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ABSTRACT: Ancient Chinese medical literature and independent studies indicate that piperine possess anticonvulsant activity and demonstrates a high degree ...

International Journal of Pharmaceutical Sciences and Nanotechnology Volume 2 • Issue 1 • April – June 2009

Research Paper

Synthesis of Novel Piperonal Derivatives and Evaluation of their Anticonvulsant Activity using A Nanoparticular Formulation *Jithan Aukunuru, Keerthana Eedula, Venkanna Pasham, Venumadhav Katla and Srinivas Reddy K Vaagdevi College of Pharmacy, Ramnagar, Hanamkonda, Warangal, 506001

ABSTRACT: Ancient Chinese medical literature and independent studies indicate that piperine possess anticonvulsant activity and demonstrates a high degree of safety. A similarity was also demonstrated with synthetic piperine derivatives as well. In this study, chalcones and Schiffs bases of piperonal with different any methyl ketones, and aromatic primary amines were synthesized using conventional and microwave-irradiation methods. Purification of these compounds was effected by recrystallization from alcohol and characterized by NMR and IR spectra. The synthesized compounds were screened for their anticonvulsant activity using a 4-aminopyridine induced model with the help of a conventional and a current nanoparticular formulation approach. A drug delivery system methodology in the form of nanoparticles for screening was adopted because it has more advantages when compared to conventional formulations in drug discovery stages. Nanoparticle formulations encapsulating selected synthesized compounds were prepared using solvent evaporation technique by taking polycaprolactone as the polymer. Few piperonal synthesized derivatives demonstrated anticonvulsant activity which was lesser than the standard phenytoin. Unfortunately, nanoparticular formulations prepared in this current study encapsulating selected synthesized compounds did not show any activity, suggesting more work is needed to demonstrate positive results.

KEYWORDS: piperonal derivatives, anticonvulsants, nanoparticles, screening, characterization, synthesis Introduction Epilepsy characterized by periodic and unpredictable occurrences of seizures is currently one of the most common disorders of the brain, affecting about 20 lakhs individuals worldwide (Mbuba CK et al., 2008). This is a chronic and progressive disorder caused by abnormal discharges of cerebral neurons. To treat epilepsy, several drugs which are called as anticonvulsants (also called antiepileptics) are available in the market. The goal of therapy with an anticonvulsant agent is to have the patient seizure free with out interfering with normal brain function (Pal SK et al., 2008). Thus, the selection of an anticonvulsant agent is primarily based on its efficacy for specific types of seizures and epilepsy. A significant proportion of patients with epilepsy suffer from intractable or drug resistant epilepsy, despite early treatment and an optimum daily dosage of an appropriate anticonvulsant agent (Pugh MJ et al., 2008). Some of the anticonvulsant drugs have significant side-effects. Thus, there is a need *For correspondence: Dr. Aukunuru Jithan, E-mail: [email protected]

for new drugs with a greater benefit as related to specificity, side effects and tolerability, even at the expense of efficacy, when compared to the existing drugs. As this quest continues, in this study we aimed at the synthesis of some novel piperine derivatives and their evaluation for the activity using conventional and nanoparticular approaches. Hooge et al., in 1996 reported anticonvulsant activity of piperine on seizures induced by excitatory amino acid receptor agonists (D’Hooge R et al., 1996). Piperine blocks convulsions induced using Kainate, but not using glutamate, NMDA or guanidinosuccinate. Further, epilepsy is treated in Chinese folk medicine with raddish and pepper, the main sources of piperine (Pie YQ et al., 1980; Pie YQ, 1983; Mori A et al., 1985). Several piperine derivatives also demonstrated significant anticonvulsant activity with a marginal safety (Pie YQ, 1983). The derivatives aimed for synthesis and screening in this study are piperonal derivatives. Piperonal, a naturally occurring derivative of piperine compound, is an aromatic aldehyde and thus, modifications

