DESC:FIELD OF INVENTION
[001] The present invention relates to a composition for treating respiratory tract infections and a method for preparation thereof. Particularly, the present invention relates to a composition exhibiting potential antiviral, anti-inflammatory, antibacterial, and antimicrobial action that effectively relieves the respiratory tract infections such as cold, bronchitis, laryngitis, tonsillitis, sinusitis, etc.
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, laryngitis, tonsillitis, sinusitis, etc. Such infections are caused a number of viruses and bacteria. Major symptoms of the respiratory tract infections include sneezing, congestion, coughing, blocked nose, headache, etc.

[003] The respiratory tract infections involve various organs of the body that take part in breathing process such as lungs, throat, sinusitis, etc. The upper respiratory tract infections are caused by various harmful pathogens that attack mucus membrane lining the nasal cavity. The pathogens fight through several physical barriers such as small hair in the nose lining, cilia lining the trachea, etc.

[004] There are various conventional methods which intend to provide relief from the respiratory tract infections. Some convention methods include the use of nasal sprays for treating cough, cold and congestion. There are several types of nasal sprays such as saline spray, steroid sprays, antihistamine spray, decongestant sprays, etc. However, such sprays are associated with a number of disadvantages such as irritation and dryness in the nose, burning sensation in nasal passage, stinging, bitter taste, etc. Other conventional methods include nasal irrigation. However, nasal irrigation is often associated with a number of side effects such as irritation in nose. Further, the saline used during the irrigation may drain some essential immune elements present in the mucus that act as first line of defense against respiratory infections. Some other conventional methods include the use of antibiotics. However, excessive consumption of the antibiotics may make the body resistant thereto. Moreover, the antibiotics are often associated with a number of side effects such as diarrhea, abdominal pain and so on. Other conventional method includes the consumption of lozenges. However, the lozenges are effective for only a short period of time and may not be effective against excessive congestion, blockage and cough. Yet other conventional methods include invasive medical treatments such as surgery for clearing blockages and opening the sinus pathways. However, such surgical treatments are associated with a number of risks such as bleeding, intracranial complications, damage to surrounding tissues or eyes, pain and numbness of face and so on.

[005] Therefore, keeping in view the problems associated with the state of the art there is a need of a technology for a safe, efficient, non-invasive means for providing relief from respiratory tract infections.

SUMMARY OF THE INVENTION
[006] The present invention relates to a composition for treating respiratory tract infections and a method for preparation thereof. The composition comprises of one or more of essential oils and one or more chemical compounds. The essential oils are present in a range 0.5 % to 24% by weight of the total composition. The organic compounds are present in a range of 23 % to 90% by weight of the total composition. The present invention also provides a method for preparation of the composition. The present invention relates to a composition exhibiting potential antiviral, anti-inflammatory, antibacterial, and antimicrobial action that effectively relieves the respiratory tract infections such as cold, bronchitis, laryngitis, tonsillitis, sinusitis, etc. The composition of the present invention may be used as steam inhalant. The composition may also be used in form of gel, cream or rub and may be applied on outer sides of nose to provide relief from nose blockage.
OBJECTIVES OF THE INVENTION
[007] A primary objective of the present invention is to provide a composition for treating respiratory tract infections and a method for preparation thereof.

[008] Another objective of the present invention is to provide a composition having therapeutic properties.

[009] Another objective of the present invention is to provide a composition exhibiting antiviral, anti-inflammatory and antibacterial properties for providing effective relief against respiratory tract infections such as such as cold, bronchitis, laryngitis, tonsillitis, sinusitis, etc.

[0010] Another objective of the present invention is to provide a composition for non-invasive administration ensuring rapid drug absorption, rapid onset of action, reduction in systemic side effects, and convenient and painless self-administration.

[0011] Yet another objective of the present invention is to provide a composition comprising a number of essential oils ensuring rapid onset of action, persisting antimicrobial action and less side effects.

[0012] Yet another objective of the present invention is to provide a steam inhalant composition which aids in thinning the mucus in the sinuses, thereby providing effective relief against the respiratory tract infections.

[0013] Other objects and advantages of the present invention will become apparent from the following description.

SOURCE OF BIOLOGICAL MATERIAL USED IN THE INVENTION
[0014] The essential oils used in present invention such as, but not limited to, Eucalyptus oil, Clove oil, Basil oil and Peppermint oil, or a combination thereof are 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 for the test strains include 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).

BRIEF DESCRIPTION OF DRAWINGS
[0015] An understanding of the present invention may be obtained by reference to the accompanying drawings, when taken in conjunction with the description herein and in which:
[0016] Figure 1 illustrates a graphical representation showing the anti-inflammatory activity exhibited by the composition;
[0017] Figure 2 illustrates the anti-inflammatory activity exhibited by the composition;
[0018] Figure 3 illustrates the anti-allergic activity exhibited by the composition;
[0019] Figure 4 illustrates the mucolytic potential exhibited by the composition; and
[0020] Figure 5 illustrates the anti-inflammatory activity exhibited by the composition.

DETAILED DESCRIPTION OF THE INVENTION
[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] It is to be understood that the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0026] 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.

[0027] Accordingly, the present invention relates to a composition for treating respiratory tract infections and a method for preparation thereof. Particularly, the present invention relates to a composition exhibiting potential antiviral, anti-inflammatory, antibacterial, and antimicrobial action that effectively relieves the respiratory tract infections such as cold, bronchitis, laryngitis, tonsillitis, sinusitis, etc.

[0028] In an embodiment, the herbal composition of the present invention comprises of one or more essential oils and one or more chemical compounds.

[0029] The essential oils are concentrated liquid extracts that are isolated from different parts of the plants. The essential oils used in the composition may be selected from a group, consisting of, such as, but not limited to, Eucalyptus oil, Clove oil, Basil oil, Peppermint oil or a combination thereof. Each essential oil used in the present invention is extracted from different part of a herb such as, Eucalyptus oil is extracted from leaves of Eucalyptus globulus, Clove oil is extracted from dried flower buds of Eugenia caryophyllus, Basil oil is extracted from leaves of Ocimum basilicum, and Peppermint oil is extracted from leaves, flowers and stems of Mentha piperita L. In an exemplary embodiment, 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 used in the present invention are present in a range of 0.5% to 24% by weight of the total composition. In an exemplary embodiment, the amount of each essential oil may vary such as Eucalyptus oil is present in a range of 2% to 24% by weight of total essential oils, Clove oil is present in a range of 0.5%-13% by weight of total essential oils, Basil oil is present in a range of 0.5%-13% by weight of total essential oils, and Peppermint oil is present in a range of 0.5%-13% by weight of total essential oils. The essential oils contain a number of chemical compounds known as phytoactive compounds. Such phytoactive compounds present in the essential oils possess antimicrobial, anti-inflammatory and antiviral properties, thereby imparting medicinal properties to the essential oils. In another exemplary embodiment, the phytoactive compound present the essential oils may include such as, but not limited to, Cineole present in Eucalyptus oil, Eugenol present in Clove oil, Chavicol present in Ajwain oil, Menthol present in Peppermint oil, Chavivol present in Tulsi oil, Alpha Copaene present in Cedarwood oil, and Aldehyde present in Cinnamon oil. In yet another exemplary embodiment, the phytoactive compound may be present in the range of 0.5% to 98% by weight of the total essential oils. The essential oils possess a wide range of healing properties as discussed below:

[0030] (a) Eucalyptus oil: The Eucalyptus oil exhibits antibacterial property used to fight germs. The antibacterial property is also effective against sinusitis, nasal congestion, asthma and aids in relieving cough, stuffiness, reducing and loosening mucus, and other respiratory ailments. The anti-inflammatory property of the Eucalyptus oil aids in fighting inflammation and promotes healing.

[0031] (b) Clove oil: The Clove oil exhibits antiviral, antifungal, antimicrobial and anti-inflammatory properties. Such properties aid in killing germs, relieving respiratory conditions such as sore throat, bronchitis, asthma, bacterial/viral cough, cold, sinusitis and arthritis. The Clove oil is also known for boosting immunity by reducing the blood’s toxin level. The antioxidant property of the clove oil acts as a scavenger against free radicals capable of causing various diseases. The Clove oil also aids in clearing nasal passage and preventing respiratory problems. Moreover, the Clove oil exhibits a cooling effect on nasal cavity and throat.

[0032] (c) Basil Oil: The Basil oil exhibits antibacterial and antiviral properties that 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. Moreover, the Basil oil also boasts the immune system..

