A low-dose phytochemical composition of Ocimum tenuiflorum for improvement of mental and physical performance

Description of the invention

Technical field of the invention

[0001] The present invention relates to a phytochemical composition from Ocimum tenuiflorum (synonym Ocimum sanctum) leaves and aerial parts useful for the improvement of mental and physical performance. More particularly, the invention relates to the phytochemical composition with a combination of five different phytoconstituents in combination for relieving anxiety, stress and other ailments related to physical and mental wellbeing and is significantly effective at very-low dose.

Background of the invention

[0002] Ocimum tenuiflorum (Ocimum sanctum), commonly known as Tulsi is an aromatic perennial plant belonging to basil family Lamiaceae. It is native to the Indian subcontinent and widespread as a cultivated plant throughout the Southeast Asian tropics.

[0003] Tulsi is cultivated for religious and traditional medicine purposes, and for its essential oil. It is widely used as herbal tea, commonly used in Ayurveda, and has a place within the Vaishnava tradition of Hinduism, in which devotees perform worship involving holy basil plants or leaves.

[0004] The genus Ocimum includes approximately 150 species, possessing a great variation in plant morphology and biology, essential oil content, and chemical composition. The economic importance and global dissemination of Ocimum, with its multiple uses including cooking and folk medicine, make it important to investigate its pharmacological and toxicological effects to ensure its efficacy and safety. In India, among the medicinal herbs known for their healing properties, the genus Ocimum is very important for its curative potential. Ocimum contain a wide range of essential oils rich in phenolic compounds and a wide array of other natural products including flavonoids and anthocyanins having great pharmacological importance.

[0005] Tulsi tastes hot and bitter and is said to penetrate the deep tissues, dry tissue secretions and normalize kapha and vata in the human body. The daily consumption of tulsi is said to prevent disease, promote general health, wellbeing and longevity and assist in dealing with the stresses of daily life. Tulsi is also credited with giving luster to the complexion, sweetness to the voice and fostering beauty, intelligence, stamina, and a calm emotional disposition.

[0006] In addition to these health -promoting properties, tulsi is recommended as a treatment for a range of conditions including anxiety, cough, asthma, diarrhea, fever, dysentery, arthritis, eye diseases, otalgia, indigestion, hiccups, vomiting, gastric, cardiac, and genitourinary disorders, back pain, skin diseases, ringworm, insect, snake and scorpion bites and malaria.

[0007] “Phyto” in the word of phytochemicals is derived from the Greek word “phyto,” which means plant. Phytochemicals are a naturally occurring group of chemicals in plants and plant-derived foods, which may function in reducing the risk of chronic diseases.

[0008] In addition to physical, toxic, and infective stress, modem living is associated with heightened levels of psychological stress caused by the many demands and fast pace of modern life. The toxic effects of chemical pollutants and the constant fear of pervasive toxic chemicals itself lead to even further stress and anxiety that may be just as toxic as the chemicals causing it. While the reality of daily chemical exposure cannot be denied, regular consumption of Ocimum not only helps to protect and detoxify the body's cells and organs, but also reduce toxic stress by relaxing and calming the mind and offering many psychological benefits including anti-depressant activity and positive effects on memory and cognitive function. [0009] While modern scientific studies suggest that Ocimum is effective in treating a range of stressful conditions, within Ayurveda, Ocimum is most commonly recommended as a preventive measure to enhance the ability to adapt to both psychological and physical stress and therefore prevent the development of stress- related diseases. To this end, many Ayurvedic practitioners recommend the regular consumption of Ocimum tea as an essential lifestyle practice.

[0010] It is an adaptogen i.e., anti-stress agent, which is known to promote mental and physical wellness. It also regulates cortisol levels and helps reduce anxiety and emotional stress by promoting homeostasis. Adaptogens reduce the intensity and negative impact of the stress caused by mental tension, emotional difficulties, poor lifestyle habits, disease, infection, pollution, and change.

[0011] The Patent Application No. IN220688A1 entitled “A herbal composition having anti-stress and adaptogenic properties and a process for the preparation thereof to a herbal composition having anti-stress and adaptogenic properties comprising organic extract as herein described of Terminalia chebula (Haritaki) 100-300 mg, organic extract as herein described of Withania somnifera (Ashwagandha) 150-500 mg, organic extract as herein described of Ocimum sanctum (Tulsi) 100-300 mg, organic extract as herein described of Tinospora cordifolia (Guduchi) 150-500 mg, with or without known additives.

