DESC:FIELD OF THE INVENTION
[0001] The present disclosure relates generally to the field of spice compositions. Particularly, the present disclosure relates to a composition for improving pulmonary and neurological conditions in a subject. The present disclosure further relates to a method of preparation of the composition.

BACKGROUND OF THE INVENTION
[0002] Background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
[0003] Chronic respiratory diseases (CRDs) are diseases of the airways and other structures of the lung. Some of the most common diseases are chronic obstructive pulmonary disease (COPD), asthma, occupational lung diseases and pulmonary hypertension. CRDs are not curable; however, various forms of treatment helps dilate major air passages and improve shortness of breath can help control symptoms and increase the quality of life for people with the disease.
[0004] Asthma is a chronic inflammatory disorder of the airways characterized by episodes of reversible breathing problems due to airway narrowing and obstruction. These episodes can range in severity from mild to life threatening. Asthma is responsible for lowering the quality of life, curtailing daily activities, reduction in lung function, and absenteeism from school or work. A large number of patients experience emotional and functional restraints due to asthma. It poses a considerable socioeconomic burden and often leads to the use of a large amount of health-care resources. The exact prevalence of asthma in India remains elusive. A large number of patients in India still consider asthma a stigma and therefore conceal the disease. The World Health Organization reports that India harbors about 15–20 million asthmatics and of these, 10%–15% are children. Almost three out of every 100 adults in India are affected with asthma. A variety of inhaled corticosteroids, ß2-agonist and combination inhalers are available at pharmacies, but these are expensive. Many believe that inhalers are habit forming and strong and poses an emotional as well as the physical burden on patients.
[0005] Neurological diseases like Alzheimer’s in India come with many taboos of superstition, stigma, being considered by others to be of weak mind and at times insane in so far that in rural areas such ailments often go untreated and the person is subject to mental trauma often leading to Anxiety and depression. India currently is going through a mental health crisis and more than 43% Indians are said to be suffering from depression and anxiety – all further catalyzed by impacts of the pandemic of 2020.
[0006] In the recent years, there has been an augmented interest globally to identify compounds isolated from natural products that are pharmacologically effective and have low or no side effects for use in preventive care and the food industry. Spices are heterogeneous collections of a wide variety of volatile and non-volatile staple dietary additives and used in culinary preparations. Spices are known to be effective as functional foods to improve health and decrease the risk of diseases. They are rich in antioxidants and comprise a diverse array of natural phytochemicals and many critical micronutrients that have complementary and overlapping actions. Several scientific studies suggest that spices are potent inhibitors of tissue damage and inflammation.
[0007] Though several different spices mixture or masalas are available in market for culinary purposes, however, none of them exhibits potential of improving the health of an individual and known to reduce the risk of such diseases, plausibly owing to disproportionate usage of different ingredients while preparing the spices mix/compositions.
[0008] Therefore, it would be desirable to develop a new and improved composition that may aid in improving pulmonary and neurological conditions. The present invention satisfies the existing needs, as well as others, and generally overcomes the deficiencies found in the prior art.

OBJECTS OF THE INVENTION
[0009] Primary object of the present invention is to provide a composition that may overcome one or more limitations associated with the conventional compositions.
[00010] It is an object of the present disclosure to provide a composition that may aid in improving pulmonary and neurological conditions.
[00011] It is an object of the present disclosure to provide a composition that is substantially devoid of any side effects.
[00012] It is an object of the present disclosure to provide a composition that exhibits synergistic activity/functional reciprocity.
[00013] It is an object of the present disclosure to provide a composition that is easy to prepare and is economical.
[00014] Another object of the present disclosure is to provide a method of preparation of the composition.

SUMMARY OF THE INVENTION
[00015] The present disclosure relates generally to the field of spice compositions. Particularly, the present disclosure relates to a composition for improving pulmonary and neurological conditions in a subject. The present disclosure further relates to a method of preparation of the composition.
[00016] An aspect of the present disclosure relates to a composition for improving pulmonary and neurology conditions, said composition including: black pepper in an amount ranging from 15 to 55% by weight of the composition; cardamom in an amount ranging from 15 to 50% by weight of the composition; star anise in an amount ranging from 5 to 30% by weight of the composition; mace in an amount ranging from 5 to 30% by weight of the composition; and cinnamon in an amount ranging from 0 to 20% by weight of the composition.
[00017] In an embodiment, the cardamom is selected from green cardamom, black cardamom and a mixture thereof. In an embodiment, the composition includes: black pepper in an amount of 40% by weight of the composition; green cardamom in an amount of 30% by weight of the composition; star anise in an amount of 15% by weight of the composition; and mace in an amount of 15% by weight of the composition.
[00018] In an embodiment, the composition includes: black pepper in an amount of 30% by weight of the composition; green cardamom in an amount of 30% by weight of the composition; black cardamom in an amount of 10% by weight of the composition; star anise in an amount of 15% by weight of the composition; and mace in an amount of 15% by weight of the composition.
[00019] In an embodiment, the composition includes: black pepper in an amount of 35% by weight of the composition; green cardamom in an amount of 25% by weight of the composition; cinnamon in an amount of 10% by weight of the composition; star anise in an amount of 15% by weight of the composition; and mace in an amount of 15% by weight of the composition.
[00020] Another aspect of the present disclosure relates to a process for preparing a composition including black pepper in an amount ranging from 15 to 55% by weight of the composition; cardamom in an amount ranging from 15 to 50% by weight of the composition; star anise in an amount ranging from 5 to 30% by weight of the composition; mace in an amount ranging from 5 to 30% by weight of the composition; and cinnamon in an amount ranging from 0 to 20% by weight of the composition, said process including the steps of:
a) dry roasting each of black pepper, cardamom, star anise, mace, and optionally cinnamon at a temperature ranging from 40°C to 100°C for a time period ranging from 1 minute to 10 minutes to obtain roasted spices;
b) grinding the roasted spices to obtain ground spices;
c) blending the ground spices to obtain a spice mixture; and
d) finely grinding the spice mixture to obtain the composition such that more than 75% particles have a size ranging from 1 micron to 1000 microns.
[00021] In an embodiment, the step of dry grinding is effected at a temperature ranging from 10°C to 100°C. In an embodiment, the ground spices have particle size ranging from 1 mm to 3 mm. In an embodiment, the step of finely grinding the spice mixture is effected at a temperature ranging from 10°C to 100°C.
[00022] Other aspects, advantages, and salient features of the invention will become apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the exemplary embodiments of the invention.

