DESC:TECHNICAL FIELD:
The invention relates to water soluble extract of Alpinia galanga which is substantially free from methyl eugenol and the compositions comprising the extract for improving mental alertness and sustained attention in humans. The invention further relates to a process for preparation of an extract of Alpinia galanga, in the form of stable, free-flowing powder, and compositions comprising the same.

BACKGROUND AND PRIOR ART:
Plants have been an essential source of medicine ever since the dawn of human cultivation. Many plant products for improving cognitive performance such as caffeine has been in use as health supplements/energy drinks.

Caffeine (1, 3, 7-trimethylxanthine) is an extensively used psycho-stimulant that produces positive effects on mental alertness and cognitive performance. These effects are largely attributed to antagonistic effect on adenosine A1 and A2A receptors in dopamine-rich brain areas; thus stimulating dopaminergic activity and resulting in increased wakefulness and pronounced motor activity.

Besides the psycho-stimulant benefits of caffeine, prolonged use of caffeine may also cause tolerance and a range of adverse effects as well which include sleep disorders such as insomnia, heart palpitations, nervousness, headaches, hypertension, occasional light headedness, jitters, gastrointestinal upset, headache, chest pain, and seizures. This altered psychosomatic state, generally termed as ‘caffeine crash’, can produce undue stress and depending upon the amount of caffeine consumed can produce troublesome social effects (failure to meet social obligations) in many individuals.Thus, high doses or even small amounts of caffeine in individuals having high sensitivity towards methyl xanthine can exacerbate this perceived stressful state.

Most over the counter medications used for mental alertness contain 200 milligrams of caffeine per tablet or capsule.

In recent times, some of the untoward occurrences have drawn regulatory agencies’ concern towards uncontrolled use of Caffeine in a variety of dietary supplements and energy drinks (James JE. Caffeine: Public Concern and the Precautionary Principle. J. Caffeine Research. May 2013, 3(1): 1-2). This situation has prompted a need for an alternative and safe ingredient which either with Caffeine or as a standalone ingredient that can provide stimulatory benefits without the crash.

Therefore, it has become an objective of the invention to provide an alternate and safe herbal product that improves mental alertness and wakefulness and yet devoid of any significant side effects.

SUMMARY OF THE INVENTION:
In line with the above objective, the present inventors have screened more than fifty medicinal plants and spices that include Alpinia galanga, to identify the natural substance/phytoconstituents which can help to improve mental alertness and wakefulness.

Alpiniagalanga (A. galanga), belongs to the family Zingiberaceae has been used as a culinary spice. Also, its traditional use is well documented for the treatment of various ailing conditions due to its anti-inflammatory, analgesic, hypoglycemic, anti-allergic, antimicrobial, gastro-protective, antioxidant, antiplatelet, anticancer, immune-stimulatory and hypo-lipidemic effects(Chudiwal AK, Jain DP, Somani RS. Alpinia galanga willd: an overview to phyto-pharmacological properties. Indian Journal of Natural products and Resources.2010; 1 (2), 143-149).

Though, methanolic extract of A.galanga has been reported to improve cognitive performance in animals (Saha S, Banerjee S. Central nervous system stimulant actions of Alpiniagalanga (L.) rhizome: a preliminary study. Indian J Exp Biol. 2013; 51(10):828-832), it has not been thoroughly studied for its psycho-stimulant effect in humans. With a principal objective of developing a safer alternative for increasing mental alertness and decreasing mental fatigue, standardised extract of A. Galanga has been prepared by specific inventive method to exclude/eliminate methyleugenol, a carcinogenic, and further studied the said extract that is free of methyleugenol for its psycho-stimulant activity in healthy young volunteers in this invention.

Methyleugenol is naturally occurring compound in many plants including Alpinia galanga. The methyl ether of eugenol with a clove-like aroma is present in many essential oils. Therefore, methyleugenol is used as a flavoring agent, as a fragrance and as an anaesthetic in rodents. However, latest studies indicate that the methyleugenol is mutagenic in animals and is reasonably anticipated to be a human carcinogen based on evidence of carcinogenicity in animals. Therefore, methyleugenol was added in California Proposition 65 list in 2001. Since, Alpinia galanga contains naturally occurring methyleugenol, the objective of the present invention is to devise an extract of Alpinia galanga that is devoid of methyleugenol and a specific process whereby the obtained extract will be free from methyl eugenol when tested by HPLC.

Various extraction and isolation processes can be used to substantially alter the
characteristics and amounts of the chemical components of the naturally occurring compositions in the plants. These processes may either result in a concentrate or remove various toxic chemicals to form non-naturally occurring novel compositions.

Thus the present invention belongs to such compositions that is devoid of methyleugenol from the compositions obtained from the plant A. Galanga and further devised a standardised process for the preparation of such composition from A. Galanga to specifically eliminate the methyleugenol and to alter the composition thus obtained to possess phytoactive matrix of polyphenols and polysaccharides in a ratio of approximately 1:1.6 to 1:8, more preferably in a ratio ranging from 1:5 to 1:6.

Accordingly, in one aspect, the present invention provides a process for preparation of water soluble extract of A. Galanga that is devoid of methyleugenol, which comprises the following steps;
a) Extracting the powder of the dried rhizome of A. Galanga with cold water extraction cycle by soaking the same for 8-10 hrs followed by hot water extraction cycle for a period of 5-6 hours at temperature of 70-80 degrees and pressure of 1-10 bars to obtain collective extract;
b) Distilling the collective extract at a temperature of 70-80 degrees under vacuum to obtain syrupy material;
c) Vacumn drying the syrupy material at 80-90 degrees to obtain the water soluble extract, that is devoid of methyl eugenol.

The process of the present invention comprises at least two or more hot water extraction cycles for a period of 5-6 hours at temperature of 70-80 degrees and pressure of 1-10 bars.

According to the process of the present invention, vacumn drying is conducted under vacumn pressure of 650 to 700 mm and for 6-8 hours.

The water soluble extract thus obtained can be pulverized optionally with excipients like silicon dioxide under controlled temperature and humidity conditions, to obtain the free-flowing, water soluble powder.

In an aspect, the aqueous media/water is added at least about 4 times by weight of the biomass (dried rhizome of A. Galanga) according to the process of the invention.The number of extraction cycles can be decided on the basis of total dissolved solids that can be leached into the aqueous solvent when employed/extracted.

The pulverization of the flakes with excipients like silicon dioxide can be conducted under controlled temperature of 25 to 45degrees and 20-50% Rh humidity conditions, to obtain the free-flowing, water soluble powder of A. Galanga that is devoid of methyl eugenol.

In an aspect, the total polyphenol content in the powdered aqueous extract is not less than 2-16% and total polysaccharide is not less than 12-96% thereby comprising a variable ratio of total polyphenol content and total polysaccharide content in the range of 1:1.6 to 1: 8.

According to another aspect, the invention provides phytochemical evaluation of the water extract of the A. Galanga and found that the extract obtained according to the process of the invention comprises; a) phyto active matrix of polyphenols and polysaccharides in variable a ratio of approximately 1:1.6 to 1:8; b) about 1% pyrocatecollic type tannins; c) galangin not more than 100 ppm; d) alkaloids not less than 0.4%; e) flavanoids not less than 3% and wherein, the extract is free from methyl eugenol.

According to another aspect, the aqueous extract is at least 90% water soluble, i.e., contains at least 90% water soluble solids. In another aspect, the aqueous extract composition contains at least 95% water soluble solids and more preferably, the aqueous extract contains at least 99% water soluble solids.

Accordingly, in another aspect, the invention provides formulations that comprise the water soluble extract of A. Galanga for oral administration.