[0012] The Patent Application No. 3323/MUM/2012 entitled “A process for the preparation of polyherbal formulation for treatment of depression ” discloses a polyherbal formulation for treatment of depression comprises the ten medicinal plants (at least six) namely Bacopa monnieri, Plumbago zeylanica, Capparis Zeylanica, Glossocardia bosvallia, Hemidesmus indicus, Ocimum sanctum, Withania somnifera, Mimosa pudica, Asparagus recemosus and Tinospora cordifolia are mixed in desired quantity to make 100 g mixture. The 10 g of above mixture has been orally administered daily morning and evening up to six months to treat depression. The polyherbal formulation is simple, easy to prepare, safer and alternative method of treatment for depression. [0013] The publication entitled “Efficacy of an Extract of Ocimum tenuiflorum (OciBest) in the Management of General Stress: A Double-Blind, Placebo- Controlled Study” by Ram Chandra Saxena et.al. discloses a randomized, doubleblind, placebo-controlled study to evaluate the efficacy of OciBest, an extract of Ocimum tenuiflorum Linn, in symptomatic control of general stress. The participants received either placebo or OciBest (1200 mg of actives per day) for six weeks. The severity of stress-related symptoms was self-evaluated by patients at weeks 0, 2, 4 and 6 of the trial periods using a symptom rating scale. After six weeks of intervention, scores of symptoms such as forgetfulness, sexual problems of recent origin, frequent feeling of exhaustion, and frequent sleep problems of recent origin decreased significantly in OciBest group as compared with placebo group. Also, the total symptom scores of OciBest group revealed significant reduction as compared to placebo group. The overall improvement in OciBest group was found to be 1.6 times or 39% more in the control of general stress symptoms with respect to placebo. No adverse events were reported during the study. The findings revealed that OciBest was found to be effective and well tolerated by all the patients over the six weeks of study period.

[0014] The publication entitled “Phytochemical, Antioxidant and Antidepressant Evaluation of Ocimum basilicum, O. tenuiflorum, O. kilimandscharicum Grown in India" discloses antioxidant activity in three Ocimum taxa viz. O. basilicum, O. tenuiflorum, O. kilimandscharicum was determined by DPPH free radical scavenging ability and reducing power assay. Total phenolic, flavonoids and tannins were determined using spectrophotometric technique and comparative antidepressant activity was evaluated using forced swim test. It was observed that all the three species of Ocimum found in western Himalayan region show significant antioxidant and antidepressant activities, however O. kilimandscharicum was found to have highest antioxidant and antidepressant activity.

[0015] Even though the state of art hints the biological activity of Ocimum in reducing stress, depression but the dosage required to provide effective biological activity is high and is the conventional extract should be administered at a dosage of greater than 1200 mg per day for effective antistress activity when given as a single herbal preparation. As a result of this high dose, it is challenging for the manufacturers of finished formulations such as capsules and tablets to incorporate the extract in their formulations as the size of the tablets or capsules have a limitation. In addition, the number of capsules needed to be taken by the consumers shall also increase in numbers and ultimately leading to non-compliance to prescribed dose.

[0016] Further, the conventional extracts are standardized to ursolic acid and oleanolic acid contents, which may affect the water solubility of the extract and also imparts bitter taste to the composition leading to non-compliance.

[0017] Hence, there is a need for a composition with effective combination of phytochemicals from Ocimum tenuiflorum, which can be administered at lower dose and also require a specific process of extraction of Ocimum tenuiflorum from leaves and aerial parts with specific processing techniques to improve the biological activity and to be administered at low dose.

Summary of the Invention

[0018] In order to overcome the drawbacks of the state of art, the present invention provides a phytochemical composition from Ocimum tenuiflorum for the improvement of physical and mental performance. More specifically, the phytochemical composition with a combination of five phytoconstituents from Ocimum tenuiflorum for improved biological activity.

[0019] The phytochemical composition of Ocimum tenuiflorum according to the present invention comprises rabdosiin at a concentration between 0.8 % to 4.0 % w/w, rosmarinic acid at a concentration between 0.7% to 4 % w/w, luteolin 7-O- beta- glucuronide at a concentration between 0 8 % to 4.0 % w/w, apigenin-7-O- beta- glucuronide at a concentration between 0.6 % to 3.0 % w/w and ociglycoside I at a concentration between 0.08 % to 0.4 % w/w. In addition, the composition also optionally comprises excipients selected from a group consisting of granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, coloring agents, flavouring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents and spheronization agents.