DETAILED DESCRIPTION OF THE INVENTION
[00023] The embodiments herein and the various features and advantageous details thereof are explained more comprehensively with reference to the non-limiting embodiments that are detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of the ways in which the embodiments herein may be practiced and to further enable those of skills in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.
[00024] Unless otherwise specified, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions may be included to better appreciate the teaching of the present invention.
[00025] As used in the description herein, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.
[00026] As used herein, the terms “comprise”, “comprises”, “comprising”, “include”, “includes”, and “including” are meant to be non- limiting, i.e., other steps and other ingredients which do not affect the end of result can be added. The above terms encompass the terms “consisting of” and “consisting essentially of”.
[00027] As used herein, the terms “composition”, “blend”, or “mixture” are all intended to be used interchangeably.
[00028] The terms “weight percent”, “vol-%”, “percent by weight”, “% by weight”, and variations thereof, as used herein, refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, “percent”, “%”, and the like are intended to be synonymous with “weight percent”, “vol-%”, etc.
[00029] The term, “black pepper” as used herein refers to a flowering vine in the family Piperaceae, cultivated for its fruit, known as a peppercorn, which is usually dried and used as a spice and seasoning. When fresh and fully mature, the fruit is about 5 mm in diameter and dark red, and contains a single seed, like all drupes.
[00030] The term, “cardamom” as used herein refers to a spice made from the pods of several plants in the genera Elettaria and Amomum in the family Zingiberaceae. Both genera are native to the Indian subcontinent and Indonesia. They are recognized by their small seed pods: triangular in cross-section and spindle-shaped, with a thin, papery outer shell and small, black seeds; Elettaria pods are light green and smaller, while Amomum pods are larger and dark brown. Green cardamom comes from the species Elettaria cardamomum, whereas Black cardamom, also known as brown, greater, large, longer, or Nepal cardamom, comes from species Amomum subulatum.
[00031] The term, “cinnamon” as used herein refers to a spice obtained from the inner bark of several tree species from the genus Cinnamomum. Cinnamon is the name for several species of trees and the commercial spice products that some of them produce. All are members of the genus Cinnamomum in the family Lauraceae. Only a few Cinnamomum species are grown commercially for spice. Cinnamomum verum is sometimes considered to be "true cinnamon", but most cinnamon in international commerce is derived from the related species Cinnamomum cassia, also referred to as "cassia." Cinnamon is used mainly as an aromatic condiment and flavouring additive in a wide variety of cuisines, sweet and savory dishes.
[00032] The term “Star Anise” as used herein refers to a seed pod from the fruit of the Illicium verum plant, an evergreen shrub native to Southwest China and Vietnam and grown in Arunachal Pradesh in India. The star anise pod, which is shaped like a star has an average of eight points, each containing a single pea-sized seed. Both the seeds and the pod are used in cooking and contain the sweet, potent anise flavor. Star anise is used in culinary applications for its distinct flavor but is also employed for its medicinal benefits.
[00033] The term, “mace” as used herein refers to a reddish-brown spice that is derived from the dried lacy coating of the nutmeg seed. The nutmeg tree (Myristica fragrans) is a tropical evergreen that produces both nutmeg and mace. Mace is the red lacy coating (called the aril) that encases the nutmeg seed. When the tree's fruit reaches maturity, it splits open and reveals the aril and seed. The fruit is harvested and the aril is removed by hand, flattened, and left to dry outside for 10 to 14 days. The red aril takes on amber-, yellow- or orange-brown color as it dries and, when left whole, is called a "blade" of mace.
[00034] In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about”. Accordingly, in some embodiments, the numerical parameters set forth in the written description are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
[00035] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
[00036] The headings and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.
[00037] The following discussion provides many example embodiments of the inventive subject matter. Although each embodiment represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus if one embodiment comprises elements A, B, and C, and a second embodiment comprises elements B and D, then the inventive subject matter is also considered to include other remaining combinations of A, B, C, or D, even if not explicitly disclosed.
[00038] The present disclosure relates generally to the field of spice compositions. Particularly, the present disclosure relates to a composition for improving pulmonary and neurological conditions in a subject. The present disclosure further relates to a method of preparation of the composition.
[00039] The present disclosure is on the premise of a surprising discovery that when black pepper, cardamom, star anise, mace, and optionally cinnamon are included as part of the composition in the weight percentages as defined herein, it exhibits exceptional functional reciprocity and synergistic activity simultaneously targeting several physiological and/or pathophysiological pathways and affords dramatic improvement in pulmonary and neurology conditions and the likes in a subject as compared to usage of individual ingredients. The observed synergistic effect is unexpected and surprising. Particularly, it could be observed that when the composition of the present disclosure is fed to Danio rerio (zebrafish models), it affords modulation of several genes (such as smad3, enos, gsdmb, ormdl3, lypd6, postn, mmp9, sgc, gpx1a and the likes) and consequently, affords restoration of apposite oxidative balance by modulating antioxidant system while aiding in maintenance of homeostasis.
[00040] Accordingly, an aspect of the present disclosure relates to a composition for improving pulmonary and neurology conditions, said composition including: black pepper in an amount ranging from 15 to 55% by weight of the composition; cardamom in an amount ranging from 15 to 50% by weight of the composition; star anise in an amount ranging from 5 to 30% by weight of the composition; mace in an amount ranging from 5 to 30% by weight of the composition; and cinnamon in an amount ranging from 0 to 20% by weight of the composition.
[00041] In an embodiment, the cardamom is selected from green cardamom, black cardamom and a mixture thereof. In an embodiment, the composition includes: black pepper in an amount of 40% by weight of the composition; green cardamom in an amount of 30% by weight of the composition; star anise in an amount of 15% by weight of the composition; and mace in an amount of 15% by weight of the composition.
[00042] In an embodiment, the composition includes: black pepper in an amount of 30% by weight of the composition; green cardamom in an amount of 30% by weight of the composition; black cardamom in an amount of 10% by weight of the composition; star anise in an amount of 15% by weight of the composition; and mace in an amount of 15% by weight of the composition.
[00043] In an embodiment, the composition includes: black pepper in an amount of 35% by weight of the composition; green cardamom in an amount of 25% by weight of the composition; cinnamon in an amount of 10% by weight of the composition; star anise in an amount of 15% by weight of the composition; and mace in an amount of 15% by weight of the composition.
[00044] In an embodiment, the composition includes: black pepper in an amount of 25% by weight of the composition; green cardamom in an amount of 25% by weight of the composition; cinnamon in an amount of 10% by weight of the composition; black cardamom in an amount of 10% by weight of the composition; star anise in an amount of 15% by weight of the composition; and mace in an amount of 15% by weight of the composition.
[00045] In an embodiment, the composition is formulated into a powder form. In an embodiment, the composition can be used as a spice mixture/seasoning. The spice mixture is useful as seasoning of food and beverages to enhance flavor and taste. The spice mixture can be used as a seasoning during preparation of the food and/or beverages or can be added after the food and/or beverage has been prepared.
[00046] In an embodiment, the spice mix naturally contains Piperine, Piperidine, Cineole, Anethole, Shikimic acid, Limonene, Caryophyllene, Estragole, Linalool, Eugenol, Elemicin, Myristene, Quercetin, Vitamin A, B1, B2, B3, B6, C, micronutrients like Cu, Fe, Mn, K, Ca, Mg, P, and Zn, antioxidants and the mixtures thereof.
[00047] Another aspect of the present disclosure relates to a process for preparing a composition including black pepper in an amount ranging from 15 to 55% by weight of the composition; cardamom in an amount ranging from 15 to 50% by weight of the composition; star anise in an amount ranging from 5 to 30% by weight of the composition; mace in an amount ranging from 5 to 30% by weight of the composition; and cinnamon in an amount ranging from 0 to 20% by weight of the composition, said process including the steps of:
a) dry roasting each of black pepper, cardamom, star anise, mace, and optionally cinnamon at a temperature ranging from 40°C to 100°C for a time period ranging from 1 minute to 10 minutes to obtain roasted spices;
b) grinding the roasted spices to obtain ground spices;
c) blending the ground spices to obtain a spice mixture; and
d) finely grinding the spice mixture to obtain the composition such that more than 75% particles have a size ranging from 1 micron to 1000 microns.
[00048] In another embodiment, the dry roasting is effected at a temperature ranging from 50°C to 100°C for a time period ranging from 2 minute to 6 minutes to obtain roasted spices. In another embodiment, the dry roasting is effected at a temperature ranging from 50°C to 90°C for a time period ranging from 2 minute to 6 minutes to obtain roasted spices.
[00049] In an embodiment, the step of dry grinding is effected at a temperature ranging from 10°C to 100°C. In an embodiment, the step of dry grinding is effected at a temperature ranging from 10°C to 50°C. In an embodiment, the step of dry grinding is effected at room temperature (35°C). In an embodiment, the ground spices have particle size ranging from 1 mm to 3 mm.
[00050] In an embodiment, the step of finely grinding the spice mixture is effected at a temperature ranging from 10°C to 100°C. In an embodiment, the step of finely grinding the spice mixture is effected at a temperature ranging from 10°C to 50°C.
[00051] The spice mixture of the present disclosure is consumed fully and no part of any spice is discarded. On the contrary, during common cooking, black pepper is consumed in whole whereas even if cinnamon, star nice, mace and cardamom are used as spices, these are used only for their volatiles that seep into the food while rest of the whole spice is discarded before consuming the food.
[00052] In an embodiment, spice mixture/seasoning has an exquisite refreshing flavor and can be used in day to day cooking of food and/or beverages, for improving respiratory and pulmonary health and for preventing/ fighting neurodegenerative ailments and depression. The spice mixture provides exquisite flavor to the food such that whole family can have common meals instead of making special bland food for subject suffering from pulmonary/respiratory and neurodegenerative diseases. The spice mixture provides better lung health, oxygen intake, healthier teeth and gums, stress relief and protection from neuro-degenerative diseases such as Alzheimer’s at no/minimal incremental cost. The spice mixture upon regular usage in food and/or beverages like tea, coffee and the like, can prevent progression of Alzheimer’s disease by reducing the amyloid plaques, can act as anti-asthmatic, assist in vasodilation, prevent infection of bronchial tracts, augment serotonin by replication of dopamine effect, and also act as anti-microbial for oral care. The spice mixture relieves the consumer from mental stress, bronchial suffrage, asthmatic affectations and improves oral health distress at nominal costs, resulting in stronger lungs, healthier minds and improved oral hygiene.
[00053] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the scope of the embodiments as described h
EXAMPLES
[00054] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
[00055] COMPOSITION
Table 1: Composition for improving Respiration
Ingredients Amount of Ingredients
(in grams)
Green Cardamom 30
Black Pepper 40
Star Anise 15
Mace 15

