eosin - Methylene Blue Agar Medium was prepared by adding 10.0 g of Pancreatic digest of gelatin, 2.0 g of dibasic potassium phosphate, 15.0 g of Agar, 10.0 g of Lactose, 400 mg of Eosin Y, and 65.0 mg of Methylene blue. Water was added to make the volume of the mixture up to 1000 ml. The pancreatic digest of gelatin, dibasic potassium phosphate and agar was dissolved in water by heating the solution. The solution was then allowed to cool. The gelled agar solution and the remaining ingredients as solutions were liquefied in a required amount before use followed by mixing the solution. For each 100 ml of the liquified agar solution 5 ml of 20% weight by volume lactose solution, 2 ml of 2% weight by volume of eosin Y solution and 2 ml of 0.33% weight by volume of methylene blue solution were used. The prepared medium may not be clear. The pH of the solution was adjusted to 7.1±0.2 after sterilization.
MacConkey Agar Medium
[0064] MacConkey Agar Medium was prepared by 17.0 g of Pancreatic digest of gelatin, 3.0 g of peptone such as meat and casein in equal proportion, 10.0 g of Lactose, 5.0 g of Sodium chloride, 1.5 g of Bile salts, 13.5 g of Agar, 30.0 mg of Neutral red, and 1.0 mg of Crystal violet. Water was added to make the volume of the mixture up to 1000 ml. The solution was boiled for 1 minute. The pH of the solution was adjusted to 7.1 ± 0.2 after sterilization.
MacConkey Broth Medium
[0065] MacConkey Broth Medium was prepared by adding 20.0 g of Pancreatic digest of gelatin, 10.0 g of Lactose, 5.0 g of dehydrated ox bile, and 10.0 mg of Bromocresol purple. Water was added to make the volume of the mixture up to 1000 ml. The pH of the solution was adjusted to 7.3 ± 0.2 after sterilization.
Mannitol-Salt Agar Medium
[0066] Mannitol-Salt Agar Medium was prepared by adding 5.0 g of Pancreatic digest of gelatin, 5.0 g of Peptic digest of animal tissue, 1.0 g of Beef extract, 10.0 g of D-Mannitol, 75.0 g of Sodium chloride, 15.0 g of Agar, and 25 mg of Phenol red. Water was added to the make the volume of the mixture up to 1000 ml. The solution was mixed by heating the solution along with constant stirring. The solution was boiled for 1 minute. The pH of the solution was adjusted to 7.4 ± 0.2 after sterilization.
[0067] Nutrient Broth Medium was prepared by adding 10.0 g of Beef extract, 10.0 g of Peptone, and 5.0 mg of Sodium chloride. Water was added to the make the volume of the mixture up to 1000 ml. The solution was mixed by heating. Adjust the pH to 8.0 to 8.4 with 5M sodium hydroxide followed by boiling the solution for 10 minutes. The solution was filtered and sterilized by heating the solution in an autoclave at a temperature of 115? for 30 minutes. The pH of the solution was adjusted to 7.3 ± 0.1.
Nutrient Agar Medium
[0068] Nutrient Agar Medium was prepared by adding 1% to 2% weight by volume of agar into the nutrient medium.
Pseudomonas Agar Medium
[0069] Pseudomonas Agar Medium for detection of Flourescein produced by species of Pseudomonas was prepared by the addition of 10.0 g of Pancreatic digest of casein, 10.0 g of Peptic digest of animal tissue, 1.5 g of Anhydrous dibasic potassium phosphate, 1.5 g of Magnesium sulfate heptahydrate, 10.0 ml of Glycerin, and 15.0 g of Agar. Water was added to make the volume of the mixture up to 1000 ml. The solution was mixed prior to adding glycerin. The solution was then heated by boiling the solution for 1 minute along with constant stirring. The pH of the solution was adjusted to 7.2 ± 0.2 after sterilization.
[0070] Pseudomonas Agar Medium for the detection of Pyocyanin produced by the species of Pseudomonas was prepared by adding 20.0 g of Pancreatic digest of gelatin, 1.4 g of anhydrous magnesium chloride, 10.0 g of anhydrous potassium sulphate, 15.0 g of Agar, and 10.0 ml of Glycerin. Water was added to make the volume of the mixture up to 1000 ml. The solution was mixed prior to adding glycerin. The solution was then heated by boiling the solution for 1 minute along with constant stirring. The pH of the solution was to 7.2 ± 0.2 adjusted after sterilization.
Sabouraud Dextrose Agar Medium
[0071] Sabouraud Dextrose Agar Medium was prepared by adding 40.0 g of Dextrose, 10.0 g of Peptic digest of animal tissue and pancreatic digest of casein (1:1), and 15.0 g of Agar. Water was added to make the volume of the mixture up to 1000 ml. The solution was mixed by heating the solution to boiling. The pH of the solution was adjusted to 5.6 ± 0.2 after sterilization.
[0072] Sabouraud Dextrose Agar Medium with antibiotics for selective isolation and cultivation of aciduric bacteria, molds and yeasts was prepared by adding 0.1 g of benzylpenicillin sodium and 0.1 g of tetracycline HCL or 50 mg of chloramphenicol in 1 liter of Sabouraud Dextrose Agar Medium immediately before use.
Enrichment medium
[0073] Selenite F Broth used as an enrichment medium for the isolation of Salmonella and Shigella species was prepared by adding 5.0 g of Peptone, 4.0 g of Lactose, 10.0 g of Disodium hydrogen phosphate, and 4.0 g of Sodium hydrogen selenite. Water was added to make the volume of the mixture up to 1000 ml. The solution was mixed in sterile container and poured into plates. The solution was sterilized by heating the solution in an autoclave for a temperature of 100? for 30 minutes.
Fluid Selenite - Cystine Medium
[0074] Fluid Selenite - Cystine Medium was prepared by adding 5.0 g of Pancreatic digest of casein, 4.0 g of Lactose, 10.0 g of Sodium phosphate, 4.0 g of Sodium hydrogen selenite, and 10.0 mg of l-Cystine. Water was added to make the volume of the mixture up to 1000 ml. The solution was mixed by heating the solution in flowing steam for 15 minutes. The pH of the solution was adjusted to 7.0 ± 0.2. Sterilization step was avoided for the preparation of Fluid Selenite - Cystine Medium.
Tetrathionate Broth Medium
[0075] Tetrathionate Broth Medium was prepared by adding 0.9 g of Beef extract, 4.5 g of Peptone, 1.8 g of Yeast extract, 4.5 g of Sodium chloride, 25.0 g of Calcium carbonate, and 40.7 g of Sodium thiosulphate. Water was added to make the volume of the mixture up to 1000 ml. The solution was mixed by heating the solution to boiling. A solution prepared by dissolving 5 g of potassium iodide and 6 g of iodine in 20 ml of water was added to the prepared solution before the use.
Tetrathionate-Bile-Brilliant Green Broth Medium
[0076] Tetrathionate-Bile-Brilliant Green Broth Medium was prepared by adding 8.6 g of Peptone, 8.0 g of dehydrated ox bile, 6.4 g of Sodium chloride, 20.0 g of Calcium carbonate, 20.0 g of Potassium tetrathionate, and 70.0 mg of Brilliant green. Water was added to the make the volume of the mixture up to 1000 ml. The solution was then boiled. Re-heating of the solution was avoided.
[0077] The pH of the solution was adjusted to 7.0 ± 0.2 after boiling the solution.

Triple Sugar - Iron Agar Medium
[0078] Triple Sugar - Iron Agar Medium was prepared by adding 3.0 g of Beef extract, 3.0 g of Yeast extract, 20.0 g of Peptone, 10.0 g of Lactose, 10.0 g of Sucrose, 1.0 g of Dextrose monohydrate, 0.2 g of Ferrous sulphate, 5.0 g of Sodium chloride, 0.3 g of Sodium thiosulphate, and 24.0 mg of Phenol red. Water was added to the make the volume of the mixture up to 1000 ml. The solution was mixed and allowed to stand for 15 minutes. The solution was then boiled and boiling point was maintained until the solution was completely homogenized and thoroughly mixed. The solution was poured into tubes and sterilized by heating the solution in an autoclave at a temperature of 121? for 15 minutes. The solution was allowed to stand in a sloped form with a 2.5 cm long bottom.
Urea Broth Medium
[0079] Urea Broth Medium was prepared by adding 9.1 g of Potassium dihydrogen orthophosphate, 9.5 g of anhydrous disodium hydrogen phosphate, 20.0 g of Urea, 0.1 g of Yeast extract, and 10.0 mg of Phenol red. Water was added to the make the volume of the mixture up to 1000 ml. The solution was mixed and sterilized by filtrating the solution. The solution was then poured aseptically into sterile containers.
Vogel-Johnson Agar Medium
[0080] Vogel-Johnson Agar Medium was prepared by adding 10.0 g of Pancreatic digest of casein, 5.0 g of Yeast extract, 10.0 g of Mannitol, 5.0 g of Dibasic potassium phosphate, 5.0 g of Lithium chloride, 10.0 g of Glycerin, 16.0 g of Agar, and 25.0 mg of Phenol red. Water was added to the make the volume of the mixture up to 1000 ml. The solution was boiled for one minute. The solution was sterilized and cooled to a temperature in a range of 45?-50?. 20 mL of 1% weight by volume sterile solution of potassium tellurite was added to the solution. The pH of the solution was adjusted to 7.0 ± 0.2 after sterilization.

