DESC:FIELD OF INVENTION
[001] The present invention relates to a herbal composition for reducing stress and a method for preparation thereof. Particularly, the present invention relates to a herbal composition comprising essential oils that relieves stress and other mental illness such as anxiety, depression and suicidal tendencies in people.

BACKGROUND OF THE INVENTION
[002] Nowadays, stress has become a part of people’s lives. Stress is one major problem faced by almost every individual in some form. Some of the reasons for stress include deadlines, pressure, liabilities, and so on. Stress may be mental or physical in nature. Mental stress includes anxiety, mood swings, irritability and compulsive behaviour. Such stress may also increase suicidal tendencies in people. Therefore, there exist a need to improve mental health condition of the people.

[003] One of the conventional method to relieve anxiety include the use of drugs. Some of the drugs used to treat anxiety includes Benzodiazepines, Buspirone, etc. However, consumption of
such drugs is associated with a number of side effects such as vision problems, headaches, dizziness, nausea, confusion and depressed feeling.

[004] Other conventional method to treat depression includes the use of antidepressants. Such antidepressants include Selective serotonin reuptake inhibitors SSR1s and Tricyclics. The antidepressants function by increasing the levels of serotonin, a neurotransmitter that affects sleep, mood, memory and appetite. However, consumption of the anti-depressants is associated with a number of side effects such as nausea, dizziness, vomiting, confusion, and blurred vision.

[005] There are several patent applications that discloses a composition for treating stress, anxiety and depression. The United States Patent Application US8329227B2 discloses formulas for producing compositions for the structural/functional nutritional support for those who struggle with poor focus, concentration and/or memory. The invention also provides compositions comprising nutritional/botanical factors helpful to those who subjectively experience transient mental fatigue or poor cognitive function. The compositions of the invention consist primarily of the following ingredients B-complex vitamins, antioxidants, minerals, phosphatidyl serine (PS), choline, dimenthyl-aminoethanol (DMAE), docosahexaenoic acid (DHA), L-pyroglutamic acid, as well as herbal extracts from Bacopa monniera, Vinca minor, and Huperzia serrata. The present invention also relates to the administration of these compounds to alleviate mental fatigue or poor cognitive function.

[006] Another Patent Application DE202004018534U1 discloses a herbal composition for avoidance and/or treatment of conditions in connection with depression. The herbal composition (I) comprises herbs (or their extracts) for: (a) promoting blood flow and reducing coagulated blood levels; (b) calming; (c) sedation; and (d) flow of 'Qi'; where (a) is one of 15 specific herbs (e.g. Salvia multiorhiza), (b) is one of 9 specific herbs (e.g. Lilium brownii), (c) is one of 4 specific herbs (e.g. Rehmannia glutinosa) and (d) is one of 20 specific herbs (e.g. Citrus cinensis).

[007] The above prior art includes oral route of administration of the herbal composition. However, the oral route of administration provides slow onset of action. Moreover, such route of administration may be inefficient due to insufficient and irregular absorption. Moreover, people may feel difficulty in administration of such composition.

[008] Therefore, keeping in view the problems associated with the state of the art there exists a need to provide an efficient, safe herbal composition that relieves stress other mental health disorders.
SUMMARY OF THE INVENTION
[009] The present invention relates to a herbal composition for reducing stress and a method for
preparation thereof. The composition comprises of one or more essential oils, at least one surfactant and water. The essential oils are present in a range of 0.1% to 25% by weight of the total composition. The surfactant is present in a range of 0.1% to 10% by weight of the total composition. Water is present in a range of 50% to 80% by weight of the total composition. The present invention also provides a method for preparation of the herbal composition. The preparation of the herbal composition employs microemulsion technique. The present invention relates to a herbal composition that relieves stress which may lead to anxiety depression and suicidal tendencies in people. The herbal composition of the present invention may be used as a spray for spraying the herbal composition on inner/outer surface of masks, any fabric, room, car or any open space, thereby relieving stress, anxiety and other mental illness, or may be used for the purpose of steam inhalation.

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

[011] Another objective of the present invention is provide an efficient, safe herbal composition that provide relief from stress other mental health disorders.

[012] Another objective of the present invention is to provide a herbal composition comprising a number of essential oils that ensures rapid onset of action.

[013] Another objective of the present invention is use essential oils containing bioactive compounds that promote mental state of a person by reliving stress and other mental illness such as anxiety, depression and suicidal tendencies in people.

[014] Another objective of the present invention is to provide sustained inhalation of therapeutically active components used in the present invention such as essential oils in order to relieve stress and provide calmness.

[015] Another objective of the present invention is to provide a herbal composition that may be sprayed on inner/outer surface of masks, fabric, room, car or any open space, thereby relieving
stress, and other mental illness such as anxiety, depression and suicidal tendencies in people.

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

SOURCES OF BIOLOGICAL MATERIAL USED IN THE PRESENT INVENTION

[017] The essential oils used in the herbal composition including such as, but not limited to, Lavender oil, Neroli oil, Sandalwood oil, and Peppermint oil, or a combination thereof are procured from local 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 include ATCC 6538 for Staphylococcus aureus, ATCC 6633 for Bacillus subtilis, ATCC 8739 for Escherichia coli and ATCC 2091, and ATCC 10231 for Candida albicans. The test strains used for determining microbial contamination including Salmonella abony 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.

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

[019] Figure 1 illustrates graphical representation depicting percentage reduction in antimicrobial activity of pathogens by time kill assay in the herbal composition;

[020] Figure 2 illustrates graphical representation depicting neuroprotective potential of the herbal composition; and

[021] Figure 3 illustrates graphical representation depicting mood elevating/stress relieving potential of the herbal composition.

DETAILED DESCRIPTION OF THE INVENTION
[022] 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.

[023] 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.

[024] 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.

[025] 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.

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

[027] 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.

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

[029] In an embodiment, the herbal composition of the present invention comprises of a one or more essential oils, at least one surfactant and water.

[030] The essential oils are aromatic compounds that are derived from different parts of herbs via distillation. The essential oils contain bioactive compounds that promote mental state of a person by reliving stress and other mental illness such as anxiety, depression and suicidal tendencies in people. The essential oils, on inhalation, pass to lungs and other body parts and ultimately to brain. On entering the brain, the essential oils affect limbic system, which is associated with emotional and behavioural responses including, stress, anxiety blood pressure, heart rate, breathing, memory and hormonal balance, thereby promoting mental health by reducing stress and other mental illness such as anxiety, depression and suicidal tendencies in people. In an exemplary embodiment, the essential oils used in the present invention may be selected from a group consisting of, such as, but not limited to, Lavender oil, Neroli oil, Sandalwood oil, Spearmint oil and Orange oil or a combination thereof. The essential oils are present in a range of 0.1% to 25% by weight of the total composition. The essential oil composition may comprise of varying amounts of different essential oils. For instance, Lavender oil is present in 0.3% to 5% by total weight of the essential oils, Neroli oil is present in a range of 0.5% to 5% by total weight of the essential oils, Sandalwood oil is present in a range of 0.1% to 5% by total weight of the essential oils, Spearmint oil is present in a range of 0.1% to 5% by total weight of the essential oils, and Orange oil is present in a range of 0.1% to 5% by total weight of the essential oils. The bioactive compounds present in the essential oils include such as, but not limited to Menthol in Spearmint, Alpha-santalol in Sandalwood oil, and Linalool in Lavender oil. In an exemplary embodiment, the bioactive compounds in the essential oils are present in a range of 1.5% to 94% by total weight of the essential oils. The essential oils possess wide range of properties that aids in reducing stress and other mental illness such as anxiety, depression and suicidal tendencies in people as described herein:

(a) Lavender oil- Lavender oil exhibits anxiolytic property that aids in relieving anxiety and induces calmness in people dealing with stress and anxiety. It also aids in relieving preoperative stress due to the presence of a bioactive compound such as Linalool in Lavender oil. Lavender oil also exhibits sedative effect and treats sleeplessness caused by stress and other mental illness.

