A nutraceutical formulation for improving qualitative and quantitative sleep

Abstract

The present invention relates to a novel composition of plant ingredients. A green process for the preparation of novel extracts that is rich in theanine, withanolides, polyphenols (Catechins, gallic acid, rutin, myricetin) and caffeine, etc. C. sinensis based extract was found and reported safe while aqueous extract of Withania somnifera leaves was found to be safe at 2000 mg / kg body weight when tested in rodents. The extracts were found to be stable up to 6 months at room temperature. Combination of extracts (1:3) i.e theanine enriched extract and aqueous extract of tea and ashwagandha were found to have sleep improvement properties such as better initiation and potentiation of sleep in terms of onset of sleep and number of sleep bouts.

Description

[0001] A NUTRACEUTICAL FORMULATION FOR IMPROVING QUALITATIVE AND QUANTITATIVE SLEEP

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a neutraceutical formulation for sleep disorders comprising (a) enriched L-theanine extract of Camellia sinensis (from spilled black tea leaves, spilled green tea leaves, dry green tea leaves, tender shoots and tea waste from tea garden and industry) and (b) aqueous extract of Withania somnifera leaves. Particularly the present invention provides a neutraceutical formulation comprising L-theanine extract of Camellia sinensis and aqueous extract of Withania somnifera leaves and contains L-theanine, polyphenols, withanones, and withanolides. Further the formulation based on this combination was found to be safe and effective in improving qualitative and quantitative sleep in mice, zebrafish and in human clinical trials.

[0004] BACKGROUND OF THE INVENTION

[0005] Sleep disorder(s) is a common health issue that affects a significant population worldwide. The global burden of insomnia is extensive both for the patient, as it affects quality of life, as well as for the society. Insomnia is linked with increased risk for neurological (like depression), occupational and physical (like hypertension) health issues. Around 10% of the global adult population develops chronic insomnia disorder, and approximately 20% suffers from intermittent insomnia symptoms. International Classification of Sleep Disorders (ICSD) identifying seven major categories that include insomnia disorders, sleep- related breathing disorders, central disorders of hypersomnolence, circadian rhythm sleepwake disorders, sleep-related movement disorders, parasomnias, and other sleep disorders. Sleep Medicine Clinics, 2022 17(2), 173-91 , demonstrated that among all the populations, aged adults, and persons with socioeconomic problems are more prone to insomnia. Sleep disorders is one the major problems world over. It is a condition characterized by a range of problems with the duration or continuity or quality of sleep.

[0006] The American Psychiatric Association in the year 2013 reported all major symptoms which include difficulty to fall asleep at bedtime, awakening during the middle of the night with difficulty of going to sleep again, or too early awakening in the morning. A lot of pharmacotherapeutic and non-pharmacological interventions are being practiced to manage insomnia.

[0007] Pharmacy and Therapeutics, 2015, 40(11), 759 discloses that Benzodiazepines class of compounds like triazolam, estazolam, temazepam, quazepam and flurazepam are primarily used for the management of insomnia. Apart from the Benzodiazepines, a few nonbenzodiazepines like zolpidem, eszopiclone, and zaleplon are also practiced for insomnia. The non-pharmacological interventions for sleep management includes sleep hygiene education, stimulus control, cognitive therapy, paradoxical intention, etc. Recently, there has been a great interest in development of nutraceutical to improve sleep for insomnia management due a several side-effects and habit-forming tendency of pharmacotherapeutic agents. Several plants and compounds like chamomile, withania, L tryptophan, L-theanine, kava kava, melatonin, cherry juice, valerian, marijuana, lemon balm, etc. have shown beneficial effects in promoting sleep.

[0008] Progress in neuro-psychopharmacology and biological psychiatry, 2008, 32(5), 1093-1105 demonstrated that out of the above compounds, Withania somnifera, is used in Ayurveda as a rasayana (rejuvenation), which promotes physical and mental health, rejuvenate the body in debilitated conditions and increase longevity.

[0009] Central Nervous System Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Central Nervous System Agents), 2010, 10(3), 238-246 discloses that the term ‘somnifera’ in its botanical name in Latin means ‘sleep inducer,’ which probably refers to its utility to treat anxiety or mental disturbances.

[0010] The root or whole plant extract of Withania somnifera has been used to induce sleep in Ayurveda. Some products from Withania somnifera are available in market viz., Stresswin and Ashwagandharista, which claims to give relief from sleep disorders. PLoS One, 2017, 12(2), e0172508 disclosed that the water extract of Ashwagandha induced significant amount of non-rapid eye movement sleep with slight change in rapid eye movement sleep.

[0011] Similarly, L-Theanine has shown to lower the synthesis of serotonin, 5-HIAA, and increases tryptophan levels in cerebral cortex. It is shown to increase the levels of gamma- aminobutyric acid (GABA), which plays an important role in various neurological disorders viz. anxiety, depression and insomnia, Tea leaves have found to be an excellent source of L-theanine. Tea leaves also contains the polyphenols that mimics the benzodiazepine receptor and helps in the management of brain disorders. Earlier, there have been some references on the use of Withania somnifera with or without L-theanine along with other herbal ingredients for improving sleep quality in several patent documents and products.

[0012] US2015 / 0320814A1 discloses nutraceutical formulation wherein combination of ashwagandha (100-500 mg) and L-theanine (50-500 mg) niacin, magnesium, ashwagandha, passion flower, skullcap, St. John's Wort, gamma aminobutryic acid, chamomile, and L-theanine has been used along for treatment of anxiety, depression and improvement of sleep.

[0013] US10517322 discloses dietary supplement formulations for promoting sleep wherein combination of ashwagandha root extract (28 mg) and L-theanine (140 mg) are present along with other naturally occurring substances.

[0014] KR10-1971574 discloses the use of Withania somnifera root extract and pure tea extract as active ingredients in a cosmetic composition for treating sleep disturbance or for sleep maintenance.

[0015] GB2510659 discloses the synergistic combination of alkylamine compounds and herbal sedatives (ashwagandha, green tea extract, theanine) for treatment of sleep disorder like insomnia. Non-patent literature from NutriScience (Scientific Assessment of the Synergistic Effects of L-Theanine and Ashwagandha) also discloses the clinical assessment of the synergistic combination of L-theanine and Ashwagandha for a potent nootropic, stress, anxiety relief formulation.