[00056] PREPARATION OF COMPOSITION
[00057] All the ingredients used in preparation of the composition are of Indian origin and are readily available commercially. Particularly, each of the ingredients was procured commercially from Chennai, India. All the ingredients were weighed accurately as per Table 1 provided herein-above and dry roasted separately at 70°C for about 5 minutes to obtain roasted spices. The roasted spices were then grinded separately at room temperature (35°C) to obtain ground spices. The ground spices were then blended and mixed thoroughly to obtain a spice mixture. The spice mixture was then finely grinded to obtain the composition such that more than 75% particles have a size below 1000 microns.
[00058] EFFICACY STUDIES
[00059] The composition of the present disclosure, prepared in accordance with Table 1 was used to assess its potential towards enhancing the respiration, and reducing the consumer’s probability to suffer from any respiratory ailment. To understand the efficacy of the composition, the swim bladder of the zebrafish models, treated with the composition (referred as treatment group) and the vehicle (without composition treatment, referred as vehicle group) were studied for generation of Reactive Oxygen Species (ROS); further RT-PCR was done to assess the oxidative stress and gene expression profiling was done to understand the physiological/patho-physiological pathways involved.
ZEBRAFISH MODEL DEVELOPMENT
Zebrafish is one of the most widely used and accepted animal for studying gene expression for several therapeutic areas including cancer and neurodegenerative disorders as they share genetic homology with the humans. To evaluate the efficacy of the composition disclosed in Table 1, freshwater zebrafish of about 4 centimeters in length were selected for establishment of models for efficacy studies. Wild type strains of Zebrafish (Danio rerio) were used that were adapted and bred at the research center. All the fishes were acclimated to constant laboratory conditions (14h light and 10h dark photoperiod, normal diet and water at 28°C) for at least one week in stock aquaria before the induction of experiment. The fishes were fed with Tetrabit commercial feed twice during the 6 hrs light cycle. Through purposive sampling, healthy adult males with no signs of infection, equivalent body size and age (around 1-2 years) were scrutinized and used for the experiment study.
Good Animal Practice as per the Institutional Animal Ethics Committee in accordance with the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India, was followed in adherence to established protocols. The washout process was carried out once in every 4 days to keep the fishes in a clean environment and to eliminate the risk of any infection.
DOSE PREPARATION
[00060] A concentrate composition was prepared using 1X Phosphate buffer (PBS) and the concentrate was diluted further using 1X PBS as mentioned herein-below in Table 2.
Table 2: Dilution of the composition
Dilution Ratio 1X PBS Composition Concentrate
1:20 2000 ul 100 ul