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in piperonal could produce compounds which have the same actions of piperine. Several groups previously reported the synthesis and pharmacological activities of piperonal derivatives. Shulgin A and Shulgin A (1991) reported the synthesis of 3,4 – methylenedioxyphenyl -2nitro propene from piperonal in presence of nitroethane and sodium hydroxide. Pavia DL et al., (1990) reported aldol condensation reaction of piperonal and acetophenone in the presence of ethanol and sodium hydroxide. Echevarria A et al. (1991) reported NMR spectroscopy, Hammett correlations and antimicrobial activity of some Schiffs bases derived from piperonal. They synthesized and characterized seven Schiff’s bases from piperonal wing different aromatic primary amines compounds. Their biological activity against microorganisms noted significant activity against Epidermophyton floccosum. The choice of piperonal for the aldehyde moiety not only stemmed from the fact that piperine like compounds could have significant anticonvulsant activity but also from the fact that many compounds containing methylenedioxy group have a variety of biological activities. Taking this into consideration it was felt worth while to synthesize piperonal derivatives and explore them for anticonvulsant activity. Application of drug delivery systems in the discovery stages is slowly gaining prominence (Aukunuru J, 2007; Shankeshi J et al., 2007; Shankeshi J and Aukunuru JV, 2008). In drug discovery process, several molecules, although promising, are dropped out in the very early phases for several reasons including insolubility, unacceptable toxicity, high dosing frequency, less flexibility for conventional formulation development, poor oral bioavailability, high systemic metabolism, etc. Thus, there is a significant issue with drop outs in a drug discovery industry. To some extent, this drop out issue can be solved using drug delivery systems. Drug delivery systems are traditionally used for either life-cycle management of their products or for generic drugs. However, a thorough scientific literature survey indicates that drug delivery systems have been adopted for screening of some drugs especially in preclinical and clinical studies with added advantages over screening by conventional formulations (Aukunuru JV et al., 2003; Saishin Y et al.,

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2003; Koushik K et al., 2004; Sankeshi J and Aukunuru J, 2009). In recent years, newer drug delivery systems are showing promising results in improving the qualities of drug candidates and reducing the visit of drop outs. For the same reason, we also used the same approach for the screening of piperonal derivatives synthesized in this study along with the usage of conventional screening method. A biodegradable nanoparticular formulation which belongs to the latest drug delivery systems methodology was opted as the drug delivery system (Rao KS et al., 2008). Nanoparticles, in general, can be used to provide targeted (cellular/ tissue) delivery of drugs, to sustain drug effect in target tissues to improve oral bioavailability to solubilize drugs for intra–vascular delivery and to improve the stability of therapeutic agents against enzymatic degradation. Nanoparticles formulated as amorphous spheres offer higher solubility than standard crystalline formulations, thus improving the poor aqueous solubility of the drug and hence its bioavailability. Nanoparticles can be formulated, as injections consisting of spherical amorphous particles which do not aggregate; hence they can be safely administered via the intravenous route. The positive and negative outcomes of such screening methodology with the help of nanoparticles that are applied in this study are discussed.

Materials and Methods Piperonal needed for synthesis of derivatives was procured from Sigma-Aldrich Chemicals. The synthesis of title compounds have been achieved in accordance with scheme-I and scheme-II. For this purpose required aromatic aldehydes (scheme-I) and aromatic amines (scheme-II) were obtained from Merck, Spectrochem, Himedia and Qualikem. As and when needed these compounds were also synthesized in our laboratory. 4aminopyridine used to induce seizures in the rats was procured from Sigma-Aldrich. Female Wister weighing about 150g rats were purchased from Mahaveer enterprises. Animals were housed under standard laboratory conditions with free access to food and water and were kept at constant temperature. Animals were acclimatized to laboratory conditions before the experiment.

Jithan Aukunuru et al. : Synthesis of Novel Piperonal Derivatives and Evaluation of their Anticonvulsant Activity…

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SCHEME-I O O

CHO

Ethanol, NaOH

O

+ O

H3C C Ar

Stirring, rt

CH=CH

O

O

I

Piperonal

C Ar

II

Ar = -C6H5 - p.OCH3.C6H4 -p.Cl.C6H4 -m.NO2.C6H4

SCHEME-II

O

CHO

+

Methanol Ar

H2N

reflux

O

Piperonal

CH=N Ar

O

O

III

IV

Ar = N

O HN

H3C

Cl

Synthesis and Characterization of Piperonal Derivatives Two schemes were used in the synthesis of piperonal derivatives. Piperonal was employed as the starting material in both Schemes. In Scheme I, piperonal was subjected to a crossed aldoil conduction different acetophenone analogues such as 4-methoxyacetophenone, 4-chloroacetophenone and mnitroacetophenone in the presence sodium hydroxide in ethanol. The reaction was conducted and using both conventional and microwave irradiation (MWI) methods and the times of the reaction and percentage yields were

O

C

N H

recorded for comparison. The experimental procedures were: A. Conventional Method: A mixture of piperonal (0.01 mole) and an appropriate aromatic methyl ketones (0.01 mole) was taken in an Erlenmeyer flask. Ethanol was then slowly added and subjected to stirring. Sodium hydroxide solution clay was added then slowly and stirred the mixture at room temperature for 8-15 hours. The progress of the mixture reaction was monitored by TLC. The solid separated was filtered, dried and purified by recrystallization from ethanol to obtain a colorless, crystalline compound.