Xylose -Lysine-Desoxycholate Agar Medium

[0081] Xylose -Lysine-Desoxycholate Agar Medium was prepared by adding 3.5 g of Xylose, 5.0 g of l-Lysine, 7.5 g of Lactose, 7.5 g of Sucrose, 5.0 g of Sodium chloride, 3.0 g of Yeast extract, 80.0 mg of Phenol red, 13.5 g of Agar, 2.5 g of Sodium desoxycholate, 6.8 g of Sodium thiosulphate, and 800 mg of Ferric ammonium citrate. Water was added to make the volume of the mixture up to 1000 ml. The solution was heated along with stirring until the boiling point is achieved. Over-heating or sterilization of the solution was avoided. The solution was heated by transferring the solution to a water-bath at a temperature of 50?. The solution was poured into plates followed by the cooling of the medium. The pH of the solution was adjusted to 7.4 ± 0.2 after sterilization. The sampling involves using 10 ml or 10 g of sample for each of the specified tests.

[0082] The precautions taken during experimentation involves performing microbial limit tests under specific conditions to prevent contamination during the tests. Such precautions taken to prevent contamination exhibit no adverse effect on microorganisms required to be detected in the tests.
Total Aerobic Microbial Count (as per API, Part-I, Vol.-IX (Extracts); Appendix -3.2, 3.2.1)

[0083] The total aerobic microbial count in the herbal composition was evaluated. The procedure for evaluating total aerobic microbial count in different types of test sample is described herein:

[0084] Water-soluble products: 10 g or 10 ml of the sample preparation to be examined was dissolved/diluted, if required, in buffered sodium chloride-peptone solution pH 7.0 or other suitable medium which exhibits no under test conditions. The solution was diluted to make the volume of the solution to 100 ml with the same medium. The pH of the suspension was adjusted to 7, if required.

[0085] Water-insoluble products (non-fatty): 10 g or 10 ml of the sample preparation to be examined, if required, was suspended, in buffered sodium chloride-peptone solution pH 7.0 or other suitable medium which exhibits no antimicrobial activity under test conditions. The solution was diluted to make the volume of the solution to 100 ml with the same medium. The prepared solution was divided, if required and mechanically homogenized. A desired surface-active agent such as 0.1% weight by volume of polysorbate 80 was added to assist the suspension of less wettable substances. The pH of the suspension was adjusted to 7, if required.

[0086] Water insoluble sample (Fatty): 10 g or 10 ml of the sample preparation to be examined, was homogenized with 5 g of polysorbate 20 or polysorbate 80. The suspension was heated, to a temperature within 400?. The suspension was thoroughly mixed while maintaining the temperature in the water-bath/oven. 85 ml of buffered sodium chloride-peptone solution of pH 7.0 or other suitable medium that exhibits no antimicrobial activity under test conditions was heated to a temperature within 400?, and added to the prepared solution. A temperature within 400? was maintained for a time period < 30 minutes required for the formation of an emulsion. The pH of the suspension was adjusted to 7, if required.

[0087] The sample was examined by determining the total aerobic microbial count in the sample using any of the following methods:

[0088] A. Membrane filtration: Membrane filters with 50 mm diameter and nominal pore size within 0.45 mm were used for retaining bacteria. A required amount such as 10 mL of each dilution containing 1g of the sample preparation to be examined was added to each of two membrane filters. The solution was immediately filtered through the membrane. The pretreated sample preparation was diluted, if required, to obtain a colony count of 10 to 100.Three or more successive quantities of a suitable liquid such as buffered sodium chloride-peptone solution pH 7.0, each about 100 ml, was filtered through the membrane.

[0089] In case of fatty sample, liquid polysorbate 20 or polysorbate 80 was used. One of the two membrane filters intended for the enumeration of bacteria was transferred to the surface of a plate of casein soyabean digest agar and the other membrane filter intended for the enumeration of fungi was transferred to the surface of a plate of Sabouraud dextrose agar with antibiotics. The plates were incubated for 5 days until a suitable count is obtained in less time duration such as 30-35? in test for bacteria and 20-25? in test for fungi. The number of colonies formed was recorded.
[0090] The number of microorganisms per g/per ml of the sample preparation to be examined was determined separately for bacteria and fungi, if required.

[0091] Plate count for bacteria: A mixture of 1ml of the pretreated sample preparation and about 15ml of liquified casein soyabean digest agar heated to a temperature within 45? was added to each petri dish. The petri dishes having diameter in a range of 9-10 cm were used. Alternatively, the pretreated sample preparation was spread on the surface of the solidified medium in a petri dish of the same diameter. The pretreated sample preparation was diluted, if required, to obtain a colony count within 300. At least two such petri dishes were prepared by using the same dilution followed by incubating the petri dishes at 30? to 35? for 5 days, until a suitable count was obtained in a less duration of time. The number of colonies formed were recorded. The results were calculated using plates with large number of colonies considering 300 colonies per plate to provide accurate results.

[0092] Plate count for fungi: The method involves similar steps as performed for evaluating plate count for bacteria except the use of Sabouraud dextrose agar with antibiotics instead of casein soyabean digest agar. The petri dishes were prepared and incubated at 20?-25? for 5 days until a suitable count was obtained in a less duration of time. The results were determined using plates within 100 colonies.

[0093] B. Multiple-tube or serial dilution method: 9.0 ml of sterile fluid soyabean casein digest medium was placed in each of fourteen test tubes with similar size. Twelve tubes were arranged in four sets of three API, Part-I, Vol.-IX (Samples); Appendices 124 tubes each. One set of three tubes were taken as control. 1 ml of the test solution was pipetted into each of three tubes of one set (“100”) and into fourth tube (A) followed by mixing the solutions. 1 ml from tube (A) was pipetted into one separate tube (B). The two tubes were containing 100 mg (or 100 µl) and 10 mg (or 10 µl) of the sample respectively. 1 ml of content from tube A was pipetted into each of second set (“10”) of three tubes. 1 ml of content from tube B was pipetted into each tube of third set (“1”). The contents of tube A and B were discarded. All tubes were properly closed and incubated. The tubes were examined for growth following the incubation period. The three control tubes were clear in color. The tubes containing the test sample were observed to determine possible number of microorganisms per g or per ml of the test sample.

[0094] Table 3 illustrates possible total microbial count by Multiple-Tube Or Serial Dilution Method:

Table 3
Tests for Specific Microorganisms (as per API, Part-I, Vol.-IX (Extracts); Appendix -3.2, 3.2.2)

[0095] Pretreatment of the test sample: The pretreatment of the test sample involves similar method as the method for evaluating total aerobic microbial count except the use of Lactose broth or other suitable medium that exhibits no antimicrobial activity under test conditions instead of buffered sodium chloride-peptone solution pH 7.0.

[0096] Test for Escherichia coli: A required amount of the pretreated test sample was placed in a sterile screw-capped container. 50 ml of nutrient broth was added to the container. The solution was mixed by shaking the container. The container containing the solution was to stand for 1 hour. The solution was mixed again by shaking the container. The cap of the container was loosened and incubated at 37? for 18-24 hours.

[0097] Primary test: 1.0 ml of the enrichment culture was added to a tube containing 5 ml of MacConkey broth. The tube was incubated in a water-bath at a temperature in a range of 36?-38? for 48 hours. A secondary test was performed in case the mixture within the tube showed acid and gas.

[0098] Secondary test: 0.1 ml of the enrichment culture was added to a tube (A) containing 5 ml of MacConkey broth and in tube (B) containing 5 ml of peptone water. Both the tubes were incubated in a water-bath at temperature in a range of 43.5? to 44.50? for 24 hours. The presence of acid and gas was determined in Tube (A) and the presence of indole was determined in tube (B). For determining the presence of indole in tube (B), 0.5 ml of Kovac’s reagent was added to the tube (B) followed by shaking the tube (B) The tube (B) was allowed to stand for 1 minute. The presence of indole was confirmed by the production of red in the reagent layer. The presence of acid and gas and of indole in the secondary test indicated the presence of Escherichia coli in the test sample. A control test was carried out by repeating the primary and secondary tests. 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 units of Escherichia coli (NCTC 9002) organisms prepared from a 24-hour culture in nutrient broth was added to 5 ml of MacConkey broth. The test was considered effective only when the results indicated the presence of Escherichia coli in control tests.