Method of preparation of Concentrate:
i. PHF Extract Preparation:
About 15 g of the sample was weighed and transferred to a clean and dry stainless-steel container. 150 ml of distilled water was added to the sample and mixed well with a ladle. The container is then closed with a lid and proceeded for steaming.
ii. Heat Steaming process:
Aluminum steam pot was used for heat steaming the sample. The lid of the steam pot was provided with a steam outlet. 1/5th of the steam pot was filled with water and pre-heated in medium flame. The container was transferred to the steam pot when the water in the vessel begins to bubble and the steam pot was closed with a lid which has a steam outlet. The steam pot was heated for about 15 minutes. After 15 minutes, the flame was switched off and the container was allowed to cool for 15 minutes (without opening the lid). After cooling, the container was removed from the steam pot.
iii. Filtration 1:
The sample in the container was filtered through a filter (large sieve) and collected in a glass bowl. The container was thoroughly rinsed with 25 ml of 2X PBS to collect the residue sticking to the wall of the container. The filtrate was transferred from the bowl to a mixer jar. The bowl was rinsed with 25ml of 2X PBS to remove the residue from the bowl and homogenized for 1 minute.
iv. Filtration 2:
The homogenized content in the jar was filtered through a filter (small sieve) and the filtrate was collected in a glass bowl. The jar was thoroughly rinsed with 50 ml PBS to collect the residue sticking to the wall of the jar. The filtrate 2 obtained is the final filtrate was used for efficacy studies. The filtrate 2 in the bowl is emptied into a clean and dry bottle, and the bowl was rinsed with 50 ml 2X PBS and transferred into the bottle.
v. Storage condition:
The final filtrate was stored at 18°C in the refrigerator. The PHF extract has a shelf life of about ten days when stored under proper storage conditions. The extract is prepared freshly every ten days considering the quality. An intra oral dosing was pursued and a single dose volume of 15 µl composition per day was administered through gavaging for 20 days, referred herein as treated group. The fishes dosed with only 1X PBS (devoid of composition), were referred herein as vehicle group. The fishes were fed with normal dry pellet feed (Tetrabit) twice in 6 hrs light cycle usually post 3hrs of oral gavage to avoid any regurgitation. Composition dosing was optimized 1000 X lesser to the relative human doses by body weight as mentioned in Table 3 herein-below:
Table 3: Dose calculation
Body weight of Zebrafish 0.5 g
Compound Composition
Dose dilution 1:20
Dose Concentration 7.5 µg/15µl

The composition dose was administered through oral gavage. A micropipette was used to manipulate the volume of the dose administered and the fishes were observed carefully for regurgitation or dispersion of dose through buccal or operculum opening before transferring to the study tank.