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B. Microwave Assisted Method: An equimolar (0.01 mole) mixture of piperonal and appropriate aromatic methyl ketones, ethanol and NaOH solution were taken into a reaction vessel and subjected to MWI at 160 watts for 4-10 minutes. The progress of the reaction was monitored by TLC. After completion of the reaction the contents were cooled to room temperature and the solid separated was filtered, dried and purified by recrystallization from ethanol. The compounds obtained in these reactions were by recrystallisation from ethanol and identified as chalcones from their analytical and spectral data (IR&1HNMR). The physical characteristics of the synthesized chalcones are recorded in Table 1. Scheme II, piperonal was treated with different aromatic amines such as 4-chloroaniline, o-toluidene, benzoic acidhydrazide and 3-hydrazono-1,3-dihydro-indol2-one using methanol as solvent. A. Conventional Method: A mixture of piperonal (0.01 mole), an aromatic primary amines (0.01 mole) and methanol was placed in a round bottomed flask (250 ml) and heated under reflux for 5-7 hours. The progress of the reaction was monitored by TLC. After completion of the reaction the contents were cooled to room temperature and poured into ice cold water (50 ml) while stirring. The solid separated was filtered, dried and purified by recrystallization using ethanol. B. Microwave Assisted Method: An equimolar (0.01 mole) mixture of piperonal, primary aromatic amines, and methanol were taken into a reaction vessel and subjected to MWI at 160 watts in a microwave oven for 6-8 minutes. The progress of the reaction was monitored using TLC. After completion of the reaction, the contents were cooled to room temperature and poured into ice cold water (50 ml) while stirring. The solid separated was filtered, dried and purified by recrystallization using ethanol. The products obtained in these reactions were identified as Schiff’s bases from their analytical and spectral data (IR&1H NMR). The physical characteristics of the synthesized Schiff’s bases were recorded and are presented in Table 2.

Development of Biodegradable Nanoparticular Formulation for Synthesized Piperonal Derivatives Biodegradable nanoparticles made out of polycaprolactone encapsulating selected three compounds synthesized in this

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study were prepared using standard protocols (Aukunuru JV et al., 2003). The nanoparticles were prepared as follows: The following solutions were prepared prior to the fabrication of nanoparticles. Sol.1: 100 mg drug was added to 400 mg polycaprolactone and then dissolved in 10 ml of dichloromethane. Sol. 2: 2% of polyvinyl alcohol was prepared by dissolving 2 gm of polyvinyl alcohol in 200 ml of water. A 5 ml of this solution was taken and was kept aside. Sol.3: From the above stock solution 100 ml was taken and kept aside. To prepare Nan particle formulations, both the solutions 1 and 2 were added into a beaker and then ultrasonicated for 10 minutes to form an emulsion. Sol 3. was added to the above solution, ultrasonicated for 5 minutes to form a w/o/w emulsion. A magnetic stirrer was taken and a bead was added and stirred for 4 hours to form nanoparticles after the evaporation of organic solvent. The three compounds synthesized in this study were further used after the preparation of nanoparticular suspensions are Ia, IIc and IId. The formulations were not characterized further for either release studies or drug-polymer interaction because the objective of this study i.e. the use of this methodology for drug screening in early discovery phases is investigational only.

Evaluation of Anticonvulsant Activity of Synthesized Piperonal Derivatives using A. Conventional Methods: In conventional method of screening, drugs are prepared as solutions, suspensions, emulsions and tablets which generally deliver the drug for maximum of one day and these formulations are administered in animal models and pharmacological activity is determined. In this study, we used oral suspensions because of the poor solubility of compounds in water to prepare the easy solution. The anticonvulsant property of piperonal derivatives was evaluated in a 4-aminopyridine induced seizure in rats. In this study, 16 male rats (150-180 gm) divided into 4 groups were selected. A solution of 4-aminopyridine was prepared and the drug was administered intraperitoneally to induce convulsions in each rat at a dose of 13.3 mg/kg. Phenytoin was used as a standard anticonvulsant drug and a 5 mg of drug was administered orally into each rat in the form of aqueous solution. The drug suspensions were prepared using 1% sodium carboxymethylcellulose and were administered orally 30 min prior to intraperitoneal administration of 4-