[0020] The composition exhibits improved biological activity, masks bitter taste and is soluble in water. The composition shall be administered at 1/5* of the dosage of the dosage of the conventional extracts.

[0021] The present invention further discloses a process for preparing the phytochemical composition from Ocimum tenuiflorum. The process of preparing the bioactive extract starts with segregation the leaves and stems from Ocimum tenuiflorum. 100 g of Ocimum tenuiflorum is used to segregate into 30g of leaves and 70g of stems. From the segregated part, 21 g i.e., 70% of leaves and 9g i.e., 30% of stems are combined to obtain 30g of the raw material. The raw material, which is a combination of leaves and stems is subjected to ethanol extraction by washing three times using 80% ethanol under vacuum 500mm Hg and at a temperature of 76+2 °C. Then the ethanol extract is segregated into two fractions namely triterpene free fraction of 1.5g and triterpene rich fraction of 1.2 g. The triterpene rich extract is discarded, and triterpene free extract is considered for further steps. During this process, triterpenes namely oleanolic acid and ursolic acid are removed completely. The final product at a concentration of 1.5g is subjected to spray drying to obtain Ocimum tenuiflorum bioactive complex, which is free from triterpenes namely oleanolic acid and ursolic acid.

[0022] The process of the present invention is unique with respect to removal of triterpenes, which enhances the solubility of the extract due to being devoid of oleanolic acid and ursolic acid. This overcomes the drawbacks of the present state of art with respect to solubility and the phytochemical composition of the present invention, which is devoid of triterpenes oleanolic and ursolic acid, shows improved biological activity and is standardized with five different constituents.

[0023] The process may also be performed by alternatively subjecting the leaves and stems separately for ethanol extraction After ethanol extraction, the triterpene rich extract, containing oleanolic and ursolic acid from both leaves and stems are discarded, and triterpene free extract is considered for further steps. The extract of leaves and stems are blended at a unique proportion with a concentration of 70% to 80% w/w and 20% to 30% w/w to obtain a final product at a concentration of 1.9g, which is subjected to spray drying to obtain Ocimum tenuiflorum bioactive complex from triterpene free extract.

[0024] The Ocimum tenuiflorum bioactive complex obtained from either of the process is rich in phytoconstituents namely rabdosiin, rosmarinic acid, luteolin 7- O-beta- glucuronide, apigenin-7-O-beta- glucuronide and ociglycoside I.

[0025] This unique process of preparation of the Ocimum tenuiflorum bioactive complex results in improved biological activity at low dose of the complex i.e., at l/5th lower dose than the conventional extract.

[0026] The present invention comprising Ocimum tenuiflorum bioactive complex is safe, tolerated and exhibited stress relieving and sleep quality improving effects in human volunteers experiencing high stress.

[0027] The present invention comprising Ocimum tenuiflorum bioactive complex shall be administered in different dosage forms such as capsules and possess mild taste, less colored and is useful in preparation of food products such as bread, nutritional bars, cookie, beverage, pizza, health drinks, biscuits, chocolates, and pasta and as food supplement.

Brief description of the drawings

[0028] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.

[0029] FIG 1 tabulates the phytochemical constituents of the composition according to an embodiment of the invention.

[0030] FIG 2 illustrates the flow chart for a process of preparation of the phytochemical composition of Ocimum tenuiflorum. [0031] FIG 3 illustrates process of preparation of Ocimum tenuiflorum bioactive complex using a separate ethanol extraction for leaves and stems.

[0032] FIG 4 depicts the presence of phytoconstituents that form the Ocimum tenuiflorum bioactive complex.

[0033] FIG 5 depicts the structures of the of phytoconstituents that form the Ocimum tenuiflorum bioactive complex.

Detailed description of the invention

[0034] Reference will now be made in detail to the description of the present subject matter, one or more examples of which are shown in figures. Each example is provided to explain the subject matter and not a limitation. Various changes and modifications obvious to one skilled in the art to which the invention pertains are deemed to be within the spirit, scope, and contemplation of the invention.

[0035] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.