SCREENING
The study groups were screened at day 3rd, 12th and 20th. Three fishes from each group were euthanized to isolate the organ for investigation on gene expression through RT PCR. Throughout the study period the groups were continuously monitored twice per day for any phenotype changes in terms of motility, visible clinical signs of redness (an indicative of inflammation) near extremities such as pelvic, pectoral, and caudal region. Fishes exhibiting abnormal phenotype changes were closely monitored and were reported in the observation log. Mortality was recorded on regular basis.
A) ANALYSIS OF OXIDATIVE STRESS IN SWIM BLADDER:
Cells under normal condition maintain a homeostasis between Reactive oxygen species (ROS) production and its elimination by antioxidation. Low levels of ROS affect the cellular signaling and intracellular reactions, whereas over production of ROS build oxidative stress which can affect the antioxidant system resulting in apoptosis and other cellular damage. Also, hypoxia induces over production of ROS which affects the mitochondrial oxidative phosphorylation.
The assay was performed to visually determine Hypoxia and ROS production in the swimm bladder by fluorescence microscopy. Hypoxia and ROS were detected by using ROS-ID Hypoxia/Oxidative stress detection kit with slight modification in manufacturer’s instruction. Pyocyanin, a physiological inducer was used as ROS inducer (referred herein as diseased group in green fluorescent expression study) and Desferrioxamine (DFO) (referred herein as diseased group in red fluorescent expression study) was used as Hypoxia inducer during the experimental study. The swim bladder tissue of the fish was gently teased out on a glass slide to breakdown the tissues. The tissues dispersed on the glass slide were re-suspended in 500 µl of distilled water to obtain a uniform individual cell suspension. Hypoxia/Oxidative stress detection mix of 100 µl was added to the cell suspension and incubated at 32° in dark for 30 mins, and 3.5 hrs for ROS and Hypoxia detection, respectively. After incubation, the cell suspension was centrifuged within the range of 2500-3000 rpm for 3 mins. After the supernatant was removed, the pellet was washed in RO water and centrifuged again. The supernatant was removed, and the pellet was re-suspended to obtain a single cell suspension. A 20 µl aliquot was suspended onto a microscope slide and analyzed under fluorescence microscope (Labomed LX 400) equipped with a standard green and red filters (490/525nm). The observation of red fluorescent product indicates the presence of Hypoxia while green fluorescent product indicates the presence of cellular production of different reactive oxygen species (ROS) and reactive nitrogen species (RNS).
Table 4: Oxidative stress in swim bladder – Relative Green Fluorescence
Screening Day 12 Day 20
Disease Group
(ROS Inducer) 142 ± 21 142 ± 21
Vehicle Group 67 ± 12 74 ± 13
Treated Group
(present composition) 44 ± 11 35 ± 7.7

It was observed that the vehicle group and the composition treated group showed a significant difference in the oxidative stress and ROS production in Zebrafish swim bladder cells when compared with disease group, determined by the relative fluorescent expression and its intensity. It was found that the , amount of ROS produced in swim bladder in composition treated group was comparatively lower than the vehicle group which translates to a reduction in ROS production which in turn shows the ability of composition to moderately alleviate the intracellular oxidative stress. The present composition was able to restore the apposite physiological conditions by maintaining the optimum oxidative balance to a certain degree.
Table 5: Oxidative stress in swim bladder – Relative Red Fluorescence
Study Groups Day 12 Day 20
Disease Group
(Hypoxia Inducer) 182 ± 23 182 ± 23
Vehicle Group 112 ± 18 91 ± 12
Treated Group
(present composition) 69 ± 14 57 ± 7.7

It was further observed that the vehicle group and the composition treated group showed a significant difference in hypoxia production in Zebrafish swim bladder cells when compared with disease group, determined by the relative red fluorescent expression and its intensity. It was further found that on the intensity of relative red fluorescence in composition treated group was significantly lower than vehicle group which stipulates oxygen depletion in short span of time, thereby perturbing the overall cellular homeostasis. The present composition was able to reduce the apposite physiological condition by substantially alleviating the hypoxic state.
B) GENE EXPRESSION BY SEMI-QUANTITATIVE RT PCR:
To harvest tissue samples, 3 fishes were randomly collected from each group and euthanized via rapid cooling using ice chips. The fishes were then dissected to isolate the whole organ tissue samples. The samples were harvested and stored in 100 µl acid phenol at -20º C until RNA extraction.
RNA Extraction
The total RNA was extracted using phenol chloroform extraction method as described in Chomczynski, P., et al, Anal Biochem. 1987 Apr;162(1):156-9 (doi: 10.1006/abio.1987.9999. PMID: 2440339). The method is based on phase separation of the sample and aids in the isolation of desired molecule. The sample was homogenized in 300 µl of Acid phenol. A volume of 250 µl of chloroform was added to the homogenate and vortexed for few seconds. The sample was incubated in the ice bath for 15 mins. After incubation, the sample was vortexed at high speed for 1 minute followed by centrifuge at high speed for 10 mins. These yielded three distinct phases as follows – clear aqueous phase containing RNA, thin inter-phase containing cellular debris and lower phase with organic substances. The aqueous phase containing RNA was transferred to a fresh centrifuge, and 100 µl of isopropanol was added to the tube followed by incubation in ice bath for 40 mins. At the end of ice incubation, the sample was centrifuged at full speed for 10 mins to precipitate RNA. The supernatant was discarded and the tube containing RNA pellet was dried in the incubator at 40°C to evaporate isopropanol. The RNA quality was evaluated with gel electrophoresis and the aliquots were directly used for reverse transcriptase reaction.
Reverse Transcriptase (RT)
cDNA was synthesized from total RNA (20 µl final reaction volume) with oligo(dT) 15 primer using AMV reverse transcriptase (First strand cDNA synthesis kit from Roche) according to manufacturer’s instruction as mentioned in Table 6. The cDNA tubes were stored at -20º C until use.
Table 6: RT Reaction Mix
RT Reaction mix Volumes
10X Reaction Buffer 2 ul
25 mM MgCl2 4 ul
Deoxynucleotide Mix 2 ul
Oligo-p(dT)15 Primer 2 ul
Rnase Inhibitor 1.0 ul
AMV Reverse Transcriptase 0.8 ul
Water, PCR Grade 8.2 ul