The formulation may be in the form of commonly used formulations, including, but not limited to: a tablet, a capsule, a lozenge, a film, a powder, a sustained release formulation, a liquid formulation. The formulation may also include commonly used nutraceutical dosage forms, including, but not limited to: functional foods and beverages, energy shots, energy bars, energy drinks, and performance supplements. The formulation may further include an excipient, such as a pharmaceutically/neutraceutically acceptable excipient.

In some embodiments, the formulation includes an effective amount of an extract of Alpinia galanga that is free of methyl eugenol and has a ratio of polyphenols to polysaccharides of about 1:1.6 to about 1:8.

In another aspect, the ratio of polyphenols to polysaccharides is about 1:1.6 to about 1:2.2.

In another aspect, the ratio of polyphenols to polysaccharides is about 1:5 to about 1:6.

In another aspect, the ratio of polyphenols of polysaccharides is about 1:5 to about 1:8.

In some embodiments, the formulation includes an effective amount of an extract of Alpinia galanga that is free of methyl eugenol and has 1% pyrocatecollic type tannins.

In some embodiments, the formulation includes an effective amount of an extract of Alpinia galanga that is free of methyl eugenol and has not more than 100 ppm galangin.

In some embodiments, the formulation includes 10–900 mg of an extract of Alpinia galanga that is free of methyl eugenol.

In other embodiments, the formulation includes 200 to 400 mg of an extract of Alpinia galanga that is free of methyl eugenol.

In other embodiments, the formulation includes 100 to 400 mg of caffeine.

In a further aspect, the formulations prepared out of the aqueous extract of A. galanga according to the invention may be free of preservatives.

In another aspect, the invention provides method of improving the mental alertness and sustained attention, which method comprises administering therapeutically effective amount of the water soluble extract or composition thereof of A. galanga in a subject in need thereof.

The effective amount of the water soluble extract composition of A. Galanga is about10 to 900 mg which can be given to the subject in need thereof.

LIST OF ABBREVIATIONS
E-AG-01: Standardized aqueous extract of Alpinia galanga prepared according to the example 1; AE: Adverse Event; ANT: Attention Network Test; BMI: Body Mass Index, GAD-7: Generalized Anxiety Disorder-7; IEC: Independent Ethics Committee; IP: Investigational Product; KSS: Karolinska sleepiness scale; PHQ-9: Patient Health Questionnaire-9; PVT: Psychomotor Vigilance Task; msec: Milisecond; SAE: Serious adverse event; SQS: Groningen’s sleep quality scale.

DESCRIPTION OF DRAWINGS:
Figure 1 shows an effect of an Alpinia galanga extract that is free of methyl eugenol and other extracts on Mean Response Time.
Figure 2 shows an effect of Alpinia galanga extract that is free of methyl eugenol and other extracts on Alertness.
Figure 3 shows an effect of Alpinia galanga extract that is free of methyl eugenol, as compared to caffeine, a composite of Alpinia galanga that is free of methyl eugenol and caffeine, and placebo on Mental Alertness.
Figure 4 shows an effect of Alpinia galanga extract that is free of methyl eugenol, as compared to caffeine, a composite of Alpinia galanga that is free of methyl eugenol and caffeine, and placebo on Sustained Attention.

DETAILED DESCRIPTION:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

Alpinia galanga is found in Indonesia, China, and Arabic gulf areas, Malaysia, Egypt, India and Sri Lanka. It grows in open sunny places, forests and brushwood. It is commonly cultivated in the mid and low-country in Sri Lanka.

Various embodiments of the present invention relates to a formulation that includes an effective amount of an extract of Alpinia galanga that is free of methyl eugenol; a method of improving cognitive performance by administering a formulation containing an effective amount of an extract of Aplinia galanga that is free of methyl eugenol; and processes for preparing an extract of Alpinia galanga that is free of methyl eugenol.

Accordingly, in an embodiment, the present invention provides water soluble extract of Alpinia galanga that is devoid of methyleugenol and a specific process whereby the obtained extract will be free from methyleugenol when tested by HPLC.

The water soluble extract as referred herein in the entire specification means and includes cold water extract at 0-25°C; hot water extract at a temperature ranging from 70 to 80°C and combination of cold water extract and hot water extract of A. Galanga.

Accordingly, the invention provides a process for preparation of water soluble extract of A. Galanga in the form of stable, free-flowing, powder which comprises the following steps;
a) Extracting the powder of the dried rhizome of A. Galanga with cold water extraction cycle by soaking the same for 8-10 hrs followed by hot water extraction cycle for a period of 5-6 hours at temperature of 70-80 degrees and pressure of 1-10 bars to obtain collective extract;
b) Distilling the collective extract at a temperature of 70-80 degrees under vacuum to obtain syrupy material;
c) Vacumn drying the syrupy material at 80-90 degrees to obtain the water soluble extract, that is devoid of methyl eugenol.

The process of the present invention comprises at least two or more hot water extraction cycles for a period of 5-6 hours at temperature of 70-80 degrees and pressure of 1-10 bars.

According to the process of the present invention, vacumn drying is conducted under vacumn pressure of 650 to 700 mm and for 6-8 hours.

As discussed above, the process for extraction of Alpinia galanga that is free of methyl eugenol may be performed initially with cold water, i.e., water at a temperature of 0-25° C and subsequently the extraction is performed using hot water at a temperature range of 70° C to 80° C.

Accordingly, water is used as an extraction solvent for Alpinia galanga. The extraction process is distinct from extractions using organic solvents. In one embodiment, cold (i.e., 0–25° C) water is used to extract Alpinia galanga. This embodiment is distinct from extractions using hot water.
The extraction process may include the steps of powdering and sieving the dried bark of Alpinia galanga. In one embodiment, the rhizome is used. The powder may then be extracted with water. The extract may then be concentrated under vacuum and dried under a controlled temperature. The dried product is pulverized under a controlled temperature and humidity in order to obtain the extract.

In further embodiments, the aqueous solution is added at four times the amount of the dried rhizome. A first extraction cycle may be performed by soaking the dried rhizome in cold water (at 0–25° C) for upto8 hours. In another embodiment, second, third and fourth extractions are performed for a period of 5–6 hours at temperature range of 70–80° C. Without wishing to be bound by any theory, Applicants believe that using the four extractions maximizes the recovery of the extracts from the dried rhizome. If necessary, additional extraction cycles may be used to recover the remaining solids that can be leached into aqueous medium. The total number of extraction cycles are calculated based on the total dissolved solids in the extract. The collected extract is then distilled at 70–80° C.

Distillation stopped after a syrupy material stops bubbling in the distillatory. The product may then be dried under vacuum at 80–90° C and at a vacuum pressure of 650 mm for 6–8 hours. The dried product may then optionally be pulverized with excipients like silicon dioxide under controlled temperature and humidity conditions, to obtain the desired powdered product.

In an aspect, the total polyphenol content in the dried aqueous extract obtained in accordance with the process of invention is not less than 2-16% and total polysaccharide is not less than 12-96% thereby comprising a variable ratio of total polyphenol content and total polysaccharide content in the range of 1:1.6 to 1: 8.

According to another aspect, the aqueous dried extract obtained in accordance with the invention is at least 90% water soluble, i.e., contains at least 90% water soluble solids. In another aspect, the aqueous extract composition is at least 95% water soluble solids and more preferably, the aqueous extract is at least 99% water soluble solids.

According to another embodiment, the invention provides phytochemical evaluation of the extract of A. Galanga and found that the extract obtained according to the process of the invention comprising; a) phytoactive matrix of polyphenols and polysaccharides in the ratio of 1:1.6 to 1:8; b) 1% pyrocatecollic type tannins; c) galangin not more than 100 ppm; d) alkaloids not less than 0.4%; e) flavanoids not less than 3% and wherein, the extract is free from methyl eugenol.

