Banisteriopsis caapi, a unique combination of MAO inhibitory and antioxidative constituents for the activities relevant to neurodegenerative disorders and Parkinson's disease

Article abstract of DOI:10.1016/j.jep.2009.10.030

Aim of the study: Parkinson's disease is a neurological disorder mostly effecting the elder population of the world. Currently there is no definitive treatment or cure for this disease. Therefore, in this study the composition and constituents of the aqueous extract of Banisteriopsis caapi for monoamine oxidases (MAO) inhibitory and antioxidant activities were assessed, which are relevant to the prevention of neurological disorders, including Parkinsonism. Materials and methods: The aqueous extract of Banisteriopsis caapi stems was standardized and then fractionated using reversed-phase (RP) chromatography. Pure compounds were isolated either by reversed-phase (RP) chromatography or centrifugal preparative TLC, using a Chromatotron. Structure elucidation was carried out by 1D and 2D NMR, Mass, IR and Circular Dichroism spectroscopy and chemical derivatization. Chemical profiling of the extract was carried out with RP-HPLC. The inhibitory activity of MAO-A, MAO-B, acetylcholinesterase, butyrylcholinesterase and catechol-O-methyl transferase enzymes, as well as antioxidant and cytotoxic activities of both Banisteriopsis caapi extract and isolated compounds was evaluated. Results: An examination of the aqueous extracts of Banisteriopsis caapi cultivar Da Vine yielded two new alkaloidal glycosides, named banistenoside A (1) and banistenoside B (2), containing "azepino[1,2-a]tetrahydro-β-carboline" unique carbon framework. One additional new natural tetrahydronorharmine (4), four known β-carbolines harmol (3), tetrahydroharmine (5), harmaline (6) and harmine (7), two known proanthocyanidines (-)-epicatechin (8) and (-)-procyanidin B2 (9), and a new disaccharide β-d-fructofuranosyl-(2 → 5)-fructopyranose (14) together with known sacharose (15) and β-d-glucose (16) were also isolated. In addition, the acetates of 1, 2, 8, 9, 14 and 15 (compounds 10-13, 17, 18) were also prepared. Harmaline (6) and harmine (7) showed potent in vitro inhibitory activity against recombinant human brain monoamine oxidase (MAO)-A and -B enzymes (IC₅₀ 2.5 and 2.0 nM, and 25 and 20 μM, respectively), and (-)-epicatechin (8) and (-)-procyanidin B2 (9) showed potent antioxidant and moderate MAO-B inhibitory activities (IC₅₀ < 0.13 and 0.57 μg/mL, and 65 and 35 μM). HPLC analysis revealed that most of the dominant chemical and bioactive markers (1, 2, 5, 7-9) were present in high concentrations in dried bark of large branch. Analysis of regular/commercial Banisteriopsis caapi dried stems showed a similar qualitative HPLC pattern, but relatively low content of dominant markers 1, 2, 7, and 9, which led to decreased MAO inhibitory and antioxidant potency. Conclusion: Collectively, these results give additional basis to the existing claim of Banisteriopsis caapi stem extract for the treatment of Parkinsonism, including other neurodegenerative disorders.

Full text of DOI:10.1016/j.jep.2009.10.030

Journal of Ethnopharmacology 127 (2010) 357–367
Contents lists available at ScienceDirect
Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jethpharm
Banisteriopsis caapi, a unique combination of MAO inhibitory and antioxidative
constituents for the activities relevant to neurodegenerative disorders and
Parkinson’s disease
Volodymyr Samoylenko , Md. Mostafizur Rahman , Babu L. Tekwani , Lalit M. Tripathia,
a
a
a,b
a
a
a,c
d
Yan-Hong Wang , Shabana I. Khan , Ikhlas A. Khan , Loren S. Miller ,
Vaishali C. Joshi , Ilias Muhammad
National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA
Department of Pharmacology, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA
Department of Pharmacognosy, Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, MS 38677, USA
Biopharm Biotech Corporation, PO Box 1071, Palo Alto, CA 94301, USA
a
a,∗
a
b
c
d
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 August 2009
Received in revised form 13 October 2009
Accepted 22 October 2009
Available online 30 October 2009
Aim of the study: Parkinson’s disease is a neurological disorder mostly effecting the elder population of the
world. Currently there is no definitive treatment or cure for this disease. Therefore, in this study the com-
position and constituents of the aqueous extract of Banisteriopsis caapi for monoamine oxidases (MAO)
inhibitory and antioxidant activities were assessed, which are relevant to the prevention of neurological
disorders, including Parkinsonism.
Materials and methods: The aqueous extract of Banisteriopsis caapi stems was standardized and then
fractionated using reversed-phase (RP) chromatography. Pure compounds were isolated either by
reversed-phase (RP) chromatography or centrifugal preparative TLC, using a Chromatotron . Structure
Keywords:
Banisteriopsis caapi (Spruce ex Griseb.)