To study the up-regulation and down-regulation properties of the present composition, nine study genes viz. smad3, enos(nos1), gsdmeb, ormdl3, lypd6, postn, mmp9, sgc and gpx1a were selected. 18s RNA was selected as control gene. Since many commonly used genes for study are known to vary under experimental conditions, so for quantifying the gene expression changes in Zebrafish, the internal control gene (18s RNA) that stably expressed under different experimental conditions was used to normalize the study genes. All the primers used in this study were designed using NCBI primer blast tool.
PCR Amplification
PCR amplification was performed using the cDNA as template with a reaction volume of 20 ul as mentioned in Table 7 and the PCR conditions were set as 94°C for 2 mins, 95°C for 5 mins and 72°C for 5 mins tabulated in Table 8. All the qPCR reactions were performed in triplicates and the negative control did not contain any sample template.

Table 7: cDNA to DNA synthesis PCR reaction mix
PCR Reaction 20 µl Volume
PCR Master Mix 10.0 µl
Primer Forward -
F (primer + TE Buffer) 2 µl
Primer Reverse -
R (primer + TE Buffer) 2 µl
PCR Grade water 4 µl
cDNA 2 µl

Table 8: PCR cycling parameters
Steps Temperature Time Cycle
Reaction heated to denature 94°C 2 min
Initial denaturation 95°C 5 min 1
Denaturation 95°C 30 seconds 30
Annealing 58°C 30 seconds 30
Extension 72°C 60 seconds 30
Final Extension 72°C 5 minutes 1

At the end of 30 cycles, a 6 µl aliquot of each sample was mixed with 2.5 µl of gel loading buffer and electrophoreses on an agarose gel for 15 mins. The gel was observed under the UV trans-illuminator for bands. A 1 ul aliquot of the sample was diluted in 2 ml distilled water and used for UV spectroscopy under 260 nm absorbance to determine the concentration of the DNA.
Profiling gene expression over time provides information about the dynamical behavior of the genes. For quantifying gene expression changes in Zebrafish, the internal control gene (18s RNA) that stably expressed under different experimental conditions was used to normalize the study gene. Table 9 herein-below shows the normalized fold change in gene expression of the nine genes under analysis.

Table 9: Normalized fold change in gene expression of the genes
Genes Day 3 Day 12
Vehicle Composition Vehicle Composition
smad3 1.0 ± 0.47 9.12 ± 0.50 1.0 ± 0.47 6.6 ± 0.59
enos (nos1) 1.0 ± 0.96 4.45 ± 0.62 1.0 ± 0.43 3.3 ± 0.99
gsdmb 1.0 ± 1.14 4.60 ± 1.00 1.0 ± 0.72 4.1 ± 0.41
ormdl3 1.0 ± 0.29 3.55 ± 0.82 1.0 ± 0.84 3.4 ±0.70
lypd6 1.0 ± 0.78 4.70 ± 0.16 1.0 ± 0.30 4.0 ± 0.69
postn 1.0 ± 0.44 4.80 ± 0.57 1.0 ± 0.61 4.0 ± 0.82
mmp9 1.0 ± 0.36 3.72 ± 0.54 1.0 ± 0.51 3.7 ± 1.28
sgc 1.0 ± 0.22 4.47 ± 0.65 1.0 ± 0.17 3.8 ± 0.88
gpx1a 1.0 ± 0.28 2.91 ± 0.36 1.0 ± 071 3.0 ± 0.60

Across all genes, the degree of fold change in present composition was higher than vehicle groups indicating a positive modulation of beneficial genes when fed with present composition. This pattern of gene expression establishes a scientific basis of substantiation into pulmonary and neurological conditions, imparted by the present composition on the consumers.
[00061] SYNERGISTIC ACTIVITY OF THE COMPOSITION
The composition, prepared in accordance with Table 10 (hereinafter referred to as PRNP - Optimum) was used for conducting experiments to assess efficacy of the composition vis-à-vis efficacy of the individual ingredients.
Table 10: Composition (PRNP - Optimum)
Ingredients Amount of ingredients
(in grams)
Green Cardamom 30
Black Pepper 40
Star Anise 15
Mace 15

DOSING
Individual ingredients dosing was optimized 1000 X lesser to the relative human doses by body weight as mentioned in the Table 11 herein-below:
Table 11: Dose calculation
Body weight of Zebrafish 0.5 g
Compound PRNP and Individual ingredients
Dose dilution 1:20
Dose Concentration 7.5 µg/15µl

