Formulation of Daidzein for Sedative-Hypnotic effect

Abstract

Formulation of Daidzein for Sedative-Hypnotic effect The present invention comprises a microemulsion of daidzein comprising oil phase, water phase, surfactant and co-surfactant. This formulation is intended for sedative-hypnotic activity. The formulated microemulsion provides enhanced bioavailability & reduced hepatic metabolism of Daidzein after oral administration compared to suspension of daidzein in oil. The invention provides an alternative with reduced adverse effects and toxicities in comparison to currently marketed synthetic sedative-hypnotic drugs.

Specification

Description:Field of the Invention
The present invention comprises a microemulsion of daidzein comprising oil phase, water phase, surfactant and co-surfactant. This formulation is intended for sedative-hypnotic activity. The formulated microemulsion provides enhanced bioavailability & reduced hepatic metabolism of Daidzein after oral administration compared to suspension of daidzein in oil. The invention provides an alternative with reduced adverse effects and toxicities in comparison to currently marketed synthetic sedative-hypnotic drugs.


Back ground of the Invention
Sedative-hypnotics are widely used in insomnia treatment, pain management, anxiety relief, and pre-anaesthetic medication. However, currently marketed synthetic sedative-hypnotics are associated with various issues such as other drug related adverse effects, drug abuse, withdrawal syndromes and severe toxicities. There is a critical need to develop potent sedative-hypnotic drugs of natural origin to overcome these drawbacks.
Daidzein is commonly found isoflavone in soybeans and other legumes. It is readily available in amorphous powdered form (CAS registry number 486-66- 8).
Daidzein [7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one] is a naturally occurring phytoestrogen fitting into the category of nonsteroidal estrogens with numerous pharmacological activitiessuch as antihaemolytic, antioxidant, anti-inflammatory anti-cancer, anti-diabetic, etc.
Daidzein exhibited good binding affinity for benzodiazepine site of GABAA receptor indicating its role in sedation and hypnosis through the molecular docking studies. Based on these findings, daidzein suspension in oil was investigated in mice for sedative and hypnotic effect. Moreover, mechanism of action was also evaluated using selective benzodiazepine antagonist flumazenil. The results confirmed the sedation and hypnosis mediated via GABAA receptor with comparable sedative-hypnotic activity to the standard drug diazepam. However, due to higher first pass metabolism leading to maximum efficacy at higher dose (400 mg/kg of body weight), microemulsion was formulated and evaluated in-vivo. Daidzein microemulsion exhibited maximum efficacy at ¼th dose (50-200 mg/kg of body weight) as that of daidzein-oil suspension.

Summary of the Invention

The present invention discloses the sedative and hypnotic activity of daidzein microemulsion. The compound has shown promising results in screening experiments, with comparable or even superior activity compared to the standard marketed medicine, diazepam. The sedative and hypnotic effects are mediated through the GABAA receptor, a primary target of these drugs, as predicted by in-silico methods and confirmed by in-vivo mechanism of action studies.

The present invention discloses microemulsion of daidzein for oral route of administration. The formulation can also be given through different routes of administration such as nasal, parenteral and rectal routes. This formulation aims to provide effective sedative and hypnotic effects with reduced dose, adverse reactions and improved pharmacokinetics and pharmacodynamics.