Jithan Aukunuru et al. : Synthesis of Novel Piperonal Derivatives and Evaluation of their Anticonvulsant Activity…

aminopyridine. Each animal was administered 1.5 mg. The onset of action was then noted and compared with that obtained after the administration of the standard. The behavioral changes of the rats were observed for 2 hours. The latency to the onset of seizures and the protection against lethality when 4-aminopyridine was administered was measured and evaluated as a percent of the control. After 4-aminopyridine was injected to the control group which received only vehicle, the onset of action indicated by jerky movements of whole body and tonic convulsions and severity of convulsions was noted. 4-aminopyridine produces powerful tonic convulsions of the body and limbs. As a result when the animal dies its body becomes opistotonus (arch-shaped).The control rat received only the vehicle. B. Nanoparticular Method: The pharmacological screening method is same as described in conventional method. However, here formulations are administered as nanoparticular systems. The formulations were administered into the animal model by IP route for only one time every two days. During the two day period, the drug is supposed to be released in a sustained manner and elicit the action. The results were taken on the second day of the administration. This approach was used for the compounds Ia, IIc and IId. Same dose as administered via oral route in conventional screening methodology (1.5 mg formulation) was administered in the form of biodegradable nanoparticles.

Results and Discussion In this study, eight piperonal derivatives (Ia-Id and IIa-IId) have been synthesized by two different Schemes one for chalcones (Scheme I) and other for Schiffs bases (Scheme II), using conventional and microwave method. Hosher percentage yields of the products were recorded in microwave irradiation method than conventional method (Tables 1, & 2). The synthesized compounds were characterized from their physical and spectral data (Tables 1-4). The synthesized compounds were screened for anticonvulsant activity using a 4-aminopyridine induced seizures model. Of the synthesized 8 compounds, 2 compounds this study IIc and IId along with piperonal demonstrated anticonvulsant activity at the selected dose, although not very significant when compared to that of

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control phenytoin. The time for noticing the convulsions in a rat after the administration of 4-aminopyridine was selected as a parameter to determine the anticonvulsant activity of the compounds. This time with control, phenytoin, piperonal, IIc and IId were 9.5, 23.6, 14.5, 14.6 and 17.5, respectively. The dose of the drugs administered was 1.5 mg. All other compounds did not demonstrate any anticonvulsant activity at the dosage selected. These results suggest that the piperonal itself has some anticonvulsant activity indicating that the piperine ring may be atleast partly responsible for anticonvulsant activity. The modifications effected to piperonal in the present study did not improve the anticonvulsant activity. Further, a novel screening methodology with the use of biodegradable nanoparticulate drug was also employed. The biodegradable nanoparticular formulations were successfully prepared using the protocol of this study. The size of the particles was less than 1000 microns suggesting the nanoparticle formation for the selected drugs. Three different compounds Ia, IIb and IIc were encapsulated into nanoparticles. All the formulations were administered at a dose of 1.5 mg formulation intraperitoneally. The evaluation of the anticonvulsant activity was done on day 2 of administration. It was speculated that the drug levels are sustained in the plasma after administration using the nanoparticular formulation. It is assumed that the drug in the plasma is still seen on day 2, and this exerts the anticonvulsant activity. Compound Ia did not show any activity with the conventional screening approach suggesting that it is not active or the dose administered to demonstrate the activity was not efficient. On the other hand, compounds IIc and IId demonstrated activity comparable to that of piperonal when administered by oral route. However, administration of nanoparticular formulation after intraperitoneal administration on day 2 did not demonstrate any activity. The dose of the formulation was the same 1.5 mg which includes the drug as well as the polymer. The encapsulation efficiency, the drug release or the drug-polymer interaction were not determined for the nanoparticular formulations. The negative results with the nanoparticular formulations suggest that drug encapsulated may be very less, release was not up to the mark or there may be some drug-polymer interaction. Further studies are necessary to establish this type of screening approach.

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Table 1. Physical data of 3-Benzo [1, 3] dioxol-5-yl-1-(N-substituted phenyl)-propenone (Chalcones)

S.No

PD

1

Ia

2

Ib

3

Ic

4

Id

%Yield

Molecular

Melting

formula

point

C16 H12O3

118-122˚C

C17H14O4

135-138˚C

C16H11O3Cl

123-128˚C

C16H11N1O5

132-136˚C

Rf (nhex:EA::3:2)

Conv.

