DESC:FIELD OF INVENTION
[001] The present invention relates to a herbal composition for nasal spray and a method for preparation thereof. Particularly, the present invention relates to a herbal composition for nasal spray having potential antibacterial, anti-inflammatory, immunomodulatory and antiviral action that effectively relieves nasal congestion and sinus.

BACKGROUND OF THE INVENTION
[002] Nasopharynx, a part of upper respiratory tract, acts as a reservoir for various pathogens such as bacteria and fungi. Such pathogens cause a number of respiratory tract infections such as common cold, mild flu, laryngitis, sinusitis, etc. During a respiratory infection caused by a virus, some of the harmful pathogens move into sinus cavity from nasopharynx and cause sinusitis. The pathogens such as virus or bacteria that line the nasal cavity causes inflammation which leads to swelling of mucous membranes surrounding the sinus cavities.

[003] Mostly, the sinus infections are viral. However, sometimes sinus infection may also be caused by bacterial infection. Sinusitis is often associated with nasal congestion. People may experience cold prior to developing sinusitis. Major viruses that cause sinus infection includes influenza viruses, parainfluenza viruses, rhinoviruses and so on. Some of the bacteria that causes sinusitis includes Streptococcus pnuemoniae, Haemophilus influenza, Moraxella catarrhalis and so on.

[004] There are various conventional methods which intend to provide relief from nasal congestion and sinusitis. Some 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 irrigation may drain some essential immune elements present in mucus which act as first line of defense against respiratory infections. Other conventional method includes the use of antibiotics. However, such antibiotics do not work when the sinus infection is caused by a virus.
[005] Further, excessive consumption of antibiotics makes body resistant to antibiotics. Moreover, antibiotics are often associated with a number of side effects such as diarrhea, abdominal pain and so on. 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.

[006] Therefore, keeping in view the problems associated with the state of the art there is a need of a technology for non-invasive, safe and efficient means for providing relief from nasal congestion and sinusitis.

OBJECTIVES OF THE INVENTION
[007] A primary objective of the present invention is to provide a herbal composition for nasal spray and a method for preparation thereof.

[008] Another objective of the present invention is to provide a herbal nasal spray composition for non-invasive intranasal administration.

[009] Another objective of the present invention is to provide antiviral, anti-inflammatory, and antimicrobial herbal composition for providing effective relief from nasal congestion and sinus.

[0010] Yet another objective of the present invention is to provide a composition comprising a number of essential oils ensuring rapid onset of action, less side effects and long lasting antimicrobial action.

[0011] Still another objective of the present invention is to provide a herbal nasal spray composition having enhanced stability and longer residual time in nasal mucosal membrane resulting in long lasting antimicrobial action.

[0012] Other objects and advantages of the present invention will become apparent from the following description.

SUMMARY OF THE INVENTION
[0013] The present invention relates to a herbal composition for nasal spray and a
method for preparation thereof. The herbal composition of the present invention comprises of one or more essential oils, one or more solubilizing agents, at least one surfactant; at least one chelating agent, a buffer solution, at least one pharmaceutically acceptable excipient, at least one polymer, salt, and water. The essential oils are present in a range of 0.1% to 20% by weight of the total herbal composition. The solubilizing agent is present in a range of 0.5% to 10% by weight of the total herbal composition. The surfactant is present in a range of 0.5% to 3% by weight of the total herbal composition. The chelating agent is present in a range of 0.01% to 5% by weight of the total herbal composition. The buffer solution is present in a range of _30- 70% by weight of the total herbal composition. The pharmaceutically acceptable excipients used in the present invention are present in a range of 0.00001% to 6% by weight of the total herbal composition. The polymers are present in a range of 0.1% to 1% by weight of the total herbal composition. Water is present in a range of 42% to 48% by weight of the total herbal composition. The present invention also provides a method for preparing herbal composition for nasal spray. The method is based on micro-emulsion technique with mucoadhesive delivery. The herbal composition of the present invention exhibits potential antibacterial, anti-inflammatory, immunomodulatory and antiviral action that effectively relieves nasal congestion and sinus.
SOURCE OF BIOLOGICAL MATERIAL USED IN THE INVENTION
[0014] The essential oils used in the herbal composition including such as, but are not limited to, Eucalyptus oil, Clove oil, Ajwain oil, Peppermint oil, Tulsi oil, Cedarwood oil, Sandalwood oil, Cinnamon 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 of the test strains includes ATCC 6538 for Staphylococcus aureus; ATCC 6633 for Bacillus subtilis; ATCC 6633 for Bacillus subtilis; ATCC 8739 for Escherichia coli; ATCC 2091 and ATCC 10231 for Candida albicans. The test strains used for determining microbial contamination including Salmonella abony is procured from National Collection of Type Cultures with NCTC number - NCTC6017 and Pseudomonas aeruginosa is procured from American Type Culture Collection 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 anti-inflammatory property of the herbal composition with reduced strength (A) by inhibition of IL-8 against TNF-a stimulation in lung cells;
[0017] Figure 2 illustrates anti-inflammatory property of the herbal composition with high strength (B) by inhibition of IL-8 against TNF-a stimulation in lung cells;
[0018] Figure 3 illustrates anti-inflammatory property of the herbal composition with reduced strength (A) by inhibition of IL-6 murine against LPS stimulation in splenocyte cells;
[0019] Figure 4 illustrates anti-inflammatory property of the herbal composition with high strength (B) by inhibition of IL-6 murine against LPS stimulation in splenocyte cells;
[0020] Figure 5 illustrates anti-inflammatory property of the herbal composition with reduced strength (A) by inhibition in levels of LTB4 against TNF-alpha stimulation in lung cells;
[0021] Figure 6 illustrates anti-inflammatory property of the herbal composition with high strength (B) by inhibition in levels of LTB4 against TNF-alpha stimulation in lung cells;
[0022] Figure 7 illustrates anti-allergic property of the herbal composition with reduced strength (A) by inhibition of histamine against compound 48/80 stimulation in immune cells;
[0023] Figure 8 illustrates anti-allergic property of the herbal composition with high strength (B) by inhibition of histamine against compound 48/80 stimulation in immune cells;
[0024] Figure 9 illustrates the mucolytic potential of the herbal composition with reduced strength (A) by inhibition of mucin (MUC5AC) levels against TNF alpha stimulation in lung cells;
[0025] Figure 10 illustrates the mucolytic potential of the herbal composition with high strength (B) by inhibition of mucin (MUC5AC) levels against TNF alpha stimulation in lung cells;
[0026] Figure 11 illustrates mucolytic potential of the herbal composition with reduced strength (A) by inhibition in egg white viscosity;
[0027] Figure 12 illustrates mucolytic potential of the herbal composition with high strength (B) by inhibition in egg white viscosity;
[0028] Figure 13 illustrates- anti-inflammatory property of the herbal composition in form of a liquid agent in nebulizer by inhibition of IL-8 against TNF-alpha stimulation in lung cells;
[0029] Figure 14 illustrates anti-inflammatory property of the herbal composition in form of a liquid agent in nebulizer by inhibition of IL- 6 against LPS stimulation in murine splenocyte cells;
[0030] Figure 15 illustrates anti-allergic potential of herbal composition in form of a liquid agent in nebulizer by inhibition of histamine against compound 48/80 stimulation in immune cells;
[0031] Figure 16 illustrates mucolytic potential of the herbal composition in form of a liquid agent in nebulizer by inhibition of mucin (MUC5AC) levels against TNF alpha stimulation in lung cells; and
[0032] Figure 17 illustrates mucolytic potential of the herbal composition in form of a liquid agent in nebulizer by inhibition in egg white viscosity in lung cells.

DETAILED DESCRIPTION OF THE INVENTION
[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] It is to be understood that the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0038] 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.

[0039] Accordingly, the herbal composition for nasal spray and a method for preparation thereof. Particularly, the present invention relates to a herbal composition for nasal spray having potential antibacterial, anti-inflammatory, immunomodulatory and antiviral action that effectively relieves nasal congestion and sinus.

[0040] In an embodiment, the herbal composition of the present invention comprises of a one or more essential oils, one or more solubilizing agents, at least one surfactant, at least one chelating agent, a buffer solution, at least one pharmaceutically acceptable excipients, at least one polymer, a salt, and water.

[0041] The herbal composition of the present invention comprises of one or more essential oils. In an exemplary embodiment, the essential oils are selected from a group consisting of, such as, but not limited to, Eucalyptus oil, Clove oil, Ajwain oil, Peppermint oil, Tulsi oil, Cedarwood oil, Sandalwood oil, Cinnamon oil or a combination thereof. The essential oils are derived from different parts of herbs such as, but not limited to, Eucalyptus oil is extracted from leaves of Eucalyptus globulus, Clove oil is extracted from dried flower buds of Eugenia caryophyllus, Ajwain oil is extracted from seeds of Trachyspermum ammi, Peppermint oil is extracted from leaves of Mentha piperita, Tulsi oil is extracted from leaves Ocimum basilicum, Cedarwood oil is extracted from leaves, needles, berries, and bark of trees from Juniperus virginiana, and Clove oil is extracted from dried flower buds of Eugenia caryophyllus. The essential oils are present in a range of 0.1% to 20% by weight of the total herbal composition. In an exemplary embodiment, each essential oil may be present in varying amount such as Eucalyptus oil is present in a range of 0.1% to 2.5% by total weight of the essential oils, Clove oil is present in a range of 0.1% to 2.5% by total weight of the essential oils, Ajwain oil is present in a range of 0.1% to 2.5% by total weight of the essential oils, Peppermint oil is present in a range of 0.1% to 2.5% by total weight of the total essential oils, Tulsi oil is present in a range of 0.1% to 2.5% by total weight of the total essential oils, Cedarwood oil is present in a range of 0.1% to 2.5% by total weight of the total essential oils, Sandalwood oil is present in a range of 0.1% to 2.5 by total weight of the essential oils, and Cinnamon oil is present in a range of 0.1% to 2.5% by total weight of the essential oils. The essential oils contain a number of chemical compounds known as phytoactive compounds, which exhibits antimicrobial, antiviral and anti-inflammatory properties. In an exemplary embodiment, the phytoactive compound present the essential oils may include such as, but not limited to, Cineole in Eucalyptus oil, Eugenol in Clove oil, Thymol in Ajwain oil, Menthol in Peppermint oil, Chavicol in Tulsi oil, Alpha Copaene in Cedarwood oil, and Aldehyde in Cinnamon oil. In an exemplary embodiment, the phytoactive compounds are present in the range of 4% to 90% by total weight of the essential oils. The essential oils possess a wide range of healing properties as discussed below:
a) Eucalyptus oil: The cineole present in Eucalyptus oil aids in the rapid healing of acute sinusitis. Eucalyptus oil exhibits antibacterial properties, which are used to fight germs. Such antibacterial properties are used in various diseases such as sinusitis, nasal congestion, asthma, and mouth odor and aid in relieving coughs by reducing and loosening mucus. The anti-inflammatory property of Eucalyptus oil aids in fighting inflammation and promotes healing. Eucalyptus vapor aids in treating bronchitis, asthma, emphysema, and whooping cough. Eucalyptus oil is employed in steam inhalation, which aids in clearing sinus and nasal congestion. The oil acts on receptors present in the nasal mucous membranes, thereby reducing stuffiness.
b) Clove oil: Clove oil exhibits antiviral, antifungal, antimicrobial, and anti-inflammatory properties that aid in killing germs and relieving respiratory conditions. The anti-inflammatory property aids in providing relief from sore throats, coughs, colds, sinusitis, and arthritis. The clove oil has a cooling effect on the throat and nasal cavity. The oil is known for boosting immunity by reducing the blood’s toxin levels. The antioxidant property of clove oil acts as a scavenger against free radicals capable of causing various diseases. Clove oil aids in clearing nasal passages, preventing respiratory problems, and also exhibits a cooling effect on the nasal cavity and throat.
c) Ajwain oil: Ajwain oil exhibits anti-inflammatory, anti-bacterial, anti-tussive, antioxidant, and anti-fungal properties. Such properties aid in providing relief from colds, coughs, fevers, and flu and help in clearing nasal blockages by discharging the mucus, making breathing easier. Ajwain oil aids in widening bronchial tubes, which is beneficial for people suffering from asthma.
d) Peppermint oil: Peppermint oil exhibits anti-inflammatory properties and aids in soothing irritation. Peppermint oil is used to treat rhinitis, which is associated with upper respiratory tract infections and allergies. Peppermint oil contains menthol, which improves the sensation of congestion and mucus buildup. The oil reduces mucus and mucus production. Peppermint also exhibits antiviral activity, which creates a cooling sensation that soothes or numbs a sore throat and helps relax the bronchial muscles of the windpipe.
e) Tulsi oil: Tulsi oil exhibits antiseptic and analgesic properties that aid in curing dry cough, bronchitis, asthma, and other lung diseases. Tulsi oil also possesses immunomodulatory properties that enhance the immune response of the body and antitussive properties that relieve coughs. Tulsi oil is rich in antioxidants, which protect against coughs and colds. Tulsi improves respiratory health by reducing congestion and blockage in the airways and lungs. Further, Tulsi boosts the production of antibodies, thereby preventing the onset of any infections. Moreover, Tulsi possesses cough-relieving properties and helps soothe the airways by coughing out sticky mucus. Furthermore, Tulsi boosts immunity and reduces the damage caused to the lungs by smoke.
f) Cedarwood oil: Cedarwood oil exhibits antiseptic, anti-inflammatory, antifungal, antispasmodic, diuretic, antibiotic, and expectorant properties. Cedarwood oil protects the body from various harmful bacteria. Cedarwood oil plays an essential role in treating various upper respiratory tract illnesses like the common cold, cough, spasms, sinusitis, and flu symptoms. The oil aids in reducing chest and nasal congestion, preventing excess catarrh deposits, and inhibiting nasal discharge. Further, Cedarwood oil is also beneficial in treating sore throats, pharyngitis, bronchitis, and asthmatic conditions.
g) Sandalwood oil: Sandalwood oil is beneficial for treating the common cold, cough, bronchitis, fever, sore mouth, and throat. Sandalwood also stimulates the production of white blood cells, boosts immunity, heals the tridoshas, treats Vata, pacifies, and balances Pitta and Kapha. The astringent and antiseptic properties of sandalwood trigger the immune system and support the body's healing process.
h) Cinnamon oil: Cinnamon oil exhibits anti-inflammatory properties that reduce inflammation in sore throats. The antimicrobial property destroys the growth of microorganisms such as bacteria, viruses, and fungi. Cinnamon oil stimulates the immune system, fights free radicals, and provides protection against germs. The antifungal property of Cinnamon oil makes it favorable for providing relief against painful throats and other throat-related infections.
[0042] The solubilizing agents used in the present invention aids in increasing the solubility of essential oils in water, thereby stabilizing the micro-emulsion. In an exemplary embodiment, the solubilizing agent used in the present invention may be selected from a group consisting of such as, but not limited to, Propylene glycol, PEG 400, 2-(2-Ethoxyethoxy)ethanol (transcutol), Labrasol, Polysorbate 80, or a combination thereof. Preferably, Propylene glycol, and PEG 400 are used are solubilizing agent in the present invention. The solubilizing agents are present in a range of 0.5% to 10% by weight of the total herbal composition. In an exemplary embodiment, the solubilizing agents used in the present invention may be present in varying amount such as PEG 400 is present in a range of 0.5% to 5% by total weight of the solubilizing agents; and Propylene Glycol is present in a range of 0.5% to 5% by total weight of the solubilizing agents.