®
␤
-Carboline alkaloids
elucidation was carried out by 1D and 2D NMR, Mass, IR and Circular Dichroism spectroscopy and
chemical derivatization. Chemical profiling of the extract was carried out with RP-HPLC. The inhibitory
activity of MAO-A, MAO-B, acetylcholinesterase, butyrylcholinesterase and catechol-O-methyl trans-
ferase enzymes, as well as antioxidant and cytotoxic activities of both Banisteriopsis caapi extract and
isolated compounds was evaluated.
Proanthocyanidines
Monoamine oxidase inhibitors
Antioxidants
Parkinson’s disease
Results: An examination of the aqueous extracts of Banisteriopsis caapi cultivar Da Vine yielded two
new alkaloidal glycosides, named banistenoside A (1) and banistenoside B (2), containing “azepino[1,2-
a]tetrahydro-ˇ-carboline” unique carbon framework. One additional new natural tetrahydronorharmine
(4), four known ˇ-carbolines harmol (3), tetrahydroharmine (5), harmaline (6) and harmine (7), two
known proanthocyanidines (−)-epicatechin (8) and (−)-procyanidin B2 (9), and a new disaccharide ˇ-d-
fructofuranosyl-(2 → 5)-fructopyranose (14) together with known sacharose (15) and ˇ-d-glucose (16)
were also isolated. In addition, the acetates of 1, 2, 8, 9, 14 and 15 (compounds 10–13, 17, 18) were also
prepared. Harmaline (6) and harmine (7) showed potent in vitro inhibitory activity against recombinant
human brain monoamine oxidase (MAO)-A and -B enzymes (IC50 2.5 and 2.0 nM, and 25 and 20 ␮M,
respectively), and (−)-epicatechin (8) and (−)-procyanidin B2 (9) showed potent antioxidant and mod-
erate MAO-B inhibitory activities (IC50 < 0.13 and 0.57 ␮g/mL, and 65 and 35 ␮M). HPLC analysis revealed
that most of the dominant chemical and bioactive markers (1, 2, 5, 7–9) were present in high concen-
trations in dried bark of large branch. Analysis of regular/commercial Banisteriopsis caapi dried stems
showed a similar qualitative HPLC pattern, but relatively low content of dominant markers 1, 2, 7, and 9,
which led to decreased MAO inhibitory and antioxidant potency.
Conclusion: Collectively, these results give additional basis to the existing claim of Banisteriopsis caapi
stem extract for the treatment of Parkinsonism, including other neurodegenerative disorders.
©
2009 Elsevier Ireland Ltd. All rights reserved.
∗ Corresponding author. Tel.: +1 662 915 1051; fax: +1 662 915 1006.
E-mail address: milias@olemiss.edu (I. Muhammad).
0
378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2009.10.030

3
58
V. Samoylenko et al. / Journal of Ethnopharmacology 127 (2010) 357–367
1
. Introduction
Banisteriopsis (Family: Malpighiaceae) is a tropical South Amer-
2. Materials and methods
2.1. General experimental procedures
ican genus with 92 species distributed mainly in Brazil, Bolivia,
Colombia, Ecuador, and Peru (Mabberley, 1997; Schultes, 1970).
Banisteriopsis caapi (Spruce ex Griseb.) Morton (Vine of the Soul)
Optical rotations were measured in CHCl3 or MeOH using
an AUTOPOL IV® instrument at ambient temperature; IR spectra
were obtained using a Bruker Tensor 27 FTIR instrument; Cir-
cular Dichroism (CD) spectra were recorded on a Olis DCM 20
CD spectrometer. The HPLC system consisted of a Model 2695
Alliance Separations Module equipped with a 2996 photodiode
array detector, and a computerized data station equipped with
Waters Empower 2 software (Waters, Milford, MA), using a Gem-
ini C18 110 Å column (Phenomenex, 150 mm × 4.6 mm I.D.; 5 ␮m
particle size; Phenomenex Inc., Torrance, CA, USA) and operated
(
Schultes and Raffauf, 1992) is an ingredient of the popular sacred
and psychoactive drinks Ayahuasca, also known as Caapi, Pinde,
Natema or Yaje, which is widely used for prophecy, divination, and
as a sacrament in the northern part of South America (Schultes,
1
970; Schultes and Siri von, 1995). However, to the best of our
knowledge, no traditional drink prepared only from Banisteriopsis
caapi has been consumed for such uses. Earlier chemical investi-
gations have reported the presence of ˇ-carboline alkaloids (ˇ-CA)
harmine, harmaline and tetrahydroharmine (THH) as the princi-
pal MAO inhibitors, together with other ˇ-CA’s, from Banisteriopsis
caapi (Hochstein and Paradies, 1957; Hashimoto and Kawanishi,
◦
at 30 C. The column was equipped with a 2 cm LC-18 guard col-
umn (Phenomenex Inc., Torrance, CA, USA). The NMR spectra were
1
acquired on a Bruker Avance DRX-400 instrument at 400 MHz ( H),
1
975, 1976; Callaway et al., 2005). In addition, two pyrrolidines,
100 (13C) in CDCl3 or CD OD, using the residual solvent as int.
