Potentiation of nootropic activity of EGCG loaded nanosuspension by piperine in swiss male albino mice

Accepted Manuscript Potentiation of nootropic activity of EGCG loaded nanosuspension by piperine in Swiss male albino mice Shakti Dahiya, Ruma Rani, Dinesh Dhingra, Sandeep Kumar, Neeraj Dilbaghi PII:

S2314-7245(18)30009-8

DOI:

https://doi.org/10.1016/j.fjps.2018.10.005

Reference:

FJPS 83

To appear in:

Future Journal of Pharmaceutical sciences

Received Date: 11 January 2018 Revised Date:

12 October 2018

Accepted Date: 31 October 2018

Please cite this article as: Dahiya S, Rani R, Dhingra D, Kumar S, Dilbaghi N, Potentiation of nootropic activity of EGCG loaded nanosuspension by piperine in Swiss male albino mice, Future Journal of Pharmaceutical sciences, https://doi.org/10.1016/j.fjps.2018.10.005. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title Page The name(s) of the author(s) - Shakti Dahiya, Ruma Rani, Dinesh Dhingra, Sandeep Kumar, Neeraj Dilbaghi A concise and informative title - Potentiation of nootropic activity of EGCG loaded

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nanosuspension by piperine in Swiss male albino mice The affiliation(s) and address (es) of the author(s) – 1.

Shakti Dahiya, PhD Scholar, Department of Bio and Nano Technology, Guru Jambheshwar University of Science & Technology, Hisar-125001.

Ruma Rani, PhD Scholar, Department of Bio and Nano Technology, Guru

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2.

Jambheshwar University of Science & Technology, Hisar-125001.

Dinesh Dhingra, Professor, Department of Pharmaceutical Sciences, Guru

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3.

Jambheshwar University of Science & Technology, Hisar-125001. 4.

Sandeep Kumar, Assistant Professor, Department of Bio and Nano Technology, Guru Jambheshwar University of Science & Technology, Hisar-125001.

5.

Neeraj Dilbaghi, Professor, Department of Bio and Nano Technology, Guru

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Jambheshwar University of Science & Technology, Hisar-125001.

The email address, and telephone number(s) of the corresponding authorProf. Neeraj Dilbaghi,

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Department of Bio and Nano Technology,

Guru Jambheshwar University of Science & Technology, Hisar-125001.

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E-mail: [email protected] Tel. 91-1662-263500, Fax. 91-1662-276240

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Potentiation of nootropic activity of EGCG loaded nanosuspension by piperine in Swiss male albino mice

Abstract

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The present study deals with preparation of nanoformulations of epigallocatchin gallate (EGCG) alone and with piperine, and evaluation of in vivo cognitive effect on Swiss albino male mice. EGCG (25 mg) was nanoencapsulated into a biocompatible and biodegradable protein nanocarrier, zein and was also separately nanoencapsulated with piperine (5 mg), a bioenhancer.

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Anti-solvent precipitation method was used for formulation of EGCG loaded nanosuspension and EGCG-Piperine nanocomplex. EGCG loaded nanosuspension (25 mg kg-1) and EGCG-

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Piperine nanocomplex (25 mg kg-1) were administered per se to mice for 3 successive weeks. The behaviour models i.e. Elevated plus maze and Morris water maze were used for assessing the cognitive behaviour. The influence of various drug treatments on locomotor activity of mice was also studied. The mice were sacrificed on 21st day followed by estimation of brain acetylcholinesterase activity. The significant reversion in scopolamine- induced amnesia in mice was observed after administration of EGCG loaded nanosuspension (25 mg kg-1) and EGCG-

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Piperine nanocomplex (25 mg kg-1). A significant reduction in brain acetylcholinesterase level in mice was observed after the administration of physostigmine (0.1 mg kg-1), EGCG loaded nanosuspension (25 mg kg-1) and EGCG-Piperine nanocomplex (25 mg kg-1). The oral administration of EGCG loaded nanosuspension for three successive weeks significantly

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improved cognitive behaviour in mice; and piperine potentiated the cognitive effect of EGCG loaded nanosuspension. Further, EGCG loaded nanosuspension showed improved cognitive

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behaviour in mice probably through inhibition of brain acetylcholinesterase activity, and facilitation of cholinergic pathways.

Keywords: Polyphenol; Nanoencapsulation; Zein; Bioenhancer; Nootropic

1. Introduction Learning is acquisition of new information whereas memory is a phenomenon of encoding, storage and retrieval of that acquired information. According to World Health

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Organization’s International Classification of Diseases (ICD-10) dementia is a spectrum of chronic progressive disorders which produces psychosocial or occupational impairment due to the dysfunctioning in one or more domains of higher cortical such as: thinking, memory, comprehension, orientation, calculation, language, learning capacity and judgment [1]. The

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decline in person’s memory is one of the most common symptoms associated with Alzheimer’s disease (AD) and it is anticipated that number of people suffering from dementia will be more than 115 million by 2050 [2]. AD may also result from the oxidative stress, reduction in activity of cholinergic neurons and accumulation of amyloid-β proteins in brain [3-5]. The cholinergic

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system in brain is playing an important role in the storage and retrieval of memory and its rate of degradation shows a direct relationship with the severity of memory impairment.