Brief Description of Drawings
Fig 1: Effect of daidzein-corn oil formulation on locomotor activity in Swiss Albino mice. * P < 0.05 significant when compared with the pre-treatment reading of the respective group.
Fig 2: Effect of daidzein-corn oil formulation on number of head dips in Swiss Albino mice. * P < 0.05 significant when compared with the pre-treatment reading of the respective group.
Fig 3: Effect of daidzein-corn oil formulation on sleep onset in Swiss Albino mice. * P < 0.05 significant when compared with the control group.
Fig 4: Effect of daidzein-corn oil formulation on sleep duration in Swiss Albino mice. * P < 0.05 significant when compared with the control group
Fig 5: Effect of daidzein-corn oil followed by flumazenil on locomotor activity in Swiss Albino mice.* P < 0.05 significant when compared with the 60 minutes reading of the respective group.
Fig 6: Effect of daidzein-corn oil followed by flumazenil on number of head dips in Swiss Albino mice. * P < 0.05 significant when compared with the 60 minutes reading of the respective group.
Fig 7: Effect of daidzein microemulsion on locomotor activity in Swiss Albino mice. * P < 0.05 significant when compared with the pre-treatment reading of the respective group.
Fig 8: Effect of daidzein microemulsion on number of head dips in Swiss Albino mice. * P < 0.05 significant when compared with the pre-treatment reading of the respective group.
Fig 9: Effect of daidzein microemulsion on sleep onset in Swiss Albino mice. * P < 0.05 significant when compared with the control group.
Fig 10: Effect of daidzein microemulsion on sleep duration in Swiss Albino mice. * P < 0.05 significant when compared with the control group.

Detailed Description of the Invention
The present invention relates to a formulation wherein the active ingredients comprise of daidzein, any edible oil or mixture of edible oils, any surfactant, co-surfactant and emulsifying agent. It also relates to the use of this formulation as a sedative, as well as hypnotic agent. In another aspect, the present invention relates to a process to produce microemulsion wherein the active ingredients comprise of daidzein solubilized in oil phase containing soya lecithin. Surfactant and co-surfactant are further added to this mixture and mixed well. Further on, distilled water is added under continuous stirring to obtain clear microemulsion.
In a preferred embodiment, phase diagram was used to define the ration of API to excipient and then trial batches were formulated using design expert software and evaluated for drug content, pH, zeta potential, particle size, stability studies. The optimized batch was further used for the in-vivo evaluation of sedative-hypnotic activity.
Formulations and Routes of Administration: In current study, daidzein suspension in oil and microemulsion of daidzein in oil along with surfactant, co-surfactant and emulsifying agent are prepared for evaluation of sedative and hypnotic effects. The formulation can be in the form for oral administration or it can be formulated as nasal preparations by using body oils forming gels and nasal drops. Also mists and sprays can be prepared for nasal use. The formulation of daidzein can also be developed for parenteral route of administration - depot formulations for intra-muscular route and emulsions, or nanosomal preparations for intravenous route. Also, the types of formulations which are given through rectal route can be prepared for administration of the drug.
Dosage Levels: The concentration of the prepared daidzein-oil suspension was 5% w/v to deliver doses of 100 mg/kg, 200 mg/kg, 400 mg/kg and 800 mg/kg to mice as per body weight. The formulated daidzein microemulsion was prepared as 1% w/v to deliver doses of 25 mg/kg, 50 mg/kg, 100 mg/kg, 150 mg/kg and 200 mg/kg to mice as per body weight. The concentration of daidzein in the formulation may vary as per body weight and species to which it is administered as a sedative and hypnotic.
Screening Experiments: In-vivo experiments were conducted in Swiss albino mice to evaluate the sedative and hypnotic activity of the formulation. The formulation demonstrated significant activity comparable to diazepam, the clinically popular sedative and hypnotic medicine.
Mechanism of Action: The in-silico study revealed that daidzein has a higher binding affinity towards the benzodiazepine (diazepam binding site) of the GABAA receptor than towards the barbiturate (phenobarbital binding site). This suggests that the sedative-hypnotic activity of daidzein may be mediated through bindingto the benzodiazepine site of the GABAA receptor, similar to diazepam.
Moreover, mechanism of action was also evaluated using selective benzodiazepine antagonist flumazenil. The results confirmed the sedation and hypnosis mediated via GABAA receptor particularly as a result of binding to its benzodiazepine site similar to diazepam.
Oral Formulation: Daidzein can be formulated as a suspension or emulsion in edible oil for oral administration. This oral formulation is suitable for inducing and maintaining sleep and providing sedative effects. Due to the sedative-hypnotic activity of the formulation, daidzein can be classified as a CNS depressant.
Novel Drug Delivery Systems: The formulation of daidzein can be incorporated into novel drug delivery systems for oral administration, including nanosomal preparations such as liposomes, phytosomes, and micelles. These drug delivery systems aim to improve the bioavailability and targeted release of daidzein, enhancing its sedative and hypnotic effects.
Nasal Formulation: For nasal administration, daidzein formulation can be formulated with body oils to create gels and nasal drops. Additionally, mists and sprays containing daidzein can be prepared for nasal use. These formulations are effective in inducing sedation and providing hypnotic effects.
Parenteral Formulation: Daidzein formulation can also be formulated for parenteral administration. For intra-muscular route, depot formulations containing daidzein can be prepared to achieve sustained release and prolonged sedative-hypnotic effects. For intravenous administration, emulsions or nanosomal preparations can be used to improve solubility and bioavailability.
Rectal Formulations: Daidzein can be formulated as nanoparticulate rectal formulations, enema, suppositories, gels, creams and ointments, which can be administered through rectal route.