[0043] In an exemplary embodiment, the surfactants are selected from a group consisting of, such as, but not limited to, Poloxomer, Sodium Bicarbonate, Sodium lauroyl Sarcosinate, Sodium lauroyl glutamate, or a combination thereof. Preferably, Poloxomer is used as a surfactant in the present invention. The surfactant is present in a range of 0.5% to 3% by weight of the total herbal composition.

[0044] The chelating agent used in the present invention aids in improving the stability of the micro-emulsion. In an exemplary embodiment, the chelating agent may be selected from a group consisting of, such as, but not limited to, 2,2',2'',2'''-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), or(ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (EGTA). Preferably, 2,2',2'',2'''-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) is used as a chelating agent in the present invention. The chelating agent is present in a range of 0.01% to 2% by weight of the total herbal composition. b

[0045] The buffer solution is used for resisting any change in the pH of the micro-emulsion on addition of any acidic or basic compounds. The buffer solution is prepared by mixing buffering agent and water. In an exemplary embodiment, the buffering agents may be selected from a group consisting of, such as, but not limited to, Disodium hydrogen phosphate, Potassium dihydrogen phosphate, or a combination thereof. The buffer solution is present in a range of 30% to 70% by weight of the total herbal composition. In another exemplary embodiment, the buffering agents are present in a range of 0.1% to 10% by total weight of the buffer solution wherein Disodium hydrogen phosphate is present in a range of 0.1% to 3% by total weight of the buffering agents and Potassium dihydrogen phosphate is present in a range of 0.1% to 4% by total weight of the buffering agents. In another exemplary embodiment, 1.8g of buffering agents are added in 100ml of water to prepare the buffer solution. In yet another exemplary embodiment, the buffer solution is prepared by mixing Disodium hydrogen phosphate and Potassium dihydrogen phosphate in a molar ratio of 1:4.

The pharmaceutically acceptable excipient used in the present invention may be selected from a group of, such as, but not limited to, anti-irritants and preservatives. The anti-irritants prevents adherence of germs, irritants, and pollutants in nasal cavity. In an exemplary embodiment, the anti-irritants used in the present invention may be selected from a group consisting of such as, but not limited to, Sodium hydrogencarbonate (Sodium bicarbonate), (2R,3r,4S)-Pentane-1,2,3,4,5-pentol (Xylitol), Zinc chloride, or a combination thereof. In an exemplary embodiment, the anti-irritants used in the present invention are in solid form including, such as, but not limited to, powder, pellet, crystal and so on. The preservatives used in the present invention prevents degradation of the herbal composition by the micro-organisms during storage. In an exemplary embodiment, the preservatives used in the present invention may be selected from a group, consisting of, such as, but not limited to, Benzalkonium chloride. In an exemplary embodiment, the preservatives used in the present invention are in liquid form such as, but not limited to, solution. The pharmaceutically acceptable excipients used in the present invention are present in a range of 0.00001% to 6% by weight of the total composition. In an exemplary embodiment, the anti-irritants used in the present invention are present in a range of 0.00001% to 4% by total weight of the pharmaceutically acceptable excipients. In another exemplary embodiment, the anti-irritants may be present in varying amount such as Sodium hydrogencarbonate is present in a range of 0.01% - 2% by weight of the total anti-irritants, xylitol is present in a range of 0.1% - 1% by weight of the total anti-irritants; and zinc chloride is present in a range of 0.00001% - 0.0005% by weight of the total anti-irritants. In another exemplary embodiment, the preservatives used in the present invention are present in a range of 0.0001% to 1.5% by total weight of the pharmaceutically acceptable excipients. In yet another exemplary embodiment, the anti-irritants may be present in varying amount such as Sodium hydrogencarbonate is used in a range of 0.01% to 2% by total weight of the pharmaceutically acceptable excipients, (2R,3r,4S)-Pentane-1,2,3,4,5-pentol (Xylitol) is present in a range of 0.01% to 1% by total weight of the pharmaceutically acceptable excipients, and zinc chloride is present in a range of 0.00001% to 0.0005% by total weight of the pharmaceutically acceptable excipients.

[0046] The polymers used in the present invention may be, selected from a group of, such as, but not limited to, mucoadhesive polymers. The mucoadhesive polymers aids help in mucoadhesive delivery of the drug to mucosal membrane. Such polymers attain adhesiveness on hydration and therefore aids in delivering a drug to a particular region. The mucoadhesive polymers interact with mucus released from the sub mucosal glands. The polymers such as hyaluronic acid increases the viscosity to optimum value for enhancing residual time in nasal mucosal membrane. In an exemplary embodiment, the mucoadhesive polymers may be selected from a group consisting of, such as, but not limited to, poloxomer or hydroxyl ethyl cellulose, or a combination thereof. Preferably, poloxomer is used as mucoadhesive polymer in the present invention. The polymers used in the present invention are present in a range of 0.1% to 1% by weight of the total herbal composition.

[0047] The salt used for preparing the herbal composition aids in treating dryness inside the nose, particularly in the nasal passage. The salt also adds moisture inside the nose that aids in dissolving and softening thick and crusty mucus. In an exemplary embodiment, the salt used in the present invention may be selected from a group consisting of, such as, but not limited to, sodium bicarbonate, or sodium chloride. Preferably, sodium bicarbonate is used as a salt in the present invention. The salt is present in a range of 0.1-0.5% by weight of the total herbal composition.

[0048] In an exemplary embodiment, water used in the present invention may include distilled water. The water is present in a range of 42% to 48% by weight of the total herbal composition.

[0049] The pH of the herbal composition prepared in the present invention may be in a range of 6.4-7.5, thus preventing the risk of irritation resulting from extreme high or extreme low pH during application.

[0050] In a preferred embodiment, the strength of the herbal composition prepared in the present invention may vary depending on the intended use. The strength of the herbal composition may increase/decrease by increasing/decreasing the amount of the compounds used during the preparation of the herbal composition. In case of less severe infections (case A), the strength of the herbal composition may be reduced by reducing the amount of different compounds used in the herbal composition. On the contrary, in case of severe infections (case B), the strength of the herbal composition may be increased by increasing the amount of different compounds used in the herbal composition.

[0051] In an embodiment, the present invention also provides a method for preparing herbal composition for nasal spray. Micro-emulsion technique is employed for the preparation of the herbal composition. The micro-emulsion is an optically transparent colloidal system involving the dispersion of tiny drops of one liquid, which is immiscible in another. The method also involves mucoadhesive delivery. Mucoadhesive delivery refers to the drug delivery system utilizing the bio-adhesive property of various mucoadhesive polymers. The polymers attain adhesiveness on hydration and thus aids in delivering a drug to a particular region. The method steps are provided herein detail:
(a) adding essential oils and solubilizing agents in an apparatus A ;
(b) preparing buffer solution by dissolving disodium Phosphate and Potassium hydrogen phosphate in distilled water;
(c) adding distilled water to the solution obtained in step (b), making up the volume of the solution to 100ml;
(d) stirring the solution obtained in step (c) until a clear solution appears.
(e) checking the pH of the solution obtained in step (d);
(f) adding pharmaceutically acceptable excipients, polymers, chelating agents; solubilizing agent; and salt one by one after complete dissolution of each compound in 20ml of distilled water contained in an apparatus (B);
(g) mixing contents of the apparatus A and apparatus B, forming a solution;
(h) adding buffer solution to the solution obtained in step (g), making the volume of the solution to 100ml;
(i) homogenizing the solution obtained in step (h) with the addition of pharmaceutically acceptable excipients for 15-20 minutes;
(j) filtering the solution obtained in step (i) after complete homogenization, resulting in a herbal composition; and
(k) checking the pH of the solution.

[0052] In an exemplary embodiment, the essential oils added in step (a) are selected from a group consisting of, such as, but not limited to, Eucalyptus oil, Clove oil, Ajwain oil, Peppermint oil, Tulsi oil, Cedarwood oil, Sandalwood oil, Cinnamon oil or a combination thereof. The essential oils are present in a range of 0.1% to 20% by weight of the total herbal composition. In an exemplary embodiment, each essential oil may be present in varying amount such as Eucalyptus oil is present in a range of 0.1% to 2.5% by total weight of the essential oils, Clove oil is present in a range of 0.1% to 2.5% by total weight of the essential oils, Ajwain oil is present in a range of 0.1% to 2.5% by total weight of the essential oils, Peppermint oil is present in a range of 0.1% to 2.5% by total weight of the total essential oils, Tulsi oil is present in a range of 0.1% to 2.5% by total weight of the total essential oils, Cedarwood oil is present in a range of 0.1% to 2.5% by total weight of the total essential oils, Sandalwood oil is present in a range of 0.1% to 2.5 by total weight of the essential oils, and Cinnamon oil is present in a range of 0.1% to 2.5% by total weight of the essential oils.

[0053] In an exemplary embodiment, the solubilizing agents added in step (a) may include a combination of Propylene glycol, and PEG 400. The solubilizing agent is present in a range of 0.5% to 13% by weight of the total herbal composition. In another exemplary embodiment, each solubilizing agent may be present in varying amount such as PEG 400 is present in a range of 0.5% to 5% by total weight of the solubilizing agents; and Propylene glycol is present in a range of 0.5% to 5% by total weight of the solubilizing agents.

[0054] In another exemplary embodiment, 1.8g of buffering agents are added in 100ml of water to prepare the buffer solution in step (b). In yet another exemplary embodiment, a molar ratio of 1(Disodium hydrogen phosphate): 4(Potassium dihydrogen phosphate) is used for preparing the buffer solution in step (b). The buffer solution in step (b) is prepared in a range of 30 - 70% by weight of the total herbal composition. In an exemplary embodiment, the buffering agents may be selected from a group consisting of, such as, but not limited to, Disodium hydrogen phosphate, Potassium dihydrogen phosphate, or a combination thereof. In another exemplary embodiment, the buffering agents are present in a range of 0.1% to 10% by total weight of the buffer solution wherein Disodium hydrogen phosphate is present in a range of 0.1% to 3% by total weight of the buffering agents and Potassium dihydrogen phosphate is present in a range of 0.1% to 4% by total weight of the buffering agents.

[0055] In an exemplary embodiment, the pharmaceutically acceptable excipients added in step (f) may be selected from a group of anti-irritants. In another exemplary embodiment, the anti-irritants may be selected from a group consisting of, such as, but not limited to, Sodium hydrogencarbonate (Sodium bicarbonate), (2R,3r,4S)-Pentane-1,2,3,4,5-pentol (Xylitol), Zinc chloride, or a combination thereof. In another exemplary embodiment, the anti-irritants used in the present invention are in solid form including, such as, but not limited to, powder, pellet, crystal and so on. In yet another exemplary embodiment, the anti-irritants used in the present invention are present in a range of 0.00001% to 4% by total weight of the pharmaceutically acceptable excipients. In yet another exemplary embodiment, the anti-irritants may be present in varying amount such as Sodium hydrogencarbonate is used in a range of 0.01% to 2% by total weight of the pharmaceutically acceptable excipients, (2R,3r,4S)-Pentane-1,2,3,4,5-pentol (Xylitol) is present in a range of 0.01% to 1% by total weight of the pharmaceutically acceptable excipients, and zinc chloride is present in a range of 0.00001% to 0.0005% by total weight of the pharmaceutically acceptable excipients.