(b) Neroli oil - Neroli oil exhibits anxiolytic properties that aids in reducing anxiety. Neroli oil also exhibits an ability to elevate the level of serotonin, thereby reducing anxiety and stress. Neroli oil, on inhalation, alleviates blood pressure by reducing cortisol level in the body. Neroli oil also aids in reducing depression, resulting from stress and anxiety.

(c) Sandalwood oil- Sandalwood oil exhibits anxiolytic properties that aids in reducing anxiety. An organic compound, alpha-santalol, present in sandalwood oil stimulates mood and improve attentiveness. Sandalwood oil also aids in reducing stress, improving sleep, as well as inducing calmness.

(d) Spearmint Oil- An organic compound, menthol, present in leaves of Spearmint, exhibits sedative effect on the body by inducing calmness and relaxing effect. Spearmint interacts with the receptors present in brain such as GABA receptor, which is a neurotransmitter and involved in alleviating nerve activity, thereby reducing stress, anxiety and inducing calmness and relaxation.

(e) Orange oil- Orange oil exhibits anxiolytic properties that aids in reducing anxiety and induces relaxing effect on the body. A bright and soothing aroma of the orange oil enhances the sensing power and also induces calmness, thereby providing a balanced state of mind.

[031] The surfactant helps in reducing oil-water interfacial tension, thereby stabilizing oil in water emulsions. The surfactant used in the present invention may be selected from a group consisting of, such as, but not limited to, emulsifier, solubilizers, or a combination thereof. The surfactants are present in a range of 0.1% to 10% by weight of the total composition. The emulsifier functions by forming physical barriers that prevents coalescing of droplets. The emulsifiers possess a hydrophilic/polar head group and a hydrophobic/nonpolar tail. The emulsifier works by surrounding the droplet of oils with the hydrophobic/nonpolar tails extending into the oil and the hydrophilic/polar head groups facing the water. In an exemplary embodiment, the emulsifiers may be selected from a group consisting of, such as, but not limited to, Sesamuls o/w, or Polysorbate 80, or a combination thereof. In another exemplary embodiment, the emulsifiers are present in a range of 0.1% to 10% by total weight of the surfactants. In yet another exemplary embodiment, the amount of each emulsifier may vary such as Sesamuls o/w is present in a range of 0.1% to 4% by total weight of the emulsifiers, Polysorbate 80 is present in a range of 0.1% to 10% by total weight of the emulsifiers. In another exemplary embodiment, the emulsifiers are used in different forms such as, but not limited to, semi-solid form. The solubilizers help in solubilizing essential oil in water. In another exemplary embodiment, the solubilizers may be selected from a group consisting of, such as, but not limited to, Propylene Glycol, PEG 400, or Transcutol P, or a combination thereof. In an exemplary embodiment, the solubilizers are present in a range of 0.1% to 25% by total weight of the surfactants. In another exemplary embodiment, the amount of each solubilizer used may vary such as Propylene glycol is present in a range of 0.2% to 10% by total weight of the solubilizers, PEG 400 is present in a range of 0.2% to 10% by total weight of the solubilizers, and Transcutol P is present in a range of 0.1% to 5% by total weight of the solubilizers. The solubilizers are used in different forms such as but not limited to liquid form.

[032] Water is present in a range of 50% to 80% by weight of the total composition.

[033] In an exemplary embodiment, Table 1 illustrates different compounds of the composition and quantity (ml and %) of each compound and bioactive compounds present in several compounds.

Sr. No. Components Quantity (ml) Quantity (in
%) Bioactive Compounds
1. Neroli oil 1.5 0.5-5 linalool -29.00%
2. Spearmint oil 1 0.1-5 L- Limonene 18 %
3. Sandalwood oil 1.5 0.1-5 Alpha-Santalol –
1.5 -2.0%
4. Lavender oil 2.5 0.3-5 Linalool 26%
5. Orange oil 1.5 0.1-5 Limonene 94.00%
6. Propylene Glycol 5 0.2-10 -
7. PEG 400 5 0.2-10 -
8. Sesamuls O/W (Cetyl alcohol+
sesame Oil+Mallic acid) 0.5 0.1-4 -
9. Polysorbate 80 8.5 0.1-10 -
10. Transcutol P
(Diethylene Glycol Monoethyl Ether) 3 0.1-5 -
11. Water 70 50-80 -
12. Total 100
Table 1
[034] In an embodiment, the present invention also provides a method for preparing the herbal composition for treating respiratory tract infections. The method involves formation of oil-in-water (o/w) emulsion wherein essential oil forms a dispersed phase and water forms continuous phase. Microemulsion technique is employed for the preparation of herbal composition. Microemulsion is an optically transparent colloidal system involving the dispersion of tiny drops of one liquid that is immiscible in another. The method discloses the following steps:
(a) adding essential oils in an apparatus (A) and heating the apparatus to a temperature ranging between 30? to 40?;
(b) adding surfactants in water contained in an apparatus (B);
(c) mixing the solution obtained in step (b);
(d) adding contents of the apparatus (A) into the apparatus (B); and
(e) homogenizing the solution obtained at step (d) using a homogenizer for 15 to 20 minutes, forming a uniform herbal solution.
[035] The essential oils added in step (a) may be selected from a group consisting of, such as, but not limited to Lavender oil, Neroli oil, Sandalwood oil, Spearmint oil and Orange oil or a combination thereof. The essential oils are present in a range of 0.1% to 25% by weight of the total herbal composition. The essential oil composition may comprise of varying amounts of different essential oils. For instance, Lavender oil is present in 0.3% to 5% by total weight of the essential oils, Neroli oil is present in a range of 0.5% to 5% by total weight of the essential oils, Sandalwood oil is present in a range of 0.1% to 5% by total weight of the essential oils, Spearmint oil is present in a range of 0.1% to 5% by total weight of the essential oils, and Orange oil is present in a range of 0.1% to 5% by total weight of the essential oils.

[036] The surfactant added in step (b) may be selected from a group consisting of, such as, but not limited to, emulsifier, solubilizers, or a combination thereof. In an exemplary embodiment, the emulsifiers may be selected from a group consisting of, such as, but not limited to, Sesamuls o/w, or Polysorbate 80, or a combination thereof. In another exemplary embodiment, the emulsifiers are present in a range of 0.1% to 10% by total weight of the surfactants. In yet another exemplary embodiment, the amount of each emulsifier may vary such as Sesamuls o/w is present in a range of 0.1% to 4% by total weight of the emulsifiers, Polysorbate 80 is present in a range of 0.1% to 10% by total weight of the emulsifiers in the present invention is selected from a group consisting of, such as, but not limited to, emulsifier, solubilizers, or a combination thereof. The surfactants are present in a range of 0.1% to 10% by weight of the total composition. . In another exemplary embodiment, the emulsifiers are used in different forms such as, but not limited to, semi-solid form. In another exemplary embodiment, the solubilizers may be selected from a group consisting of, such as, but not limited to, Propylene Glycol, PEG 400, or Transcutol P, or a combination thereof. In an exemplary embodiment, the solubilizers are present in a range of 0.1% to 25% by total weight of the surfactants. In another exemplary embodiment, the amount of each solubilizer used may vary such as Propylene glycol is present in a range of 0.2% to 10% by total weight of the solubilizers, Propylene glycol is present in a range of 0.2% to 10% by total weight of the solubilizers, and Transcutol P is present in a range of 0.1% to 5% by total weight of the solubilizers. The solubilizers are used in different forms such as but not limited to liquid form.