[0016] The two products Schiff® Sleep and Shine Sleep Relaxed (Reckitt Benckiser Healthcare) and ZenX (88Herbs) claims synergistic blend of Ashwagandha and L-theanine in combination with polymers, binders, processing aids and other anticaking agents. However, these products used inorganic L-theanine not derived from Camellia sinensis.

[0017] Proceedings of the National Academy of Sciences, India Section B: Biological Sciences, 2023 93(2), 397-408 discloses maximum synergistic antioxidant potential of Camellia sinensis and Withania somnifera with DPPH assay, superoxide radical scavenging and hydrogen peroxide scavenging assay. Molecules, 2021 , 26(15), 4593 uses individual compositions of methanolic extracts of Camellia and Withania for antioxidative and chemoprotective potential.

[0018] Journal of Pharmacognosy and Phytochemistry, 2015, 4(2), 282-291 describes the tea infusion using Withania somnifera stems, Cinnamon bark, Tinospora cordifolia stems, Terminalia arjuna bark, Green tea and mixture of these herbs to assess their potential for new herbal tea development. These formulations use roots or plant parts of W. somnifera.

[0019] OBJECTIVES OF THE INVENTION

[0020] The main objective of the present invention is to develop a nutraceutical formulation that induces qualitative and quantitative sleep in humans. Yet another objective of the present invention is to develop process for the preparation of L-theanine enriched extract from Camellia sinensis (from spilled black tea leaves, spilled green tea leaves, dry green tea leaves and tea waste from tea garden and industry / orthodox black and green teas) through green process.

[0021] Yet another objective of the present invention is to develop a process for the preparation of aqueous extract of Withania somnifera leaves.

[0022] SUMMARY OF THE INVENTION

[0023] Accordingly the present invention provides a nutraceutical formulation for the sleep disorders, comprising aqueous extract of Withania somnifera leaves in a range from 45-60 wt% and enriched theanine extract of Camellia sinensis leaves / shoots in a range from 15- 20 wt%, along with standard excipients ranges from 0-35 wt%.

[0024] In an embodiment of present invention the standard excipients may comprise fillers, disintegrant, glidants, lubricant and preservatives.

[0025] In another embodiment of present invention the filler are selected from lactose anhydrous and / maltodextrin at concentration in the range of 5-30 wt% and disintegrant are selected from carboxymethyl cellulose sodium (CMC Na) at concentration in the range of 0.0- 2.0wt%.

[0026] In yet another embodiment of present invention the glidant are selected from talc and aerosil at concentration of 1 -5 wt% and lubricant is selected from magnesium stearate at concentration of 1-5 wt% and the preservatives are selected from mixture comprising Sodium methyl-paraben and Sodium propyl-paraben at concentration in range of 0.0-0.5 wt%.

[0027] In yet another embodiment of present invention the said nutraceutical formulation may be in the form of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, lyophilized preparations, etc.

[0028] In yet another embodiment of present invention the said formulation improved the sleep latency period by 30.33%, total sleep time by 10.30 % percentage sleep efficiency percentage by 11 .45% and wake after sleep by 17.39% in humans. In yet another embodiment of present invention the said formulation provide 0.54-0.70 fold increase in onset of sleep bouts in zebrafish and 5.5 to 10.40 fold increase in number of sleep bouts in zebrafish.

[0029] In still another embodiment, the present invention provides a process of preparation of nutraceutical formulation wherein the said process comprise steps of, i. preparation of Camellia sinensis leaves extract by placing the decoction of tea leaves and tea leaves waste in solvent selected from methanol, ethanol or various ethanol and water combination at temperature in the range of 60-100 °C pH in range of 4-6 followed by filtration and passing the extract through the cationic resin, ii. the extract obtained in step (i) was passed through series of column chromatography (Column-I & II) with water on adsorbent Ambrolite IR20 / IR120, XAD 7HP / 15HP and polyamide either individually or one after one or parallels was spray dried to obtain Camellia sinensis leaves extract to obtain the enriched theanine extract, iii. the theanine rich extract of step (ii) was spray dried to obtain a powder, iv. aqueous extract of Withania somnifera leaves was prepared by shade drying 5 kg leaf material to obtain 650 gm dried leaves, v. 100 gm of dried leaves obtained in step (iv) were macerated to fine powder for making aqueous extract in one litre sterile distilled water (10% w / v), the mixture was incubated overnight at 50eC with gentle shaking, extracts were centrifuged at 4000 rpm at 25eC for 20 min; pellet was extracted using 500 ml of sterile water in the similar manner for 4 h supernatant was collected and filtered through first muslin cloth and then a sieve gauge containing 1 no Whatman filter paper; vi. 1.2 L filtrate obtained in step (v) was concentrated by using rotary evaporator at 50 eC and the concentrated extract was lyophilized to get a free flowing dry Withania somnifera extract, vii. aqueous extract of Withania somnifera leaves as obtained in step (vi) , enriched theanine extract as obtained in step (iii) were mixed with standard excipients of fillers, disintegrant, glidants, lubricant and preservatives in a ratio of 3:1 to obtain nutraceutical formulation. In still another embodiment of present invention the solvent used for extraction is performed with methanol, ethanol or various ethanol and water combinations ranging from (0-100%).

[0030] In still another embodiment of present invention the tea leaves were selected from spilled black tea leaves, spilled green tea leaves, dry green tea leaves, tender shoots of tea plant, tea waste from tea garden and tea industry, orthodox green tea, or orthodox black tea.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1. The representative figure of plants used in the formulation, the extracts produced and the HPLC profile of the marker compounds.

[0033] Figure 2. The representative figure of TLC and NMR for the identification of L-theanine from Cammelia leaves.

[0034] Figure 3. UPLC-PDA Chromatogram of L-theanine, withanolides and polyphenols of standards and plant extracts.

[0035] Figure 4: Effect of Withania somnifera (WS) extract on the onset and total duration of the loss of diazepam-mediated righting reflex as an index of sleep.aP < 0.05 in comparison to vehicle control group and;bP < 0.05 in comparison to WS-50 (Withania somnifera 50 mg / kg) group.

[0036] Figure 5: Effect of L-Theanine(Lt) extract on the onset and total duration of the loss of diazepam-mediated righting reflex as an index of sleep.aP < 0.05 in comparison to vehicle control group and;bP < 0.05 in comparison to Lt-10(L-Theanine 10 mg / kg) group.