[0033] (d) Peppermint oil: The Peppermint oil exhibits antimicrobial property against various harmful pathogens which enters the body through mouth. The antibacterial property of the Peppermint oil also aids in fighting off harmful bacteria which triggers congestion. The Peppermint oil also possesses antiseptic and analgesic properties that aids in curing dry cough, bronchitis, asthma and other lung diseases. The Peppermint oil also exhibits immunomodulatory property that enhances immunity as well as antitussive property that relieves cough.

[0034] The chemical compounds used in the present invention may be selected from a group of such as, but not limited to, organic compounds. In an exemplary embodiment, the organic compounds may be selected from a group consisting of, such as, but not limited to, camphor, menthol, or a combination thereof. Camphor is a solid, transparent, waxy organic compound exhibiting anti-inflammatory, anti-microbial, anti-viral and analgesic properties, providing relief against respiratory infections such as cold, cough, etc. Menthol is a waxy, clear/white, crystalline organic compound exhibiting anti-inflammatory, anti-bacterial, anti-fungal, antiviral and analgesic properties, providing relief against cold, cough sore throat, etc. In an exemplary embodiment, the chemical compounds used in the present invention are present in solid form such as, but not limited to, powder, pellet and crystal. The chemical compounds used in the present in a range of 23% to 90% by weight of the total composition. In yet another exemplary embodiment, the amount of chemical compound may vary such as camphor is present in a range of 23% to 45% by weight of the total chemical compounds; and menthol is present in a range of 23% to 45% by weight of the total chemical compounds. The chemical compounds used in the present invention possess various medicinal properties as discussed below:

[0035] (a) Camphor: Camphor possesses anti-inflammatory, anti-bacterial, anti-fungal, antiviral and analgesic properties. Due to the presence of such properties, camphor acts as a decongestant to reduce blocked nose and a cough suppressant. camphor also helps in overcoming sleep difficulty in patients suffering from respiratory tract infections.

[0036] (b) Menthol: Menthol exhibits anti-inflammatory, anti-bacterial, anti-fungal, antiviral and analgesic properties. Such properties aid in treating cold, cough, irritation in throat, sore throat and other respiratory tract infections. Menthol also provides cooling effect that opens up the cavities and suppresses cough.

[0037] Table 1 illustrates different compounds and quantity (ml and %) of each compound used in the composition of the present invention.

Sr. No. Compounds Quantity (ml)
Quantity (%)
1. Camphor 33.3 23%-45%
2. Menthol 33.3 23%-45%
3. Eucalyptus Oil 13.3 2%-24%
4. Clove Oil 6.6 0.5%-13%
5. Basil Oil 6.6 0.5%-13%
6. Peppermint Oil 6.9 0.5%-13%
Total 100
Table 1

[0038] In an embodiment, the present invention also provides a method for preparing a composition for treating respiratory tract infections. The method discloses the following steps:

(a) adding essential oils in an apparatus;
(b) adding chemical compounds in the apparatus;
(c) heating the mixture obtained in step (b) at a temperature ranging between 30?-40? until the chemical compounds completely melts; and
(d) allowing the solution obtained in step (c) to cool down to room temperature.

[0039] The essential oils added in step (a) may be selected from such as, but are not limited to, Eucalyptus oil, Clove oil, Basil oil, Peppermint oil or a combination thereof. The essential oils are present in a range of 0.5% to 24% by weight of the total composition. In an exemplary embodiment, the amount of each essential oil may vary such as Eucalyptus oil is present in a range of 2% to 24% by weight of the total essential oils, Clove oil is present in a range of 0.5% to 13% by weight of the total essential oils, Basil oil is present in a range of 0.5% to 13% by weight of the total essential oils, and Peppermint oil is present in a range of 0.5% to 13% by weight of the total essential oils.

[0040] The chemical compounds added in step (b) may be selected from a group of such as, but not limited to, organic compounds. In an exemplary embodiment, the organic compounds may be selected from a group consisting of, such as, but not limited to, camphor, menthol, or a combination thereof. The organic compounds are present in a range of 23% to 90% by weight of the total composition. In an exemplary embodiment, the amount of organic compounds may vary such as camphor is present in a range of 23% to 45% by weight of the total chemical compounds, and menthol is present in a range of 23% to 45% by weight of the total chemical compounds.

[0041] The composition prepared in the present invention may be stored in an air tight container/bottle and kept in a dry and cool place.

[0042] In an embodiment, the composition may be used as an inhalant by adding few drops of the composition prepared in the present invention in water followed by boiling the water, which may further be used for the purpose steam inhalation.

[0043] In another embodiment, the composition prepared in the present invention may be used as gel, cream or rub, which may be applied on outer sides of nose to provide relief from nose blockage. The gel, cream, or rub may be prepared by adding a thickening agent such as, but not limited to, gelling agent or wax. The thickening agent is used in a range of 1%-10% by weight of the total composition. In an exemplary embodiment, the wax may be, such as, but not limited to beeswax, petroleum jelly, carnauba wax, or a combination thereof. In another exemplary embodiment, the may be, such as, but not limited to, pectin, gelatin, tragacanth, starch, sodium alginate, carbomer, polyvinyl alcohol clays, cellulose derivatives, or a combination thereof.

[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 composition of the present invention were evaluated. Such characteristics include color and odor of the composition. The herbal composition was perceived to be pale yellow in color. The herbal composition exhibited a characteristic odor.

2. Weight per milliliter
[0046] The weight per milliliter of the composition of the present invention was evaluated. A clean and dry pycnometer was selected. The pycnometer was calibrated by filling the pycnometer with recently boiled and cooled water at 25? followed by weighing the contents. The capacity of the pycnometer was determined by considering the weight of 1 ml of water at 25? when weighed in air of density 0.0012 gram per ml to be 0.99602 gram. The results remain unaffected by minor deviations in the density of air from the given value. The temperature of the liquid sample to be examined was adjusted to about 20? and the pycnometer was filled with the liquid sample. The temperature of the filled pycnometer was adjusted to 25? followed by removal of excess of the liquid sample. The filled pycnometer was then weighed. The tare weight of the pycnometer was subtracted from the weight of filled pycnometer. The weight per milliliter was determined by dividing the weight of the quantity of liquid sample in air (g) which fills the pycnometer at the specified temperature by the capacity (ml) of the pycnometer at the same temperature.

3. Specific Gravity
[0047] The specific gravity of the composition of the present invention was evaluated. A clean and dry pycnometer was selected. The pycnometer was calibrated by filling the pycnometer with boiled and cooled water at 25? followed by weighing the pycnometer filled with water. The capacity of the pycnometer was determined by considering the weight of 1 ml of water at 250 when weighed in air of density 0.0012 gram per ml to be 0.99602 gram. The results remain unaffected by minor deviations in the density of air from the given value. The temperature of the liquid sample to be examined was adjusted to about 20? and the pycnometer was filled with the liquid sample. The temperature of the filled pycnometer was adjusted to 25? followed by removal of excess of the liquid sample. The filled pycnometer was then weighed. The tare weight of the pycnometer was subtracted from the weight of filled pycnometer. The weight per milliliter was determined by dividing the weight of the quantity of liquid sample in air (g) which fills the pycnometer at the specified temperature by the capacity (ml) of the pycnometer at the same temperature. The specific gravity of the liquid sample was determined by dividing the weight of the liquid sample contained in the pycnometer at 25? by the weight of water contained in the pycnometer at 25?.
4. Refractive Index
[0048] The refractive index of the composition of the present invention was evaluated. The refractive index was determined (as per API part I, vol. VI, appendix 3, 3.1.1) at 25? (±0.5) with reference to wavelength of the D line of sodium (? 589.3 nm). The temperature was carefully adjusted and maintained to avoid any variation in the refractive index since refractive index significantly varies with the temperature.
5. pH
[0049] The pH of the composition of the present invention was evaluated. The pH meter was calibrated using buffer solution before use. The pH of the composition was determined in triplicate and average of the triplicate values was determined.