[0056] The polymer added in step (f) may be selected from a group of mucoadhesive polymers. In an exemplary embodiment, the polymer may be selected from a group consisting of, such as, but not limited to, poloxomer or hydroxyl ethyl cellulose, or a combination thereof. Preferably, poloxomer is used as mucoadhesive polymer in the present invention. The polymers used in the present invention are present in a range of 0.1% to 1% by weight of the total herbal composition.

[0057] In an exemplary embodiment, the chelating agent added in step (f) may be selected from a group consisting of, such as, but not limited to, 2,2',2'',2'''-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA), or(ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (EGTA). Preferably, 2,2',2'',2'''-(Ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA) is used as a chelating agent in the present invention. The chelating agent is present in a range of 0.01% to 5% by weight of the total herbal composition.

[0058] In an exemplary embodiment, the surfactant added in step (f) may include poloxomer. The surfactant is present in a range of 0.5% to 3% by weight of the total herbal composition.

[0059] In an exemplary embodiment, the salt added in step (f) may be selected from a group consisting of, such as, but not limited to, sodium bicarbonate, or sodium chloride. Preferably, sodium bicarbonate is used as a salt in the present invention. The salt is present in a range of 0.1-0.5% by weight of the total herbal composition.

[0060] In an exemplary embodiment, the pharmaceutically acceptable excipients added in step (i) may be selected from a group of preservatives. In another exemplary embodiment, the preservatives used in the present invention may be selected from a group, consisting of, such as, but not limited to, Benzalkonium chloride. In another exemplary embodiment, the preservatives used in the present invention are in liquid form such as, but not limited to, solution. In yet another exemplary embodiment, the preservatives are present in a range of 0.0001% to 1.5% by total weight of the pharmaceutically acceptable excipients.

[0061] In an exemplary embodiment, water used in the preparation of the herbal composition may include distilled water. The water is present in a range of 42% to 48% by weight of the total herbal composition.

[0062] The pH of the herbal composition prepared in the present invention may be in a range of 6.4 to 7.5, thus preventing the risk of irritation resulting from extreme high or extreme low pH during application.

[0063] In case the pH in step (e) and step (k) is not in a range of 6.4-7.4 then phosphate buffer solution is added to maintain the desired pH.

[0064] In a preferred embodiment, the herbal composition prepared in the present invention may be used as nasal spray to get relief from nasal congestion and sinus. In yet another embodiment, the herbal composition prepared in the present invention may optionally be used as nasal wash or drops which may be directly added inside the nose or may be added in a steamer. The herbal composition of the present invention may also be optionally be used as a liquid agent in nebulizer, providing relief from relieves nasal congestion and sinus and other respiratory infections such as, but not limited to, asthma, and bronchitis. The herbal composition in nebulizer is vaporized, leaving no residue.

[0065] The following illustrates experimental data of the present invention and should not be construed to limit the scope of the invention.
Experimental Data

[0066] Several experiments were performed to evaluate general parameters, weight per millilitre, specific gravity, peroxide value (as per API Part I, Vol. VI, appendix 3, 3.10.), acid value (as per API Part I, Vol. VI, appendix 3, 3.9.), Iodine value, pH (as per API, Part I, Vol. IX, Appendix-2, 2.1.10.), saponification, viscosity (as per API Part-I, Vol. VI, Appendix 3, 3.4), microbial contamination, heavy metal analysis, pesticide residue, aflatoxins, and germ killing efficacy of the herbal composition for reduced strength (A) and high strength (B).
1. General Parameters
[0067] The general parameters of the herbal composition of the present invention were evaluated. Such characteristics include the colour and odour of the composition. The herbal composition was perceived to be off-white in colour. The herbal composition exhibited a characteristic odour.
2. Weight per millilitre
[0068] The weight per millilitre of the herbal composition was evaluated. A clean and dry pycnometer was selected. The pycnometer was calibrated by filling it with boiled and cooled water at 25 °C. The contents were weighed. Considering the weight of 1 ml of water at 25 °C when weighed in air with a density of 0.0012 g per ml, it is 0.99602 g. The capacity of the pycnometer was calculated. The results remain unaffected by the minor deviations in the density of air from the given value. The temperature of the substance to be examined was adjusted to about 20 °C, followed by filing the substance in the pycnometer. The temperature of the filled pycnometer was adjusted to 250. The excess of the substances was removed, and the pycnometer was then weighed again. The tare weight of the pycnometer was subtracted from the filled weight of the pycnometer. The weight per millilitre was calculated by dividing the weight (g) of the quantity of liquid that fills the pycnometer at the specified temperature in air by the capacity (ml) of the pycnometer at the same temperature.
3. Specific Gravity
[0069] The specific gravity of the herbal composition in liquid form was evaluated. A clean and dry pycnometer was selected. The pycnometer was calibrated by filling it with boiled and cooled water at 25 °C. The contents were weighed. Considering that the weight of 1 ml of water at 25 °C when weighed in air of density 0.0012 g per ml is 0.99602 g. The capacity of the pycnometer was calculated. The results remain unaffected by the minor deviations in the density of air from the given value. The temperature of the substance to be examined was adjusted to about 20 °C, followed by filing the substance in the pycnometer. The temperature of the filled pycnometer was adjusted to 250. The excess substances were removed, and the pycnometer was then weighed again. The tare weight of the pycnometer was subtracted from the filled weight of the pycnometer. The weight per millilitres was calculated by dividing the weight (g) of the quantity of liquid that fills the pycnometer at the specified temperature in air by the capacity (ml) of the pycnometer at the same temperature. The specific gravity of the liquid was calculated by dividing the weight of the liquid contained in the pycnometer by the weight of water contained, both determined at 25 °C unless otherwise directed in the individual monograph.
4. Peroxide Value

[0070] The peroxide value of the herbal composition was evaluated. 5 g of the substance to be examined was accurately weighed and poured into a 250-ml glass-stoppered conical flask. 30 ml of a mixture of 3 volumes of glacial acetic acid and 2 volumes of chloroform were added to the flask, and the mixture was stirred until dissolved. 0.5 ml of saturated potassium iodide solution was added to the mixture. The mixture was allowed to stand for one minute after shaking it. 30 ml of water was added to the mixture, and the mixture was then titrated with constant and vigorous shaking with 0.01M sodium thiosulfate until the yellow color disappeared. 0.5 ml of starch solution was added to the mixture. The titration was then resumed by simultaneously shaking the mixture vigorously until the blue color disappeared. The process was then repeated without the substance being tested. The volume of 0.01M sodium thiosulfate in the blank determination was within 0.1 ml. The peroxide value was calculated as given below:
Peroxide value = 10 (a-b)/W
Wherein,
W = weight of the substance (g)
[0071] The refractive index of the herbal composition was evaluated. The refractive index was calculated to be 250 (0.5) with reference to the wavelength of the D line of sodium (? 589.3 nm). The temperature was carefully adjusted and maintained to avoid variations in the refractive index.
5. Acid value
[0072] The acid value of the herbal composition was evaluated. About 10 g of the substance (1 to 5) in the case of a resin was accurately weighed and poured into a 250-ml flask. 50 ml of a mixture of equal volumes of alcohol and solvent ether, neutralized by the addition of 1 ml of a solution of phenolphthalein, was added to the flask. The flask was heated gently on a water bath, if required, until the substance was completely melted. The solution obtained was titrated with 0.1 N potassium hydroxide along with constant shaking of the flask until a pink color was obtained. The pink color persists for fifteen seconds. The required amount of potassium hydroxide (ml) was recorded. The acid value was calculated as given below:
Acid Value = a × 0.00561 × 1000
W
Wherein,
a = the required amount of 0.1N potassium hydroxide (ml); and
W = weight of the substance taken (g)
6. Iodine Value
[0073] The Iodine value of the herbal composition was evaluated using the iodine monochloride method. The test sample to be examined was accurately weighed and placed in a dry iodine flask. 10 ml of carbon tetrachloride was added and dissolved in the flask. 20 ml of Iodine monochloride solution was added to the flask. A stopper previously moistened with a solution of potassium iodine was inserted in the flask. The solution in the flask was allowed to stand in a dark place at a temperature of about 17? or for 30 minutes. 15 ml of a solution of potassium iodine and 100 ml of water were added to the flask and mixed by shaking the flask. The solution in the flask was titrated with 0.1 N sodium thiosulfate using a solution of starch as an indicator. The required amount of sodium thiosulfate (ml) was recorded. The process was repeated, but without the substance to be tested. The required amount of 0.1 N sodium thiosulfate (ml) was recorded. The iodine value was calculated as below:
Iodine value = (b-a) × 0.01269 × 100
W
Wherein,
W = weight of the substance taken (g)
[0074] The approximate weight of the substance to be taken (g) is calculated by dividing 20 by the highest expected iodine value. In cases where more than half of the available halogen is absorbed, the test is repeated with a smaller quantity of the test sample.
[0075] Iodine monochloride Solution: The solution is prepared by any of the following methods:
[0076] A. (1) 13 g of iodine was dissolved in a mixture of 300 ml of carbon tetrachloride and 700 ml of glacial acetic acid. 15 ml of a solution of potassium iodide and 100 ml of water were added to 20 ml of the prepared solution. The prepared solution was then titrated with 0.1 nM sodium thiosulfate. Washed and dried chlorine was passed through the remainder of the iodine solution until the amount of 0.1 N sodium thiosulfate required for titration was approximately, but more than, doubled.
[0077] (2) 8g of iodine trichloride was dissolved in about 200 ml of glacial acetic acid. 9g of iodine was dissolved in 300 ml of carbon tetrachloride. The two prepared solutions were mixed, and glacial acetic acid was added to make the volume of the solution 1000 ml. Iodine monochloride Solution was stored in a stopper bottle in a cool place so as to protect the solution from light.
[0078] Pyridine Bromide Method: An accurately weighed substance was placed in a dry iodine flask. 10 ml of carbon tetrachloride was added and dissolved in the flask. 25 ml of pyridine bromide solution were added to the flask. The solution in the flask was allowed to stand for ten minutes in a dark place and complete the determination described under the iodine monochloride method, beginning with the words. 15 ml of a solution of potassium iodide and 100 ml of water were added to 20 ml of the prepared solution. The above-mentioned process was repeated. The approximate weight of the substance (g) to be taken was calculated by dividing 12.5 by the highest expected iodine value. In cases where more than half of the available halogen is absorbed, the test is repeated with a smaller quantity of the substance.

[0079] Pyridine bromide Solution: 8 g of pyridine and 10 g of sulfuric acid were dissolved in 20 ml of glacial acetic acid. 8 g of bromine dissolved in 20 ml of glacial acetic acid was added to the prepared solution and diluted to 100 ml with glacial acetic acid. The pyridine bromide solution was freshly prepared.
7. pH
[0080] The pH of the herbal composition was evaluated. The pH meter was calibrated using buffer solution before being used. The pH of the herbal composition in liquid form was determined in triplicate, and the average value was calculated. The pH of the herbal composition was calculated to be 6.4.
8. Saponification Value
[0081] 35–40 g of potassium hydroxide were dissolved in 20 ml of water. A sufficient amount of alcohol was added to the prepared solution to make the volume of the solution 1000 ml. The solution was allowed to stand overnight. The clear liquid was poured off. Accurately weighing about 2 g of the substance, it was poured into a tared 250-ml flask, followed by 25 ml of the alcoholic solution of potassium hydroxide. A reflux condenser was attached and boiled in a water bath for one hour. The flask was rotated to cool the contents inside. 1 ml of phenolphthalein solution was added to the flask, and excess alkali was titrated with 0.5 N hydrochloric acid. The required amount of 0.5 N hydrochloric acid (ml) was recorded (a). The process was repeated with the same reagents in the same amount without the substance to be tested. The required amount of 0.5 N hydrochloric acid (ml) was recorded (b). The saponification value was calculated as given below:
Saponification Value = (b-a) × 0.02805 × 1.000
W
Wherein,
W = weight of the substance taken (g)
9. Viscosity:
[0082] The viscosity of the herbal composition in liquid form was evaluated. The test liquid was filled in a U-tube viscometer in accordance with the expected viscosity of the liquid so that the fluid level stood within 0.2 mm of the filling mark of the viscometer when the capillary was vertical and the specified temperature was attained by the test liquid. The test liquid was pipetted or 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 calculated as given below:
Kinematic viscosity = kt
Wherein,
k = the constant of the viscometer tube determined by observing liquids of known kinematic viscosity; and
t = time (s) for the meniscus to pass through the two specified marks.
[0083] The viscosity of the herbal composition was evaluated at 5.04 cps.
10. Microbial Contamination
[0084] The microbial contamination of the herbal composition was evaluated. A number of tests were performed for the estimation of different viable aerobic microorganisms present in the test sample and the detection of specific microbial species present in the sample.