The amounts of polyphenols and polysaccharides produced by each method of the invention are shown below in table 1.

Table 1
Extraction Process Polyphenols Polysaccharides
Hot water (70° C) 8.09% 13.09%
Hot water (80° C) 7.19% 16.13%
Cold water (0–25° C)
Batch 253 4.89% 24.90%
Cold water (0–25° C) batch 264 5.20% 26.91%
Cold water (0–25° C) batch 327 5.86% 35.65%
Methanol 3.23% 38.34%

As shown by these data, the ratio of polyphenols to polysaccharides is markedly different when cold water is used as the extraction solvent as compared to when hot water is used. In particular, the polyphenol content is relatively low while the polysaccharide content is significantly higher.
Therefore, the ratio of about 1:5 is different using cold water as compared to a ratio of 1:1.6 or 1:2.2 using hot water. The use of methanol as an extraction solvent results in a ratio of 1:11.9. Accordingly, using cold water, the extract of Alpinia galanga results in a ratio of polyphenols to polysaccharides that is distinct from the ratio derived from using other methods.

In another embodiment, the extract of Alpinia galanga is free from methyl eugenol. In one aspect, the extract does not include any detectable amount of methyl eugenol. In another aspect, the extract includes less than 1 ppm of methyl eugenol.

In the additional embodiment of the present invention, a formulation containing an effective amount of the extract of Aplinia galanga that is free of methyl eugenol and further comprising polyphenol content to polysaccharide content in a ratio of 1:1.6 to 1:8 is administered to improve cognitive performance. An improvement in cognitive performance includes, but is not limited to: improved mental alertness, increased attention, decreased mean response time, decreased mental fatigue, impeded caffeine crash, improved sustained wakefulness, and combinations thereof. Therefore, any administration of an effective amount of an extract of Alpinia galanga that is free of methyl eugenol, having polyphenol content to polysaccharide content in a ratio of 1:1.6 to 1:8 that results in the foregoing effects, or similar effects, is encompassed by the present disclosure.

According to a study described below, in Example 5, the water-soluble extract of Aplinia galanga that is free of methyl eugenol shows increased improvement of cognitive performance as compared to a methanol extract. The data further shows the superiority of the water-soluble extract of Alpinia galanga that is free of methyl eugenol as compared to caffeine, placebo, and extracts of other herbs. The mean response time for subjects administered the water-soluble extract of Alpinia
galanga that is free of methyl eugenol improved after one hour and after three hours. Further, the water-soluble extract of Alpinia galanga that is free of methyl eugenol improved alertness time after one hour and after three hours.

Without wishing to be bound by any theory, the Applicants believe that the components of the water-soluble extract of Alpinia galanga that is free of methyl eugenol are responsible for the beneficial results. In particular, the specific ratio of polyphenols, polysaccharides, and pyrocatecollic type tannins present in Alpinia galangal as shown in table 2 is responsible for the beneficial results. The water-extraction method of the present invention produced these compounds in ratios and amounts that are distinct from the naturally occurring powder or from an alcoholic extraction. Therefore, the superior improvement of cognitive performance may be due to the presence these components in a specific ratio/amounts.

In another embodiment, the invention provides a formulation that includes an effective amount of an extract of Alpinia galanga that is free of methyl eugenol and a pharmaceutically acceptable excipient.

The formulation may be any type of pharmaceutically acceptable formulation that is used to dose an active agent. Examples of pharmaceutically acceptable formulations include: tablets, capsules, lozenges, films, powders, sustained release formulations, liquid formulations. In one embodiment, the formulations include oral dosage forms, i.e., tablets, capsules, and sustained release formulations. In another embodiment, the formulation is a capsule. In one aspect, the capsule may include granules of Alpinia galanga that is free of methyl eugenol and a granulating polymer.

The pharmaceutically acceptable excipient/agent may be a binder, a diluent, a filler, a disintegrant, a lubricant, a glidant, a coating, a flavoring, a buffer, or combinations thereof. In one embodiment, the pharmaceutically acceptable excipient is microcrystalline cellulose. In another embodiment, the pharmaceutically acceptable excipient is maltodextrin.

The formulation may also include commonly used nutraceutical dosage forms, including, but not limited to: dietary and nutritional supplements, functional foods and beverages, energy shots, energy bars, energy drinks, performance supplements, and sports products.

In one embodiment, the effective amount of the extract of Alpinia galanga that is free of methyl eugenol is 10 to 900 mg. However, the safe and effective amount of the extract of Alpinia galanga that is free of methyl eugenol includes any amount that has the desired therapeutic effect, i.e., improvement in cognitive performance.

In other embodiments, the extract of Alpinia galanga that is free of methyl eugenol may include one or more of the following characteristics: having a ratio of polyphenols to polysaccharides of about 1:1.6 to about 1:8; including 1% pyrocatecollic type tannins; including not more than 100 ppm galangin; or combinations of these characteristics.

The Alpinia galanga extract that is free of methyl eugenol includes polyphenolic compound and polysaccharides. In one embodiment, the ratio of polyphenols to polysaccharides is about 1:1.6. In another embodiment, the ratio of polyphenols to polysaccharides is about 1:2.2. In another embodiment, the ratio of polyphenols to polysaccharides is about 1:5. In yet another embodiment, the ratio of polyphenols to polysaccharides is about 1:6. In a further embodiment, the ratio of polyphenols to polysaccharides is about 1:8.

In yet another embodiment, the invention provides method of improving the mental alertness and sustained attention, which method comprises administering therapeutically effective amount of the aqueous extract composition of A. galanga in a subject in need thereof.

In another embodiment, the composition is suitable for oral administration and thus can be formulated in variety of oral dosage forms such as tablets, capsules, granules, powders, caplets, pellets etc. using suitable excipients and conventional methods.

In a further embodiment, the invention provides evaluation of the extract for its safety and efficacy in improving the mental alertness and wakefulness in a subject. Accordingly, in a clinical trial consisting of a batch of 5 healthy human subjects each, the aqueous extract as per the invention and the methanolic extract as shown in table 2 were tested for improving mental alertness. While the aqueous extract showed effect in improving alertness; the methanolic extract did not show any effect on mental alertness.

Based on the above results, the extract has been standardised to comprise phytoactive matrix of polyphenols and polysaccharides in the ratio of about 1: 1.6 to 1:8.

Encouraged with the small clinical trial, the inventors have conducted larger study in healthy human subjects on specific parameters related to attention network and sleep architecture using standardized tests and questionnaires. The objective of the clinical study is to determine the effect of aqueous extract of Alpinia galanga on mental alertness and sustained attention in comparison with caffeine and Placebo in subjects with habitual Caffeine intake.

The study designed was randomized, double dummy, double blind, placebo controlled, cross-over study.Mental alertness (assessed by Attention Network Test) was the outcome measure of prime importance for this study. In addition, sustained attention, mental fatigue, sustained wakefulness and sleep pattern were also assessed. Further, safety and tolerability was assessed by blood pressure measurement and reported adverse events.

Fifty nine healthy male and female computer literate subjects in the age group of 18-40 years with moderate caffeine consumption were randomized in the study. The subjects had mean body mass index (BMI) of = 18.5 and < 25.00 kg/m2 and had scores of = 7 and = 14 on Generalized Anxiety Disorder (GAD-7) score and Patient Health Questionnaire-9 (PHQ-9) scale subsequently. The alertness score on Jin Fan’s Attention Network Test (ANT, version 1.3.0) was recorded to be 50 ± 20 in at least two out of three sessions during screening visit. Subjects were administered one of the interventional products (E-AG-01(aqueous extract of Alpinia galanga), caffeine, combination of E-AG-01and caffeine or placebo) followed by sequential administration of remaining interventions in consecutive visits.

