DESC:FIELD OF INVENTION
The present invention relates to a medicinal composition prepared from Withania somnifera extract for the treatment of sleep disorders among the mammals, preferably humans, more especially Withania somnifera extract composition in an effective dosage form comprising total withanolides for reducing the time of onset of sleep and prolongation of total sleep duration among mammals, preferably humans and method of preparation of the same.
BACKGROUND
Sleep is a vital physiological process that takes up one-third of our lives on an average and is essential for maintaining our physical, mental, and emotional well-being. A complex interplay between chronological age, stage of development, genetic, behavioural, environmental, and social factors affects sleep patterns and requirements. Adults should get at least 7 hours of sleep each night to maintain good health. Over time, population studies have revealed a steadily increasing prevalence of adults sleeping less than 6 hours per night as well as children and adolescents.
In recent times, Insomnia or sleeplessness has become a common disorder that is affecting a large global population and impairing the general health and mental wellbeing. Using information from national polls in both Canada and the US reveal that more than 20% of adults overall experience this condition of sleeplessness, and during the next years, this number is anticipated to rise many years. Given the continuous worldwide population ageing, sleep issues may represent an underappreciated public health burden in many emerging nations, especially among older persons.
A growing number of literatures have reported on the COVID-19 pandemic's effects on sleep quality and factors connected to those effects. In a cohort study, patients with COVID-19 also reported having trouble sleeping and experiencing psychological anguish. Six months after the acute infection, patients with COVID-19 reported having trouble sleeping, depression, and anxiety.
Sleep also has immune-supportive functions. Impairments of the immune-inflammatory system are a possible mechanism mediating the detrimental effects of sleep deprivation on health, particularly its role in the risk and outcomes of chronic diseases. In fact, epidemiological and experimental studies show the link between sleep deprivation and the risk of metabolic (obesity, type 2 diabetes (T2DM)), cardiovascular (CV) (hypertension, coronary artery disease), and other diseases. Additionally, research suggests that lack of sleep increases the risk of stroke, cancer, and neurological illnesses (NDDs). Likewise, sleep loss raises the risk for depression and psychopathological and psychiatric illnesses such as psychosis, anxiety, suicidal ideation, and negative mood and mood control.
Sleep plays a critical role in the brain’s ability to form new memories. The Common causes of insomnia include stress, an irregular sleep schedule, poor sleeping habits, mental health disorders like anxiety and depression, physical illnesses and pain, medications, neurological problems, and specific sleep disorders.
Improvements in sleep quality and quantity as well as reduction of daytime impairments are the two main goals of treatment for sleep disorders, regardless of the therapeutic modality used. A behavioural intervention, such as stimulus control therapy or relaxation therapy, is typically included in the initial phases of treatment. In certain cases, pharmacotherapy is necessary.
Most currently available drugs for insomnia develop dependency and/or adverse effects. Currently, benzodiazepines (BZDs) and non-benzodiazepines based drugs are most frequently prescribed for the treatment of sleep problems. They both interact with gamma-aminobutyric acid (GABA) A receptors (GABAAR) to exert their respective effects. However, the use of both classes of drugs for clinical purposes is associated with unfavourable side effects, including addiction, systemic fatigue, loss of concentration, headache and digestive problems.
Several psychiatric disorders, such as anxiety disorders and depression, have shown strong relationships with insomnia. The synthetic drugs /sleeping pills for insomnia are available in the market but may have so many adverse effects. A large volume of melatonin was used as insomnia drug. It is worth noting that there are many after-effects that can occur with melatonin consumption, such as short-term depression, dizziness, headaches, and irritability. Consuming excessive melatonin can impact the natural circadian rhythm over time.
With comprehension that the synthetic prescription medicines for sleeplessness also have negative side effects, efforts to find new hypnotic drugs with minimal side effects continue. Alternative herbal treatments are becoming more popular as people look for all-natural ingredients that will improve sleep quality and reduce adverse effects. Hence natural therapies could be an alternative choice of treatment for insomnia.
Withania somnifera (Ashwagandha) has been used in ancient Indian medicine for treating sleep disorders, rheumatism, chronic fatigue, impotency, and to increase libido. Many studies have been conducted on the root extracts of Ashwagandha for treating sleep disorders. However, the active component for somnogenic activity is not yet known.