[0085] Preliminary Testing: Prior to performing preliminary tests, the diluted samples to be examined were inoculated with separate viable cultures of Escherichia coli, Salmonella abony, Pseudomonas aeruginosa, and Staphylococcus aureus in order to ensure that the test sample prevents the inhibition of multiplication under the test conditions of microorganisms that may be present. The inoculation was performed by adding 1 ml of 24-hour broth culture containing at least 1000 microorganisms to the first dilution (in buffer solution with pH 7.2 using fluid soyabean-casein digest medium or fluid lactose medium) of the test sample and following the test procedure. In the event that the organisms fail to grow in the relevant medium, the volume of the diluent is increased with the quantity of the test sample remaining the same, or a sufficient amount of a desired inactivating agent is incorporated into the diluents or a combination thereof to permit the growth of the organisms in the medium. In cases where inhibitory substances are present in the test sample, 0.5% soy lecithin and 4% polysorbate 20 were added to the culture medium. Alternatively, the test was repeated using fluid casein digest, soy lecithin, and polysorbate 20 medium to demonstrate neutralization of preservatives or other antimicrobial agents in the test sample. In cases where inhibitory substances are contained in the test samples and the test sample is soluble, the membrane filtration method is used. The failure to isolate the inoculated organism even after the incorporation of desired inactivating agents and/or a substantial increase in the volume of diluent and/or the sample being 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 the spectrum of inhibition and bactericidal activity of the sample.
[0086] A different culture medium was prepared for evaluating microbial contamination, or a dehydrated culture medium was used such that the medium possessed similar ingredients when reconstituted as directed by the manufacturer and/or yielded media comparable to the media obtained from the formulae given below. If agar is used in the preparation of culture medium, then the moisture content of the agar is within 15%. Purified water was used in the culture medium if required. The medium was sterilized by heating it in an autoclave (15 psi) at 121 °C 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 0.1 N hydrochloric acid or 0.1 N sodium hydroxide was added to yield the required pH in the medium before use. The pH of the medium was calculated at 25 ± 2 °C.
[0087] The procedure for preparing different culture mediums is described herein:
[0088] 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 bring the volume of the solution up to 1000 ml. The solution was heated and stirred, followed by boiling for 1 minute. The solution was then sterilized and cooled to a temperature in the range of 45°C–50°C. 10 ml of a 1% weight-by-volume solution of sterile potassium tellurite and 50 ml of egg yolk emulsion were added to the mixture. The solution was thoroughly mixed and poured onto 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 a ratio of 3:7 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 after sterilization to 6.80.2.
[0089] Bismuth Sulfite 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 increase the volume of the solution to 1000 ml. The solution was dissolved with the help of heat and sterilized by maintaining 115 °C for 30 minutes. Solution 2 was prepared by adding 3.0 g of Ammonium bismuth citrate, 10.0 g of Sodium sulfite, 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 100 ml. The solution was mixed and heated to boiling. The solution was then allowed to cool to room temperature. 1 part of solution 2 was added to 10 parts of solution 1 previously melted and cooled to a temperature of 55 °C and poured into plates. The Bismuth sulfite Agar Medium was stored at 2°C to 8°C for 5 days before use.
[0090] 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 increase 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 it at 115 °C for 30 minutes, followed by mixing the solution. The solution was then poured onto plates.
[0091] 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 increase the volume of the mixture to 1000 ml. 0.1 to 1.0% weight by volume of Polysorbate 20 or Polysorbate 80 was added to the solution. The solution was sterilized by heating it in an autoclave at 121 °C for 15 minutes.
[0092] 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 1000 ml. The pH of the medium was adjusted after sterilization to 7.3 ± 0.2.
[0093] 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 sulfate, 0.3 g of Cetrimide, 13.6 g of Agar, and 10.0 g of Glycerin. The water was added to bring the volume of the mixture to 1000 ml. The solution was then heated by boiling for 1 minute and shaking the solution. The pH of the medium was adjusted to achieve a pH of 7.0 to 7.4 after sterilization.
[0094] 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 thiosulfate, 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 bring 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 it was completely homogenized. The solution was then cooled to 80 °C and mixed. The solution was then poured onto plates. Overheating of Desoxycholate Citrate Agar was avoided during the preparation of the medium. Re-melting of the solution was also avoided. The surfaces of the plates were dried before use.
[0095] Fluid Casein Digest, Soya Lecithin, and Polysorbate 20 Medium was prepared by adding 20.0 g of Pancreatic digest of casein, 5.0 g of soy lecithin, and 40.0 ml of Polysorbate 20. The water was added to bring the volume of the mixture to 1000 ml. The pancreatic digest of casein and soy lecithin was dissolved in water, followed by heating in a water bath at 480 to 500 for about 30 minutes. Polysorbate 20 was added to the solution and mixed, followed by dispensing it to the user as per the requirement.
[0096] 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 after sterilization to 6.9 ± 0.2.
[0097] 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 increase the volume of the solution to 1000 ml. The pH of the medium after sterilization was 6.9 ± 0.2.
[0098] Levine Eosin-MMethylene 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 increase 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 gelledagar solution and the remaining ingredients as solutions were liquefied in the required amounts before use, followed by mixing the solution. 5 ml of a 20% weight by volume solution of lactose, 2 ml of a 2% weight by volume solution of Eosin Y, and 2 ml of a 0.33% weight by volume solution of methylene blue were used for each 100 ml of the liquified agar solution. The finished medium may not be clear. The pH of the medium was adjusted after sterilization to 7.3 ± 0.2.
[0099] 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 bring 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 after sterilization to 7.3 ± 0.2.
[00100] 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 increase the volume of the mixture to 1000 ml. The pH of the medium was adjusted after sterilization to 7.3 ± 0.2.
[00101] Mannitol-Salt Agar Medium was prepared with 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 increase the volume of the mixture to 1000 ml. The solution was mixed and heated, along with stirring the solution. The solution was then boiled for one minute. The pH of the medium was adjusted after sterilization to 7.3 ± 0.2.
[00102] Nutrient Agar Medium was prepared by adding 1% to 25% weight by volume of agar to nutrient broth.
[00103] 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 bring the volume of the mixture to 1000 ml. The mixture 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 it at 115 °C for 30 minutes. The pH of the medium was adjusted after sterilization to 7.3 ± 0.2.
[00104] 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 sulfate heptahydrate, 10.0 ml of Glycerin, and 15.0 g of Agar. Water was added to increase the volume of the mixture to 1000 ml. The solid components were dissolved in water before adding glycerin. The solution was mixed by heating it to boiling for 1 minute. The pH of the medium was adjusted after sterilization to 7.3 ± 0.2.
[00105] 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 sulfate, 15.0 g of Agar, and 10.0 ml of Glycerin. The water was added to bring the volume of the mixture to 1000 ml. The solid components were dissolved in water before adding glycerin. The solution was mixed by heating it to boiling for 1 minute. The pH of the medium was adjusted after sterilization to 7.3 ± 0.2.
[00106] 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 increase the volume of the mixture to 1000 ml. The solution was then mixed and boiled. The pH of the medium was adjusted after sterilization to 5.6 ± 0.2.
[00107] Sabouraud Dextrose Agar Medium with Antibiotics was prepared by adding 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 was added to 1 liter of Sabouraud Dextrose Agar Medium immediately before use.
[00108] 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 increase the volume of the mixture to 1000 ml. The mixture was dissolved and distributed in sterile containers. The solution was then sterilized by maintaining it at 100 °C for 30 minutes.
[00109] 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 increase 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. The sterilization step was avoided for the preparation of Fluid Selenite-Cystine Medium.
[00110] 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 thiosulfate. Water was added to increase 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.
[00111] 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 increase 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.
[00112] 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 thiosulfate, and 24.0 mg of Phenol red. Water was added to increase 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 the solution was completely homogenized. The solution was thoroughly mixed and distributed in tubes, followed by sterilization at 121 °C for 15 minutes. The solution was allowed to stand in a sloped form with a 2.5 cm-long bottom.
[00113] 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 bring the volume of the mixture to 1000 ml. The solution was then mixed and sterilized, followed by distribution aseptically in sterile containers.
[00114] 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 increase 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 of 450–500. 20 ml of a 1% weight by volume sterile solution of potassium tellurite was added to the solution. The pH of the medium was adjusted after sterilization to 7.0 ± 0.2.
[00115] 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 thiosulfate, and 800 mg of Ferric ammonium citrate. Water was added to increase the volume of the mixture to 1000 ml. The mixture of solids and water was heated to boiling. Overheating and sterilization were avoided. The solution was poured into a water bath maintained at about 500 and poured onto plates just after the medium was cooled. Then the final pH of the medium was adjusted to 7.4 ± 0.2.
[00116] The sampling involves using 10 ml or 10 g of sample for each of the specified tests.
[00117] Precautions taken during experimentation involve 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.
[00118] Total Aerobic Microbial Count: The total aerobic microbial count in the herbal composition was evaluated. The procedure for evaluating the total aerobic microbial count in different types of test samples is described herein:
[00119] Water-soluble sample: 10 g or 10 ml of the sample to be examined was dissolved or diluted in buffered sodium chloride-peptone solution pH 7.0 or another suitable medium that exhibits no antimicrobial activity under the test conditions. The solution was diluted to make the volume of the solution 100 ml with the same medium. The pH of the solution was adjusted to around 7 using buffers, if required.
[00120] 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 that exhibits no antimicrobial activity under test conditions. The solution was diluted to make the volume of the solution 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 poorly wettable substances. The pH of the suspension was adjusted to around 7.
[00121] 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 of 400. The solution was mixed while maintaining the temperature in the water bath or in an oven. 85 ml of buffered sodium chloride-peptone solution pH 7.0 or other suitable medium that exhibits no antimicrobial activity under test conditions heated within 400 was added to the prepared solution, if required. The temperature was maintained for the time period required for the formation of an emulsion. However, such a time period may not exceed 30 minutes. The pH of the solution was adjusted to around 7 using buffers, if required.
[00122] The sample was examined by determining the total aerobic microbial count in the sample using any of the following methods:
[00123] Membrane filtration: Membrane filters with a 50 mm diameter and a nominal pore size within 0.45 mm were used for retaining bacteria. 10 ml, or the required amount of each dilution containing 1g of the sample to be examined, was added to each of the 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.
[00124] In the case of the 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 a shorter time period, such as at 30°C to 35°C in the test for bacteria and 200 to 250 in the test for fungi. The number of colonies formed was counted. The number of microorganisms per gram or per ml of the sample to be examined was calculated separately for bacteria and fungi, if required.
[00125] Plate count for bacteria: Petri dishes with a diameter of 9cm to 10 cm were used. A mixture of 1 ml of the pretreated sample preparation and about 15 ml of liquified casein soyabean digest agar at 45 °C 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 a colony count within 300. At least two such petri dishes were prepared using the same dilution and incubated at 300 to 350 for 5 days, unless a reliable count was obtained in a shorter time period. The number of colonies formed was counted.
[00126] The results were calculated using plates with the highest number of colonies, considering 300 colonies per plate to provide maximum results.
[00127] Plate count for fungi: Sabouraud dextrose agar with antibiotics was used, and the steps were the same as those followed in the test for bacteria. The petri dishes were incubated at 200 to 250 for 5 days unless a reliable count was obtained in a shorter time period. The results were calculated using plates, not colonies within 100.
[00128] 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 (Samples), 124 tubes each. One set of three tubes was taken as a control. 1 ml of the solution of suspension of the test sample was pipetted into each of the three tubes of one set ("100") and into the 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 l) and 10 mg (or 10 l) of the specimen, respectively. 1 ml from tube A was pipetted into each of the second set ("10") of three tubes. 1 ml from tube B was pipetted into each tube of the third set ("1"). The remaining contents of tubes 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 were observed to determine the probable number of microorganisms per gram or per ml of the test sample.
[00129] Table 2 illustrates probable number of microorganisms (per g or per ml) of the test sample.