[0099] Alternative test: A portion from the enrichment culture obtained in the previous test was streaked on the surface of MacConkey agar medium by using inoculating loop. The dishes were covered and inverted followed by incubating the dishes. In case during examination none of the colonies were brick-red in colour and showed a surrounding zone of precipitated bile (API, Part-I, Vol.-IX (Extracts); Appendices 125) then the sample was considered to fulfil the requirements of the test for the absence of Escherichia coli. In case the colonies were present then the detected colonies were individually transferred to the surface of Levine eosin - methylene blue agar medium plated on petri dishes. The plates were covered and inverted followed by incubating the plates. In case, during examination, none of the colonies exhibited a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light then the sample was considered to fulfil the requirements of the test for the absence of Escherichia coli. The presence of Escherichia coli was further confirmed by a suitable cultural and biochemical tests.

[00100] Test for Salmonella: A required amount of the pretreated sample preparation to be examined containing 1 g or 1 ml of the sample was transferred to a 100 ml of nutrient broth in a sterile screw-capped jar followed by mixing the solution by shaking the jar. The solution was allowed to stand for 4 hours followed by mixing the solution by shaking the jar. The cap of the jar was loosened and incubated at a temperature in a range of 35? to 37? for 24 hours.

[00101] Primary test: 1.0 ml of the enrichment culture was added to a tube (A) containing 10 ml of selenite F broth and tube (B) containing tetrathionate-bile brilliant green broth. Both the tubes were incubated at a temperature in a range of 36?-38? for 48 hours. A subculture was prepared from each of the two prepared cultures on at least two of the following four agar media including bismuth sulphate agar, brilliant green agar, deoxycholate citrate agar and xylose-lysine deoxycholate agar. The plates were incubated at temperature in a range of 36?-38? for 18 to 24 hours. In case during examination none of the colonies conformed to the description provided in Table 3 then the sample was considered to fulfil the requirements of the test indicating the absence of the genus Salmonella. Secondary test was performed in case the colonies conforming to the description in Table 3 were formed.

[00102] Secondary test: The colonies showing the characteristics provided in Table 4 were subcultured in a Triple Sugar Iron Agar by inoculating the surface of the slope and then making a s tab culture with the same inoculating needle. Simultaneously, a tube of urea broth was inoculated. The tube was incubated at a temperature in a range of 36? to 38? for 18 to 24 hours. The formation of acid and gas in the stab culture with/without concomitant blackening and the absence of acidity from the surface growth in the Triple Sugar Iron Agar together with the absence of a red colour in urea broth indicated the presence of Salmonella in the test sample. In case only acid was produced without the production of gas in the cultures then the presence of Salmonella was confirmed by agglutination tests. The control tests were performed by repeating the primary and secondary tests using 1.0 ml of the enrichment culture and a volume of broth containing 10 to 50 units of Salmonella abony (NCTC 6017) organisms prepared from a 24-hour culture in nutrient broth for the inoculation of the tubes (A and B). The test was considered effective only when the results indicated the presence of Salmonella in control tests.

S.No. Medium Description of Colony
1 Bismuth Sulphite Agar Black or green
2 Brilliant Green Agar Small, transparent, and colourless, or opaque, pinkish or white (Frequently surrounded by a pink or
red zone)
3 Deoxycholate Citrate Agar Colourless or opaque, with or without black centre
4 Xylose- Lysine- Deoxycholate
Agar Red with or without black centre
Table 4

[00103] Test for Pseudomonas aeruginosa: The sample preparation to be examined was preheated. 100 ml of fluid soyabean-casein digest medium was inoculated with a required amount of the solution/suspension/emulsion thus obtained containing 1 g or 1 ml of the sample preparation to be examined. The solution was mixed and incubated at a temperature in a range of 35? to 37? for 24 to 48 hours.

[00104] The medium was examined for growth. In case the growth was indicated in the medium, a portion of the medium was streaked on the surface of cetrimide agar medium, each plated on Petri dishes. The dishes were covered and inverted at a temperature in a range of 35? to 37? for 18 to 24 hours. In case during examination, none of the plates showed colonies exhibiting the characteristics provided in the Table 3 for the media used, the sample was considered to fulfil the requirement for the absence of Pseudomonas aeruginosa. In case any colonies [API, Part-I, Vol.- IX (Extracts); Appendices 126] conforming to the description provided in Table 3 were formed then oxidase and pigment tests were performed. The detected colonies were streaked from the agar surface of cetrimide agar on the surfaces of Pseudomonas agar medium for detection of fluorescein and Pseudomonas agar medium for detection of pyocyanin contained in Petri dishes. The inoculated medium was covered and inverted, followed by incubating at a temperature in a range of 33? to 37? for at least 3 days. The streaked surfaces were examined under ultra-violet light. The plates were examined to determine if the indicated colonies conformed to the conditions provided in Table 3. In case the growth of said colonies were detected on the plates then 2 or 3 drops of a freshly prepared 1% weight by volume of N,N,N´,N´-tetramethyl-4-phenylenediamine dihydrochloride solution was placed on filter paper and smeared with the colony. In the absence of production of pink color turning to purple, the sample was considered to fulfil the requirements of the test indicating absence of Pseudomonas aeruginosa.

[00105] Test for Staphylococcus aureus: The test for Staphylococcus aureus involves a method similar to the method for testing the presence of Pseudomonas aeruginosa. In case, during examination of incubated plates, none of the plates showed colonies exhibiting characteristics provided in the Table 3 for the media used, the sample was considered to fulfil the requirements for the absence of Staphylococcus aureus. In case the growth of the said colonies was detected on the plates then coagulase test was performed. The detected colonies were transferred from the agar surface of any of the media provided in Table 3 to individual tubes, each containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with or without additives. The tubes were incubated in water-bath at a temperature of 37? followed by examining the tubes for 3 hours and subsequently at suitable intervals up to 24 hours. In case any level of coagulation was not detected then the sample was considered to fulfil the requirements of the test indicating the absence of Staphylococcus aureus.
Validity of the tests for total aerobic microbial count (API, Part-I, Vol.-IX (Extracts); Appendices, 126):
[00106] A number of test strains were separately grown in tubes containing fluid soyabean-casein digest medium at a temperature in a range of 30? to 35? for 18 to 24 hours or at a temperature of 20? for 48 hours. The test strains include Staphylococcus aureus (ATCC 6538; NCTC 10788); Bacillus subtilis (ATCC 6633; NCIB 8054); Escherichia coli (ATCC 8739; NCIB 8545); and Candida albicans (ATCC 2091; ATCC 10231).
[00107] The portions of each of the cultures were diluted using buffered sodium chloride peptone solution of pH 7.0 to make test suspensions containing about 100 units of viable microorganisms per ml. The suspension of each of the microorganisms was used separately as a control of the counting methods in the presence and absence of the sample preparation to be examined, if required. A count for any of the test organisms differing by a factor within 10 from the [API, Part-I, Vol.-IX (Extracts); Appendices 127] calculated value for the inoculum was required Total aerobic microbial count method using sterile buffered sodium chloride peptone solution of pH 7.0 as the test preparation was performed to test the sterility of the medium and the diluent along with aseptic performance of the test. Absence of the growth of microorganisms was required to confirm the validity of the tests for total aerobic microbial count.
Validity of the tests for specified microorganisms (as per API, Part-I, Vol.-IX (Extracts); Appendices, 127):

[00108] The test strains of Staphylococcus aureus and Pseudomonas aeruginosa were separately grown in fluid soyabean-casein digest medium and Escherichia coli and Salmonella typhimurium at a temperature in a range of 30? to 35? for 18 to 24 hours. The portions of each of the cultures were diluted by using buffered sodium chloride-peptone solution pH 7.0 to make test suspensions contain about 103 units of viable microorganisms per ml. Equal volume of each suspension was mixed and 0.4 ml i.e. approximately 102 units of microorganisms of each strain was used as an inoculum in the test for E. coli, S. typhimurium, P. aeruginosa and S. aureus, in the presence and absence of the sample preparation to be examined, if required. A positive result for the respective strain of microorganism was recommended.