Studies have revealed that it has rejuvenating, immunomodulatory, anti-cancer, anti-stress, anti-oxidant, anti-inflammatory, and hemopoietic activities. The majority of Ashwagandha's biological properties are thought to be attributed to withanolides, which are the primary physiologically active components of the plant's roots and leaves. The failure of an ethanol extract with a high concentration of withanolides to induce sleep in rats, according to recent research, suggests that withanolides may not be a factor in sleep promotion. Alcoholic leaf extract (with high ratio of Withanone to Withaferin A) or water extract (contains Withaferin A and Withanone), were ineffective to induce sleep in mice. A more recent study found, Triethylene glycol (TEG), a purified water-based component has been linked to sleep in mice. Available information about TEG is limited to animal studies, except few case reports of accidental consumption of TEG. Various animal studies showed little or no TEG toxicity, suggesting that its lower volumes are safe in biological system, but higher volumes could be dangerous. Triethylene glycol is thought to be metabolized by alcohol dehydrogenase to acidic products resulting in the production of a metabolic acidemia.
Before totally ruling out withanolides and their derivatives for their ability to induce sleep, specific withanolides must, however, be evaluated directly. Research on the effect of Ashwagandha on insomnia in humans is limited. As a result, assessing the effects and safety of Ashwagandha on sleep can provide a foundation for its use.
US patent 9375463 provides a supplemental composition for a nutraceutical formulation and methods for administering the same to a user for inducing or maintaining sleep as well as for alleviating pain to improve sleep. Formulation of ingredients comprising of extract of ashwagandha, extract of lavender, extract of valerian, extract of hops, melatonin, magnesium, vitamin B12, zinc, and/or devil's claw, bromelain and boswellia. The activity mentioned in US 9375463 is due to the combined action of extract of ashwagandha, lavender, valerian, hops and melatonin, all of which have the property of promoting sleep and reducing anxiety.
SUMMARY OF THE INVENTION
Reducing the time of onset of sleep and prolonging total sleep duration are both goals related to improving the quality and quantity of sleep. Reducing the time of onset of sleep refers to the amount of time it takes for an individual to fall asleep after lying down to sleep. If someone has a long onset time, it might indicate difficulty in initiating sleep, which could be related to factors such as stress, anxiety, insomnia, or other sleep disorders. Prolongation of total sleep duration involves increasing the overall amount of time spent asleep during a night. If a person is not getting enough sleep, it can lead to various health issues and impact overall well-being. Increasing total sleep duration can be beneficial for physical and mental health. Improving both the onset of sleep and total sleep duration is often a goal in addressing sleep-related issues or disorders.
Present invention discloses a medicinal composition prepared from Withania somnifera extract in an effective dosage form comprising total withanolides for reducing the time of onset of sleep and prolongation of total sleep duration and method of preparation of the same. Said medicinal composition prepared from Withania Somnifera extract is comprising of 8-15% total Withanolides; 6-10% total Glycowithanolides and 2-5% of total Aglycones wherein 2-5% of Withaferin A is present within the total Aglycones present in the composition.
We also disclose a method of preparing said medicinal composition comprising the steps of: 1, comprising the steps of: a) cleaning the roots and leaves of Withania somnifera with water, drying of water and powdering to form a powder of Withania somnifera; b) extracting by refluxing the powder of Withania somnifera with 70% methanol at 95oC for 120 minutes to obtain first residue and first supernatant; c) extracting the resultant first residue in a similar way as in step b) to obtain second residue and second supernatant; d) extracting the resultant second residue in a similar way as in step b) to obtain third residue and third supernatant; e) pooling together all the three supernatants and concentrating the same in an Agitated thin film evaporator (ATFE) at a temperature of 60oC for 60 minutes to form a concentrated methanol extract; f) extracting the resultant concentrated methanol extract with a mixture of Chloroform and methanol in 1:1 ratio at a temperature of 60oC for 60 minutes to obtain fourth residue and fourth supernatant; g) pooling together all the supernatants and concentrating the same in an Agitated thin film evaporator (ATFE) to form a concentrated Chloroform and methanol extract and h) vacuum drying the resultant concentrated Chloroform and methanol extract and drying to form powdered extract of Ashwagandha having said medicinal composition.
The medicinal composition in the effective dosage in its formulated form contains at least one additional ingredient selected from pharmaceutically acceptable excipient, pharmaceutically acceptable diluent and pharmaceutically acceptable carrier.