MWI

92%

95%

0.28

88%

91%

0.24

82%

85%

0.26

78%

80%

0.30

PD: piperine derivative

Table 2. Physical data of 3-Benzo [1, 3] dioxol-5-yl-1-(N-substituted phenyl)amine (Schiffs bases)

S.No

PD

Molecular formula

Melting point

C14 H10O2NCl

82-86˚C

C15H13O2N

98-102˚C

C15H12N2O3

138-144˚C

C16H11N3O3

202-208˚C

%Yield Conv.

1

IIa

2

IIb

3

IIc

4

IId

MWI

Rf (nhex:EA::3:2)

50%

60%

0.35

92%

95%

0.4

78%

83%

0.4

75%

75%

0.48

PD: piperine derivative

Table 3. IUPAC names and Spectral Data of Synthesized Chalcones PD

IUPAC NAME

IR Spectral Data -1

3100 cm (Aromatic CH stretch), 1659.5 cm-1 1a

3-Benzo [1, 3] dioxol -5-yl-1-

(

C=O stretch), 1590.06 cm-1 (Aromatic C=C

phenyl-propenone

stretch), 1253 cm-1 (C-O stretch), 1630 cm-1 (Aliphatic C=C stretch)

1b

3-Benzo [1, 3] dioxol-5-yl-1-

3100 cm-1 (Aromatic CH stretch), 1653 cm-1

(4-methoxyphenyl)-

(C=O stretch), 1583 (C=C stretch), 1253 cm-1

propenone

(C-O stretch), 1037.61 cm-1

(OCH3 stretch)

-1

3-Benzo [1, 3] dioxol-5-yl-11c

(4-chlorophenyl)-propenone

3100cm (Aromatic CH stretch), 1656 cm-1(C=O stretch), 1596 cm-1(C=C stretch) 1253, cm-1(C-O stretch) 1640 cm-1(Aliphatic C=C, stretch) 3100 cm-I (Aromatic CH stretch), 1662 cm-1(C=O

1d

3-Benzo [1, 3] dioxol-5-yl-1-

stretch), 1609.64cm-1(Aliphatic C=C stretch),

(3-nitrophenyl) – propenone

1589cm-1 (Aromatic C=C stretch), 1253 cm-1 (C-O stretch)

Jithan Aukunuru et al. : Synthesis of Novel Piperonal Derivatives and Evaluation of their Anticonvulsant Activity…

441

Table 4. IUPAC names and Spectral Data of Synthesized Schiffs Base PD

IIa

IUPAC NAME

IR Spectral Data -1

-

Benzo[1,3] dioxol-5-ylmethylene-(4chlorophenyl)-amine

3100cm (Aromatic CH stretch), 1640 cm 1 (C=N stretch), 1620 cm-1 (Aromatic C=C -1 stretch), 1253 cm (C-O stretch), 1700 cm-1 (C=O stretch)

Benzo [1, 3] dioxol-5-ylmethylene-o-tolyl)-amine

3100cm-1 (Aromatic CH stretch), 1650 cm-1 (C=N Stretch), 1620 cm-1 (Aromatic C=C stretch), 1253 cm-1 (C-O stretch), 1700 cm-1 (C=O stretch)

IIb

-1

IIc

IId

Benzoic acid benzo [1, 3] dioxol-5-yl-methylenehydrazide 3-(benzo [1, 3] dioxol-5yl-methylenehydrazono)-[1, 3] dihydro-indol-2-one.

-1

3416.69cm (NH stretch), 1600 cm (C=N Stretch), 1590 cm-1 (Aromatic C=C -1 stretch), 1253 cm (C-O stretch), -1 1637.78cm (C=O Stretch) -1 (NH stretch), 1617 cm-1 (C=N 3200cm -1 (Aromatic CH stretch), 3100 cm -1 stretch), 1253 cm (C-O stretch), 1726cm-1 (C=O stretch)

Table 5. Onset Time of Convulsions for Promising Derivatives Compound

Compound

II (c)

II (d)

15.10

14.0

18.10

23.0

13.0

15.15

17.20

9.20

22.10

14.15

13.20

16.40

10.30

24.00

16.0

16.20

18.30

9.5

23.65

14.56

14.63

17.5

0.88

1.46

1.29

1.31

0.86

S.No

Control

Phenytoin

Piperonal

1

10.10

25.5

2

8.40

3 4 Mean STDEV

Acknowledgement The authors of this work would like to acknowledge the management of Vaagdevi College of Pharmacy, Warangal, for providing infrastructure useful in the conduction of this work. Dr. A.V. Jithan would like to acknowledge the Department of Science and Technology, India for providing financial assistant ship for this project. This work was partly funded under a SERC-DST Young Investigator project to Dr. Aukunuru Jithan (Dr. A.V. Jithan)

References Aukunuru J. In the Era of Nanotechnology: Are Drug Delivery Systems Dawning a New Dress (Part I). Pharmabuzz. 2: 23-30 (2007).