3
shihunine and (S)-(+)-dihydroshihunine (Kawanishi et al., 1982),
and terpenoids (Aquino et al., 1991) were also reported. The alka-
loid content of Banisteriopsis caapi was determined previously by
GC/MS (Rivier and Lindgren, 1972), LC/MS (Kawanishi et al., 1998),
and HPLC (Serrano-Due n˜ as et al., 2001), suggesting the content of
harmine is highest among ˇ-CA’s, followed by THH and harma-
line.
Parkinson’s disease (PD) is caused by a loss of neurons from
substantia nigra of the brain. Once damaged, these neurons stop
producing dopamine and compromise the brain’s ability to con-
trol movement. It is not known what damages certain neurons
in PD patients. One reason is that free radicals/toxic particles
normally deactivated in the body are responsible, which can
be controlled by antioxidants as adjuvant with dopamine ago-
nist or MAO inhibitors. The usefulness of Banisteriopsis caapi
was established for alleviating symptoms of PD (Serrano-Due n˜ as
et al., 2001), which contains MAO inhibitor harmine as active
constituent used in PD treatment (Sánchez-Ramos, 1991). A
double-blind, randomized placebo-controlled trial of Banisteriopsis
standard; multiplicity determinations (DEPT) and 2D NMR spectra
(COSY, HMQC, HMBC and NOESY) were obtained using standard
Bruker pulse programs; HRMS were obtained by direct injection
using a Bruker Bioapex-FTMS with electro-spray ionization (ESI).
®
Plant material was extracted by either Coffee Maker (Mr. Coffee ,
®
ISX-43) or Accelerated Solvent Extractor (Dionex , ASE-200) using
H O as a solvent. Water extracts were freeze-dried using Freeze
2
®
Dry System (Labconco , Freezone 4.5). TLC was carried out on
either reversed phase silica gel (Analtech , RP18 SiO , 150 ␮m,
®
2
UV254) with MeCN–H O (9:1) or acetone–H O–NH ·H O (7:3:0.1),
2
2
3
2
silica gel (EMD® Chemicals Inc., SiO 60 F ) using CHCl –MeOH
2
254
3
(7:3) or alumina plates (EMD® Chemicals Inc., Al O3 60 F254) using
2
CH Cl –MeOH (9:1) as solvent system; centrifugal preparative TLC
2
2
®
(CPTLC) was performed by a Chromatotron (Harrison Research
Inc., model 8924), tagged with a fraction collector (SpectralChrom
CF-1) on a 1, 2 or 4 mm silica gel rotors (Analtech , SiO coated with
®
®
2
F254 indicator); flash CC was carried out over reversed phase sil-
®
ica gel (J.T. Baker, Bakerbond C18 prep LC SiO , 40 ␮m). Samples
2
were dried using a Savant Speed Vac Plus SC210A Concentrator.
The isolated compounds were visualized by observing under UV
light at 254 or 365 nm, followed by spraying separately with Dra-
caapi, using
ment in motor function of PD patients (Serrano-Due n˜ as et al.,
001). Tests for MAO inhibition using liver homogenate showed
a single dose, revealed a significant improve-
2
gendorff’s and/or 1% vanillin-H SO4 spray reagents. The reference
2
that Banisteriopsis caapi stem extract and harmine showed a
concentration-dependent inhibition of MAO-A, and an increase
in release of dopamine from rat striatal slices (Schwarz et al.,
standards of harmol, harmine, harmaline, and (−)-epicatechin were
purchased from Sigma–Aldrich (St. Louis, MO), while others were
available in our laboratories.
2
003).
