DESC:Technical field of the invention
The present invention relates to herbal compositions comprising combination of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical in said extract and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; for stress relief and promoting sleep.

Background of the invention
Stress is the root cause of many diseases and is induced by various factors. Mental stress is a complex psychological reaction resulting in an emotional state of tension in response to different stimuli or stressors, viz., physical, psychological, or social, and intensities, acute versus chronic and high versus low intensity. Various harmful effects of stress include body’s weakened immunity and other conditions such as headache, migraine, hypertension, dyspepsia, fatigue, depression, reduced stamina, lowered mood, male impotence, hair loss, rough skin, stomach ulcers, etc.

Nervousness, tension, anxious thoughts, and physical changes like increased blood pressure characterize anxiety. Anxiety is considered one of the symptoms of clinical depression. Depression is a disorder that changes the function of the brain and controls emotions. Together, anxiety and depression are linked to attention deficit hyperactivity disorder (ADHD).

Sleep is a complex, vital, physiological process inherent in each individual. During sleep, the brain relieves mental and physical fatigue acquired during work and processes information to strengthen cognitive functions, including memory. The need for sleep is a biological drive similar to thirst or hunger. Various factors, such as stress, tension, fear, and anxiety, cause sleep disturbances or disorders. Sleep is affected by individual experiences, especially the demands from social and organizational contexts, which result in high stress levels and poor sleep quality. In addition, organic disorders have harmful effects on the body and are associated with sleep quality. Reduction of sleep quality leads to insomnia and several behavioral changes such as pain, fatigue, mood, etc.

Benzodiazepines such as diazepam, lorazepam, clonazepam, alprazolam etc., are some of the drugs available for treatment. However, excessive use of these drugs inhibits GABA abnormally and leads to several side effects.

Hence, there is an unmet remedial need for developing safe herbal solutions for stress relief and promoting sleep for physical and mental well-being.

Patent publication no. 3662/DELNP/2009 disclosed an acetone extract of Rosemary leaves for use as a medicament for the treatment of a disorder connected to reduced neurotransmission and a process comprising the following steps: a) grinding dried leaves of Rosmarinus officinalis; b) extracting the leaves of step a) with acetone to obtain an acetone extract; c) optionally filtrating the acetone extract; d) concentrating the acetone extract by (partly, preferably nearly completely) removing the acetone; e) optionally spray-drying the thus concentrated acetone extract.

Another patent publication, CN110283226 (A), disclosed Rosmarinus officinalis extraction, particularly a method for extracting antioxidant components from Rosmarinus officinalis. The method of extracting the antioxidant components from the Rosmarinus officinalis comprises the following steps: using a solvent to extract the Rosmarinus officinalis, concentrating an extracting solution, centrifuging and collecting a centrifuged supernatant and centrifuged precipitate respectively, and performing macroporous resin purification, concentration and drying on the centrifuged supernatant to separate rosmarinic acid; dissolving the centrifuged precipitate, using trapped fluid obtained by organic nanofiltration to separate ursolic acid and oleanolic acid, and using membrane passing liquid obtained by the organic nanofiltration to separate carnosic acid and carnosol. According to the method provided by the invention, water-soluble and fat-soluble antioxidant components such as rosmarinic acid, ursolic acid, oleanolic acid, carnosic acid, and carnosol can be extracted simultaneously so that the utilization rate of the Rosmarinus officinalis is improved.

The other patent publication no. US2006141063 (A1) disclosed an herbal composition that is useful as a nutrient, anti-stress, and adaptogenic agent. The composition comprises of plant juice or extracts of plants selected from the group consisting of Mangifera indica, Evolvulus alsinoides, Withania somnifera, Asparagus racemosus, and Amaranthus hypochondriacus, together with excipients/additives.

Therefore, the present inventors aim to develop safe and efficient herbal compositions to relieve stress and anxiety and promote sleep.

Objective of the invention
Therefore, the primary objective of the present invention is to provide synergistic and safe herbal compositions comprising combination of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof in said extract; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; process for their preparation and method of treatment for stress relief and promoting sleep.

Summary of the invention
Accordingly, in one aspect, the present invention provides herbal composition comprising combination of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof in said extract; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; for stress relief and promoting sleep.

In an aspect, the present invention provides a herbal composition for stress relief and sleep promotion comprising the extract of Rosmarinus officinalis in the range of about 10% to 90% and the extract of Nelumbo nucifera in the range of about 90% to 10%, wherein the extract of Rosmarinus officinalis comprises of magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid and oleanolic acid or mixtures thereof in said extract.

In another aspect, the present disclosure provides formulation of the synergistic herbal compositions comprising combination of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof in said extract; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents.

In another aspect, the present disclosure provides a process for the preparation of compositions comprising combinations of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof in said extract; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents.

In a further aspect, the present disclosure provides methods of obtaining at least one health benefit selected from relief from stress, depression, anger, anxiety, negative emotions, nervousness, tension, and/or fatigue; elevation of mood, thinking ability, concentration and/or decision making; promoting relaxation, sleep and sleep quality in a human subject; wherein the method comprises supplementing the said human subject with an effective dose of a composition comprising combination of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof in said extract; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents.

In a further aspect, the present disclosure provides use of the present herbal compositions comprising a combinations of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof in said extract; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; and optionally containing at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents; for obtaining at least one health benefit selected from relief from stress, depression, anger, anxiety, negative emotions, nervousness, tension, and fatigue; elevation of mood, thinking ability, concentration, and decision making; promoting relaxation, sleep, and sleep quality in a human subject.

Brief Description of Drawings/Figures:
Figure 1: depicts the chemical structures of carnosic acid, rosmarinic acid, ursolic acid and oleanolic acid.

Detailed description of the invention
The invention will now be described in detail in connection with certain preferred and optional embodiments so that various aspects thereof may be more fully understood and appreciated.

Unless specified otherwise, all technical and scientific terms, such as but not limited to chemical structures, process steps, or materials disclosed herein are intended, but also include equivalents thereof as commonly understood by those ordinarily skilled in the art to which this invention belongs. To describe the invention, certain terms are defined herein specifically as follows.

The term “about” is used herein to refer to a degree of deviation and means approximately, in the region of, roughly, or round. The chemical compounds may be identified by their chemical structure, chemical name, or common name as used herein. The compounds used herein may contain one or more chiral centers and/or double bonds and, therefore may exist as isomers. The terms “metal salt”, “metal complex” and “metal chelate” convey the same meaning and are used interchangeably in the specification. Thus, Rosmarinus officinalis extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid; Rosmarinus officinalis extract containing magnesium complex of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid; Rosmarinus officinalis extract containing magnesium chelate of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid; convey the same meaning and are interchangeably used throughout in the specification. The plant names Rosemary, Rosmarinus officinalis, and Salvia rosmarinus convey the same meaning and are interchangeably used throughout the specification.

The term “methods of obtaining health benefit” refers to inhibiting, preventing, or arresting the development of pathology (disease, disorder, or condition) and/or causing the reduction, remission, or regression of a pathology. Those with skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission, or regression of a pathology.

The source of the herbs used in the invention is as follows:
1. Rosmarinus officinalis leaves were procured from a vendor located in Erode, Tamil Nadu.
2. Nelumbo nucifera seed was procured from a vendor located in Mumbai, Maharashtra.

Rosmarinus officinalis: It is an evergreen perennial shrub native to the Mediterranean and Europe, cultivated in many parts of the world. The leaves are commonly used as a condiment to season food and possess a variety of pharmacological properties, including anti-aging, hepatoprotective, anti-bacterial, anti-thrombotic, anti-ulcerogenic, diuretic, anti-diabetic, antioxidant, anti-nociceptive, anti-inflammatory, and anti-depressant activities. Carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid are the major phytochemicals in Rosmarinus officinalis leaf extract and are responsible for pronounced biological activities (Figure-1). Other constituents present in Rosmarinus officinalis include flavonoids such as homoplantaginin, cirsimaritin, genkwanin, gallocatechin, nepetrin, hesperidin, 6-hydroxyluteolin-7-glucoside, luteolin-3'-glucuronide, and luteolin-3'-O-(O-acetyl)-ß-D-glucuronide.

Nelumbo nucifera also known as sacred lotus, Indian lotus, or simply lotus, is one of two extant species of aquatic plant in the family Nelumbonaceae. It is sometimes colloquially called a water lily, though this more often refers to members of the family Nymphaeaceae. It has a very wide native distribution, ranging from central and northern India through northern Indochina and East Asia with isolated locations at the Caspian Sea. The species also occurs in southern India, Sri Lanka, virtually all of Southeast Asia, New Guinea, and northern and eastern Australia,

Some of the molecules containing carboxylic acid functional groups are known to exhibit sub-optimal physicochemical or biopharmaceutical properties. These properties can be enhanced simply by converting the carboxylic acid group into metal salt, complex or chelate. Therefore, most of the drugs containing the carboxylic acid group are available as metal salts/complexes/chelates.

Magnesium is the fourth most abundant essential mineral in the body, half of which is distributed in the bone and the other half in the muscle and other soft tissues. Magnesium is one of the minerals responsible for managing bone metabolism, nerve transmission, cardiac excitability, neuromuscular conduction, muscular contraction, vasomotor tone, and blood pressure. Magnesium also plays a significant role in glucose-insulin metabolism and immune response.

Thus, given the importance of magnesium, the inventors of the present invention prepared magnesium salt of Rosmarinus officinalis extract, and this extract contains magnesium salt of carnosic acid, the magnesium salt of rosmarinic acid, the magnesium salt of ursolic acid, and magnesium salt of oleanolic acid.

Rosmarinus officinalis extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid and oleanolic acid: Rosmarinus officinalis 90% aq. ethanol extract was prepared, and the extract was treated with magnesium oxide to form magnesium salts of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid. The typical extract thus obtained contains 8.79% carnosic acid, 2.78% rosmarinic acid, 16.64% ursolic acid, 4.42% oleanolic acid, and 0.45% magnesium (Example-1). For comparison, Rosmarinus officinalis 90% aq. ethanol extract without magnesium salt was also prepared (Comparative example-1A). Similarly, Rosmarinus officinalis 80% aq. ethanol extract, ethanol extract, methanol extract, and acetone extracts were prepared and treated with magnesium oxide to form magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (Examples-2, 3, 4, and 5).

Salt or complex formation: Major constituents of Rosmarinus officinalis, i.e., carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid, can form metal salts with a base as each of these phytochemicals contain a carboxylic acid group (see Figure-1). Rosmarinus officinalis extract, when reacted with metal oxides, metal hydroxides, or metal carbonates, etc., forms metal salts of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid. For example, treatment of Rosmarinus officinalis extract with magnesium oxide results Rosmarinus officinalis extract comprising magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid. The process typically employed a fine filtration step to remove unreacted magnesium oxide, which is practically insoluble either in aq. alcohol or in water. Hence, the only probability of magnesium in the product is that it forms salts of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid. The presence of magnesium in ICP-MS confirms the formation of magnesium salt (Table 1).

Further, Rosmarinus officinalis extract comprising magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid showed a pH of about 5.6. In contrast, Rosmarinus officinalis extract without magnesium salt showed a pH of about 4.3. The difference in pH values further confirms the formation of magnesium salts of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid.

