DESC:FIELD
The present disclosure relates to a pharmaceutical composition and a process for its preparation.
DEFINITIONS
As used in the present disclosure, the following terms are generally intended to have the meaning as set forth below, except to the extent that the context in which they are used indicates otherwise.
Fixed oil refers to oils that are non-volatile and remain in a liquid state at room temperature. Typically, it consists of oil-soluble components derived from botanicals, which includes, fatty acid esters, free fatty acids, lecithins, waxes, and polysaccharides.
Volatile oil refers to oils that readily evaporate into the air at room temperature due to their low boiling points.
BACKGROUND
The background information herein below relates to the present disclosure but is not necessarily prior art.
Black cumin (Nigella sativa) seed was extracted by various known extraction techniques such as alcoholic extraction, solvent extraction, mechanical extraction, cold pressing, enzymatic extraction, and supercritical fluid extraction. Black cumin seed oil is known for its medicinal properties, used for various indications in Ayurveda and Unani. The pharmacological effects of black cumin are specifically attributed to the components in the volatile/essential oil.
The major active constituents present in black cumin seed essential oil are thymoquinone which constitutes about 30-48%, p-cymene (7-15%), carvacrol (6-12%), 4-terpineol (2-7%), t-anethole (1-4%), sesquiterpene longifene (1-8%), etc. Thymoquinone (TQ) which is chemically called 2-isopropyl-5-methyl-1, and 4-benzoquinone, is the most pharmacologically active constituent found in black cumin oil.
These volatile oils by their specific nature, readily evaporate, difficult to maintain their chemical constituent levels for a prolonged period of time during their shelf life. The active constituents in the volatile/essential oil are not readily soluble in water and have poor bioavailability when consumed by humans. When consumed, because of the poor bioavailability of black cumin volatile oil, it does not provide the desired health benefits. Moreover, the black cumin volatile oil is not palatable directly due to its bitter taste and strong aroma.
The black cumin volatile oil is often mixed with other synthetic agents to stabilize and make it palatable. The use of synthetic ingredients in nutraceuticals and functional foods is restricted by various regulatory authorities across the world.
The present disclosure provides a pharmaceutical composition comprising black cumin volatile oil along with naturally occurring substances to enhance the stability and bioavailability and use a simple process.
Therefore, there is a need for a new pharmaceutical composition that is made of natural ingredients, capable of enhancing the bioavailability of the active chemical constituents in black cumin oil to provide desired health benefits.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein satisfies, are as follows:
An object of the present disclosure is to ameliorate one or more problems of the background or to at least provide a useful alternative.
Another object of the present disclosure is to provide a pharmaceutical composition.
Yet another object of the present disclosure is to provide a black cumin seed extract composition with enhanced bioavailability.
Still another object of the present disclosure is to provide a pharmaceutical composition comprising food-grade ingredients (GRAS - Generally Recognized as Safe).
Still another object of the present disclosure is to provide a simple and efficient process for the preparation of a pharmaceutical composition.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
The present disclosure relates to a pharmaceutical composition and the process for its preparation. In an aspect, the pharmaceutical composition comprises a) at least one volatile oil in an amount in the range of 5 mass% to 15 mass% with respect to the total mass of the composition; b) at least one fixed oil in an amount in the range of 30 mass% to 60 mass% with respect to the total mass of the composition; c) at least one extract in an amount in the range of 20 mass% to 40 mass% with respect to the total mass of the composition; d) at least one phospholipid in an amount in the range of 2 mass% to 20 mass% with respect to the total mass of the composition; e) optionally at least one antioxidant in an amount in the range of 1 mass% to 5 mass% with respect to the total mass of the composition; and f) optionally at least one emulsifying agent in an amount in the range of 1 mass% to 10 mass% with respect to the total mass of the composition.
In another aspect, the process for the preparation of a pharmaceutical composition comprises blending a predetermined amount of mechanically pressed oil with a predetermined amount of extract for a first predetermined time period to obtain a first mixture. The first mixture is blended with a predetermined amount of phospholipid followed by heating at a predetermined temperature for a second predetermined time period to obtain a homogeneous mixture. The homogeneous mixture is optionally blended with a predetermined amount of anti-oxidant and emulsifying agent to obtain the pharmaceutical composition.