[037] Water is present in a range of 50% to 80% by weight of the total composition.

[038] In an embodiment, the herbal composition prepared in the present invention may be used in form of a spray for spraying the herbal composition on inner/outer surface of a mask, fabric, room, car or any open space; or for the purpose of steam inhalation by adding few drops of the herbal composition in a steamer, so as to provide relief from stress and other mental illness such as anxiety, depression leading to suicidal tendencies in people.

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

EXPERIMENTAL DATA

1. General Parameters
[040] The general parameters of the herbal composition prepared in the present invention were evaluated such as color and odor. The herbal composition perceived to be white in color and with a characteristic odor.

2. Weight per milliliter

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

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

4. Refractive Index (as per API - I Vol VI, Appendix 3, 3.1.1)
[043] The refractive index of the herbal composition prepared in the present invention was evaluated. The refractive index was determined at 25? (±0.5) with reference to the wavelength of D line of sodium (? 589.3 nm). The temperature was carefully adjusted and maintained to avoid any variation in the refractive index since refractive index significantly varies with the temperature.

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

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

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

7. Viscosity
[046] The viscosity of the herbal composition prepared in the present invention was evaluated. The sample liquid to be examined was filled in a U tube viscometer in accordance with the expected viscosity of the liquid so that the level of fluid was within 0.2 mm of the filling mark of the viscometer when the capillary was vertical and specified specific temperature was attained by the liquid sample. The liquid sample was pipetted/blown to the specified weight of the viscometer and the time taken for the meniscus to pass the two specified marks was measured. The kinematic viscosity (in centistokes) was determined using the following equation:

Kinematic viscosity = kt

Wherein,
k = constant of the viscometer tube determined by observing liquids of known kinematic viscosity; and
t = time for meniscus to pass through the two specified marks (seconds).

8. Microbial Contamination (as per API, Part-I, Vol.-IX (Extracts); Appendices)
[047] The microbial contamination of the herbal composition prepared in the present invention was evaluated. The number of viable aerobic microorganisms present in the test sample and the presence of specific microbial species in the test sample was estimated by the following tests:

[048] 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 samples prevent the inhibition of multiplication under the test conditions of microorganisms that may be present. The inoculation was performed by adding 1 ml of 24 hours broth culture containing at least 1000 microorganisms to the first dilution (in buffer solution with pH 7.2 using fluid soyabean- casein digest medium or fluid lactose medium) of the test samples and following the test procedure. In case, the organisms fail to grow in the relevant medium then the volume of the diluent was increased with the quantity of the test sample remaining the same or a sufficient amount of a desired inactivating agent in the diluents was incorporated or a combination thereof to permit the growth of the organisms in the medium. In case, the inhibitory substances were present in the test samples then 0.5% of soya lecithin and 4% of polysorbate 20 was added to the culture medium. Alternatively, the same procedure was repeated using fluid casein digest soya lecithin-polysorbate 20 medium for demonstrating neutralization of preservatives or other antimicrobial agents in the test sample. In case, the inhibitory substances were present in the test samples and the test samples were soluble then membrane filtration method was used. The failure to isolate the inoculated organism even after the incorporation of a desired inactivating agents and/or substantial increase in the volume of diluent and/or the sample was not suitable for applying the membrane filtration method, may be due to the bactericidal activity of the sample. Such failure indicates a probability that the sample may not be contaminated with the given species of microorganisms. The monitoring of the sample was resumed to establish a spectrum of inhibition and bactericidal activity of the sample.

Culture Media (as per API, Part-I, Vol.-IX (Extracts); Appendices)
[049] Different culture mediums were prepared for evaluating microbial contamination or a dehydrated culture media was used such as that the medium exhibits similar ingredients when reconstituted as directed by the manufacturer and/or yield media comparable to the media obtained from the formula given below to prepare different mediums. Agar with moisture content within 15% was used for the preparation of the culture medium, if required. Purified water was also used in the culture medium, if required. The medium was sterilized by heating in an autoclave at 15 psi and at 121? for 15 minutes. The preparation of culture medium by using the formula given below involves dissolving soluble solids in water by using heat, if required. A required amount of solutions of 0.1N hydrochloric acid or 0.1N sodium hydroxide was added to yield the required pH in the medium before the use. The pH of the medium was determined at 25° ± 2°. The procedure for preparing different culture mediums is described herein:

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

[051] Bismuth Sulphite Agar Medium was prepared by forming two solutions. Solution 1 was prepared by mixing 6.0 g of Beef extract, 10.0 g of Peptone, 24.0 g of Agar, 0.4 g of Ferric citrate, and 10.0 mg of Brilliant green. Water was added to make the volume of the mixture to 1000 ml. The solution was mixed with the help of heat and sterilized by maintaining at 115? for 30 minutes. Solution 2 was prepared by mixing 3.0 g of Ammonium bismuth citrate, 10.0 g of Sodium sulphite, 5.0 g of anhydrous disodium hydrogen phosphate, and 5.0 g of Dextrose monohydrate. Water was added to make the volume of the mixture to 100 ml. The solution was mixed and heated to boiling. The solution was then allowed to cool at room temperature. 1 part of solution 2 was added to 10 parts of solution 1 previously melted and cooled to a temperature of 55? followed by pouring the prepared solution into plates. The Bismuth Sulphite Agar Medium was stored at 2? to 8? for 5 days before use.

[052] Brilliant Green Agar Medium was prepared by mixing 10.0 g of Peptone, 3.0 g of Yeast extract, 10.0 g of Lactose, 10.0 g of Sucrose, 5.0 g of Sodium chloride, 80.0 g of Phenol red, 12.5 mg of Brilliant green, 12.0 g of Agar, and 5.0 g of Sodium chloride. Water was added to make the volume of the mixture to 1000 ml. The solution was mixed and allowed to stand for 15 minutes. The solution was then sterilized by maintaining at 115? for 30 minutes followed by mixing the solution. The prepared solution was then poured into plates.

[053] Buffered Sodium Chloride - Peptone Solution pH 7.0 was prepared by mixing 3.56 g of Potassium dihydrogen phosphate, 7.23 g of Disodium hydrogen phosphate, 4.30 g of Sodium chloride, and 1.0 g of Peptone (meat or casein). Water was added to make the volume of the mixture to 1000 ml. 0.1% to 1.0% weight by volume of Polysorbate 20 or Polysorbate 80 was added and sterilized by heating the prepared solution in an autoclave at 121? for 15 minutes. 0.1% to 1.0% weight by volume of Polysorbate 20 or polysorbate 80 was added to the solution. The solution was sterilized by heating the solution in an autoclave at 121? for 15 minutes followed by pouring the solution into the plates.

[054] Casein Soyabean Digest Agar Medium was prepared by mixing 15.0 g of Pancreatic digest of casein, 5.0 g of Papaic digest of soyabean meal, 5.0 g of Sodium chloride; and 15.0 g of Agar. The water was added to the mixture to make the volume of the solution to 1000 ml. The prepared solution was then poured into the plates. The pH of the medium was adjusted to 7.3 ± 0.2 after sterilization.

[055] Cetrimide Agar Medium was prepared by mixing 20.0 g of Pancreatic digest of gelatin, 1.4 g of Magnesium chloride, 10.0 g of Potassium sulphate, 0.3 g of Cetrimide, 13.6 g of Agar, and 10.0 g of Glycerin. The water was added to make the volume to the mixture to 1000 ml. The solution was then heated by boiling the prepared solution for 1 minute along with shaking the solution. The prepared solution was pourned into the plates. The pH of the medium was adjusted to 7.0 to 7.4 after sterilization.