6. Saponification Value
[0050] About 35 to 40 gram of potassium hydroxide was dissolved in 20 ml water, forming a solution. A required amount of alcohol was added to the prepared solution to make the volume of the solution to 1000 ml. The solution was allowed to stand overnight followed by pouring off the clear liquor. Accurately weighed 2 g of the sample was added in a tared 250 ml flask. 25 ml of the alcoholic solution of potassium hydroxide was added into the flask. A reflux condenser was attached to the flask followed by boiling the flask on a water-bath for about one hour. The flask was then rotated in order to cool the solution within the flask. 1 ml of solution of phenolphthalein was added to the flask. The excess of alkali was titrated with 0.5 N hydrochloric acid. The required volume of hydrochloric acid (a) was recorded. The same procedure was repeated with similar quantities of the same reagents without using the sample to be examined. The required volume of hydrochloric acid (b) was recorded. The saponification value was determined using the following equation:

Saponification Value = (b–a) × 0.02805 × 1.000
W
Wherein,
W= weight of the sample taken (grams)

7. Viscosity
[0051] The viscosity of the composition of the present invention was evaluated. The sample liquid to be examined was filled in a U tube viscometer in accordance with the expected viscosity of the liquid so that the level of fluid was within 0.2 mm of the filling mark of the viscometer when the capillary was vertical and the specified temperature was attained by the liquid sample. The liquid sample was pipetted/blown to the specified weight of the viscometer and the time taken for the meniscus to pass the two specified marks was measured. The kinematic viscosity (in centistokes) was determined using 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).
8. Microbial Contamination
[0052] The microbial contamination of the composition was evaluated. The number of viable aerobic microorganisms present in the test sample and the presence of specified microbial species in the test sample was estimated by the following tests:

[0053] Preliminary Testing: Prior to performing preliminary tests, the diluted samples to be examined was inoculated with separate viable cultures of Escherichia coli, Salmonella species, 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. 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 samples and following the test procedure. In case the organisms fail to grow in the relevant medium then the volume of the diluent was increased with the quantity of test sample remaining the same or a sufficient amount of a desired inactivating agent in the diluents was incorporated or a combining thereof to permit the growth of the organisms in the medium. In case the inhibitory substances are present in the test samples then 0.5% of soya lecithin and 4% of polysorbate 20 was added to the culture medium. Alternatively, the same procedure may be repeated using fluid casein digestsoya lecithin-polysorbate 20 medium for demonstrating neutralization of preservatives or other antimicrobial agents in the test sample. In case the inhibitory substances are present in the test samples and the test sample was soluble then membrane filtration method was used. The failure to isolate the inoculated organism even after the incorporation of a desired inactivating agents and/or substantial increase in the volume of diluent and/or the sample is not suitable for applying the membrane filtration method, may be due to the bactericidal activity of the sample. Such failure may indicate a probability that the 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.

Culture Media

[0054] Different culture medium were prepared for evaluating microbial contamination or a dehydrated culture media was used such as 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. Agar with moisture content within 15% was used in the preparation of the culture medium, if required. Purified water was used in the culture medium, if required. The medium was sterilized by heating in an autoclave at 15 psi and at 121? for 15 minutes. The preparation of culture medium by using the formulas given below involves dissolving soluble solids in water by using heat, if required. A required amount of solutions of 0.1N hydrochloric acid or 0.1N sodium hydroxide was added to yield the required pH in the medium before the use. The pH of the medium was determined at 250 ± 20. The procedure for preparing different culture mediums is described herein:

[0055] 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 volume of the solution up to 1000ml. The solution was heated to boiling for 1 minute along with stirring the solution. The solution was then sterilized and cooled to a temperature in a range of 45?-50?. 10 ml of a 1% weight by volume of solution of sterile potassium tellurite and 50 ml of egg yolk emulsion was added to the solution. The solution was thoroughly and gently mixed followed by pouring the solution into plates. Egg-yolk emulsion was prepared by disinfecting the surface of whole shell eggs followed by aseptically cracking the eggs and separating out intact yolks into a sterile graduated cylinder. A sterile saline solution was added to the egg yolk to obtain ratio of 3:7 of egg-yolk to saline. The solution was added to a sterile blender cup and agitated at high speed for 5 seconds. The pH of the medium was adjusted to 6.8 ± 0.2 after sterilization.

[0056] Bismuth Sulphite Agar Medium was prepared by forming two solutions. Solution 1 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, and 10.0 mg of Brilliant green. Water was added to make the volume of the solution to 1000ml. The solution was dissolve with the help of heat and sterilized by maintaining at 115? for 30 minutes. Solution 2 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 to 100 ml. The solution was mixed and heated to boiling. The solution was then allowed to cool at room temperature. 1 part of solution 2 was added to 10 parts of solution 1 previously melted and cooled to a temperature of 55? and poured into plates. The Bismuth Sulphite Agar Medium was stored at 2? to 8? for 5 days before use.

[0057] Brilliant 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 make the volume of the mixture to 1000 ml. The solution was then mixed and allowed to stand for 15 minutes. The solution was then sterilized by maintaining at 115? for 30 minutes followed by mixing the solution. The solution was then poured into plates.

[0058] 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 make the volume of the mixture to 1000 ml. 0.1% to 1.0% weight by volume Polysorbate 20 or Polysorbate 80 may be added. Sterilize by heating in an autoclave at 121? for 15 minutes. 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 121? for 15 minutes.

[0059] Casein Soyabean Digest Agar Medium was prepared by adding 15.0 g of Pancreatic digest of casein, 5.0 g of Papaic digest of soyabean meal, 5.0 g of Sodium chloride; and 15.0 g of Agar. The water was added to the mixture to make the volume of the solution to 1000 ml. The pH of the medium was adjusted to 7.3 ± 0.2 after sterilization.
[0060] 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. The water was added to make the volume to the mixture to 1000 ml. The solution was then heated by boiling for 1 minute along with shaking the solution. The pH of the medium was adjusted to 7.0 to 7.4 after sterilization.

[0061] 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. The water was added to make the volume of the mixture to 1000 ml. The solution was mixed and allowed to stand for 15 minutes. The solution was gently boiled with continuous stirring until solution was completely homogenized. The solution was then cooled to 80? and mixed. The solution was then poured into plates. Overheating of Desoxycholate Citrate Agar was avoided during the preparation of the medium. Re-melting of the solution was also avoided. The surface of the plates were dried before use.

[0062] 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. The water was added to make the volume of the mixture to 1000 ml. The pancreatic digest of casein and soya lecithin was dissolved in water followed by heating in a water-bath at 48? to 50? for about 30 minutes. Polysorbate 20 was added to the solution and mixed followed by dispensing the solution as per the requirement.

[0063] 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 1000 ml. The solution was rapidly cooled after sterilization. The pH of the medium was adjusted to 6.9 ± 0.2 after sterilization.

[0064] 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 solution to 1000 ml. The pH of the medium to 6.9 ± 0.2 after sterilization.

[0065] 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 solution 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. 5 ml of 20% weight by volume of solution of lactose, 2 ml of a 2% weight by volume of solution of Eosin Y and 2 ml of a 0.33% weight by volume of solution of methylene blue was used for each 100 ml of the liquified agar solution. The prepared medium may not be clear. The pH of the medium was adjusted to 7.1±0.2 after sterilization.

[0066] MacConkey Agar Medium was prepared by adding 17.0 g of Pancreatic digest of gelatin, 3.0 g of Peptone (meat and casein, equal parts), 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 final volume of solution to 1000 ml. The mixture of solids and water was boiled for 1 minute. The pH of the medium was adjusted to 7.1 ± 0.2 after sterilization.

[0067] 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 to 1000 ml. The pH of the medium was adjusted to 7.3 ± 0.2 after sterilization.

[0068] Mannitol-Salt Agar Medium was prepared by 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 make the volume of the mixture to 1000 ml. The solution was mixed by heating the solution to boiling for 1 minute along with stirring the solution. The pH of the medium was adjusted to 7.4 ± 0.2 after sterilization.

[0069] Nutrient Agar Medium was prepared by adding 1% to 2% weight by volume of agar into nutrient broth.

[0070] 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 make the volume of mixture to 1000 ml. The solution was dissolved with the help of heat. The pH of the solution was adjusted 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 maintaining at 115? for 30 minutes. The pH of the medium was adjusted to 7.3 ± 0.1 after sterilization.

[0071] Pseudomonas Agar Medium for Detection of Flourescein was prepared by adding 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 sulphate hepta hydrate, 10.0 ml of Glycerin, and 15.0 g of Agar. Water was added to make the volume of the mixture to 1000 ml. The solid components were dissolved in water before adding glycerin. The solution was mixed by heating the solution to boiling for 1 minute. The pH of the medium was adjusted to 7.2 ± 0.2 after sterilization.

[0072] Pseudomonas Agar Medium for Detection of Pyocyanin 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. The water was added to make the volume of the mixture to 1000 ml. The solid components were dissolved in water before adding glycerin. The solution was mixed by heating the solution to boiling for 1 minute. The pH of the medium was adjusted to 7.2 ± 0.2 after sterilization.