The inventors surprisingly found that the Rosmarinus officinalis extract comprising magnesium salts of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1) exhibited better efficacy in stress relief and reduced anxiety, compared to the Rosmarinus officinalis extract without magnesium (RO-1) as shown below.

Evaluation of the Rosmarinus officinalis leaves extract containing magnesium and Rosmarinus officinalis leaves extract without magnesium for relief from stress and anxiety: The efficacy of these extracts was evaluated in an in vivo experiment in chronic restraint-stressed (CRS) ICR mice (animals) as shown in example 21.

Stress is the major risk factor associated with diverse diseases like depression and anxiety, diabetes, obesity, Alzheimer etc. An in vivo experiment was conducted to evaluate the efficacy of the inventive Rosemary extract.

In the elevated plus maze and light-dark transition test, the stress and anxiety were significantly reduced in the mice supplemented with Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salts of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), when compared to the Rosmarinus officinalis leaves 90% aq. ethanol extract without magnesium (RO-1). As shown in Table 3, the percent increase of time spent in open arm (stress relief) by the ROMg-1 supplemented mice (G3 group) showed a 24.09% increase over the chronic resistance-stressed (CRS) control animals (G1), in the elevated plus maze. In contrast, the RO-1-supplemented mice (G2 group) showed only 15.61% increase over G1 mice.

Similarly, the ROMg-1 supplemented mice (G3 group) showed a 20% reduction of anxiety over the CRS animals (G1). In contrast, the RO-1-supplemented mice (G2 group) showed a 10% reduction only (Table 4).

In another test, the percent increase in the number of entries into light (stress relief) of the ROMg-1 supplemented mice (G3 group) is 21.94% over the CRS animals (G1). In contrast, the RO-1-supplemented mice (G2 group) showed only a 10.14% increase over the CRS animals (G1) (Table 5).

Increased serum level of a potential biomarker of stress and anxiety, such as dopamine, in the experimental mice, also confirms the efficacy of supplementation. ROMg-1-supplemented mice (G3 group) showed a 6.35% increase in the serum dopamine level over the CRS animals (G1); in contrast, the RO-1-supplemented mice (G2 group) showed only a 4.44% increase over the CRS animals (G1) (Table 6).

Hence, the inventive Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical in the extract selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof; surprisingly showed better efficacy in reducing stress and anxiety compared to the regular extract without a magnesium salt.

Compositions
Encouraged by the increased stress relief efficacy of Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical in the extract selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof; the inventors prepared several compositions of Rosmarinus officinalis extract containing magnesium in combination with at least one ingredient selected from the extracts, fractions, phytochemicals or mixtures thereof derived from Nelumbo nucifera. Thus, various solvent extracts of Nelumbo nucifera were prepared with different solvents.

Nelumbo nucifera seed extract: N. nucifera seeds were pulverized, and the powder was extracted with 90% aqueous ethanol followed by water. The two extracts were combined and evaporated to get N. nucifera seed extract blend of 90% aq. ethanol extract and water extract (NN-1), and the details are summarized in example 7.
Similarly, other solvents such as 90% aqueous ethanol, 50% aqueous ethanol, water and methanol were used to obtain N. nucifera seed 90% aqueous ethanol extract (NN-2), 50% aqueous ethanol extract (NN-3), water extract (NN-4) and methanol extract (NN-5) respectively (examples 8 & 9). These extracts were standardized to total saponins by gravimetry, polysaccharides by UV and proanthocyanidins by UV as summarized in Table 2.

Then, various compositions (C1–C29) of Rosmarinus officinalis extract containing magnesium in combination with at least one ingredient selected from the extracts, fractions, phytochemicals, or mixtures thereof derived from Nelumbo nucifera were prepared as described in examples 11–19.

Acetylcholinesterase (AChE): Acetylcholinesterase (AChE) is a cholinergic enzyme primarily found at the neuromuscular junctions, especially in muscles and nerves. AChE hydrolyzes Acetylcholine (Ach) into acetic acid and choline. The primary role of AChE is to terminate neuronal transmission and signaling between synapses. The link between chronic or marked acute psychological stress and depression is strong. Activation of AChE decreases the Ach levels in the brain, which is implicated in the pathophysiology of neurodegenerative disorders, including major depressive disorders (MDD). Hence, inhibition of AChE reduces Ach breakdown and improves its levels in the brain. Antidepressant-like effects of the AChE inhibitor (AChEI), Huperzine A, have been reported in rats exposed to unpredictable chronic stress. Several AChEIs are currently used world-wide to reduce stress and depression; and to elevate mood. Several studies have demonstrated that chronic administration of AChEIs such as Donepezil, Rivastigmine, and Galantamine have demonstrated clinical efficacy in improving depressive symptomology.

The compositions mentioned above (compositions 1 to 29) were evaluated for acetylcholinesterase (AChE) inhibitory activity in comparison with the corresponding individual ingredients (Example 22). The data from in vitro AChE assay revealed that these compositions unexpectedly showed better efficacy in enzyme inhibition when compared to their corresponding individual ingredients, suggesting that the Rosmarinus officinalis leaves extract comprising magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid in combination with Nelumbo nucifera seed extracts tend to exhibit synergism.

For example, Rosmarinus officinalis leaves 90% aq. ethanol extract with magnesium (ROMg-1) at 8.33 µg/mL and a blend of Nelumbo nucifera seed 90% ethanol extract and spent water extract (NN-1) at 4.17 µg/mL showed 10.21% and 3.82% AChE inhibition respectively. The composition-2 containing these two extracts at a 2:1 ratio showed 22.07% AChE inhibitory activity at 12.5 ?g/mL concentration, which is significantly better than the additive effect (10.21%+3.82% = 14.03%) from these two ingredients, suggesting a synergistic effect between Rosmarinus officinalis leaves 90% aq. ethanol extract with magnesium (ROMg-1) and Nelumbo nucifera seed extract blend (NN-1) to inhibit AChE. The compositions -1, 3, 4 and 5 obtained when combining these two ingredients at ratios 3:1, 1:1, 1:2, and 1:3, respectively, also showed synergism in inhibiting AChE compared to the inhibitions shown by their corresponding individual ingredients as summarized in Table 7. While the similar composition-2A of Rosmarinus officinalis leaves 90% aq. ethanol extract without magnesium (RO-1) and Nelumbo nucifera seed extract blend (NN-1) in the same ratio of 2:1 showed a 10.27% increase at 12.5µg/mL, which is much lower than the similar composition containing the magnesium complex. This increased inhibition of AChE of the inventive composition is surprising and unexpected.

Similarly, the other compositions (6-29) also showed synergism in acetylcholinesterase inhibition compared to the inhibitions shown by their corresponding individual ingredients, as summarized in Tables 8-11.

MAO in Stress and Sleep: Stress is one of the primary risk factors contributing to the development of many psychiatric disorders, including depressive disorders, post-traumatic stress disorder, and anxiety disorders. Stress and sleep are inter-related; higher stress levels interfere with sleep patterns. It is possible that different types of stressors, such as psychosocial, environmental, exercise etc. may bring with them different impacts on sleep. Monoamine oxidases (MAO-A and -B) play an important role in the catabolism of catecholamines, including serotonin (MAO-A), and regulating their levels in the brain. Inhibition of MAOs will improve serotonin levels in the brain and reduce stress, which could help improve sleep quality and duration.

The present inventive compositions (compositions-1 to 29) were evaluated for MAO-A and B inhibitory activity compared with the corresponding individual ingredients (Example 23). The data from in vitro MAO-A and B assay showed that these compositions unexpectedly showed better efficacy in the inhibition when compared to their corresponding individual ingredients, suggesting that the Rosmarinus officinalis leaves extract comprising magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid in combination with Nelumbo nucifera seed extracts tend to exhibit synergism.

For example, Rosmarinus officinalis leaves 90% aq. ethanol extract with magnesium (ROMg-1) at 0.67 µg/mL and Nelumbo nucifera seed extract blend (NN-1) at 0.33 µg/mL showed 9.85% and 1.91% MAO-A inhibition, respectively. The composition-2 containing these two extracts at 2:1 ratio showed 21.02% MAO-A inhibition at 1 ?g/mL concentration, which is significantly better than the additive effect (9.85% + 1.91% = 11.76%) from these two ingredients, suggesting a synergistic effect between Rosmarinus officinalis leaves 90% aq. ethanol extract with magnesium (ROMg-1) and Nelumbo nucifera seed extract blend (NN-1) in inhibiting MAO-A. The compositions-1, 3, 4 and 5 obtained when combining these two ingredients at ratios 3:1, 1:1, 1:2, and 1:3, respectively, also showed synergism in MAO-A inhibition compared to the inhibitions shown by their corresponding individual ingredients as summarized in Table 12. While the similar composition-2A of Rosmarinus officinalis leaves 90% aq. ethanol extract without magnesium (RO-1) and Nelumbo nucifera seed extract blend (NN-1) in the same ratio of 2:1 showed a 11.37% inhibition at 1 µg/mL, which is much lower than the MAO-A inhibition exhibited a similar composition containing the magnesium complex. This increased inhibition of MAO-A of the inventive composition is surprising and unexpected. Similarly, the other compositions (6-29) also showed synergism in MAO-A inhibition compared to the inhibitions shown by their corresponding individual ingredients as summarized in Tables 13-16.

Similarly, Rosmarinus officinalis leaves 90% aq. ethanol extract with magnesium (ROMg-1) at 0.67 µg/mL and Nelumbo nucifera seed extract (NN-1) at 0.33 µg/mL showed 4.18% and 4.74% MAO-B inhibition respectively. The composition-2 containing these two extracts at a 2:1 ratio showed 17.43% MAO-B inhibition at 1 ?g/mL concentration, which is significantly better than the additive effect 4.18% + 4.74% = 8.92%) from these two ingredients, suggesting a synergistic effect between Rosmarinus officinalis leaves 90% aq. ethanol extract with magnesium (ROMg-1) and Nelumbo nucifera seed extract blend (NN-1) in inhibiting MAO-B. The compositions-1, 3, 4 and 5, which were obtained when combining these two ingredients at ratios 3:1, 1:1, 1:2, and 1:3, respectively, also showed synergism in MAO-B inhibition compared to the inhibitions shown by their corresponding individual ingredients, as summarized in Table 17. While the similar composition-2A containing Rosmarinus officinalis leaves 90% aq. ethanol extract without magnesium (RO-1) and Nelumbo nucifera seed extract blend (NN-1) in the same ratio of 2:1 showed a 8.91% increase at 1 µg/mL, which is much lower than the inhibition shown by a similar composition containing the magnesium complex. This increased inhibition of MAO-B of the inventive composition is surprising and unexpected. Similarly, the other compositions (6-29) also showed synergism in MAO-A inhibition compared to the inhibitions offered by their corresponding individual ingredients, as summarized in Tables 18-21.