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Acetylcholinesterase (AChE), an enzyme responsible for metabolism of brain acetylcholine and hence it is assumed that inhibition of AChE elevates acetylcholine level in brain and is one of key strategies used for the treatment of AD [6]. The imbalance between the antioxidants and free radicals generation is also considered one of the causes responsible for occurrence of AD [7]. A number of anti-cholinesterase drugs like donepezil, galantamine, rivastigmine and tacrine have been reported to be used for treatment of cognitive disorders. However, these drugs have side

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effects such as anorexia, nausea, diarrhea vomiting, and insomnia [8, 9]. Physostigmine, a popular cholinesterase inhibitor has been reported to improve memory of human beings, but short half-life and variable bioavailability narrows its therapeutic index [10]. Epigallocatchin gallate (EGCG), a polyphenol found mainly in green tea, has gained a lot

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of importance due to its potent antioxidant, anti-inflammatory, anti-microbial and anticancer activities [11-14]. EGCG has also been reported to slow-down or prevent AD and enhance

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learning and memory by reduction of oxidative stress [15]. Ferrous ion (Fe2+) is a cofactor responsible for the functioning of many proteins involved in neuronal function. Fenton reaction catalyzes the reduction of ferric (Fe3+) to ferrous (Fe2+) in brain. Increase in Fe2+ concentration occurs in some areas of brain during aging which is the key factor responsible for neurodegenerative diseases (Alzheimer’s, Parkinson’s and Huntington’s disease). The Fe2+ accumulation leads to ROS generation which abolishes neurons activity. EGCG, a strong radical scavenger scavenge the ROS and also chelates divalent transition metal ions. The property of chelation of divalent metals inhibits the accumulation of Fe2+ ions in neurons, thereby stops the Fe2+ mediated DNA damage [16]. EGCG undergoes methylation, glucuronidation and sulfation 2

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in gastro-intestinal tract, which limits its in vivo bioavailability and inefficient systemic delivery [17, 18]. Piperine, a dietary modulator, prevents the glucuronidation of EGCG in small intestine, resulting in increased absorption. Piperine also slows down the release of EGCG from gastrointestinal transit, which increases its absorption rate in intestine by increasing its residence

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time in intestine [19].

To attain maximum outcome from a neuroprotective drug, novel approaches which enhance the oral bioavailability of herbal bioactive compounds are required. Now a day, various novel approaches have greatly established for the enhancement of bioavailability and therapeutic

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effect of herbal compounds [20-22]. The use of nanoencapsulation technique for enhancement of oral bioavailability of herbal compounds has attracted substantial attention [19]. Numerous

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nanoformulations of EGCG using various biodegradable polymeric nanocarriers have been reported which enhanced its in vitro as well as in vivo oral bioavailability [23-26] but EGCG nanoformulations using zein as a nanocarrier in combination with piperine have not yet been studied. Zein, a nanocarrier used in the present study is water insoluble protein which gained enormous attention as drug delivery carrier due to its desirable attributes such as biocompatibility, biodegradability, hydrophobicity, unique solubility, low toxicity and it’s

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generally recognized as safe (GRAS) status for human applications by the Food and Drug Administration (FDA). In addition, zein complexes with other compounds quite easily, which make it’s a reliable candidate for drug delivery [27]. Thus, the present study was undertaken to prepare EGCG loaded nanosuspension as well as EGCG-Piperine nanocomplex using zein as a

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nanocarrier, and evaluation of their effect on cognitive behaviour of mice using behavioral models. The outcome of various drug treatments on acetylcholinesterase activity of brain in mice

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was also estimated.

2. Materials and Methods 2.1. Materials

Epigallocatechin gallate, piperine (MP Biomedicals Private Limited, Mumbai, India), zein, scopolamine hydrobromide (Sigma Chemicals Co., St. Louis, MO, USA), Lecithin, Tween -80, physostigmine, 5, 5 -dithiobis-2-nitrobenzoic acid and acetylthiocholine iodide (Hi-Media

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Lab Pvt. Ltd., Mumbai, India) were used in current study. Analytical grade chemicals were used other than these chemicals.

2.2.

Methods

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2.2.1. Preparation of EGCG loaded nanosuspension and EGCG-Piperine nanocomplex

The EGCG loaded nanoparticles encapsulated into zein nanocarrier were prepared by anti-solvent precipitation method [28]. The organic phase containing zein (polymer) and lecithin (stabilizer) was dissolved in 70% (v/v) ethanol and filtered by 0.22-µm micro filter for

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elimination of impurities. The antisolvent phase contains tween-80 dissolved in double distilled water. The immediate addition of organic phase into anti-solvent phase under vigorous stirring at

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1200 rpm resulted into the formation of blank nanoparticles (Fig. 1).