BRIEF DESCRIPTION OF THE DRAWINGS
The present disclosure will now be described with the help of the accompanying drawing, in which:
Figure 1 illustrates the effect of Control, stress-induced group, BCO-5, and SCO-5 on locomotor activity. Values are expressed as mean ± SD of six rats each. The values which significantly differ at P < 0.05 are marked with */#;
Figure 2 illustrates the effect of the Control, stress-induced group, BCO-5, and SCO-5 on plasma orexin concentration. Values are expressed as mean ± SD of six rats each. The values which significantly differ at P < 0.05 are marked with */#;
Figure 3 illustrates the effect of Control, stress-induced group, BCO-5, and SCO-5 on plasma corticosterone levels. Values are expressed as mean ± SD of six rats each. The values which significantly differ at P < 0.05 are marked with */#;
Figure 4 illustrates the effect of the Control, stress-induced group, BCO-5, and SCO-5 on serum melatonin concentration. Values are expressed as mean ± SD of six rats each. The values which significantly differ at P < 0.05 are marked with */#;
Figure 5 illustrates the plasma concentration of chemical constituents a) thymoquinone; b) a-thujene; c) carvacrol and d) p-cymene;
Figure 6 illustrates the PQSI score of BCO-5, SCO-5, and placebo at baseline, after the 45th day and 90th day. The values are expressed as mean ± SD;
Figure 7 illustrates salivary cortisol levels of BCO-5, SCO-5, and placebo at baseline, after the 45th day and 90th day. The values are expressed as mean ± SD;
Figure 8 illustrates salivary melatonin levels of BCO-5, SCO-5, and placebo at baseline, after 45th day and 90th day. The values are expressed as mean ± SD;
Figure 9 illustrates plasma orexin concentration of BCO-5, SCO-5, and placebo at baseline, after the 45th day and 90th day. The values are expressed as mean ± SD; and
Figure 10 illustrates immunoglobulin levels a) IgM and b) IgG values of BCO-5, SCO-5, and placebo at baseline, after 45th day and 90th day. The values are expressed as mean ± SD.
DETAILED DESCRIPTION
The present disclosure relates to a pharmaceutical composition and a process for its preparation.
Embodiments, of the present disclosure, will now be described with reference to the accompanying drawing.
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and methods, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known processes, well-known apparatus structures, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms "a,” "an," and "the" may be intended to include the plural forms as well, unless the context suggests otherwise. The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the method and process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed elements.
The terms first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used to distinguish one element, component, region, layer, or section from another component, region, layer or section. Terms such as first, second, third etc., when used herein do not imply a specific sequence or order unless clearly suggested by the present disclosure.
These volatile oils by their specific nature, readily evaporate, difficult to maintain their chemical constituent levels for a prolonged period of time during their shelf life. The active constituents in the volatile/essential oil are not readily soluble in water and have poor bioavailability when consumed by humans. When consumed, because of the poor bioavailability of black cumin volatile oil, it does not provide the desired health benefits. Moreover, the black cumin volatile oil is not palatable directly due to its bitter taste and strong aroma.
The black cumin volatile oil is often mixed with other synthetic agents to stabilize and make it palatable. The use of synthetic ingredients in nutraceuticals and functional foods is restricted by various regulatory authorities across the world.
The present disclosure provides a pharmaceutical composition that can mitigate the above-mentioned problems.
In an aspect of the present disclosure, there is provided a pharmaceutical composition.
The pharmaceutical composition comprising a) at least one volatile oil in an amount in the range of 5 mass% to 15 mass% with respect to the total mass of the composition; b) at least one fixed oil in an amount in the range of 30 mass% to 60 mass% with respect to the total mass of the composition; c) at least one extract in an amount in the range of 20 mass% to 40 mass% with respect to the total mass of the composition; d) at least one phospholipid in an amount in the range of 2 mass% to 20 mass% with respect to the total mass of the composition; e) at least one antioxidant in an amount in the range of 1 mass% to 5 mass% with respect to the total mass of the composition; and f) optionally at least one emulsifying agent in an amount in the range of 1 mass% to 10 mass% with respect to the total mass of the composition.
In an embodiment of the present disclosure, the volatile oil is black cumin (Nigella sativa) volatile oil.
In an embodiment of the present disclosure, the fixed oil is at least one oil selected from the group of oils consisting of oils rich in medium chain triglycerides, and oils rich in polyunsaturated fatty acids.
In an embodiment of the present disclosure, the oils rich in polyunsaturated fatty acids is at least one selected from the group consisting of black cumin (Nigella sativa) oil, olive oil, grape seed oil, pomegranate seed oil, fish oil, flax seed oil, palmetto oil, and sea buckthorn oil.
In an embodiment of the present disclosure, oils rich in medium chain triglycerides is coconut oil.
In an embodiment of the present disclosure, the extract is an alcoholic extract of black cumin seeds (Nigella sativa).
In an embodiment of the present disclosure, the extract is a supercritical fluid extract of black cumin seeds (Nigella sativa).