Biological Evaluation: The sedative and hypnotic activity of daidzein formulation was studied using commonly employed animal experimental models. In an in-silico study, daidzein demonstrated a higher binding affinity towards the benzodiazepine site of the GABAA receptor, the major target of these drugs, compared to the barbiturate site. This suggests that the sedative-hypnotic activity of daidzein may be mediated through binding to the same site as diazepam.
The locomotor activity test in mice showed significant inhibition of locomotor activityat 60, 90, and 120 minutes after oral administration of daidzein-corn oil suspension and at 30, 60, 90 and 120 minutes after oral administration of daidzein microemulsion. The hole-board test demonstrated decreased explorative behavior and curiosity in mice, more prominently than with diazepam. Additionally, daidzein reduced the timerequired for the onset of thiopentone-induced sleep and increased the duration of sleep compared to the control group.
The action of daidzein as a sedative-hypnotic mediated via GABAA receptor (benzodiazepine binding site) was confirmed using selective benzodiazepine antagonist flumazenil. The results depicted reversal of sedation after flumazenil administration concluding the action of daidzein mediated via benzodiazepine site of GABAA receptor. However, the results were not statistically significant as the dose of daidzein is very high (400 mg/kg) as compared to diazepam (1 mg/kg) to be reversed by flumazenil (5 mg/kg) as it is a competitive antagonist of benzodiazepine site of GABAA receptor with short half-life of 4-11 minutes.
Locomotor Activity Test: Daidzein-corn oil suspension and daidzein microemulsion was orally administered to mice, and the percentage inhibition of locomotor activity was measured at various time intervals. It was found that the highest inhibition of locomotor activity occurred at 90 and 120 minutes after administration.
Hole-Board Test: The explorative behavior and curiosity of mice were assessed using the hole-board test. Daidzein formulations reduced explorative behavior and curiosity more significantly than diazepam, indicating a stronger sedative effect.
Thiopentone Induced Sleep Test: Daidzein formulations reduced time required for the onset of thiopentone-induced sleep and increased the duration of sleep, compared to the control group. This demonstrates the hypnotic effect of daidzein.
Mechanism of Action Study: The action of daidzein as a sedative-hypnotic mediated via GABAA receptor (benzodiazepine binding site) was confirmed using selective benzodiazepine antagonist flumazenil. The results depicted reversal of sedation after flumazenil administration concluding the action of daidzein mediated via benzodiazepine site of GABAA receptor.
The current invention discloses the sedative and hypnotic effects of daidzein formulations. It provides a safer and effective alternative to currently marketed synthetic sedative-hypnoticdrugs with reduced adverse effects. The invention can be formulated for various routes of administration, making it a versatile and valuable addition to the field of pharmaceuticals.