The similar procedure used for the synthesis of blank nanoparticles was followed for the formation of drug loaded nanoparticles. The bioactive compound, EGCG (25mg) and EGCG + piperine (25 mg + 5 mg) were dissolved in the organic phase which lead to the formation of EGCG loaded nanosuspension and EGCG-Piperine nanocomplex respectively. The prepared nanosuspensions were lyophilized in laboratory model freeze dryer (Freezone 6-Plus Labconco,

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USA) using 1% (w/v) mannitol as cryoprotectant for long term stability of nanoparticles. The synthesized nanoformulations were evaluated for various physiochemical characterization techniques such as particle size, polydispersity index and zeta potential.

In vivo studies on cognitive effects

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2.3.

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2.3.1. Experimental animals

Swiss albino male mice (weight ˜ 25– 30 g; 3 months old) were obtained from Disease

Free Small Animal House, LUVAS, Hisar, Haryana, India. Six mice were sheltered per cage (cage size: 29 cm × 22 cm × 14 cm) under standard environmental conditions with interchanging 12-h light/dark cycle. The animals were freely accessible to food and water throughout the study. The experimental procedure was approved by Institutional Animals Ethics Committee (IAEC) in its 30th meeting held on 12th August, 2016 (Endst. No. IAEC/2016/10-17, dated 5-9-2016). The 4

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maintenance of animals was done by following the rules made by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment, Forests and Climate Change, Government of India, New Delhi (Registration no.

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0436/PO/Re/S/2001).

2.4. Behavioral models employed for evaluation of effects of drugs on cognitive effects in mice 2.4.1. Elevated Plus Maze

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The effects of drugs on cognitive behaviour in mice was evaluated by employing elevated plus maze model as described earlier [29, 30]. Elevated plus maze made of two open (16 cm × 5cm) and two closed arms (16 cm × 5 cm × 15 cm) elongated from a central platform (5cm× 5

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cm) and raised to a height of 25 cm from the ground was employed. During 1st day of experiment, every single mouse was kept at open arm end diverting out of way from central platform followed by recording transfer latency (TL). The time taken by mice to move from open arms to one of the closed arms using its four paws in the closed arm is termed as TL. TL was noted on 1st day (i.e., 20th day of drug administration) for each animal. The TL was recorded 90

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sec, if the mice didn’t enter into one of the closed arm within 90 sec than it was pushed gently into any closed arms. The mice were permitted to walk around for another 2 minutes into the maze followed by returning to its home cage. The retention of learned-task (memory) was

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evaluated 24h (i.e. on 21st day of drug administration) after performing the acquisition trial.

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2.4.2. Morris Water Maze

The procedure for testing cognitive behaviour of mice using Morris water maze were

same as described earlier [31, 32]. The water maze comprised of a rounded pool (60 cm × 25 cm) filled with water maintained at 25◦C to 20 cm depth made opaque using milk powder. The water tank was split into four equal quadrants using two threads erected at right angle to each other. A submerged platform (white, top surface of 6 cm × 6 cm) was kept inside the target quadrant (Q4 in current study) 1 cm below the surface of water in the pool. During whole training session, platform was retained at the same position. Four successive trials were performed on each mouse after 5 min time gap for four successive days (i.e. from 17th day of drug administration to 20th 5

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day), in which mice were permitted to locate the hidden platform and remain there for 20s. During each training session, the mice was gently released in water facing the pool wall by changing drop location at each trial followed by locating allowed submerged platform in 120 sec. If the mice were unable to locate platform in 120 s, it was gently directed towards platform and

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permitted for 20 s to remain there. Time taken by each mouse for locating the hidden platform in target quadrant from the starting quadrant is defined as escape latency (EL). EL was recorded on 17th, 18th, 19th and 20th days. Each mouse was exposed to four training trials for four successive days by changing the starting point after every trial as described below and position of the target

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quadrant (Q4 in current study) remained same during the training period. 1st Day Q1 Q2 Q3 Q4 2nd Day Q2 Q3 Q4 Q1

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3rd Day Q3 Q4 Q1 Q2

4th Day Q4 Q1 Q2 Q3

On 5th day (i.e., 21st day of drug administration), hidden platform was withdrawn from the pool and animal was gently dropped into any of the three quadrants and were permitted to locate target quadrant within 300s. The time taken by mouse to find hidden platform in target

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quadrant is considered as an index of retrieval or memory. The position of the observer and water maze remains same throughout the study.

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2.4.3. Estimation of Locomotor Activity

In order to evaluate the effects of physostigmine, EGCG, EGCG loaded nanosuspension,

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EGCG-Piperine nanocomplex, blank nanosuspension and piperine on locomotor activity, number of horizontal locomotor activity of test and control mice were recorded for 5 min using Medicraft Photoactometer (Model number 600-4D, INCO, Ambala, India).