At the end of the study, the alertness score was increased by 11.65, 12.50 and 12.62 msecs from baseline at one, three and five hours respectively in E-AG-01group which indicates its efficacy to enhance mental alertness. The increase when compared to placebo was statistically significant at three hour (p = 0.042). Mean response time was significantly reduced by 15.55 msecs (p = 0.026)at three hours in the composite group inferring improvement in sustained attention as compared to placebo.

A. galanga water soluble extract (E-AG-01) was found to induce beneficial effect on mental alertness and a combination of A. galanga with Caffeine was able to impede the Caffeine crash and improve sustained attention at three hours. Thus, the sustained stimulant effects of A. galanga water soluble extract opens up new avenue as an important ingredient in energy drinks or similar products.

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

EXAMPLES
Example 1:
Process for manufacture of aqueous extract of A. Galanga (E-AG-01)
Powdered dried rhizome (100gm) of A. galanga was subjected to cold water (400ml)extractionby soaking for 8-10 hrs at room temperature and pressure of 1-10 bar. This was followed by subjected to second, third and fourth hot water extraction cycles for a period of 5-6 hours at temperature of 70-80 degrees and pressure of 1-10 bar. These consecutive extractions were to ensure complete extraction of biomass as confirmed by total dissolved solids. The combined extracts were concentrated under vacuum by distilling collective extract at a temperature of 70-80 degrees to obtain syrupy material, followed by vacuum drying under controlled temperature of at 80-90 degrees and at a vacuum of 650 mm and for 6-8 hours to obtain the desired extract as flakes(yield:10 gms).

The water extract thus obtained comprises the following phytoconstituents such as total flavanoinds, total polyphenols, total polysaccharides and galangin in the following amounts when compared to methanol extract, as shown in table 2.

Table 2:
Extract Flavanoids Polyphenols Polysaccharides Galangin
Alpinia galanga water extract as per the process of the present invention 4.6% 4.89% 24.90% 100ppm
Alpinia galanga methanolic extract 9.11% 3.23% 38.34% 200 ppm

These flakes are optionally pulverized with excipients like silicon dioxide under controlled temperature of 25-45degrees and relative humidity of 20-50% to obtain the free-flowing, water soluble powder of the desired extract.

Example 2
Phytochemical evaluation of the extract:
The extract thus obtained from example 1 was subjected to phytochemical evaluation by HPLC and found that the extract comprises a) phytoactive matrix of polyphenols and polysaccharides in the ratio of about 1:5.1 b) 1% pyrocatecollic type tannins c) galangin not more than 100 ppm; d) alkaloids not less than 0.4% e) flavanoids not less than 3% and wherein the extract is free from methyleugenol.

Example 3
For comparison purpose different process conditions (at high temperatures) are used to obtain the aqueous extracts which give different results. The process is devoid of cold extraction cycle at room temperature. The data is provided in the table 3 below.

The below table 3 shows how the composition differs using the same solvent (water for extraction) with different temperature process conditions.

Table 3:
Extract Flavanoids Polyphenols Polysaccharides
AG only hot water extraction (80 deg) 7.51% 7.19% 16.13%
AG only hot water extraction (70 deg) 8.56% 8.09% 13.09%

Example 4
Composition containing the aqueous extract of A. galanga (E-AG-01)
a) Alpinia galanga extract – 95-100%
b) Silicon dioxide – 0-5%

Example 5
The effect of the water-soluble extract of Alpinia galanga that is free of methyleugenol (E-AG-01) as compared to a water-insoluble extract, caffeine, and other herbs:

The water insoluble extract as referred herein means and includes methanolic extract as depicted in table 2.

The following study compared the effects Cymbopogon flexuosus (lemongrass, LG), Alpinia galanga (AG1 and AG2), and Glycyrrhiza glabra (licorice, GG1 and GG2) on improving brain performance, mainly by enhancing attention network-related functioning.

Participants:
Seventy subjects (male and female) between 18–40 years of age with a body mass index (BMI) of 18–25 kg/m², a resting blood pressure <140/90 mmHg, and habituated to average caffeine consumption were considered eligible for inclusion in the study. Only right-handed subjects were included in the study to avoid spatial bias. Subjects had to refrain from caffeine products and vigorous physical activity 12 h prior to the study. As caffeine abstinence tends to increase sleepiness, consequently reducing the alertness score, the included subjects had to have an Epworth’s sleeping scale = 10 indicating low mental alertness during general situations in day-to-day activities at screening and at all visits. The subjects had to refrain from smoking 24 h before study onset and refrain from alcohol intake throughout the study. Subjects with a history or presence of clinically important cardiac, renal, hepatic, endocrine (including diabetes mellitus), pulmonary, biliary, gastrointestinal, pancreatic, or neurological disorders and uncontrolled hypertension were excluded from the study. For each subject, the study was terminated after data collection on the assessment day.

Interventions:
On day 1 of assessment, subjects were randomized in one of the arms to receive either the investigational product (IP), comparator (caffeine: CF), or the placebo. The IPs included Cymbopogon flexuosus (LG), water-soluble and water-insoluble extracts of Alpinia galanga (AG1 and AG2 respectively), and water-soluble and water-insoluble extracts of Glycyrrhiza glabra (GG1 and GG2 respectively). All interventions were administered to subjects in the form of identical capsules that had been packed in duly labeled HDPE bottles. The double-blinded nature of the study was ensured and strictly followed. All IPs were standardized for their activity, and the extracts were prepared by elimination of impurities using modern manufacturing techniques. The manufacture extracts and packaging of the finished product were carried out at a GMP-certified contract manufacturing facility in India. To maintain blinding, all the capsules were averaged to a weight of 500 mg. The composition details of all treatments are shown in table 4 below:

Table 4

Test Visit Procedure
On day 1 of the assessment, subjects reported to the clinic during the morning hours, and testing began at an early time of day (8:00–9:00 a.m.) for each visit to avoid influence of daily challenges (related to mental and physical stress) on outcomes. The time of day was matched for all visits to reduce variability due to the diurnal pattern. Subjects reported to the clinic after 24 hours of abstinence from caffeine and caffeine-containing products or any psychostimulants prior to all visits during the study. The subjects were also instructed to get sufficient sleep during the night prior to testing, which was self-reported by the participants in a sleep diary. Upon arrival at the clinic, subjects were asked to rest quietly for 15–20 minutes, after which physical examination was done, caffeine history was recorded, and the subjects were asked to fill out an Epworth’s sleep scale questionnaire to rate their sleepiness. A standardized meal of approximately 200 calories was provided to control variations from possible digestion confounds. Baseline data on ANT was collected 30 minutes after breakfast followed by administration of the IP. Each dose consisted of two capsules of the product with a glass of water, which was administered to subject by the trial coordinator at the investigational site. After ingestion of IP, data were collected at 1, 3, and 5 hours and controlled for mean response time, alertness, orientation, and executive attention before and after IP administration. No other food or calorie containing beverages were provided during the five-hour period. The subjects were allowed to drink water as desired and allowed to relax in an isolated room at a comfortable temperature and were free to use a computer, listen to music, or read magazines during the clinic stay period.

From days 2 to 6, participants were advised to take two capsules daily until day 6. On the day 7 visit, subjects followed the exact same schedule as that of day 1: after ingestion of two capsules, the subjects were allowed to rest for 30 minutes and last time point data were collected by ANT. To ensure treatment compliance, product accountability was monitored by a clinical research coordinator on the
following visit.