GENE EXPRESSION BY SEMI-QUANTITATIVE RT PCR
SCREENING
The study groups were screened at day 12. Throughout the study period the study groups were continuously monitored twice per day for phenotype changes in terms of motility, visible clinical signs of redness (an indicative of inflammation) near extremities such as pelvic, pectoral and caudal region. Fishes exhibiting abnormal phenotype changes were closely monitored and were reported in the observation log. Mortality was recorded on regular basis. Three fishes were randomly selected from each group and were euthanized via rapid cooling using ice chips. The fishes were then dissected to isolate the organ samples for studying the gene expression via RT PCR. The samples were harvested and stored in 150 µl acid phenol at -20º C until RNA extraction.
RNA Extraction
The total RNA was extracted using phenol chloroform extraction method as described in Chomczynski, P., et al, Anal Biochem. 1987 Apr;162(1):156-9 (doi: 10.1006/abio.1987.9999. PMID: 2440339). The method is based on phase separation of the sample and aids in the isolation of desired RNA. The sample was homogenized in 300 µl of Acid phenol. A volume of 250 µl of chloroform was added to the homogenate and vortexed for few seconds. The sample was incubated in the ice bath for 15 mins. After incubation, the sample was vortexed at high speed for 1 minute followed by centrifuge at high speed for 10 mins. This yielded three distinct phases as follow – clear aqueous phase containing RNA, thin interphase containing cellular debris and lower phase with organic substances. The aqueous phase containing RNA was transferred to a fresh centrifuge, and 100 µl of isopropanol was added to the tube followed by incubation in ice bath for 40 mins. At the end of ice incubation, the sample was centrifuged at full speed for 10 mins to precipitate RNA. The supernatant was discarded and the tube containing RNA pellet was dried in the incubator at 40°C to evaporate isopropanol. The RNA quality was evaluated with gel electrophoresis and the aliquots were directly used for reverse transcriptase reaction.
Reverse Transcriptase
cDNA was synthesized from total RNA (20 µl final reaction volume) with oligo(dT) 15 primer using AMV reverse transcriptase (First strand cDNA synthesis kit from Roche) according to manufacturer’s instruction as mentioned in (Table 12). The cDNA tubes were stored at -20º C until use.
Table 12: RT Reaction Mix
RT Reaction mix Volumes Concentration
10X Reaction Buffer 2 ul 1X
25 mM MgCl2 4 ul 5 mM
Deoxynucleotide Mix 2 ul 1 mM
Oligo-p(dT)15 Primer 2 ul 0.04 A260 units (1.6 g)
Rnase Inhibitor 1.0 ul 50 units
AMV Reverse Transcriptase 0.8 ul >=20 units
Water, PCR Grade 8.2 ul -

To study the up-regulation and down-regulation properties of composition and the individual ingredients, nine study genes viz. smad3, enos(nos1) and gpx1a were selected. 18s RNA was selected as control gene. Since many commonly used study genes are known to vary with experimental conditions, for quantifying the gene expression changes in Zebrafish, the internal control gene (18s RNA) that stably expressed under different experimental conditions was used to normalize the study gene. All the primers used in the study were designed using NCBI primer blast tool.
PCR Amplification
PCR amplification was performed using the cDNA as template with a reaction volume of 20 µl as mentioned in Table 13 and the PCR conditions were set as 94°C for 2 mins, 95°C for 5 mins and 72°C for 5 mins tabulated in table 14. All the qPCR reactions were performed in triplicates and the negative control did not contain any sample template.
Table 13: cDNA to DNA synthesis PCR reaction mix
PCR Reaction 20 µl Volume Final Concentration
PCR Master Mix 10 µl 12.5 ul
Primer Forward -F (primer + TE Buffer) 2 µl 200 nm
Primer Reverse -R (primer + TE Buffer) 2 µl 200 nm
PCR Grade water 4 µl -
cDNA 2 µl -

Table 14: PCR cycling parameters
Steps Temperature Time Cycle
Reaction heated to denature 94°C 2 min
Initial denaturation 95°C 5 min 1
Denaturation 95°C 30 seconds 30
Annealing 58°C 30 seconds 30
Extension 72°C 60 seconds 30
Final Extension 72°C 5 minutes 1

At the end of 30 cycles, a 6 µl aliquot of each sample was mixed with 2.5 µl of gel loading buffer and electrophoresed on an agarose gel for 15 mins. The gel was observed under the UV transilluminator for bands. A 1 ul aliquot of the sample was diluted in 2 ml distilled water and used for UV spectroscopy under 260 nm absorbance to determine the concentration of the DNA.
Profiling gene expression over time provides information about the dynamical behavior of the genes. For quantifying gene expression changes in zebrafish, the internal control gene (18s RNA) that stably expressed under different experimental conditions was used to normalize the study gene. Table 15 shows the normalized fold change in gene expression of three genes under analysis.
Table 15: Normalized fold change in gene expression of three genes
Fold Change in Gene Expression
smad3 Enos gpx1a
PRNP – Optimum 6.6 3.3 3.0
Placebo 1.000 1.000 1.000
Green Cardamom 0.576 -1.186 1.354
Black Pepper 0.756 -1.056 -1.330
Star Anise 0.686 -1.044 -1.655
Mace 0.723 -1.313 0.948
Cinnamon 0.760 -1.106 1.005