[0073] 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 to 1000 ml. The solution was then mixed by boiling the solution. The pH of the medium was adjusted to 5.6 ± 0.2 after sterilization.

[0074] Sabouraud Dextrose Agar Medium with Antibiotics was prepared was adding 0.1 g of benzylpenicillin sodium and 0.1 g of tetracycline HCL to 1 liter of Sabouraud Dextrose Agar Medium. Alternatively, 50 mg of chloramphenicol may be added to 1 liter of Sabouraud Dextrose Agar Medium immediately before use.

[0075] Selenite F Broth 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 to 1000 ml. The mixture was dissolved and distributed in sterile containers. The solution was then sterilized by maintaining at 100? for 30 minutes.

[0076] 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 to 1000 ml. The solution was mixed and heated in flowing steam for 15 minutes. The final pH of the medium was added to 7.0 ± 0.2. Sterilization step was avoided for the preparation of Fluid Selenite - Cystine Medium.

[0077] 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 to 1000 ml. The solid components were dissolved in water followed 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 before use.

[0078] 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 make the volume of the mixture to 1000 ml. The solution was heated to boiling. Reheating of the solution was avoided. The pH was adjusted to achieve a pH of 7.0 ± 0.2 after heating.

[0079] 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 make the volume of the mixture to 1000 ml. The solution was mixed and allowed to stand for 15 minutes. The solution was then boiled and the boiling point was maintained until solution was completely homogenous. The solution was thoroughly mixed and distributed in tubes followed by sterilization by maintaining at 121? for 15 minutes. The solution was allowed to stand in a sloped form with about 2.5 cm long bottom.

[0080] 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. The water was added to make the volume of the mixture to 1000 ml. The solution was then mixed and sterilized followed by distributing the solution aseptically in sterile containers.

[0081] 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 make the volume of the mixture to 1000 ml. The solution of solids was boiled for 1 minute. The solution was then sterilized and cooled to a temperature in a range of 45?-50?. 20 ml of 1% weight by volume of sterile solution of potassium tellurite was added to the solution. The pH of the medium was adjusted to 7.0 ± 0.2 after sterilization.

[0082] Xylose -Lysine-Desoxycholate Agar Medium 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 to 1000 ml. The mixture of solids and water was heated to boiling. Overheating and sterilizing was avoided. The solution was to a water-bath maintained at about 50? and poured into plates just after the medium was cooled. The final pH of the medium was adjusted to 7.4 ± 0.2.

[0083] The sampling involves using 10 ml or 10 g of sample for each of the specified tests.

[0084] Precautions taken during experimentation involves performing the microbial limit tests under specific conditions designed to prevent accidental contamination during the test. The precautions taken to prevent contamination during the test exhibit no adverse effect on microorganisms that are required to be revealed in the test.

[0085] Total Aerobic Microbial Count: The total aerobic microbial count in the herbal composition was evaluated. The procedure for evaluating total aerobic microbial count in different types of test samples is described herein:

[0086] Water-soluble sample: 10 g or 10 ml of the sample to be examined was dissolved/diluted in buffered sodium chloride-peptone solution pH 7.0 or other suitable medium which exhibits no antimicrobial activity under the test conditions. The solution was diluted to make the volume of the solution to 100 ml with the same medium. The pH of the medium was adjusted to around 7, if required.

[0087] Water-insoluble sample (non-fatty): 10 g or 10 ml of the sample to be examined was suspended in a 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 suspension was divided, if required and homogenized mechanically. A suitable surface-active agent such as 0.1% weight by volume of Polysorbate 80 was added to assist the suspension of less wettable sample. The pH of the suspension was adjusted to around 7.

[0088] Water insoluble sample (Fatty): 10 g or 10 ml of the sample to be examined was homogenized with 5 g of polysorbate 20 or polysorbate 80. The sample was heated to a temperature within 40?. The solution was mixed while maintaining the temperature in the water-bath or in an oven. 85 ml of a buffered sodium chloride-peptone solution pH 7.0 or other suitable medium that exhibits no antimicrobial activity under test condition heated within 40? was added to the prepared solution, if required. The temperature was maintained for time period required for the formation of an emulsion. However, such time period may not exceed 30 minutes. The pH of the solution was adjusted to around 7, if required.

[0089] The sample was examined by determining the total aerobic microbial count in the sample using any of the following methods:

[0090] (i) Membrane filtration: Membrane filters with 50 mm diameter and nominal pore size within 0.45 mm was used for retaining bacteria. 10 ml or a required amount of each dilution containing 1g of the sample to be examined wad 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. Each membrane was washed by filtering three or more successive quantities each of about 100 ml, of a suitable liquid such as buffered sodium chloride-peptone solution pH 7.0 through the membrane.

[0091] 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. 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 unless a reliable count was obtained in less time period such as 300 to 350 in the test for bacteria and 200 to 250 in the test for fungi. The number of colonies formed were counted. The number of microorganisms per g or per ml of the sample to be examined was determined separately for bacteria and fungi, if required.

[0092] Plate count for bacteria: Petri dishes with diameter of 9 cm to 10 cm were used. A mixture of 1ml of the pretreated sample preparation and about 15 ml of liquified casein soyabean digest agar within 45? was added to each petri dish. 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 as described above was diluted, if required to obtain colony count within 300. At least two such petri dishes were prepared using the same dilution and incubated at 30? to 35? for 5 days until a reliable count was obtained in less time period. The number of colonies formed were counted.

[0093] The results were evaluated using plates with the highest number of colonies considering 300 colonies per plate to provide maximum results.

[0094] 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 incubated at 20? to 25? for 5 days until a reliable count was obtained in less time period. The results were evaluated using plates with colonies within 100.

[0095] B. Multiple-tube or serial dilution: 9.0 ml of sterile fluid soyabean casein digest medium was placed in each of fourteen test tubes of similar size. Twelve of the tubes were arranged in four sets of three 124 tubes each. One set of three tubes were taken as controls. 1 ml of the solution of suspension of the test sample was pipetted into each of three tubes of one set (“100”) and into fourth tube (A). The solution was mixed in each test tube. 1 ml of the contents of tube (A) was pipetted into the one remaining tube (B) which was not included in the set. The solution in tube B was mixed. These two tubes contained 100 mg (or 100 µl) and 10 mg (or 10 µl) of the specimen respectively. 1 ml from tube A was pipetted into each of the second set (“10”) of three tubes pipette. 1 ml from tube B was pipetted into each tube of the third set (“1”). The remaining contents of tube A and B were discarded. All the tubes were properly closed and incubated. The tubes were examined for growth after the incubation period. The three control tubes were clear. The tubes containing the test sample was observed to determine probable number of microorganisms per g or per ml of the test sample.

[0096] Table 2 illustrates possible number of microorganisms per g or per ml of the test sample.

Table 2

Tests for Specified Microorganisms:
[0097] Pretreatment of the test sample: The pretreatment of 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.

[0098] Test for Escherichia coli: A required amount of pretreated test sample was placed in a sterile screw-capped container. 50 ml of nutrient broth was added to the container and mixed by shaking the container. The container containing the mixture was allowed to stand for 1 hour. However, the time period may vary to 4 hours for gelatin. The mixture was again mixed by shaking the container. The cap of the container was loosened followed by incubating at 37? for 18-24 hours.

[0099] Primary test: 1.0 ml of the enrichment culture was added to a tube containing 5 ml of MacConkey broth. The mixture was incubated in a water-bath at 36?-38? for 48 hours. In case, the mixture within the tube showed acid and gas then secondary test was performed.

[00100] 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 43.5? - 44.5? for 24 hours. Tube (A) was examined for acid and gas. Tube (b) was examined for indole. To examine Tube (B) for Indole, 0.5 ml of Kovac’s reagent was added to the tube (B) and content of tube (B) were mixed by shaking the tube. The tube was allowed to stand for 1 minute. The presence of Indole was indicated by the formation of red colour the reagent layer. The presence of acid and gas and indole in the secondary test indicated the presence of Escherichia coli. A control test was performed by repeating the primary and secondary tests. 1.0 ml of the enrichment culture and a suitable volume of broth containing 10 units to 50 units of Escherichia coli 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 that the control contains Escherichia coli.

[00101] Alternative test (as per API, Part-I, Vol.-IX (Extracts); Appendices 125): A portion from the enrichment culture obtained in the previous test was streaked on the surface of MacConkey agar medium by means of an inoculating loop. The dishes were covered and incubated. In case, during examination none of the colonies were brick-red in colour and showed A surrounding zone of precipitated bile then the sample was considered to fulfil the requirements of the test for absence of Escherichia coli. In case, the colonies as mentioned above were present then the suspected 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 incubation 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 suitable cultural and biochemical tests.