In an embodiment of the present disclosure, the phospholipid is at least one selected from the group consisting of lecithin, phosphatidylcholine, phosphatidylserine, lecithin-fatty oil derivatives, glycerine-lecithin blends or its derivatives, and vitamin E succinimide esters.
In an embodiment of the present disclosure, the emulsifying agent is at least one selected from the group consisting of glycerol esters of fatty acids, polyglycerol esters of fatty acids, glycerin, propylene glycol, polysorbates, castor oil derivatives and polyglyceryl esters of fatty acids.
In an embodiment of the present disclosure, the antioxidant is at least one selected from the group consisting of tocopherol and rosemary extract.
In an embodiment of the present disclosure, the tocopherol is isolated from sunflower oil, synthetic tocopherol, and its derivatives, wherein said derivative is tocopherol acetate.
In an embodiment of the present disclosure, the composition comprises thymoquinone in the range of 3 vol% to 10 vol %, a-thujene in the range of 0.5 vol% to 2 vol%, carvacrol in the range of 0.5 vol% to 2 vol%, and p-cymene in the range of 0.5 vol% to 2 vol% with respect to the total volume of the composition.
In an embodiment of the present disclosure, the composition comprises thymoquinone, a-thujene, carvacrol, and p-cymene in a volume ratio in the range of 2:1:1:1 to 10:1:1:1.
In another aspect of the present disclosure, there is provided a process for the preparation of the pharmaceutical composition.
The process is described in detail.
In a first step, a predetermined amount of mechanically pressed oil is mixed with a predetermined amount of extract and blended at a speed in the range of 1000 rpm to 6000 rpm for a first predetermined time period to obtain a first mixture.
In an embodiment of the present disclosure, the first predetermined time period is in the range of 30 minutes to 180 minutes.
In a second step, a predetermined amount of phospholipid is mixed with the first mixture followed by heating at a predetermined temperature for a second predetermined time period to obtain a homogeneous mixture.
In an embodiment of the present disclosure, the predetermined temperature is in the range of 45°C to 65°C.
In an embodiment of the present disclosure, the second predetermined time period is in the range of 30 minutes to 90 minutes.
In the final step, the homogeneous mixture is optionally mixed with a predetermined amount of anti-oxidant and an emulsifying agent followed by blending at a speed in the range of 1000 rpm to 5000 rpm to obtain the pharmaceutical composition.
In an embodiment of the present disclosure, the present disclosure provides a method of treating stress, sleep/wake cycle, body weight, and improving immunity in a subject administering to a subject in need thereof a therapeutically effective amount of composition.
In an embodiment of the present disclosure, the present disclosure provides a method of maintaining lipid profile in a subject comprising administering to a subject in need thereof a therapeutically effective amount of composition of the present disclosure, wherein said lipid profile is maintained by reducing low-density lipoprotein (LDL) cholesterol, triglycerides and increasing high-density lipoprotein (HDL) cholesterol.
In an embodiment of the present disclosure, the method of treating stress, sleep/wake cycle through
a. antagonist activity on orexin receptor;
b. amelioration of at least one marker selected from cortisol and melatonin.
The present disclosure provides the pharmaceutical composition, which enhances the bioavailability of the black cumin oil, specifically the chemical constituents of the oil, uses only natural ingredients in the preparation of the composition, and decreases the dosage of the black cumin. Further, the experimental studies revealed that the composition of the present disclosure effectively modulates neurotransmitters to provide health benefits including sleep, stress, lipid profile, immunity, and body weight.
The foregoing description of the embodiments has been provided for purposes of illustration and not intended to limit the scope of the present disclosure. Individual components of a particular embodiment are generally not limited to that particular embodiment, but, are interchangeable. Such variations are not to be regarded as a departure from the present disclosure, and all such modifications are considered to be within the scope of the present disclosure.
The present disclosure is further illustrated herein below with the help of the following experiments. The experiments 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 skill in the art to practice the embodiments herein. Accordingly, the experiments should not be construed as limiting the scope of embodiments herein. These laboratory scale experiments can be scaled up to industrial/commercial scale and the results obtained can be extrapolated to industrial/commercial scale.
EXPERIMENTAL DETAILS
EXPERIMENT 1: Mechanical extract of the Black cumin seed
100 kg of cleaned black cumin seeds (having a moisture content in the range of 6 w/w to 8% w/w) was charged into the reactor (hopper) of a mechanical pressing machine having a jet mill and stainless steel press. The black cumin seeds are powered and have a particle size in the range of 0.5 mm to 1 mm through the pressing machine at a speed in the range of 3 m/s. The black cumin seed powder was maintained at a temperature range of 25°C to 30°C during mechanical pressing to obtain the black cumin seed oil and the residue. During the mechanical pressing cold water having a temperature in the range of 6°C to 8°C was circulated around the mechanical pressing machine. The so obtained black cumin seed oil was filtered through a nylon cloth have a 300-micron size to remove the particulate material. Further, the filtered black cumin seed oil is passed through the anhydrous sodium sulfate to remove the moisture content and obtain the black cumin seed oil (black cumin oil A, mechanically pressed).