[056] Desoxycholate-Citrate Agar Medium was prepared by mixing 5.0 g of Beef extract, 5.0 g of Peptone, 10.0 g of Lactose, 8.5 g of Trisodium citrate, 5.4 g of Sodium thiosulphate, 1.0 g of Ferric citrate, 5.0 g of Sodium desoxycholate, 0.02 g of Neutral red, and 12.0 g of Agar. The water was added to make the volume of the mixture to 1000 ml. The solution was mixed and allowed to stand for 15 minutes followed by gently boiling the solution with continuous stirring until solution was completely homogenized. The solution was then cooled to 80? and followed by mixing the solution. The prepared solution was poured into plates. The surface of the plates was dried before use. Overheating of Desoxycholate Citrate Agar was avoided during the preparation of the medium. Re-melting of the solution was also avoided.

[057] Fluid Casein Digest - Soya Lecithin - Polysorbate 20 Medium was prepared by mixing 20.0 g of Pancreatic digest of casein, 5.0 g of Soya lecithin, and 40.0 ml of Polysorbate 20. The water was added to make the volume of the mixture to 1000 ml. The pancreatic digest of casein and soya lecithin was dissolved in water followed by heating the solution in a water-bath at 48? to 50? for about 30 minutes. Polysorbate 20 was added to the solution followed by mixing the prepared solution. The solution was then dispensed into the plates.

[058] Fluid Lactose Medium was prepared by mixing 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 prepared solution was then poured into the plates. The pH of the medium was adjusted to 6.9 ± 0.2 after sterilization.

[059] Lactose Broth Medium was prepared by mixing 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 to 1000 ml. The prepared solution was then poured into the plates. The pH of the medium was adjusted to 6.9 ± 0.2 after sterilization.

[060] Levine Eosin - Methylene Blue Agar Medium was prepared by mixing 10.0 g of Pancreatic digest of gelatin, 2.0 g of dibasic potassium phosphate, 15.0 g of Agar, 10.0 g of Lactose, 400 mg of Eosin Y, and 65.0 mg of Methylene blue. Water was added to make the volume of the mixture to 1000 ml. The pancreatic digest of gelatin, dibasic potassium phosphate and agar was dissolved in water by heating the solution. The solution was then allowed to cool. The gelled agar solution and the remaining ingredients of the solutions were liquefied in a required amount before use followed by mixing the solution. 5 ml of 20% weight by volume of solution of lactose, 2 ml of a 2% weight by volume of solution of Eosin Y and 2 ml of a 0.33% weight by volume of solution of methylene blue was used for each 100 ml of the liquified agar solution. The prepared medium may not appear to be clear. The prepared solution was then poured into the plates. The pH of the medium was adjusted to 7.1±0.2 after sterilization.

[061] MacConkey Agar Medium was prepared by mixing 17.0 g of Pancreatic digest of gelatin, 3.0 g of Peptone (meat and casein, equal parts), 10.0 g of Lactose, 5.0 g of Sodium chloride, 1.5 g of Bile salts, 13.5 g of Agar, 30.0 mg of Neutral red, and 1.0 mg of Crystal violet. Water was added to make the final volume of mixture to 1000 ml. The mixture of solids and water was boiled for 1 minute. The prepared solution was then poured into the plates. The pH of the medium was adjusted to 7.1 ± 0.2 after sterilization.

[062] MacConkey Broth Medium was prepared by mixing 20.0 g of Pancreatic digest of gelatin, 10.0 g of Lactose, 5.0 g of dehydrated ox bile, and 10.0 mg of Bromocresol purple. Water was added to make the volume of the mixture to 1000 ml. The prepared solution was then poured into the plates. The pH of the medium was adjusted to 7.3 ± 0.2 after sterilization.

[063] Mannitol-Salt Agar Medium was prepared by mixing 5.0 g of Pancreatic digest of gelatin, 5.0 g of Peptic digest of animal tissue, 1.0 g of Beef extract, 10.0 g of D-Mannitol, 75.0 g of Sodium chloride, 15.0 g of Agar, and 25 mg of Phenol red. Water was added to make the volume of the mixture to 1000 ml. The solution was mixed by heating the solution to boiling for 1 minute along with stirring the solution. The prepared solution was then poured into the plates. The pH of the medium was adjusted to 7.4 ± 0.2 after sterilization.

[064] Nutrient Agar Medium was prepared by adding 1% to 2% weight by volume of agar into nutrient broth. Nutrient Broth Medium was prepared by mixing 10.0 g of Beef extract, 10.0 g of Peptone, and 5.0 mg of Sodium chloride. Water was added to make the volume of mixture to 1000 ml. The solution was dissolved with the help of heat. The pH of the solution was adjusted to 8.0 to 8.4 with 5M sodium hydroxide followed by boiling the solution for 10 minutes. The solution was filtered and sterilized by maintaining at 115? for 30 minutes. The prepared solution was then poured into the plates. The pH of the medium was adjusted to 7.3 ± 0.1 after sterilization.

[065] Pseudomonas Agar Medium for Detection of Flourescein was prepared by mixing 10.0 g of Pancreatic digest of casein, 10.0 g of Peptic digest of animal tissue, 1.5 g of anhydrous dibasic potassium phosphate, 1.5 g of Magnesium sulphate hepta hydrate, 10.0 ml of Glycerin, and 15.0 g of Agar. Water was added to make the volume of the mixture to 1000 ml. The solid components were dissolved in water before adding glycerin. The solution was mixed by heating the solution to boiling for 1 minute. The prepared solution was then poured into the plates. The pH of the medium was adjusted to 7.2 ± 0.2 after sterilization.

[066] Pseudomonas Agar Medium for Detection of Pyocyanin was prepared by mixing 20.0 g of Pancreatic digest of gelatin, 1.4 g of anhydrous magnesium chloride, 10.0 g of anhydrous potassium sulphate, 15.0 g of Agar, and 10.0 ml of Glycerin. The water was added to make the volume of the mixture to 1000 ml. The solid components were dissolved in water before adding glycerin. The solution was mixed by heating the solution to boiling for 1 minute. The prepared solution was then poured into the plates. The pH of the medium was adjusted to 7.2 ± 0.2 after sterilization.

[067] Sabouraud Dextrose Agar Medium was prepared by mixing 40.0 g of Dextrose, 10.0 g of Peptic digest of animal tissue and pancreatic digest of casein (1:1), and 15.0 g of Agar. Water was added to make the volume of the mixture to 1000 ml. The solution was then mixed by boiling the solution. The prepared solution was then poured into the plates. The pH of the medium was adjusted to 5.6 ± 0.2 after sterilization.

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

[069] Selenite F Broth was prepared by mixing 5.0 g of Peptone, 4.0 g of Lactose, 10.0 g of Disodium hydrogen phosphate, and 4.0 g of Sodium hydrogen selenite. Water was added to make the volume of the mixture to 1000 ml. The mixture was dissolved and distributed in sterile containers. The solution was then sterilized by maintaining at 100? for 30 minutes.

[070] Fluid Selenite - Cystine Medium was prepared by mixing 5.0 g of Pancreatic digest of casein, 4.0 g of Lactose, 10.0 g of Sodium phosphate, 4.0 g of Sodium hydrogen selenite, and 10.0 mg of l-Cystine. Water was added to make the volume of the mixture to 1000 ml. The solution was mixed and heated in a flowing steam for 15 minutes. The prepared solution was then poured into the plates. The final pH of the medium was adjusted to 7.0 ± 0.2. Sterilization step was avoided for the preparation of Fluid Selenite - Cystine Medium.