2.5.

Experimental Design

2.5.1. Groups for Elevated Plus Maze (EPM) Group 1 to 7: Normal saline (10 ml kg-1), physostigmine (0.1 mg kg-1), EGCG (25 mg kg-1), EGCG loaded nanosuspension (containing 25 mg kg-1 of EGCG), EGCG-Piperine nanocomplex 6

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(containing 25 mg kg-1 of EGCG and 5 mg kg-1 of piperine), blank nanosuspension (25 mg kg-1) and piperine (5 mg kg-1) respectively were administered intraperitoneally (i.p) for 3 successive weeks. After 30 minutes of 20th day of drug administration (learning) transfer latency (TL) was recorded and followed by evaluating retention on 21st day i.e. after 24 h of learning trial. On 21st

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day, 1h later to elevated plus maze, mice were examined for locomotor activity.

Group 8 to 11: Normal saline (10 ml kg-1), EGCG (25 mg kg-1), EGCG loaded nanosuspension (containing 25 mg kg-1 of EGCG) and EGCG-Piperine nanocomplex (containing 25 mg kg-1 of

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EGCG and 5 mg kg-1 of piperine) respectively were administered intraperitoneally (i.p) for 3 successive weeks. On 20th day, TL was recorded after 45 min of drugs administration. On the

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21st day, scopolamine (0.4 mg kg-1, i.p.) was administered after 30 min inoculation of normal saline, EGCG, EGCG loaded nanosuspension and EGCG-Piperine nanocomplex respectively according to protocol of previous studies [30, 33, 34].. After 45 min of scopolamine injection TL was recorded.

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2.5.2. Groups for Morris Water Maze

Group 12 to 18: Normal saline (10 ml kg-1), physostigmine (0.1 mg kg-1), EGCG (25 mg/kg), EGCG loaded nanosuspension (containing 25 mg kg-1 of EGCG), EGCG-Piperine nanocomplex (containing 25 mg kg-1 of EGCG and 5 mg kg-1 of piperine), blank nanosuspension (25 mg kg-1)

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and piperine (5 mg kg-1) respectively were administered intraperitoneally for 3 successive weeks. After 45 min of drug administration, escape latency (EL) was recorded from 17th day to 20th day.

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On 21st day, time spent in target quadrant (TSTQ) was recorded 45 min after drug administration.

Group 19 to 21: Normal saline (10 ml kg-1), EGCG (25 mg kg-1), EGCG loaded nanosuspension (containing 25 mg kg-1 of EGCG) and EGCG-Piperine nanocomplex (containing 25 mg kg-1 of EGCG and 5 mg kg-1 of piperine) respectively were administered intraperitoneally for 3 successive weeks. After 45 min of drug administration, escape latency (EL) was recorded from 17th day to 20th day. On 21st day, scopolamine (0.4 mg kg-1, i.p.) was inoculated 30 min after administration of normal saline, EGCG, EGCG loaded nanosuspension and EGCG-Piperine 7

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nanocomplex respectively according to protocol of previous studies [30, 33, 34]. The time spent in target quadrant (TSTQ) was recorded 45 min after inoculation of scopolamine.

2.6.

Biochemical Estimation

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2.6.1. Brain sample collection and assessment of brain acetylcholinesterase activity

After behavioral testing of mice of groups 1 to 7 on 21st day, animal’s sacrification was performed by giving light anesthesia to animal using diethylether followed by their cervical

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dislocation. The whole brain of animal was removed very carefully, weighed and homogenized in phosphate buffer (pH 8, 0.1 M) in an ice bath. The obtained homogenate was centrifuged at

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3000 rpm for 10 min at 4oC using refrigerated centrifuge (C-30 Plus, Remi Centrifuge) and cloudy supernatant was used for evaluation of brain acetyl cholinesterase activity [35]. The resultant supernatant (0.4 mL) was taken into a test tube containing phosphate buffer (2.6 ml) followed by adding 0.1 mL of 5, 5-dithiobis-2-nitrobenzoic acid (DTNB) reagent and absorbance was recorded at 412 nm via UV-Vis Spectrophotometer (SPECTROstar® Nano, BMG LABTECH, Germany). Acetylthiocholine iodide (0.02 mL) solution was added followed by

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measuring the absorbance again after 15 min. The change in absorbance was noted every minute. The hydrolysis rate of substrate was estimated using formula: R = change in absorbance/min×5.74 × 10−4/C0, Where, R = rate of hydrolysis of acetylthiocholine iodide/min/mg tissue,

Statistical analysis

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2.7.

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C0 =weight of tissue homogenate in mg/mL.

The data analysis was performed by analysis of variance (ANOVA) and Tukey–Kramer

multiple comparison test in Graph Pad Instat package, version 3.1 and expressed as mean ± SEM and. p< 0.05 was considered as statistically significant. Statistical significance of existing experimental data was measured at confidence level of 0.05.