[0037] Figure 6. Effect of Withania somnifera and L-theanine alone and in different combinations on the onset of sleep bouts in zebrafish. Each value is shown as a fold change over Normal (Vehicle) control group. Normal (Vehicle): Group kept in fish water; WS (200 ug / mL): Group exposed to 200 pg / mL concentration of Withania somnifera; LT (85 ug / mL): Group exposed to 85 pg / mL concentration of L-theanine; WS+LT (50:50): Group exposed to Withania somnifera and L-theanine at 50 : 50 ratio of their respective effective concentrations; WS+LT (75:25): Group exposed to Withania somnifera and L- theanine at 75 : 25 ratio of their respective effective concentrations; WS+LT (25:75): Group exposed to Withania somnifera and L-theanine at 25 : 75 ratio of their respective effective concentrationsand; Diazepam: Reference standard group exposed to 12.5 pM concentration of diazepam, ns: Insignificant, p-value is less than 0.05 was indicated as significant. Figure 7. Effect of Withania somnifera and L-theanine alone and in different combinations on the number of bouts in zebrafish during the test period. Each value is shown as a fold change over Normal (Vehicle) control group. Normal (Vehicle): Group kept in fish water; WS (200 ug / mL): Group exposed to 200 pg / mL concentration of Withania somnifera; LT (85 ug / mL): Group exposed to 85 pg / mL concentration of L- theanine; WS+LT (50:50): Group exposed to Withania somnifera and L-theanine at 50 : 50 ratio of their respective effective concentrations; WS+LT (75:25): Group exposed to Withania somnifera and L-theanine at 75 : 25 ratio of their respective effective concentrations; WS+LT (25:75): Group exposed to Withania somnifera and L-theanine at 25 : 75 ratio of their respective effective concentrationsand; Diazepam: Reference standard group exposed to 12.5 pM concentration of diazepam, p-value is less than 0.05 was indicated as significant.

[0038] DETAILED DESCRIPTION OF THE INVENTION

[0039] The preferred embodiments are provided so that the disclosure will be thorough and will fully convey the scope to those who are skilled in the art. Various specific details are set as specific components to provide an overall understanding of the preferred embodiments of the present disclosure. It will be apparent to those skilled in the art that the specific details need not be employed and the embodiments may be embodied in many different forms and the steps followed do not limit the scope of the disclosure.

[0040] It is also to be understood that the terminology used herein is for the purpose of describing only the particular embodiments of the invention and is not intended to limit the scope of the invention in any manner. It must also be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0041] In an embodiment the present invention provides a nutraceutical oral capsule formulation for treating the sleep disorders, comprising of: i. 150 mg of aqueous extract of Withania somnifera leaves; ii. 50 mg of enriched theanine extract of tea (Camellia sinensis) leaves / shoots; iii. wherein the ration of aqueous extract of Withania somnifera leaves, and enriched theanine extract of tea leaves / shoots is 3:1 ; iv. and standard excipients.

[0042] As used herein the term “enriched theanine extract of tea (Camellia sinensis) leaves / shoots” refers to the preparation of extract from the leaves / shoots of plant Camellia sinensis using a simple blender to grind the dried leaves / shoots with water followed by a series of centrifugations to remove the solid waste, finally a series of filtrations to have a sterile aqueous extract in the form of dried powder containing higher proportion of theanine. As used herein the term “aqueous extract of Withania somnifera leaves” refers to preparation of extract using water followed by a series of centrifugation and filtration to eliminate solid waste. The supernatant containing soluble metabolites were dried and collected in the form of dried powder. The protocol for extracting soluble compounds from Withania somnifera leaves was performed using a simple blender to grind the dried leaves with water followed by a series of centrifuges to remove the solid waste, finally a series of filtrations to have sterile aqueous extract in the form of solid dried powder.

[0043] The plant resource used in experiments are Withania somnifera which has been cultivated in the Experimental Farm of CSIR-lndian Institute of Integrative Medicine, Chatha, Jammu, UT of Jammu and Kashmir, India. The plant resource Camellia sinensis used in present invention has been cultivated in the Banuri Tea Experimental Farm of CSIR-lnstitute of Himalayan Bioresourse Technology at Palampur, Kangra, Himachal Pradesh, India.

[0044] The aqueous extract of Withania somnifera leaves is hygroscopic in nature. The invention provides the process for preparing the oral capsule formulation, wherein the method involves a geometric dilution of excipients followed by addition of phyto constituent enriched extracts.

[0045] As used herein the term “phytoconstituent enriched extracts” refers to the extract which is having large amount of bioactive compounds present in leaves. The phytoconstituents / bioactive compounds are generally synthesized in plants and many of them may be grouped as active drug constituents and inert non-drug constituents. The excipients used may comprise: at least one / two fillers, at least one / or no disintegrant, at least one / two g lidants, at least lubricant and preservatives.

[0046] The present invention provides a nutraceutical formulation for the sleep disorders, comprising aqueous extract of Withania somnifera leaves in a range from 45-60 wt% and enriched theanine extract of Camellia sinensis leaves / shoots in a range from 15-20 wt%, along with standard excipients ranges from 0-35 wt%.

[0047] In an embodiment of the present invention the excipient composition comprises 5-30 wt% of lactose anhydrous and / maltodextrin, 0.0-2 wt% of carboxymethyl cellulosesodium, 1-5 wt% of talc and / aerosil, 1 -5 wt% of magnesium stearate and 0.0-0.5 wt% of sodium methylparaben and sodium propylparaben. In an another embodiment the nutraceutical product may be in the form of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, lyophilized preparations, etc.

[0048] In an another embodiment the present invention also provides a process for the preparation of theanine enriched extract of Camellia sinensis comprising the following steps;

[0049] (i) collecting and weighing 5 Kg of Camellia sinensis (spilled black tea leaves, spilled green tea leaves, dry green tea leaves and tea waste from tea garden and industry); (ii) material was decocted / hot percolated (2 percolation) in extraction vessel; (iii) extract temperature was measured at 80°C and then filtered;(iv) adjusting the pH of the extract to 4-6; (v) passing the extract through the cationic resin and spray dried.

