Development and characterization of morin hydrate loaded microemulsion for the management of Alzheimer’s disease

Figures & data

Figure 1. Experiment procedure and treatment schedule. CI: cannula implantation; ICV-STZ: intracerebroventricular streptozotocin; ORT: object recognition task; MWM: Morris water maze.

Table 1. Solubility of morin hydrate in oils, surfactants and co-surfactants.

Type	Vehicle	Solubility (mg/ml)
Oil	Capmul MCM	14.7 ± 0.58
Oil	Olive oil	4.2 ± 0.16
Oil	Castor oil	2.8 ± 0.11
Oil	Oleic acid	3.1 ± 0.12
Oil	Isopropyl myristate	2.1 ± 0.08
Surfactant	Cremophor EL	10.5 ± 0.42
Surfactant	Span 20	5.2 ± 0.20
Surfactant	Tween20	3.6 ± 0.14
Surfactant	PEG-400	9.6 ± 0.38
Surfactant	PG	6.3 ± 0.25
Surfactant	Labrasol	1.7 ± 0.06

Bold Values Represents that Morin Hydrate is highly Soluble in Oil Capmul MCM and in surfactant Cremophor EL, PEG-400, respectively.

Figure 2. Pseudoternary phase diagram with varying ratios of the investigated quaternary system water/Cremophor EL/PEG-400/Capmul MCM with (a) Smix in the ratio of 5:5, (b) Smix in the ratio of 6:4, (c) Smix in the ratio of 9:1, (d) Smix in the ratio of 7:3, and (e) Smix in the ratio of 8:2. The shaded areas indicate the clear w/o microemulsion system. The phase study revealed that the maximum emulsification region was found to be 7:3 surfactant/co-surfactant ratio.

Table 2. Composition of optimized blank microemulsion.

Category	Name	% w/w
Oil	Capmul MCM	60
Aqueous phase	Water	10
Surfactant	Cremophor EL	21
Co-surfactant	PEG-400	9

Table 3. Physicochemical properties of morin hydrate-loaded microemulsion.

Formulation	Mean diameter size (nm)	Polydispersity index	Zeta potential (mV)	Viscosity (Pa.s)	pH	Solubility (mg/ml)
F1	203.4 ± 5.1	0.312 ± 0.05	−9.23 ± 0.9	18.61 ± 0.4	7.3	–
F2	227.5 ± 6.8	0.323 ± 0.09	−10.61 ± 0.31	22.81 ± 0.6	6.1	9.04 ± 0.36

Figure 3. Size and size distribution images of optimized morin hydrate loaded microemulsion. Globule size and polydispersity index of optimized (F2) formulation were found to be 227.5 and 0.323 nm, respectively.

Figure 4. Zeta potential of optimized morin hydrate loaded microemulsion. Zeta potential of optimized morin loaded microemulsion (F2) was found to be (−10.61 mV).

Figure 5. Percentage in vitro release profile of morin hydrate loaded microemulsion. Each point represents mean ± SD (n = 3). The release profile of formulation (F2) showing 94.05% release in 240 min.

Table 4. Physical stability studies of morin loaded microemulsion.

	Optimized of microemulsion after 3 months of stability study
Tests	Initial	4 ± 2 °C	25 ± 2 °C
Drug content	98.2 ± 0.4	92.34 ± 0.02	91.95 ± 0.04
% Transmittance	95.03 ± 2	92.90 ± 0.5	90.20 ± 0.6
Particle size (nm)	227.5 ± 0.02	245.3 ± 0.05	256.5 ± 0.03

Figure 6. Effect of morin hydrate on memory performance in object recognition test in ICV-STZ infused rats (a) acquisition phase, trail-1 and (b) retention phase, trail-2. Values are expressed as ^aP < 0.05 versus sham control, ^bP < 0.05 versus STZ-; ^cP < 0.05 versus STZ + morin hydrate.

Figure 7. Effect of morin hydrate loaded microemulsion (ME) on mean latency in Morris water maze (MWM) task in ICV-STZ infused rats. ^aP < 0.05 versus sham control; ^bP < 0.05 versus STZ; ^cP < 0.05 versus STZ + morin hydrate.

Table 5. Various parameters of oxidative stress.

Treatment	MDA (nmol of MDA/mg protein)	GSH (μmol of GSH/mg protein	Nitrite (μg/ml)	Acetylcholinesterase (μM/mg protein)
Sham control	0.216 ± 0.136	0.0324 ± 0.0062	72.17 ± 23.57	0.097 ± 0.04
STZ	4.785 ± 0.108^a	0.0020 ± 0.0008^a	320 ± 47.0.5^a	0.357 ± 0.03^a
STZ + morin hydrate	3.218 ± 0.184^b	0.0204 ± 0.0051^b	220.8 ± 29.60^b	0.243 ± 00.56^b
STZ + morin hydrate ME	1.819 ± 0.121^b^,^c	0.0305 ± 0.0056^b^,^c	160.2 ± 26.17^b^,^c	0.121 ± 0.03^b^,^c

^a P < 0.05 versus sham control.

^b P < 0.05 versus STZ.

^c P < 0.05 versus STZ + morin hydrate.

Table 6. Concentration of drug reaching the blood at different time intervals.

Time (h)	Conc. of morin hydrate(plain) (μg/ml)	Conc. of morin loadedmicroemulsion (μg/ml)
1	0.001 ± 0.0002	0.004 ± 0.001
2	0.0013 ± 0.0001	0.0072 ± 0.0002
3	0.0023 ± 0.00021	0.0094 ± 0.00015
4	0.0010 ± 0.00019	0.0073 ± 0.00015
5	0.0007 ± 0.000208	0.0042 ± 0.000204

Figure 8. Overlay chromatogram of morin hydrate in plasma.

Figure 9. Standard curve of morin hydrate in plasma.

Figure 10. Concentration of morin hydrate in brain versus time for plain morin hydrate (IN) and morin hydrate loaded microemulsion (IN).