3. Results 3.1.

Preparation of EGCG loaded nanosuspension and EGCG-Piperine nanocomplex

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EGCG- loaded nanosuspension and EGCG- Piperine nanocomplex using protein nanocarrier were prepared using anti-solvent precipitation method and optimized using full factorial design expert software (Version 8.0.7.1, Stat-Ease Inc, Minneapolis, MN). The particle size, polydispersity index and zeta potential of EGCG loaded nanosuspension and EGCG-

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piperine nanocomplex was found to be 118.3 nm, 0.125, -36.4 mV and 184.2 nm, 0.231, -38.3 mV respectively.

3.2.

Behavioral models employed for evaluation of effect of drugs on cognitive behaviour of

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mice

3.2.1. Effect of different drug treatments on transfer latency (TL) of mice employing elevated

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plus maze

Physostigmine (0.1 mg kg-1, i.p.), EGCG loaded nanosuspension (containing 25 mg kg-1 of EGCG, i.p.) and EGCG-Piperine nanocomplex (containing 25 mg kg-1 of EGCG and 5 mg kg1

of piperine, i.p.) administered per se for 3 successive weeks significantly (p<0.01, p<0.01 and

p<0.001 respectively) decreased TL of mice on 20th day in comparison to vehicle treated control,

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indicating improvement of learning. But EGCG, blank nanosuspension and piperine did not show any significant affect on TL of mice on 20th day in comparison to vehicle treated control, indicating their non-significant effect on learning. On analyzing ‘q’ value, the order of improvement of learning was EGCG-Piperine nanocomplex (q=13.143) > physostigmine

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(q=6.303) > EGCG loaded nanosuspension (q=6.035). Physostigmine, EGCG, EGCG loaded nanosuspension and EGCG-Piperine nanocomplex

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significantly (p<0.001, p<0.05, p<0.001 and p<0.001 respectively) declined TL of mice on 21st day (24 h after acquisition trial) in comparison to vehicle treated control, indicating significant improvement in cognitive behaviour. On analyzing ‘q’ value, the order of improvement of cognitive effect was EGCG-Piperine nanocomplex (q=14.958) > physostigmine (q=12.973) > EGCG loaded nanosuspension (q=11.905) > EGCG (q= 5.495). The blank nanosuspension and piperine did not significantly decrease TL of mice on 21st day (24 h after acquisition trial) in comparison to vehicle treated control. Scopolamine (0.4 mg/kg, i.p.) administered 3o min before retention trial significantly (p<0.001) increased TL of mice in comparison to vehicle treated control, demonstrating its amnesic effect. EGCG, EGCG loaded nanosuspension and EGCG9

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piperine

nanocomplex

significantly

(p<0.001)

reversed

scopolamine-induced

memory

impairment in mice as to the scopolamine treated animals. On the basis of ‘q’ value, the order of reversal of scopolamine-induced memory impairment was EGCG-Piperine nanocomplex

-----------Table 1-----------

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(q=20.757) > EGCG loaded nanosuspension (q=11.600) > EGCG (q=10.226) (Table 1).

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(TSTQ) in mice employing Morris Water Maze

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3.2.2. Effect of different treatments on escape latency (EL) and Time spent in Target Quadrant

All drug treatments except piperine and blank nanosuspension significantly declined escape latency on 20th day of drug administration, indicating significant improvement of learning. But physostigmine, EGCG loaded nanosuspension and EGCG-Piperine nanocomplex also significantly (p<0.01, p<0.01 and p<0.001 respectively) declined escape latency on 19th day of drug administration. On the basis of ‘q’ value, the order of significance for improvement of

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learning on 20th day of drug administration was EGCG-Piperine nanocomplex (q=13.217) > EGCG loaded nanosuspension (q=10.590) > EGCG (q=5.500). Physostigmine, EGCG, EGCG loaded nanosuspension and EGCG-Piperine nanocomplex significantly (p<0.001, p<0.05, p<0.001 and p<0.001 respectively) increased TSTQ by mice on 21st day of drug administration

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in comparison to vehicle treated control, indicating significant improvement in cognitive potential. On the basis of ‘q’ value, the order of significance for improvement in cognitive

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behaviour was EGCG-Piperine nanocomplex (q=16.127) > Physostigmine (q= 13.806) > EGCG loaded nanosuspension (q=12.237) > EGCG (q=5.020). The single dosage administration of scopolamine (0.4 mg/kg, i.p.) 30 minutes prior to

recording of TSTQ on 21st day significantly (p<0.05) reduced TSTQ by mice in Morris Water Maze, indicating its amnesic effect. Various treatments such as EGCG, EGCG loaded nanosuspension and EGCG-Piperine nanocomplex for 3 successive weeks significantly (p<0.01, p<0.001 and p<0.001 respectively) reversed scopolamine-induced decrease in TSTQ, indicating significant reversal of scopolamine-induced memory impairment (Table 2). On the basis of ‘q’ value, the order of reversal of scopolamine-induced memory impairment was EGCG-Piperine 10

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nanocomplex (q=15.751) > EGCG loaded nanosuspension (q=12.425) > EGCG (q=6.024). Blank nanosuspension and piperine did not significantly increase TSTQ in comparison to respective vehicle treated control, indicating their non-significant effect on cognition in mice.