[0036] The term "Adaptogen" refers to herb that stabilizes the physiological processes and promotes homeostasis.

[0037] The term “Antidepressant” refers to medications used to treat major depressive disorder, some anxiety disorders, some chronic pain conditions, and to help manage some addictions.

[0038] The term “ Phytochemical” refers to bioactive nutrient plant chemicals in fruits, vegetables, grains, and other plant foods that may provide desirable health benefits beyond basic nutrition to reduce the risk of major chronic diseases.

[0039] The present invention overcomes the drawback of the existing state-of-the- art technologies by providing a phytochemical composition from Ocimum tenuiflorum for the improvement of physical and mental performance. More particularly, the present invention discloses a combination of five phytochemicals from Ocimum tenuiflorum that acts together to reduce the dose required than the conventional extracts.

[0040] The present invention also discloses a process of extraction of the phytochemicals from leaves and aerial parts of Ocimum tenuiflorum and process to prepare a specific phytochemical composition that exhibits improved biological activity in enhancing the wellbeing and mental health.

[0041] The composition of the present invention comprises a combination of five phytochemicals namely rabdosiin, rosmarinic acid, luteolin 7-O-beta- glucuronide, apigenin-7-O-beta- glucuronide and ociglycoside I from Ocimum tenuiflorum which is soluble in water due to being devoid of ursolic acid and oleanolic acid and possess mild taste and less colour compared to the conventional extracts.

[0042] FIG 1 tabulates the phytochemical constituents of the composition according to an embodiment of the invention. The phytochemical composition of Ocimum tenuiflorum according to the present invention comprises rabdosiin at a concentration between 0.8 % to 4.0 % w/w, rosmarinic acid at a concentration between 0.7 % to 4 % w/w, luteolin 7-O-beta- glucuronide at a concentration between 0.8 % to 4.0 % w/w, apigenin-7-O-beta- glucuronide at a concentration between 0.6 % to 3.0 % w/w and ociglycoside I at a concentration between 0.08 % to 0.4 % w/w.

[0043] The composition also comprises excipients selected from a group consisting of granulating agents, binding agents, lubricating agents, disintegrating agents, sweetening agents, coloring agents, flavouring agents, coating agents, plasticizers, preservatives, suspending agents, emulsifying agents and spheronization agents.

[0044] The composition is prepared as an extract, which is standardized with the five phytochemical constituents. As a result, and due to absence of ursolic acid and oleanolic acid, the extract is soluble in water and also masks the bitter taste.

[0045] The composition is effective, and exhibits improved biological activity due to the specific combination of the phytoconstituents and unique process of standardization imparts improved biological activity. [0046] The phytochemical composition of the present invention is used in multiple dosage forms such as capsules, tablets, syrups, lozenges, troches, elixirs, phytoceuticals and nutraceuticals.

[0047] The phytochemical composition of the present invention comprising specific five constituents is recommended at a dosage of l/5 ^th of the recommended dosages of the conventional extracts thus improving the patient compliance and is also cost effective.

[0048] The present invention also discloses a process for preparing the phytochemical composition according to an embodiment of the invention. The process is a unique extraction procedure from the leaves and aerial parts of Ocimum tenuiflorum. The use of specific steps of extraction, improves the structural features of the composition and improved the biological activity.

[0049] FIG 2 illustrates the flow chart for preparation of the phytochemical composition of Ocimum tenuiflorum. The process (200) of preparing the extract starts at step (201) of segregating the leaves and stems from Ocimum tenuiflorum. 100 g of Ocimum tenuiflorum is used to segregate into 30g of leaves and 70g of stems. At step (202), 21 g i.e., 70% of leaves and 9g i.e., 30% of stems are considered and combined to obtain 30g of the raw material. At step (203), the raw material with a combination of leaves and stems is subjected to ethanol extraction. The ethanol extraction is achieved by washing three times using 80% ethanol under vacuum 500mm Hg and at a temperature of 76±2°C. At step (204), the ethanol extract is segregated into two fractions namely triterpene free fraction of 1.5g and triterpene rich fraction of 1.2 g. The triterpene rich extract is discarded, and triterpene free extract is considered for further steps. The process results in removal of triterpenes namely oleanolic acid and ursolic acid. At step (205), the final product at a concentration of 1.5g is subjected to spray drying to obtain Ocimum tenuiflorum bioactive complex from triterpene free extract.