[071] Tetrathionate Broth Medium was prepared by mixing 0.9 g of Beef extract, 4.5 g of Peptone, 1.8 g of Yeast extract, 4.5 g of Sodium chloride, 25.0 g of Calcium carbonate, and 40.7 g of Sodium thiosulphate. Water was added to make the volume of the mixture to 1000 ml. The solid components were dissolved in water followed by heating the solution to boiling. A solution prepared by dissolving 5 g of potassium iodide and 6 g of iodine in 20 ml of water was added to the prepared solution before using the prepared solution. The prepared solution was then poured into the plates.

[072] Tetrathionate-Bile-Brilliant Green Broth Medium was prepared by mixing 8.6 g of Peptone, 8.0 g of dehydrated ox bile, 6.4 g of Sodium chloride, 20.0 g of Calcium carbonate, 20.0 g of Potassium tetrathionate, and 70.0 mg of Brilliant green. Water was added to make the volume to the mixture to 1000 ml. The solution was heated to boiling. Reheating of the solution was avoided. The prepared solution was then poured into the plates. The pH was adjusted to achieve a pH of 7.0 ± 0.2 after heating.

[073] Triple Sugar - Iron Agar Medium was prepared by mixing 3.0 g of Beef extract, 3.0 g of Yeast extract, 20.0 g of Peptone, 10.0 g of Lactose, 10.0 g of Sucrose, 1.0 g of Dextrose monohydrate, 0.2 g of Ferrous sulphate, 5.0 g of Sodium chloride, 0.3 g of Sodium thiosulphate, and 24.0 mg of Phenol red. Water was added to make the volume of the mixture to 1000 ml. The solution was mixed and allowed to stand for 15 minutes. The solution was then boiled and the boiling point of the solution was maintained until solution turned out to be completely homogenous. The solution was then thoroughly mixed and distributed in tubes followed by sterilization by maintaining at 121? for 15 minutes. The solution was allowed to stand in a sloped form with approximately 2.5 cm long bottom.

[074] Urea Broth Medium was prepared by mixing 9.1 g of Potassium dihydrogen orthophosphate, 9.5 g of anhydrous disodium hydrogen phosphate, 20.0 g of Urea, 0.1 g of Yeast extract, and 10.0 mg of Phenol red. The water was added to make the volume of the mixture to 1000 ml. The solution was then mixed and sterilized followed by distributing the solution aseptically in sterile petri plates.

[075] Vogel-Johnson Agar Medium was prepared by mixing 10.0 g of Pancreatic digest of casein, 5.0 g of Yeast extract, 10.0 g of Mannitol, 5.0 g of dibasic potassium phosphate, 5.0 g of Lithium chloride, 10.0 g of Glycerin, 16.0 g of Agar, and 25.0 mg of Phenol red. Water was added to make the volume of the mixture to 1000 ml. The solution of solids was boiled for 1 minute. The solution was then sterilized and cooled to a temperature in a range of 45?-50?. 20 ml of 1% weight by volume of sterile solution of potassium tellurite was added to the solution. The prepared solution was then poured into the plates. The pH of the medium was adjusted to 7.0 ± 0.2 after sterilization.

[076] Xylose -Lysine-Desoxycholate Agar Medium was prepared by mixing 3.5 g of Xylose, 5.0 g of l-Lysine, 7.5 g of Lactose, 7.5 g of Sucrose, 5.0 g of Sodium chloride, 3.0 g of Yeast extract, 80.0 mg of Phenol red, 13.5 g of Agar, 2.5 g of Sodium desoxycholate, 6.8 g of Sodium thiosulphate, and 800 mg of Ferric ammonium citrate. Water was added to make the volume of the mixture to 1000 ml. The mixture of solids and water was heated to boiling. Overheating and sterilization of the solution was avoided. The solution was heated to a water-bath maintained at about 50? and poured into plates just after the medium was cooled. The final pH of the medium was adjusted to 7.4 ± 0.2.

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

[078] Precautions taken during experimentation involves performing the microbial limit tests (as per As per API Part-I, Vol. IX, Appendix 3, 3.2.) under specific conditions designed to prevent accidental contamination of the medium during the test. The precautions taken to prevent contamination during the test exhibit no adverse effect on detection of the microorganisms during the tests (if present).

[079] Total Aerobic Microbial Count (as per API Part-I, Vol. IX, Appendix 3, 3.2.1): 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 detail:

[080] Water-soluble sample: 10 g or 10 ml of the sample to be examined was dissolved/diluted in a buffered sodium chloride-peptone solution pH 7.0 or other suitable medium which exhibits no antimicrobial activity under the test conditions. The solution was diluted to make the volume of the solution to 100 ml with the same medium. The pH of the medium was adjusted near7, if required.

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

[082] Water insoluble sample (Fatty): 10 g or 10 ml of the sample to be examined was homogenized with 5 g of polysorbate 20 or polysorbate 80. The sample was heated to a temperature within 40?. The solution was mixed while maintaining the temperature in the water-bath or in an oven. 85 ml of buffered sodium chloride-peptone solution pH 7.0 or other suitable medium that exhibits no antimicrobial activity under test condition, heated to a temperature within 40? was added to the prepared solution, if required. The temperature was maintained for the time period (within 30 minutes) required for the formation of an emulsion. The pH of the solution was adjusted near 7, if required.

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

[084] (i) Membrane filtration: Membrane filters with 50 mm diameter and nominal pore size within 0.45 mm was used for retaining bacteria. 10 ml or a required amount of each dilution containing 1g of the sample to be examined wad added to each of 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.

[085] In case of fatty sample, liquid polysorbate 20 or polysorbate 80 was used. One of the two membrane filters intended for the enumeration of bacteria was transferred to the surface of a plate of casein soyabean digest agar. The other membrane filter intended for the enumeration of fungi was transferred to the surface of a plate of Sabouraud dextrose agar with antibiotics. The plates were incubated for 5 days unless a reliable count was obtained in less time period such as 300 to 350 in test for bacteria and 200 to 250 in the test for fungi. The number of colonies formed were recorded. The number of microorganisms per g or per ml of the sample to be examined was determined separately for bacteria and fungi, if required.

[086] Plate count for bacteria: Petri dishes with diameter of 9 cm 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 within 45? was added to each petri dish. Alternatively, the pretreated sample preparation was spread on the surface of the solidified medium in a petri dish of the same diameter. The aforementioned pretreated sample preparation was diluted, if required to obtain colony count within 30°. At least two such petri dishes were prepared using the same dilution and incubated at 30? to 35? for 5 days until a reliable count was obtained in less time period. The number of colonies formed were recorded.

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

[088] Plate count for fungi: The method involves similar steps as performed for evaluating plate count for bacteria except the use of Sabouraud dextrose agar with antibiotics instead of casein soyabean digest agar. The petri dishes were incubated at 20? to 25? for 5 days until a reliable count was obtained in less time period. The results were evaluated by colony count method using plates with colonies within 100.

[089] 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 tubes each. One set of three tubes were taken as controls. 1 ml of the solution of suspension of the test sample was pipetted into each of three tubes of one set (“100”) and into 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 µl) and 10 mg (or 10 µl) of the specimen respectively. 1 ml from tube A was pipetted into each tube of the second set (“10”) of three tubes pipette. 1 ml from tube B was pipetted into each tube of the third set (“1”). The remaining contents of tube A and B were discarded. All the tubes were properly closed and incubated. The tubes were examined for growth after the incubation period. The three control tubes were reported to be clear. The tubes containing the test sample was observed to determine probable number of microorganisms per g or per ml of the test sample.