Table 2

Tests for Specified Microorganisms
[00130] Pretreatment of the test sample: Lactose broth or other suitable medium that exhibits no antimicrobial activity under test conditions, and the same steps were performed as followed while evaluating the total aerobic microbial count.
[00131] Test for Escherichia coli: A required amount of pretreated test sample was placed in a sterile screw-capped container. 50 ml of nutrient broth were added to the container and mixed by shaking it. The container containing the mixture was allowed to stand for an 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 incubation at 37 °C for 18–24 hours.
[00132] 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°C–38°C for 48 hours. In the event that the contents of the tube showed acid and gas, a secondary test was performed.
[00133] Secondary test: 0.1 ml of the enrichment culture was added to a tube (A) containing 5 ml of MacConkey broth and a tube (B) containing 5 ml of peptone water. Both tubes were incubated in a water bath at 43.5°C–44.50°C 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 tube (B), and the contents of tube (B) were mixed by shaking the tube. The tube was allowed to stand for one minute. The presence of Indole was indicated by the formation of a red colour in the reagent layer. The presence of acid, 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 to 50 units of Escherichia coli organisms prepared from a 24-hour culture in nutrient broth were added to 5 ml of MacConkey broth. The test was considered effective only when the results indicated that the control contained Escherichia coli.
[00134] Alternative test (as per API, Part-I, Vol.-IX (Extracts), Appendix 3): A portion of 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. If, 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 the absence of Escherichia coli. In the event that the colonies mentioned above were present, 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. If, 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.
[00135] Test for Salmonella abony: 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 °C for 24 hours.
[00136] Primary test: 1.0 ml of the enrichment culture was added to a tube (A) containing 10 ml of selenite-F broth and a tube (B) containing tetrathionate-bile brilliant green broth. The tubes were incubated at 36–38 °C for 48 hours. A subculture was prepared from each of the two prepared cultures on at least two of the agar media, including bismuth sulfate agar, brilliant green agar, deoxycholate citrate agar, and xylose-lysine deoxycholate agar. The plates were incubated at 36–38 °C for 18–24 hours. If, during examination, none of the colonies conforms to the description provided in Table 2, then the sample is considered to fulfil the requirements of the test, indicating the absence of the genus Salmonella. A secondary test was performed in cases where colonies conforming to the description in Table 2 were formed.
[00137] 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 360 to 380 for 18 to 24 hours. The presence of Salmonella abony was indicated by the formation of acid and gas in the stab culture with or 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 the urea broth. However, if only acid is produced without the production of gas in the cultures, then the presence of Salmonella abony 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 tubes (A) and (B). The test was considered effective only when the results indicated that the control contained Salmonella abony.
[00138] 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 the required amount of the solution, suspension, or emulsion thus obtained, containing 1 g or 1 ml of the preparation to be examined. The solution was mixed and incubated at 350 to 370 for 24 to 48 hours.
[00139] The medium was examined for growth. In case growth was indicated in the medium, a portion of the medium was streaked on the surface of cetrimide agar medium, each plated on Petri dishes. The petri dishes were covered and incubated at 35°C to 37°C for 18 to 24 hours. If, 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 (Extracts) conforming to the description as provided in Table 2 were formed, 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 inverted, followed by incubation at 330 to 370 for at least 3 days. The streaked surfaces were examined under ultraviolet light. The plates were examined to determine the presence of colonies conforming to the description in Table 2, if any. If 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 were put on filter paper and smeared with the colony. In the absence of the production of pink turning purple, the sample was considered to fulfil the requirements of the test, indicating the absence of Pseudomonas aeruginosa.
[00140] Test for Staphylococcus aureus: The same procedure is followed for testing the presence of Staphylococcus aureus as for Pseudomonas aeruginosa. In the event that, 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 the event that growth was detected, a 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 or without additives. The tubes were incubated in the water bath at 37°C. The tubes were examined for 3 hours and subsequently at 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), Appendix 3):
[00141] A number of test strains were grown separately in tubes containing fluid soyabean-casein digest medium at 30°C to 35°C for 18 to 24 hours. However, in the case of Candida albicans, the test strains were grown separately in tubes containing fluid soyabean-casein digest medium at 20 °C for 48 hours. The test strains include Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Candida albicans.
[00142] 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 for 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 of 10 from the calculated value for the inoculum was required. The sterility of the medium and diluent and the aseptic performance of the test were tested by performing the total aerobic microbial count method using sterile buffered sodium chloride peptone solution pH 7.0 as the test preparation. The absence of microorganism growth was required to confirm the validity of the tests for total aerobic microbial count.
Validity of the tests for specified microorganisms:
[00143] The test strains of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Salmonella abony were grown separately in fluid soyabean-casein digest medium at 30–35 °C for 18–24 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 volumes of each suspension were mixed, and 0.4 ml (approximately 102 microorganisms of each strain) was used as an inoculum in the test for E. coli, S. abony, 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 methods, as mentioned below:

(a) Determination of Lead (Pb) by Graphite Oven Method:
[00144] The following conditions were taken as reference for determining the presence of lead (Pb) using Graphite Oven Method:
• dry temperature: 100-1200 maintained for 20 seconds;
• ash temperature: 400-7500 maintained for 20-25 seconds;
• atomic temperature: 1700-21000 maintained for 4-5 seconds;
• measurement wavelength: 283.3 nm; and
• background calibration: deuterium lamp (D lamp) or Zeeman effect.
[00145] 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 a 2% nitric acid solution containing 1 µg per ml lead. The solution was stored at 0°C–5°C.
[00146] Preparation of the calibration curve: A required amount of lead standard stock solutions was accurately measured and diluted with 2% nitric acid solution to a concentration of 0, 5, 20, 40, 60, and 80 ng per ml. 1 ml of 1% ammonium dihydrogen phosphate and 0.2% magnesium nitrate were added to accurately measure 1 ml of the prepared solutions, followed by mixing the solution. Accurately measured 20 µl of the prepared solutions were pipetted into the atomic generator of the graphite oven. The absorbance of solutions was determined, followed by preparing the calibration curve with absorbance as the vertical axis and concentration as the horizontal ordinate.
[00147] Preparation of the test solution: 0.5 gm 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) were added to the flask. A small hopper was placed on top of the flask. The mixture was macerated overnight, followed by heating and boiling the mixture to slake on the electric hot plate. A required amount of the above mixture was added to the solution, which 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, after turning colorless and transparent or yellowish, was cooled and transferred into a 50-ml volumetric flask. The container was washed with a 2% nitric acid solution, and the washing solution was added to the same volumetric flask, followed by diluting with the same solvent to make the volume of the washing solution 50 50ml. The solution was mixed properly by shaking the flask. Simultaneously, a reagent blank solution was prepared according to the procedure mentioned above.
[00148] Determination of Lead: The presence of lead was determined by accurately measuring 1 ml of the test solution and the corresponding reagent blank solution. 1 ml of solution containing 1% ammonium dihydrogen phosphate and 0.2% 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 solutions according to the steps followed for preparing the calibration curve. The content of lead (Pd) in the test solution was calculated from the calibration curve.
(b) Determination of Cadmium (Cd) by Graphite Oven Method:

[00149] The following conditions were taken as reference for determining the presence of Cadmium (Cd) using Graphite Oven Method:
• dry temperature: 100-1200 maintained for 20 seconds;
• ash temperature: 300-5000 maintained for 20-25 seconds;
• atomic temperature: 1500-19000 maintained for 4-5 seconds;
• measurement wavelength: 228.8 nm; and
• background calibration: deuterium lamp (D lamp) or Zeeman effect.

[00150] Preparation of Cadmium (Cd) Standard Stock Solution: A required amount of cadmium single-element standard solution was accurately measured for preparing a standard stock solution with 2% nitric acid containing 0.4 µg per ml cadmium. The solution was stored at 0–5 °C.

[00151] Preparation of the calibration curve: A required amount of cadmium standard stock solutions was accurately measured and diluted to concentrations 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 the vertical axis and concentration as the horizontal axis.

[00152] Preparation of test solution: The preparation of a test solution of cadmium involves the same steps as those provided for the preparation of a test solution of lead.
[00153] Determination of Cadmium: The presence of cadmium was determined by accurately measuring 10–20 µl of the test solution and the corresponding reagent blank solution. The absorbance of the solutions was determined by performing the same steps as for the preparation of the calibration curve. In case of any kind of discrepancy in results, 1 ml of the standard solution, blank solution, and test solution were separately weighed. 1 ml of a solution containing 1% ammonium dihydrogen phosphate and 0.2% 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 calculated by following the steps mentioned above. The content of cadmium (Cd) in the test solution was calculated from the calibration curve.

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

[00154] 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.

[00155] 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 0-5?.

[00156] Preparation of the calibration curve: A required amount of Arsenic standard stock solutions was accurately measured and diluted with 2% nitric acid to concentrations of 2, 4, 8, 12 and 16 mg 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 in a range of 20ml - 100ml to the volume. 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 flask was then cooled. The required amount of each solution was transferred into the hydride generator device. The absorbance of the prepared solutions was determined and a calibration curve was plotted with a peak area (absorbance) as vertical axis and concentration as horizontal ordinate.

[00157] Preparation of test solution: The preparation of test solution of arsenic involves same steps as those provided for the preparation of test solution of lead.

[00158] Determination of Arsenic: The presence of arsenic was determined by measuring 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 same steps were followed as for preparing the calibration curve. The content of arsenic in the test solution was calculated from the calibration curve.
(4) Determination of Mercury (Hg) by Cold Absorption Method:

[00159] 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 percent sodium borohydride and 0.1 per cent 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.

[00160] 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 0-5?.

[00161] Preparation of calibration curve: Accurately measured 0, 0.1, 0.3, 0.5, 0.7 and 0.9 ml of mercury standard stock solution were transferred into separate 50 ml volumetric flasks. 40 ml of 4% sulphuric acid solution and 0.5 ml of 5% potassium permanganate solution was added to each flask. The solution within each flask was mixed by shaking the flask. A drop of 5% hydroxylamine hydrochloride solution was added into each flask until the violet red disappeared. Each solution was diluted with 4% sulfuric acid solution to bring the volume of the solution to 50ml followed by mixing the solutions by shaking the flasks properly. The required amount of each solution was injected into 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.

[00162] 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 25 ml. The solution was mixed by shaking the flask. The solution was then centrifuged, if required. The supernatant obtained was used as the test solution. The blank reagent solute was simultaneously prepared by following the same steps.

[00163] 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. Same steps were followed as the steps followed for preparing calibration curve in case of arsenic. The content of mercury in the test solution was calculated from the calibration curve.

12. Pesticide Residue

[00164] Limits: The sample to be examined as required to compile with the limits indicated in Table 2.

[00165] 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 are calculated 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)

[00166] 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 are calculated using the following expression:
(ADI x M x E)/ MDD x 100

Wherein,

E =experimentally determined extraction factor of the method of preparation,

[00167] Higher limits may also be permitted in cases where a plant requires a particular cultivation method or exhibits a metabolism or 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.

[00168] 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.

[00169] Size of sampling: The samples from each container as indicated above were taken in case the number of containers was 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.
[00170] 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.

[00171] Reagents: All reagents and solvents used were free from contaminants majorly pesticides that may interfere with the analysis. High quality solvents or solvents that were recently re-distilled in an apparatus made of glass were used. In any case, suitable blank tests were performed

[00172] Apparatus: The apparatus and glassware were thoroughly cleaned to keep them pesticide free, Cleaning procedure may involve soaking the apparatus and/or glassware 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.

12. Aflatoxins

[00173] 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 types of aflatoxins are as follows:

[00174] 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.

[00175] Sodium Chloride Solution: 5 g of sodium chloride was dissolved in in 50 ml of purified water.

[00176] 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 in 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.

[00177] Test Solution 2: In case the solution contains interfering plant pigments then the following procedure is followed which involves collecting 100 ml of the filtrate from the start of the flow and transferring it 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, was mixed 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. Same steps were followed as the steps followed for preparing the test solution 1.

[00178] 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 a layer of 1.5 g of anhydrous sodium sulfate was added to the top of the column. 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.

[00179] 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.

[00180] 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.

13. 99.9% Germ Killing Efficacy:

[00181] The procedure for evaluating germ killing efficacy is as per ASTM E2315-016 and determines change in a population of aerobic microorganisms within a specified sampling time when antimicrobial test materials were present. Several options for organism selection and growth, inoculum preparation, sampling times and temperatures were provided. When the technique was performed as a specific test method then standardization of the variables was required. Antimicrobial activity of specific materials, as measured by the technique, may vary significantly depending on variables selected. The test results of microorganisms requiring growth supplements or special incubation conditions may not be directly comparable to organisms evaluated without the stated conditions.

[00182] 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 calculated. The percent and/or log10 reduction was calculated by comparison with the microbial population.

14. Dispensing Volume

[00183] The amount of nasal spray solution released per pump/spray is termed as Dispensing volume per pump/spray which is calculated by pumping the nasal spray from pump 10 times and calculating the total volume of solution collected and taking its average for per spray.

Dispensing volume = Total volume of 10 pumps/10

[00184] In an exemplary embodiment, Table 11 illustrates the experimental results for different tests performed on the herbal nasal spray composition with reduced strength (A).