5. Heavy metal Analysis (API, Part-I, Vol.-IX (Extracts); Appendix-3, 3.1):

[00109] The presence of heavy metals in the herbal composition of the present invention was evaluated using different methods as provided herein:

(a) Determination of Lead (Pb) by Graphite Oven Method:

[00110] The following conditions were taken as reference for determining the presence of lead (Pb) using Graphite Oven Method:

• dry temperature: 100-1200 maintain for 20 seconds;
• ash temperature: 400-7500 maintain for 20-25 seconds;
• atomic temperature: 1700-21000 maintain for 4-5 seconds;
• measurement wavelength: 283.3 nm; and
• background calibration: deuterium lamp (D lamp) or Zeeman effect.

[00111] Preparation of lead standard stock solution: A required amount of lead single-element standard solution was accurately measured for preparing standard stock solution with 2% nitric acid solution containing 1 µg per ml lead. The solution was stored at 0?-5?.

[00112] Preparation of calibration curve: A required amount of lead standard stock solution was measured and diluted with 2% nitric acid solution to the concentration of 0, 5, 20, 40, 60, 80 ng per ml. 1 ml of 1% ammonium dihydrogen phosphate and 0.2% magnesium nitrate was added to accurately measured 1 ml of the prepared solutions. The solutions were properly mixed and accurately measured. 20 µl of the prepared solutions were put into the atomic generator of graphite oven. The absorbance of the solutions was determined followed by preparing the calibration curve with absorbance as vertical axis and concentration as horizontal ordinate.

[00113] Preparation of test solution: 0.5 g of the coarse powder of the sample to be examined was accurately weighed and transferred into a casparian flask. 5-10 ml of the mixture of nitric acid and Perchloric acid (4:1) was added into the flask. A small hopper was placed on top of the flask. The solution was kept overnight for maceration followed by heating the solution to boiling to slake on the electric hot plate. In case a brownish-black color appeared in the solution, a required amount of the prepared mixture was added to the solution followed by constantly heating the solution until the solution turned transparent and was clean. The temperature was then raised and continuously heated to thicken smoke until white smoke dispersed and the slaked solution turned colourless, and transparent or yellowish in colour. The solution was allowed to cool and then transferred into a 50 ml volumetric flask followed by washing the container with 2% nitric acid solution. The washing solution was added into the same volumetric flask and diluted with the same solvent to the volume followed by thoroughly mixing the solution by shaking the flask. Similarly, a reagent blank solution was simultaneously prepared. The preparation of the reagent blank solution involves similar steps as performed for preparing the test solution.

[00114] Determination of the Lead: The presence of lead was determined by accurately measuring 1 ml of the test solution and the corresponding reagent blank solution. 1 ml of solution containing 1% of ammonium dihydrogen phosphate and 0.2% of magnesium nitrate was added separately to the test solution and blank solution. The prepared solutions were properly mixed by shaking the solutions. Accurately measured 10-20 µl of both the test solution and blank solution were pipetted
to determine the absorbance of both the solutions according to the method for preparation of calibration curve. The content of lead (Pd) in the test solution was determined from the calibration curve.

(b) Determination of Cadmium (Cd) by Graphite Oven Method:

[00115] The following conditions were taken as reference for determining the presence of cadmium (Cd) using Graphite Oven Method:

• dry temperature: 100-1200 maintain for 20 seconds;
• ash temperature: 300-5000 maintain for 20-25 seconds;
• atomic temperature: 1500-19000 maintain for 4-5 seconds;
• measurement wavelength: 228.8 nm; and
• background calibration: deuterium lamp (D lamp) or Zeeman effect.

[00116] Preparation of cadmium (Cd) standard stock solution: A required amount of cadmium single-element standard solution accurately was measured for preparing standard stock solution with 2% nitric acid containing 0.4 µg per ml cadmium. The solution was and stored at 0?-5?.

[00117] Preparation of calibration curve: A required amount of cadmium standard stock solutions was accurately measured and diluted to the concentration of 1.6, 3.2, 4.8, 6.4 and 8.0 ng per ml with 2% nitric acid. Accurately measured 10 µl of each prepared solution was pipetted and injected into the graphite oven. The absorbance of solutions was determined followed by preparing the calibration curve with absorbance as vertical axis and concentration as horizontal ordinate.

[00118] Preparation of test solution: The test solution of cadmium was prepared by a method similar to the method for preparation of test solution of lead.

[00119] Determination of Cadmium: The presence of cadmium was determined by accurately measuring 10-20 µl of the test solution and corresponding reagent blank solution. The absorbance of the solutions was determined by a method similar to the method for preparation of the calibration curve. In case of interference, 1 ml of the standard solution, 1 ml of blank solution and 1 ml of test solution were accurately weighed followed by addition of 1 ml of a solution containing 1% ammonium dihydrogen phosphate and 0.2% magnesium nitrate into each solution. The prepared solutions were thoroughly mixed by shaking the solutions. The absorbance of the prepared solutions was determined by following the steps performed for preparing the calibration curve followed by calculating the content of cadmium in the test solution from the calibration curve.

(c) Determination of Arsenic (As) by Hydride Method:

[00120] The following conditions were considered for determining the presence of Arsenic (As) by Hydride Method:
• Apparatus: a suitable hydride generator device;
• a reducing agent: a solution containing 1% sodium borohydride and 0.3% sodium hydroxide;
• a carrier liquid: 1% hydrochloric acid;
• a carrier gas: nitrogen;
• a measurement wavelength: 193.7 nm; and
• a background calibration: a deuterium lamp (D lamp) or Zeeman effect.

[00121] Preparation of Arsenic (As) standard stock solution: A required amount of arsenic single- element standard solution accurately weighed for preparing standard stock solution with 2% nitric acid solution containing 1.0 µg/ml arsenic. The solution was stored at 0?-5?.

[00122] Preparation of calibration curve: A required amount of arsenic standard stock solution was accurately measured and diluted with 2% nitric acid to concentration of 2, 4, 8, 12 and 16 ng per ml. Accurately measured 10 ml of each stock solution was transferred into 25 ml volumetric flask followed by adding 1 ml of 25% potassium iodide solution prepared prior to use into each prepared solution. The solutions were thoroughly mixed by shaking the flask. 1 ml of ascorbic acid solution prepared prior to use was added into each solution. The solutions were mixed thoroughly by shaking the flask and diluted with hydrochloric acid solution to the volume. The solutions were again mixed by properly shaking the flask followed by closing the stopper and immersing the flask in a water bath at a temperature of 80? for 3 minutes. The solutions were allowed to cool down and a required amount of each solution was transferred separately into the hydride generator device. The absorbance of the prepared solutions was determined followed by plotting the calibration curve with peak area (absorbance) as vertical axis and concentration as horizontal ordinate.

[00123] Preparation of test solution: The test solution of arsenic was prepared by a method similar to the method for preparation of test solution of lead.

[00124] Determination of Arsenic: The presence of arsenic was determined by pipetting accurately measured 10 ml of the test solution and the corresponding reagent separately. 1ml of 25% potassium iodide solution prepared prior to use was added to each solution. The method further involves similar steps as performed for preparation of the calibration curve. The content of arsenic in the test solution was determined from the calibration curve.

(d) Determination of Mercury (Hg) by Cold Absorption Method:
The following conditions were considered for determining the presence of Mercury (Hg) by Hydride Method
• Apparatus: a suitable hydride generator device;
• a reducing agent: a solution containing 0.5 per cent sodium borohydride and 0.1 per cent sodium hydroxide;
• a carrier liquid: 1 per cent hydrochloric acid;
• a carrier gas: nitrogen;
• a measurement wavelength: 253.6 nm; and
• background calibration: deuterium lamp (D lamp) or Zeeman effect.

[00125] Preparation of mercury standard stock solution: A required amount of mercury single element standard solution was accurately measured for preparing standard stock solution with 2% nitric acid solution containing 1.0µg per ml mercury. The solution was stored at 0?-5?.

[00126] Preparation of calibration curve: Accurately measured 0, 0.1, 0.3, 0.5, 0.7 and 0.9 ml of mercury standard stock solution was transferred into a 50 ml volumetric followed by adding 40 ml of 4% sulphuric acid solution and 0.5 ml of 5% potassium permanganate solution into each flask. The prepared solutions were thoroughly mixed by shaking the flasks. 5% hydroxylamine hydrochloride solution was added dropwise into each flask until the violet red disappeared followed by diluting the solutions with 4% sulfuric acid solution to the volume. The prepared solutions were thoroughly mixed by shaking the flasks. A required amount of each solution was injected to the hydride generator device. The absorbance of each solution was determined followed by plotting the calibration curve with peak area (absorbance) as vertical axis and concentration as horizontal ordinate.