GABAA- receptor-a in Stress and Sleep: Gamma-aminobutyric acid (GABA) type A receptor (GABAA) is one of the major neurotransmitter receptors responsible for sleep regulation. GABA is an inhibitory neurotransmitter that dampens the excitatory neurons in the central nervous system (CNS) and peripheral nervous system (PNS). GABA binds to its receptor to exert its physiological actions. Therefore, an increase in GABA receptors is crucial for enhanced GABA signaling. Modulation of the GABAA receptor is one of the key strategies that are approved for developing pharmacological therapies for insomnia and sleep disorders. Allosteric modulators, such as benzodiazepines, barbiturates, and neurosteroids, can enhance GABAA receptor function. However, excessive use of these drugs can lead to side effects, including psychomotor impairment i.e., loss of muscle control, sedation, memory loss etc. Hence, safer therapeutic strategy including the development of herbal remedies that increase GABAergic neurotransmission through GABAA receptor activation, are warranted to relieve stress and anxiety and promote sleep.

The present inventive compositions (compositions-1 to 29) were tested for GABAA-Ra (GABA type A receptor, subunit a) expression in comparison with the corresponding individual ingredients (Example 24). The data from in vitro GABAA-Ra assay showed that these compositions unexpectedly increased the receptor expressions when compared to their corresponding individual ingredients, suggesting that the Rosmarinus officinalis leaves extract comprising magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid in combination with Nelumbo nucifera seed extracts tend to exhibit synergism.

For example, Rosmarinus officinalis leaves 90% aq. ethanol extract with magnesium (ROMg-1) at 66.67 ng/mL and Nelumbo nucifera seed extract blend (NN-1) at 33.33 ng/mL showed 0.39- and 0.19- units of GABAA-Ra expressions, respectively. The composition-2 containing these two extracts at a 2:1 ratio showed a 0.86unit of GABAA-Ra expression at 100 ng/mL concentration, which is significantly better than the additive effect (0.39 + 0.19 = 0.58 unit) from these two ingredients, which is 48.3% better that the additive effect, suggesting a synergistic effect between Rosmarinus officinalis leaves 90% aq. ethanol extract with magnesium (ROMg-1) and Nelumbo nucifera seed extract blend (NN-1) to increase GABAA-Ra. Similarly, the compositions-1, 3, 4 and 5, which were obtained when combining these two ingredients at ratios 3:1, 1:1, 1:2, and 1:3, respectively, also showed synergism in increasing GABAA-Ra expression when compared to the expressions (% increase) shown by their corresponding individual ingredients as summarized in Table 22. In contrast, the composition-2A containing Rosmarinus officinalis leaves 90% aq. ethanol extract without magnesium (RO-1) and Nelumbo nucifera seed extract blend (NN-1) in the same ratio of 2:1 showed a 0.53 units GABAA-Ra of expression at 100 ng/mL, which is much lower (12.7% increase as compared to 48.3% increase) than that exhibited by a similar composition containing the magnesium complex (Comp-2). This increased GABAA-Ra expression by the inventive composition is surprising and unexpected. Similarly, the other compositions (6-29) also showed synergism in GABAA-Ra overexpression as compared to their corresponding individual ingredients, as summarized in Tables 23-26.

Furthermore, the observations from the in vivo preclinical efficacy experiments clearly establish that composition containing Rosmarinus officinalis extract containing magnesium (ROMg-1) and Nelumbo nucifera seed extract blend (NN-1) confer synergistic benefits in reducing chronic stress and anxiety in the experimental mice. Importantly, these benefits are greater than the corresponding composition without magnesium (Tables 3-26).

Formulations: The synergistic herbal compositions comprising combination of the first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof in said extract; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; may be formulated with at least one component selected from pharmaceutically or nutraceutically or dietically acceptable excipients, carriers and diluents.

The synergistic compositions may be formulated using pharmaceutically or nutraceutically or dietically acceptable excipients, carriers, and diluents; monosaccharides, disaccharides, polysaccharides, dextrins, polyhydric alcohols or sugar alcohols, cellulose-based derivatives, silicates, metallic stearates, organic acids, fatty acid esters, calcium pantothenate, amino acids, proteins, organic metal salts, natural pigments, flavours, Class I & Class II preservatives and mixtures thereof.

Monosaccharides are selected from but not limited to glucose, dextrose, fructose, galactose; Disaccharides are selected but not limited to sucrose, maltose, lactose, lactulose, trehalose cellobiose, chitobiose; Polysaccharides are selected from but not limited to starch and modified starch such as sodium starch glycolate, pre-gelatinized starch, soluble starch, ultrasperse A and other modified starches; Dextrins are selected from but not limited to yellow dextrin, white dextrin, maltodextrin, glucidex 12D, rice maltodextrin, Tapioca/Cassava Maltodextrin; Polyhydric alcohols or sugar alcohols are selected but not limited to sorbitol, mannitol, inositol, xylitol, isomalt; Cellulose based derivatives are selected from but not limited to microcrystalline cellulose, hydroxy propyl methyl cellulose, hydroxy ethyl cellulose; Silicates are selected but not limited to neusilin, veegum, talc, colloidal silicon dioxide, syloids; Metallic stearates are selected from but not limited to calcium stearate, magnesium stearate, zinc stearate; Organic acids are selected from but not limited to citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid; Fatty acid esters are selected but not limited to acacia, carrageenan, guar gum, xanthan gum; Proteins are selected from but not limited to whey protein, whey isolate, casein, gelatin, pectin, agar; Organic metal salts are selected from but not limited to sodium chloride, calcium chloride, dicalcium phosphate, zinc sulphate, zinc chloride.

For example, composition 30 was prepared by combining 2 parts of Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1) and 1 part of Nelumbo nucifera seed extract blend (NN-1) along with excipients such as soluble starch and colloidal silicon dioxide in aqueous ethanol (Example 20).

After identifying the increased efficacy of Rosmarinus officinalis extract containing magnesium, the compositions comprising Rosmarinus officinalis extract containing magnesium with Nelumbo nucifera extract blend (NN-1) along with excipients (comp - 30) was evaluated for relief from stress and anxiety in an animal model.

Evaluation of the composition containing Rosmarinus officinalis extract containing magnesium salt (ROMg-1) and Nelumbo nucifera extract blend (NN-1) along with excipients for stress relief and anxiety in chronic restraint-stressed mice: The efficacy of the composition-30 was evaluated in an in vivo experiment performed in chronic restraint-stressed ICR mice (example 25). The data from in vivo study unexpectedly showed better efficacy for the composition-30 in reducing stress and anxiety. The comp-30 at 150 mg dose showed significant reduction in stress as indicated by a 44.32% increase over the control animals (G1) in the time spent in open arm in the elevated plus maze and light-dark transition test (stress and anxiety) (Table-27). In addition, composition-30 at 150 mg dose showed 55.9% increase over the control animals (G1) in the number of entries into the light (Table 29), further supporting the stress reduction in supplemented animals. These results further indicate reduction in anxiety in the experimental animals supplemented with comp-30 as supported by a 43.6% reduction in anxiety index.

Further, the serum levels of some critical neurohormones that regulate stress and sleep, such as dopamine, serotonin, and corticosterone, were also measured to evaluate the efficacy of the compositions.
Dopamine: Dopamine and serotonin play a pivotal role in the sleep-wake cycle. Dopamine has a dynamic role in regulating circadian rhythm through its activity in the pineal gland. Impaired circadian rhythm provokes severe behavioral disorders. It essentially wakes up the brain by inhibiting norepinephrine. Norepinephrine enhances melatonin production, facilitating sleep, whereas dopamine mitigates melatonin during the onset of the day, promoting the wake-up process. Thus, regulating circadian rhythm through dopamine opens many novel therapeutic avenues. In line with this, disturbance in circadian rhythm can be reduced by modulating dopamine activity, and the quality of sleep can be promoted.

Serotonin: Serotonin, a well-known neurohormone mediator in the central nervous system, plays a vital role in sleep regulation. It is a precursor of melatonin and plays a vital role in sleep-wake cycles. Serotonin primarily promotes wakefulness. However, there is also evidence for its possible role in enhancing sleep. The multifarious receptor types regulate sleep or activity promotion since they have specific roles in enzyme activities, ion channel regulation etc. It is also evident that serotonin imbalance induces depression and sleep disorder symptoms. Entry of serotonin into the neuron can initiate multifarious pathways. Acceptance or rejection of serotonin into the neuronal cell modulates the brain's activities, leading to happiness or depression. A similar tendency is also observed with sleep induction and wakefulness. This emphasizes that serotonin and serotonin receptors play a key role in sleep regulation.

Corticosterone: Corticosterone, an adrenal steroid, is an essential component of the stress response in rodents. It primarily plays a crucial role in rapid eye movement (REM) sleep. Increased levels of corticosterone result in enhanced sleep latency, increased waking time after sleep onset, the number of awakening incidences, and reduced time of non-rapid eye movement (NREM) sleep. The circadian clock mediates the secretion of both serotonin and corticosterone in the suprachiasmatic nucleus. Various studies have established that temporal control of the hypothalamic-pituitary-adrenal axis regulates the circadian rhythms of circulating corticosterone.

The data on the serum levels of the neurohormones unexpectedly showed improved efficacies in the animals supplemented with comp-30, compared to the vehicle control (CMC-Na) supplemented animals. For example, comp-30 at 150 mg dose showed 8.4% increase in serum dopamine levels over the control animals (G1) (Table 30). Similarly, comp-30 also showed a 13.4% increase in serum serotonin levels compared to the control animals (G1) (Table 31). Further, the serum corticosterone levels were reduced by 28.7% in animals supplemented with comp-30, compared to the control animals (G1) (Table 32).

Evaluation of the composition 30 for sleep latency and duration in Thiopental-induced mice:
The efficacy of the comp-30 was evaluated in an in vivo experiment performed in Thiopental-induced mice (example 26). The animals supplemented with composition-30 unexpectedly showed better efficacy in promoting sleep. The mean sleep onset time in the mice supplemented with in the 50- and 100- mg/kg of comp-30 was reduced by 19.22% and 29.81%, respectively, compared to the control mice (G1). These reductions are significantly better than the control animals. Similarly, the 50 and 100- mg/kg of comp-30 supplemented groups (G2 and G3) showed considerable improvements in sleeping time or sleep duration by 32.33% and 45.54%, respectively, compared to the control animals. Overall, supplementation of both doses of comp-30 significantly and dose-dependently improved the sleep parameters in the experimental animals (Table 33).

In conclusion, the foregoing observations on the behavioral changes in the elevated plus maze and light-dark transition experiments, modulations of the serum levels of the endocrine factors and improvement in sleep parameters suggest that the present inventive herbal compositions reduced anxiety and stress in the experimental animals. Given that increased stress and anxiety worsen sleep quality, these botanical compositions are expected to improve sleep quality or mitigate sleep disorders. Interestingly, supplementation of these compositions positively modulated the endocrine factors regulating sleep quality and stress levels. Together, these observations suggest that the disclosed herbal compositions have the potential to relieve stress, anxiety, and depression, elevate mood, and improve sleep quality.

Process: The process for the preparation of compositions comprising a combination of first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof in said extract; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; which comprises the following steps of;
(i) Extracting Rosmarinus officinalis powder with a suitable solvent;
(ii) Treating the extract with a compound of magnesium source;
(iii) Filtering, evaporating the solvent and drying the residue to obtain Rosmarinus officinalis extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid;
(iv) Blending the extract of step (iii) with at least one extract derived from Nelumbo nucifera; in the presence of a suitable solvent; and optionally with suitable excipients; and
(v) Drying the product to obtain the composition.