The volatile oil content was 10% to 20%, and fixed oil content was 80% to 90%; wherein the oil contains thymoquinone 3% to 6%; a-thujene 0.4 to 0.9%; carvacrol 0.6% to 1.1%; and p-cymene 0.7% to 1.2%.
EXPERIMENT 2: Solvent extraction
50 kg of the residue obtained in Experiment 1 was charged into a reactor with condenser, mechanical stirrer, and steam jacket, followed by adding 200 liters of aqueous ethyl alcohol (95:5 v/v alcohol to water) to the residue and stirred at a speed of 40 rpm for 2 hours to obtain the crude extract. The so obtained crude extract was filtered, followed by heating at 40oC to 45oC under vacuum for 2.4 hrs to obtain black cumin alcohol extract.
The volatile oil content was 10-25%, alcohol extractives were 75 to 90%; wherein the alcohol extract contains 8% to 10% of thymoquinone; 0.3 to 0.6 % of a-thujene; 0.3% to 0.45 of carvacrol; and 0.6% to 0.8% of p-cymene.
EXPERIMENT 3: Supercritical fluid extract
50 kg of the residue obtained in Experiment 1 was charged into an extractor followed by adding followed by pumping 60 kg of micro-filtered supercritical CO2 from the bottom of the at a pressure range of 100 bar to 180 bar for 1 hour to obtain the first mixture. The CO2 stored in cylinders was passed to the chiller and chilled at 3.5°C to 6.5°C followed by pressurizing using a plunger pump (100 bar to 150 bar) to obtain the supercritical CO2. The so obtained first mixture was heated at 70°C using a hot water jacket followed by the separation of CO2 to obtain crude extract. The separated CO2 was sent back to the chiller unit in gaseous form. The crude extract was passed through anhydrous sodium sulphate to remove moisture and obtain black cumin supercritical fluid extract.
The black cumin supercritical fluid extract contains a volatile oil content of 25-30 % and fixed oil content of 70-75%; whereas the supercritical fluid extract contains 9% to 12% thymoquinone; 0.3 to 0.6% of a-thujene; 0.09% to 0.2 of carvacrol; and 1.3% to 1.7% of p-cymene.
EXPERIMENT 4: Preparation of pharmaceutical composition in accordance with the present disclosure
1.5 kg of black cumin seed oil obtained in Experiment 1 and 1 kg of black cumin alcohol extract obtained in Experiment 2 were charged into a vessel followed by stirring for 60 minutes to obtain a first mixture. The first mixture is a free flowing oil that does not show any sedimentation on standing at >15°C.
To the first mixture, 100 g of lecithin was added under stirring followed by heating at 50°C for 60 minutes to obtain a homogeneous mixture. To the homogeneous mixture, 7.3 g of tocopherol and 3.1 g of rosemary extract (having a carnosic acid content >10% w/w) were added under stirring to obtain a pharmaceutical composition.
The pharmaceutical composition after consumption, forms a microemulsion upon contact with water or any gastrointestinal fluid, which helps to increase the absorption.
The pharmaceutical composition has a volatile oil content of 14.8 %, fixed oil content of 49%, and alcohol extractives of 32%; lecithin content of 3.8%, anti-oxidants of 0.4 %. wherein the composition contains 5.9% of thymoquinone; 0.61% of a-thujene; 0.63 of carvacrol; and 0.7% of p-cymene.
EXPERIMENT 5: Preparation of pharmaceutical composition in accordance with the present disclosure
1 kg of black cumin seed oil obtained in Experiment 1 and 1 kg of black cumin supercritical fluid extract obtained in Experiment 3 were charged into a vessel followed by stirring for 60 minutes to obtain a first mixture. The first mixture is a free flowing oil that does not show any sedimentation on standing at >15°C.
To the first mixture, 360 g of lecithin was added under stirring followed by heating at 50°C for 60 minutes to get a homogeneous mixture. The homogeneous mixture was then cooled to room temperature and blended with 130 g of glycerol-polysorbate blend and 40 g of antioxidants (28 g natural mixed tocopherol derived from sunflower and 12 g rosemary extract) and stirred at 3000 rpm for 1 hour, followed by filtration to obtain a pharmaceutical composition.