Outcome Measures:
The Attention Network Test (ANT) examines the effects of cues and targets within a single reaction time task in order to explore the efficiency of the alerting, orienting, and executive control networks of attention and mean response time with respect to different psychological and physiological states. It also provides an opportunity to examine the brain activity of these three networks as they operate in a single integrated task.

An adaptation of ANT, Centre for Research on Safe Driving Attention Network Task (CRSD-ANT), was used to evaluate the effect of IP on outcome measures.

Primary efficacy variables:
CRSD-ANT was utilized to assess the effect of IPs on the mean response time (MRT) in milliseconds. Data were used to capture superiority within groups from baseline to each time point on the same day and intergroup comparisons.
Secondary efficacy variables:
Effects of IPs on different aspects of the attention network, such as alertness, orientation, and executive control, were also assessed by ANT. These parameters are collectively responsible for achieving and maintaining vigilance and alertness while performing a continuous task.

Statistical Analysis:
The sample size was calculated using PS: Power and Sample Size Calculation version 3.1.2 (2014). Continuous response variables were analyzed from matched pairs of subjects. In absence of prior data, an assumption was made that the difference in the response of matched pairs is normally distributed with a standard deviation of 2. Considering the mean response of matched pairs is two, at least eight subjects per arm were targeted to reject the null hypothesis that this response difference is zero with a probability (power) of 0.8. The Type I error probability associated with the test of this null hypothesis was 0.1.

All values are presented as means ± standard deviation (SD). Statistical analyses were carried out on the data characterized by 95% confidence interval. Variables were tested for normality followed by student’s paired t test for intergroup comparisons at different time points. p values of M 0.05 were considered statistically significant.

Results:
70 subjects were recruited, of which 64 completed the study and 6 were withdrawn due to non-compliance. Analysis was performed on the intention to treat (ITT) population, which included subjects who were administered at least a single dose and for whom at least a single post-baseline assessment was available.
None of the treatment groups showed any significant reduction in response time at 1 h after IP administration. However, subjects in the LG group showed a significant increase in the MRT at 1 h (p = 0.01), indicating delayed response. The caffeine group showed an improvement in the MRT at 3 h after IP administration (p = 0.06), followed by a reversed trend in response time. The change in the response time in various groups is presented in Figure 1. None of the treatment groups showed a significant effect on response time at 5 h after administration and after 7 days of exposure to IP.

Alertness:
Among all the treatment groups, only the AG1 group showed a consistent improvement in the alertness time at 1 h (p = 0.07) and 3 h (p = 0.07) and achieved statistical significance at 5 h (p = 0.007), which can be attributed to the reduction in the alertness score in placebo group at 5 h.
However, the GG1 group exhibited a significant increase in alertness at 3 h, which slightly dropped at 5h. Changes in the other groups were not as appreciable. Figure 2 presents these data, revealing the superiority of the AG1 group in increasing alertness over the remaining groups. At day 7 after IP, the caffeine group exhibited a decrease in alertness compared to the placebo group (p = 0.1). None of the treatment groups were effective in improving alertness at this point.

Orientation:
None of the investigated IPs and the comparator demonstrated any significant effect on orientation compared to the baseline. At day 7 after administration of IP, none of the products demonstrated a better effect than placebo.

Executive attention:
None of the interventional groups showed any significant improvement in executive attention compared to placebo at 1, 3, and 5 hours after administration. At day 7, none of the groups showed significant effect on executive attention.

Discussion and Conclusion:
The above data were used to evaluate the efficacy and safety of natural product extracts in subjects with caffeine dependence. Several extracts were screened to elucidate the psychostimulant potential of the selected natural sources. Unlike studies which report findings on the basis of subjective feelings, these data were used to compare the effects of the IPs on different aspects of the attention network using the ANT, which is the only tool to independently analyze the different aspects of the attention network.

Although, change in caffeine group at 3h did not reach statistical significance, it indicates caffeine’s efficacy in reducing the mean response time, thus ascertaining its literature reported effect.

This finding also validated the reliability of the ANT as an assessment tool for attention-related studies.

Among the extracts (LG, AG1, AG2, GG1, and GG2), AG1 showed a consistent
improvement in alertness at 1, 3, and 5 h as compared to baseline and placebo. This can be attributed to the water-soluble compounds such as polyphenols, pyrocatecollic type tannins, and polysaccharides, abundantly present in A. galanga, which were skillfully extracted in the water-soluble extract.

The neurocognitive effect responsible for improving alertness (observed in the AG1 group) cannot be solely attributed to purine-like alkaloids such as caffeine or theacrine, as AG1 has a rich phytochemical profile in addition to alkaloids. Moreover, the polyphenols and flavonoids of AG1 appear to be helpful in combating the caffeine-like “crash.”

Example 6
Effect of the extract of Alpinia galanga that is free of methyl eugenol as compared to caffeine and a composite for increasing mental alertness and decreasing mental fatigue
Data were collected to assess the stimulant effect of A. galanga that is free of methyl eugenol in healthy young volunteers on specific parameters related to attention network and sleep architecture using standardized tests and questionnaires.

The efficacy of the extract of Alpinia galanga that is free of methyl eugenol was analysed with a principal objective of developing a safe alternative to caffeine for increasing mental alertness and decreasing mental fatigue. The data were used to assess the extract of Alpinia galanga that is free of methyl eugenol as a psycho-stimulant in healthy young volunteers using specific parameters related to important aspects of attention networks and using standardized tests and questionnaires.

Subjects, Methods and study design
Subjects were derived through multiple sources including a healthy volunteer’s database and a consumer group’s survey agency and enrolled in the study after screening. Eighteen to forty years old healthy, teetotalers and nonsmoking male and female subjects with minimal computer literacy were enrolled in the study. All subjects were predominantly right handed with a history of moderate caffeine consumption (2-4 cups of caffeinated beverages per day). The caffeine history was taken to ensure that the participants were acquainted with caffeine’s stimulant effect and were not caffeine-sensitive. The subjects with body mass index (BMI) between18.50 and 25.00 kg/m2 and a resting blood pressure = 140/90 mm Hg were considered eligible. To avoid subjects with psychotic disorders such as anxiety, depression, ADD or ADHD, subjects with Generalized Anxiety Disorder Screening (GAD-7) score = 7 & Patient Health Questionnaire-9 (PHQ-9) score =14 were excluded from the study. Subjects with alertness score (Jin Fan’s Attention Network Test, version 1.3.0) of 50 ± 20 milliseconds at screening visit were considered eligible. Concomitant therapy was strictly prohibited during the course of the study to exclude any significant effect on study results. All the subjects were instructed thoroughly on the study procedures.

The study was a single center, randomized, double dummy, double-blind, placebo-controlled, cross-over group study.

Interventional products (IPs):
Subjects were randomized and allocated to interventional products in each visit, divided in four treatment arms based on randomization chart [SPSS version 10.0, IBM] and similar trend was followed for subsequent visits. The interventional products (IP) include: Alpinia galanga proprietary extract (E-AG-01), caffeine, a combination of E-AG-01with Caffeine as a composite (300mg Alpiniagalanga Rhizome Extract that is free of Methyl Eugenol plus Caffeine 200mg) and a placebo. All these treatments were administered to subjects in the form of capsules which were identical in appearance and packed in duly labelled HDPE bottles using the blind codes. As subjects in one arm received a combination of caffeine and A. galanga, the subjects receiving solely A. galanga or caffeine or placebo were co-assigned to additional placebo capsule to achieve double dummy design of the study.