The effective dosage form of the medicinal composition contains 200mg to 2000mg of medicinal composition. Said effective dosage of its formulation form contains at least one additional ingredient selected from pharmaceutically acceptable excipient, pharmaceutically acceptable diluent, and pharmaceutically acceptable carrier. Effective dosage can be made in the dispensable form such as capsule, tablet, powder, granule, soft gel capsule, suspension, dispersion, elixir, paste, gel, liquid, and dragees.
In one embodiment, on administration of the effective dosage form of said composition of Withania somnifera extract to mammals, preferably humans reduce the time of onset of sleep to about 50% to 90% and increases total sleep duration to about 400% to 900%.
Administration of the effective dosage form of said composition to mammals, preferably humans, reduces the time of onset of sleep up to 90% and increases total sleep duration up to 900% (up to 10-fold). During the evaluation of the efficacy of the medicinal composition prepared from Withania somnifera extract in an effective dosage form when administered to mammals and humans, the results indicated that there is a decrease in Sleep-onset latency time and increase in Sleep efficiency. There is a decrease in Pittsburgh Sleep Quality Index (PSQI) Score and Insomnia Severity Index (ISI) Score. Athens Insomnia scale score decreased after 30 days treatment.
DETAILED DESCRIPTION OF THE INVENTION
Currently, sleep disturbances are rather common and are becoming a bigger public health issue. More than 27% of people worldwide are thought to experience difficulties falling asleep or staying asleep due to sleep disorders.
The present invention is directed towards a medicinal composition/ formulation prepared from Withania somnifera extract for the treatment of sleep disorders, more especially Withania somnifera extract composition in an effective dosage form wherein itis comprising of total withanolides for reducing the onset of sleep and prolongation of total sleep duration in a subject in need thereof.
The word “Withania somnifera” and “Ashwagandha” mean the same thing and may be used interchangeably throughout the specification. The term “Withania” referred to plants from the group but not limited to Acnistus, Datura, Deprea, Dunalis, Iochroma, Jaborosa, Lycium, Nicandra, Physalis, Salpichroa, Tubocapsicum, Discopodium, Trechonaetes, Withania, Witheringia, and any plant from the genus withania or a plant that contains Withanolides.
As used herein, the term "subject" refers to the target of administration. It can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and new-born subjects, whether male or female, are intended to be covered unless otherwise specified.
As used herein, the terms "effective amount" and "effective dose" refer to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The effective amount and the therapeutic effect of it will be different for different subjects based on their age, weight and other health condition.
“Glycowithanolides” means “Withanolide glycosides” in this document and may be used interchangeably. Withaferin A stated in several places in this specification is a Withanolideaglycone. “Total Withanolides” or Withnolides means Withanolide glycosides and Withanolideaglycones combined.
Effective dosage form of Withania somnifera extract composition comprising total withanolides. Total withanolides comprising glycowithanolides and aglycones. Total withanolides in the Withania somnifera extract composition is about 8-15%. Total glycowithanolides in the composition is about 6-10% and total aglycones in the composition is about 2-5%. Aglycones in the composition comprises about 2-5% Withaferin A.
In another embodiment Withania somnifera extract composition comprising 8-15% total withanolides, 6-10% glycowithanoides, 2-5% aglycones, 3-9% polyphenol, 2.5-7% triterpenoids and 4.5-9% flavonoids.
In an embodiment, a Withania somnifera extract with 14.6% w/w of total withanolides, 9.9% glycowithanoilides and 4.9% aglycones is disclosed. The composition further contains 7.1% polyphenol, 4.2% triterpenoids and 6.04% flavonoids.
In one embodiment the effective dosage form of Withania somnifera extract composition optionally contains at least one additional ingredient selected from pharmaceutically acceptable excipient, pharmaceutically acceptable diluent and pharmaceutically acceptable carrier. Pharmaceutically acceptable excipient includes but not limited to starch, modified starch, cellulose ethers, cellulose esters, microcrystalline cellulose, carboxy methyl cellulose, polyvinyl pyrrolidone, polyethylene glycol, propylene glycol, mineral oils, glycerine, lipids, fatty alcohols, carbohydrates, calcium phosphates, calcium carbonates, magnesium stearate, lactose, sucrose, glucose, mannitol, sorbitol, fumed silica, antioxidants and flavouring agents.