[00102] 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 100 ml of nutrient broth in a sterile screw-capped jar. The mixture within the jar was mixed by shaking the jar followed by allowing the jar to stand for 4 hours. The mixture within the jar was again mixed by shaking the jar. The cap of the jar was loosened and incubated at 35?-37? for 24 hours.

[00103] 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. The tubes were incubated at 36?-38? for 48 hours. A subculture was prepared from each of the two prepared cultures on at least two of the agar media including bismuth sulphate agar, brilliant green agar, deoxycholate citrate agar and xylose-lysine deoxycholate agar. The plates were incubated at 36?-38? for 18 to 24 hours. In case, during examination, none of the colonies conforms to the description provided in Table 2 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 colonies conforming to the description in Table 2 were formed.

[00104] Secondary test: The colonies showing the characteristics given in Table 2 were subcultured in a Triple Sugar - Iron Agar Medium by inoculating the surface of the slope followed by making a stab culture with the same inoculating needle. Simultaneously, a tube of urea broth was also inoculated. Incubation was performed at 36? to 38? for 18 to 24 hours. The presence of Salmonella was indicated by 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 Medium along with the absence of a red colour in urea broth. However, in case only acid is produced without the production of gas in the cultures, then the presence of Salmonella was confirmed by agglutination tests. The control test was performed by repeating the primary and secondary tests using 1.0 ml of the enrichment culture and a suitable volume of broth containing 10 to 50 Salmonella abony prepared from a 24-hour culture in nutrient broth for inoculating tube (A) and tube (B). The test was considered effective only when the results indicated that the control contains Salmonella.

[00105] Test for Pseudomonas aeruginosa: The sample preparation to be examined was pretreated as described above. 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 preparation to be examined. The solution was mixed and incubated at 35? to 37? for 24 to 48 hours.

[00106] The medium was examined for growth of Pseudomonas aeruginosa (as per API, Part-I, Vol.-IX (Extracts); Appendices 126). In case, growth was indicated in the medium then a portion of the medium was streaked on the surface of cetrimide agar medium, each plated on petri dishes. The petri dishes were covered and incubated at 35? to 37? for 18 to 24 hours. In case, during examination, none of the plates showed colonies exhibiting the characteristics listed in Table 2 for the media used, then the sample was considered to fulfil the requirement for the absence of Pseudomonas aeruginosa. In case, any colonies conforming to the description as provided in Table 2 were formed then oxidase and pigment tests were performed. The suspected colonies from the agar surface of cetrimide agar were streaked on the surface of Pseudomonas agar medium for detection of fluorescein and pyocyanin contained in Petri dishes. The inoculated media was covered and inverter followed by incubating at 33? to 37? for at least 3 days. The streaked surfaces were examined under ultra-violet light. The plates were examined to determine the presence of colonies conforming to the description in Table 2, if any. In case, the growth of suspected colonies occurred 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 put on filter paper and smeared with the colony. The absence of the production of pink colour turning to purple, the sample was considered to fulfil the requirements of the test indicating the absence of Pseudomonas aeruginosa.

[00107] Test for Staphylococcus aureus: The test for Staphylococcus aureus involves 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 listed in Table 2 for the media used, the sample was considered to fulfil the requirements for the absence of Staphylococcus aureus. In case, the growth was detected then coagulase test was performed. The suspected colonies were transferred from the agar surface of the media listed in Table 2 to individual tubes, each containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with/without additives. The tubes were incubated in the water-bath at 37?. The tubes were examined for 3 hours and subsequently at a suitable intervals up to 24 hours. The presence of any level of coagulation indicates that the sample fulfilled the requirements of the test indicating the absence of Staphylococcus aureus.

Validity of the tests for total aerobic microbial count (as per API, Part-I, Vol.-IX (Extracts); Appendices 127):
[00108] A number of test strains were grown separately in tubes containing fluid soyabean-casein digest medium at 30? to 35? for 18 to 24 hours. However, in case of Candida albicans, the test strains were grown separately in tubes containing fluid soyabean-casein digest medium at 20? for 48 hours. The test strains include the following:

• Staphylococcus aureus
• Bacillus subtilis
• Escherichia coli
• Candida albicans

[00109] The portions of each of the cultures were diluted using buffered sodium chloride-peptone solution pH 7.0 to make test suspensions containing about 100 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 preparation to be examined, if required. A count for any test organisms differing by a factor within 10 from the calculated value for the inoculum was required. The sterility of the medium, diluent and the aseptic performance of the test was evaluated by performing total aerobic microbial count method using sterile buffered sodium chloride peptone solution pH 7.0 as the test preparation. Absence of the growth of microorganism was required to confirm the validity of the tests for total aerobic microbial count.

Validity of the tests for specified microorganisms:
[00110] The test strains of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Salmonella typhimurium were grown separately in fluid soyabean-casein digest medium and at 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 containing about 103 viable microorganisms per ml. Equal volume of each suspension was mixed and 0.4 ml (approximately 102 micro-organisms 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 specific strain of microorganism was required to confirm the validity of the tests for specified microorganisms.

11. Heavy Metal Analysis: The presence of heavy metals in the herbal composition was evaluated using different method provided herein:
(a) Determination of Lead (Pb) by Graphite Oven Method:

[00111] The following conditions were taken as reference for determining the presence of lead (Pb) using Graphite Oven Method:
• dry temperature: 100?-120? maintained for 20 seconds;
• ash temperature: 400?-750? maintained for 20-25 seconds;
• atomic temperature: 1700?-2100? maintained for 4-5 seconds;
• measurement wavelength: 283.3 nm; and
• background calibration: deuterium lamp (D lamp) or Zeeman effect.

[00112] Preparation of lead (Pb) 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? to 50?.

[00113] Preparation of calibration curve: A required amount of lead standard stock solutions were accurately measured and diluted with 2% of nitric acid solution to concentration of 0, 5, 20, 40, 60, 80 ng per ml. 1 ml of 1% of ammonium dihydrogen phosphate and 0.2% of magnesium nitrate was added to accurately measured 1 ml of the prepared solutions followed by mixing the solution. Accurately measured 20 µl of the prepared solutions was pipetted into the atomic generator of graphite oven. The absorbance of solutions was determined followed by preparing the calibration curve with absorbance as vertical axis and concentration as horizontal ordinate.

[00114] 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 to the flask. A small hopper was placed on the top of the flask. The mixture was macerated overnight followed by heating to boiling the mixture to slake on the electric hot plate. A required amount of above mixture was added in the solution turned brownish-black in color followed by constantly heating the solution until a clean and transparent solution appeared. The temperature was then raised and the solution was constantly heated to thicken the smoke until white smoke dispersed. The slaked solution on turning colourless and transparent or yellowish was cooled and transferred it into a 50 ml volumetric flask. The container was washed with 2% nitric acid solution and the washing solution was added into the same volumetric flask followed by diluting with the same solvent to make the volume of the solution to 50ml. The solution was mixed properly by shaking the flask. Simultaneously, a reagent blank solution was prepared according to the procedure mentioned above.

[00115] Determination of Lead: The presence of lead was determined by accurately measuring 1 ml of the test solution and corresponding reagent blank solution. 1 ml of solution containing 1% 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.10-20 µl of both the test and blank solutions were accurately measured to determine the absorbance of both the solutions according to the steps followed for preparing the 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:
[00116] The following conditions were taken as reference for determining the presence of Cadmium (Cd) using Graphite Oven Method:
• dry temperature: 100?-120? maintained for 20 seconds;
• ash temperature: 300?-500? maintained for 20-25 seconds;
• atomic temperature: 1500?-1900? maintained for 4-5 seconds;
• measurement wavelength: 228.8 nm; and
• background calibration: deuterium lamp (D lamp) or Zeeman effect.

[00117] Preparation of Cadmium (Cd) standard stock solution: A required amount of cadmium single-element standard solution was accurately measured for preparing standard stock solution with 2% nitric acid containing 0.4 µg per ml cadmium. The solution was stored at 0? to 50?.

[00118] 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 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.

[00119] Preparation of test solution: Test solution of cadmium was prepared by a method similar to the method for preparation of test solution of lead.