The suitable solvent used in the process for the preparation of Rosmarinus officinalis extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid, and Nelumbo nucifera extract according to the present invention wherein the suitable solvent is selected from but not limited to C1-C5 alcohols, like ethanol, methanol, n-butanol, water, and mixtures thereof; ketones selected from acetone, methylisobutyl ketone, chlorinated solvents selected from methylene dichloride and chloroform; water and mixtures thereof; C1-C7 hydrocarbons such as but not limited to hexane, pentane; esters such as but not limited to ethyl acetate and mixtures thereof.

The magnesium source used in the process is selected from the oxides, hydroxides, or carbonates; examples include magnesium oxide, magnesium hydroxide, and magnesium carbonate.

Thus, the present synergistic herbal compositions comprising combination of first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical in the extract selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; relieve stress and anxiety and promote sleep.

Therefore, in an important embodiment, the present invention provides synergistic herbal compositions comprising the combination of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical in the extract selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; for stress relief and promoting sleep.

In an embodiment, the present invention discloses a herbal composition for stress relief and sleep promotion comprising the extract of Rosmarinus officinalis in the range of about 10% to 90% and extract of Nelumbo nucifera in the range of about 90% to 10%, wherein the extract of Rosmarinus officinalis comprises of magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid and oleanolic acid or mixtures thereof.

In one preferred embodiment, the present invention provides synergistic herbal compositions comprising the combination of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical in the extract selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; further containing optionally at least one additional component selected from the group consisting of biological agent(s), and Nootropic agent(s); pharmaceutically acceptable active ingredients, vitamins, minerals; pharmaceutically or nutraceutically or dietically acceptable, excipients, carriers or diluents.

In one preferred embodiment, the present invention provides synergistic herbal compositions comprising combination of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical in the extract selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; wherein the concentration of Rosmarinus officinalis extract in the composition varies in the range of 10%–90% by weight, and Nelumbo nucifera in the composition varies in the range of 90%–10% by weight.

In another embodiment, the present invention discloses synergistic herbal compositions as described above, wherein the Rosmarinus officinalis extract in the composition is standardized to carnosic acid, rosmarinic acid, ursolic acid, oleanolic acid and/or a total of these four markers; and magnesium; and Nelumbo nucifera extract in the composition is standardized to at least one phytochemical reference marker compound or pharmacologically active marker but not limited to total saponins, polysaccharides or proanthocyanidins.

In another embodiment, the seed extract of Nelumbo nucifera comprises of saponins, polysaccharides or proanthocyanidins or mixtures thereof.

In another embodiment, the present invention discloses synergistic herbal compositions as described above, wherein the Rosmarinus officinalis extract in the composition is standardized to carnosic acid in the range of 2–20%, rosmarinic acid is in the range of 0.5–6%, ursolic acid is in the range of 2–30%, oleanolic acid is in the range of 1.0–10%, and magnesium is in the range of 0.1–2%.

In another embodiment, the present invention discloses synergistic herbal compositions as described above, wherein the Nelumbo nucifera extract, fraction, or mixtures thereof in the composition are standardized to at least one phytochemical reference marker compound or pharmacologically active marker, wherein phytochemical marker compound or group of phytochemical compounds is in the concentration range of 0.01% to 50% by weight of the extract.

In another embodiment, the compositions as disclosed above, wherein the phytochemical reference marker compound or pharmacologically active marker or group of markers or its magnesium salt, may be estimated by several analytical techniques known in the art such as but not limited to HPLC, gravimetry, UV, GC and ICP-mass.

In yet another embodiment, the present invention provides synergistic herbal compositions as described above, wherein the Rosmarinus officinalis and Nelumbo nucifera extracts or fractions are obtained from at least one plant part selected from the group comprising leaves, stems, tender stems, tender twigs, aerial parts, whole fruit, fruit peel, fruit rind, immature fruit, seeds, flower heads, root, bark, hardwood, rhizome or whole plant or mixtures thereof.

In another embodiment, the present invention provides synergistic herbal compositions as disclosed above, wherein the Rosmarinus officinalis and Nelumbo nucifera extracts or fractions or mixtures thereof are produced using at least one solvent selected from C1–C5 alcohols selected from ethanol, methanol, n-propanol, isopropyl alcohol; ketones selected from acetone, methyl isobutyl ketone, chlorinated solvents selected from methylene dichloride and chloroform; water and mixtures thereof; C1–C7 hydrocarbons such as but not limited to hexane, pentane; esters such as but not limited to ethyl acetate and mixtures thereof.

The process of extraction of Rosmarinus officinalis and Nelumbo nucifera involves the use of known techniques such as maceration, digestion, decoction, infusion, percolation, Soxhlet extraction, refluxing, continuous, ultrasound-assisted, microwave-assisted, pressurized liquid/fluid extraction, supercritical carbon dioxide extraction. Further, the extracts or fractions may be purified using column chromatography or resin chromatography using a microporous resin.

In another embodiment, the present invention provides a process for the preparation of compositions comprising combination of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical in the extract selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; further containing optionally at least one additional component selected from the group consisting of biological agent(s), and Nootropic agent(s); pharmaceutically acceptable active ingredients, vitamins, minerals; pharmaceutically or nutraceutically or dietically acceptable, excipients, carriers or diluents.

The suitable solvent used in the process for the preparation of Rosmarinus officinalis extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid, according to the present invention, is selected from but not limited to C1–C5 alcohols, like ethanol, methanol, n-butanol and mixtures thereof. The magnesium source used in the process is selected from the oxides, hydroxides, or carbonates; examples include magnesium oxide, magnesium hydroxide, and magnesium carbonate.

In yet another embodiment, the present invention provides methods of obtaining at least one health benefit selected from relief from stress, depression, anger, anxiety, negative emotions, nervousness, tension, and fatigue; elevation of mood, thinking ability, concentration and decision making; promoting relaxation, sleep and sleep quality in a human subject; wherein the method comprises supplementing the said human subject with an effective dose of a composition comprising combination of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical in the extract selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; further containing optionally at least one additional component selected from the group consisting of biological agent(s), and Nootropic agent(s); pharmaceutically acceptable active ingredients, vitamins, minerals; pharmaceutically or nutraceutically or dietically acceptable, excipients, carriers or diluents.

In yet another embodiment, the present disclosure provides use of the present herbal composition comprising combination of a first ingredient selected from Rosmarinus officinalis extract containing magnesium salt or complex or chelate of at least one phytochemical in the extract selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof; and a second ingredient selected from extract, fraction, phytochemical or mixtures thereof derived from Nelumbo nucifera; further containing optionally at least one additional component selected from the group consisting of biological agent(s), and Nootropic agent(s); pharmaceutically acceptable active ingredients, vitamins, minerals; pharmaceutically or nutraceutically or dietically acceptable, excipients, carriers or diluents; obtaining at least one health benefit selected from relief from stress, depression, anger, anxiety, negative emotions, nervousness, tension, and fatigue; elevation of mood, thinking ability, concentration, and decision making; promoting relaxation, sleep and sleep quality in a human subject.

In another embodiment of the invention, the composition as disclosed above is formulated into a dosage form selected from dry powder form, liquid form, beverage, food product, dietary supplement, or any suitable form such as a tablet, a capsule, a soft chewable tablet, gummies or gummy bar.

In another embodiment of the invention, the composition as disclosed above is formulated into a nutritional/dietary supplement that can be contemplated/made into the dosage form of healthy foods or food for specified health uses, such as solid food like chocolate or nutritional bars, semisolid food like cream, jam, or gel or beverage such as refreshing beverage, lactic acid bacteria beverage, drop, candy, chewing gum, gummy candy, yogurt, ice cream, pudding, soft adzuki bean jelly, jelly, cookie, tea, soft drink, juice, milk, coffee, cereal, snack bar.

In another embodiment of the invention, the composition disclosed above is formulated into a controlled-release tablet, using controlled-release polymer-based coatings by techniques including nanotechnology, microencapsulation, colloidal carrier systems, and other drug delivery systems for obtaining the desired therapeutic benefit.

Those of ordinary skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments or examples disclosed herein but is intended to cover modifications within the objectives and scope of the present invention as defined in the specification. The examples are given solely for illustration and are not to be construed as limitations of the present disclosure, as many variations thereof possible without departing from the spirit of the disclosure.

Examples

Example 1: Preparation of Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1).

Dried Rosemary leaf raw material was pulverized, and the powder (200 g) was extracted with 90% aq. ethanol (1.2 L) at hot conditions for 2 h. The mixture was filtered, and the extraction process was repeated twice with 90% aq. ethanol (2 × 800 mL). The combined 90% aq. ethanol extract was evaporated to 400 mL of volume and magnesium oxide (400 mg) was Added. The mixture was stirred at ambient temperature for 2 h, and evaporated under reduced pressure to a syrupy mass. The syrupy mass was dried at 70–75oC under vacuum for 12 h to give the product a pale green color solid (ROMg-1, 47 g).

Comparative Example 1A (RO-1): The comparative extract (RO-1) was prepared as described in example 1, without using magnesium oxide.

Example 2: Preparation of Rosmarinus officinalis leaves 80% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-2).

Rosmarinus officinalis leaves 80% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid was prepared as described in example-1 employing 80% aq. ethanol in place of 90% aq. ethanol.

Example 3: Preparation of Rosmarinus officinalis leaves ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-3).

Rosmarinus officinalis leaves ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid was prepared as described in example-1 employing ethanol in place of 90% aq. ethanol.

Example 4: Preparation of Rosmarinus officinalis leaves methanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-4).

Rosmarinus officinalis leaves methanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid was prepared as described in example-1 employing methanol in place of 90% aq. ethanol.

Example 5: Preparation of Rosmarinus officinalis leaves acetone extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-5).

Rosmarinus officinalis leaves acetone extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid was prepared as described in example-1 employing acetone in place of 90% aq. ethanol.

Example 6: Standardization of Rosmarinus officinalis leaves extracts
The various extracts of Rosmarinus officinalis leaves were standardized to carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid by analytical HPLC and Magnesium by ICP-MS, and the results are summarized in Table 1.

Table 1: Analytical results of Rosmarinus officinalis leaf extracts
Ex. No. Extract code Extraction solvent HPLC Magnesium by ICP-MS
Carnosic acid Rosmarinic acid Ursolic acid Oleanolic acid
1 ROMg-1 90% aq. ethanol 8.79% 2.78% 16.64% 4.42% 0.45%
1A RO-1 90% aq. ethanol 9.43% 2.33% 14.79% 4.45% ---
2 ROMg-2 80% aq. ethanol 5.87% 2.96% 8.62% 3.86% 0.35%
3 ROMg-3 Ethanol 11.79% 2.07% 14.8% 4.88% 0.3%
4 ROMg-4 Methanol 8.77% 2.19% 16.12% 5.72% 0.3%
5 ROMg-5 Acetone 21.49% 1.01% 11.08% 6.49% 0.33%

Example 7: Preparation of Nelumbo nucifera seed extract blend (NN-1).