Sr No. Test Result Standard Limits
1. General Description
1.1. Color Off White Off White
1.2. Odour Characteristic Characteristic
2. Weight per ml 1.0120 gm/ml 0.5-1.5 gm/ml
3. Specific Gravity 1.0217 1.01-1.03
4. Peroxide Value 0.42 -
5. Refractive Index 1.3453 1.0-1.5
6. Acid Value 0.38 0.1-0.5
7. Iodine Value 0.48 -
8. pH 6.4 6.0-6.5
9. Saponification 6.10 -
10. Viscosity 5.04 cps 5-10 cps
11. Microbial Contamination
11.1. Total Microbial Count, cfu/g Less than 10 Not more than 100000
11.2. Total Yeast and Mould, count/g Less than 10 Not more than 1000
11.3. Escherichia coli/g Absent Should be absent
11.4. Pseudomonas aeruginosa /g Absent Should be absent
11.5. Salmonella abony/10g Absent Should be absent
11.6. Staphylococcus aureus/g Absent Should be absent
12. Heavy metal Analysis
12.1. Mercury Below Quantification Limit 0.20-1.0ppm
12.2. Cadmium Below Quantification Limit 0.20-0.30ppm
12.3. Lead Below Quantification Limit 0.20-10.0ppm
12.4. Arsenic Below Quantification Limit 0.20-3.0ppm
13. Pesticide Residue
13.1. Alchalor Below Quantification Limit 0.0050-0.020mg/kg
13.2. Malathoin Below Quantification Limit 0.0050-1.0mg/kg
13.3. Phoslane Below Quantification Limit 0.0050-0.10mg/kg
13.4. Pyrethrins Below Quantification Limit 0.0050-3.0mg/kg
13.5. Ethion Below Quantification Limit 0.0050-2mg/kg
13.6. Chlorpyrifos Below Quantification Limit 0.0050-0.20mg/kg
13.7. Methyl Parathion Below Quantification Limit 0.0050-0.20mg/kg
13.8. Lindane (Gamma - HCH) Below Quantification Limit 0.0050-0.60mg/kg
13.9. Aldrin and dieldrin (sum of) Below Quantification Limit 0.0050-0.50mg/kg
13.10. Azinophos – methyl Below Quantification Limit 0.0050-1.0mg/kg
13.11. Bromopropylate Below Quantification Limit 0.0050-3.0mg/kg
13.12. Chlorpyrifos-methyl Below Quantification Limit 0.0050-0.10mg/kg
13.13. Deltamethrin Below Quantification Limit 0.0050-0.50mg/kg
13.14. Diazinon Below Quantification Limit 0.0050-0.10mg/kg
13.15. Dichlorvos Below Quantification Limit 0.0050-1.0mg/kg
13.16. Endrin Below Quantification Limit 0.0050-0.050mg/kg
13.17. Fonofos Below Quantification Limit 0.0050-0.050mg/kg
13.18. Methidathion Below Quantification Limit 0.0050-0.20mg/kg
13.19. Parathion Below Quantification Limit 0.0050-0.50mg/kg
13.20. Permethrin Below Quantification Limit 0.0050-1.0mg/kg
13.21. Perimiphos-methyl Below Quantification Limit 0.0050-4.0mg/kg
13.22. Fentrothion Below Quantification Limit 0.0050-0.50mg/kg
13.23. Cypermethrin and isomers Below Quantification Limit 0.0050-1.0mg/kg
13.24. Hexachlorocyclohexane Below Quantification Limit 0.0050-0.30mg/kg
13.25. Piperonyl-butoxide Below Quantification Limit 0.0050-3.0mg/kg
13.26. Heptachlor Below Quantification Limit 0.0050-0.050mg/kg
13.27. Fenvalerate Below Quantification Limit 0.0050-1.50mg/kg
13.28. Endosulfan Below Quantification Limit 0.0050-3.0mg/kg
13.29. DDT Below Quantification Limit 0.0050-1.0mg/kg
13.30. Hexachlorobenzene Below Quantification Limit 0.0050-0.10mg/kg
13.31. Chlorfenvinphos Below Quantification Limit 0.0050-0.50mg/kg
13.32. Dithiocarbamates Below Quantification Limit 0.0050-2.0mg/kg
13.33. Chlordane Below Quantification Limit 0.0050-0.50mg/kg
13.34. Quintozene Below Quantification Limit 0.0050-0.10mg/kg
14. Aflatoxin
14.1. Total Aflatoxin Below Quantification Limit Not more than 5mcg/Kg
14.2. Aflatoxin B1 Below Quantification Limit 1-2mcg/Kg
14.3. Aflatoxin B2 Below Quantification Limit 1-2mcg/Kg
14.4. Aflatoxin G2 Below Quantification Limit 1-2mcg/Kg
14.5. Aflatoxin G1 Below Quantification Limit 1-2mcg/Kg
15. 99% Germ Killing Efficacy
15.1. 99% Germ Killing Efficacy (For 30 Sec) % Reduction
15.1.1. Salmonella abony 99.9593 Not less than 99%
15.1.2. Staphylococcus aureus 99.9618 Not less than 99%
15.1.3. Escherichia coli 99.9139 Not less than 99%
15.1.4. Aspergillus brasilensis 99.9786 Not less than 99%
15.1.5. Candida albicans 99.9742 Not less than 99%
15.1.6. Listeria monocytogenes 99.9909 Not less than 99%
15.1.7. Staphylococcus epidermis 99.8841 Not less than 99%
15.1.8. Pseudomonas aerginosa 99.9497 Not less than 99%
15.2. 99% Germ Killing Efficacy (For 60 Sec) % Reduction
15.2.1. Salmonella abony 99.9831 Not less than 99%
15.2.2. Staphylococcus aureus 99.9957 Not less than 99%
15.2.3. Escherichia coli 99.9877 Not less than 99%
15.2.4. Aspergillus brasilensis 99.9858 Not less than 99%
15.2.5. Candida albicans 99.9954 Not less than 99%
15.2.6. Listeria monocytogenes 99.9968 Not less than 99%
15.2.7. Staphylococcus epidermis 99.9838 Not less than 99%
15.2.8. Pseudomonas aerginosa 99.9899 Not less than 99%
Table 11

[00185] In an exemplary embodiment, Table 2 illustrates the experimental results for different tests performed on the herbal nasal spray composition with high strength (B)

Sr No. Test Result Standard Limits
1. General Description
1.1. Color Off White Off White
1.2. Odour Characteristic Characteristic
2. Weight per ml 1.0102 gm/ml gm 0.5-1.5 gm/ml gm
3. Specific Gravity 1.0129 1.01-1.02
4. Peroxide Value 0.34
5. Refractive Index 1.3507 1.0-1.5
6. Acid Value 0.36 0.1-0.5
7. Iodine Value 3.93
8. pH 6.2 6.0-6.5
9. Saponification 3.04 -
10. Viscosity 5.26 cps 5-10 cps
11. Microbial Contamination
11.1. Total Microbial Count, cfu/g Less than 10 Not more than 100000
11.2. Total Yeast and Mould, count/g Less than 10 Not more than 1000
11.3. Escherichia coli/g Absent Should be absent
11.4. Pseudomonas aeruginosa /g Absent Should be absent
11.5. Salmonella abony/10g Absent Should be absent
11.6. Staphylococcus aureus/g Absent Should be absent
12. Heavy metal Analysis
12.1. Mercury Below Quantification Limit 0.20-1.0ppm
12.2. Cadmium Below Quantification Limit 0.20-0.30ppm
12.3. Lead Below Quantification Limit 0.20-10.0ppm
12.4. Arsenic Below Quantification Limit 0.20-3.0ppm
13. Pesticide Residue
13.1. Alchalor Below Quantification Limit 0.0050-0.020mg/kg
13.2. Malathoin Below Quantification Limit 0.0050-1.0mg/kg
13.3. Phoslane Below Quantification Limit 0.0050-0.10mg/kg
13.4. Pyrethrins Below Quantification Limit 0.0050-3.0mg/kg
13.5. Ethion Below Quantification Limit 0.0050-2mg/kg
13.6. Chlorpyrifos Below Quantification Limit 0.0050-0.20mg/kg
13.7. Methyl Parathion Below Quantification Limit 0.0050-0.20mg/kg
13.8. Lindane (Gamma - HCH) Below Quantification Limit 0.0050-0.60mg/kg
13.9. Aldrin and dieldrin (sum of) Below Quantification Limit 0.0050-0.50mg/kg
13.10. Azinophos – methyl Below Quantification Limit 0.0050-1.0mg/kg
13.11. Bromopropylate Below Quantification Limit 0.0050-3.0mg/kg
13.12. Chlorpyrifos-methyl Below Quantification Limit 0.0050-0.10mg/kg
13.13. Deltamethrin Below Quantification Limit 0.0050-0.50mg/kg
13.14. Diazinon Below Quantification Limit 0.0050-0.10mg/kg
13.15. Dichlorvos Below Quantification Limit 0.0050-1.0mg/kg
13.16. Endrin Below Quantification Limit 0.0050-0.050mg/kg
13.17. Fonofos Below Quantification Limit 0.0050-0.050mg/kg
13.18. Methidathion Below Quantification Limit 0.0050-0.20mg/kg
13.19. Parathion Below Quantification Limit 0.0050-0.50mg/kg
13.20. Permethrin Below Quantification Limit 0.0050-1.0mg/kg
13.21. Perimiphos-methyl Below Quantification Limit 0.0050-4.0mg/kg
13.22. Fentrothion Below Quantification Limit 0.0050-0.50mg/kg
13.23. Cypermethrin and isomers Below Quantification Limit 0.0050-1.0mg/kg
13.24. Hexachlorocyclohexane Below Quantification Limit 0.0050-0.30mg/kg
13.25. Piperonyl-butoxide Below Quantification Limit 0.0050-3.0mg/kg
13.26. Heptachlor Below Quantification Limit 0.0050-0.050mg/kg
13.27. Fenvalerate Below Quantification Limit 0.0050-1.50mg/kg
13.28. Endosulfan Below Quantification Limit 0.0050-3.0mg/kg
13.29. DDT Below Quantification Limit 0.0050-1.0mg/kg
13.30. Hexachlorobenzene Below Quantification Limit 0.0050-0.10mg/kg
13.31. Chlorfenvinphos Below Quantification Limit 0.0050-0.50mg/kg
13.32. Dithiocarbamates Below Quantification Limit 0.0050-2.0mg/kg
13.33. Chlordane Below Quantification Limit 0.0050-0.50mg/kg
13.34. Quintozene Below Quantification Limit 0.0050-0.10mg/kg
14. Aflatoxin
14.1. Total Aflatoxin Below Quantification Limit Not more than 5mcg/Kg
14.2. Aflatoxin B1 Below Quantification Limit 1-2mcg/Kg
14.3. Aflatoxin B2 Below Quantification Limit 1-2mcg/Kg
14.4. Aflatoxin G2 Below Quantification Limit 1-2mcg/Kg
14.5. Aflatoxin G1 Below Quantification Limit 1-2mcg/Kg
15. 99% Germ Killing Efficacy
15.1. 99% Germ Killing Efficacy (For 30 Sec) % Reduction
15.1.1. Salmonella abony 99.9661 Not less than 99%
15.1.2. Staphylococcus aureus 99.9979 Not less than 99%
15.1.3. Escherichia coli 99.9636 Not less than 99%
15.1.4. Aspergillus brasilensis 99.9098 Not less than 99%
15.1.5. Candida albicans 99.9843 Not less than 99%
15.1.6. Listeria monocytogenes 99.9934 Not less than 99%
15.1.7. Staphylococcus epidermis 99.9735 Not less than 99%
15.1.8. Pseudomonas aerginosa 99.9803 Not less than 99%
15.2. 99% Germ Killing Efficacy (For 60 Sec) % Reduction
15.2.1. Salmonella abony 99.9929 Not less than 99%
15.2.2. Staphylococcus aureus 99.9994 Not less than 99%
15.2.3. Escherichia coli 99.9921 Not less than 99%
15.2.4. Aspergillus brasilensis 99.9749 Not less than 99%
15.2.5. Candida albicans 99.9956 Not less than 99%
15.2.6. Listeria monocytogenes 99.9993 Not less than 99%
15.2.7. Staphylococcus epidermis 99.9934 Not less than 99%
15.2.8. Pseudomonas aerginosa 99.9899 Not less than 99%
Table 2
[00186] Several experiments were performed on herbal composition when utilized as agent in nebulizer using similar methods as used for herbal composition A and B to evaluate general parameters, weight per millilitres, specific gravity, peroxide value (as per API Part I, Vol. VI, appendix 3, 3.10.), acid value (as per API Part I, Vol. VI, appendix 3, 3.9.), Iodine value, pH (as per API, Part I, Vol. IX, Appendix-2, 2.1.10.), saponification, viscosity (as per API Part-I, Vol. VI, Appendix 3, 3.4), microbial contamination, heavy metal analysis, pesticide residue, Aflatoxins and germ killing efficacy of the herbal composition used as an agent for nebulizer.

[00187] Table 3 illustrates the experimental results for different tests performed on the herbal composition utilized as an agent in nebulizer.