It should be noted that the identities of different Baniste-
2.2. Plant material
riopsis species are incompletely known due to the paucity of
fertile collections and lack of detailed taxonomic study. There
are at least thirty different Banisteriopsis caapi that natives of
Amazon have knowledge of and have different uses (Schultes
and Hofmann, 1992). During the course of chemical and biolog-
ical standardization of Banisteriopsis caapi under an NIH funded
investigation at the NCNPR for neurological disorders relevant
to Parkinsonism, an extract of Banisteriopsis caapi cultivar Da
Vine, collected in Oahu, Hawaii, demonstrated potent in vitro
MAO-A inhibitory and antioxidant activities. This led to the
bioassay-guided isolation of two new ˇ-carboline alkaloidal gly-
cosides, banistenoside A (1) and banistenoside B (2), a new
tetrahydronorharmine (4), four known ˇ-carbolines harmol (3),
tetrahydroharmine (5), harmaline (6) and harmine (7), two proan-
thocyanidines (−)-epicatechin (8) and (−)-procyanidin B2 (9), and
a new disaccharide ˇ-d-fructofuranosyl-(2 → 5)-fructopyranose
Fresh leaves, stems, and large branches of Banisteriopsis caapi
cultivar Da Vine (Miller, 1986) were collected from the island of
Oahu, Hawaii, in August and November 2007, and June 2008, as
well as from Hilo (Big island), Hawaii, USA, in October, 2007. A
reference specimen was collected from Oahu, and a voucher speci-
men (HLA # 7835) was deposited at the Herbarium of Harold Lyon
Arboretum, University of Hawaii. The collector of the voucher spec-
imen of the plant at Lyon Arboretum was Dr. Kenneth M. Nagata
(Accession # L-81.0727; collector # 2789; dated 03/13/1984). The
regular Banisteriopsis caapi (mature stems) samples analyzed in this
study (BCEx-1–BCEx-4) were procured/obtained from commercial
(via internet) and NCNPR sources during 2005–2009. In addition, all
samples used in this work are preserved using the standard proce-
dures for collection, drying, grinding and packaging at the NCNPR.
(
14) (Fig. 1), using regular and RP silica gel chromatography. In this
2.3. Extraction of plant material
paper, we report the isolation, characterization and bioactivities of
isolated compounds, and HPLC analysis of Banisteriopsis caapi cul-
tivar Da Vine and two regular/commercial samples of Banisteriopsis
caapi.
2.3.1. Preparation of extracts from different plant parts
The different plant parts (leaves, young and mature stems, bark
and debarked stems, and large branches (diameter: 3–8 cm)) of

V. Samoylenko et al. / Journal of Ethnopharmacology 127 (2010) 357–367
359
Fig. 1. Chemical structures of compounds isolated from Banisteriopsis caapi.
Banisteriopsis caapi, fresh and dried, were extracted using hot water
This gave strong yellow-brown colored water extract (ca 20 mL in
total).
by two different methods. A portion of each fresh sample was dried
◦
◦
at 45 C for 2–4 days, to get dried material. Before processing, the
All water extracts were cooled, frozen at −20 C and freeze-
◦
fresh plant material was cut into small pieces, while dried plant
material was ground.
dried by Freeze Dry System (−44 C, 1.6 Pa). After freeze-drying
all samples were transferred into the vials and sealed with
®
(
A) “Hot maceration”: Water extracts were prepared by simmer-
Parafilm .
ing with boiled water using a Coffee Maker. Plant material was
placed onto a coffee filter and extracted successively with distilled
H O for three times (30–70 g/100 mL; H O 3×). This gave strong
2.3.2. Preparation of standardized extract of Banisteriopsis caapi
(Da Vine) whole stem
2
2
yellow colored extracts.
Based on the MAO-A and antioxidant activity of initial extracts of
different plant parts, prepared by using different extraction meth-
ods, the “most active” extracts were identified (Table 1). These are
hot aqueous extracts from dried powdered samples of large branch,
whole mature/big stem and mature stem bark prepared by method
A (using a coffee maker). In order to proceed for bioassay-guided
isolation of active constituents and markers, bulk extraction of
dried powdered large branch (471 g) was executed using the stan-
(
B) “ASE extraction”: Water extracts were prepared by a semi-
automated Accelerated Solvent Extractor (ASE-200). Plant material
was packed into a steel 33 mm cells and extracted successively with
◦
distilled H O for four times (15–20 g/5 mL H O 4×; 100 C, 3.45 MPa
2
2
of N , 30 min). Next optimal method was developed and used: pre-
heat time = 0 min, pressure = 3.45 MPa, heat time = 5 min, T = 100 C,
2
◦
static time = 30 min, flush = 100 vol%, purge time = 2 min, cycles = 4.

Table 1
MAO-A and MAO-B inhibitory, and antioxidant and cytotoxic activities of representative Banisteriopsis caapi extracts and compounds.