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

Table 2

Tests for Specified Microorganisms (as per API Part-I, Vol. IX, Appendix 3, 3.2.2):

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

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

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

[094] Secondary test: 0.1 ml of the enrichment culture was added to a tube (A) containing 5 ml of MacConkey broth and in tube (B) containing 5 ml of peptone water. Both the tubes were incubated in a water-bath at 43.5? - 44.5? for 24 hours. Tube (A) was examined for acid and gas. Tube (b) was examined for indole. To examine Tube (B) for Indole, 0.5 ml of Kovac’s reagent was added to the tube (B) and content of tube (B) were mixed by shaking the tube. The tube was allowed to stand for 1 minute. The presence of Indole group was indicated by the formation of red colour reagent layer. The presence of acid and gas and indole in the secondary test indicated the presence of Escherichia coli. A control test was performed by repeating the primary and secondary tests. 1.0 ml of the enrichment culture and a suitable volume of broth containing 10 to 50 Escherichia coli (NCTC 9002) organisms prepared from a 24-hour culture in nutrient broth was added to 5 ml of MacConkey broth. The test was considered effective only when the results indicated that the control contains Escherichia coli.

[095] Alternative test: A portion from the enrichment culture obtained in the previous test was streaked on the surface of MacConkey Agar medium by means of an inoculating loop. The dishes were covered and incubated. In case, during examination, none of the colonies were brick-red in colour and showed surrounding zone of precipitated bile then the sample was considered to fulfil the requirements of the test for the absence of Escherichia coli. In case, the colonies as mentioned above were present then the suspected colonies were individually transferred to the surface of Levine eosin - methylene blue agar medium plated on petri dishes. The plates were covered and inverted followed by incubation. .In case, during examination, none of the colonies exhibited a characteristic metallic sheen under reflected light and a blue-black appearance under transmitted light then the sample was considered to fulfil the requirements of the test for the absence of Escherichia coli. The presence of Escherichia coli was further confirmed by suitable cultural and biochemical tests.

[096] 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? for 24 hours.

[097] Primary test: 1.0 ml of the enrichment culture was added to a tube (A) containing 10 ml of selenite F broth and tube (B) containing tetrathionate-bile brilliant green broth. The tubes were incubated at 36?-38? for 48 hours. A subculture was prepared from each of the two prepared cultures on at least two of the agar media including bismuth sulphate agar, brilliant green agar, deoxycholate citrate agar and xylose-lysine deoxycholate agar. The plates were incubated at 36?- 38? for 18 to 24 hours. In case, during examination, none of the colonies conforms to the description provided in Table 2 then the sample was considered to fulfil the requirements of the test indicating the absence of the genus Salmonella abony. Secondary test was performed in case colonies conforming to the description in Table 2 were formed.

[098] Secondary test: The colonies showing the characteristics given in Table 2 were subcultured in a Triple Sugar - Iron Agar medium by inoculating the surface of the slope followed by making a stab culture with the same inoculating needle. Simultaneously, a tube of urea broth was also inoculated. Incubation was performed at 36? to 38? for 18 to 24 hours. The presence of Salmonella abony was indicated by the formation of acid and gas in the stab culture with/without concomitant blackening and the absence of acidity from the surface growth in the Triple Sugar - Iron Agar medium along with the absence of red colour in urea broth. However, in case, only acid is produced without the production of gas in the cultures, then the presence of Salmonella 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 (NCTC 6017) prepared from a 24-hour culture in nutrient broth for inoculating tube (A) and tube (B). The test was considered effective only when the results indicated that the control contains Salmonella abony.

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

[100] The medium was examined for the growth of Pseudomonas aeruginosa In case, growth was indicated in the medium then a portion of the medium was streaked on the surface of cetrimide agar medium, each plated on petri dishes. The petri dishes were covered and incubated at 35? to 37? for 18 to 24 hours. In case, during examination, none of the plates showed colonies exhibiting the characteristics listed in Table 2 for the media used, then the sample was considered to fulfil the requirement for the absence of Pseudomonas aeruginosa. In case, any colonies conforming to the description as provided in Table _2_ were formed then oxidase and pigment tests were performed. The suspected colonies from the agar surface of cetrimide agar were streaked on the surface of Pseudomonas agar medium for detection of fluorescein and pyocyanin contained in petri dishes. The inoculated media was covered and inverted followed by incubating the media at 33? to 37? for at least 3 days. The streaked surfaces were examined under ultra-violet light. The plates were examined to determine the presence of colonies conforming to the description in Table 2, if any. In case, the growth of suspected colonies occurred on the plates then 2 or 3 drops of a freshly prepared 1% weight by volume of N,N,N´,N´-tetramethyl-4-phenylenediamine dihydrochloride solution was put on filter paper and smeared with the colony. In case of absence of the development of pink colour turning into purple, the sample was considered to fulfil the requirements of the test indicating the absence of Pseudomonas aeruginosa.

[101] Test for Staphylococcus aureus: The test for Staphylococcus aureus involves method similar to the method for testing the presence of Pseudomonas aeruginosa. In case, during examination of incubated plates, none of the plates showed colonies exhibiting characteristics listed in Table 2 for the media used, the sample was considered to fulfil the requirements for the absence of Staphylococcus aureus. In case, the growth was detected, then coagulase test was performed. The suspected colonies were transferred from the agar surface of the media listed in Table 2 to individual tubes, each containing 0.5 ml of mammalian, preferably rabbit or horse, plasma with/without additives. The tubes were incubated in the water-bath at 37?. The tubes were examined for 3 hours and subsequently at a suitable intervals up to 24 hours. The presence of any level of coagulation indicates that the sample fulfilled the requirements of the test indicating the absence of Staphylococcus aureus.

Validity of the tests for total aerobic microbial count (as per API, Part-I, Vol.-IX (Extracts); Appendices): :

[102] A number of test strains were grown separately in tubes containing fluid soyabean-casein digest medium at 30? to 35? for 18 to 24 hours. However, in case of Candida albicans, the test strains were grown separately in tubes containing fluid soyabean-casein digest medium at 20? for 48 hours. The test strains include Staphylococcus aureus, Bacillus subtilis, Escherichia coli,
and Candida albicans.

[103] The portions of each of the cultures were diluted using buffered sodium chloride-peptone solution pH 7.0 to make test suspensions containing about 100 viable microorganisms per ml. The suspension of each of the microorganisms was used separately as a control of the counting methods in the presence and absence of the preparation to be examined, if required. A count for any test organisms differing by a factor within 10 from the calculated value for the inoculum was required. The sterility of the medium, diluent and the aseptic performance of the test was tested by performing total aerobic microbial count method using sterile buffered sodium chloride peptone solution pH 7.0 as the test preparation. The absence of the growth of microorganism was required to confirm the validity of the tests for total aerobic microbial count.

[104] Table 3 illustrates result of test performed to evaluate microbial contamination in the herbal composition against contamination by yeast, mould, and bacteria.
S No Test Result Standard Limits
1 Total Microbial Count, cfu/g Less than 10 Not more than 100000
2 Total Yeast and Mould, count/g Less than 10 Not more than 1000
3 Escherichia coli/g Absent Should be absent
4 Pseudomonas aeruginosa /g Absent Should be absent
5 Salmonella abony/10g Absent Should be absent
6 Staphylococcus aureus/g Absent Should be absent
Table 3
Validity of the tests for specified microorganisms (as per API, Part-I, Vol.-IX (Extracts); Appendices):
[105] The test strains of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Salmonella abony were grown separately in fluid soyabean-casein digest medium at 30? to 35? for 18 to 24 hours. The portions of each of the cultures were diluted by using buffered sodium chloride-peptone solution pH 7.0 to make test suspensions containing about 103 viable microorganisms per ml. Equal volume of each suspension was mixed and 0.4 ml (approximately 102 micro-organisms of each strain) was used as an inoculum in the test for E. coli, S. typhimurium, P. aeruginosa and S. aureus, in the presence and absence of the sample preparation to be examined, if required. A positive result for the specific strain of microorganism was required to confirm the validity of the tests for specified microorganisms.