The pharmaceutical composition has a volatile oil content of 14 %, fixed oil content of 59.5% (obtained from supercritical fluid extract and mechanical extract) and lecithin of 18%, emulsifier of 6.5%, and antioxidant of 2%, wherein the composition contains 6.48% of thymoquinone; 0.64% of a-thujene; 0.67% of carvacrol; and 0.72% of p-cymene.
EXPERIMENT 6: In-vitro study
The efficacy of the composition of the present disclosure was evaluated in a stress-induced model of the rats. Twenty-four male rats were grouped into four groups (six animals/group) wherein Group I – Sham (control), Group II – Stress-induced group, Group III - SCO-5 treated stress-induced animals (20 mg/kg b. wt.; p.o), Group IV - BCO-5 (pharmaceutical composition of the present disclosure, Experiment 4) treated stress-induced animals (20 mg/kg b. wt.; p.o).
The stress and sleep disorder in the rats was induced by the ‘Gentle handling’ method. The gentle handling involves shaking and tapping the cage, touching the animal with a soft brush or with hands, and introducing novel objects into the cage for 6 hours. Alternatively, the animals were exposed to environmental noises ranging from 1 to 25 Hz for 3 minutes in every 30-minute interval. Sound Wave Tone Generator (Fire Shooters, Tsim Sha Tsui, Hong Kong) was used to generate noises.
At the baseline, the body weight of the animals was taken and every alternative day before the drug administration. The duration of the study was 14 days.
The study design is provided below for ready reference.

After the completion of the study the locomotor activity, plasma orexin A, serum corticosterone, and serum melatonin concentration were evaluated. The results are provided below.
Effect of BCO-5 on locomotor activity (Open field test)
The locomotor activity was assessed 24 hours after the administration of the last dose of treatment (on day 15). The locomotor activity was performed using the Open field test (OFT), to evaluate the animal behavioral studies. The test evaluates the locomotion with reference to the number of lines crossed by the rat over a 5-minute interval, which is a reaction of an animal to an unknown environment and stress stimuli. The values are recorded and analyzed.
The results showed a significant increase in locomotor activity among stress-induced Group II animals (P < 0.05), as compared to that of control animals in Group I (21 ± 2.83 in Group I vs 53 ± 6.74 in Group II). Also, a significant increase was observed in Group III (SCO-5 treated stress-induced rats) as compared to Group I (P < 0.05). Stress induced animals with supplementation with BCO-5 in Group IV animals (stress-induced) demonstrated a significant reduction as compared to Group II (53 ± 6.74 for Group II vs 26 ± 3.54 for Group IV; P < 0.05). The locomotor activity observed in Group IV showed no significant difference with Group I (Figure 1).

Effect of BCO-5 on plasma Orexin-A
When animals are exposed to stress stimuli, generate orexin and the orexin-receptor interaction induces wakefulness, while the dual orexin receptor antagonists (DORA) block the downstream signaling pathway and inhibit wakefulness.
Plasma Orexin-A was found to be significantly increased among stress-induced Group II animals compared to the Control (P < 0.05). However, administration of BCO-5 to stress-induced animals (Group IV) was found to offer a significant reduction in the Orexin-A with respect to Group II (47 %, P < 0.05). No significant change was observed between Groups I and IV (P > 0.05). The results are illustrated in Figure 2. Though a reduction was observed in SCO-5 treated group III, it was not as significant as in group IV.
Effect of BCO-5 on serum corticosterone
Cortisol (corticosterone in rodents) is one of the major hormones released in response to stress and is known to be circadian dependent and hence affect sleep patterns and duration.
Serum corticosterone was found to increase significantly among the stress-induced Group II animals compared to control (Group I) (P<0.05; 69%). The supplementation of BCO-5 produced a significant reduction in stress-induced animals in Group IV in comparison with Group II animals (37.81%, P < 0.05), the values are close to the control group. There was no significant change between Group I and Group IV (P >0.05). It is evident from the results that a significant increase was observed between Groups I and III (P < 0.05). Thus, it indicates that the composition of the present disclosure significantly reduces corticosterone levels. The results are illustrated in Figure 3.

Effect of Pharmaceutical composition of the present disclosure (BCO-5) on serum melatonin
Melatonin is a hormone that is responsible for regulating sleep-wake cycles in humans. Melatonin is primarily associated with sleep regulation; it can also affect stress levels.
Serum melatonin showed a significant decrease among the stress-induced animals in Group II compared to Control animals in Group I (P < 0.05; 16.6 %), whereas, the BCO-5 treated stress-induced rats (Group IV) showed a significantly high concentration compared to Group II (P < 0.05; 35 %). The observed melatonin concentration in Group IV was not statistically different with respect to Group I. While an improvement was observed in Group III treated with SCO in comparison to Group II, there was no statistically significant difference. The results are illustrated in Figure 4.