Sample name
Samples collected
Code #
Yielda (%)
MAO-A IC50 (␮g/mL)
MAO-B
Antioxidant
Cytotoxicity
IC50 (␮g/mL)
(␮g/mL)
% inhibition
Fresh stem
Dried stem
August, 2007; Oahu
fBCDVS-1
BCDMS
BCBYS
fBCDVBm-1
BCBMS
fBCDVDB-1
DDYS
BCDMDS
fBCDVL-1
BCDL
fBCDVS-2
fBCDVL-2
BCDVFYSO
BCDVDYSO
BCDVFLO
BCDVDLO
BCDVFYSAO
BCDVFLAO
BCfBS
0.85
12.74
10.08
2.16
14.29
2.67
9.76
10.12
0.49
10.06
11.7
5.28
0.80
7.28
0.60
4.08
6.29
8.35
3
0.2–0.67
0.04
0.1
0.015–0.025
0.033
0.025–0.035
0.08–0.043
0.05
0.125
0.6
0.9
0.2
0.58
100
100
100
100
100
100
100
100
100
100
100
100
>100
>100
>100
>100
>100
>100
100
43
38
46
23
45
27
45
57
41
39
16
30
ND
ND
ND
ND
ND
ND
51
55
34
46
ND
ND
ND
ND
NT
NT
1.6
NT
2.2
NT
4.0
4.0
NT
5.9
NT
NT
NT
NT
NT
NT
NT
NT
0.4
3.0
1.9
2.0
NT
NT
7.8
1.0
NT
NT
NC
NT
NC
NT
NC
NC
NT
NC
NT
NT
NT
NT
NT
NT
NT
NT
NC
NC
23
Dried young stem bark
Fresh mature stem bark
Dried mature stem bark
Fresh debarked young stem
Dried debarked young stem
Dried debarked mature stem
Fresh leaves
Dried leaves
b
Fresh stem
Fresh leavesb
October, 2007; Big Islandc
Fresh young stem
Dried young stem
Fresh leaves
1.13
1.35
0.33
0.24
Dried leaves
b
Fresh young stem
Fresh leavesb
0.22
Fresh bark large branch
Fresh debarked large branch
Dried bark large branch
Dried debarked large branch
Fresh big stems
November, 2007; Oahu
July 2008; Big Island
0.045
0.035
0.03
BCfDS
BCDBS
BCDDS
fBCDVSbig
BCDVSbig
BCEx-1
5.2
18.1
7.6
100
100
100
>100
>100
>100
>100
0.02
NC
NT
NT
NC
29
2.9
0.034
0.028
0.32
Dried big stems
9.56
15.43
9.80
Dried matured stemd
Dried matured stemd
BCEx-4
0.385
Pure compd/standards
MAO-A IC50 (nM)
MAO-B IC50 (␮M)
Antioxidant
Cytotox.
Pure compd/standards
MAO-A IC50 (nM)
MAO-B IC50 (␮M)
Antioxidant
Cytotox.
IC50 (␮g/mL)
IC50 (␮g/mL)
3
3
Banistenoside A (1)
Banistenoside B (2)
Harmol (3)
THH (5)
Harmaline (6)
Harmine (7)
4.9 × 10
>100
>100
NT
>100
25
NA
NT
NT
NA
NA
NA
>12.5
7.8
NT
NC
25
Epicatechin (8)
Procyanidin B2 (9)
Clorgyline
Deprenyl
Vitamin C
51.7 × 10
65
35
1.8
0.040
NT
<0.13
0.57
NT
NT
1.34
NT
6.3
NC
NT
NT
NT
3
3
21.5 × 10
18
8.5 × 10
1.6
9.0
NT
NT
74
2.5
2.0
20
NC
Doxorubicin
NT
0.2–0.35
a
Yield was measured by weight.
Extracted with water by automated ASE-200 extractor.
b
c
Plants from this collection are 3 years old. NT = not tested. ND = not detected (tested up to maximum concentration of 100 ␮g/mL). NA = no antioxidant activity upto 31.25 ␮g/mL. NC = no cytotoxicity to HL-60 cells up to
3
1.25 ␮g/mL.
d
Regular/commercial Banisteriopsis caapi sample.

V. Samoylenko et al. / Journal of Ethnopharmacology 127 (2010) 357–367
361
dardized method, as described above, which yielded 60 g of dried
pale-yellow powder.
2R,3R-epicatechin, and its literature values (Korver and Wilkins,
1971).
(
−)-2R,3R-Epicatechin-4ˇ,8-(−)-2R,3R-epicatechin
[(−)-
2.4. Bioassay-guided fractionation and characterization of
procyanidin B2] (9): The physical and spectral data of 9 were
isolated compounds
identical to those reported in the literature (Korver and Wilkins,
1
971; Barrett et al., 1979; Khan et al., 1997). In addition, the CD
The freeze-dried powdered aqueous extract (53.7 g), obtained
by bulk isolation from large branch, was subjected to RPCC over C-
data [(in MeOH) ꢀmax, nm ([ꢂ] in mdeg): 221 (−16.24), 243 (52.58),
276 (−3.64)] was also in agreement with those reported in the
literatures (Barrett et al., 1979).