11. Heavy Metal Analysis (as per API Part-I, Vol. IX, Appendix 3, 3.1):
[106] The presence of heavy metals in the herbal composition was evaluated using different method provided herein:

[107] Determination of Lead (Pb) by Graphite Oven Method:

[108] 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.

[109] Preparation of lead (Pb) standard stock solution: A required amount of lead single-element standard solution was accurately measured for preparing standard stock solution with 2% nitric acid solution containing 1 µg per ml lead. The solution was stored at 0?-50?.

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

[111] Preparation of test solution: 0.5 g of the coarse powder of the sample to be examined was accurately weighed and transferred into a casparian flask. 5-10 ml of the mixture of nitric acid and perchloric acid (4:1) was added into the flask. A small hopper was placed on the top of the flask. The mixture was macerated overnight followed by heating the mixture by boiling the mixture to slake on an electric hot plate. A required amount of above mixture was added in the solution turned brownish- black in color followed by constantly heating the solution until a clean and transparent solution appeared. The temperature was then raised and the solution was constantly heated to thicken the smoke until white smoke dispersed. The slaked solution on turning colourless and transparent or yellowish was cooled and transferred into a 50 ml volumetric flask. The container was washed with 2% nitric acid solution and the washing solution was added into the same volumetric flask followed by diluting with the same solvent to 2% of the volume. The solution was mixed properly by shaking the flask. Simultaneously, a reagent blank solution was prepared according to the procedure mentioned above.

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

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

[113] 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.

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

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

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

[117] Determination of Cadmium: The presence of cadmium was determined by accurately measuring 10-20µl of the test solution and corresponding reagent blank solution. The absorbance of the solution was determined by a method similar to the method for preparation of the calibration curve. In case of interference, 1 ml of the standard solution, blank solution and test solution were separately weighed. 1 ml of a solution containing 1% of ammonium dihydrogen phosphate and 0.2% of magnesium nitrate was added to the prepared solutions separately. Each prepared solution was properly mixed by shaking the solutions. The absorbance of the prepared solutions was determined by following the steps mentioned above. The content of cadmium (Cd) in the test solution was determined from the calibration curve.

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

[118] 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.

[119] 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?- 50?.

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

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

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

(d) Determination of Mercury (Hg) by Cold Absorption Method:

[123] 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 per cent 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.

[124] 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?-50?.

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

[126] 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 an 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 violet red colour disappeared. The solution was then diluted with 4% sulphuric acid solutions to make the volume to 25 ml. The solution was mixed by shaking the flask. The solution was then centrifugated, if required. The supernatant obtained was used as the test solution. Similarly, the reagent blank solution was simultaneously prepared by performing the same steps.

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

[128] Table 4 illustrates experimental result of test performed to determine contamination of the herbal composition against heavy metals
S No Test Result Standard Limits
1 Mercury Below Quantification Limit 0.20-1.0ppm
2 Cadmium Below Quantification Limit 0.20-0.30ppm
3 Lead Below Quantification Limit 0.20-10.0ppm
4 Arsenic Below Quantification Limit 0.20-3.0ppm
Table 4

12. Pesticide Residue (as per API Part-I, Vol. IX, Appendix 3, 3.3)
[129] Limits: The sample to be examined is required to compile with the limits indicated in Table 5 The limits applying to the suspected pesticides not provided in Table 5may comply with the limits set by European Community directives 76/895 and 90/642, including their annexes and successive updates. The limits for suspected pesticides not provided in Table 5or in European Community were determined using the following expression:

(ADI x M)/MDD x 100
Wherein,
ADI = Acceptable Daily Intake (mg/kg of body mass), as published by FAO-WHO;
M = body mass in kilograms (60 kg); and
MDD = daily dose of the drug (Kg)

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

(ADI x M x E)/ MDD x 100
Wherein,
E =experimentally determined extraction factor of the method for preparation

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

[132] 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.

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

[134] 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.

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

[136] Apparatus: The apparatus and glasswares were thoroughly cleaned to keep them pesticide free. Cleaning procedure may involve soaking the apparatus and/or glasswares in a solution of phosphate-free detergent for at least 16 hours followed by rinsing with large quantities of distilled water and washing with acetone and hexane or heptane.

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

13. Aflatoxins (as per API Part-I, Vol. IX, Appendix 3, 3.4)
[138] The presence of different types of aflatoxins including, B2, G1 and G2 in the herbal composition prepared in the present invention was evaluated. The procedure for evaluating the presence of different type of aflatoxins are as follows:
[139] 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.
[140] Sodium Chloride Solution: 5 g of sodium chloride was dissolved in 50 ml of purified water.
[141] Test Solution 1: About 200 g of plant material was grinded to form 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 125 ml conical flask. The organic solvent was allowed to evaporate to dryness on a water bath. The residue was then cooled. The residue obtained was dissolved in 0.2 ml of a mixture of chloroform and acetonitrile (9.8:0.2) followed by shaking the mixture by mechanical means, if required.
[142] Test Solution 2: In case, the solution possesses interfering plant pigments then the following procedure was followed which involves collecting 100 ml of the filtrate from the start of the flow and transferring to a 250 ml beaker. 20 ml of Zinc Acetate-Aluminum Chloride Reagent and 80 ml of water was added to the filtrate. The solution was stirred and allowed to stand for 5 minutes. 5 g of required filtering aid, such as diatomaceous earth, mix was added into the solution followed by filtering the solution. The first 50 ml of the filtrate was discarded and the next 80 ml portion of the filtrate was collected. The filtrate was transferred to a separatory funnel. 40 ml of sodium chloride solution and 25 ml of hexane was added into the filtrate followed by shaking the solution for 1 minute. The method further involves similar steps as performed for preparing test solution 1.
[143] Cleanup Procedure: In case, interferences exist in the residue prepared in test solution 1, following procedure was followed which involves placing a medium-porosity sintered-glass disk or a glass wool plug at the bottom of a 10 mm x 300 mm chromatographic tube. A slurry of 2 g of silica gel with a mixture of ethyl ether and hexane (3: 1) was prepared and poured into a column followed by washing with 5 ml of the same solvent mixture. The absorbent was allowed to settle and added to the top of the column layer of 1.5 g of anhydrous sodium sulfate. The residue obtained was dissolved in 3 ml of methylene chloride and transferred to the column. The flask was rinsed twice with 1 ml portions of methylene chloride and the rinses were transferred to the column and eluted at a rate of 1 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 elute 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.
[144] 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.
[145] Procedure: 2.5 µl, 5 µl, 7.5 µl and 10 µl of the flatoxin 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.
[146] Table 6 illustrates experimental result of test performed to evaluate presence of aflatoxins in the herbal composition.
S No Test Result Standard Limits
1 Mercury Below Quantification Limit 0.20-1.0ppm
2 Cadmium Below Quantification Limit 0.20-0.30ppm
3 Lead Below Quantification Limit 0.20-10.0ppm
4 Arsenic Below Quantification Limit 0.20-3.0ppm
Table 6
[147] Table 7 illustrates the experimental result of different tests performed on the herbal composition prepared in the present invention.