[0050] The term “theanine enriched extract of Camellia sinensis" refers to the preparation of extract from the leaves / shoots of plant Camellia sinensis using a simple blender to grind the dried leaves / shoots with water followed by a series of centrifuges to remove the solid waste, finally a series of filtrations to have a sterile aqueous extract in the form of dried powder containing higher proportion of theanine. The term “decocted / hot percolated” refers to a technique of extraction of desirable constituents from a plant source at high temperature (80°C) or simply by boiling or heating it. The term “extract temperature” refers to the temperature maintained (80°C ) during the extract preparation. The term “cationic resin” refers to the resins consist of combinations of insoluble molecules bearing fixed negative charges, that can loosely bind with positively charged ions (cations). These readily exchange with cations present in the extract depending upon their affinity towards the resin and their concentration. Ambrolite IR 20 / 120 resin (Highly cationic resin) was used to get the important constituents from the extract. The term “spray dried” refers to a process of preparation of a solid dry powder from a liquid or slurry material by spraying the liquid / slurry material into a hot drying medium. Here the extracts were concentrated using spray-dried technique with a pilot scale spray dryer and recovered powder around 3-5% in yield- 15%)

[0051] In an embodiment the present invention also provides a process for the preparation of aqueous extract of Withania somnifera leaves comprising the following steps;

[0052] (i) Collecting and weighing 5 Kg of Withania somnifera fresh leaves; (ii) 5 kg leaf material was shade dried and about 650 gm dried leaves were obtained; (iii) 100 gm of dried leaves were macerated to fine powder using pestle and mortar for making aqueous extract; (iv) one litre sterile distilled water (10% w / v) was added to the leaf powder and the mixture was incubated overnight at 50eC with gentle shaking (v) extracts were centrifuged at 4000 rpm at 25eC for 20 min; (vi) pellet was again extracted using 500 ml of sterile water in the similar manner for 4 h; (vii) supernatant was collected and filtered through first muslin cloth and then a sieve gauge containing 1 no Whatman filter paper; (viii) filtrate (about 1 .2 L) was concentrated by using rotary evaporator at 50eC; (ix) concentrated extract was lyophilized to get a free flowing extract; (x) final product (dried extract) was kept in air tight container at 4eC till use.

[0053] The term “aqueous extract of Withania somnifera leaves” refers to preparation of extract using water followed by a series of centrifugation and filtration to eliminate solid waste. The supernatant containing soluble metabolites were dried and collected in the form of dried powder.

[0054] The present invention also studies the effect of individual extracts and their combinations in different ratios on the mice and zebrafish models for sleep related disorders.

[0055] The present invention pertains to the use of Camellia sinensis (spilled black tea leaves, spilled green tea leaves, dry green tea leaves and tea waste from tea garden and industry) and Withania somnifera (leaves) for the preparation of extract, and thereof proving its efficacy in the improvement of sleep in zebrafish and mice model.

[0056] The invention provides for chemically characterize both the extracts using various spectroscopic techniques including thin layer chromatography, HPLC and NMR.

[0057] The effective combination used to prepare the formulation (capsule) for treatment of sleep disorders in humans is provided. A total of 40 subjects were enrolled after they met the inclusion and exclusion criteria. Written informed consent was obtained from the enrolled subjects on Day -7 as well as physical examination, vital signs, medication history and past medical history, and assessment of physician global assessment of insomnia, insomnia severity index (ISI), and self-reported questionnaire by the study subjects were recorded on Day -7.

[0058] The study protocol was performed for a duration of 8 days administered at a frequency of two capsules per day taken with lukewarm water at night on all the enrolled subjects on Period I (Visit 1 and Visit 2) and Period II (Visit 1 and Visit 2). The study analysis reveals that all the study medications were equally well tolerated and there were no adverse events reported. Overall, all the study medications were safe and well tolerated. The human intervention studies using the formulation and a control placebo suggested an improvement of 30.28% in the mean physician global index score, 41 .65% in the mean insomnia severity index, and 30.83% the mean score of self-reported questionnaire. Furthermore, an improvement of 30.33% in the mean sleep latency period, 10.30% in total sleep time and 11 .45% in sleep efficiency was also observed using polysomniography was also observed. The mean wake after sleep onset score also improved by 17.39% in formulation treated group.

[0059] Among the serum biomarkers, the mean serum melatonin level at the baseline was 256.69 and at the end of 8 days of treatment was 309.02. The mean serum dopamine level at the baseline was 135.31 and at the end of 8 days of treatment was 147.23. The mean serum cortisol level at the baseline was 606.99 and at the end of 8 days of treatment was 416.84. The mean serum serotonin level at the baseline was 157.26 and at the end of 8 days of treatment was 181 .49.

[0060] In conclusion, this study revealed that treatment with formulation of a combination of Withania leaf extract and theanine enriched Camellia extracts has improved the various parameters of insomnia in a positive manner in relation to the sleep quality, sleep efficiency and sleep duration as assessed by the efficacy parameters (table 1).

[0061] Table 1. Percentage improvement in sleep parameters after use of developed formulation than the placebo in humans clinical trials.

[0062] The present invention evaluates the individual ingredients and their combination for sleep potentiation in mice and zebrafish models. The results showed better onset and duration of sleep in terms of onset of sleep and number of sleep bouts (table 2(a) and 2(b)). Furthermore, chemically characterized extracts were combined in optimized ratio and utilized to prepare formulation.

[0063] Table 2 (a) represents the effect of Withania somnifera extract and L-theanine alone and in different combinations on the onset of sleep bouts in zebrafish during the test period. Each value is represented as fold change over Normal (Vehicle) control group.

[0064] Table 2 (b) represents the effect of Withania somnifera and L-theanine alone and in different combinations on the number of bouts in zebrafish during the test period. Each value is represented as fold change over Normal (Vehicle) control group.

[0065] EXAMPLES

[0066] The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention.

[0067] EXAMPLE - 1 Illustrates the process for the preparation of enriched theanine extract of Camellia sinenisis

[0068] Different parts of Camellia sinenisis (Syamaparni / Caha)such as spilled black tea leaves / spilled green tea leaves / dry green tea leaves / tender shoots and tea waste from tea garden and industry) or made teas (orthodox green tea / orthodox black tea) were used individually or in combination as a raw material to produce Theanine enriched extract of C. sinenisis. The raw material was checked for authenticity and quality (table 3 and table 4). The quality C. sinenisis materials defines by the range of theanine and that should be in the range of 1 - 2% or >2% (Figure-2). Further, the material is processed to prepare enriched theanine extract (Schemel -3).