[00127] Preparation of test solution: Accurately weighed 1 g of the coarse powder of the sample to be examined was transferred into a casparian flask. 5-10 ml of the mixture solution of nitric acid and perchloric acid (4:1) was added followed by mixing the solution thoroughly within the flask. A small hopper was fixed on the -top of the flask. The solution was macerated overnight and heated to slake on the electric hot plate at 120?-140? for 4-8 hours until the solution was completely slaked. The mixture solution was then cooled followed by adding a required amount of 4% sulfuric acid solution and 0.5 ml of 5% potassium permanganate solution into the solution. The solution was then mixed by shaking the flask. A drop of 5% hydroxylamine hydrochloride solution was added into the solution until violet red colour disappeared. The solution was diluted with 4% sulphuric acid solutions to make the volume to 25 ml followed by mixing the solution by shaking the flask. The solution was centrifugated, if required, and the supernatant obtained was used as the test solution. Simultaneously, the reagent blank solution was simultaneously prepared. The preparation of the reagent blank solution involves similar steps as performed for preparing the test solution.

[00128] Determination of Mercury: An accurately measured required amount of the test solution and corresponding reagent blank solution was pipetted in separate flaks. 1ml of 25% potassium iodide solution prepared prior to use was added to each solution. The method further involves similar steps as performed for preparation of the calibration curve in case of Arsenic. The content of mercury in the test solution was determined from the calibration curve.

Table 5 illustrates _experimental results of the test performed for heavy metal analysis of the herbal composition.

S No Test Result Standard Limits
1 Mercury Below Quantification Limit 0.20-1.0ppm
2 Cadmium Below Quantification Limit 0.20-0.30ppm
3 Lead Below Quantification Limit 0.20-10.0ppm
4 Arsenic Below Quantification Limit 0.20-3.0ppm
Table 5

6. Pesticide Residue (as per API, Part-I, Vol.-IX (Extracts); Appendix-3, 3.3):

[00129] Limits: The sample to be examined required to comply with the limits indicated in the Table 4. The limits applying to the indicated pesticides not provided in Table 4 may comply with the limits set by European Community directives 76/895 and 90/642, including their annexes and successive updates. Limits for pesticides not provided in the Table 4 or European Community were determined using the following expression:

(ADI x M)/MDD x 100

Wherein,
ADI = Acceptable Daily Intake (mg/kg of body mass), as published by FAO-WHO; M = body mass in kilograms (60 kg); and
MDD = daily dose of the drug (Kg)

[00130] In case the drug is intended for the preparation of extracts, tinctures or other pharmaceutical forms, preparation method of which modifies the content of pesticides in the finished product, the limits were determined using the following expression:

(ADI x M x E)/ MDD x 100

Wherein,

E = experimentally determined Extraction factor of the method of preparation

[00131] Higher limits may also be permitted in cases majorly where a plant requires a particular cultivation method or exhibits a metabolism or a structure that gives rise to a higher content of pesticides as compared to the normal content of the pesticide. The competent authority may grant total or partial exemption from the test when the complete history (nature and quantity of the pesticides used, date of each treatment during cultivation and after the harvest) of the treatment of the batch is known and may be checked precisely.

[00132] Sampling Method: One sample from the total content suitable for performing the tests was taken and thoroughly mixed for containers up to 1 kg. Three samples, equal in volume, from the upper, middle and lower parts of the container, each being suitable to perform the tests were taken in case of containers between 1 kg and 5 kg. The samples were thoroughly mixed and a suitable amount to perform the tests was taken from the mixture. Three samples, each of at least 250 g from the upper, middle and lower parts of the container were taken for the containers within 5 kg. The samples were thoroughly mixed and a required amount was taken from the mixture to perform the tests.

[00133] Size of sampling: The samples were taken from each container as indicated in above method in case few or at least three containers were used. n+1 samples were taken for containers as indicated in above method in case large number of containers were used. The value was rounded up to the nearest unit, if required. The samples were to be analyzed immediately to avoid possible degradation of the residues. The samples were stored in air-tight containers suitable for food contact, at a temperature below 00 and protected from light in case of inability to immediately analyze the sample.

[00134] Size of sampling: The samples from each container as indicated above were taken in case the number of containers were three or fewer. In case the number of containers were more than three, n+1 samples (rounded up to the nearest unit, if required) were taken.

[00135] The samples were required to be analyzed immediately to avoid degradation of the residues. In case of delay in analyzing the samples, the samples were stored in air-tight containers suitable for food contact at a temperature below 0? and were protected from light.

[00136] Reagents: All reagents and solvents used were free from contaminants, majorly pesticides that may interfere with the analysis. High quality solvents were used or solvents that were recently re-distilled in an apparatus made of glass were used. In any case, a suitable blank test was performed.

[00137] Apparatus: The apparatus, majorly glasswares were cleaned to ensure that the apparatus was free from pesticides. For example, the apparatus was soaked for at least 16 hours in a solution of phosphate-free detergent followed by rinsing the apparatus with large quantities of distilled water and washing with acetone and hexane or heptane.

Table 6 illustrates __experimental results of the test performed for pesticide analysis of the herbal composition.
S No Test Result Standard Limits
1 Alachlor Below Quantification Limit 0.0050-0.020mg/kg
2 Malathion Below Quantification Limit 0.0050-1.0mg/kg
3 Phosalone Below Quantification Limit 0.0050-0.10mg/kg
4 Pyrethrins Below Quantification Limit 0.0050-3.0mg/kg
5 Ethion Below Quantification Limit 0.0050-2mg/kg
6 Chlorpyrifos Below Quantification Limit 0.0050-0.20mg/kg
7 Methyl Parathion Below Quantification Limit 0.0050-0.20mg/kg
8 Lindane (Gamma - HCH) Below Quantification Limit 0.0050-0.60mg/kg
9 Aldrin and dieldrin (sum of) Below Quantification Limit 0.0050-0.050mg/kg
10 Azinophos - methyl Below Quantification Limit 0.0050-1.0mg/kg
11 Bromopropylate Below Quantification Limit 0.0050-3.0mg/kg
12 Chlorpyrifos-methyl Below Quantification Limit 0.0050-0.10mg/kg
13 Deltamethrin Below Quantification Limit 0.0050-0.50mg/kg
14 Diazinon Below Quantification Limit 0.0050-0.50mg/kg
15 Dichlorvos Below Quantification Limit 0.0050-1.0mg/kg
16 Endrin Below Quantification Limit 0.0050-0.050mg/kg
17 Fonofos Below Quantification Limit 0.0050-0.050mg/kg
18 Methidathion Below Quantification Limit 0.0050-0.20mg/kg
19 Parathion Below Quantification Limit 0.0050-0.50mg/kg
20 Permethrin Below Quantification Limit 0.0050-1.0mg/kg
21 Pirimiphos-methyl Below Quantification Limit 0.0050-4.0mg/kg
22 Fenitrothion Below Quantification Limit 0.0050-0.50mg/kg
23 Cypermethrin & isomers Below Quantification Limit 0.0050-1.0mg/kg
24 Hexachlorocyclohexane Below Quantification Limit 0.0050-0.30mg/kg
25 Piperonyl-butoxide Below Quantification Limit 0.0050-3.0mg/kg
26 Heptachlor Below Quantification Limit 0.0050-0.050mg/kg
27 Fenvalerate Below Quantification Limit 0.0050-1.50mg/kg
28 Endosulfan Below Quantification Limit 0.0050-3.0mg/kg
29 DDT Below Quantification Limit 0.0050-1.0mg/kg
30 Hexachlorobenzene Below Quantification Limit 0.0050-0.10mg/kg
31 Chlorfenvinphos Below Quantification Limit 0.0050-0.50mg/kg
32 Dithiocarbamates Below Quantification Limit 0.0050-2.0mg/kg
33 Chlordane Below Quantification Limit 0.0050-0.050mg/kg
34 Quintozene Below Quantification Limit 0.0050-1.0 mg/kg

Table 6
7. Aflatoxins (as per API, Part-I, Vol.-IX (Extracts); Appendix-3, 3.4):