1. Microbial Contamination
1.1. Total Microbial Count, cfu/g Less than 10 Not more than 100000
1.2. Total Yeast and Mould, count/g Less than 10 Not more than 1000
1.3. Escherichia coli/g Absent Should be absent
1.4. Pseudomonas aeruginosa /g Absent Should be absent
1.5. Salmonella abony/10g Absent Should be absent
1.6. Staphylococcus aureus/g Absent Should be absent
2. Heavy metal Analysis
2.1. Mercury Below Quantification Limit 0.20-1.0ppm
2.2. Cadmium Below Quantification Limit 0.20-0.30ppm
2.3. Lead Below Quantification Limit 0.20-10.0ppm
2.4. Arsenic Below Quantification Limit 0.20-3.0ppm
3. Pesticide Residue
3.1. Alchalor Below Quantification Limit 0.0050-0.020mg/kg
3.2. Malathoin Below Quantification Limit 0.0050-1.0mg/kg
3.3. Phoslane Below Quantification Limit 0.0050-0.10mg/kg
3.4. Pyrethrins Below Quantification Limit 0.0050-3.0mg/kg
3.5. Ethion Below Quantification Limit 0.0050-2mg/kg
3.6. Chlorpyrifos Below Quantification Limit 0.0050-0.20mg/kg
3.7. Methyl Parathion Below Quantification Limit 0.0050-0.20mg/kg
3.8. Lindane (Gamma - HCH) Below Quantification Limit 0.0050-0.60mg/kg
3.9. Aldrin and dieldrin (sum of) Below Quantification Limit 0.0050-0.50mg/kg
3.10. Azinophos – methyl Below Quantification Limit 0.0050-1.0mg/kg
3.11. Bromopropylate Below Quantification Limit 0.0050-3.0mg/kg
3.12. Chlorpyrifos-methyl Below Quantification Limit 0.0050-0.10mg/kg
3.13. Deltamethrin Below Quantification Limit 0.0050-0.50mg/kg
3.14. Diazinon Below Quantification Limit 0.0050-0.10mg/kg
3.15. Dichlorvos Below Quantification Limit 0.0050-1.0mg/kg
3.16. Endrin Below Quantification Limit 0.0050-0.050mg/kg
3.17. Fonofos Below Quantification Limit 0.0050-0.050mg/kg
3.18. Methidathion Below Quantification Limit 0.0050-0.20mg/kg
3.19. Parathion Below Quantification Limit 0.0050-0.50mg/kg
3.20. Permethrin Below Quantification Limit 0.0050-1.0mg/kg
3.21. Perimiphos-methyl Below Quantification Limit 0.0050-4.0mg/kg
3.22. Fentrothion Below Quantification Limit 0.0050-0.50mg/kg
3.23. Cypermethrin and isomers Below Quantification Limit 0.0050-1.0mg/kg
3.24. Hexachlorocyclohexane Below Quantification Limit 0.0050-0.30mg/kg
3.25. Piperonyl-butoxide Below Quantification Limit 0.0050-3.0mg/kg
3.26. Heptachlor Below Quantification Limit 0.0050-0.050mg/kg
3.27. Fenvalerate Below Quantification Limit 0.0050-1.50mg/kg
3.28. Endosulfan Below Quantification Limit 0.0050-3.0mg/kg
3.29. DDT Below Quantification Limit 0.0050-1.0mg/kg
3.30. Hexachlorobenzene Below Quantification Limit 0.0050-0.10mg/kg
3.31. Chlorfenvinphos Below Quantification Limit 0.0050-0.50mg/kg
3.32. Dithiocarbamates Below Quantification Limit 0.0050-2.0mg/kg
3.33. Chlordane Below Quantification Limit 0.0050-0.50mg/kg
3.34. Quintozene Below Quantification Limit 0.0050-0.10mg/kg
4. Aflatoxin
4.1. Total Aflatoxin Below Quantification Limit Not more than 5mcg/Kg
4.2. Aflatoxin B1 Below Quantification Limit 1-2mcg/Kg
4.3. Aflatoxin B2 Below Quantification Limit 1-2mcg/Kg
4.4. Aflatoxin G2 Below Quantification Limit 1-2mcg/Kg
4.5. Aflatoxin G1 Below Quantification Limit 1-2mcg/Kg
5. 99% Germ Killing Efficacy
5.1. 99% Germ Killing Efficacy (For 30 Sec) % Reduction
5.1.1. Salmonella abony 99.9593 Not less than 99%
5.1.2. Staphylococcus aureus 99.9618 Not less than 99%
5.1.3. Escherichia coli 99.9139 Not less than 99%
5.1.4. Aspergillus brasilensis 99.9786 Not less than 99%
5.1.5. Candida albicans 99.9742 Not less than 99%
5.1.6. Listeria monocytogenes 99.9909 Not less than 99%
5.1.7. Staphylococcus epidermis 99.8841 Not less than 99%
5.1.8. Pseudomonas aerginosa 99.9497 Not less than 99%
5.2. 99% Germ Killing Efficacy (For 60 Sec) % Reduction
5.2.1. Salmonella abony 99.9831 Not less than 99%
5.2.2. Staphylococcus aureus 99.9957 Not less than 99%
5.2.3. Escherichia coli 99.9877 Not less than 99%
5.2.4. Aspergillus brasilensis 99.9858 Not less than 99%
5.2.5. Candida albicans 99.9954 Not less than 99%
5.2.6. Listeria monocytogenes 99.9968 Not less than 99%
5.2.7. Staphylococcus epidermis 99.9838 Not less than 99%
5.2.8. Pseudomonas aerginosa 99.9899 Not less than 99%
Table 3

[00188] ACUTE INTANASAL TOXICITY STUDY

[00189] An experimental study was performed to evaluate the acute inhalation toxicity of the herbal composition with reduced strength (A) and high strength (B) through an intra-nasal route in Wistar Rats.
G1 served as control group and was treated with 0.9% normal saline and G2-G4 served as treated groups and were treated with the herbal composition of the reduced strength (A) and the herbal composition with the high strength (B), respectively. The test composition was administered drop by drop twice a day via intranasal route in both the nostrils with the help of micropipette at the dose volume of 75µL of 0.9% normal saline in G1 and, 25µL, 50µL and 75µL of test composition in G1-G4 respectively. Post dosing, the Wistar rats were observed once in a day, for up to 14 days.
[00190] Results:

[00191] Clinical Signs – The Wistar rats were examined for clinical signs of toxicity and mortality at 30 min post dosing and later at least once daily for 14 days. No clinical signs of evident toxicity were reported in any of the Wistar rats of any group (G1-G4) throughout the observation period as shown in Table 4 and Table 5 for herbal composition with reduced strength (A) and herbal composition with high strength (B), respectively.

[00192] Table 4 illustrates clinical signs results for herbal composition with reduced strength (A).
Group Animal No. Day
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Mor Eve
G1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
G2 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
G3 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
G4 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
Mor: Morning; Eve: Evening; 1 indicates normal clinical sign
Table 4
[00193] Table 5 illustrates clinical signs results for herbal composition with high strength (B).
group Animal No. Day
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Mor Eve
G1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
G2 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
G3 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
G4 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
Mor: Morning; Eve: Evening; 1 indicates normal clinical sign
Table 5

[00194] Mortality/Morbidity - All the Wistar rats were observed for mortality/morbidity daily, once a day, throughout the experimental period. No mortality/morbidity was reported in any of the Wistar rat of any group (G1-G4) throughout the observation period as shown in Table 6 and Table 7 for herbal composition with reduced strength (A) and herbal composition with high strength (B), respectively.

[00195] Table 6 illustrates mortality/morbidity data for herbal composition with reduced strength (A).

Group Animal No. Day Mortality/Morbidity
G1 1 1-15 Nil
2 1-15 Nil
3 1-15 Nil
G2 1 1-15 Nil
2 1-15 Nil
3 1-15 Nil
4 1-15 Nil
G3 1 1-15 Nil
2 1-15 Nil
3 1-15 Nil
4 1-15 Nil
G4 1 1-15 Nil
2 1-15 Nil
3 1-15 Nil
4 1-15 Nil
Key: Nil = No morbidity/mortality
Table 6
[00196] Table 7 illustrates mortality/morbidity data for herbal composition with high strength (B).

Group Animal No. Day Mortality/Morbidity
G1 1 1-15 Nil
2 1-15 Nil
3 1-15 Nil
G2 1 1-15 Nil
2 1-15 Nil
3 1-15 Nil
4 1-15 Nil
G3 1 1-15 Nil
2 1-15 Nil
3 1-15 Nil
4 1-15 Nil
G4 1 1-15 Nil
2 1-15 Nil
3 1-15 Nil
4 1-15 Nil
Key: Nil = No morbidity/mortality
Table 7

[00197] Body Weight- The body weight of all the Wistar rats was recorded on the day 1 (prior to dosing), day 8 and day 15. The percentage change in body weight was calculated on day 1 to 8, day 8 to 15 and day 1 to 15. The percentage body weight change for each group was calculated as per the given formula:

[00198] An increase in the body weight of all the Wistar rats was reported on day 8, day 15 as compared to day 1 as shown in Table No 10 and Table No. 11 for herbal composition with reduced strength (A) and herbal composition with high strength (B), respectively.

[00199] Table 8 illustrates body weight data for herbal composition with reduced strength (A).

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
1 178.10 191.13 198.53 7.32 3.87 11.47
2 191.89 199.02 201.76 3.72 1.38 5.14
3 192.77 201.70 205.60 4.63 1.93 6.66
Mean 187.59 197.28 201.96 5.22 2.39 7.76
SEM 4.75 3.17 2.04 1.08 0.76 1.91
G2
1 193.22 196.25 198.75 1.57 1.27 2.86
2 193.28 206.98 207.98 7.09 0.48 7.61
3 197.05 199.21 201.99 1.10 1.40 2.51
4 190.57 199.61 200.75 4.74 0.57 5.34
Mean 193.53 200.51 202.37 3.62 0.93 4.58
SEM 1.33 2.28 1.99 1.41 0.24 1.19
G3
1 206.74 216.38 222.14 4.66 2.66 7.45
2 196.51 209.77 216.57 6.75 3.24 10.21
3 198.22 219.33 222.98 10.65 1.66 12.49
4 207.62 209.88 214.43 1.09 2.17 3.28
Mean 202.27 213.84 219.03 5.79 2.43 8.36
SEM 2.86 2.40 2.09 2.00 0.34 1.98
G4
1 253.31 255.36 261.96 0.81 2.58 3.41
2 180.58 197.98 199.38 9.64 0.71 10.41
3 194.44 203.17 212.98 4.49 4.83 9.54
4 187.11 192.61 197.06 2.94 2.31 5.32
Mean 203.86 212.28 217.85 4.47 2.61 7.17
SEM 16.72 14.52 15.12 1.88 0.85 1.67

Table 8

[00200] Table 9 illustrates body weight data for herbal composition with high strength (B).

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
1 191.50 199.67 208.77 4.27 4.56 9.02
2 209.24 215.18 227.14 2.84 5.56 8.55
3 209.95 215.17 220.50 2.49 2.48 5.03
Mean 203.56 210.01 218.80 3.20 4.20 7.53
SEM 6.04 5.17 5.37 0.54 0.91 1.26
G2
1 195.93 199.87 205.39 2.01 2.76 4.83
2 207.21 215.87 228.51 4.18 5.86 10.28
3 212.51 219.16 228.51 3.13 4.27 7.53
4 235.65 241.71 244.28 2.57 1.06 3.66
Mean 218.46 225.58 233.77 3.29 3.73 7.16
SEM 8.73 8.12 5.26 0.47 1.41 1.92
G3
1 197.80 210.20 217.39 6.27 3.42 9.90
2 206.53 215.19 223.69 4.19 3.95 8.31
3 218.98 227.16 234.44 3.74 3.20 7.06
4 232.29 239.43 245.60 3.07 2.58 5.73
Mean 219.27 227.26 234.58 3.67 3.24 7.03
SEM 7.44 7.00 6.33 0.32 0.40 0.74
G4
1 201.35 211.17 218.01 4.88 3.24 8.27
2 202.79 207.28 213.88 2.21 3.18 5.47
3 220.38 227.41 236.12 3.19 3.83 7.14
4 230.20 236.53 241.89 2.75 2.27 5.08
Mean 217.79 223.74 230.63 2.72 3.09 5.90
SEM 8.02 8.64 8.54 0.28 0.45 0.63
Table 9

[00201] Gross Pathology - On day 15, Wistar rats were humanely scarified by overdose of Thiopentone sodium injection by intraperitoneal route and subjected to gross pathological examination. . All gross pathological changes were recorded for each Wistar rat with particular attention to any changes in the respiratory tract. No external abnormalities were reported in any of the Wistar rats of control (G1) and treatment groups (G2-G4). Further, no internal abnormalities were reported in any of the Wistar rats of control (G1) and treatment groups (G2-G4) as shown in Table No. 12 and Table No. 13 for herbal composition with reduced strength (A) and herbal composition with high strength (B), respectively.

[00202] Table 10 illustrates gross pathology data for external and internal abnormalities for herbal composition with reduced strength (A).

Group
Animal No. External Examination Internal Examination
G1
1 NAD NAD
2 NAD NAD
3 NAD NAD
G2
1 NAD NAD
2 NAD NAD
3 NAD NAD
4 NAD NAD
G3
1 NAD NAD
2 NAD NAD
3 NAD NAD
4 NAD NAD
G4
1 NAD NAD
2 NAD NAD
3 NAD NAD
4 NAD NAD
NAD = No Abnormality Detected
Table 10

[00203] Table 11 illustrates gross pathology data for external and internal abnormalities for herbal composition with high strength (B)
Group
Animal No. External Examination Internal Examination
G1
1 NAD NAD
2 NAD NAD
3 NAD NAD
G2
1 NAD NAD
2 NAD NAD
3 NAD NAD
4 NAD NAD
G3
1 NAD NAD
2 NAD NAD
3 NAD NAD
4 NAD NAD
G4
1 NAD NAD
2 NAD NAD
3 NAD NAD
4 NAD NAD
NAD = No Abnormality Detected
Table 11

[00204] Conclusion:

[00205] The herbal composition “A” and “B” were applied twice a day in morning and evening (single day dosing) through intra-nasal route at a dose volume of 25µL, 50µL and 75µL in the Wistar rats. No mortality, no sign of toxicity, and no gross pathological changes were reported in the Wistar rats. Hence, the herbal composition “A” and “B” were found to be safe for intra-nasal route in a single day dosing.