500 g of dried Nelumbo nucifera seeds were ground to powder and extracted with 90% aqueous ethanol (2500 mL) at ambient temperature for 2 h. The 90% aqueous ethanol extract was separated, and the remaining material was extracted with 90% aqueous ethanol (2×2000 mL) under similar extraction conditions. The combined 90% aqueous ethanol extracts was filtered on a supercell and evaporated to a volume of 200 mL under reduced pressure. After 90% aqueous ethanol extraction, the spent material was further extracted with water (2250 mL) at ambient temperature for 2 h. The mixture was filtered, and the extraction process was repeated twice with water (2×1250 mL) under similar extraction conditions. The combined water layer was filtered on a supercell and evaporated to a volume of 200 mL under reduced pressure. The 90% aqueous ethanol extract and water extract were combined and concentrated under vacuum to obtain the Nelumbo nucifera seed extract blend as a pale brown color powder (NN-1, 93.5 g).

Example 8: Preparation of Nelumbo nucifera seed 90% aqueous ethanol extract (NN-2)
To Nelumbo nucifera seed powder (100 g), 90% aqueous ethanol (600 mL) was added, and the mixture was heated at 65-70oC for 2 h. The mixture was filtered, and the extraction process was repeated with 90% aqueous ethanol (2 x 400 mL). The combined 90% aqueous ethanol extract was evaporated under reduced pressure to give the product as a pale green color solid (NN-2, 11.5 g).

Example 9: Preparation of Nelumbo nucifera seed 50% aqueous ethanol extract, water extract, and methanol extract.
The 50% aqueous ethanol extract (NN-3; 14.0 g), water extract (NN-4; 7.5 g), and methanol extract (NN-5; 11.0 g) of Nelumbo nucifera seed were obtained by adopting a similar extraction procedure described above using 50% aqueous ethanol, water, and methanol as extraction solvents respectively.

Example 10: Standardization of Nelumbo nucifera seed extracts
The various extracts of Nelumbo nucifera seed were standardized to Total saponins by gravimetry, Polysaccharides by UV, and Proanthocyanidins by UV, and the results are summarized in Table 2.
Table 2: Analytical results of Nelumbo nucifera seed extracts
S. No. Extract code Extraction solvent Total saponins by gravimetry Polysaccharides by UV Proantho-cyanidins
by UV
1 NN-1 90% ethanol followed by water 47.85% 51.64% 0.93%
2 NN-2 90% ethanol 60.60% 64.62% 16.11%
3 NN-3 50% ethanol 69.01% 77.15% 7.40%
4 NN-4 Water 48.24% 35.99% 1.26%
5 NN-5 Methanol 61.51% 70.23% 18.37%

Example 11: Preparation of various compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1) and Nelumbo nucifera seed extract blend (NN-1).
Comp-1: C-1 was prepared by combining ROMg-1 and NN-1 in a ratio of 3:1.
Comp-2: C-2 was prepared by combining ROMg-1 and NN-1 in a ratio of 2:1.
Comp-3: C-3 was prepared by combining ROMg-1 and NN-1 in a ratio of 1:1.
Comp-4: C-4 was prepared by combining ROMg-1 and NN-1 in a ratio of 1:2.
Comp-5: C-5 was prepared by combining ROMg-1 and NN-1 in a ratio of 1:3.
Composition for comparison (comp-2A): Comp-2A was prepared by combining Rosmarinus officinalis leaves 90% aq. ethanol extract without magnesium (RO-1) and Nelumbo nucifera seed extract blend (NN-1) in a ratio of 2:1.

Example 12: Preparation of various compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed 90% ethanol extract (NN-2).
Comp-6: C-6 was prepared by combining ROMg-1 and NN-2 in a ratio of 2:1.
Comp-7: C-7 was prepared by combining ROMg-1 and NN-2 in a ratio of 1:1.
Comp-8: C-8 was prepared by combining ROMg-1 and NN-2 in a ratio of 1:2.

Example 13: Preparation of various compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed 50% ethanol extract (NN-3).
Comp-9: C-9 was prepared by combining ROMg-1 and NN-3 in a ratio of 2:1.
Comp-10: C-10 was prepared by combining ROMg-1 and NN-3 in a ratio of 1:1.
Comp-11: C-11 was prepared by combining ROMg-1 and NN-3 in a ratio of 1:2.

Example 14: Preparation of various compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed water extract (NN-4).
Comp-12: C-12 was prepared by combining ROMg-1 and NN-4 in a ratio of 2:1.
Comp-13: C-13 was prepared by combining ROMg-1 and NN-4 in a ratio of 1:1.
Comp-14: C-14 was prepared by combining ROMg-1 and NN-4 in a ratio of 1:2.

Example 15: Preparation of various compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed methanol extract (NN-5).
Comp-15: C-15 was prepared by combining ROMg-1 and NN-5 in a ratio of 2:1.
Comp-16: C-16 was prepared by combining ROMg-1 and NN-5 in a ratio of 1:1.
Comp-17: C-17 was prepared by combining ROMg-1 and NN-5 in a ratio of 1:2.

Example 16: Preparation of various compositions containing Rosmarinus officinalis leaves 80% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-2) and Nelumbo nucifera seed extract blend (NN-1).
Comp-18: C-18 was prepared by combining ROMg-2 and NN-1 in a ratio of 2:1.
Comp-19: C-19 was prepared by combining ROMg-2 and NN-1 in a ratio of 1:1.
Comp-20: C-20 was prepared by combining ROMg-2 and NN-1 in a ratio of 1:2.

Example 17: Preparation of various compositions containing Rosmarinus officinalis leaves ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-3) and Nelumbo nucifera seed water extract (NN-4).
Comp-21: C-21 was prepared by combining ROMg-3 and NN-4 in a ratio of 2:1.
Comp-22: C-22 was prepared by combining ROMg-3 and NN-4 in a ratio of 1:1.
Comp-23: C-23 was prepared by combining ROMg-3 and NN-4 in a ratio of 1:2.

Example 18: Preparation of various compositions containing Rosmarinus officinalis leaves methanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-4) and Nelumbo nucifera seed 50% aq. ethanol extract (NN-3).
Comp-24: C-24 was prepared by combining ROMg-4 and NN-3 in a ratio of 2:1.
Comp-25: C-25 was prepared by combining ROMg-4 and NN-3 in a ratio of 1:1.
Comp-26: C-26 was prepared by combining ROMg-4 and NN-3 in a ratio of 1:2.

Example 19: Preparation of various compositions containing Rosmarinus officinalis leaves acetone extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-5) and Nelumbo nucifera seed methanol extract (NN-5).
Comp-27: C-27 was prepared by combining ROMg-5 and NN-5 in a ratio of 2:1.
Comp-28: C-28 was prepared by combining ROMg-5 and NN-5 in a ratio of 1:1.
Comp-29: C-29 was prepared by combining ROMg-5 and NN-5 in a ratio of 1:2.

Example 20: Formulation of the compositions
Comp-30: A mixture of Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1, 600 g) and 70% aqueous ethanol (900 mL) was stirred at ambient temperature for 10–15 min. Added Nelumbo nucifera seed extract blend (NN-1, 300 g) to the mixture slowly and stirred for further 10–15 min. R.O water (2700 mL) was added and mixed the contents thoroughly, then soluble starch (80 g) was added and stirred further for 20–30 min. The contents were dried at ambient temperature under reduced pressure to give the product as flakes. These flakes are homogenously blended with colloidal silicon dioxide (20 g) in a polyethylene bag or any suitable blender. The material was pulverized further and sieved through the #40 mesh to get the composition as a fine powder (Comp-30).

Example 21: Evaluation of the rosemary extracts for relief from stress and anxiety in chronic restraint-stressed ICR mice
Treatments: Eighteen chronic restraint-stressed (CRS) ICR mice were randomly assigned to three groups, G1 to G3 (n=6). The animals were supplemented (using oral gavage) with one of 0.1% w/v CMC-Na (G1 group), 150 mg/kg of RO-1 (G2), and 150 mg/kg of ROMg-1 (G3) in 10 mL of 0.1% CMC-Na in water, for 15 days. The elevated plus maze and light-dark transition test were performed on day 15 and day 16, respectively. On day 17, after 1hr of supplementation, blood samples were collected from the retro-orbital plexus of all animals under mild isoflurane anesthesia. Serum was separated by centrifuging the blood samples at 3200g for 15 minutes at 4ºC and stored at -80ºC until analysis. Post blood collections on day 17, all surviving animals were euthanized using CO2 asphyxiation followed by exsanguination and necropsy.

Test parameters: The elevated plus maze and light-dark transition test were conducted as per the procedures disclosed in the literature, and the results are presented in Tables 3–5. In the elevated plus maze experiment, time spent in the open arm and the number of entries in the open arm were recorded, and the anxiety index was calculated using the following formula.

Anxiety index=[1-{(open arm time÷total time on maze)+(open arm entry÷total entry)}]÷2

Dopamine, a bio-marker related to stress and anxiety, its serum levels were evaluated using a commercially available ELISA kit, and the results are presented in Tables 6.

Table 3: Percent increase of time spent in open arm (stress relief) by Rosmarinus officinalis extracts (ROMg-1 or RO-1)-supplemented chronic restraint-stressed (CRS) mice
Groups Supplementation Mean ± SD of time spent in open arm (sec.) Percent increase in time (from G1)
G1 CMC-Na 10.25±1.259 --
G2 Rosmarinus officinalis leaves 90% aq. ethanol extract without magnesium (RO-1) 11.85±1.02 15.61
G3 Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium (ROMg-1) 12.72±1.63 24.09
Note: The increase in percentage time indicates better efficacy

Table 4: Percent reduction of anxiety in Rosmarinus officinalis extracts (ROMg-1 or RO-1)-supplemented chronic restraint-stressed (CRS) mice
Groups Supplementation Anxiety index
(Mean ± SD) Percent reduction of anxiety (from G1)
G1 CMC-Na 0.40±0.05 --
G2 Rosmarinus officinalis leaves 90% aq. ethanol extract without magnesium (RO-1) 0.36±0.04 10.0
G3 Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium (ROMg-1) 0.32±0.02 20.0
Note: The decrease in percentage indicates better efficacy

Table 5: Percent increase of entries in light by Rosmarinus officinalis extracts (ROMg-1 or RO-1)-supplemented chronic restraint-stressed (CRS) mice
Groups Supplementation Number of entries in light (Mean ± SD) Percent increase in the number of entries (from G1)
G1 CMC-Na 12.03±0.77 --
G2 Rosmarinus officinalis leaves 90% aq. ethanol extract without magnesium (RO-1) 13.25±1.02 10.14
G3 Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium (ROMg-1) 14.67±1.14 21.94
Note: The increase in percentage indicates better efficacy

Table 6: Percent increase of serum dopamine levels in Rosmarinus officinalis extracts (ROMg-1 or RO-1)-supplemented chronic restraint-stressed (CRS) mice
Groups Supplementation Mean ± SD of serum Dopamine concentration (ng/mL) Percent increase in Dopamine (from G1)
G1 CMC-Na 106.26±2.63 --
G2 Rosmarinus officinalis leaves 90% aq. ethanol extract without magnesium (RO-1) 110.98±2.03 4.44
G3 Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium (ROMg-1) 113.01±1.54 6.35
Note: The increase in percentage indicates better efficacy

Example 22: Procedure for Acetylcholinesterase inhibitory activity
Acetylcholinesterase activity was measured using the substrate acetylthiocholine iodide, which converts to thiocholine. The reaction of thiocholine with the chromogenic substrate Dithionitrobenzoic acid (DTNB) leads to the formation of a yellow anion of 2-thio- 2-Nitrobenzoic acid, which absorbs strongly at 412 nm.
The AChE assay was performed following the method of Ellman et. al, with minor modifications, using acetylthiocholine iodide as a substrate. Ellmans reaction mixture contains 1.0 mM acetylthiocholine iodide and 0.5 mM 5, 5’-dithio-bis-(2-nitrobenzoic acid) in a 50 mM sodium phosphate buffer (pH 8.0). The assay mixture contained 50 µ1 of 50 mM phosphate buffer at pH - 8.0, 30 µl of test substance at various concentrations, and 20 µl of (100 mU/mL) enzyme. For blanks, the enzyme was replaced with phosphate buffer. The reaction mixture was mixed thoroughly, 100 µl of Ellman's reagent was added and incubated at room temperature for 10 min. The absorbance was measured at 412 nm using a microplate reader. The percentage inhibition of enzyme activity was calculated by comparing the optical densities (ODs) of the test wells with that of control wells using the following formula.