[00138] The presence of different types of aflatoxins including B1, B2, G1 and G2 in the herbal composition of the present invention was evaluated. The procedure for evaluating the presence of different types of aflatoxins are described herein:
[00139] Zinc Acetate – Aluminum Chloride Reagent: 20 g of zinc acetate and 5 g of aluminum chloride was dissolved in water to make the volume of the solution to 100 ml.
[00140] Sodium Chloride Solution: 5 g of sodium chloride was dissolved in 50 ml of purified water.
[00141] Test Solutions:
• Test Solution 1: About 200 g of plant material was grinded to a fine powder. Accurately weighed 50 g of the powdered material was transferred to a glass-stoppered flask. 200 ml of a mixture of methanol and water (17: 3) was added followed by vigorous shaking of the solution by mechanical means for at least 30 minutes. The solution was then filtered. The first 50 ml of the filtrate was discarded and the next 40 ml portion was collected. The filtrate was then transferred to a separatory funnel. 40 ml of sodium chloride solution and 25 ml of hexane was added into the filtrate followed by mixing the solution by shaking the flask for 1 minute. The layers were allowed to separate and the lower aqueous layer was transferred to a second separatory funnel. The aqueous layer was extracted in the separatory funnel twice, each time with 25 ml of methylene chloride by shaking the solution for 1 minute. The layers were allowed to separate each time followed by separating the lower organic layer and collecting the combined organic layers in 125 ml conical flask. The organic solvent was allowed to evaporate to dryness on a water bath. The residue was then cooled. The residue obtained was dissolved in 0.2 ml of a mixture of chloroform and acetonitrile (9.8:0.2) followed by shaking the mixture by mechanical means, if required.
• Test Solution 2: In case the solution possesses interfering plant pigments then the following procedure was followed which involves collecting 100 ml of the filtrate from the start of the flow and transferring to a 250 ml beaker. 20 ml of Zinc Acetate-Aluminum Chloride Reagent and 80 ml of water was added to the filtrate. The solution was stirred and allowed to stand for 5 minutes. 5 g of a required filtering aid, such as diatomaceous earth, mix was added into the solution followed by filtering the solution. The first 50 ml of the filtrate was discarded and the next 80 ml portion of the filtrate was collected. The filtrate was transferred to a separatory funnel. 40 ml of sodium chloride solution and 25 ml of hexane was added into the filtrate followed by shaking the solution for 1 minute. The method further involves similar steps as performed for preparing test solution 1.

[00142] Cleanup Procedure: In case interferences exist in the residue prepared in test solution 1, following procedure was followed which involves placing a medium-porosity sintered-glass disk or a glass wool plug at the bottom of a 10 mm x 300 mm chromatographic tube. A slurry of 2 g of silica gel with a mixture of ethyl ether and hexane (3: 1) was prepared and poured into a column followed by washing with 5 ml of the same solvent mixture. The absorbent was allowed to settle and added to the top of the column a layer of 1.5 g of anhydrous sodium sulfate. The residue obtained was dissolved in 3 ml of methylene chloride and transferred to the column. The flask was rinsed twice with 1 ml portions of methylene chloride and the rinses were transferred to the column and eluted at a rate within ml per minute. 3 ml of hexane, 3 ml of diethyl ether and 3 ml of methylene chloride was added successively to the column followed by eluting at a rate within 3 ml per minute. The eluates were discarded. 6 mL of a mixture of methylene chloride and acetone (9:1) was added to the column and eluted at a rate within 1 ml per minute preferably without the aid of vacuum. The eluate was collected in a small vial. A boiling chip was added, if required and allowed to evaporate to dryness on a water bath. The residue was dissolved in 0.2 ml of a mixture of chloroform and acetonitrile (9.8:0.2) followed by shaking the mixture by mechanical means, if required.

[00143] Aflatoxin Solution: Accurately weighed quantities of aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 were dissolved in a mixture of chloroform and acetonitrile (9.8:0.2) to obtain a solution having concentrations of 0.5 µg /per ml each for aflatoxin B1 and G1 and 0.1µg per ml each for aflatoxins for B2 and G2.

[00144] Procedure: 2.5 µl, 5 µl, 7.5 µl and 10 µl of the Aflatoxin Solution and three 10 µl applications of either test solution 1 or test solution 2 were separately applied to a suitable thin- layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture. 5 µl of the aflatoxin solution was superimposed on one of the three 10 µl applications of the test solution. The spots were allowed to dry and the chromatogram was developed in an unsaturated chamber containing a solvent system consisting of a mixture of chloroform, acetone and isopropyl alcohol (85:10:5) until the solvent front moved at least 15 cm from the origin. The plate was removed from the developing chamber followed by marking the solvent front and allowing the plate to air-dry. The spots were located on the plate by examination under UV light at 365 nm. The four applications of the aflatoxin solution appeared as four clearly separated blue fluorescent spots. The spot obtained from the test solution that was superimposed on the aflatoxin solution was no more intense than that of the corresponding aflatoxin solution. No spot from any of the other test solutions corresponded to any of the spots obtained from the applications of the aflatoxin solution. In case any spot of aflatoxins was obtained in the test solution then the position of each fluorescent spot of the test solution was compared with the spots of the aflatoxin solution to identify the type of aflatoxin present. The intensity of the aflatoxin spot, if present in the test solution, when compared with that of the corresponding aflatoxin in the aflatoxin solution provided an approximate concentration of aflatoxin in the test solution.
Table 7 illustrates experimental results for the tests for trace metal analysis of the herbal composition.
S No Test Result Standard Limits
1 Total Aflatoxin Below Quantification Limit Not more than 5mcg/Kg
2 Aflatoxin B1 Below Quantification Limit 1-2mcg/Kg
3 Aflatoxin B2 Below Quantification Limit 1-2mcg/Kg
4 Aflatoxin G2 Below Quantification Limit 1-2mcg/Kg
5 Aflatoxin G1 Below Quantification Limit 1-2mcg/Kg
Table 7
8. 99.9% Germ Killing Efficacy

[00145] The germ Killing Efficacy was evaluated as per ASTM E2315-016. The procedure for evaluating germ killing efficacy measures the changes in a population of aerobic microorganisms within a specified sampling time when antimicrobial test materials were present. Several options for organism selection and growth, inoculum preparation, sampling times and temperatures were provided. When the technique was performed as a specific test method then standardization of the variables was required. Antimicrobial activity of specific materials, as measured by the technique, may vary significantly depending on variables selected. The test results of microorganisms requiring growth supplements or special incubation conditions may not be directly comparable to organisms evaluated without the stated conditions.

[00146] The test material or a dilution of the test material was brought into contact with a known population of microorganisms for a specified period of time at a specified temperature. An appropriate and specified neutralization technique was applied to quench the antimicrobial activity of the test material at specified sampling interval, for example 30 seconds or 60 seconds or any range covering several minutes or hours and the surviving microorganisms were determined. The percent and/or log10 reduction was determined by comparing with the microbial population.

[00147] Table 8 illustrates the experimental results for different tests performed on the herbal composition of the present invention:

Sr No. Test Result Standard Limits
1. General Description
1.1. Color Dark brown Dark brown
1.2. Odour Characteristic Characteristic
2. pH 8.85 8.0-10.0
3. Viscosity 10.3 cps 7-12 cps
4. Microbial Contamination
4.1. Total Microbial Count, cfu/g Less than 10 Not more than 100000
4.2. Total Yeast and Mould, count/g Less than 10 Not more than 1000
4.3. Escherichia coli/g Absent Should be absent
4.4. Pseudomonas aeruginosa /g Absent Should be absent
4.5. Salmonella/10g Absent Should be absent
4.6. Staphylococcus aureus/g Absent Should be absent
5. Heavy metal Analysis
5.1. Mercury Below Quantification
Limit 0.20-1.0ppm
5.2. Cadmium Below Quantification
Limit 0.20-0.30ppm
5.3. Lead Below Quantification
Limit 0.20-10.0ppm
5.4. Arsenic Below Quantification
Limit 0.20-3.0ppm
6. Pesticide Residue
6.1. Alchalor Below Quantification
Limit 0.0050-
0.020mg/kg
6.2. Malathoin Below
Quantification Limit 0.0050-
1.0mg/kg
6.3. Phoslane Below Quantification
Limit 0.0050-
0.10mg/kg
6.4. Pyrethrins Below Quantification
Limit 0.0050-
3.0mg/kg
6.5. Ethion Below
Quantification Limit 0.0050-2mg/kg
6.6. Chlorpyrifos Below Quantification
Limit 0.0050-
0.20mg/kg
6.7. Methyl Parathion Below Quantification
Limit 0.0050-
0.20mg/kg
6.8. Lindane (Gamma - HCH) Below
Quantification Limit 0.0050-
0.60mg/kg
6.9. Aldrin and dieldrin (sum of) Below
Quantification Limit 0.0050-
0.50mg/kg
6.10. Azinophos - methyl Below
Quantification Limit 0.0050-
1.0mg/kg
6.11. Bromopropylate Below Quantification
Limit 0.0050-
3.0mg/kg
6.12. Chlorpyrifos-methyl Below Quantification
Limit 0.0050-
0.10mg/kg
6.13. Deltamethrin Below
Quantification Limit 0.0050-
0.50mg/kg
6.14. Diazinon Below Quantification
Limit 0.0050-
0.10mg/kg
6.15. Dichlorvos Below
Quantification Limit 0.0050-
1.0mg/kg
6.16. Endrin Below Quantification
Limit 0.0050-
0.050mg/kg
6.17. Fonofos Below
Quantification Limit 0.0050-
0.050mg/kg
6.18. Methidathion Below Quantification
Limit 0.0050-
0.20mg/kg
6.19. Parathion Below Quantification
Limit 0.0050-
0.50mg/kg
6.20. Permethrin Below
Quantification Limit 0.0050-
1.0mg/kg
6.21. Perimiphos-methyl Below Quantification
Limit 0.0050-
4.0mg/kg
6.22. Fentrothion Below
Quantification Limit 0.0050-
0.50mg/kg
6.23. Cypermethrin and isomers Below Quantification
Limit 0.0050-
1.0mg/kg
6.24. Hexachlorocyclohexane Below
Quantification Limit 0.0050-
0.30mg/kg
6.25. Piperonyl-butoxide Below Quantification
Limit 0.0050-
3.0mg/kg
6.26. Heptachlor Below Quantification
Limit 0.0050-
0.050mg/kg
6.27. Fenvalerate Below Quantification
Limit 0.0050-
1.50mg/kg
6.28. Endosulfan Below
Quantification Limit 0.0050-
3.0mg/kg
6.29. DDT Below Quantification
Limit 0.0050-
1.0mg/kg
6.30. Hexachlorobenzene Below
Quantification Limit 0.0050-
0.10mg/kg
6.31. Chlorfenvinphos Below Quantification
Limit 0.0050-
0.50mg/kg
6.32. Dithiocarbamates Below
Quantification Limit 0.0050-
2.0mg/kg
6.33. Chlordane Below Quantification
Limit 0.0050-
0.50mg/kg
6.34. Quintozene Below Quantification
Limit 0.0050-
0.10mg/kg
7. Aflatoxin
7.1. Total Aflatoxin Below Quantification
Limit Not more than 5mcg/Kg
7.2. Aflatoxin B1 Below
Quantification Limit 1-2mcg/Kg
7.3. Aflatoxin B2 Below Quantification
Limit 1-2mcg/Kg
7.4. Aflatoxin G2 Below Quantification
Limit 1-2mcg/Kg
7.5. Aflatoxin G1 Below
Quantification Limit 1-2mcg/Kg
8. Germ Killing Efficacy
8.1. Germ Killing Efficacy
(For 30 Sec) Percent
Reduction
8.1.1.
Salmonella abony 99.9695 Not less than 99%
8.1.2.
Staphylococcus aureus 99.9856 Not less than 99%
8.1.3.
Escherichia coli 99.8510 Not less than 99%
8.1.4.
Aspergillus brasilensis 78.4489 Not less than 99%
8.1.5.
Candida albicans 99.9495 Not less than 99%
8.1.6.
Listeria monocytogenes 99.9909 Not less than 99%
8.1.7. Staphylococcus epidermis 99.9238 Not less than 99%
8.1.8. Pseudomonas aerginosa 99.9278 Not less than 99%
8.2. Germ Killing Efficacy
(For 60 Sec) Percent
Reduction
8.2.1.
Salmonella abony 99.9881 Not less than 99%
8.2.2.
Staphylococcus aureus 99.9948 Not less than 99%
8.2.3.
Escherichia coli 99.9877 Not less than 99%
8.2.4.
Aspergillus brasilensis 86.9691 Not less than 99%
8.2.5.
Candida albicans 99.9956 Not less than 99%
8.2.6.
Listeria monocytogenes 99.9963 Not less than 99%
8.2.7. Staphylococcus epidermis 99.9838 Not less than 99%
8.2.8. Pseudomonas aerginosa 99.9899 Not less than 99%

Table 8

9. EVALUATION OF ACUTE ORAL MUCOSAL IRRITATION CAUSED BY THE HERBAL COMPOSITION IN WISTAR RAT

[00148] Another experimental test was performed to evaluate acute oral mucosal irritation caused by the herbal composition prepared in the present invention, administered in Wistar Rats via by intraoral instillation.

Experimental Procedure:

[00149] Test Item Preparation-
5mL of the herbal composition was mixed with 10mL of lukewarm water as recommended for clinically usage.

[00150] Procedure: The Wistar rats were acclimatized for 7 days prior to starting of the experiment. The Wistar rats were randomly grouped into four groups (G1-G4) as shown in the Table 1 based on their body weight. The test item was applied on the Wistar rats twice a day, primarily, morning and evening (single day dosing) into oral cavity of the Wistar rats using micropipette at a dose volume of 125µL in G2, 250µL in G3 and 500µL in G4. The control group of the Wistar rats were dosed with 0.9% normal saline, twice a day, at a dose volume of 500µL. After Instillation, each rat was stable for about 20-30 seconds, after that the Wistar rat was slightly reversed to spit out the test composition from the oral mucosa.
Table 9 illustrates the random grouping of Wistar rats in four groups based on their body weights.

Group & Treatment Dose, Regimen & ROA No. of animals

G1 500 µL, twice a day (Morning & Evening) in a single day, Intraoral 3
G2 125 µL, twice a day (Morning & Evening) in a single day, Intraoral 4
G3 250 µL, twice a day (Morning & Evening) in a single day, Intraoral 4
G4 500µL, twice a day (Morning & Evening) in a single day, Intraoral 4
Table 9

[00151] Observations:
Clinical Signs- Wistar rats were examined for clinical signs at 1 hour post dosing and later once a day throughout experimental period.

Mortality/Morbidity- All the Wistar rats were observed for mortality/morbidity twice daily throughout the experimental period.

Body Weight-
The Body weight of all the Wistar rats were recorded on day 1 (i.e. prior to dosing), day 8 and day 15. The percentage change in body weights were calculated on day 1 to 8, day 8 to 15 and day 1 to 15.

Macroscopic Observation- The macroscopic observation of oral mucosa was performed in all the Wistar rats after 1 hour of test item application and thereafter daily, once a day, throughout the experimental period. The observation site included tongue dorsum, labial mucosa and incisor gingiva of mandibular oral vestibule. The oral mucosal irritation was graded as per Table 2.

Table 10 illustrates oral mucosal irritation grades by macroscopic observation

Observation site Reaction Grade
Oral Mucosa (Tongue dorsum) Negative 0
Discoloration, slight Sloughing 1
Sloughing in several areas 2
Ulceration 3
Oral Mucosa (Mandibular oral vestibule: Labial mucosa and incisor gingiva Negative 0
Slight Redness, sloughing, dryness 1
Skin rough and brittle, small sores 2
Cracking and bleeding 3
Table 10

[00152] The irritancy scores were calculated by taking an average of the combined daily grades for the tongue dorsum, labial mucosa and incisor gingiva of the mandibular oral vestibule. Based on the irritancy scores, the irritation potential of test formulation was classified according to Table 3.

Table 11 illustrates classification of the irritancy scores by macroscopic observation
Irritancy Scores Classification
0 Non-Irritant
> 0 and < 0.4 Very mild irritant
= 0.4 and < 1.0 Mild irritant
= 1.0 and < 2.0 Moderate irritant
= 2.0 Severe Irritant
Table 11

[00153] Gross Pathology:
All the surviving Wistar rats were sacrificed by CO2 asphyxiation and gross pathological examination was performed for external and internal on day 15.

Result:

Clinical signs- No clinical signs of evident toxicity were reported in the Wistar rats throughout the observation period as shown in Table 4.

Table 12 illustrates Clinical Signs in individual Wistar rat
Group Animal No. Day
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Mor 1h Eve 1h
G1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
G2 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
7 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
G3 8 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
9 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
10 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
11 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
G4 12 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
13 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
14 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
15 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Mor: Morning; Eve: Evening; 1=Normal
Table 12
[00154] Mortality/Morbidity- No mortality/morbidity was reported in the Wistar rats throughout the observation period as shown in Figure 5.

Table 13 illustrates Mortality/Morbidity testing in each individual female Wistar rat
Group Animal No. Day Mortality/Morbidity
G1 1 1-15 Nil
2 1-15 Nil
3 1-15 Nil
G2 4 1-15 Nil
5 1-15 Nil
6 1-15 Nil
7 1-15 Nil
G3 8 1-15 Nil
9 1-15 Nil
10 1-15 Nil
11 1-15 Nil
G4 12 1-15 Nil
13 1-15 Nil
14 1-15 Nil
15 1-15 Nil

Nil = No morbidity/mortality
Table 13

[00155] Body Weight: An increase in the body weight of all the Wistar rats was reported on day 8, day 15 as compared to day 1 as seen in Table 6.