Example
The suspension of daidzein in edible oil and microemulsion of daidzein is prepared for inducing sedation and hypnotic effects. The daidzein was prepared as a 5% w/v suspension with corn oil and was administered at three different dose levels based on body weight (100 mg/kg; 200 mg/kg; 400 mg/kg for sedation experiment and 200 mg/kg; 400 mg/kg; 800 mg/kg for hypnosis experiment). The formulated daidzein microemulsion was prepared as 1% w/v to deliver doses of 25 mg/kg, 50 mg/kg, 100 mg/kg, 150 mg/kg and 200 mg/kg for sedation and hypnosis experiments.
The formulation with any pharmaceutically acceptable carrier can be in the form for oral administration or it can be formulated as nasal preparations in the form of gels or nasal drops or mists or sprays for nasal use or for parenteral route of administration or emulsions or nanosomal preparations for intravenous route and also can be formulated in any type of rectal use formulations.

Example
The biological evaluation of the daidzein formulation was performed for sedative and hypnotic effect using Swiss albino mice as an animal model.
The locomotor activity test in mice showed significant inhibition of locomotor activity at 60, 90, and 120 minutes after oral administration of daidzein-corn oil suspension and at 30, 60, 90 and 120 minutes after oral administration of daidzein microemulsion. It was found that the highest inhibition of locomotor activity occurred at 90 and 120 minutes after administration. The hole-board test demonstrated decreased explorative behavior and curiosity in mice, more prominently than with diazepam. Additionally, daidzein reduced the time required for the onset of thiopentone-induced sleep and increased the duration of sleep compared to the control group.
The action of daidzein as a sedative-hypnotic mediated via GABAA receptor (benzodiazepine binding site) was confirmed using selective benzodiazepine antagonist flumazenil. The results depicted reversal of sedation after flumazenil administration concluding the action of daidzein mediated via benzodiazepine site of GABAA receptor. However, the results were not statistically significant as the dose of daidzein is very high (400 mg/kg) as compared to diazepam (1 mg/kg) to be reversed by flumazenil (5 mg/kg) as it is a competitive antagonist of benzodiazepine site of GABAA receptor with short half-life of 4-11 minutes.
Locomotor Activity Test: Daidzein-corn oil suspension and daidzein microemulsion was orally administered in different dosage in mice, and the locomotor activity counts and percentage inhibition of locomotor activity was measured at various time intervals. The effect of administration of corn oil or pseudo microemulsion and diazepam were also studied. It was found that the highest inhibition of locomotor activity occurred at 90 and 120 minutes after administration. Effect of daidzein-corn oil formulation and daidzein microemulsion treatment on locomotor activity of mice is tabulated in Table 1 and Table 2 respectively.

Table 1: Effect of daidzein-corn oil formulation on locomotor activity of mice.
Treatment Dose Pretreatment LocomotorActivity Counts
[% inhibition]
30 min 60 min 90 min 120 min
Control
(Corn oil) 0.1
ml/mouse 521.5 ± 80.3 365.5 ± 67.8**
[29.9] 292.5 ± 31.8
[43.9] 320.6 ± 53.6*
[38.5] 325.8 ± 62.3*
[37.5]
Diazepam 1 mg/kg 440.6 ± 48.9 349.5 ± 72.8
[20.7] 127.2 ± 41.6***
[71.1] 66.8 ± 21.9**
[84.8] 86.3 ± 12.7**
[80.4]
Daidzein- corn oil formulation 100 mg/kg 844.0 ± 153.2 666.2 ± 149.9
[21.1] 445.5 ±140.4***
[47.2] 296.3 ± 92.1**
[64.9] 308.3 ± 121.8**
[63.5]
200 mg/kg 666.2 ± 115.9 399.6 ± 70.7
[40.0] 243.0 ± 52.3**
[63.5] 144.6 ± 22.1*
[78.3] 208.3 ± 74.2*
[68.7]
400 mg/kg 793.0 ± 116.5 483.3 ± 155.5
[39.1] 340.5 ± 142.2*
[57.1] 214.3 ± 101.8*
[73.0] 189.5 ± 61.3**
[76.1]
Values are presented as Mean ± SEM. Number of animals in each group (n) = 6. Statistical analysis done using two-way ANOVA followed by Dunnett's multiple comparisons test.
* p< 0.05, ** p< 0.01, *** p< 0.001.
Table 2: Effect of daidzein microemulsion on locomotor activity of mice.