% inhibition of AChE activity= [(control- sample)/control] x 100.

The results of acetylcholinesterase inhibitory activity of the individual extracts and their compositions are summarized in Tables 7-11.

Table 7: Acetylcholinesterase inhibitory activities of the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract with magnesium (ROMg-1) or without magnesium (RO-1) and Nelumbo nucifera seed extract blend (NN-1) and their individual ingredients.
Comp # ROMg-1 NN-1 Ratio Comp Dose
µg/mL % Inhibition of AChE
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-1 9.38 11.50 3.13 2.86 3:1 12.5 14.36 19.94
C-2 8.33 10.21 4.17 3.82 2:1 12.5 14.03 22.07
C-3 6.25 7.66 6.25 5.72 1:1 12.5 13.38 23.33
C-4 4.17 5.11 8.33 7.62 1:2 12.5 12.74 18.33
C-5 3.13 3.83 9.38 8.58 1:3 12.5 12.41 17.67
RO-1 NN-1
C-2A 8.33 8.26 4.17 3.82 2:1 12.5 12.08 10.27

Table 8: Acetylcholinesterase inhibitory activities of the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed 90% ethanol extract (NN-2); and ROMg-1 and Nelumbo nucifera seed 50% ethanol extract (NN-3).

Comp # ROMg-1 NN-2 Ratio Comp Dose
µg/mL % Inhibition of AChE
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-6 8.33 10.21 4.17 3.94 2:1 12.5 14.16 21.84
C-7 6.25 7.66 6.25 5.91 1:1 12.5 13.57 26.91
C-8 4.17 5.11 8.33 7.87 1:2 12.5 12.99 22.44
ROMg-1 NN-3
C-9 8.33 10.21 4.17 4.58 2:1 12.5 14.79 19.16
C-10 6.25 7.66 6.25 6.86 1:1 12.5 14.53 21.64
C-11 4.17 5.11 8.33 9.15 1:2 12.5 14.26 19.66

Table 9: Acetylcholinesterase inhibitory activities of the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed water extract (NN-4); and ROMg-1 and Nelumbo nucifera seed methanol extract (NN-5).
Comp # ROMg-1 NN-4 Ratio Comp Dose
µg/mL % Inhibition of AChE
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-12 8.33 10.21 4.17 2.20 2:1 12.5 12.41 17.70
C-13 6.25 7.66 6.25 3.30 1:1 12.5 10.96 18.58
C-14 4.17 5.11 8.33 4.39 1:2 12.5 9.51 17.55
ROMg-1 NN-5
C-15 8.33 10.21 4.17 6.89 2:1 12.5 17.10 27.83
C-16 6.25 7.66 6.25 10.33 1:1 12.5 17.99 25.59
C-17 4.17 5.11 8.33 13.76 1:2 12.5 18.88 28.44

Table 10: Acetylcholinesterase inhibitory activities of the compositions containing Rosmarinus officinalis leaves 80% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-2) and Nelumbo nucifera seed extract blend (NN-1); Rosmarinus officinalis leaves ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMG-3) and Nelumbo nucifera seed water extract (NN-4).
Comp # ROMg-2 NN-1 Ratio Comp Dose
µg/mL % Inhibition of AChE
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-18 8.33 6.26 4.17 3.82 2:1 12.5 10.08 14.12
C-19 6.25 4.70 6.25 5.72 1:1 12.5 10.42 19.48
C-20 4.17 3.14 8.33 7.62 1:2 12.5 10.76 14.80
ROMg-3 NN-4
C-21 8.33 4.39 4.17 2.20 2:1 12.5 6.59 16.02
C-22 6.25 3.29 6.25 3.30 1:1 12.5 6.59 12.76
C-23 4.17 2.20 8.33 4.39 1:2 12.5 6.59 13.36

Table 11: Acetylcholinesterase inhibitory activities of the compositions containing Rosmarinus officinalis leaves methanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-4) and Nelumbo nucifera seed 50% aq. ethanol extract (NN-3); compositions containing Rosmarinus officinalis leaves acetone extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-5) and Nelumbo nucifera seed methanol extract (NN-5).
Comp # ROMg-4 NN-3 Ratio Comp Dose
µg/mL % Inhibition of AChE
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-24 8.33 3.66 4.17 4.58 2:1 12.5 8.24 15.08
C-25 6.25 2.74 6.25 6.86 1:1 12.5 9.61 19.20
C-26 4.17 1.83 8.33 9.15 1:2 12.5 10.98 16.95
ROMg-5 NN-5
C-27 8.33 4.52 4.17 6.89 2:1 12.5 11.41 21.61
C-28 6.25 3.39 6.25 10.33 1:1 12.5 13.72 22.20
C-29 4.17 2.26 8.33 13.76 1:2 12.5 16.03 23.46

Example 23: Procedure for Monoamine oxidase (MAO) Assay
Monoamine oxidase (MAO) assay was performed using the rat liver mitochondrial fraction as a source of the MAO enzyme.
Mitochondrial fraction preparation: Rat liver tissue (400-500 mg) was homogenized in 30 mL ice cold 1xPBS using a hand-held homogenizer (20-30 cycles). The tissue lysate was centrifuged at 1000g for 10 min. at 4°C. The supernatant was collected in new tubes and centrifuged at 12000g for 15 min. at 4°C. The pellet was washed twice with 1 mL 1xPBS and centrifuged at 12000g. The resultant pellet consisting of mitochondrial fraction was resuspended in 2-4 mL 1xPBS, and 200 µL aliquots were stored at -80°C.
Assay procedure: Protein was quantified from freshly thawed rat liver mitochondrial fractions using a Pierce BCA protein assay kit (Thermo Scientific, Cat# 23225). Black, U-bottom opaque plates (Thermo-Fisher Scientific, Cat# 267342) were used for the assay. The samples/controls were prepared in an assay buffer (1xPBS). Controls/samples (in 20 µL volume) were added into the respective wells. Five micrograms of mitochondrial protein lysate (~80µL/well) was added, except to blank wells. For blank wells, 100 µL/well assay buffer was added. After 5 min., using a multichannel pipette, 100µL MAO reaction mix [Tyramine (20.8mg/ml in DMSO for MAO-A) or 200ul of Benzylamine (17.2mg/ml in water for MAO-B) + 200U HRP + 20µl of Amplex Red (39.9mM) in 10 ml assay buffer (1xPBS)] was added to each well. The fluorescence intensities were measured at Ex: 530 nm/Em: 585 nm in a kinetic mode set at 37°C for 60 min, using a multimode reader (SpectramaxM5e, Molecular Devices, USA). The MAO enzyme inhibition was calculated using the following formula:

The results of MAO-A or B inhibitory activity of the individual extracts and their compositions are summarized in Tables 12-21.
Table 12: MAO-A inhibitory activities of the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1) and Nelumbo nucifera seed extract blend (NN-1).
Comp # ROMg-1 NN-1 Ratio Comp Dose
µg/mL % Inhibition of MAO-A
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-1 0.75 11.03 0.25 1.45 3:1 1 12.47 29.58
C-2 0.67 9.85 0.33 1.91 2:1 1 11.76 21.02
C-3 0.5 7.35 0.5 2.90 1:1 1 10.25 18.00
C-4 0.33 4.85 0.67 3.88 1:2 1 8.73 22.35
C-5 0.25 3.68 0.75 4.34 1:3 1 8.02 21.75
RO-1 NN-1
C-2A 0.67 7.26 0.33 1.91 2:1 1 9.17 11.37

Table 13: MAO-A inhibitory activities of the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed 90% ethanol extract (NN-2); and ROMg-1 and Nelumbo nucifera seed 50% ethanol extract (NN-3).
Comp # ROMg-1 NN-2 Ratio Comp Dose
µg/mL % Inhibition of MAO-A
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-6 0.67 9.85 0.33 7.23 2:1 1 17.08 25.28
C-7 0.5 7.35 0.5 10.96 1:1 1 18.31 29.62
C-8 0.33 4.85 0.67 14.69 1:2 1 19.54 22.28
ROMg-1 NN-3
C-9 0.67 9.85 0.33 2.06 2:1 1 11.91 28.31
C-10 0.5 7.35 0.5 3.12 1:1 1 10.47 16.49
C-11 0.33 4.85 0.67 4.18 1:2 1 9.03 27.86

Table 14: MAO-A inhibitory activities of the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed water extract (NN-4); and ROMg-1 and Nelumbo nucifera seed methanol extract (NN-5).
Comp # ROMg-1 NN-4 Ratio Comp Dose
µg/mL % Inhibition of MAO-A
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-12 0.67 9.85 0.33 3.85 2:1 1 13.70 19.84
C-13 0.5 7.35 0.5 5.83 1:1 1 13.18 29.89
C-14 0.33 4.85 0.67 7.81 1:2 1 12.66 24.82
ROMg-1 NN-5
C-15 0.67 9.85 0.33 1.60 2:1 1 11.45 19.86
C-16 0.5 7.35 0.5 2.42 1:1 1 9.77 17.11
C-17 0.33 4.85 0.67 3.24 1:2 1 8.09 18.68

Table 15: MAO-A inhibitory activities of the compositions containing Rosmarinus officinalis leaves 80% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-2) and Nelumbo nucifera seed extract blend (NN-1); Rosmarinus officinalis leaves ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-3) and Nelumbo nucifera seed water extract (NN-4).
Comp # ROMg-2 NN-1 Ratio Comp Dose
µg/mL % Inhibition of MAO-A
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-18 0.67 13.34 0.33 1.91 2:1 1 15.25 24.13
C-19 0.5 9.96 0.5 2.90 1:1 1 12.85 17.05
C-20 0.33 6.57 0.67 3.88 1:2 1 10.45 20.28
ROMg-3 NN-4
C-21 0.67 8.39 0.33 3.85 2:1 1 12.24 18.70
C-22 0.5 6.26 0.5 5.83 1:1 1 12.09 17.37
C-23 0.33 4.13 0.67 7.81 1:2 1 11.94 19.48