[0050] The process of preparation of the bioactive complex is simple, unique and is free from triterpenes especially oleanolic acid and ursolic acid. The removal of oleanolic acid and ursolic acid enhances the efficacy of the bioactive complex and the complex is soluble in water due to absence of triterpenes.

[0051] This unique process of preparation of the bioactive complex results in improved biological activity at very low dose of the complex i.e., at 1/5* lower dose than the conventional extract.

[0052] According to another embodiment of the invention, the process of preparation of extract is also achieved by an alternate process as explained in the example.

[0053] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner.

Example 1: A process of preparation of Ocimum tenuiflorum bioactive complex using a separate ethanol extraction for leaves and stems

[0054] FIG 3 illustrates process of preparation of Ocimum tenuiflorum bioactive complex using a separate ethanol extraction for leaves and stems. The process (300) of preparing the extract starts at step (301) of segregating the leaves and stems from Ocimum tenuiflorum. 100 g of Ocimum tenuiflorum is used to segregate into 30g of leaves and 70g of stems. At step (302), 21 g i.e., 70% of leaves, 9g i.e., 30% of stems are considered and combined to obtain 30g of the raw material. At step (303), the raw materials leaves, and stems are subjected to ethanol extraction separately. The ethanol extraction is achieved by washing three times using 80% ethanol under vacuum 500mm Hg and at a temperature of 76±2°C. At step (304), the ethanol extract is segregated into two fractions namely triterpene free fraction of 1.8 g and triterpene rich fraction of 1.8 g separately for ethanol extract obtained from leaves and triterpene free fraction of 2.1 g and triterpene rich fraction of 2.1 g from stems. The triterpene rich extract from both leaves and stems are discarded, and triterpene free extract is considered for further steps. The process results in removal of triterpenes namely oleanolic acid and ursolic acid. At step (305), the extract of leaves and stems are blended at a unique proportion with a concentration of 70% to 80% w/w and 20% to 30% w/w respectively. At step (306), the final product at a concentration of 1.9g is subjected to spray drying to obtain Ocimum tenuiflorum bioactive complex from triterpene free extract.

[0055] The Ocimum tenuiflorum bioactive complex obtained from either of the process is rich in phytoconstituents namely rabdosiin, rosmarinic acid, luteolin 7- O-beta- glucuronide, apigenin-7-O-beta- glucuronide and ociglycoside I. The presence of these phytoconstituents is confirmed by using High Performance Liquid Chromatography (HPLC).

[0056] FIG 4 depicts the presence of phytoconstituents form the Ocimum tenuiflorum bioactive complex. The Ocimum tenuiflorum bioactive complex is subjected to HPLC to confirm the presence of phytoconstituents.

[0057] FIG 5 depicts the structures of the of phytoconstituents form the Ocimum tenuiflorum bioactive complex. The phytoconstituents include rabdosiin (FIG 5a), rosmarinic acid (FIG 5b), luteolin 7-O-beta- glucuronide (FIG 5c), apigenin-7-O- beta- glucuronide (FIG 5d) and Ociglycoside I (FIG 5e).

[0058] The Ocimum tenuiflorum bioactive complex of the present invention is devoid of triterpenes namely oleanolic acid and ursolic acid and hence is water soluble thus enhancing the compatibility and stability of the bioactive complex.

[0059] The efficacy of the Ocimum tenuiflorum bioactive complex of the present invention is analyzed for its activity on stress, sleep, mood and quality of life parameters in adults experiencing high stress.

Example 2: Effect of Ocimum tenuiflorum bioactive complex on stressrelieving effects in adults experiencing high stress