1
8 silica gel (1:15) and eluted with MeCN (8 L) and then MeCN–H O
2
mixture with increasing concentrations of H O (1% → 10%), and
(−)-ˇ-d-Fructofuranosyl-(2 → 5)-fructopyranose
(Di-d-
−55.8 (c 0.78, MeOH);
2
23.5
5
0 mL fractions were collected. Fractions were pooled by TLC,
Fructose) (14): Colorless solid; [˛]D
1
combined, and then evaporated under reduced pressure (total
8.65 g from 14 combined fractions, i.e., fractions A–N). Fraction
H NMR (pyridine-D5, J in Hz) ıH 5.60 (brs, OH), 5.12 (1H, d, 8.0,
ꢀ
1
H-3), 5.04 (1H, t, 8.0, H-4), 4.82 (1H, brd, 12.0, H-4 ), 4.62 (1H, dd,
ꢀ
ꢀ
A (325 mg) was purified by CPTLC, using 2 mm silica gel rotor with
8.0, 12.0, H-3 ), 4.59 (1H, m, H-5), 4.49 (1H, dd, 1.0, 12.0, H-6 ), 4.39
ꢀ
ꢀ
a
ꢀ
a
CHCl –MeOH (9:1) as a solvent, which afforded 8 (110 mg), fol-
(1H, d, 8.0, H-1 ), 4.38 (1H, m, H-5 ), 4.29 (m, H-1 ), 4.29 (m, H-6),
3
a
lowed by 9 (24 mg), while fraction B (125 mg) yielded 5 (38 mg),
followed by 4 (24 mg), by using a similar CPTLC procedure. Frac-
tion D (118 mg) was also separated by CPTLC, using 1 mm silica
4.24–4.26 (m, H-1), 4.24 (1H, d, 12.0, H-6), 4.11 (1H, dd, 4.0, 12.0,
H-6 ); C NMR (pyridine-D5) ıC 103.6 (s, C-2), 99.3 (s, C-2 ), 84.3
ꢀ
13
ꢀ
ꢀ
ꢀ
(d, C-5), 78.0 (d, C-3), 77.0 (d, C-4), 73.3 (d, C-3 ), 71.7 (d, C-5 ), 70.0
ꢀ
ꢀ
ꢀ
1
gel rotor with CHCl –MeOH (9:1) as a solvent, which afforded 7
(d, C-4 ), 66.2 (t, C-1 ), 64.9 (t, C-1), 64.2 (t, C-6 ), 63.8 (t, C-6); H
3
b
ꢀ
b
(
56 mg), followed by 6 (17 mg), while fraction H (276 mg) was sep-
NMR (CD OD, J in Hz) ıH 5.51 (brs, OH), 4.62 (m, H-1 ), 4.62 (m,
3
c
H-1), 4.49 (d, 8.0, H-1), 4.49c (d, 8.0, H-1 ), 4.06 (m, H-3), 4.06
ꢀ
d
d
arated by a 2 mm silica gel CPTLC rotor, using CHCl –MeOH (9:1)
3
ꢀ ꢀ ꢀ
(m, H-5 ), 4.02 (1H, dd, 4.0, 12.0, H-6 ), 3.86 (1H, brs, H-3 ), 3.78
e
as solvent, which gave 14 (20 mg), and a mixture H-1 (90 mg), con-
taining 9 and 14. Fraction I (489 mg) was also separated by CPTLC,
e
ꢀ
e
(m, H-4), 3.78 (m, H-4 ), 3.73 (1H, m, H-5), 3.62 (dd, 4.0, 12.0,
e
ꢀ
a–e
using 4 mm silica gel rotor [CHCl –MeOH (9:1) as solvent], which
H-6), 3.62 , (dd, 4.0, 12.0, H-6 ), 3.49 (1H, brd, 8.0, H-6) ( signals
3
13
yielded 7 (29 mg), followed by another mixture I-1 (317 mg), which
are superimposed on each other); C NMR (CD OD) ı 101.7 (s,
3 C
ꢀ ꢀ
C-2), 97.8 (s, C-2 ), 81.3 (d, C-5), 76.6 (d, C-5 ), 75.4 (d, C-3), 70.4 (d,
was further purified by 2 mm silica gel CPTLC rotor [CHCl –MeOH
3
ꢀ
ꢀ
ꢀ
ꢀ
(
(
9:1) as solvent] to yield compounds 15 (sacharose; 79 mg) and 16
ˇ-d-glucose; 55 mg). Finally, fraction L (2.0 g) was separated by RP-
C-4), 69.8 (d, C-3 ), 68.3 (d, C-4 ), 64.5 (t, C-6 ), 62.9 (t, C-1 ), 62.9 (t,
C-1), 61.2 (t, C-6).
flash chromatography over C-18 silica gel, eluted with increasing
Acetylation of fraction L-2: Fraction L-2 (355 mg) containing
the mixture 1, 2 and 15 was dissolved in pyridine (1 mL) and
treated with acetic anhydride (2 mL). The reaction mixture was
stirred overnight at room temperature, and then dried under vac-
uum to give the solid residue. The residue was subjected to CPTLC
concentrations of H O in acetone (1:9 → 1:1, 2 L), which afforded 1
2
(
60 mg), followed by 2 (20 mg) as well as fractions L-1 (0.3 g) and
L-2 (1.3 g). Fraction L-2 consisted of mixtures of 1, 2 and 15, while
fraction L-1 was the mixture of 6 and 7.