The results of the present study suggest that BCO-5 has the potential to function as a natural Dual Orexin Receptor Antagonist (DORA) in alleviating stress-induced insomnia in adult male Sprague Dawley rats.
Experiment 7: Bioavailability study
The bioavailability of the composition of the present disclosure (BCO-5, Example 5), was compared with supercritical extracted black cumin oil with 5% TQ (SCO-5), and Cold pressed oil with 1% TQ (CPO) was carried out. Twelve healthy subjects (n=12) aged 22 to 55 were selected and administered 200 mg of black cumin oil extract disclosed in the present invention (BCO-5), 200 mg of supercritical extracted black cumin oil (SCO-5), and 1000 mg Cold pressed oil (CPO) in a randomized double-blind cross-over study. After the study of each composition, the subject was allowed a washout period of 10 days followed by the administration of other compositions. Thus, each of the subjects was administered with all types of compositions. These pharmaceutical compositions (oils) were administered as soft gelatin capsules with 200 mL of drinking water, after 30 min of having the breakfast.
For each of the compositions, the blood samples (3 mL) were collected before administering the first dose, corresponding to 0 time points and at 0.5, 1, 2, 3, 4, 5, 6, 8, and 12 hr post-administration. The blood samples were collected into vacuum tubes containing K2EDTA and subsequently centrifuged at 3500 rpm at 4 °C for 10 min within 60 min of collection. Following centrifugation, the plasma was separated, frozen, and stored at 80°C.
The active compounds from the samples were extracted from plasma by adding 1 ml of acidified methanol (0.1% acetic acid) to 1 ml of sample, followed by high-speed mixing for 1 minute and centrifuged at 9000 for 10 min at 4 °C to obtain a top layer and bottom layer. The top layer was collected in a micro centrifuging tube. The procedure was performed in duplicate and the obtained top layers were mixed to obtain the supernatant mixture and filtered using a PVDF syringe filter (0.45 µm) to obtain the sample. The so obtained samples (20 µL) were evaluated using GC-MS (Gas chromatography–mass spectrometry).
The results are provided in Table 1 below and Figure 5.
Table 1: Plasma concentration values of the chemical constituents

BCO-5
Chemical constituent Cmax (ng/mL) Tmax (h) T1/2 (h) AUC (ng/ mL.h)
TQ 143.7 ± 44.20 3.14 ± 0.42 4.01 ± 0.36 372.3 ± 84.32
AT 26.10 ± 11.79 2.09 ± 0.17 2.99 ± 0.21 66.13 ± 17.50
CV 27.05 ± 10.79 2.11 ± 0.30 3.75 ± 0.25 89.32 ± 23.81
PC 101.56 ± 37.33 3.12 ± 0.20 4.27 ± 0.35 277.5 ± 43.81
SCO - 5
TQ 38.99 ± 19.06 1.08 ± 0.21 1.95 ± .033 65.11 ± 20.75
AT 11.94 ± 8.55 1.07 ± 0.12 1.66 ± 0.14 14.18 ± 5.09
CV 5.78 ± 2.05 1.04 ± 0.09 1.89 ± 0.14 9.166 ± 2.17
PC 35.03 ± 18.15 1.11 ± 0.09 1.04 ± 0.09 77.08 ± 15.81
CPO (Cold press oil)
TQ 15.58 ± 7.36 1.10 ± 0.14 1.36 ± 0.23 29.72 ± 11.24
AT 9.63 ± 3.742 0.99 ± 0.17 1.54 ± 0.20 9.576 ± 2.21
CV 3.95 ± 2.67 1.04 ± 0.16 1.43 ± 0.19 5.801 ± 2.53
PC 28.01 ± 7.612 1.12 ± 0.26 1.75 ± 0.31 48.73 ± 14.77
*AT - a-thujene, TQ – thymoquinone, CV- carvacrol and PC – p-cymene
It is evident from the above results that thymoquinone bioavailability from BCO-5 has a Cmax of 143.7 ng/mL, with AUC 372.3 ng/mL.h at a Tmax of 3h. BCO-5 is 5.71 times more bioavailable than SCO-5 and 12.52 times more than CPO.
a-thujene bioavailability from BCO-5 has a Cmax of 26.10 ng/mL, with an AUC of 66.13 ng/mL.h at a Tmax of 3h. BCO-5 is 4.66 times more bioavailable than SCO-5 and 6.90 times more than CPO
Carvacrol bioavailability from BCO-5 has a Cmax of 27.05 ng/mL, with an AUC of 89.32 ng/mL.h at a Tmax of 3h. BCO-5 is 9.74 times more bioavailable than SCO-5 and 15.39 times more than CPO
P-cymene bioavailability from BCO-5 has a Cmax of 101.56 ng/mL, with an AUC of 277.5 ng/mL.h at a Tmax of 3h. BCO-5 is 3 times more bioavailable than SCO-5 and 5.69 times more than CPO.