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3.2.3. Effect of different treatments on locomotor activity of mice

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But piperine potentiated the cognitive behaviour of EGCG loaded nanosuspension (Table 2).

Physostigmine, EGCG, EGCG loaded nanosuspension, EGCG-Piperine nanocomplex,

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blank nanosuspension and piperine did not reveal any significant change in spontaneous locomotor activity scores of mice in comparison to vehicle treated control (Fig.2).

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3.3. Effect of different treatments on brain acetylcholinesterase (AChE) activity of mice

Administration of physostigmine, EGCG, EGCG loaded nanosuspension and EGCGPiperine nanocomplex for 3 successive weeks resulted in significant (p<0.001, p<0.01, p<0.001, and p<0.001 respectively) decrease in brain AChE activity of mice in comparison to vehicle

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treated control. On analyzing ‘q’ value, the order of significance for decrease of brain acetylcholinesterase activity was EGCG-Piperine nanocomplex (q=12.947) > Physostigmine

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(q=11.206) > EGCG loaded nanosuspension (q=8.921) > EGCG (q=5.549). Blank nanosuspension and piperine did not reveal any significant decrease in brain AChE activity in comparison to control. But piperine potentiated brain acetyl cholinesterase inhibitory activity of EGCG loaded nanosuspension (Fig. 3).

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4. Discussion

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The present study was attempted to examine the effect of EGCG, EGCG loaded nanosuspension and EGCG-Piperine nanocomplex on nootropic potential of normal and cognitive deficit mice. This is the first study demonstrating improvement of cognitive potential in mice by EGCG loaded nanosuspension and indicating superior cognitive effects after

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incorporation of piperine (a bioenhancer). EGCG loaded nanosuspension and EGCG- Piperine nanocomplex using protein nanocarrier were prepared by employing anti-solvent precipitation method. Zein, a corn protein was employed as a nanocarrier due to its non-toxicity, biocompatibility, and biodegradability; and it is generally recognized as safe by FDA for human

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applications [27].

Standard behavioral models like elevated plus maze (EPM) and Morris water maze

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(MWM) were used to validate the effect of various drugs on cognitive behaviour. Elevated plus maze (EPM) is predominantly employed model for estimation of anxiety. Besides this, it has also been used as a model for assessment of learning and memory behaviour in rodents [36]. The decrease in transfer latencies on 1st day and 2nd day (i.e., 24 h later the trial) indicates improvement in cognitive behaviour respectively and vice-versa. Administration of EGCG, EGCG loaded nanosuspension and EGCG-Piperine nanocomplex per se for 3 successive weeks

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significantly improved cognitive potential in mice, as shown by decrease in TL on 21st day of drug administration. EGCG loaded nanosuspension and EGCG-Piperine nanocomplex also significantly enhanced learning of mice, as determined by decline in TL on 20st day of drug administration.

EGCG-Piperine

nanocomplex

produced

better

cognitive

effect

than

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physostigmine and EGCG loaded nanosuspension. Thus, piperine potentiated cognitive effect of EGCG loaded nanosuspension. The cognitive potential of EGCG-Piperine nanocomplex was

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even better that physostigmine, the standard anticholinesterase drug employed. Improvement in cognition behaviour by EGCG is in line with earlier studies [37, 38]. To corroborate the results obtained in elevated plus maze, the second laboratory model,

Morris water maze (MWM) was employed. It is also a commonly used test model for assessing the effect of drugs on cognitive behaviour of rodents. A decline in escape latency at the time of training period and increase in time spent in target quadrant (TSTQ) over retention trial indicates enhancement of cognitive potential respectively. EGCG, EGCG loaded nanosuspension and EGCG-piperine nanocomplex significantly decreased escape latency on 3rd and 4th day of training period and increased TSTQ, indicating their improvement in cognitive behaviour. In this 12

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model also, EGCG-Piperine nanocomplex produced better cognitive effect than physostigmine and EGCG loaded nanosuspension. Thus, piperine potentiated cognitive behaviour of EGCG loaded nanosuspension. No significant effect on locomotor activities of mice after administration of various drug treatments was evident indicating that cognitive behaviour of EGCG, EGCG

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loaded nanosuspension and EGCG-Piperine nanocomplex was specific and not false positive. The regulation in cognitive function is guarded by the central cholinergic system. Cholinergic drugs reversed the effect of amnesia induced by scopolamine in laboratory animals. Scopolamine, a cholinergic muscarinic receptor antagonist, produced cognitive deficit in animals

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and is used as a demonstrative model to study the possible contribution of cholinergic system for memory improving effect of drugs [33, 39]. EGCG, EGCG loaded nanosuspension and EGCG-

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Piperine nanocomplex administered per se for 3 successive weeks markedly antagonized the effect of scopolamine-induced amnesia, indicating that these drugs produced improvement in cognitive behaviour possibly through enhancement of cholinergic activity. EGCG-Piperine nanocomplex produced better reversal of scopolamine-induced memory impairment than physostigmine and EGCG loaded nanosuspension, indicating potentiation of cognition of EGCG loaded nanosuspension by piperine.