Table 5: Composition details of IPs
Ingredients E-AG-01(mg/Capsule) Caffeine
(mg/Capsule) Placebo
(mg/Capsule)
Alpiniagalanga Rhizome Extract that is free of Methyl
Eugenol 300 - -
Caffeine (Anhydrous) - 200 -
Microcrystalline Cellulose (MCC) 250 350 550
Total 550 550 550

Test Visit Procedure:
Subjects reported to the clinic during the morning hours and testing began at early time of day (8:00-9.00 a.m.) for each visit to avoid influence of daily challenges (related to mental and physical stress) on study outcomes and the time of day was matched for all three visits to reduce variability in response due to diurnal pattern. Subjects reported to the clinic following a 24 hours abstinence from caffeine and caffeine-containing products or any psycho-stimulants prior to all visits to site during the study. Subjects were also instructed to obtain sufficient sleep during the night prior to testing which was confirmed using sleep diary. Upon arrival at clinic, subjects were asked to rest quietly for 15-20 minutes after which vital parameters were measured and a standardized meal of approximately 200 calories was provided to control variations from possible digestion confounds. Baseline data was collected 30 minutes post breakfast followed by administration of IP, wherein one dose of the product was administered to subject by trial coordinator at investigational site.

Post ingestion of IP, data was collected at 1, 3 and 5 hours. No other food or calorie-containing beverages were provided during the study period. Subjects were allowed to drink water as desired. Subjects were allowed to relax in an isolated room at a comfortable temperature and free to use the computer, listen to music or read magazines during the clinic stay period. To analyze the effect on IP on sleep architecture, second dose was supplied in a labeled bottle to be taken before dinner in the night of the same day and subjects were asked to record all the details pertaining to sleep quality and duration in sleep diary. To ensure treatment compliance, product accountability was monitored by clinical research coordinator on the following visit.

Outcome Measures:
The study was mainly designed to elucidate investigational product’s effect on various psychoactive measures in habitual caffeine consumers. Also, the likelihood of desired synergistic effect to reduce ‘caffeine crash’ was explored by consuming E-AG-01 and caffeine together.

The primary efficacy variable was mental alertness. Alertness is defined as achieving and maintaining a state of high sensitivity to incoming stimuli. The alerting network is theorized to be responsible for achieving and maintaining vigilance and alertness during the performance of a continuous task. The Attention network test (ANT) was implemented for this purpose as it provides a behavioural measure of the efficiency of the different components of attention networks separately within a single task. The downloaded JAVA version of ANT 1.3.0 (Fan et al., 2002, 2005) was used to conduct the trial. The subject was seated in a silent and secluded room. All external distractions were avoided and subjects were asked to give complete attention to the task at hand. Mental alertness in ANT was calculated in terms of difference score quantified in milliseconds and calculated by subtracting average double-cue RTs from the no-cue RTs. Higher score indicates more efficient functioning of the alerting system.

The secondary efficacy variables namely sustained attention [assessed by Psychomotor Vigilance Task (PVT)], mental fatigue [assessed by Karolinska Sleepiness Scale (KSS) Score], sustained wakefulness (assessed by sleep duration) and sleep pattern [assessed by Groningen’s Sleep Quality Scale (SQS) and sleep diary] were also assessed.

Sustained attention to the environment is impacted by multiple underlying brain processes and related psychological constructs, out of which the sleep–wake state, dependent on multiple brain-stem–thalamo–cortical pathways, is an important aspect. The Psychomotor Vigilance Test (PVT) (according to the criteria reported in Basner and Dinges) objectively assesses sustained attention which might be associated with fatigue-related changes in alertness due to sleep loss, extended wakefulness, circadian misalignment and time on task. The study assessed effect of IP on mean response time using psychomotor vigilance test. In this study, 10-minutes computer-based PVT was performed. Subjects were asked to respond by clicking the mouse on first appearance of red colored digit on screen for the duration of 10 minutes. The mean RT was calculated in milliseconds which is the time a subject took to press the response button as soon as each stimulus appeared, in order to keep RT as low as possible. Lower MRT thus indicates more efficient sustained attention.

Mental fatigue is an indicator of individual’s mental or physical performance capability and can impair an individual’s alertness and ability to perform a continuous task. The subjects were asked to complete a standardized Karolinska sleepiness scale (KSS) questionnaire [Nordin M, Åkerstedt T, Nordin S. Psychometric evaluation and normative data for the Karolinska Sleep Questionnaire. Sleep and Biological Rhythms. 2013; 11 (4):216-226] for assessment of mental fatigue at first visit to site and then prior to all sessions of ANT and PVT. The questionnaire is a self-defined 10 grade KSS score scale where lower score implies low mental fatigue and higher alertness.

Wakefulness in terms of sleep quality was assessed by Groningen’s sleep quality score questionnaire and sleep diary [Carney CE, Buysse DJ, Ancoli-Israel S, Edinger JD, Krystal AD, Lichstein KL, Morin CM. The consensus sleep diary: standardizing prospective sleep self-monitoring. Sleep. 2012;35(2):287-302] in which sleep quality was graded in the form of a score (SQS) on the scale of 0-14 score. Lower score on this scale indicates higher subjective quality of sleep and vice versa. Subject was asked to fill SQS at all visit to site. They were provided with a sleep diary to fill in the morning immediately on waking up and SQS on the day after IP administration at night. The effect of IP on wakefulness was also assessed by recording the duration of sleep between two assessment sessions. Subject were asked to start the stop watch before taking a nap and to stop it once they are awake or when they are woken up by the investigator for assessment. The stop watch was lapped and re-started if the subject wanted to nap again.

Safety parameters were also evaluated by measurement of vitals and incidence of adverse events to assure safe use of the interventional products.

Power Calculation:
The trial was designed to demonstrate efficacy of E-AG-01 over placebo. Internal
unpublished data show at least 12±3% increase in alertness score in the active arm compared to placebo arm with a type I error (T) of 5%. A sample size of 60 subjects was used to achieve 90% study power, accounting for dropouts &withdrawals (~ 20%) as well as non-evaluable subjects for the primary efficacy outcome (~25 %).

Statistical Analysis:
All values are presented as means ± standard deviation (SD) in the tables as well as in figures. Statistical analyses were carried out using SPSS 11.0 for Windows (Chicago, IL, USA) on the data characterized by 95 % confidence interval. Variables were tested for normality using the Shapiro-Wilk test followed by Multivariate ANOVA to evaluate amongst group statistical significance. Further, Student’s paired t-test was used for inter-group comparisons at different time points. p values of = 0.05 were considered statistically significant.

Continuous variables (Age, height, weight and BMI) were summarized by treatment group using summary statistics (number of observations, mean and standard deviation). ANOVA was applied to prove the insignificance in demographic characteristics across the four groups. Data pertaining to PHQ-9 and GAD-7 parameters was also evaluated statistically by ANOVA for assessment of within group significance level.

Final statistical analyses were carried out only on the data from the subjects whose baseline alertness score for the day matches with protocol defined alertness score.

When the statistical significance was acquired, effect size was calculated by “Cohen’s d” method for determination of clinical relevance of the observed effects. A value between 0.20 and 0.49 is considered as a small effect, whereas a value between 0.5 and 0.79 is considered as a medium effect and ‘d’ greater than or equal to 0.8 indicates a large effect size.[Cohen J. Statistical power for the behavioural sciences, 2nd ed., Hillsdale & Lawrence Erlbaum: Hillsdale, NJ, USA, 1998]

Study Population:
Out of the124 subjects screened for this study, 59 subjects met the protocol-defined inclusion-exclusion criteria and were enrolled in the study. Six subjects out of 59 were dropped out during the study, primarily due to consent withdrawal (4/6) or were lost to follow-up (2/6). Two of the withdrawals were attributed to safety concerns.