Treatment

Dose

Pretreatment Locomotor Activity Counts
(% inhibition)

30 min 60 min 90 min 120 min
Control
(Pseudo
Microemulsion) 0.1
ml/mouse 317.3 ± 39.0
214.8 ± 25.1
(32.3) 193.8 ± 25.6
(38.9) 160.0 ± 19.5
(49.6) 132.7 ± 14.2*
(58.2)
Diazepam 1mg/kg 347 ± 27.0 229.2 ± 26.4*
(34.0) 175.3 ± 21.5**
(49.5) 149.7 ± 19.6***
(56.9) 96.2 ± 13.1**
(72.3)



Daidzein
microemulsion 25 mg/kg 294.0 ± 18.8 189.0 ± 8.8*
(35.7) 151.0 ± 11.4**
(48.6) 153.8 ± 14.8***
(47.7) 136.5 ± 18.2**
(53.6)
50 mg/kg 301.0 ± 27.9 184.2 ± 27.9*
(38.8) 124.8 ± 19.7**
(58.5) 71.3 ± 10.6***
(76.3) 58.3 ± 9.6**
(80.6)
100 mg/kg 402.3 ± 44.4 220.8 ± 29.2**
(45.1) 133.3 ± 24.4**
(66.9) 87.7 ± 17.3**
(78.3) 69.0 ± 21.4**
(82.9)
150 mg/kg 356.0 ± 30.5 232.0 ± 33.5*
(34.9) 171.8 ± 32.3**
(51.7) 112.7 ± 16.3**
(68.4) 87.2 ± 21.4****
(75.5)
200 mg/kg 307.2 ± 18.3 152.3 ± 9.6**
(50.4) 67.5 ± 9.9***
(78.0) 56.5 ± 8.9***
(81.6) 46.8 ± 8.0****
(84.8)
Values are presented as Mean ± SEM. Number of animals in each group (n) = 6. Statistical analysis done using two-way ANOVA followed by Tukey's multiple comparisons test.
* p< 0.05, ** p< 0.01, *** p< 0.001, **** p < 0.0001.
Hole-Board Test: The explorative behaviour and curiosity of mice were assessed using the hole-board test. The administration of the formulation of daidzein-corn oil and daidzein microemulsion reduced explorative behaviour and curiosity more significantly in comparison to only corn oil/pseudo microemulsion and the conventional sedative drug diazepam, indicating a stronger sedative effect. The results of the test are tabulated in in Table 3 for daidzein-corn oil formulation and in Table 4 for daidzein microemulsion.


Table 3: Effect of daidzein-corn oil formulation on number of head dips in mice.
Treatment Dose Pretreatment Responses (45min)
Number of head
dips %inhibition
Control
(Corn oil) 0.1 ml/mouse 47.6 ± 6.1 30.8 ± 2.79** 35.3
Diazepam 1 mg/kg 48.8 ± 2.08 26.8 ± 4.14*** 45.1
Daidzein- corn oil formulation 100 mg/kg 30.6 ± 5.08 18.8 ± 4.85 38.6
200 mg/kg 27.0 ± 6.1 13.3 ± 3.2* 50.7
400 mg/kg 35.2 ± 4.1 5.5 ± 2.0**** 84.4

Values are presented as Mean ± SEM. Number of animals in each group (n) = 6. Statistical analysis done using two-way ANOVA followed by Sidak's multiple comparisons test.
* p< 0.05, ** p< 0.01, *** p< 0.001, **** p < 0.0001.


Table 4: Effect of daidzein microemulsion on number of head dips in mice.