Aukunuru JV, Ayalasomayajula SP and Kompella UB. Nanoparticle formulation enhances the delivery and activity of a vascular endothelial growth factor antisense oligonucleotide in human retinal pigment epithelial cells. J Pharm Pharmacol. 55: 1199-1206 (2003). D'Hooge R, Pei YQ, Raes A, Lebrun P, van Bogaert PP and de Deyn PP. Anticonvulsant activity of piperine on seizures induced by excitatory amino acid receptor agonists. Arzneimittelforschung. 46: 557-560 (1996). Echevarria A, Nascimento MD, Gerônimo V, Miller J and Giesbrecht A. NMR Spectroscopy, Hammett Correlations and Biological Activity of Some Schiff Bases Derived from Piperonal. J. Braz. Chem. Soc. 10: 60-64 (1999).

HEC1

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Koushik K, Dhanda DS, Cheruvu NP and Kompella UB. Pulmonary delivery of deslorelin: large-porous PLGA particles and HPbetaCD complexes. Pharm Res. 21: 1119-1126 (2004). Mbuba CK, Ngugi AK, Newton CR and Carter JA. The epilepsy treatment gap in developing countries: a systematic review of the magnitude, causes, and intervention strategies. Epilepsia. 49: 1491-1503 (2008). Mori A, Kabuto H and Pei YQ. Effects of piperine on convulsions and on brain serotonin and catecholamine levels in E1 mice. Neurochem Res. 10:1269-75 (1985). Pal SK, Sharma K, Prabhakar S and Pathak A. Psychosocial, demographic and treatment seeking strategic behavious, including faith healing practices, among patients in northwest India. Epilepsy Behav. 13: 323-332 (2008). Pavia DL, Lampman GM, Kriz GS and Engel RG. Introduction to organic laboratory techniques: a smallscale approach. 2nd ed. Brooks/Cole-Thomson Learning, Belmont (CA), 2005, pp 212. Pei YQ, Yue W, Cui JR and Yao HY. A study of the central pharmacological action of piperine and its derivatives. Yao Xue Xue Bao. 15: 198-205 (1980). Pei YQ. A review of pharmacology and clinical use of piperine and its derivatives. Epilepsia. 24: 177-182 (1983).

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Pugh MJ, Van Cott AC, Cramer JA, Knoefel JE, Amuan ME, Tabares J, Ramsay RE and Berlowitz DR. Treatment In Geriatric Epilepsy Research (TIGER) team. Trends in antiepileptic drug prescribing for older patients with new-onset epilepsy: 2000-2004. Neurology. 70: 2171-2178 (2008). Rao KS, Reddy MK, Horning JL and Labhasetwar V. TAT-conjugated nanoparticles for the CNS delivery of anti-HIV drugs. Biomaterials. 29: 4429-4438 (2008). Saishin Y, Silva RL, Saishin Y, Callahan K, Schoch C, Ahlheim M, Lai H, Kane F, Brazzell RK, Bodmer D and Campochiaro PA. Periocular injection of microspheres containing PKC412 inhibits choroidal neovascularization in a porcine model. Invest Ophthalmol Vis Sci. 44: 4989-4993 (2003). Shankeshi J and Aukunuru J. Preparation, Characterization and Evaluation of Antidiabetic Activity of a Liposomal Formulation Encapsulating Curcumin, an Ayurvedic Natural Product. The Pharmacist. 3: 1-5 (2008). Shankeshi J, Aukunuru J and Vanga MR. Preclinical Assessment of Sustained Release Biodegradable Curcumin Microspheres in Diabetes. Proc. A.P. Akademi of Sciences, Hyderabad. 11: 180-187 (2007). Shulgin AT and Shulgin A. Phenethylamines I Have Known And Loved: A Chemical Love Story. Transform Press. Lafayette, California, 1991, pp100.

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