[00120] Determination of Cadmium: The presence of cadmium was determined by accurately measuring 10-20µl 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 any discrepancy in results, 1 ml of the standard solution, blank solution and test solution were separately weighed. 1 ml of a solution containing 1% of ammonium dihydrogen phosphate and 0.2% of magnesium nitrate was added to the prepared solutions separately. Each prepared solution was properly mixed by shaking the solutions. The absorbance of the prepared solutions was determined by performing same steps as performed for calculating absorbance in case of lead. The content of cadmium (Cd) in the test solution was determined from the calibration curve.
(c) Determination of Arsenic (As) by Hydride Method:

[00121] The following conditions were considered for determining the presence of Arsenic (As) by Hydride Method:
• apparatus: suitable hydride generator device,
• reducing agent: a solution containing 1% of sodium borohydride and 0.3% of sodium hydroxide;
• carrier liquid: 1% of hydrochloric acid;
• carrier gas: nitrogen;
• measurement wavelength: 193.7 nm; and
• background calibration: deuterium lamp (D lamp) or Zeeman effect.

[00122] Preparation of Arsenic (As) standard stock solution: A required amount of Arsenic (As) single element standard solution was accurately weighed for preparing standard stock solution with 2% nitric acid solution containing 1.0 µg per ml Arsenic (As). The solution was stored at 15? to 30?.

[00123] Preparation of calibration curve: A required amount of Arsenic standard stock solutions was accurately measured and diluted with 2% of nitric acid to concentration of 2, 4, 8, 12 and 16 ng per ml. Accurately measured 10 ml of each prepared solution was transferred into 25 ml volumetric flask. 1 ml of 25% potassium iodide solution prepared prior to use was added to each prepared solution. The solutions were mixed properly by shaking. 1 ml of ascorbic acid solution prepared prior to use was added to each solution. The solutions were mixed properly by shaking. The prepared solutions were diluted with hydrochloric acid solution to the volume in range of 20 ml to 100 ml. The prepared solutions were properly mixed by shaking. The stopper of the flask was closed and the flask was immersed in a water bath at 80? for 3 minutes. The solution within the flask was then cooled. A required amount of each solution was transferred into the hydride generator device. The absorbance of the prepared solutions were determined and a calibration curve was plotted with a peak area (absorbance) as vertical axis and concentration as horizontal ordinate.

[00124] Preparation of test solution: Test solution of arsenic was prepared by a method similar to the method for preparation of test solution of lead.

[00125] Determination of Arsenic: The presence of arsenic was determined by pipetting accurately measured 10 ml of the test solution and corresponding reagent blank solution 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:
[00126] The following conditions were considered for determining the presence of Mercury (Hg) by Hydride Method:
• apparatus: suitable hydride generator device;
• reducing agent: a solution containing 0.5% sodium borohydride and 0.1% sodium hydroxide;
• carrier liquid: 1 per cent hydrochloric acid;
• carrier gas: nitrogen;
• measurement wavelength: 253.6 nm; and
• background calibration: deuterium lamp (D lamp) or Zeeman effect.

[00127] Preparation of Mercury (Hg) standard stock solution: A required amount of Mercury (Hg) single-element standard solution was accurately weighed for preparing standard stock solution with 2% nitric acid solution containing 1.0 µg per ml Mercury (Hg). The solution was stored at 15? to 30?.

[00128] 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 separately 50 ml volumetric flasks. 40 ml of 4% sulphuric acid solution and 0.5 ml of 5% potassium permanganate solution was added into each flask. The solution within each flask was mixing by shaking the flask. A drop 5% hydroxylamine hydrochloride solution was added into each flask until the violet red disappeared. Each solution was diluted with 4% sulfuric acid solution to make the volume of the solution to 50 ml followed by mixing the solutions by shaking the flasks properly. A required amount of each solution was injected to the hydride generator device. The absorbance of each solution was determined and then the calibration curve was plotted with peak area (absorbance) as vertical axis and concentration as horizontal ordinate.

[00129] Preparation of test solution: Accurately weighed 1 g of the 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 into the flask followed by mixing the solution thoroughly within the flask. A small hopper was fixed on the flask-top. 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 solution was then cooled followed by adding a required amount of 4% sulfuric acid solution and 0.5 ml of 5% potassium permanganate solution. The solution was then mixed by shaking the flask. A drop of 5% hydroxylamine hydrochloride solution was added into the solution until the violet red colour disappeared. The solution was then diluted with 4% sulphuric acid solutions to make the volume to 25 ml. The solution was mixed by shaking the flask. The solution was then centrifugated, if required. The supernatant obtained was used as the test solution. Similarly, the reagent blank solution was simultaneously prepared by performing the same steps.

[00130] Determination of Mercury: 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.
12. Pesticide Residue
[00131] Limits: The sample to be examined required to compile with the limits indicated in Table 3.

[00132] The limits applying to the suspected pesticides not provided in table may comply with the limits set by European Community directives 76/895 and 90/642, including their annexes and successive updates. Limits for suspected pesticides not provided in Table or in 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)

[00133] 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

[00134] Higher limits may also be permitted in cases where a plant requires a particular cultivation method or exhibit a metabolism or a structure that gives rise to a higher content of pesticides. 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 can be checked precisely.

[00135] Sampling Method: One sample from the total content 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 were taken for containers between 1 kg and 5 kg. The samples were thoroughly mixed and a required amount was taken from the mixture to perform the tests. Three samples, each of at least 250 g from the upper, middle and lower parts of the container were taken for containers of more than 5 kg. The samples were thoroughly mixed and a required amount was taken from the mixture to perform the tests.

[00136] 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.

[00137] 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.
[00138] 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, suitable blank tests were performed.

[00139] Apparatus: The apparatus and glasswares were thoroughly cleaned to keep them pesticide free, Cleaning procedure may involve soaking the apparatus and/or glasswares in a solution of phosphate-free detergent for at least 16 hours followed by rinsing with large quantities of distilled water and washing with acetone and hexane or heptane.
13. Aflatoxins
[00140] The presence of different types of aflatoxins including, B2, G1 and G2 in the herbal composition of the present invention was evaluated. The procedure for evaluating the presence of different type of aflatoxins are as follows:

[00141] 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.

[00142] Sodium Chloride Solution: 5 g of sodium chloride was dissolved in 50 ml of purified water.

[00143] Test Solution 1: About 200 g of test sample 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 into the flask. The solution within the flask was mixed by shaking the flask vigorously by mechanical means for at least 30 minutes followed by filtering the solution. First 50 ml of the filtrate was discarded and next 40 ml portion 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 layers were allowed to separate. The lower aqueous layer was transferred to a second separatory funnel. The aqueous layer in the separatory funnel was extracted twice, each time with 25 ml of methylene chloride along with shaking the solution for 1 minute. The layers were then allowed to separate followed by separating the lower organic layer and collecting the combined organic layers in a 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.

[00144] Test Solution 2: In case the solution possess 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.

[00145] 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.

[00146] Aflatoxin Solution: Accurately weighed quantities of aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2 was 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.

[00147] 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.
14. 99.9% Germ Killing Efficacy:
[00148] 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 the 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.
[00149] 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.

[00150] Table 3 illustrates the experimental results for different tests performed on the composition of the present invention.