[0069] The process described below can be scaled up to a larger quantity of extract. The details provided for preparation of the following summaries reveal the presently used method for extract preparations and should not be considered as limiting. The quantities and times described below can be varied substantively to provide a suitable extract of respective material in accordance with the invention.

[0070] Table 3. Raw material quality

[0071] Table 4. Specification of material

[0072] Process : The production process involves 6 unit processes that are linear viz.,

[0073] 1) Drying of input materials in an industrial tray dryer

[0074] 2) Pulverizing or grinding of raw materials -

[0075] 3) Extraction (Percolation / stirred tank extraction)

[0076] 4) Filtration

[0077] 5) Pass the filter from column 1 & 2

[0078] 6) Vacuum concentration

[0079] 7) Spray drying

[0080] 8) Packing

[0081] A detailed description of herbal extract preparation (3 methods) Method A

[0082] Camellia sinensis tea shoots either fresh or dried were used for the extraction of theanine. 5 Kg material was decocted / hot percolated (2 percolation) in extraction vessel with distilled water (1 :20) at temperature 90-95 °C for 45 min. Extract temperature was measured 80°C and then filter with sparkler filter to remove the particles. Filtrate (TSS:0.9 Brix (Below 1° Brix) was concentrated with rota evaporator apparatus to reduce its volume upto 1 / 3rd. pH of the extract was found 4-6. Extract was passed from the Ambrolite IR 20 / IR 120 resin (Highly cationic resin) and then from Ambrolite XAD 7HP resin. Thereafter, pH was measured, and material was concentrated (TSS:10-15Brix (spray dried at temperature inlet 180-120°C, outlet 70-95°C (BuchiPVt Ltd / other trade mark). Yield was 19 gm of enriched Theanine extract i.e. 0.38% (5 kg batch) and 0.57 % (12.5 kg batch) Theanine was enriched upto 30-50%, and polyphenols were upto 30-50%.

[0083] Method B

[0084] 25 Kg material was decocted / hot percolated (2 percolation) in extraction vessel with distilled water (1 :10 / 20) at temperature 90-95°C for 45 min. Extract temperature was measured 80°C and then filter with sparkler filter to remove the particles. Filtrate was concentrated with rotaevaporator apparatus to reduce its volume upto 1 / 3rd. pH of the extract was found 4-6. Extract was passed from the Ambrolite IR 20 / 120 resin (Highly cationic resin)

[0085] Thereafter, pH was measured, and spray dried at temperature inlet 140 °C, outlet 80 °C (Shachiengineering- Fabricated as required). Yield of enriched Theanine extract was 2.9 % (25 kg batch). Theanine and polyphenols were enriched in the extract.

[0086] Method C

[0087] 25 Kg material was decocted / hot percolated (2 percolation) in extraction vessel with distilled water (1 :20 / 10) at temperature 90-95°C for 45 min. Extract temperature was measured 80°C and then filter with sparkler filter to remove the particles. Filtrate was concentrated with rotaevaporator apparatus to reduce its volume upto 1 / 3rd. pH of the extract was found 4.5-6.0. Extract was passed from the XAD 7HP resin (approx, neutral PH) / IR 20 / both and after first passing the extract was again passed through the respective resin and washed with water. Thereafter, pH was measured and spray dried at temperature inlet 140°C, outlet 80°C (Shachi engineering- Fabricated as required). Yield of enriched theanine extract was 5.0 % (25 kg batch). Theanine and polyphenols were enriched in the extract. a) Extraction process (Stirred tank extractor)

[0088] • First percolation

[0089] In the first percolation, Load 100 kg pre-mixed herbs in the extraction vessel and add 10 times RO water (1000 liters) in the extraction vessel and heat it at 75-80 °C for 30 minutes with proper agitation 10-15 rpm.

[0090] Parameters used:

[0091] ❖ RO water: Material ratio(10:1 )

[0092] ❖ Vessel Temperature: 75-80°C

[0093] ❖ Extraction time: 30 min.

[0094] ❖ Tank agitation: 10-15rpm

[0095] • Second percolation

[0096] In the second percolation, load the residual meal obtained from 1stextraction and add 10 times RO water (1000 liters) in the extraction vessel and heat it at 75-80 °C for 30 minutes with proper agitation 10-15 rpm.

[0097] • Third percolation In the third percolation, load the residual meal obtained from 2nc* extraction in extraction vessel and add 10 times RO water (1000 liters) in the extraction vessel, and heat it at 75-80 °C for 30 minutes with proper agitation 10-15 rpm.

[0098] Parameters used:

[0099] ❖ RO water: Material ratio( 10:1 )

[0100] ❖ Vessel Temperature:75-80 °C

[0101] ❖ Extraction time: 30minutes

[0102] ❖ Tank agitation: 10-15rpm a) Mixing process

[0103] Collection of extract percolated from extractions

[0104] Extract collected from first, second and third percolation are mixed together total quantity 2700 liters’ total extract.

[0105] Parameters:

[0106] ❖ TSS: 0.9 Brix (Belowl ° Brix)

[0107] □ pH: 5.5 (varies from 4.5-6.0)

[0108] □ TDS:103

[0109] ❖ Taste: Acceptable (with fresh tea extract aroma) b) Filtration process

[0110] Extract collected from first and second percolation are mixed together and filtered with one time with 10-micron muslin cloth and stored in Stainless steel or food grade HDPE container / vessel c) Column chromatography

[0111] Pass the liquid extract through Column -I and II using adsorbents [ Ambrolite IR 20 / 120 / XAD 7HP / 15HP / polyamide (4:1 )]. i.e Similar adsorbent in columns or different adsorbents. After passing from column l&ll extract was filtered or nonfiltered and further subjected for concentration. d) Concentration process

[0112] Filtered extract pass through online concentrator (Equipment name: Centitherm / reducer) and again stored concentrated herbal extract in Stainless steel or food-grade HDPE container / vessel

[0113] Parameters used: • Before concentration herbal extract, TSS:0.8 Brix (Belowl Brix)

[0114] • T emperature: 60 °C to 650

[0115] • Set Vacuum:500mmHg

[0116] • After Concentration herbal extract TSS:10-15Brix

[0117] • Concentrated herbal extract quantity around 200liters e) Spray drying process

[0118] • Concentrated extract spray-dried with a pilot scale spray dryer. And recovered powder around 3-5% in yield-15%)

[0119] Parameters used:

[0120] • Concentrated extract TSS:10-15 Brix Inlet temperature: 120-140 / 140-160 / 160- 180 / 180-183°C

[0121] • Outlet temperature: 80 / 90 °C - 95°C

[0122] • Received spray dried powder and packed at HDPE container with inner polybag

[0123] • Final product Yield: 3-5 % (approx.)