[00206] An experimental study was conducted to evaluate acute inhalation toxicity of the composition used in form of a liquid agent for nebulizer in Wistar rats. The Wistar rats were made to inhale the herbal composition using clinical nebulizer. The study was performed in accordance to the method utilized for herbal composition A and B to evaluate acute intranasal toxicity tests such as Clinical signs, Mortality/ Morbidity, Change in body weight, Gross pathology (as shown in Table 12 -21)

[00207] Table 12-17 illustrates acute intranasal toxicity test results in the female Wistar rats :
Group Animal No. Day
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Mor Eve
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
Mor: Morning; Eve: Evening; 1=Normal
Table 12

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
Key: Nil = No morbidity/mortality
Table 13

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
1 175.87 184.63 195.68 4.98 5.98 11.26
2 213.49 217.22 223.59 1.75 2.93 4.73
3 213.96 222.36 236.05 3.93 6.16 10.32
4 226.06 232.93 256.10 3.04 9.95 13.29
5 230.21 240.25 256.28 4.36 6.67 11.32
Mean 211.92 219.48 233.54 3.61 6.34 10.19
SEM 9.59 9.59 11.32 0.56 1.12 1.45
G2
6 186.13 196.55 211.39 5.60 7.55 13.57
7 200.20 213.45 239.42 6.62 12.17 19.59
8 224.49 242.70 261.65 8.11 7.81 16.55
9 225.28 227.38 238.18 0.93 4.75 5.73
10 230.29 237.81 238.54 3.27 0.31 3.58
Mean 213.28 223.58 237.84 4.91 6.52 11.80
SEM 8.57 8.41 7.97 1.27 1.95 3.09

Table 14

Group
Animal No. External Examination Internal Examination
G1
1 NAD NAD
2 NAD NAD
3 NAD NAD
4 NAD NAD
5 NAD NAD
G2
6 NAD NAD
7 NAD NAD
8 NAD NAD
9 NAD NAD
10 NAD NAD

Table 15

[00208] Table 16-21 illustrates acute intranasal toxicity test results in Male Wistar rats :

Group Animal No. Day
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Mor Eve
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
Mor: Morning; Eve: Evening; 1=Normal
Table 16
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

Table 17

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
1 222.21 239.91 243.01 7.97 1.29 9.36
2 244.90 265.48 267.22 8.40 0.66 9.11
3 248.85 259.36 282.20 4.22 8.81 13.40
4 261.10 277.60 280.58 6.32 1.07 7.46
5 266.22 273.46 298.89 2.72 9.30 12.27
Mean 248.66 263.16 274.38 5.93 4.23 10.32
SEM 7.67 6.61 9.32 1.09 1.98 1.09
G2
6 235.04 243.52 249.12 3.61 2.30 5.99
7 239.05 246.45 259.34 3.10 5.23 8.49
8 250.06 253.37 266.71 1.32 5.27 6.66
9 254.68 266.33 270.35 4.57 1.51 6.15
10 269.43 283.29 297.76 5.14 5.11 10.51
Mean 249.65 258.59 268.66 3.55 3.88 7.56
SEM 6.09 7.32 8.13 0.66 0.82 0.86
Table 18
Group
Animal No. External Examination Internal Examination
G1
1 NAD NAD
2 NAD NAD
3 NAD NAD
4 NAD NAD
5 NAD NAD
G2
6 NAD NAD
7 NAD NAD
8 NAD NAD
9 NAD NAD
10 NAD NAD
NAD = No Abnormality Detected
Table 19

Several experiments were performed to evaluate in-vitro efficacy of herbal composition A and B as well as the herbal composition utilized as agent in nebulizer.
[00209] Anti-inflammatory property of the herbal composition with reduced strength (A):
[00210] As shown in Figure 1, anti-inflammatory property of the herbal composition with reduced strength (A) was evaluated in lung cells by inhibition in level of IL-8 against TNF-a stimulation. A maximum inhibition of 68.8% of IL-8 against TNF-a stimulation in lung cells was reported, this indicating anti-inflammatory property of the herbal composition (A).

[00211] Anti-inflammatory property of the herbal composition with high strength (B):
[00212] As shown in Figure 2, anti-inflammatory property of the herbal composition with high strength (B) was evaluated in lung cells by inhibition in level of IL-8 against TNF-a stimulation. A maximum inhibition of 63.6% of IL-8 against TNF-a stimulation was reported in lung cells, thus indicating anti-inflammatory property of the herbal composition (B).

[00213] Anti-inflammatory property of the herbal composition with reduced strength (A):
[00214] As shown in Figure 3, anti-inflammatory property of the herbal composition with reduced strength (B) was evaluated in immune cells, particularly murine splenocyte cells by inhibition in level of IL-6 against LPS stimulation. A maximum inhibition of 59.3% of IL-6 against LPS stimulation was reported in murine splenocyte cells, thus indicating anti-inflammatory property of the herbal composition (A).

[00215] Anti-inflammatory property of the herbal composition with high strength (B):
[00216] As shown in Figure 4, anti-inflammatory property of the herbal composition with high strength (A) was evaluated in immune cells, particularly murine splenocyte cells by inhibition in level of IL-6 against LPS stimulation. A maximum inhibition of 93.9% of IL-6 against LPS stimulation was reported in murine splenocyte cells, thus indicating anti-inflammatory property of the herbal composition (B).

[00217] Anti-inflammatory property of the herbal composition with reduced strength (A):
[00218] As shown in Figure 5, anti-inflammatory property of the herbal composition with reduced strength (A) was evaluated in lung cells by inhibition in level of LTB4 against TNF-a stimulation. A maximum inhibition of 53.4% of LTB4 against TNF-a stimulation was reported in lung cells, thus indicating anti-inflammatory property of the herbal composition (A).

[00219] Anti-inflammatory property of the herbal composition with high strength (B):
[00220] As shown in Figure 6, anti-inflammatory property of the herbal composition with high strength (B) was evaluated in lung cells by inhibition in level of LTB4 against TNF-a stimulation. A maximum inhibition of 53.7% of LTB4 against TNF-a stimulation was reported in lung cells, thus indicating anti-inflammatory property of the herbal composition (B).

[00221] Anti-allergic property of the herbal composition with reduced strength (A):
[00222] As shown in Figure 7, anti-allergic property of the herbal composition with reduced strength (A) was evaluated in immune cells by inhibition in level of histamine against compound 48/80 stimulation. A maximum inhibition of 45.7% of histamine against compound 48/80 stimulation was reported in immune cells, thus indicating anti-allergic property of the herbal composition (A).

[00223] Anti-allergic property of the herbal composition with high strength (B):
[00224] As shown in Figure 7, anti-allergic property of the herbal composition with high strength (B) was evaluated in immune cells by inhibition in level of histamine against compound 48/80 stimulation. A maximum inhibition of 48% of histamine against compound 48/80 stimulation was reported in immune cells, thus indicating anti-allergic property of the herbal composition (B).

[00225] Mucolytic potential of the herbal composition with reduced strength (A):
[00226] As shown in Figure 9, mucolytic potential of the herbal composition with reduced strength (A) was evaluated in lung cells by inhibition in level of mucin (MUC5AC) against TNF-a stimulation in lung cells. A maximum inhibition of 92.2% of mucin (MUC5AC) against TNF-a stimulation was reported in lung cells, thus indicating mucolytic potential of the herbal composition (A).

[00227] Mucolytic potential of the herbal composition with high strength (B)
[00228] As shown in Figure 10, mucolytic potential of the herbal composition with high strength (B) was evaluated in lung cells by inhibition in level of mucin (MUC5AC) against TNF-a stimulation in lung cells. A maximum inhibition of 100% of mucin (MUC5AC) against TNF-a stimulation was reported in lung cells, thus indicating mucolytic potential of the herbal composition (B).

[00229] Mucolytic potential of the herbal composition with reduced strength (A):
[00230] As shown in Figure 10, mucolytic potential of the herbal composition with reduced strength (A) was evaluated by inhibition in egg white viscosity. A maximum inhibition of 77.6% of egg white viscosity was reported, thus indicating mucolytic potential of the herbal composition (A).

[00231] Mucolytic potential of the herbal composition with high strength (B):
[00232] As shown in Figure 12, mucolytic potential of the herbal composition with high strength (B) was evaluated by inhibition in egg white viscosity. A maximum inhibition of 76.6% in egg white viscosity was reported, thus indicating mucolytic potential of the herbal composition (B).

[00233] Anti-inflammatory property of the herbal composition in form of liquid agent in nebulizer:
[00234] As shown in Figure 13, anti-inflammatory property of the herbal composition in form of a liquid agent in nebulizer was evaluated by inhibition of IL-8 against TNF-a stimulation in lung cells. A maximum inhibition of 43.3% of IL-8 against TNF-a stimulation was reported in lung cells, thus indicating anti-inflammatory property of the herbal composition in form of a liquid agent in nebulizer.

[00235] Anti-inflammatory property of the herbal composition in form of liquid agent in nebulizer:
[00236] As shown in Figure 14, anti-inflammatory property of the herbal composition in form of a liquid agent in nebulizer was evaluated by inhibition of IL-6 against LPS stimulation in murine splenocyte cells. A maximum inhibition of 53.2% of IL-6 against LPS stimulation was reported in murine splenocyte cells, thus indicating anti-inflammatory property of the herbal composition in form of a liquid agent in nebulizer.

[00237] Anti-allergic property of the herbal composition in form of liquid agent in nebulizer:
[00238] As shown in Figure 15, anti-allergic property of the herbal composition in form of a liquid agent in nebulizer was evaluated by inhibition of histamine against compound 48/80 stimulation in immune cells. A maximum inhibition of 50.4% of histamine against compound 48/80 stimulation was reported in immune cells, thus indicating anti-allergic property of the herbal composition in form of a liquid agent in nebulizer.

[00239] Mucolytic potential of the herbal composition in form of liquid agent in nebulizer:
[00240] As shown in Figure 16, mucolytic potential of the herbal composition in form of a liquid agent in nebulizer was evaluated by inhibition of mucin (MUC5AC) level against TNF-a stimulation in lung cells. A maximum inhibition of 73.8% of mucin (MUC5AC) against TNF-a stimulation was reported in lung cells, thus indicating mucolytic property of the herbal composition in form of a liquid agent in nebulizer.

[00241] Mucolytic potential of the herbal composition in form of liquid agent in nebulizer:
[00242] As shown in Figure 17, mucolytic potential of the herbal composition in form of a liquid agent in nebulizer was evaluated by inhibition in egg white viscosity in lung cells. A maximum inhibition of 78.5% in egg white viscosity in lung cells, thus indicating mucolytic property of the herbal composition in form of a liquid agent in nebulizer.

[00243] The herbal composition for nasal spray is used as non-invasive route of administration. Such route of administration possesses a number of advantages such as fast onset of action, rapid absorption of drug, decrease in systemic side effects, painless and convenient, self/home administration. Intranasal delivery method avoids first pass metabolism and degradation of drug in gastrointestinal tract, thus resulting in increased bioavailability.

[00244] While this invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the scope of the appended claims.
,CLAIMS:WE CLAIM:
1. A herbal composition for nasal spray, comprising:
a. one or more essential oils in a range of 0.1% to 20% by weight of total herbal composition;
b. one or more solubilizing agents in a range of 0.5% to 10% by weight of total herbal composition;
c. at least one surfactant in a range of 0.5% to 3% by weight of the total herbal composition;
d. at least one chelating agent in a range of 0.01% to 2% by weight of the total herbal composition;
e. a buffer solution in a range of 30 -70 %by weight of the total herbal composition;
f. at least one pharmaceutically accepted excipient in a range of 0.00001% to 6% by weight of the total herbal composition;
g. at least one polymer in a range of 0.1% to 1% by weight of the total herbal composition;
h. a salt in a range of 0.1-0.5% by weight of the total herbal composition; and
i. water in a range of 42% to 48% by weight of the total herbal composition.

2. The herbal composition as claimed in claim 1, wherein the essential oil is selected from a group consisting of Eucalyptus oil, Clove oil, Ajwain oil, Peppermint oil, Tulsi oil, Cedarwood oil, Sandalwood oil, Cinnamon oil or a combination thereof.

3. The herbal composition as claimed in claim 1, wherein the solubilizing agent is selected from a group consisting of Propylene glycol, PEG 400, 2-(2-Ethoxyethoxy)ethanol (transcutol), Labrasol, Polysorbate 80 or a combination thereof.

4. The herbal composition as claimed in claim 1, wherein the surfactant is selected from a group of Poloxomer, Sodium Bicarbonate, Sodium lauroyl Sarcosinate, Sodium lauroyl glutamate, or a combination thereof.

5. The herbal composition as claimed in claim 1, wherein the chelating agent is selected from a group consisting of 2,2',2'',2'''-(Ethane-1,2-diyldinitrilo) tetraacetic acid (EDTA), (ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid) (EGTA) or a combination thereof.

6. The herbal composition as claimed in claim 1, wherein the pharmaceutically accepted excipient is selected from a group of anti-irritant, or preservatives or a combination thereof.

7. The herbal composition as claimed in claim 7, wherein the anti-irritant is selected from a group consisting of Sodium hydrogencarbonate (Sodium bicarbonate), (2R,3r,4S)-Pentane-1,2,3,4,5-pentol (Xylitol), Zinc chloride or a combination thereof.

8. The herbal composition as claimed in claim 7, wherein the preservative is Benzalkonium chloride.

9. The herbal composition as claimed in claim 1, wherein the polymer is selected from a group of mucoadhesive polymer.

10. A method for preparing a herbal composition for nasal spray as claimed in claim 1, comprising steps of:
(a) adding essential oils and solubilizing agents in an apparatus A ;
(b) preparing buffer solution by dissolving disodium Phosphate and Potassium hydrogen phosphate in distilled water;
(c) adding distilled water to the solution obtained in step (b), making up the volume of the solution to 100ml;
(d) stirring the solution obtained in step (c) until a clear solution appears.
(e) checking the pH of the solution obtained in step (d);
(f) adding pharmaceutically acceptable excipients, polymers, chelating agents; solubilizing agent; and salt one by one after complete dissolution of each compound in 20ml of distilled water contained in an apparatus (B);
(g) mixing contents of the apparatus A and apparatus B, forming a solution;
(h) adding buffer solution to the solution obtained in step (g), making the volume of the solution to 100ml;
(i) homogenizing the solution obtained in step (h) with the addition of pharmaceutically acceptable excipients for 15-20 minutes;
(j) filtering the solution obtained in step (i) after complete homogenization, resulting in a herbal composition; and
(k) checking the pH of the solution.