On completion of the study, further statistical analyses were performed on Per Protocol (PP) and Intended to Treat (ITT) populations. PP group represents the subjects whose baseline matched with protocol specified screening criteria on each baseline alertness score and completed the study successfully. ITT group included the subjects who met all inclusion/exclusion criteria and received at least one dose of each investigational product. Only the results pertaining to PP group are presented and considered to obtain clinically significant outcomes.

Varying number of subjects for some efficacy parameters in PP group is accountable to discarded data for the subjects for whom baseline did not match to protocol specified inclusion-exclusion criteria. Hence, the corresponding data was excluded from the statistical analysis, which did not affect the minimum study power (90 %).

All subjects were screened for demographic parameters (Age, Gender, Height, Weight and BMI) at screening visit to confirm compliance with the protocol. Analysis of demographic characteristics of all subjects confirmed that there was no significant standard deviation (p > 0.05) among the treatment groups. Analysis of baseline characteristics of all subjects analyzed by PHQ-9 and GAD-7 questionnaires confirmed that none of the groups had PHQ-9 and GAD-7 scores beyond the specified range, assuring that none of the subjects of study population was suffering from any mental disorders such as anxiety and depression. The groups did not differ statistically from each other (p > 0.05). Data pertaining to these measures is presented in Table 6.

Table 6: Demographics and Baseline Characteristics.
Variables E-AG-01
(N=34)
Mean ± SD Caffeine
(N=35)
Mean ± SD Composite
(N=38)
Mean ± SD Placebo
(N=38)
Mean ± SD p value
Male 17 16 19 19 -
Female 17 19 19 19 -
Age
(years ± months) 22 ± 5 22 ± 5 22 ± 6 22 ± 6 0.982
Height (m) 1.66 ± 0.10 1.64 ± 0.11 1.66 ± 0.11 1.65 ± 0.12 0.920
Weight (kg) 59.67 ± 8.13 57.66 ± 8.19 58.54 ± 7.84 58.87 ± 9.15 0.805
BMI (kg/m2) 21.63 ± 1.93 21.26 ± 1.91 21.28 ± 1.95 21.45 ± 2.02 0.859
PHQ-9 1.8 ± 1.6 1.5 ± 1.6 1.6 ± 1.6 1.5 ± 1.6 0.919
GAD-7 2.1 ± 2.1 1.5 ± 1.6 1.9 ± 2.3 1.9 ± 2.3 0.663

Effect on Outcome Measures:
Mental alertness was considered as primary outcome measure based on its importance in the assessment of cognitive performance. Data obtained for alerting network is expressed as alertness score (Table 7 and Figure 3).

Table 7: Effect of Interventions on Mental Alertness with Intergroup Analysis

Note: *MANOVA test applied across 4 groups to get the p values, ** paired t test applied between two groups; #Decreased significantly as compared to baseline; ## Statically significant difference in score across the groups; ## Significant change in score as compared to placebo, BL: Baseline value; 1H: Value at 1 hour; 3H: Value at 3 hours; 5H: Value at 5 hours.

In the placebo group, there was a statistically insignificant increase in alertness score of 5.05 ± 19.71, 2.61 ± 20.66, and 9.79 ± 20.07 ms from baseline at one, three and five hours, respectively.

In E-AG-01 administered group, significant increase in alertness score of 11.65 ± 23.94, 12.50 ± 19.73, and 12.62 ± 24.71 msecs from baseline at one, three and five hours respectively was observed. As the changes across the groups were significant at three hours (p=0.036), the groups were individually compared to placebo using student’s paired t-test. E-AG-01 group demonstrated statistically significant improvement in alertness score (p = 0.042).

In the caffeine group, alertness score as compared to baseline increased significantly by 8.97 ± 18.20 ms (95 % CI: -4.96 -12.80, p = 0.006) at one hour; however, the score decreased by 1.23 ± 18.60 ms (95 %CI: -13.04 -5.37, p = 0.698) from the baseline at three hours, indicating a caffeine crash.
Also, in composite group, the score significantly increased by 10.27 ± 20.34 ms(95 %CI: -4.96 - 12.80, p = 0.004) from baseline at one hour followed by the return approximately to the baseline score with decrease of 0.68 ± 21.87 (95% CI: -13.01 to 6.43, p = 0.074) at three hours.

As the changes across the groups were significant at three hours (p=0.03), they were individually compared to the placebo using a student’s paired t-test, wherein the E-AG-01 group demonstrated a statistically significant improvement in the alertness score (p = 0.04).

At five hours, all groups demonstrated an increase in the alertness score, owing to the logistic factors of the study and asymptomatic performance improvement due to the diurnal pattern of the alertness, however the increase in the alertness score was maximum in the E-AG-01 group (12.62 ± 0.68 ms from baseline).

As the result was statistically significant for E-AG-01, the effect size was calculated in terms of a Cohen’s d value in comparison with placebo. The derived value of d = 0.59 for the EAG-01 group as against d= 0.08 for the caffeine group confirmed a significant medium effect size in alertness score in E-AG-01 group compared to a remarkably small effect size in caffeine group.

Effect on Secondary Outcome Measures
The data were used to assess the effect of IP on the sustained attention by using the PVT and the results were expressed as MRT in msecs. As evident from the results, the E-AG-01 group did not demonstrate any significant improvement in the MRT whereas the results from the caffeine group suggested a declining trend in the MRT till 3 hours after which it showed an increase which may be attributed to the crash effect. The composite group exhibited a trend similar to the caffeine group but achieved a within-group statistical significance at 5 hours interval as compared to baseline (p = 0.02). In addition, the intergroup analysis for the composite group revealed a significant decrease in MRT compared to the placebo at 1 hour (p = 0.01), 3 hours (p = 0.04) and 5 hours (p = 0.01).The mean response time as computed by PVT is expressed in the following table 8 and in Figure 4:

Table 8:

Note: *MANOVA test applied across 4 groups to get the p values, ** Student’s paired t-test applied between two groups; # Decreased significantly as compared to baseline; ### Significant change in score as compared to placebo, BL: Baseline value; 1H: Value at 1 hour; 3H: Value at 3 hours; 5H: Value at 5 hours.

After the statistical evaluation and interpretation of the data obtained for the mental fatigue, it was found that none of the groups showed significant reduction in mental fatigue as assessed by KSS. Intergroup analysis also confirmed the statistically insignificant results in this outcome parameter. We also assessed the effect of IP on wakefulness and sleep quality and the results obtained for all four
groups imply no statistically significant change in these outcome measures as well. The data show the effect of an extract of Alpinia galanga that is free of methyl eugenol in comparison with the caffeine (comparator) and the placebo (control) on various aspects of attention network. Consistent with caffeine’s well known effect on the alerting network, alertness score increased till one hour followed by a reduction, probably due to a caffeine crash at three hours. At the same time, the extracts of Alpinia galanga that is free of methyl eugenol showed an improvement in alertness score until five hours interval. In the composite group, alertness score increased significantly at one hour followed by a reduction, indicating a caffeine crash, which was less than that observed in the caffeine group. Hence, it can be hypothesized that the extract of Alpinia galanga that is free of methyl eugenol is able to impede the caffeine crash as evident from three hour and five hour alertness score.

The alerting network recruits a distributed network of brain regions, primarily the thalamus and bilateral frontal and parietal brain regions. Given the dense dopaminergic innervation of the human thalamus and prefrontal cortex and that the caffeine is generally thought to up regulate the dopaminergic availability, the present results are consistent with the theorized effects of caffeine on the CNS function. Based on these facts, it can be postulated that the extract of Alpinia galanga that is free of methyl eugenol also improves alertness in a similar way as that of caffeine by enhancing the dopaminergic activity.
The effect size for this efficacy parameter was calculated in terms of standardized mean effect, (denoted as Cohen’s d) which expresses the mean difference between two groups in the standard deviation units. The results suggest that in terms of probability of superiority of treatments, there is a 66% chance that a randomly selected subject from the E-AG-01 group will exhibit a definite improvement in the mental alertness than a randomly selected subject from the placebo group.