Experiment 8: In-vivo study
The pharmaceutical composition of the present disclosure (BCO-5, Experiment 5) and supercritical fluid extract of the black cumin oil (SCO) was evaluated for its efficacy in the stress-sleep-immunity axis.
In this study, 48 healthy human volunteers were enrolled in the study, divided into three groups i.e. Placebo (n=16), SCO-5 (n=16), and BCO-5 (n=16). The subjects were administered with a dose of 200 mg/day of either placebo, SCO-5, or BCO-5. The duration of the study was 90 days. At the base, all the subjects were evaluated with the PQSI questionnaire, salivary cortisol, orexin concentration, immunoglobulin, and serum IgM levels. The PQSI and the salivary cortisol and melatonin were evaluated after 45 days and at the end of the study at 90 days.
PSQI score
The PSQI is a 19-item self-reported questionnaire designed to determine the overall sleep quality and disturbances over a period of one-month tenure.
Intra-group comparison (baseline versus day 45) of the PSQI total score revealed a significant reduction (31.66%; P < 0.001) in the BCO-5 group and a non-significant reduction in both placebo and SCO-5.
By the end of the study period (day 90), BCO-5 supplementation revealed a continuous improvement in sleep parameters with a significant reduction in the PSQI total score (42.78%) and component scores on both intra and inter-group comparisons. The total PSQI score of BCO-5 was significantly lower than that at baseline (P < 0.001). No significant change was observed in SCO-5. The results are illustrated in Figure 6.
Salivary cortisol
The intra- and inter-group analysis of cortisol at the end of the 45th day exhibited a significant decrease (28.22%; P < 0.001) for BCO-5, while placebo and SCO-5 showed no significant change (P > 0.05).
At the end of the study (day 90), BCO-5 participants showed a significant decrease (39.28%; P < 0.001), while the placebo and SCO-5 groups exhibited no significance (P > 0.05) with respect to the baseline. Inter-group comparison at the end of the study also revealed a significant decrease in cortisol levels among BCO-5 participants compared to placebo. The results are illustrated in Figure 7.
Salivary melatonin
The intra- and inter-group analysis of melatonin at the end of the 45th day exhibited a significant increase (from 41.84 ± 15.03 to 50.78 ± 16.65; 21.36%; P < 0.05) for BCO-5, while SCO-5 and placebo showed no significant change (P > 0.05).
At the end of the study (day 90), the subjects of BCO-5 showed a significant increase (from 41.84 ± 15.03 to 69.59 ± 18.69;66.32%; P < 0.001), while the SCO-5 and placebo group exhibited no significance (P > 0.05) with respect to the baseline. Inter-group comparison at the end of the study also revealed a significant increase in melatonin levels among BCO-5 participants compared to placebo. The results are illustrated in Figure 8.
Orexin concentration
At the end of the study, the plasma concentration of orexin in the BCO-5 group, decreased significantly upon both intra- and inter-group comparisons (P < 0.001), while the placebo and SCO-5 exhibited no significant change. The results are illustrated in Figure 9.
Immunoglobulin levels
Immunoglobulins, also known as antibodies, are glycoprotein molecules produced by plasma cells (white blood cells). They act as a critical part of the immune response by specifically recognizing and binding to particular antigens, such as bacteria or viruses, and aiding in their destruction.
Intra-group analysis of IgM and IgG at the end of the study period revealed a significant increase compared to baseline (P < 0.008 and P < 0.001, respectively), whereas the placebo and SCO-5 showed no significant effect. Inter-group analysis of IgM and IgG levels also showed a significant increase compared to the placebo. The results are illustrated in Figures 10a and 10b.
TECHNICAL ADVANCEMENTS
The present disclosure described hereinabove has several technical advantages including, but not limited to, the realization of a pharmaceutical composition that:
• enhances the bioavailability of black cumin oil;
• is safe without any adverse effects;
• uses only natural substances;
• reduces the therapeutic dose of black cumin oil;
• is having significant brain health functions including the regulation of Orexin receptors, modulation of neurotransmitters to provide health benefits including sleep, stress, and cognition;
• is having a significant effect on metabolic disorders and body weight;
• contains natural ingredients that are regarded as generally safe (GRAS); and
• retains the chemical constituents in the volatile oil, thus enhancing the shelf-life.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step,” or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results. While certain embodiments of the inventions have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Variations or modifications to the formulation of this invention, within the scope of the invention, may occur to those skilled in the art upon reviewing the disclosure herein. Such variations or modifications are well within the spirit of this invention.