[0124] Other lab scale process: Enriched theanine extract was prepared according to the scheme-2 a & b and parameters were same as industrial method and Method A-C.

[0125] EXAMPLE - 2

[0126] Illustrates the chemical composition of enriched theanine extract of Camellia sinenisis. The chemical composition of extract was analyzed using HPLC / UPLC-PDA. The analysis revealed the presence of theanine and polyphenols (Catechins) (table 5 and table 6). These chemical compositions may vary or depend on the conditions of the raw material and their processing.

[0127] I) Standard Test Procedures (protocol analytical)

[0128] Analytical method for the qualitative and quantitative estimation of Quality parameters.

[0129] TLC method

[0130] Amino acid (Theanine)

[0131] Solvent system: n-butanol: acetone: acetic acid: water (14:14: 4: 2.2)

[0132] Derivatizing agent: Ninhydrine reagent

[0133] Polyphenols (Catechins)

[0134] Solvent system: Toluene: acetone: formic acid (18:18:4.2)

[0135] Derivatizing agent: Anasaldehyde sulphuric acid reagent (Kumar et aL, 2016)

[0136] NMR method: Qualitative and quantitative estimation of Theanine and polyphenols has been carried out NMR spectroscopic method as earlier reported by Kumar et al., 2016. UPLC-PDA

[0137] • UHPLC-PDA method (10 min) was developed for the qualitative and quantitative analysis of Theanine in the plant extracts, Theanine enriched extract / final product. a) Theanine estimation

[0138] Method A:

[0139] Instrument system - Waters ACQUITY UPLC, H-Class

[0140] Detector: ACQUITY UPLC PDA

[0141] Column: Waters BEH RP C18, 100 mm

[0142] Inj. Vol.: 1 pl

[0143] Flow rate: 0.5 ml / min

[0144] The analytical column used was C18 column (2.1 mm, 100mm). The gradient elution system was used, mobile phase A contained sodium acetate buffer, mobile phase B was acetonitrile (ACN). The gradient was as follows: 0-0.54 min, 0.1% B, hold at same gradient for 0.54 min.; then 0.54-5.74 min, linear gradient from 0.1% B to 9.1% B; 5.74-6.74 min., 21.2% B; 6.74-8.12 min, linear gradient from 21.2% B to 39.5% B; 8.12-9.04 min, 59.6% B; 9.04-10.0 min, 90.0% B; 10.0-10.64 min, 90% B; 10.64-10.73 min, 0.1% B; 10.73-12.0 min, 0.1% B. Elution was performed at a solvent flow rate of 0.5 mL / min.

[0145] Method B

[0146] Instrument system - Waters ACQUITY UPLC, H-Class

[0147] Detector - ACQUITY UPLC PDA

[0148] Column - Agilent Eclipse Plus RRHD C18

[0149] Inj. Vol. - 2 pl

[0150] Flow rate - 0.16 ml / min

[0151] The analytical column used was Agilent Eclipse plus, RRHD C18 column (2.1 mm, 150mm, 1.8 pm). The gradient elution system was used, mobile phase A contained 0.1% Formic acid in water, mobile phase B was pure methanol (MeOH). The gradient was as follows: 0- 2 min, 3% B, 2-5 min, linear gradient from 3% B to 20% B; 5-7 min., 40% B; 7-8 min, linear gradient from 40% B to 3% B; 8-10 min, 3% B. Elution was performed at a solvent flow rate of 0.16 mL / min. Detector used was ACQUITY UPLC PDA at wavelength 210 nm.

[0152] Polyphenol estimation (Catechins)

[0153] Instrument system - Shimadzu, HPLC

[0154] Column - RP C18

[0155] Flow rate - 10 ml / min

[0156] The analytical column used was RP C18 column (4.6mm 150 mm, 5 mm). The column was equilibrated under starting condition for 5 min. Detection wavelength was set at 210 nm. The gradient elution system was used, mobile phase A contained 0.1% OPA in water, mobile phase B was pure acetonitrile (ACN). The gradient started from 0 min. at 10% B; then from 0-10 min, 10% B to 30% B; 10-15 min, linear gradient from 30% B to 35% B; 15-18 min, 20% B; 18-20 min, linear gradient from 20% B to 10% B. Elution was performed at a solvent flow rate of 10 mL / min.

[0157] Table 5 Determination of theanine

[0158] Table 6 Composition of other compounds in theanine enriched extract

[0159] EXAMPLE - 3 Illustrates the process for the preparation of lyophilized aqueous extract of Withania somnifera leaves.

[0160] Withania somnifera fresh leaves were used for the preparation of extract. 5 kg leaf material was shade dried and about 650 gm dried leaves were obtained. 100 gm of dried leaves were macerated to fine powder using pestle and mortar for making aqueous extract. One litre sterile distilled water (10% w / v) was added to the leaf powder and the mixture was incubated overnight at 50eC with gentle shaking. After incubation the extracts were centrifuged at 4000 rpm at 25eC for 20 min. The pellet was again extracted using 500 ml of sterile water in the similar manner for 4 h.The supernatant was collected and filtered through first muslin cloth and then a sieve gauge containing 1 no Whatman filter paper. The filtrate (about 1 .2 L) was concentrated by using rotary evaporator at 50eC.

[0161] The concentrated extract was lyophililized to get a free flowing extract. The final product (dried extract) was kept in airtight container at 4eC till use.

[0162] EXAMPLE - 4

[0163] Illustrates the chemical composition of aqueous extract of Withania somnifera leaves.

[0164] HPLC-PDA method for the estimation Withania component. The method of 40 min. was developed for the qualitative and quantitative analysis of Withaferin A, Withanolide A and Withanone in the plant extracts / spray dried extract / final product. Sample preparation: Sample for HPLC estimation were prepared using 10 mg lyophilized aqueous extract in 1 ml of methanol and water: methanol (1 :1). It was filtered through 0.22 pm PTFE filter for injecting into column.