The plant parts used for making the composition can be derived from roots, stems, flowers, leaves and seeds.
The effective dosage form of Withania somnifera extract composition is a capsule, tablet, powder, granule, soft gel capsule, suspension, dispersion, elixir, paste, gel, liquid, dragees, or combinations thereof.
The effective dosage of the oral composition of the extract of Withania somnifera has a dosage ranging from about 200 mg to about 2000 mg of the extract of Withania somnifera.
The effective dosage form of Withania somnifera extract composition reduces the onset of sleep up to 90% and increases total sleep duration up to 900% (up to 10-fold) in a subject in need thereof.
In one embodiment, on administration of the effective dosage form of Withania somnifera extract composition to mammals, preferably humans reduces the time of onset of sleep to about 50% to 90% and increases total sleep duration to about 400% to 900%.
In one embodiment method of preparation of Withania somnifera extract composition is disclosed. Roots and leaves of Withania somnifera are powdered and extracted three times with 95% methanol. The residue and supernatants are separated. All the supernatants are pooled and concentrated to form a concentrated methanol extract. Concentrated methanol extract is further extracted with a mixture of chloroform and methanol in 1:1 ratio. Supernatant obtained is vacuum dried to form a powdered extract of Withania somnifera. Sleep latency, or sleep onset latency, is the time it takes a person to fall asleep after turning the lights out. Different people fall asleep in different amounts of time, on average, a healthy person takes between 10 and 20 minutes to fall asleep. Sleep latency is an important measure because it can reflect a person’s overall sleepiness and provide insight into sleep quality.
In one embodiment the effect of Withania somnifera extract composition on Pentobarbital Sodium/ Thiopental Sodium induced sleep activity in swiss albino mice models is evaluated. The dosage administered to animals ranges from 50mg/Kg to 100mg/Kg bodyweight (equivalent human dose for an average human body weight of 60Kg is 244mg to 488mg). Administration of Withania somnifera composition in effective dosage decreases onset time of sleep and increase the duration of sleep.
Withania somnifera extract composition at 50mg/Kg and 100mg/Kg in pentobarbital model showed 54% and 63% decrease in the onset time of sleep and 700% (8 fold) and 850% (9.5 fold) increase in the duration of sleep compared to control group. Animals administered with Melatonin showed only 29% decrease in onset time of sleep and 500% (6 fold) increase in duration of sleep compared to control group.
Withania somnifera extract composition at 50mg/Kg and 100mg/Kg in thiopental model showed 75% and 88% decrease in the onset time of sleep and 445% (5.5 fold) and 581% (6.8 fold) increase in the duration of sleep compared to sleep depressive control group. Animals administered with Melatonin showed only 44% decrease in the onset time of sleep and 109% increase in duration of sleep compared to control group.
In another embodiment, efficacy of Withania somnifera extract composition in the management of Insomnia in mammals and humans is evaluated. Dosage administered is 500mg capsule once daily for 30 days. Mean change in Sleep-onset latency time is measured at baseline, day 15 and day 30. Other parameters analysed at baseline, day 15 and day 30 are Mean change in Sleep efficiency (total sleep time divided by time allotted for sleep as measured from lights out to arising time), Mean change in Pittsburgh Sleep Quality Index (PSQI) Score, Mean change in Insomnia Severity Index (ISI) Score, Mean change in Athens Insomnia scale score.
Pittsburgh Sleep Quality Index (PSQI)
The PSQI is a self-rating questionnaire resulting in a global score between 0 and 21, which consists of seven sub scores. A PSQI global score > 5 indicates that a subject is having severe difficulties in at least two areas, or moderate difficulties in more than three areas.
Insomnia Severity Index (ISI)
The Insomnia Severity Index has seven questions. The seven answers are added up to get a total score ISI >14which is considered to be chronic insomnia.
Athens Insomnia Scale (AIS)
The AIS is a self-assessment psychometric instrument designed for quantifying sleep difficulty based on the ICD-10 criteria. It consists of eight items: the first five pertain to sleep induction, awakenings during the night, final awakening, total sleep duration, and sleep quality; while the last three refer to well-being, functioning capacity, and sleepiness during the day. AIS>10 indicates significant insomnia.
The results indicated that there is a decrease in Sleep-onset latency time and increase in Sleep efficiency. There is a decrease in Pittsburgh Sleep Quality Index (PSQI) Score and Insomnia Severity Index (ISI) Score. Athens Insomnia scale score decreased after 30 days treatment.