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Alzheimer’s disease primarily affects the functioning of cholinergic neurons resulting in decreased activity of acetylcholinesterase [40]. Anti-cholinesterase agents can be employed as one of the strategies for the treatment of dementia. EGCG, EGCG loaded nanosuspension and EGCG-Piperine nanocomplex significantly inhibited brain AChE activity in compassion to

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control. It can be concluded that inhibiting the AChE activity led to rise in brain acetylcholine levels which might be responsible for cognitive effect shown by EGCG, EGCG loaded

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nanosuspension and EGCG-Piperine nanocomplex. Brain acetylcholinesterase inhibiting activity of EGCG-Piperine nanocomplex was better than physostigmine and EGCG loaded nanosuspension. This indicated that piperine potentiated brain acetylcholinesterase inhibiting activity of EGCG loaded nanosuspension. Acetylcholinesterase inhibitory activity of EGCG has been reported in literature [41]. Factors like low bioavailability and short half-life, low penetration into blood–brain barrier of natural polyphenols curtails their therapeutic efficacy [25, 42]. Catechins, a sub-class of polyphenols present in green tea and out of all the catechins, EGCG is least bioavailable [43]. Activity of EGCG was lost as it undergoes many bioconversions like methylation, sulfonation 13

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and glucuronidation in liver and intestine [44]. Therefore, large concentrations of EGCG cannot be attained and retained in plasma and tissues. This narrows the therapeutic potential of EGCG and causes primary hindrance of its application in clinical research. Hence, the use of bioavailability enhancers is one of approach to overcome the limitation associated with low

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bioavailability of EGCG as well as to potentiate its therapeutic utility too.

In the present study, piperine was co-encapsulated along with EGCG to improve its bioavailability. Piperine potentially inhibits the process of glucuronidation in liver and intestine Further, piperine has been known to improve the bioavailability of epigallocatechin-3-gallate by

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inhibiting its glucuronidation and increasing its absorption in small intestine [19]. It was concluded from the current study that encapsulation of EGCG into a protein nanocarrier

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significantly improved the potency of EGCG which increased its bioavailability via enhancing the absorption of drug, as indicated by better cognitive effect of EGCG nanosuspension than EGCG alone. Further, combination of EGCG with a bioenhancer i.e. EGCG-Piperine nanocomplex displayed superior cognitive effect than EGCG nanosuspension and this potentiation effect by piperine might be due to its improvement of bioavailability of EGCG.

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5. Conclusion

The administration of EGCG, EGCG loaded nanosuspension and EGCG-piperine nanocomplex for 3 successive weeks significantly improved cognitive behaviour in mice. Order of improvement in cognitive effect was EGCG-piperine nanocomplex > Physostigmine > EGCG

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loaded nanosuspension>EGCG. The improvement of cognitive potential in mice by EGCG and its nanoformulations might be due to inhibition of brain acetylcholinesterase activity and also

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due to its antioxidant activity. Thus, EGCG-Piperine nanocomplex may be further explored for its efficacy in cure of cognitive disorders.

Acknowledgments

The first author, Shakti Dahiya (IF 130021) thanks Department of Science &

Technology, Government of India for providing financial assistance in the form of “INSPIRE” Fellowship. The authors thank DST-Nanomission for providing infrastructural facility for execution of the study.

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Disclosure The authors report no conflicts of interest in this work.

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ACCEPTED MANUSCRIPT Table 1 Effect of different drug treatments on transfer latency (TL) of mice employing elevated plus maze

Piperine Blank nanosuspension Scopolamine EGCG + Scopolamine on 21st day EGCG loaded nanosuspension + Scopolamine on 21st day EGCG-Piperine nanocomplex + Scopolamine on 21st day

TL Sec (on 21st day) 32.5 ± 0.76 18.3 ± 1.05c 26.5 ± 1.18 a 19.5 ± 1.12c

21.5 ± 0.76c

16.2 ± 1.49c

33.8 ± 0.60 39.7 ± 1.05 36.1 ± 1.30 31.6 ± 1.12a

29.8 ± 0.60 34.5 ± 1.38 42.5 ± 0.76c 31.3 ± 0.84d

29.6 ± 1.30b

29.8 ± 1.45d

19.5 ± 0.76c

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NPs weight equivalent to 25 mg kg-1 of EGCG+ 0.4 mg