Sr. No. Test Result Standard Limits
1. General Description
1.1. Color Pale yellow Pale yellow
1.2. Odour Characteristic Characteristic
2. Weight per ml 0.9252 gm/ml 0.5-1.5 gm/ml
3. Refractive Index 1.4704 1.0-1.5
4. pH 4.39 -
5. Saponification 0.77 -
6. Viscosity 10.6 cps 7-12 cps
7. Microbial Contamination
7.1. Total Microbial Count, cfu/g Less than 10 Not more than 100000
7.2. Total Yeast and Mould, count/g Less than 10 Not more than 1000
7.3. Escherichia coli/g Absent Should be absent
7.4. Pseudomonas aeruginosa /g Absent Should be absent
7.5. Salmonella/10g Absent Should be absent
7.6. Staphylococcus aureus/g Absent Should be absent
8. Heavy metal Analysis
8.1. Mercury Below Quantification Limit 0.20-1.0ppm
8.2. Cadmium Below Quantification Limit 0.20-0.30ppm
8.3. Lead Below Quantification Limit 0.20-10.0ppm
8.4. Arsenic Below Quantification Limit 0.20-3.0ppm
9. Pesticide Residue
9.1. Alchalor Below Quantification Limit 0.0050-0.020mg/kg
9.2. Malathoin Below Quantification Limit 0.0050-1.0mg/kg
9.3. Phoslane Below Quantification Limit 0.0050-0.10mg/kg
9.4. Pyrethrins Below Quantification Limit 0.0050-3.0mg/kg
9.5. Ethion Below Quantification Limit 0.0050-2mg/kg
9.6. Chlorpyrifos Below Quantification Limit 0.0050-0.20mg/kg
9.7. Methyl Parathion Below Quantification Limit 0.0050-0.20mg/kg
9.8. Lindane (Gamma - HCH) Below Quantification Limit 0.0050-0.60mg/kg
9.9. Aldrin and dieldrin (sum of) Below Quantification Limit 0.0050-0.50mg/kg
9.10. Azinophos - methyl Below Quantification Limit 0.0050-1.0mg/kg
9.11. Bromopropylate Below Quantification Limit 0.0050-3.0mg/kg
9.12. Chlorpyrifos-methyl Below Quantification Limit 0.0050-0.10mg/kg
9.13. Deltamethrin Below Quantification Limit 0.0050-0.50mg/kg
9.14. Diazinon Below Quantification Limit 0.0050-0.10mg/kg
9.15. Dichlorvos Below Quantification Limit 0.0050-1.0mg/kg
9.16. Endrin Below Quantification Limit 0.0050-0.050mg/kg
9.17. Fonofos Below Quantification Limit 0.0050-0.050mg/kg
9.18. Methidathion Below Quantification Limit 0.0050-0.20mg/kg
9.19. Parathion Below Quantification Limit 0.0050-0.50mg/kg
9.20. Permethrin Below Quantification Limit 0.0050-1.0mg/kg
9.21. Perimiphos-methyl Below Quantification Limit 0.0050-4.0mg/kg
9.22. Fentrothion Below Quantification Limit 0.0050-0.50mg/kg
9.23. Cypermethrin and isomers Below Quantification Limit 0.0050-1.0mg/kg
9.24. Hexachlorocyclohexane Below Quantification Limit 0.0050-0.30mg/kg
9.25. Piperonyl-butoxide Below Quantification Limit 0.0050-3.0mg/kg
9.26. Heptachlor Below Quantification Limit 0.0050-0.050mg/kg
9.27. Fenvalerate Below Quantification Limit 0.0050-1.50mg/kg
9.28. Endosulfan Below Quantification Limit 0.0050-3.0mg/kg
9.29. DDT Below Quantification Limit 0.0050-1.0mg/kg
9.30. Hexachlorobenzene Below Quantification Limit 0.0050-0.10mg/kg
9.31. Chlorfenvinphos Below Quantification Limit 0.0050-0.50mg/kg
9.32. Dithiocarbamates Below Quantification Limit 0.0050-2.0mg/kg
9.33. Chlordane Below Quantification Limit 0.0050-0.50mg/kg
9.34. Quintozene Below Quantification Limit 0.0050-0.10mg/kg
10. Aflatoxin
10.1. Total Aflatoxin Below Quantification Limit Not more than 5mcg/Kg
10.2. Aflatoxin B1 Below Quantification Limit 1-2mcg/Kg
10.3. Aflatoxin B2 Below Quantification Limit 1-2mcg/Kg
10.4. Aflatoxin G2 Below Quantification Limit 1-2mcg/Kg
10.5. Aflatoxin G1 Below Quantification Limit 1-2mcg/Kg
11. 99% Germ Killing Efficacy
11.1. 99% Germ Killing Efficacy (For 30 Sec) % Reduction
11.1.1. Salmonella abony 99.9593 Not less than 99%
11.1.2. Staphylococcus aureus 99.9925 Not less than 99%
11.1.3. Escherichia coli 99.9503 Not less than 99%
11.1.4. Aspergillus brasilensis 98.8473 Not less than 99%
11.1.5. Candida albicans 99.9495 Not less than 99%
11.1.6. Listeria monocytogenes 99.9239 Not less than 99%
11.1.7. Staphylococcus epidermis 99.9371 Not less than 99%
11.1.8. Pseudomonas aerginosa 99.9278 Not less than 99%
11.2. 99% Germ Killing Efficacy (For 60 Sec) % Reduction
11.2.1. Salmonella abony 99.9881 Not less than 99%
11.2.2. Staphylococcus aureus 99.9988 Not less than 99%
11.2.3. Escherichia coli 99.9940 Not less than 99%
11.2.4. Aspergillus brasilensis 99.4988 Not less than 99%
11.2.5. Candida albicans 99.9956 Not less than 99%
11.2.6. Listeria monocytogenes 99.9929 Not less than 99%
11.2.7. Staphylococcus epidermis 99.9927 Not less than 99%
11.2.8. Pseudomonas aerginosa 99.9899 Not less than 99%

Table 3
IN VITRO EFFICACY STUDIES
[00151] (a) Anti-inflammatory activity

[00152] Anti-inflammatory activity was evaluated by inhibition of inflammatory cytokine (IL-8) in lung cells and cytokine (IL-6) in Immune cells as shown in Figure 1 and Figure 2. The Immune & Lung cells were counted and plated in 24 well culture plates at a density of 1 x 106 cells/well in DMEM +10% FBS and incubated overnight in 5% CO2 incubator at 37°C. After overnight incubation, the cells were serum starved with DMEM + 0% FBS for 24 hours. After incubation, the cells were treated with different concentrations of the composition for 24 hours. Following 24 hours treatment, cells were stimulated with LPS (Immune cells) & TNF-a (Lung cells) respectively for 24 hours. The untreated cells were taken as negative control. The cells treated with LPS (Immune cells) & TNF-a (Lung cells) were taken as control. The cells treated with Dexamethasone were taken as positive control. After incubation, the cell supernatants were collected and stored at -20°C until analyzed. The level of cytokine in supernatants of Immune & lung cells was evaluated by employing commercially available kit as per manufacturer’s instructions. It was reported that the treatment of lung cells and immune cells with the composition resulted in inhibition of TNF-a stimulated levels of IL-8 in Lung cells (A549) by a maximum of 95% as compared to TNF-a stimulated group and inhibition of LPS stimulated levels of IL-6 in Immune cells (Splenocytes) by a maximum of 90.6% as compared to LPS stimulated group

[00153] Anti-allergic activity - Anti-allergic activity of the composition was evaluated by inhibition of histamine in immune cells (as shown in Figure 3). KU812 cells were counted and plated (10,000 cells/well/180µl) in 96 well plate. The cells were treated with the composition at different concentration for 24 hours. The cells corresponding to the positive control group were treated with Epinephrine. After 24 hours, the cells were pelleted at 1500 rpm for 5 min and re- suspended in 180 µl of tyrode buffer. The cells stimulated with compound 40/80 for 15 minutes at a final concentration of 25 µg/ml (20 µl was added from 10x stock) were taken as control. The unstimulated cells were taken as negative control. After stimulation with compound 48/80, the above reaction was terminated by incubating the plate on ice for 15 min. The cell suspension was centrifuged at 400 g for 5 min. The supernatant was collected from each well carefully for estimation of the levels of histamine released from cells. The supernatant obtained above was mixed with NaOH and o-phthalaldehyde dye as following:
Supernatant (160 µl) + 1N NaOH (32µl) + OPT (8µl)
(Ratio- 20:4:1)

The fluorescence of the above mixture was evaluated at 360 nm excitation/ 460 nm emission at sensitivity 50.

[00154] Further, an inhibition of 29.8% of histamine in immune cells (THP-1) stimulated with compound 40/80 was reported. Mucolytic activity

[00155] Mucolytic activity of the composition of was evaluated by inhibition of Mucin (MUC5AC) in lung cells (A549) and inhibition of egg viscosity. A549 Cell suspension in DMEM + 10% FBS was plated at a density of 80,000 cells/48 well cell culture plates and incubated at 37°C for 24 hours. The cells were sera starved by replacing medium with DMEM+ 0% FBS for 24 hours. After incubation, the cells were treated with the composition at different concentrations for 24 hours. After pretreatment with the composition, the cells were stimulated with Hu-TNF-a (5µg/ml) for another 24 hours. The cells treated with Hu-TNF-a alone were taken as control. The cells treated with Dexamethasone + Hu-TNF-a were taken as positive control. The untreated cells were taken as a negative control.Subsequent to 24 hour stimulation with TNF-a, the culture supernatants were collected from each well and stored at -20 °C until analysis. The level of Mucin (MUC5AC) in the culture supernatants of A549 cells were evaluated using ELISA as per manufacturer’s instructions. An inhibition of 88.8 % of TNF-a stimulated levels of mucin (MUC5AC) in lung cells (A549) (as shown in Figure 4) was reported and a decrease by 82.8% in viscosity of egg white solution (as shown in Figure 5).