Table 16: MAO-A inhibitory activities of the compositions containing Rosmarinus officinalis leaves methanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-4) and Nelumbo nucifera seed 50% aq. ethanol extract (NN-3); Rosmarinus officinalis leaves acetone extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-5) and Nelumbo nucifera seed methanol extract (NN-5).
Comp # ROMg-4 NN-3 Ratio Comp Dose
µg/mL % Inhibition of MAO-A
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-24 0.67 10.39 0.33 2.06 2:1 1 12.45 19.05
C-25 0.5 7.76 0.5 3.12 1:1 1 10.88 20.21
C-26 0.33 5.12 0.67 4.18 1:2 1 9.30 18.38
ROMg-5 NN-5
C-27 0.67 8.52 0.33 1.60 2:1 1 10.11 19.46
C-28 0.5 6.36 0.5 2.42 1:1 1 8.78 15.44
C-29 0.33 4.19 0.67 3.24 1:2 1 7.44 13.47

Table 17: MAO-B inhibitory activities of the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1) and Nelumbo nucifera seed extract blend (NN-1).
Comp # ROMg-1 NN-1 Ratio Comp Dose
µg/mL % Inhibition of MAO-B
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-1 0.75 4.68 0.25 3.59 3:1 1 8.27 15.58
C-2 0.67 4.18 0.33 4.74 2:1 1 8.92 17.43
C-3 0.5 3.12 0.5 7.18 1:1 1 10.30 15.96
C-4 0.33 2.06 0.67 9.61 1:2 1 11.67 15.07
C-5 0.25 1.56 0.75 10.76 1:3 1 12.32 28.77
RO-1 NN-1
C-2A 0.67 3.69 0.33 4.74 2:1 1 8.43 8.91

Table 18: MAO-B inhibitory activities of the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed 90% ethanol extract (NN-2); and ROMg-1 and Nelumbo nucifera seed 50% ethanol extract (NN-3).
Comp # ROMg-1 NN-2 Ratio Comp Dose
µg/mL % Inhibition of MAO-B
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-6 0.67 4.18 0.33 2.14 2:1 1 6.32 18.66
C-7 0.5 3.12 0.5 3.24 1:1 1 6.36 20.46
C-8 0.33 2.06 0.67 4.33 1:2 1 6.39 16.23
ROMg-1 NN-3
C-9 0.67 4.18 0.33 1.55 2:1 1 5.73 11.70
C-10 0.5 3.12 0.5 2.35 1:1 1 5.47 12.33
C-11 0.33 2.06 0.67 3.14 1:2 1 5.20 13.06

Table 19: MAO-B inhibitory activities of the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed water extract (NN-4); and ROMg-1 and Nelumbo nucifera seed methanol extract (NN-5).
Comp # ROMg-1 NN-4 Ratio Comp Dose
µg/mL % Inhibition of MAO-B
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-12 0.67 4.18 0.33 2.23 2:1 1 6.41 14.34
C-13 0.5 3.12 0.5 3.38 1:1 1 6.50 12.85
C-14 0.33 2.06 0.67 4.52 1:2 1 6.58 11.33
ROMg-1 NN-5
C-15 0.67 4.18 0.33 0.81 2:1 1 4.99 16.72
C-16 0.5 3.12 0.5 1.22 1:1 1 4.34 17.99
C-17 0.33 2.06 0.67 1.63 1:2 1 3.69 12.17

Table 20: MAO-B inhibitory activities of the compositions containing Rosmarinus officinalis leaves 80% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-2) and Nelumbo nucifera seed extract blend (NN-1); Rosmarinus officinalis leaves ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-3) and Nelumbo nucifera seed water extract (NN-4).
Comp # ROMg-2 NN-1 Ratio Comp Dose
µg/mL % Inhibition of MAO-B
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-18 0.67 12.26 0.33 4.74 2:1 1 17.00 23.43
C-19 0.5 10.62 0.5 7.18 1:1 1 17.80 22.30
C-20 0.33 8.97 0.67 9.61 1:2 1 18.58 26.88
ROMg-3 NN-4
C-21 0.67 16.97 0.33 2.23 2:1 1 19.20 28.84
C-22 0.5 12.67 0.5 3.38 1:1 1 16.04 24.64
C-23 0.33 8.36 0.67 4.52 1:2 1 12.88 31.75

Table 21: MAO-B inhibitory activities of the compositions containing Rosmarinus officinalis leaves methanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-4) and Nelumbo nucifera seed 50% aq. ethanol extract (NN-3); Rosmarinus officinalis leaves acetone extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-5) and Nelumbo nucifera seed methanol extract (NN-5).
Comp # ROMg-4 NN-3 Ratio Comp Dose
µg/mL % Inhibition of MAO-B
µg/mL % Inhibition µg/mL % Inhibition Additive (Calculated) Observed
C-24 0.67 12.56 0.33 1.55 2:1 1 14.11 19.86
C-25 0.5 9.38 0.5 2.35 1:1 1 11.72 18.65
C-26 0.33 6.19 0.67 3.14 1:2 1 9.33 17.09
ROMg-5 NN-5
C-27 0.67 9.77 0.33 0.81 2:1 1 10.57 17.41
C-28 0.5 7.29 0.5 1.22 1:1 1 8.51 15.25
C-29 0.33 4.81 0.67 1.63 1:2 1 6.45 14.85

Example 24: Procedure for evaluating GABAA receptor-a expressions using Western blot assay
Cell culture and treatments: Human SHSY5Y neuroblastoma cells (400,000 cells/well in 3mL) were seeded in 6-well cell culture plates and maintained in DMEM medium containing 10% FBS at 37°C in a humidified atmosphere of 5% dCO2. After 48 hrs, cells were replaced with serum-free DMEM medium. After 17 hours of serum starvation, cells were treated with test samples (100ng/ml diluted in serum-free DMEM medium) for 6 hours. The untreated SHSY5Y cells served as vehicle control.

Western blot: After 6 hours, cell culture plates were placed on an ice tray and washed twice with 1X PBS. Sixty microliters of lysis buffer (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 10 µg/mL Aprotinin, 10 µg/mL Leupeptin, 1% Triton X-100, 1 mM NaF, 1 mM Na3VO4, 0.5% Sodium deoxycholate, and 1 µM Pepstatin) was added to each well and cell lysates were collected. The cell lysates were centrifuged at 13000xg for 10 minutes at 4°C and the protein content in the clarified supernatants was estimated using a Pierce BCA protein assay kit (Thermo Scientific, Cat# 23225). An equal amount of cell lysate proteins was resolved in SDS-PAGE, the resolved proteins were electro-blotted onto nitrocellulose (NC) membrane strips using standard procedures. After blocking the non-specific sites, the NC membranes were reacted with GABAA receptor-a (Millipore Cat# 06-868; 1:2000 dilution) antibody overnight. The membranes were washed, unbound antibodies were removed, and further reacted with horseradish peroxidase (HRP) tagged anti-rabbit IgG (Jackson Immuno Research laboratories, Cat# 111-035-045). Finally, the specific immunoreactive GABAA receptor-a (GABAA-Ra) protein bands were detected with a chemiluminescent substrate (Thermo Scientific, Cat# 34080), and protein band images were captured using a Bio-Rad Molecular imager (Model: ChemiDOC XRS+). The stripped membranes were reprobed with anti-ß-actin antibody (Sigma, Cat# A4700-100µL; 1:10000 dilution), and the actin protein expression was used to assess equal cell-lysates protein loading and for normalization of the GABAA-Ra expression. The normalization of expression and the densitometric analysis of the protein bands were performed using Carestream MI software. The normalized values (arbitrary units) were measured using the following formula:
Normalized GABAA-Ra expression = density of GABAA-Ra ÷ density of actin

Percent increases of the GABAA-Ra expression in the compositions-treated cell lysates (observed vs. additive) are presented in Tables 22-26.

Table 22: GABAA-Ra overexpression by the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1) and Nelumbo nucifera seed extract blend (NN-1).
Comp # ROMg-1 NN-1 Ratio Comp Dose
ng/mL GABAA-Ra expression
ng/mL GABAA-Ra expression ng/mL GABAA-Ra expression Additive (Calculated) Observed % increase
C-1 75 0.44 25 0.14 3:1 100 0.58 0.70 20.7
C-2 66.67 0.39 33.33 0.19 2:1 100 0.58 0.86 48.3
C-3 50 0.29 50 0.29 1:1 100 0.58 0.94 62.1
C-4 33.33 0.19 66.67 0.38 1:2 100 0.57 0.85 49.1
C-5 25 0.15 75 0.43 1:3 100 0.57 0.79 38.6
RO-1 NN-1
C-2A 66.67 0.28 33.33 0.19 2:1 100 0.47 0.53 12.7

Table 23: GABAA-Ra overexpression by the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed 90% ethanol extract (NN-2); and ROMg-1 and Nelumbo nucifera seed 50% ethanol extract (NN-3).
Comp # ROMg-1 NN-2 Ratio Comp Dose
ng/mL GABAA-Ra expression
ng/mL GABAA-Ra expression ng/mL GABAA-Ra expression Additive (Calculated) Observed % increase
C-6 66.67 0.39 33.33 0.24 2:1 100 0.62 0.91 46.8
C-7 50 0.29 50 0.36 1:1 100 0.65 0.99 52.3
C-8 33.33 0.19 66.67 0.47 1:2 100 0.67 0.81 20.9
ROMg-1 NN-3
C-9 66.67 0.39 33.33 0.20 2:1 100 0.59 0.70 18.6
C-10 50 0.29 50 0.30 1:1 100 0.59 0.76 28.8
C-11 33.33 0.19 66.67 0.40 1:2 100 0.59 0.74 25.4
Table 24: GABAA-Ra overexpression by the compositions containing Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1), and Nelumbo nucifera seed water extract (NN-4); and ROMg-1 and Nelumbo nucifera seed methanol extract (NN-5).
Comp # ROMg-1 NN-4 Ratio Comp Dose
ng/mL GABAA-Ra expression
ng/mL GABAA-Ra expression ng/mL GABAA-Ra expression Additive (Calculated) Observed % increase
C-12 66.67 0.39 33.33 0.21 2:1 100 0.60 0.88 46.7
C-13 50 0.29 50 0.31 1:1 100 0.60 0.96 60.0
C-14 33.33 0.19 66.67 0.41 1:2 100 0.61 0.95 55.7
ROMg-1 NN-5
C-15 66.67 0.39 33.33 0.22 2:1 100 0.61 0.82 34.4
C-16 50 0.29 50 0.34 1:1 100 0.63 0.98 55.6
C-17 33.33 0.19 66.67 0.45 1:2 100 0.64 0.96 50.0

Table 25: GABAA-Ra overexpression by the compositions containing Rosmarinus officinalis leaves 80% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-2) and Nelumbo nucifera seed extract blend (NN-1); Rosmarinus officinalis leaves ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-3) and Nelumbo nucifera seed water extract (NN-4).