[0060] A double-blind placebo controlled study is conducted to analyze the effect of Ocimum tenuiflorum bioactive complex on stress, sleep, mood and quality of life parameters in adults experiencing high stress. The study was conducted as two-arm, parallel-group, 8-week, randomized, double-blind, placebo-controlled trial with 50 volunteers per group. The test group was administered capsule containing Ocimum tenuiflorum bioactive complex and placebo group was administered with microcrystalline cellulose. All volunteers were instructed to take 2 capsules, twice daily with or without food for 8 weeks, delivering 250 mg daily of the present invention containing Ocimum tenuiflorum bioactive complex. The primary outcome analyzed was stress level using Perceived Stress Scale (PSS). The secondary outcome analyzed were mood, insomnia, sleep quality, sleep time, sleep efficiency, blood pressure, salivary cortisol, salivary alpha amylase and hair cortisol concentration. After 8 weeks, the parameters were analyzed, and the results indicated that there was statistically significant reduction in stress level as analyzed using PSS scale in Ocimum tenuiflorum group than the placebo group. A statistically significant improvement was observed with respect to insomnia parameters including sleep duration, sleep quality in Ocimum tenuiflorum bioactive complex group than the placebo group. The levels of salivary cortisol, salivary alpha amylase, blood pressure was significantly reduced in Ocimum tenuiflorum bioactive complex group than the placebo group. The levels of hair cortisol is low in Ocimum tenuiflorum bioactive complex group as compared to placebo group at week 8. The Ocimum tenuiflorum bioactive complex also demonstrated an improvement of quality-of-life parameters measured using PROMIS scale. However, no serious adverse events were reported by participants and the frequency of adverse effects was similar in the two groups.

[0061] The study interprets that the administration of Ocimum tenuiflorum bioactive complex is associated with significantly greater improvements in PSS scores compared to the placebo. Based on the PSS, there was a 38% decrease in perceived stress after 8 weeks of intervention in participants taking Ocimum tenuiflorum bioactive complex compared to a 19% decrease in people taking the placebo.

[0062] Further, the reduction of hair cortisol concentrations in participants taking Ocimum tenuiflorum bioactive complex suggests that the complex Ocimum dampen HPA-axis activity in the long-term. Ocimum tenuiflorum bioactive complex was well tolerated with no significant adverse effects reported by the volunteers. Example 3: Efficacy of Ocimum tenuiflorum bioactive complex on acute stress in rat model

[0063] The efficacy of Ocimum tenuiflorum bioactive complex is analyzed on active stress in albino Wistar rat model. The experiment includes three groups of rat model namely vehicle control group I administered with 0.5% CMC, group II administered with 500 mg/day, which is a human equivalent dose of 2.5% regular Ocimum and group III administered with 250 mg/day (Human equivalent dose) of Ocimum tenuiflorum bioactive complex. The test compounds are administered orally for 8 days for all the three groups. After 8 days, the albino Wistar rats were analyzed for immobility time in forced swim test (FST) as a stressor and behavioural parameter for 5 minutes. This is followed by analysis of corticosterone levels in blood as a parameter for stress. The results indicated that the Ocimum tenuiflorum bioactive complex is effective in coping up the acute stress compared to the group treated with regular Ocimum.

[0064] Ocimum tenuiflorum bioactive complex of the present invention is unique with respect to its activity and can be administered 1/5* of the dose of the conventional extract for stress-relieving and sleep enhancing activity. Ocimum tenuiflorum bioactive complex is soluble in water.

[0065] The Ocimum tenuiflorum bioactive complex possess mild taste, less colored and is useful in preparation of food products such as bread, nutritional bars, cookie, beverage, pizza, health drinks, biscuits, chocolates, and pasta and as food supplement.

[0066] The Ocimum tenuiflorum bioactive complex shall be administered in different dosage forms such as capsules, tablets, syrups, lozenges, troches, elixirs, phytoceuticals and nutraceuticals.

[0067] The process employed in the present invention imparts specific features to the Ocimum tenuiflorum bioactive complex prepared. The process is unique, simple, cost effective with respect to removal of triterpene fractions thus reducing the ursolic acid and oleanolic acid contents making the complex water soluble thus enhancing the patient compliance upon administration. The complex prepared by the process possess superior consistency, batch to batch and possibly with biological quality control.

Example 4: The efficacy of Ocimum tenuiflorum bioactive complex on acute stress in rat model

[0068] The efficacy of Ocimum tenuiflorum bioactive complex is analyzed on stress in Swiss albino mice model. The experiment includes two groups of mice namely vehicle control group I administered with 0.5% CMC; group II administered with a very low-dose of 4 mg/kg orally. The test compound is administered orally for 10 days for both the groups. After 10 days, swimming time till exhaustion was recorded in minutes for each animal. The results indicated an approximately 2-fold increase in swim time for d Ocimum tenuiflorum bioactive complex compared to vehicle control.

[0069] The Ocimum tenuiflorum bioactive complex forms the major phytochemical composition that attributes to efficacy.