[0165] Instrument system - Agilentl 260 Infinity Quaternary LC System

[0166] Detector: PDA

[0167] Column: LiChrosorb RP-18 HPLC Column (4.6 x250 mm; 5 pm)

[0168] Inj. Vol.: 10ul

[0169] Flow rate: 0.5 ml / min

[0170] Solvent: MeOH:water (60:40)

[0171] HPLC-PDA method (40 min) was developed for the qualitative and quantitative analysis of withanolides namely Withaferin A, Withanolide A and Withanone in the lyophilized plant extracts. An isocratic elution protocol was used with mobile phase Methanol and water mixture (60:40) for 40 min.

[0172] EXAMPLE - 5

[0173] Illustrates efficacy testing of Withania somnifera extract

[0174] Withania somnifera extract was dissolved in the fish water to achieve a final concentration of 200 ug / mL and temperature was set at 28.5°C and fish behaviour was noted. The onset of bouts and number of bouts were recorded during the test duration of 1 h with diazepam 12.5pM as a reference standard. The bouts were considered as an index of sleep-like behaviour. Fold change in the onset and number of bouts over normal vehicle control group was calculated. There was a significant fold change decrease in the onset (p = 0.003) and increase in the number (p < 0.001 ) of sleep bouts over normal vehicle control group when compared using One-way analysis of variance followed by Student-Newman- Keuls post hoc test. EXAMPLE - 6

[0175] Illustrates efficacy testing of L-Theanine

[0176] L-Theanine was dissolved in the fish water to achieve a final concentration of 85 ug / mL and temperature was set at 28.5°C and fish behaviour was noted. The onset of bouts and number of bouts were recorded during the test duration of 1 h with diazepam 12.5pM as a reference standard. The bouts were considered as an index of sleep-like behaviour. Fold change in the onset and number of bouts over normal vehicle control group was calculated. There was a significant fold change decrease in the onset (p = 0.033) and increase in the number (p < 0.001 ) of sleep bouts over normal vehicle control group when compared using One-way analysis of variance followed by Student-Newman-Keuls post hoc test.

[0177] EXAMPLE - 7

[0178] Illustrates efficacy testing of 50:50 combination of Withania somnifera : L-Theanine

[0179] A combination of 50:50 combination of Withania somnifera : L-Theanine was prepared i.e., Withania somnifera (100 ug / mL) extractand L-Theanine(42.5 ug / mL). The combinations were dissolved in the fish water, temperature was set at 28.5°C and fish behaviour was noted. Fold change in the onset and number of bouts over normal vehicle control group was calculated. There was a significant fold change decrease in the onset (p = 0.024) & increase in the number (p = 0.002) of sleep bouts over normal vehicle control group when compared using one-way analysis of variance followed by Student-Newman-Keuls post hoc test.

[0180] EXAMPLE - 8

[0181] Illustrates efficacy testing of 75:25 combination of Withania somnifera : L-Theanine

[0182] A combination of 75:25 combination of Withania somnifera: L-Theanine was prepared i.e., Withania somnifera (150 ug / mL) extract and L-Theanine(21 .25 ug / mL). The combinations were dissolved in the fish water and temperature was set at 28.5°C and fish behaviour was noted. Fold change in the onset and number of bouts over normal vehicle control group was calculated. There was a significant fold change decrease in the onset (p = 0.029) & increase in the number (p < 0.001 ) of sleep bouts over normal vehicle control group when compared using One-way analysis of variance followed by Student-Newman-Keuls post hoc test.

[0183] EXAMPLE - 9

[0184] Illustrates efficacy testing of 25:75 combination of Withania somnifera : L-Theanine

[0185] A combination of 25:75 combination of Withania somnifera : L-Theanine was prepared i.e., Withania somnifera (50 ug / mL) extract and L-Theanine(63.75 ug / mL). The combinations were dissolved in the fish water and temperature was set at 28.5°C and fish behaviour was noted. Fold change in the onset and number of bouts over normal vehicle control group was calculated. There was an insignificant change in the onset (p = 0.07), but significant increase in the number (p < 0.001) of sleep bouts over normal vehicle control group when compared using One-way analysis of variance followed by Student-Newman-Keuls post hoc test.

[0186] EXAMPLE - 10

[0187] Illustrates formulation development in the capsule formulations, excipients comprise at least one / two fillers, at least one / or no disintegrant, at least one / two glidants, at least lubricant and preservatives.

[0188] The filler used in the formulation composition includes, the lactose anhydrous and / maltodextrin. In the composition, the concentration of fillers is used in the range of 5-30 wt%.

[0189] The disintegrant used in tablets and capsules to ensure the rapid breakdown into their primary particles, facilitating the dissolution or release of the active ingredients. The disintegrant includes carboxymethyl cellulose sodium (CMC Na) in the concentration range of 0.0-2.0wt%.

[0190] Further, oral formulation composition included talc and / aerosil as glidant and magnesium stearate as lubricant, whereas, glidants are agents that we use to increase the flow ability of a powder, and lubricants are used to reduce friction between machine parts and the product to make the process smooth. The glidant and lubricant used in the formulations are in the concentration range of 1 -5 wt%. The formulation composition of the present invention also comprises preservatives including a mixture of Sodium methyl-paraben and Sodium propyl-paraben. The concentration range of preservatives used in the formulations is 0.0-0.5 wt%.

[0191] In another embodiment, the present invention discloses the process for the preparation of the capsule formulation comprises geometric dilution of excipients followed by the addition of phytoconstituent enriched extracts.

[0192] The formulation development was performed in the form of capsules comprising 150 mg of extract of Withania somnifera and 50 mg of enriched theanine extract of tea (Camellia sinensis) per capsule. Formulation comprises a filler, a disintegrant, a glidant, a lubricant and preservatives. Excipients were mixed by geometric dilution and mixing was performed using a mortar and pestle. Subsequently, Withania somnifera extract wastriturate properly with excipient and then tea extract was added to it with trituration in the mortal pestle. The table 7 shows the compositions of capsule formulations (F1 , F2, F3, and F4). The procedure of formulation preparation was similar in all formulations, but differs only in the excipient’s composition.