Example
Method of preparation of Withania somnifera extract composition.
100Kg of roots and leaves of Withania somnifera were collected, cleaned and dried. After drying, the plant parts were powdered to form a powder of Withania somnifera. The powder of Withania somnifera was extracted with 70% methanol. The powder was refluxed with 70% methanol (700L) at 95oC for 120 minutes to obtain the first residue and the first supernatant. The first residue was then further extracted two more times (for a total of 3 times) with methanol at each time resulting in second and third supernatants. The residue and supernatants were separated. All the supernatants were pooled and concentrated in an Agitated thin film evaporator (ATFE) to form a concentrated methanol extract (Sample 1).
Sample 1 was again extracted with a mixture of chloroform and methanol in 1:1 ratio at a temperature of 600C for 60 minutes to obtain fourth residue and fourth supernatant. The fourth residue was then further extracted with chloroform and methanol resulting in fifth supernatant. All the supernatants were pooled and concentrated under vacuum to form a concentrated chloroform- methanol extract. Concentrated extract was dried under vacuum to form powdered extract of Ashwagandha (Sample 2).
The total withanolides content of Sample 2 was about 14.6% by weight, glycowithanolides 9.9% by weight and aglycones 4.9% by weight.
Evaluation of the effect of Withania somnifera extract composition on pentobarbital induced sleep activity in experimental mice
Swiss albino mice weighing 25 – 30 gm were used for the study. The animals were randomized into five groups consisting of six animals.
? Group I–Sleep depressive control treated with 0.5% carboxymethylcellulose (CMC),
? Group II - positive control and treated with Diazepam (1 mg/kg w/w),
? Group III - treatment group fed with melatonin at the dose level of 1.02mg/kg body weight.
? Group IV - treatment groups fed with Withania somnifera extract composition at the dose level of 50mg/kg body weight, and
? Group V - treatment groups and fed with Withania somnifera extract composition at the dose level of 100mg/kg body weight.
Thirty minutes later, pentobarbital 40 mg/kg body weight was administered to each mouse to induce sleep. The onset time of sleep was recorded for all the animals. After induction of sleep, mice were placed in the inverted position and when sedation was over, the time point when the mice returned to their normal posture was noted. The time interval between dosing and the start of sleep was recorded as latency time.
Table:
Groups Onset time of sleep(latency time) (min) Percentage decrease in onset time of sleep Duration of sleep Percentage increase in duration of sleep Fold enhancement of duration of sleep
Group I 59 10
Group II 21 ?65% 108 ?980% 10.8
Group III 42 ?29% 60 ?500% 6
Group IV
(50mg/kg) 27 ?54% 80 ?700% 8
Group V
(100mg/kg) 22 ?63% 95 ?850% 9.5
From the table, the mouse administered with Withania somnifera extract composition at 50mg/Kg and 100mg/Kg showed 54% and 63% decrease in the onset time of sleep and 700% (8 fold) and 850% (9.5 fold) increase in the duration of sleep compared to control group. Group III animals administered with Melatonin showed only 29% decrease in onset time of sleep and 500% (6-fold) increase in duration of sleep compared to control group. Group II animals treated with Diazepam showed 65% decrease in onset time of sleep and 980% (10.8-fold) increase in duration of sleep. The mouse administered with Withania somnifera extract composition at 100mg/Kg showed almost comparable result in onset time of sleep and duration of sleep with control group treated with diazepam.
Evaluation of the effect of Withania somnifera extract composition on thiopental sodium induced hypnosis in swiss albino mice.
Swiss albino mice weighing 25 – 30 gm were used for the study. The animals were randomized into five groups consisting of six animals.
? Group I- Sleep depressive control treated with 0.5% carboxymethylcellulose (CMC).
? Group II - positive control and treated with Diazepam (1 mg/kg w/w).
? Group III - treatment group fed with melatonin at the dose level of 1.02mg/kg body weight.
? Group IV - treatment groups fed with Withania somnifera extract composition at the dose level of 50mg/kg body weight, and
? Group V - treatment groups and fed with Withania somnifera extract composition at the dose level of 100mg/kg body weight.