The numerical values given for various physical parameters, dimensions and quantities are only approximate values and it is envisaged that the values higher than the numerical value assigned to the physical parameters, dimensions and quantities fall within the scope of the invention unless there is a statement in the specification to the contrary.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation. ,CLAIMS:WE CLAIM:

1. A pharmaceutical composition comprising
a. at least one volatile oil in an amount in the range of 5 mass% to 15 mass% with respect to the total mass of the composition;
b. at least one fixed oil in an amount in the range of 30 mass% to 60 mass% with respect to the total mass of the composition;
c. at least one extract in an amount in the range of 20 mass% to 40 mass% with respect to the total mass of the composition;
d. at least one phospholipid in an amount in the range of 2 mass% to 20 mass% with respect to the total mass of the composition;
e. optionally at least one antioxidant in an amount in the range of 1 mass% to 5 mass% with respect to the total mass of the composition; and
f. optionally at least one emulsifying agent in an amount in the range of 1 mass% to 10 mass% with respect to the total mass of the composition.
2. The composition as claimed in claim 1, wherein said volatile oil is black cumin (Nigella sativa).
3. The composition as claimed in claim 1, wherein said fixed oil is at least one oil selected from the group of oils consisting of oils rich in medium chain triglycerides and oils rich in polyunsaturated fatty acids.
4. The composition as claimed in claim 3, wherein said oils rich in polyunsaturated fatty acids is at least one selected from the group consisting of black cumin (Nigella sativa) oil, olive oil, grape seed oil, pomegranate seed oil, fish oil, flax seed oil, palmetto oil, and sea buckthorn oil.
5. The composition as claimed in claim 3, wherein oils rich in medium chain triglycerides is coconut oil.
6. The composition as claimed in claim 1, wherein said extract is an alcoholic extract of black cumin seeds (Nigella sativa).
7. The composition as claimed in claim 1, wherein said extract is a supercritical fluid extract of black cumin seeds (Nigella sativa).
8. The composition as claimed in claim 1, wherein said phospholipid is at least one selected from the group consisting of lecithin, phosphatidylcholine, phosphatidylserine, lecithin-fatty oil derivatives, glycerine-lecithin blends or its derivatives, and vitamin E succinimide esters.
9. The composition as claimed in claim 1, wherein said emulsifying agent is at least one selected from the group consisting of glycerol esters of fatty acids, polyglycerol esters of fatty acids, glycerine, propylene glycol, polysorbates, and castor oil derivatives.
10. The composition as claimed in claim 1, wherein said antioxidant is at least one selected from the group consisting of tocopherol and rosemary extracts.
11. The composition as claimed in claim 1, wherein said composition comprises thymoquinone in the range of 3 vol% to 10 vol %, a-thujene in the range of 0.5 vol% to 2 vol%, carvacrol in the range of 0.5 vol% to 2 vol%, and p-cymene in the range of 0.5 vol% to 2 vol% with respect to the total volume of the composition.
12. The composition as claimed in claim 1, wherein said composition comprises thymoquinone, a-thujene, carvacrol, and p-cymene in a volume ratio in the range of 2:1:1:1 to 10:1:1:1.
13. A process for the preparation of a pharmaceutical composition, said process comprising the following steps
a. blending a predetermined amount of mechanically pressed oil with a predetermined amount of extract for a first predetermined time period to obtain a first mixture;
b. blending said first mixture with a predetermined amount of phospholipid followed by heating at a predetermined temperature for a second predetermined time period to obtain a homogeneous mixture; and
c. blending homogeneous mixture with a predetermined amount of anti-oxidant and optionally emulsifying agent to obtain a pharmaceutical composition.
14. The process as claimed in claim 13, wherein said first predetermined time period is in the range of 30 minutes to 180 minutes.
15. The process as claimed in claim 13, wherein said predetermined temperature is in the range of 45°C to 65°C.
16. The process as claimed in claim 13, wherein said second predetermined time period is in the range of 30 minutes to 90 minutes.

Dated this 11th day of September, 2023

_______________________________
MOHAN RAJKUMAR DEWAN, IN/PA – 25
of R.K.DEWAN & CO.
Authorized Agent of Applicant

TO,
THE CONTROLLER OF PATENTS
THE PATENT OFFICE, AT CHENNAI