Table 15 shows fold change in gene expression of smad3, enos(nos1) and gpx1a in adult Zebrafish post 12 days treatment with present composition and its individual ingredients (Green cardamom, Black pepper, Star Anise, Mace and Cinnamon) against Placebo. It could be noted that the fold change in the expression of smad3, enos(nos1) and gpx1a in adult Zebrafish treated with the composition of the present disclosure was several folds higher than in zebrafish treated with individual ingredients i.e. expression levels of all the three target genes were down-regulated in Green cardamom, Black pepper, Star Anise, Mace and Cinnamon alone as compared to the composition of the present disclosure. Accordingly, it could be concluded that composition of the present disclosure, exhibiting synergistic effect, offers improved respiration/pulmonary to the consumer as compared to usage of the individual ingredients.
[00062] To further assess effect of the composition of present at different dosages, further efficacy studies were conducted. Particularly, for the purpose of these studies, two different compositions (“PRNP – Low” and “PRNP – High”) were realized, details whereof are provided in Table 16 and Table 17.
Table 16: Composition PRNP - Low
Ingredients PRNP - Low
(grams)
Green Cardamom 15
Black Pepper 15
Star Anise 5
Mace 5

Table 17: Composition PRNP – High
Ingredients PRNP - High
(grams)
Green Cardamom 50
Black Pepper 55
Star Anise 30
Mace 30
Cinnamon 20

[00063] The same study protocol, as detailed above (establishing synergism for composition details whereof are provided in Table 10 above), was used for both of these studies. Study outcomes are provided in Table 18 comparing the efficacy of the composition at different dosage levels (PRNP – Low, PRNP - Optimum and PRNP – High) vis-à-vis that of individual ingredients.

Table 18: Comparison of the efficacy of Composition at different dosage levels
Fold Change in Gene Expression
Composition/ingredients smad3 Enos gpx1a
PRNP – High 7.767 5.262 5.804
PRNP – Optimum 6.6 3.3 3.0
PRNP – Low 2.986 2.946 2.671
Placebo 1.000 1.000 1.000
Green Cardamom 0.576 -1.186 1.354
Black Pepper 0.756 -1.056 -1.330
Star Anise 0.686 -1.044 -1.655
Mace 0.723 -1.313 0.948
Cinnamon 0.760 -1.106 1.005

[00064] The aforesaid efficacy studies establish that the composition of the present disclosure affords several folds higher expression of smad3, enos(nos1) and gpx1a in adult Zebrafish treated with the composition even at varying concentration/dosage levels of the ingredients Green cardamom, Black pepper, Star Anise, Mace and Cinnamon alone. Accordingly, it could be concluded that composition of the present disclosure, exhibiting synergistic effect at low, optimum and high dosage, offers improved respiration/pulmonary to the consumer as compared to usage of the individual ingredients.

ADVANTAGES
[00065] The present disclosure provides a composition that overcomes the limitations associated with the conventional compositions.
[00066] The present disclosure provides a composition that improves pulmonary and neurology conditions and other problems associated with them.
[00067] The present disclosure provides a composition that is substantially devoid of any side effects.
[00068] The present disclosure provides a composition that exhibits synergistic activity/functional reciprocity.
[00069] The present disclosure provides a composition that is easy to prepare and is economical.
[00070] The present disclosure provides a method of preparation of the composition.

,CLAIMS:1. A composition for improving pulmonary and neurology conditions, said composition comprising:
black pepper in an amount ranging from 15 to 55% by weight of the composition;
cardamom in an amount ranging from 15 to 50% by weight of the composition;
star anise in an amount ranging from 5 to 30% by weight of the composition;
mace in an amount ranging from 5 to 30% by weight of the composition; and
cinnamon in an amount ranging from 0 to 20% by weight of the composition.
2. The composition as claimed in claim 1, wherein the cardamom is selected from green cardamom, black cardamom and a mixture thereof.
3. The composition as claimed in claim 1, wherein the composition comprises:
black pepper in an amount of 40% by weight of the composition;
green cardamom in an amount of 30% by weight of the composition;
star anise in an amount of 15% by weight of the composition; and
mace in an amount of 15% by weight of the composition.
4. The composition as claimed in claim 1, wherein the composition comprises:
black pepper in an amount of 30% by weight of the composition;
green cardamom in an amount of 30% by weight of the composition;
black cardamom in an amount of 10% by weight of the composition;
star anise in an amount of 15% by weight of the composition; and
mace in an amount of 15% by weight of the composition.
5. The composition as claimed in claim 1, wherein the composition comprises:
black pepper in an amount of 35% by weight of the composition;
green cardamom in an amount of 25% by weight of the composition;
cinnamon in an amount of 10% by weight of the composition;
star anise in an amount of 15% by weight of the composition; and
mace in an amount of 15% by weight of the composition.
6. A process for preparing a composition comprising black pepper in an amount ranging from 15 to 55% by weight of the composition; cardamom in an amount ranging from 15 to 50% by weight of the composition; star anise in an amount ranging from 5 to 30% by weight of the composition; mace in an amount ranging from 5 to 30% by weight of the composition; and cinnamon in an amount ranging from 0 to 20% by weight of the composition, said process comprising the steps of:
a) dry roasting each of black pepper, cardamom, star anise, mace, and optionally cinnamon at a temperature ranging from 40°C to 100°C for a time period ranging from 1 minute to 10 minutes to obtain roasted spices;
b) grinding the roasted spices to obtain ground spices;
c) blending the ground spices to obtain a spice mixture; and
d) finely grinding the spice mixture to obtain the composition such that more than 75% particles have a size ranging from 1 micron to 1000 microns.
7. The process as claimed in claim 6, wherein the step of dry grinding is effected at a temperature ranging from 10°C to 100°C.
8. The process as claimed in claim 6, wherein the ground spices have particle size ranging from 1 mm to 3 mm.
9. The process as claimed in claim 6, wherein the step of finely grinding the spice mixture is effected at a temperature ranging from 10°C to 100°C.