Treatment

Dose

Pretreatment Number of Head Dips
(% inhibition)

30 min 60 min 90 min 120 min
Control
(Pseudo
microemulsion) 0.1
ml/mouse 32.2 ± 5.7
15.8 ± 2.0
(50.8) 18.2 ± 4.3
(43.5) 15.2 ± 5.4
(52.8) 13.2 ± 1.6
(59.1)
Diazepam 1mg/kg 46.2 ± 5.1
24.3 ± 6.1*
(47.3) 18.7 ± 4.4***
(59.6) 10.3 ± 3.4***
(77.6) 10.0 ± 3.0**
(78.3)


Daidzein microemulsion 25 mg/kg 30.8 ± 1.9

16.2 ± 1.8**
(47.6)
13.3 ± 2.3**
(56.8) 13.8 ± 3.9*
(55.1) 15.5 ± 3.3**
(49.7)
50 mg/kg 40.0 ± 7.3
23.2 ± 6.8**
(42.1) 8.8 ± 2.3*
(77.9) 4.2 ± 1.1*
(89.6) 3.8 ± 1.4*
(90.5)
100mg/kg 40.5 ± 4.0
22.2 ± 5.6**
(45.3) 13.7 ± 4.3****
(66.3) 9.3 ± 2.5**
(77.0) 10.2 ± 5.0****
(74.8)
150 mg/kg 34.7 ± 5.9
17.7 ± 5.9**
(49.0) 8.7 ± 2.1*
(75.0) 5.0 ± 1.9**
(85.6) 5.5 ± 2.8**
(84.1)
200 mg/kg 24.2 ± 3.0
11.0 ± 1.5*
(54.5) 1.8 ± 1.5**
(92.4) 1.2 ± 0.7**
(95.2) 2.0 ± 0.8**
(91.7)

Values are presented as Mean ± SEM. Number of animals in each group (n) = 6. Statistical analysis done using two-way ANOVA followed by Tukey's multiple comparisons test.
* p< 0.05, ** p< 0.01, *** p< 0.001, **** p < 0.0001.

Thiopentone Induced Sleep Test: The formulation of daidzein-corn oil and daidzein microemulsion reduced the time required for the onset of thiopentone-induced sleep and increased the duration of sleep, compared to the control group (Table 5 and 6). This demonstrates the hypnotic effect of daidzein-corn oil formulation and daidzein microemulsion.

Table 5: Effect of daidzein-corn oil formulation on sleep onset and duration.
Treatment Dose Onset of sleep
(min) Duration of sleep
(min)
Control (Corn oil) 0.1 ml/mouse 2.333 ± 0.21 7.0 ± 0.63
Diazepam 1 mg/kg 2.167 ± 0.40 60.0±21.19*
Daidzein-corn oil formulation 200 mg/kg 1.667 ± 0.33 24.0 ± 2.1
400 mg/kg 1.167 ± 0.16* 35.83 ± 8.9*
800 mg/kg 1.167 ± 0.16* 23.67 ± 5.4

Values are presented as Mean ± SEM. Number of animals in each group (n) = 6. Statistical analysis done using Kruskal-Wallis non-parametric test followed by Dunn's multiple comparisons test. * p < 0.05 significant

Table 6: Effect of daidzein microemulsion on sleep onset and duration.

Treatment Dose Onset of Sleep (min) Duration of Sleep (min)
Control (pseudo microemulsion) 0.1 ml/mouse 3.5 ± 0.24 6.3 ± 0.78
Diazepam 1mg/kg 2 ± 0.28*** 56.8 ± 3.9****
Daidzein microemulsion 50mg/kg 1.7 ± 0.23**** 16.5 ± 2.20**
100mg/kg 1.7 ± 0.23**** 25.5 ± 1.9****
150mg/kg 1.8 ± 0.33*** 34.3 ± 1.0****



Values are presented as Mean ± SEM. Number of animals in each group (n) = 6. Statistical analysis done using one way ANOVA followed by Dunnett's multiple comparisons test.
** p< 0.01, *** p< 0.001, **** p < 0.0001.

Mechanism of Action Study: The action of daidzein as a sedative-hypnotic mediated via GABAA receptor (benzodiazepine binding site) was confirmed using selective benzodiazepine antagonist flumazenil using the locomotor activity test and hole board test. The results depicted reversal of sedation after flumazenil administration at 75 minutes after administration of daidzein-corn oil formulation by decreasing the inhibition of locomotor activity and increasing the number of head dips, thus concluding the action of daidzein mediated via benzodiazepine site of GABAA receptor (Table 7 and 8). However, the results were not statistically significant as the dose of daidzein is very high (400 mg/kg) as compared to diazepam (1 mg/kg) to be reversed by flumazenil (5 mg/kg) as it is a competitive antagonist of benzodiazepine site of GABAAreceptor.