Thirty minutes later, thiopental sodium 40mg/kg body weight was administered to each mouse to induce sleep. The onset time of sleep was recorded for all the animals. After induction of sleep, mice were placed in the inverted position and when sedation was over, the time point when the mice returned to their normal posture was noted. The time interval between dosing and the start of sleep was recorded as latency time.
Groups Onset time of sleep (latency time) (min) Percentage decrease in onset time of sleep Duration of sleep Percentage increase in duration of sleep Fold enhancement of duration of sleep
Group I 61 11
Group II 8 ?87% 79 ?618% 7.2
Group III 34 ?44% 23 ?109% 2.1
Group IV
(50mg/kg) 15 ?75% 60 ?445% 5.5
Group V
(100mg/kg) 7 ?88% 75 ?581% 6.8
From the table, the mouse administered with Withania somnifera extract composition at 50mg/Kg and 100mg/Kg showed 75% and 88% decrease in the onset time of sleep and 445% (5.5 fold) and581% (6.8 fold) increase in the duration of sleep compared to sleep depressive control group. Group III animals administered with Melatonin showed only 44% decrease in the onset time of sleep. and 109% increase in duration of sleep compared to control group. The mouse administered with Withania somnifera extract composition at 100mg/Kg showed almost comparable result in onset time of sleep and duration of sleep with control group treated with diazepam. Group II animals treated with Diazepam showed 87% decrease in onset time of sleep and 618% (7.2 fold) increase in duration of sleep.
The above examples clearly show the effect of Withania somnifera extract composition in thiopental sodium and pentobarbital models. The mouse administered with Withania somnifera extract composition showed almost comparable results with control group treated with diazepam. Onset time of sleep is decreased and duration of sleep is increased after administering Withania somnifera extract composition.
We have brought out the novel features of the invention by explaining some of the preferred embodiments under the invention, enabling those in the art to understand and visualize our invention. Although the invention has been described in considerable detail with reference to certain preferred embodiments thereof, various modifications can be made without departing from the scope of the invention as described herein above and as defined in the following claims. ,CLAIMS:1. A medicinal composition prepared from Withania somnifera Extract comprising:8-15% total withanolides; 6-10% total glycowithanolides and 2-5% of total aglycones wherein 2-5% of Withaferin A is present within the total aglycones of the composition.
2. The medicinal composition as claimed in claim 1, wherein the effective dosage in its formulated form contains at least one additional ingredient selected from a pharmaceutically acceptable excipient, pharmaceutically acceptable diluent and pharmaceutically acceptable carrier.
3. The medicinal composition as claimed in claim 2, wherein the effective dosage form contains 200mg to 2000mg of the medicinal composition.
4. The medicinal composition as claimed in claims 1 to 3, wherein it is capable of reducing the time of onset of sleep up to 90%, once the effective dosage form is administered to a mammal, preferably a human.
5. The medicinal composition as claimed in claim 1, wherein it is capable of increasing total sleep duration up to 900% (up to 10 fold), once the effective dosage form is administered to a mammal, preferably a human.
6. The medicinal composition as claimed in claim 1, wherein said composition in its effective dosage form made in the dispensable form such as capsule, tablet, powder, granule, soft gel capsule, suspension, dispersion, elixir, paste, gel, liquid, and dragees.
7. A method of preparing the medicinal composition as claimed in claim 1, comprising the steps of:
a) cleaning the roots and leaves of Withania somnifera with water, drying of water and powdering to form a powder of Withania somnifera;
b) extracting by refluxing the powder of Withania somnifera with 70% methanol at 95oC for 120 minutes to obtain first residue and first supernatant;
c) extracting the resultant first residue in a similar way as in step b) to obtain second residue and second supernatant;
d) extracting the resultant second residue in a similar way as in step b) to obtain third residue and third supernatant;
e) pooling together all the three supernatants and concentrating the same in an Agitated thin film evaporator (ATFE) at a temperature of 600C for 60 minutes to form a concentrated methanol extract;
f) extracting the resultant concentrated methanol extract with a mixture of chloroform and methanol in 1:1 ratio at a temperature of 600C for 60 minutes to obtain fourth residue and fourth supernatant;
g) pooling together all the supernatants and concentrating the same in an Agitated thin film evaporator (ATFE) to form a concentrated Chloroform and methanol extract and
h) vacuum drying the resultant concentrated chloroform and methanol extract and drying to form powdered extract of Ashwagandha having said medicinal composition.