Using the above procedures, bulk quantities of 1–9 (i.e.,
over 2 mm silica gel rotor [CHCl –MeCN (9:1) as solvent], which
3
8
0–100 mg) were isolated from aqeuous stem extracts of samples
afforded 10 (28 mg) and 18 (127 mg), together with fraction L-2-1
collected in 2007 and 2008. Compound 3 was identified by HPLC
using direct comparison with an authentic sample of harmol. Com-
pounds 5–9, 15 and 16 were identified by comparison of physical
and spectral data with those reported in the literatures, and by
direct comparison with their authentic samples as THH, harmaline,
harmine, epicatechin and procyanidin B2, sacharose and glucose,
respectively.
(127 mg), which was further separated by CPTLC, using 1 mm silica
gel rotor with CHCl –MeCN (19:1) as a solvent to yield compounds
3
11 (20 mg) and additional amounts of 18 (37 mg). Compound 18
was identified as sacharose octaacetate by physical and NMR data.
Banistenoside A heptaacetate (10): Colorless solid; [˛]D28 −8.7
(c 0.6, CHCl ); CD (in MeOH) ꢀmax, nm ([ꢂ] in mdeg): 316 (13.03),
3
−
1
361 (−0.85), 369 (−0.79); IR (film) ꢁmax, cm : 3455 (OH, NH),
Banistenoside A (1): Colorless solid; UV (MeOH) ꢀmax, nm:
3382 (OH, NH), 2920, 2850, 1804, 1752, 1631, 1426, 1371, 1225,
−
1
1
13
2
2
26, 269 and 290; IR (film) ꢁmax, cm : 3382 (NH), 2920,
1072, 1037, 910, 733; H and C NMR data, see Table 2; HRESIMS
850, 1804, 1752, 1631, 1225, 1037, 733; 1H and
data, see Table 2; HRESIMS m/z 367.1522 [MH+H O-Glucosyl]
calcd. for C17H23N O7, 367.1505); 529.2013 [MH+H O] (calcd. for
13
C NMR
m/z 805.2635 [MH] (calcd. for C37H45N O18, 805.2667); 827.2454
+
2
+
[MNa]+ (calcd. for C37H44N O18Na, 827.2487).
2
2
+
28
(
Banistenoside B heptaacetate (11): Colorless solid; [˛]D +31.1
2
2
C23H33N O12, 529.2033).
(c 0.4, CHCl ); CD (in MeOH) ꢀmax, nm ([ꢂ] in mdeg): 311 (21.39);
2
3
−
1
Banistenoside B (2): Colorless solid; UV (MeOH) ꢀmax, nm:
IR (film) ꢁmax, cm : 3448 (OH, NH), 3021, 2923, 2851, 1749, 1692,
−
1
1
13
2
2
26, 269 and 290; IR (film) ꢁmax, cm : 3448 (NH), 3021, 2923,
1434, 1371, 1221, 1044, 757; H and C NMR data, see Table 2;
+
851, 1749, 1692, 1632, 1221, 1044, 757; HRESIMS m/z 367.1491
HRESIMS m/z 805.2693 [MH] (calcd. for C37H45N O18, 805.2667);
2
+
827.2518 [MNa]+ (calcd. for C37H44N O18Na, 827.2487).
[
MH+H O-glucosyl] (calcd. for C17H23N O7, 367.1505); 529.2028
2
2
2
+
[
MH+H O] (calcd. for C23H33N O12, 529.2033).
Acetylation of (−)-epicetechin (8): Epicatechin
8 (27 mg,
2
2
Tetrahydronorharmine (4): Colorless solid; UV (MeOH) ꢀmax,
93 mmol) was dissolved in pyridine (1 mL). Acetic anhydride (1 mL)
was added dropwise to the solution while stirring. The reaction
mixture was stirred overnight at ambient temperature. The reac-
tion mixture was dried under vacuum to give the solid residue
which was further purified by flash chromatography over silica gel
to yield compound 12 (39 mg, yield 84%) as a pale solid. Physical and
Spectral data of 12 were identical to those reported in the literature
(Castillo et al., 2004).
nm: 226, 269 and 290; 1H NMR (CDCl , J in Hz) ıH 7.10 (1H, d,
3
7
.5, H-5), 6.67 (1H, brs, H-8), 6.49 (1H, brd, 7.5, H-6), 4.15 (2H, m,
13
H-1), 3.58 (3H, s, OCH ), 3.30 (2H, m, H-3), 2.81 (2H, m, H-3);
C
3
NMR (CDCl ) ıC 156.9 (s, C-7), 137.8 (s, C-8b), 124.0 (s, C-9a), 120.8
3
(
s, C-4b), 118.2 (d, C-5), 109.2 (d, C-6), 105.8 (s, C-4a), 94.5 (d, C-8),
5
2
4.7 (q, OCH ), 42.6 (t, C-3), 41.0 (t, C-1), 18.5 (t, C-4); HRESIMS m/z
03.1176 [MH] (calcd. for C12H15N O, 203.1184).