TL Sec (on 20th day) 37.8 ± 2.09 30 ± 1.41b 32.1 ± 1.17 30.3 ± 1.41b

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Dose (kg-1) 10 ml 0.1 mg 25 mg NPs weight equivalent to 25 mg kg-1 of EGCG NPs weight equivalent to 25 mg kg-1 of EGCG and 5 mg kg-1 of piperine 5 mg 25 mg 0.4 mg NPs weight equivalent to 25 mg kg-1 of EGCG+ 0.4 mg NPs weight equivalent to 25 mg kg-1 of EGCG+ 0.4 mg

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Treatments Control Physostigmine EGCG EGCG loaded nanosuspension EGCG-Piperine nanocomplex

19.8 ± 0.95d

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n = 6 in each group. Values are interpreted as Mean ± SEM. Data analysis was done by one-way ANOVA and Tukey-kramer multiple comparison test. F (10, 55) = 24.856; p <0.0001 (20th day); F (10, 55) = 55.398; p <0.0001 (21st day); a p < 0.05, bp < 0.01 and cp < 0.001 in comparison to vehicle treated control; d p < 0.001 in comparison to scopolamine treated group

ACCEPTED MANUSCRIPT Table 2 Effect of different drug treatments on escape latency (EL) and time spent in target quadrant (TSTQ) of mice employing Morris water maze Treatments Control

Dose (kg-1)

EL (sec) Day-17

EL (sec) Day-18

EL (sec) Day-19

EL (sec) Day-20

10 ml

108.8± 2.01

99.3 ± 2.85

94.8 ± 3.09 b

TSTQ (sec) Day- 21

90 ± 2.76

119.5±0.76c

0.1 mg

101.5± 2.99

89.8 ± 1.28

79. 8 ±2.36

EGCG

25 mg

102.3± 1.36

92.3 ± 2.23

84.8 ± 2.85

78.8 ±1.40a

96.2 ± 1.30a

EGCG loaded nanosuspension

25 mg kg-1 EGCG

100.5 ±2.01

90.5 ± 2.57

80.8 ± 2.23b

68.5 ±2.19c

115.3± 4.27c

EGCG-Piperine nanocomplex

25 mg kg-1 EGCG

101± 1.63

86.5 ± 1.50

76.5 ± 3.76c

63.2 ±1.74c

125.6± 3.49c

Piperine

5 mg

109.5± 0.76

93.2 ± 0.83

91.8 ± 2.02

88.5 ± 1.71

79.8 ± 0.95

Blank nanosuspension

25 mg

103.8± 0.60

100.7 ± 2.93

93.2 ± 1.92

89.5 ± 1.80

81.3 ± 3.59

Scopolamine

0.4 mg

106.2± 1.35

98.6 ± 3.13

85.7 ± 2.64

79.8 ±2.63a

69.8 ± 3.88d

+

25 mg kg-1 + 0.4 mg

104.8± 0.95

94.5 ± 2.95

86.7 ± 1.91

80 ± 2.24a

85.8 ± 2.57e

EGCG loaded nanosuspension + Scopolamine

25 mg kg-1 + 0.4 mg

103.7± 1.36

92.5 ± 1.73

79.5 ±2.32b

67.5 ±2.20c

102.8± 2.65f

EGCG-Piperine nanocomplex + Scopolamine

25 mg kg-1 + 0.4 mg

103.1± 2.83

87.2 ± 5.18

76.8 ± 1.01c

64.8 ±1.64c

111.7± 1.28f

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Physostigmine

EGCG Scopolamine

62.8 ±1.54

82.8 ± 0.95 c

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n = 6 in each group. Values are interpreted as Mean ± SEM. Data analysis was done by one-way ANOVA and Tukey-kramer multiple comparison test. F (10, 55) = 2.823; p = 0.0067 (17th day); F (10, 55) = 3.063; p = 0.0037 (18th day); F (10, 55) = 6.968; p < 0.0001 (19th day); F (10, 55) = 28.238; p < 0.0001 (20th day); F (10, 55) = 49.846; p <0.0001 (21st day) a p < 0.05, bp < 0.01 and cp < 0.001 in comparison to vehicle treated control; d p < 0.05, ep < 0.01 and fp < 0.001 in comparison to scopolamine treated group

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Fig. 1. Synthesis of zein encapsulated nanoparticles using anti-solvent precipitation

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methodology

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Fig. 2. Effect of various treatments on locomotor activity of mice

n = 6 in each group. Values are expressed as Mean ± SEM. Data were analyzed by one-way

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ANOVA followed by Tukey–kramer multiple comparison test. F (6, 35) = 5.145; p>0.05.

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Fig. 3. Effect of various treatments on brain AChE activity of mice n = 6 in each group. Values are expressed as Mean ± SEM. Data were analyzed by one-way ANOVA followed by Tukey–kramer multiple comparison test. F (6, 35) = 24.430; p <

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0.0001; a p< 0.01 and bp < 0.001 as compared to vehicle treated control.