Table 14 illustrates body weight and dose volume data for individual Wistar Rat

Groups Animal No. Body Weight (g) Percentage (%)
change in Body Weight
Day 1 Day 8 Day 15 Day 1-8 Day 8-15 Day 1-15
G1 1 181.02 181.59 186.24 0.31 2.56 2.88
2 185.32 187.40 189.11 1.12 0.91 2.05
3 225.63 227.18 229.60 0.69 1.07 1.76
Mean 197.32 198.72 201.65 0.71 1.51 2.23
SEM 14.21 14.33 14.00 0.23 0.53 0.34
G2 4 176.58 178.17 182.14 0.90 2.23 3.15
5 187.72 188.58 195.75 0.46 3.80 4.28
6 201.12 203.61 211.24 1.24 3.75 5.03
7 234.51 234.09 231.08 -0.18 -1.29 -1.46
Mean 199.98 201.11 205.05 0.60 2.12 2.75
SEM 12.55 12.17 10.52 0.31 1.19 1.46
G3 8 177.54 181.27 181.62 2.10 0.19 2.30
9 186.52 190.91 199.61 2.35 4.56 7.02
10 226.45 227.75 227.80 0.57 0.02 0.60
11 227.53 227.62 228.75 0.04 0.50 0.54
Mean 204.51 206.89 209.45 1.27 1.32 2.61
SEM 13.11 12.17 11.48 0.57 1.08 1.52
G4 12 174.52 173.28 176.48 -0.71 1.85 1.12
13 199.63 200.67 204.39 0.52 1.85 2.38
14 198.89 204.94 208.04 3.04 1.51 4.60
15 235.12 236.67 237.05 0.66 0.16 0.82
Mean 202.04 203.89 206.49 0.88 1.34 2.23
SEM 12.47 12.98 12.39 0.78 0.40 0.86
Table 14

[00156] Macroscopic Observation- No macroscopic lesions were observed at observation site in any of the treatment and control groups Wistar R. Irritancy scores was calculated by taking an average of the combined daily grades for the tongue dorsum, labial mucosa and incisor gingiva of the mandibular oral vestibule. Based on the irritancy score “0”, the irritation potential of test item was classified as “Non-Irritant” as shown in Table 7.

Table 15 illustrates macroscopic observation of oral mucosa on tongue dorsum
Group Animal No. Observation site: Tongue dorsum/Days
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Mor 1h Eve 1h
G1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
G2 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
G3 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
G4 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
13 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
15 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Reaction: Negative=0, Discoloration, slight Sloughing = 1, Sloughing in several areas=2, Ulceration=3
Table 15

Table 16 illustrates macroscopic observation of oral mucosa on Labial mucosa and incisor gingiva

Group Animal No. Observation site: Mandibular oral vestibule: Labial mucosa and incisor gingiva /Days
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Mor 1h Eve 1h
G1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
G2 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
G3 8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
G4 12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
13 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
15 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Reaction: Negative=0, Slight Redness, sloughing, dryness = 1, Skin rough and brittle, small sores =2, Cracking and bleeding =3
Table 16

[00157] Gross Pathology- No internal or external abnormalities were reported in any of the Wistar rats of treated or control groups as shown in Table 8.

Table 17 illustrates Gross Pathological Findings in an individual female Wistar rat

Group
Animal No. External Examination Internal Examination
G1 1 NAD NAD
2 NAD NAD
3 NAD NAD
G2 4 NAD NAD
5 NAD NAD
6 NAD NAD
7 NAD NAD
G3 8 NAD NAD
9 NAD NAD
10 NAD NAD
11 NAD NAD
G4 12 NAD NAD
13 NAD NAD
14 NAD NAD
15 NAD NAD

Table 17

NAD = No Abnormalities Detected
Conclusion: The Wistar rats showed no mortality, no sign of toxicity, no sign of any irritation in oral mucosal cavity and no gross pathological changes. Hence, the test item i.e. the herbal composition was reported to be safe for oral gargle route in a single day dosing.

10. Anti-microbial property of the herbal composition
As shown in Figure 1, the time kill analysis may monitor the effect of various concentrations of an antimicrobial agent i.e. herbal composition over time in relation to the stages of the growth of the bacteria. The herbal composition demonstrated >97% germ-kill efficacy against various pathogenic micro-organisms i.e., Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermis, Listeria monocytogene, Candida albicans, Escherichia coli and Salmonella abony, at intervals of 30 and 60 seconds. The herbal composition also shows over 78% reduction in Aspergillus brasilensis at 30 second and 86 % reduction at 60 second time interval.

11. Anti-inflammatory property of the herbal composition
[00158] As shown in Figure 2, LPS is an inflammatory stimulus which activates cells of the innate immune system, such as macrophages and neutrophils, which synthesize pro-inflammatory factors, such as IL-1ß and TNF, MMPs and free radicals that lead to dramatic secondary inflammation in tissues. Immune cells were stimulated using LPS and anti-inflammatory effect of herbal composition was reported on the cells. The herbal composition showed a maximum of 100% inhibition in IL-6 levels of LPS stimulated immune cells.

12. Anti-allergic property of the herbal composition
[00159] As shown in Figure 3, Compound 48/80 causes mast cell degranulation and considered to be a powerful promoter of the release of histamine. Immune cells (THP-1) were stimulated with Compound 48/80 for 15-20 mins to induce the release of histamine in cells mimicking allergic condition. The herbal composition showed an anti-allergic potential by 53% inhibition in the levels of histamine released in THP-1.

13. Analgesic property of the herbal composition
[00160] As shown in Figure 4, COX-2 inhibitors are a type of nonsteroidal anti-inflammatory drug (NSAID) that directly target cyclooxygenase-2, COX-2, an enzyme responsible for inflammation. The herbal composition exhibited a maximum of 83.4% inhibition of COX-2 enzyme activity depicting analgesic potential of the herbal composition.

14. Mucolytic property of the herbal composition
[00161] As shown in Figure 5, N-acetylcysteine is a classic antioxidant that may reduce mucus viscosity and promote mucus discharge. It may also significantly improve lung function, reducing mucus viscosity and enhancing small airway function. The herbal composition showed 84.5% decrease in the viscosity of egg white solution, indicating mucolytic potential of the herbal composition.

[00162] The herbal composition for treating sore throat related infections has a number of advantages described herein:
(a) The herbal composition prepared in the present invention is safe and comprises of natural components with negligible side effects.
(b) The herbal composition possesses potential antiviral, anti- inflammatory, antibacterial, and antimicrobial action that effectively relieves sore throat and other throat related infections.

[00163] While this invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the scope of the appended claims.
,CLAIMS:WE CLAIM:

1. A herbal composition for treating throat related infections and a method for preparation thereof, comprising:
• one or more essential oils in a range of 1-12% by weight of total herbal composition;
• one or more herbal extracts in a range of 1% to 20% by weight of the total composition;
• at least one solubilizing agent in a range of 1% to 16% by weight of the total herbal composition;
• a salt in a range of 8% to12% by weight of the total herbal composition;
• one or more additives in a range of 11% by 30% by weight of the total herbal composition; and
• water in a range of 25% to 40% by weight of the total herbal composition.

2. The herbal composition as claimed in claim 1, wherein the essential oils are selected from a group consisting of Lemon oil, Rosemary oil, Ginger oil, Peppermint oil, Cinnamon oil, Basil oil, Clove oil, or a combination thereof.

3. The herbal composition as claimed in claim 1, wherein the herbal extracts are selected from a group consisting of, Kalmegh extract, Licorice extract, Ginseng extract, Tea extract, or a combination thereof.

4. The herbal composition as claimed in claim 1, wherein the solubilizing agent is selected from a group consisting of,EG 400, Propylene glycol, Poloxomer 188 and Polysorbate 80, or a combination thereof.

5. The herbal composition as claimed in claim 1, wherein the salt is sodium bicarbonate.

6. The herbal composition as claimed in claim 1, wherein the additive is selected from a group of sweeteners, or solubilizers, or a combination thereof.

7. A method for preparing the herbal composition as claimed in claim 1, comprising steps of:
(a) adding salt in water contained in apparatus (A) followed by dissolving the salt thoroughly in the water;
(b) heating the solution obtained in step (a) at a temperature ranging between 60-70?;
(c) adding herbal extracts to the solution obtained in step (b);
(d) stirring the solution prepared in step (c);
(e) filtering the solution obtained in step (d);
(f) adding solubilizing agent and essential oils in an apparatus (B);
(g) stirring the solution obtained in step (f);
(h) adding additives and water to the solution obtained in step (e);
(i) mixing the solution obtained in step (h) and the solution obtained in step (g), forming another solution; and
(j) homogenizing the solution obtained in step (i), forming a herbal composition.