[00156] Acute Inhalation Toxicity

[00157] The study was performed to evaluate the acute inhalation toxicity of the composition by inhalation route in Wistar Rats using Clinical Steamer.

[00158] Inhalation route via steam vaporizer machine was selected as route of administration. Prior to experimentation, forty drops of the test formulation were mixed in 250ml of Milli Q water in the reservoir of steamer, to generate the steam. Three different concentrations of the composition was formulated in water. Steam inhalation was provided to all the Wistar rats until the entire composition cleared from the reservoir. G1 served as vehicle control group and was treated with milli Q water whereas G2 served as treated group and was treated with the prepared formulation. The prepared formulation was administered by inhalation route using clinical steamer. The steam inhalation of the prepared formulation was provided to the Wistar rats until the entire formulation cleared from the reservoir. Prior to administration, forty drops of the prepared formulation were mixed in 250mL of Milli Q Water in the reservoir of steamer. The prepared formulation was administered twice a day (single day dosing) into the Wistar rats. The vehicle control group was treated with Milli Q water in the same manner. Post dosing, the Wistar rats were observed, for once, 1 hour after the exposure on day of treatment and was repeated daily up to 14 days. On day 15, the Wistar rats were humanely scarified by overdose of Thiopentone sodium and subjected to gross pathological examination.

[00159] No clinical signs of evident toxicity were observed in any of the Wistar rat of control and treatment groups throughout the observation period. There was a progressive increase in the body weight of all the Wistar rats in both the sex of control and treatment groups on day 8 and day 15 as compared to day 1. After completion of experiment, all the surviving Wistar rats were terminally sacrificed by the overdose of thiopental sodium by intraperitoneal route method of euthanasia.

[00160] OBSERVATIONS

[00161] Clinical Signs -

[00162] The Wistar rats were examined for clinical signs at 1 hour post dosing and later once a day throughout study period.

[00163] Mortality/Morbidity -

[00164] All the Wistar rats were observed for mortality/morbidity once daily throughout the experimental period.

[00165] Body Weights-

[00166] Body weight of all the Wistar rats was recorded on the day of doing, day 1 (prior to dosing), day 8 and day 15. The percentage change in body weight was evaluated on day 1 to 8, day 8 to 15 and day 1 to 15. The percentage change in body weight for each group was evaluated using the following formula:
Percentage Body Weight Change =

[00167] Gross Pathology -

[00168] All the survived Wistar rats were euthanized by overdose of Thiopentone sodium injection by intraperitoneal route. All gross pathological changes were recorded for each Wistar rat with particular attention to any changes in the respiratory tract. No external and internal adverse lesions were observed in any of the Wistar rat of control and treated groups.

[00169] RESULTS

[00170] Clinical Signs –

[00171] No clinical signs of evident toxicity were observed in Wistar rats throughout the observation period of study in both the sex of G1 and G2 as shown in Table 4 and Table 5.

[00172] Mortality/Morbidity -

[00173] No mortality/morbidity was observed in both G1 and G2 groups throughout the observation period as shown in Table 6 and Table 7.

[00174] Body Weight -

[00175] There was progressive increase in the body weight of all the Wistar rats in control (G1) and treatment (G2) group on day 8, day 15 as compared to day 1 as shown in Table 6.

[00176] Gross Pathology -

[00177] External Findings -

[00178] No external abnormalities were observed in any of the Wistar rats of the both the groups.

[00179] Internal Findings -

[00180] No internal abnormalities were observed in any of the Wistar rats of the both the groups (as shown in Table 8).

[00181] DISCUSSION AND CONCLUSION -

[00182] Based on the findings of the clinical signs, body weight and gross pathology, it was concluded that the composition of the present invention in a single day dosing was found to be safe to be used in a clinical steamer in both the sex of Wistar Rats.

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
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
G2 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
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
Table 4
Group Animal No. Day Mortality/Morbidity
G1 1 1-15 Nil
2 1-15 Nil
3 1-15 Nil
4 1-15 Nil
5 1-15 Nil
G2 6 1-15 Nil
7 1-15 Nil
8 1-15 Nil
9 1-15 Nil
10 1-15 Nil
Nil = No morbidity/mortality
Table 5

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
1 128.83 150.56 178.52 16.87 18.57 38.57
2 178.87 196.15 206.33 9.66 5.19 15.35
3 180.43 189.56 201.19 5.06 6.14 11.51
4 193.97 197.54 199.18 1.84 0.83 2.69
5 203.76 214.23 215.98 5.14 0.82 6.00
Mean 177.17 189.61 200.24 7.71 6.31 14.82
SEM 12.92 10.58 6.16 2.61 3.25 6.33
G2
6 163.44 175.95 176.76 7.65 0.46 8.15
7 172.09 175.34 176.02 1.89 0.39 2.28
8 185.57 194.63 196.74 4.88 1.08 6.02
9 191.06 203.95 200.21 6.75 -1.83 4.79
10 206.61 216.67 218.64 4.87 0.91 5.82
Mean 183.75 193.31 193.67 5.21 0.20 5.41
SEM 7.51 8.02 7.98 0.99 0.53 0.95
Table 9
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
1 230.21 232.12 249.25 0.83 7.38 8.27
2 251.90 268.87 278.73 6.74 3.67 10.65
3 259.27 263.17 285.74 1.50 8.58 10.21
4 276.41 280.04 307.36 1.31 9.76 11.20
5 285.47 297.47 328.83 4.20 10.54 15.19
Mean 260.65 268.33 289.98 2.92 7.98 11.10
SEM 9.67 10.78 13.44 1.12 1.20 1.13
G2
6 247.83 264.20 269.86 6.61 2.14 8.89
7 249.18 269.01 280.39 7.96 4.23 12.53
8 261.45 279.70 287.76 6.98 2.88 10.06
9 272.61 291.88 298.70 7.07 2.34 9.57
10 306.02 336.14 341.36 9.84 1.55 11.55
Mean 267.42 288.19 295.61 7.69 2.63 10.52
SEM 10.65 12.90 12.37 0.58 0.45 0.67
Table 10

Group
Animal No. External Examination Internal Examination (Male) Internal
Examination
(Female)
G1
1 NAD NAD NAD
2 NAD NAD NAD
3 NAD NAD NAD
4 NAD NAD NAD
5 NAD NAD NAD
G2
6 NAD NAD NAD
7 NAD NAD NAD
8 NAD NAD NAD
9 NAD NAD NAD
10 NAD NAD NAD
NAD = No Abnormality Detected
Table 8
[00183] The composition prepared in the present invention possesses a number of advantages:
• The present invention provides a composition for non-invasive administration
• The non-invasive route of administration ensures rapid drug absorption, rapid onset of action, reduction in systemic side effects, and convenient and painless self-administration
• The essential oils used in the present invention exhibits antimicrobial action, antibacterial, antiviral and antifungal properties. Such properties of essential oils also aids in treating cough, cold, sore throat and also provides relief from difficulty in breathing due to congestion
• The essential oils ensures rapid onset of action, persisting antimicrobial action, less side effects, and enhanced efficacy against respiratory tract infections
• The chemical compounds used in the present invention possess anti-inflammatory, anti-bacterial, anti-fungal, antiviral and analgesic properties. Such properties aids in treating cold, cough, irritation in throat, sore throat and other respiratory tract infections
• The composition of the present invention may be used as a steam inhalant and provides relief from irritation in throat and swollen blood vessels in the nasal passages along with thinning of mucus in the sinuses

[00184] 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 composition for treating respiratory tract infections, comprising:
a. one or more of essential oils in a range of 0.5% to 90% by weight of total composition; and
b. one or more organic compounds in a range of 23% to 90% by weight of total composition; and
c. optionally, a thickening agent in a range of 1-10% by weight of total composition. .

2. The composition as claimed in claim 1, wherein the essential oil is selected from a group consisting of eucalyptus oil, clove oil, basil oil and peppermint oil or a combination thereof.

3. The composition as claimed in claim 1, wherein the organic compound is selected from a group consisting of camphor, menthol or a combination thereof.

4. The composition as claimed in claim 1, wherein thickening is selected from a group consisting of gelling agent, or wax.

5. A method for preparing the composition as claimed in claim 1, , comprising steps of:
(a) adding essential oils in an apparatus;
(b) adding chemical compounds in the apparatus;
(c) heating the mixture obtained in step (b) at a temperature ranging between 30?-40? until the chemical compounds completely melts; and

(d) allowing the solution obtained in step (c) to cool down to room temperature.