Comp # ROMg-2 NN-1 Ratio Comp Dose
ng/mL GABAA-Ra expression
ng/mL GABAA-Ra expression ng/mL GABAA-Ra expression Additive (Calculated) Observed % increase
C-18 66.67 0.19 33.33 0.19 2:1 100 0.38 0.65 71.1
C-19 50 0.15 50 0.29 1:1 100 0.43 0.72 67.4
C-20 33.33 0.10 66.67 0.38 1:2 100 0.48 0.80 66.7
ROMg-3 NN-4
C-21 66.67 0.29 33.33 0.21 2:1 100 0.49 0.84 71.4
C-22 50 0.21 50 0.31 1:1 100 0.52 0.97 86.5
C-23 33.33 0.14 66.67 0.41 1:2 100 0.56 0.86 53.6

Table 26: GABAA-Ra overexpression by the compositions containing Rosmarinus officinalis leaves methanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-4) and Nelumbo nucifera seed 50% aq. ethanol extract (NN-3); Rosmarinus officinalis leaves acetone extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-5) and Nelumbo nucifera seed methanol extract (NN-5).
Comp # ROMg-4 NN-3 Ratio Comp Dose
ng/mL GABAA-Ra expression
ng/mL GABAA-Ra expression ng/mL GABAA-Ra expression Additive (Calculated) Observed % increase
C-24 66.67 0.25 33.33 0.20 2:1 100 0.45 0.76 68.9
C-25 50 0.19 50 0.30 1:1 100 0.48 0.82 70.8
C-26 33.33 0.12 66.67 0.40 1:2 100 0.52 0.96 84.6
ROMg-5 NN-5
C-27 66.67 0.32 33.33 0.22 2:1 100 0.55 0.80 45.5
C-28 50 0.24 50 0.34 1:1 100 0.58 0.85 46.6
C-29 33.33 0.16 66.67 0.45 1:2 100 0.61 1.00 63.9

Example 25: Evaluation of the composition-30 for relief from stress and anxiety in chronic restraint-stressed ICR mice
Treatments: Twelve chronic restraint stressed (CRS) ICR mice were randomly assigned to two groups (n=6). The animals were supplemented (using oral gavage) with 0.1% w/v CMC-Na (G1 group) and 150 mg/kg of comp-30 (G2) in 10 mL of 0.1% CMC-Na in water for 15 days. The elevated plus maze and light-dark transition tests were performed on days 15 and 16, respectively. On day 17, after 1hr of supplementation, blood samples were collected from the retro-orbital plexus of all animals under mild isoflurane anesthesia. The serum was separated by centrifuging the blood samples at 3200g for 15 minutes at 4ºC and stored at -80ºC until analysis. After blood sample collection on day 17, all surviving animals were euthanized using CO2 asphyxiation followed by exsanguination and necropsy.

Test parameters: The elevated plus maze and light-dark transition tests were conducted as per the procedures disclosed in the literature, and the results are presented in Tables 27–29. In the elevated plus maze experiment, time spent in the open arm and the number of entries into the open arm were recorded. The anxiety index was calculated using the following formula.

Anxiety index=[1-{(open arm time÷total time on maze)+(open arm entry÷total entry)}]÷2

Bio-markers related to stress and anxiety, such as dopamine, serotonin, and corticosterone, were evaluated utilizing commercially available ELISA kits, and the results are presented in Tables 30-32.
Table 27: Percent increases of time spent in the open arm by the stressed (CRS) mice supplemented with compositions of Rosmarinus officinalis extract containing magnesium (ROMg-1) and Nelumbo nucifera extract blend (NN-1) combined with excipients.
Groups Supplementation Mean ± SD of time spent in open arm (sec.) Percent increase in time spent (from G1)
G1 CMC-Na 18.25±2.25 --
G2 Comp-30: Combining ROMg-1 and NN-1 in the ratio of 2:1 and 10% excipients 26.34±1.08 44.32
Note: The increase in percentage time indicates better efficacy

Table 28: Percent reductions of anxiety in the stressed (CRS) mice supplemented with compositions of Rosmarinus officinalis extract containing magnesium (ROMg-1) and Nelumbo nucifera extract blend (NN-1) combined with excipients.
Groups Supplementation Anxiety index
(Mean ± SD) Percent Anxiety reduction (from G1)
G1 CMC-Na 0.39±0.05 --
G2 Comp-30: Combining ROMg-1 and NN-1 in the ratio of 2:1 and 10% excipients 0.22±0.09 43.6
Note: The decrease in percentage indicates better efficacy

Table 29: Percent increase of entries in light by the stressed (CRS) mice supplemented with compositions of Rosmarinus officinalis extract containing magnesium (ROMg-1) and Nelumbo nucifera extract blend (NN-1) combined with excipients.
Groups Supplementation Number of entries in light (Mean ± SD) Percent increase of entries (from G1)
G1 CMC-Na 11.63±1.77 --
G2 Comp-30: Combining ROMg-1 and NN-1 in the ratio of 2:1 and 10% excipients 18.13±3.31 55.9
Note: The increase in percentage indicates better efficacy

Table 30: Percent increase of serum dopamine levels in the stressed (CRS) mice supplemented with compositions of Rosmarinus officinalis extract containing magnesium (ROMg-1) and Nelumbo nucifera extract blend (NN-1) combined with excipients.
Groups Supplementation Mean ± SD of Dopamine concentration (ng/mL) Percent increase in dopamine levels (from G1)
G1 CMC-Na 108.46±2.63 --
G2 Comp-30: Combining ROMg-1 and NN-1 in the ratio of 2:1 and 10% excipients 117.60±1.75 8.4
Note: The increase in percentage indicates better efficacy

Table 31: Percent increase of serum serotonin levels in stressed (CRS) mice supplemented with compositions of Rosmarinus officinalis extract containing magnesium (ROMg-1) and Nelumbo nucifera extract blend (NN-1) combined with excipients.
Groups Supplementation Mean ± SD of Serotonin concentration (ng/mL) Percent increase in serotonin levels (from G1)
G1 CMC-Na 120.15±1.24 --
G2 Comp-30: Combining ROMg-1 and NN-1 in the ratio of 2:1 and 10% excipients 136.31±1.88 13.4
Note: The increase in percentage indicates better efficacy

Table 32: Percent decrease of serum corticosterone levels in stressed (CRS) mice supplemented with compositions of Rosmarinus officinalis extract containing magnesium (ROMg-1) and Nelumbo nucifera extract blend (NN-1) combined with excipients.
Groups Supplementation Mean ±SD of Corticosterone concentrations (ng/mL) Percent decrease in corticosterone levels (from G1)
G1 CMC-Na 198.30±3.31 --
G2 Comp-30: Combining ROMg-1 and NN-1 in the ratio of 2:1 and 10% excipients 141.41±1.06 28.7
Note: More decrease indicates better efficacy
Example 26: Evaluation of the composition-30 for sleep latency and duration in Thiopental-induced mice
The effect of comp 30 on sleep induction was evaluated in Thiopental sodium-treated male CD-1 mice. Eighteen mice (average body weight 18-22 g) were randomly distributed in to three groups (n =6). The vehicle control (0.5% Na-CMC) and the composition comp-30 at doses 50 and 100 mg/kg body weight were orally administered in groups 1, 2, and 3 mice, respectively. Thirty minutes later, thiopental sodium (50 mg/kg, i.p.) was administered to each mouse to induce sleep. The animals were observed for the latent period for sleep (time between thiopental sodium administration to loss of righting reflex) and duration of sleep (time between the loss and recovery of reflex). The latent period for sleep is considered as sleep onset time. The sleep onset and sleep duration are measured in minutes and the results are presented in Tables 33.

Table 33: Percent reduction in sleep onset and percent increase in sleep duration in mice supplemented with composition-30 comprising Rosmarinus officinalis leaves 90% aq. ethanol extract containing magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid (ROMg-1) and Nelumbo nucifera seed extract blend (NN-1) in 2:1 ratio combined with excipients.

Groups Supplementation Sleep onset (min)
Mean ± SD Sleep Duration (min) Mean ± SD % reduction in sleep onset (from G1) % increase in sleep duration (from G1)
G1 Control + Thiopentone 50 mg/kg 4.63 ± 1.02 55.92 ± 12.27 - -
G2 Comp-30: 50 mg/kg + Thiopentone 50 mg/kg) 3.74 ± 0.96 74.0 ± 9.51 19.22 32.33
G3 Comp-30: 100 mg/kg + Thiopentone 50 mg/kg 3.25 ± 0.88 81.39 ± 10.13 29.81 45.54

Although the invention has been described in detail in the foregoing for the purpose of illustration, it is to be understood that such detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims.
,CLAIMS:
1. A herbal composition for stress relief and sleep promotion comprising combination of first ingredient selected from leaf extract of Rosmarinus officinalis in the range of about 10% to 90% and second ingredient selected from seed extract of Nelumbo nucifera in the range of about 90% to 10%, wherein the leaf extract of Rosmarinus officinalis comprises of magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid and oleanolic acid or mixtures thereof in said extract.

2. The herbal composition as claimed in claim 1, wherein the leaf extract of Rosmarinus officinalis and seed extract of Nelumbo nucifera are in the ratio of about 3:1 to 1:3.

3. The herbal composition as claimed in claim 1, wherein the leaf extract of Rosmarinus officinalis comprises of magnesium salt of carnosic acid, rosmarinic acid, ursolic acid and oleanolic acid.

4. The herbal composition as claimed in claim 1, wherein the leaf extract of Rosmarinus officinalis comprises of magnesium salt of carnosic acid, rosmarinic acid, ursolic acid and oleanolic acid and has a pH of about 5.6.

5. The herbal composition as claimed in claim 1, wherein the leaf extract of Rosmarinus officinalis comprises of about 5% to 12% of carnosic acid, about 1% to 5% of rosmarinic acid, about 12% to 22% of ursolic acid, about 1% to 8% of oleanolic acid, and about 0.1% to 2% of magnesium.

6. The herbal composition as claimed in claim 1, wherein the seed extract of Nelumbo nucifera comprises of saponins, polysaccharides or proanthocyanidins or mixtures thereof.

7. The herbal composition as claimed in claim 1, wherein the herbal composition further optionally comprises of least one pharmaceutically or nutraceutically or dietically acceptable excipient.

8. A process for preparing herbal composition comprising of leaf extract of Rosmarinus officinalis comprising magnesium salt or complex or chelate of at least one phytochemical selected from carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid or mixtures thereof in the extract, and seed extract of Nelumbo nucifera, comprising the steps of;
(i) extracting Rosmarinus officinalis powder with a suitable solvent;
(ii) treating the extract with a compound of magnesium source;
(iii) filtering, evaporating the solvent and drying the residue to obtain Rosmarinus officinalis extract comprising magnesium salt of carnosic acid, rosmarinic acid, ursolic acid, and oleanolic acid;
(iv) blending the extract of step (iii) with at least one extract derived from Nelumbo nucifera in the presence of a suitable solvent and optionally with suitable excipients and
(v) drying the product to get the composition.

9. The process as claimed in claim 8 wherein the solvent is C1-C5 alcohols selected from ethanol, methanol, n-butanol, water, and mixtures thereof; or ketones selected from acetone, methylisobutyl ketone; chlorinated solvents selected from methylene dichloride and chloroform; water and mixtures thereof.

10. The process as claimed in claim 8, wherein the magnesium source is selected from magnesium oxide, magnesium hydroxide, and magnesium carbonate.

11. A method of obtaining at least one health benefit selected from relief from stress, depression, anger, anxiety, promoting relaxation, sleep, and sleep quality in a human subject, wherein the method comprises supplementing the said human subject with an effective dose of a herbal composition as claimed in any one of the claims 1 to 10.

12. Use of an herbal composition as claimed in any one of the claims 1 to 10 for obtaining at least one health benefit selected from relief from stress, depression, anger, anxiety, promoting relaxation, sleep, and sleep quality in a human subject.