SUPPLEMENTARY MATERIAL
Pistagremic acid, a novel β-secretase enzyme (BACE1)inhibitor from Pistacia integerrima
Stewart
Abdur Rauf *a, Ghias Uddina, Ajmal Khanb, Bina S. Siddiquib, Mohammad Arfana, Kourosh
Dalvandic, Taibi Ben Hadda*
a
Institute of Chemical Sciences, University of Peshawar, Peshawar (25120), KPK, Pakistan
b
H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan
c
Dr.Panjwani Centre for Molecular Medicine and Drug Research, International Centre for
Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan
d
Mohammed First University, Faculty of Sciences Materials Chemistry Laboratory Oujda-60000.
Morocco
Abstract
A new triterpenic compound named pistagremic acid (PA) was once again isolated from Pistacia
integerima. The compound (PA) was found significantly active against β- secretase enzyme
(BACE1) with IC50 value of 350 ± 2 nM in comparison to the standard inhibitors [Asn670,
Sta671, Val672]-Amyloid-β/A4 Precursor Protein 770 Fragment 662-675 (IC50 = 290.71 ± 1
nM). The selectivity of this compound was also evaluated against the acetylcholinesterase and
butyrylcholinesterase enzymes. Interestingly compound (PA) was found to be inactive against
them and showed selectivity towards β- secretase enzyme (BACE1).
Keywords: Pistacia integerrima; pistagremic acid; triterpenoid; β- secretase, selectivity,
acetylcholinesterase and butyrylcholinesterase.
*Corresponding author: Abdur Rauf Institute of Chemical Sciences, University of Peshawar,
Peshawar-25120, KPK, Pakistan; E-mail: mashaljcs@yahoo.com
Experimental
Plant material
P. integrrima galls were collected from Toormang, Razagram area of district Dir, Khyber PukhtunKhawa
province of Pakistan in the month of February, 2010. The plant material was
identified by Prof. Dr.
Abdur Rashid of the department of Botany, University of Peshawar, Pakistan. A voucher specimen no
(RF-895) was deposited in the herbarium of the mentioned department.
Extraction and isolation
Shade dried and crushed bark of Pistacia integerrima (Stewart) (14 kg) was subjected to cold extraction
with MeOH. MeOH extract (600g) was suspended in water and successively partitioned with hexane,
CHCl3, EtOAc and BuOH. EtOAC fraction (30g) was subjected to Column chromatography on silica gel
(Merck Silica gel 60(0.063-0.200mm), 5  60 cm). Column was first eluted with hexane-Acetone (100:0 →
0:100) as a solvent system. A total of 33 fractions, RF-1 to RF-33 were obtained based on TLC profiles.
Fraction RF-20 obtained at (100:0 → 15:100: Hexane-Acetone) gradient contained colorless crystals of
various sizes and was separated from the solution by decantation. The melting point was recorded as 139141 ºC. The crystals were washed with n- hexane for several times. To obtain pure and larger crystals, these
crystals were re-grown from a mixture of hexane-acetone and chloroform (70:20:10) and thus obtained a
compound named Pistagremic acid (50 mg). Based on the above arguments and single crystal x-rays
crystallographic studies, the structure of (PA) was assigned as 2-Methyl-6-(4,4,10,13,14-pentamethyl-3-O2,3,4,5,6,7,10,11,12,13,14,15,16,-17-tetradecahydro-1H-cyclopenta[a]-phenanthren-17-yl)hept-2-enoic
acid (Arfan et al., 2011).
Beta secretase FRET assay
82.5 µL assay buffer (50 mM sodium acetate buffer with pH= 4.5) add into each (inhibitor) in each well,
and then 2.5 µL from 20U/100µL solution of enzyme (0.5 U) should be added. After that 20 min
incubation time is required (25°C). Reaction will be started by adding 62.5 nM from 12500 nM/1mL
solution of substrate; it should be incubated for 60 min at 37°C in the dark condition (Citron, 2002). After
well of 96 black plate, then 1000 nM from 100 µM /1mL stock solution of compound incubation time
plate should be read by the fluorometer machine (CHAMELEON TM -HIDEX). Emission and excitation
for Mca are 325 and 400 nm respectively.
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Chemical structure of Pistagremic acid (PA)
Crystallographic image of Pistagremic acid (PA)