Sr No. Test Result Standard Limits
1. General Description
1.1. Color White White
1.2. Odour Characteristic Characteristic
2. Weight per ml 0.9889 gm/ml 0.5-1.5 gm/ml
3. Specific Gravity 1.0035 1.00-1.01
4. Refractive Index 1.3699 1.0-1.5
5. pH 4.57 4.0-8.0
6. Saponification 0.93 -
7. Viscosity 8.40 cps 7-12 cps
8. Microbial Contamination
8.1. Total Microbial Count, cfu/g Less than 10 Not more than 100000
8.2. Total Yeast and Mould, count/g Less than 10 Not more than 1000
8.3. Escherichia coli/g
Absent Should be absent
8.4. Pseudomonas aeruginosa /g Absent Should be absent
8.5. Salmonella abony/10g Absent Should be absent
8.6. Staphylococcus aureus/g Absent Should be absent
9. Heavy metal Analysis
9.1. Mercury Below Quantification
Limit 0.20-1.0ppm
9.2. Cadmium Below Quantification
Limit 0.20-0.30ppm
9.3. Lead Below Quantification
Limit 0.20-10.0ppm
9.4. Arsenic Below Quantification
Limit 0.20-3.0ppm
10. Pesticide Residue
10.1. Alchalor Below Quantification
Limit 0.0050-
0.020mg/kg
10.2. Malathoin Below Quantification
Limit 0.0050-
1.0mg/kg
10.3. Phoslane Below
Quantification Limit 0.0050-
0.10mg/kg
10.4. Pyrethrins Below Quantification
Limit 0.0050-
3.0mg/kg
10.5. Ethion Below
Quantification Limit 0.0050-2mg/kg
10.6. Chlorpyrifos Below Quantification
Limit 0.0050-
0.20mg/kg
10.7. Methyl Parathion Below
Quantification Limit 0.0050-
0.20mg/kg
10.8. Lindane (Gamma - HCH) Below
Quantification Limit 0.0050-
0.60mg/kg
10.9. Aldrin and dieldrin (sum of) Below Quantification
Limit 0.0050-
0.50mg/kg
10.10. Azinophos - methyl Below Quantification
Limit 0.0050-
1.0mg/kg
10.11. Bromopropylate Below
Quantification Limit 0.0050-
3.0mg/kg
10.12. Chlorpyrifos-methyl Below
Quantification Limit 0.0050-
0.10mg/kg
10.13. Deltamethrin Below
Quantification Limit 0.0050-
0.50mg/kg
10.14. Diazinon Below Quantification
Limit 0.0050-
0.10mg/kg
10.15. Dichlorvos Below Quantification
Limit 0.0050-
1.0mg/kg
10.16. Endrin Below
Quantification Limit 0.0050-
0.050mg/kg
10.17. Fonofos Below Quantification
Limit 0.0050-
0.050mg/kg
10.18. Methidathion Below
Quantification Limit 0.0050-
0.20mg/kg
10.19. Parathion Below Quantification
Limit 0.0050-
0.50mg/kg
10.20. Permethrin Below
Quantification Limit 0.0050-
1.0mg/kg
10.21. Perimiphos-methyl Below Quantification
Limit 0.0050-
4.0mg/kg
10.22. Fentrothion Below Quantification
Limit 0.0050-
0.50mg/kg
10.23. Cypermethrin and isomers Below
Quantification Limit 0.0050-
1.0mg/kg
10.24. Hexachlorocyclohexane Below Quantification
Limit 0.0050-
0.30mg/kg
10.25. Piperonyl-butoxide Below
Quantification Limit 0.0050-
3.0mg/kg
10.26. Heptachlor Below Quantification
Limit 0.0050-
0.050mg/kg
10.27. Fenvalerate Below
Quantification Limit 0.0050-
1.50mg/kg
10.28. Endosulfan Below Quantification
Limit 0.0050-
3.0mg/kg
10.29. DDT Below Quantification
Limit 0.0050-
1.0mg/kg
10.30. Hexachlorobenzene Below Quantification
Limit 0.0050-
0.10mg/kg
10.31. Chlorfenvinphos Below
Quantification Limit 0.0050-
0.50mg/kg
10.32. Dithiocarbamates Below Quantification
Limit 0.0050-
2.0mg/kg
10.33. Chlordane Below
Quantification Limit 0.0050-
0.50mg/kg
10.34. Quintozene Below Quantification
Limit 0.0050-
0.10mg/kg
11. Aflatoxin
11.1. Total Aflatoxin Below Quantification
Limit Not more than 5mcg/Kg
11.2. Aflatoxin B1 Below Quantification
Limit 1-2mcg/Kg
11.3. Aflatoxin B2 Below Quantification
Limit 1-2mcg/Kg
11.4. Aflatoxin G2 Below Quantification
Limit 1-2mcg/Kg
11.5. Aflatoxin G1 Below Quantification
Limit 1-2mcg/Kg
Table 7
[148] 14. Antimicrobial property of the herbal composition:
[149] As shown in Figure 1, the time kill analysis monitors the effect of various concentrations of an antimicrobial agent over time in relation to the stages of the growth of the bacteria. The herbal composition showed >90% germ-kill efficacy against various pathogenic micro-organisms including Pseudomonas aeruginosa, Staphylococcus epidermis, Listeria monocytogene, Candida albicans, Aspergillus brasilensis, Escherichia coli and Salmonella abony, at intervals of 30 and 60 seconds. The herbal composition also showed over 70% reduction in Staphylococcus aureus at 60 second time interval.

[150] 15. Neuroprotective potential of the herbal composition:
[151] As shown in Figure 2, the neuroprotective potential of herbal composition was determined by evaluating its effect on restoration of cell viability against neurotoxin induced damage in neuronal cells. The herbal composition showed a maximum of 30.6% restoration of cell viability against MPP+ induced damage in neuronal cells.

[152] 16. Mood elevating/stress relieving potential of the herbal composition:
[153] As shown in Figure 3, the mood elevating/ stress relieving potential of the herbal composition was determined by evaluating its effect on release of serotonin in neuronal cells. The herbal composition showed a maximum of 31.2% increase in serotonin levels in neuronal cells as compared to untreated control cells.

[154] The herbal composition prepared in the present invention possesses a number of advantages:
• The present invention provides a safe and efficient herbal composition for reducing stress.
• The essential oils used in the present invention contains bioactive compounds that aids in relieving stress and anxiety, resulting in depression and other mental illness, thereby promoting mental health of a person.

[155] 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 reducing stress, comprising:
• one or more essential oils in a range of 0.1% to 25% by weight of the total composition;
• at least one surfactant in a range of 0.1% to 10% by weight of the total composition; and
• water in a range of 50% to 80% by weight of the total composition.

2. The herbal composition as claimed in claim 1, wherein the essential oils are selected from a group consisting of Lavender oil, Neroli oil, Sandalwood oil, Spearmint oil and Orange oil, or a combination thereof.

3. The herbal composition as claimed in claim 1, wherein the surfactant is selected from a group of emulsifier, solubilizers, or a combination thereof.

4. The herbal composition as claimed in claim 3, wherein the emulsifier is selected from a group consisting of Sesamuls o/w and Polysorbate 80, or a combination thereof.

5. The herbal composition as claimed in claim 3, wherein the solubilizer is selected from a group consisting of, Propylene Glycol, PEG 400 and Transcutol P, or a combination thereof.

6. A method for preparing herbal composition as claimed in claim 1, comprising steps of:
(a) adding essential oils in an apparatus (A) and heating the apparatus to a temperature ranging between 30? to 40?;
(b) adding surfactants in water contained in an apparatus (B);
(c) mixing the solution obtained in step (b);
(d) adding contents of the apparatus (A) into the apparatus (B); and
(e) homogenizing the solution obtained at step (d) using a homogenizer for 15 to 20 minutes, forming a uniform herbal solution.