[0193] Table 7

[0194] Ingredients Quantity (wt%)

[0195] F1 F2 F3 F4

[0196] Withania somnifera extract 49.995 60 60 57.7

[0197] Enriched theanine extract of tea 16.665 20 20 19.2

[0198] (Camellia sinensis)

[0199] Maltodextrin 13.07

[0200] Starch - - - 12.82

[0201] Microcrystalline cellulose - - - 7.7 phosphate

[0202] Anhydrous lactose 13.07 10.8 11.8

[0203] CMC Na 1 - - -

[0204] Magnesium stearate 2 4 4 2.05

[0205] Talc 4 4 4

[0206] Aerosil - 1 -

[0207] Preservatives 0.2 0.2 0.2 0.51

[0208] EXAMPLE - 11

[0209] Characterization of developed formulation

[0210] The moisture content of the powdered capsule formulation was determined using Karl Fischer method. The Karl Fischer titration is a technique for the determination of the moisture content of pharmaceutical products. The powdered extractcomposition was filled in a capsule and stored in airtight container kept at 30°C ± 2°C / 65% ± 5 humidity conditions. A separate container was prepared for each test interval. At each test interval (0, 1 , and 2 months) samples were visually examined for physical changes and moisture content. Moisture content of F1 is presented in a table 8

[0211] TABLE 8

[0212] Time interval (month) Moisture content (%)

[0213] 0 4.34

[0214] 1 3.02

[0215] 2 3.70

[0216] The formulations prepared in F1 , F2 and F3 were assessed for flow property by measuring angle of repose. The angle of repose was determined by the funnel method described in United State Pharmacopoeia (USP).. Table 9 shows the flow behaviour of the prepared formulations.

[0217] TABLE 9

[0218] Formulations Angle of Repose Flow property (USP)

[0219] “Fl 38.28°±3.89° Fair 27.01 °±4.20° Excellent

[0220] F3 26.00°± 0.80° Excellent

[0221] ADVANTAGES OF THE INVENTION

[0222] • The enriched theanine extract from C. sinenisis and aqueous extract of Withania somnifera leaves are prepared by green process with standardized chemical composition.

[0223] • The raw material in both the extract is the unused / least parts of both the plants.

[0224] • Both the extracts were evaluated for sleep improvement activity and found to have significant effect. • Different compositions of both the extracts were evaluated for sleep improvement activity and found significant but the ratio 1 :3 (theanine extract: withania extract) was found to be the most effective combination.

[0225] • Most significant combination was used to develop various formulations such as capsules, tablets, syrup, sachet etc. • The extracts, their combination and product have sleep improvement properties and can be used for various sleep related disorders.

[0226] • The extracts and combinations can be utilized for various health and wellness products.

Claims

WE CLAIM1. A nutraceutical formulation for the sleep disorders, comprising aqueous extract of Withania somnifera leaves in a range from 45-60 wt% and enriched theanine extract of Camellia sinensis leaves / shoots in a range from 15-20 wt%, along with standard excipients ranges from 0-35 wt%.

2. The nutraceutical formulation as claimed in claim 1 , wherein the standard excipients may comprise fillers, disintegrant, glidants, lubricant and preservatives.

3. The nutraceutical formulation as claimed in claim 1 , wherein the filler are selected from lactose anhydrous and / maltodextrin at concentration in the range of 5-30 wt% and disintegrant are selected from carboxymethyl cellulose sodium (CMC Na) at concentration in the range of 0.0-2.0wt%.

4. The nutraceutical formulation as claimed in claim 1 , wherein the glidant are selected from talc and aerosil at concentration of 1-5 wt% and lubricant is selected from magnesium stearate at concentration of 1-5 wt% and the preservatives are selected from mixture comprising Sodium methyl-paraben and Sodium propyl-paraben at concentration in range of 0.0-0.5 wt%.

5. The nutraceutical formulation as claimed in claim 1 , wherein the said nutraceutical formulation may be in the form of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, nonaqueous solutions, lyophilized preparations, etc.

6. The nutraceutical formulation as claimed in claim 1 , wherein the said formulation improved the sleep latency period by 30.33%, total sleep time by 10.30 % percentage sleep efficiency percentage by 11 .45% and wake after sleep by 17.39% in humans.

7. The nutraceutical formulation as claimed in claim 1 , wherein the said formulation provide 0.54-0.70 fold increase in onset of sleep bouts in zebrafish and 5.5 to 10.40 fold increase in number of sleep bouts in zebrafish.

8. A process of preparation of nutraceutical formulation as claimed in claim 1 wherein the said process comprise steps of, i. preparation of Camellia sinensis leaves extract by placing the decoction of tea leaves and tea leaves waste in solvent selected from methanol, ethanol or various ethanol and water combination at temperature in the range of 60-100 °C pH inrange of 4-6 followed by filtration and passing the extract through the cationic resin, ii. the extract obtained in step (i) was passed through series of column chromatography (Column-1 & II) with water on adsorbent Ambrolite IR20 / IR120, XAD 7HP / 15HP and polyamide either individually or one after one or parallels was spray dried to obtain Camellia sinensis leaves extract to obtain the enriched theanine extract, iii. the theanine rich extract of step (ii) was spray dried to obtain a powder, iv. aqueous extract of Withania somnifera leaves was prepared by shade drying 5 kg leaf material to obtain 650 gm dried leaves, v. 100 gm of dried leaves obtained in step (iv) were macerated to fine powder for making aqueous extract in one litre sterile distilled water (10% w / v), the mixture was incubated overnight at 50eC with gentle shaking, extracts were centrifuged at 4000 rpm at 25eC for 20 min; pellet was extracted using 500 ml of sterile water in the similar manner for 4 h supernatant was collected and filtered through first muslin cloth and then a sieve gauge containing 1 no Whatman filter paper; vi. 1.2 L filtrate obtained in step (v) was concentrated by using rotary evaporator at 50eC and the concentrated extract was lyophilized to get a free flowing dry Withania somnifera extract, vii. aqueous extract of Withania somnifera leaves as obtained in step (vi) , enriched theanine extract as obtained in step (iii) were mixed with standard excipients of fillers, disintegrant, glidants, lubricant and preservatives in a ratio of 3:1 to obtain nutraceutical formulation.

9. The process as claimed in claim 8, wherein the solvent used for extraction is performed with methanol, ethanol or various ethanol and water combinations ranging from (0- 100%).

10. The process as claimed in claim 8, wherein the tea leaves were selected from spilled black tea leaves, spilled green tea leaves, dry green tea leaves, tender shoots of tea plant, tea waste from tea garden and tea industry, orthodox green tea, or orthodox black tea.

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