Alternatively, it can be stated that the effect size represented as Cohen’s d value indicates that 73 % subjects from the E-AG-01 group (d = 0. 599) would exhibit higher alertness than the subjects in the placebo group as compared to the caffeine group wherein only 47% subjects would have a better alertness.

The sustained attention was assessed by the PVT, which generally reflects the arousal and attention state of an individual. Caffeine appears to exhibit dose-dependent performance improvement in a variety of basic psychomotor tasks as a direct result of altered CNS activity and is well reported by a number of studies. Some studies also suggest that the extended vigilance is generally improved following caffeine consumption at a dose of ~ 400 mg and performance diminishes with very high dose of caffeine (e.g. 600 mg). In agreement with these reported findings, the data show that neither caffeine at 200 mg nor the extract of Alpinia galanga that is free of methyl eugenol at 300 mg independently exhibited significant reduction in the MRT. However, the combination of these ingredients at the same dose was found to be effective in improving the sustained attention as indicated by statistically significant data obtained for the composite group. It has been reported that the relationship between the sustained attention and the task performance follows an inverted U-curve, i.e. poor performance can occur due to both under- and over-arousal. This can be one of the reasons for wide-spread range of observations in MRT, leading to the bigger SDs and insignificant p values.

However, derived p values showed a positive trend in the reduction of MRT in the caffeine and composite groups. Hence, an attempt was made to analyze the treatment groups individually in comparison with placebo by student’s t-test which showed that the improvement in sustained attention was statistically significant in comparison with placebo, implying the superiority of the composite group in enhancing the sustained attention and arousal state. Thus, co-administration of the extract of Alpinia galanga that is free of methyl eugenol with caffeine may modulate the neural activity in the cerebral regions related to the sustained attention.

Within and between group analyses provided the statistically significant results, which serve as a constructive evidence for the beneficial effect of the extract of Alpinia galanga that is free of methyl eugenol on enhancement of mental alertness and sustained attention.

Example 7
The effect of E-AG-01 on the focalized attention in comparison with caffeine and placebo in moderate caffeine-habituates
The objective of this study was to evaluate whether E-AG-01 is capable of improving the mental sharpness.

A total of 59 caffeine-habituated and healthy young adults (18-40 years) with BMI of 18.50 -25.00 kg/m2 were crossed over in four interventional groups as, placebo, E-AG-01, caffeine and a combination of caffeine and E-AG-01. All participants completed the attention network test (ANT) which measured the accuracy and speed factors of the alerting, orienting and executive control networks.

Results:
The results of accuracy parameter in terms of percentage (%) error rate showed a remarkable difference between E-AG-01 and rest of the treatment groups, wherein the error rate was dropped by 1.71% (1 h), 1.4% (3 h) and 0.86% (5 h) from baseline. Caffeine group demonstrated a decrease of 0.37% (1 h) and 0.44% (3 h), followed by an increase of 0.2% (5 h)] whereas the error rate of subjects in Caffeine + E-AG-01 group decreased by 0.24% (1 h) and 0.27% (3 h) followed by an increase of 0.2% at 5 hour. Placebo group exhibited an increase of 0.15% (3 h) & 0.78% (5 h).

Conclusion:
These results conclude that E-AG-01 exhibited selectively enhanced focalized attention to a higher extent in comparison with caffeine and placebo.

It should be apparent from the foregoing description that various embodiments of the disclosed formulation, extraction process, and use may be implemented to provide the various types of formulations described herein. The description further implements methods of administering an extract of Alpinia galanga that is free of methyl eugenol to improve cognitive performance as well as a process for preparing an extract of Alpinia galanga that is free of methyl eugenol.

Although the various embodiments have been described in detail with particular reference to certain aspects thereof, it should be understood that the disclosed formulation, extraction process, and use are capable of other embodiments and its details are capable of modifications in various obvious respects. As is readily apparent to those skilled in the art, variations and modifications can be affected while remaining within the spirit and scope of the disclosed formulation, extraction process, and use.

Accordingly, the foregoing disclosure and description are for illustrative purposes only and do not in any way limit the disclosed formulation, extraction process, and use, which are defined only by the claims.
,CLAIMS:
1. A process for preparation of water soluble extract of A. Galanga that is devoid of methyleugenol, which comprises the following steps;
a) Extracting the powder of the dried rhizome of A. Galanga with cold water extraction cycle by soaking the same for 8-10 hrs followed by hot water extraction cycle for a period of 5-6 hours at temperature of 70-80 degrees and pressure of 1-10 bars to obtain collective extract;
b) Distilling the collective extract at a temperature of 70-80 degrees under vacuum to obtain syrupy material;
c) Vacumn drying the syrupy material at 80-90 degrees to obtain the water soluble extract, that is devoid of methyl eugenol.

2. The process as claimed in claim 1, wherein, the process comprises at least two or more hot water extraction cycles for a period of 5-6 hours at temperature of 70-80 degrees and pressure of 1-10 bars.

3. The process as claimed in claim 1, wherein, the ratio of dried rhizome of A. Galanga to water is at least about 1:4.

4. The process as claimed in claim 1, wherein, the vacumn drying is conducted under vacumn pressure of 650 to 700 mm and for 6-8 hours.

5. The process as claimed in claim 1, wherein, the dried extract is optionally pulverized with excipients like silicon dioxide under controlled temperature and humidity conditions, to obtain the free-flowing, water soluble powder.

6. The process as claimed in claim 4, wherein, the pulverization of the extract can be conducted under controlled temperature of 25 to 45 degrees and 20-50% Rh humidity conditions, to obtain the free-flowing, water soluble powder of A. Galanga.

7. The process as claimed in claim 1, wherein, the water soluble extract is standardised to comprise phytoactive matrix of polyphenols and polysaccharides in a ratio of approximately 1:1.6 to 1:8.

8. The process as claimed in claim 7, wherein, the water soluble extract is standardised to comprise phytoactive matrix of polyphenols and polysaccharides in a ratio of approximately 1:5 to 1:6.

9. Standardised water soluble extract of A. Galanga obtained in accordance with the process as claimed in claim 1, wherein, the extract is characterized to comprise a) phytoactive matrix of polyphenols and polysaccharides in a variable ratio of approximately 1:1.6 to 1:8; b) about 1% pyrocatecollic type tannins; c) galangin not more than 100 ppm; d) alkaloids not less than 0.4%; flavanoids not less than 3% and wherein, the extract is free from methyl eugenol.

10. A formulation comprising water soluble extract of A. Galanga in an effective amount of 10mg to 900mg along with a pharmaceutical/neutraceutical excipient/carrier, wherein, the extract is standardised to comprise phytoactive matrix of polyphenols and polysaccharides in a ratio of approximately 1:1.6 to 1:8.

11. The formulation as claimed in claim 10, wherein, the standardised extract comprise phytoactive matrix of polyphenols and polysaccharides in a ratio of approximately 1:5 to 1:6.

12. The formulation as claimed in claim 10, wherein, the formulation comprises an effective amount of water soluble extract of Alpinia galanga that is free of methyl eugenol and has not more than 100 ppm of galangin.

13. The formulation as claimed in claim 10, wherein, the formulation comprises an effective amount of water soluble extract of Alpinia galanga that is free of methyl eugenol and has about 1% pyrocatecollic type tannins.

14. The formulation as claimed in claim 10, wherein the amount of Alpinia galanga extract is about 200 to about 400 mg.

15. The formulation as claimed in claim 10, optionally comprises 100-400 mg caffeine.