Table 7: Effect of daidzein-corn oil followed by flumazenil on locomotor activity in mice.
Treatment Dose Pretreatment Locomotor Activity Counts
(% inhibition)
30 min 60 min 80 min 90 min 105 min
Control
(corn oil) 0.1 ml/mouse 341 ± 36.7** 187.2 ± 37.8 (45.1) 151.5 ± 24.0
(55.6) 200.8 ± 22.5
(41.1) 128.5 ± 13.8
(62.3) 120.7 ± 13.8
(64.6)
Diazepam 1 mg/kg 213.2 ± 35.3* 127.7 ± 14.4**
(40.1) 57.3 ± 5.1
(73.1) 113.3 ± 14.7*
(46.8) 72.8 ± 8.7
(65.8) 51.2 ± 8.8
(76.0)
Daidzein-corn oil formulation 400 mg/kg 287.2 ± 38.9* 152.8 ± 21.7*
(46.8) 90.8 ± 29.5
(68.4) 128.8 ± 31.1
(55.1) 90.3 ± 27.7
(68.5) 75.0 ± 24.4
(73.9)

Values are presented as Mean ± SEM. Number of animals in each group (n) = 6. Statisticalanalysis done using two-way ANOVA followed by Dunnett's multiple comparisons test.
* p< 0.05, ** p< 0.01.

Table 8: Effect of daidzein-corn oil followed by flumazenil on number of head dips in mice.
Treatment Dose Pretreatment Number of Head Dips
(% inhibition)
35 min 85 min
Control
(corn oil) 0.1 ml/mouse 25.8 ± 5.3 5.5 ± 0.8
(78.7) 10.7 ± 3.1
(58.5)
Diazepam 1mg/kg 20.2 ± 3.0 5.2 ± 1.9
(74.4) 10.3 ± 2.4
(48.8)
Daidzein-corn oil formulation 400 mg/kg 20.2 ± 3.5 3.7 ± 1.4
(81.8) 6.2 ± 1.4
(69.4)

Values are presented as Mean ± SEM. Number of animals in each group (n) = 6. Statisticalanalysis done using two-way ANOVA followed by Dunnett's multiple comparisons test.

It was observed that control group mice (received corn oil/pseudo microemulsion without daidzein (placebo)) showed significant sedative activity compared to pretreatment. However, the locomotor activity, head dips and sleep duration in control group mice were non-significant compared to diazepam or daidzein formulation treated animals.

It is understood that the above description is merely exemplary of the invention and that variations may be made by those skilled in the art without departing from the spirit and scope of the invention. All such modifications and variations are intended to be included within the scope of the appended claims.



, Claims:We Claim:

1] A Daidzein Microemulsion for inducing sedation and hypnosis effects comprising Daidzein & other excipients and pharmaceutically acceptable carrier.
2] A pharmaceutical formulation of claim 1 consisting of Daidzein with other excipients like Tween 80, PEG 400 & soyabean oil in specific proportion. The proportion of the incorporated ingredients can be varied to get stable microemulsion with enhanced drug release & required particle size.
3] A pharmaceutical formulation as claimed in claim 1 wherein the formulation is in the form of solution or emulsion or gel.
4] A pharmaceutical formulation as claimed in claim 1 wherein the formulation is in the form of microemulsion. It can be prepared by using various pharmaceutically acceptable oils, surfactants, co - surfactants and other excipients.
5] A pharmaceutical formulation as claimed in claim 1 wherein the formulation is for oral route of administration or intravenous administration or intra-muscular administration or nasal administration or rectal route of administration.
6] A process for preparation of formulation of Daidzein Microemulsion is by phase titration method or any other pharmaceutically acceptable method(s) to deliver desired dose of 25mg/kg, 50mg/kg, 100mg/kg, 150mg/kg & 200mg/kg depending on body weight.

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