3
+
2
(
−)-2R,3R-Epicatechin (8): The physical and spectral data of
Acetylation of fraction H-1: Fraction H-1 (77 mg), containing the
mixture 9 and 14, was dissolved in pyridine (1 mL) and treated with
acetic anhydride (2 mL). The reaction mixture was stirred overnight
at room temperature, and dried under vacuum to give the solid
residue, which was subjected to CPTLC, using 1 mm silica gel rotor
8
2
(
were identical to those reported in the literature (Sun et al.,
006; Balde et al., 1991). The CD data [(in MeOH); ꢀmax, nm
[ꢂ] in mdeg): 244 (21.58), 269 (−5.83), 290 (−3.46)] was in
agreement with those recorded for an authentic sample of (−)-

Table 2
1
H and 13C NMR data of 1, 10 and 11.
#
1
H
1
10
H
10
C
11
H
11
C
1
13
1
13
1
13
C
HMBC
HMBC
1
3
4
4.44 m
3.31 brs 3.05 m
2.51 m
56.0 (d)
40.8 (t)
16.0 (t)
5.98 d (4.3)
4.06 dd (2.0, 12.4), 3.42 m
2.81 m
49.4
41.9
21.8
C-3, 4a, 9a, 10, 11, 14
C-14
C-4a
5.08 d (10.0)
52.2
38.3
20.7
C-3, 4a, 9a, 10, 11, 14
–
C-4a
a
4.98 dd (5.6, 11.6), 2.81 m
a
2.81 m
4
4
5
6
7
8
8
9
1
1
1
1
1
1
2
3
4
5
6
a
b
–
–
109.6 (s)
119.9 (s)
118.0 (d)
108.3 (d)
158.5 (s)
94.6 (d)
137.0 (s)
120.0 (s)
73.0 (d)
79.3 (d)
68.9 (d)
70.9 (d)
175.3 (s)
94.4 (d)
71.8 (d)
71.7 (d)
66.1 (d)
72.3 (d)
61.5 (t)
–
–
109.6
120.4
118.9
109.1
156.8
94.8
137.2
126.7
72.9
77.2
72.5
71.2
170.6
96.7
–
–
–
–
110.2
120.0
119.1
109.8
156.9
95.2
137.1
126.7
81.2
69.5
71.1
73.4
164.4
101.6
71.5
73.3
67.4
–
–
6.90b
7.34 d (8.8)
6.77 dd (2.0, 8.8)
–
6.81 d (2.0)
–
C-4a, 4b, 7, 8a
C-4b, 7, 8
–
C-4b, 6, 7, 8a
–
–
7.34 d (8.4)
6.76 dd (2.4, 8.8)
–
6.92 d (2.4)
–
C-4a, 4b, 7, 8a
C-4b, 7, 8
–
C-4b, 6, 7, 8a
–
–
6.19 brd (8.8)
–
6.35 d (2.8)
–
–
4.23 d brs
4.44 m
4.09 brs
4.04 brs
–
a
a
0
1
2
3
–
–
ꢀ
ꢀ
4.32 d (4.3)
5.20 d (8.4)
5.61 t (8.0, 8.4)
5.72 d (8.0)
–
C-1, 1 , 9a, 12
3.98 d (10.0)
5.77 d (4.4)
5.32 t (4.4, 4.8)
5.87 d (4.8)
–
C-1, 1 , 9a, 11, 12
C-10, 12, 13
C-10, 11, 13
C-12
C-10, 12, 13
C-10, 11, 13
C-12, 14
4
–
ꢀ
ꢀ
ꢀ
4.44 m
4.54 d (4.8)
C-2 , 10
–
–
C-6
–
C-4
4.91 d (3.6)
C-2 , 10
–
–
C-6
–
C-4
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
a
4.94–5.0 ma
5.11–5.17 ma
4.36 m
71.4
72.1
68.6
72.8
4.38 ma
4.31 m
4.94–5.0 m
a
5.11–5.17 m
a
4.94–5.0 ma
ꢀ
ꢀ
5.11–5.17 ma
ꢀ
ꢀ
a
3.82 m
3.55 m
4.30 dd (5.2, 12.0), 4.12 m
3.81 dd (2.4, xx)
4.38 dd (4.0,12.4), 4.0 dd
72.5
61.5
3.81 ma 3.31 m
62.2
(
1.2, 12.4)
7
-OMe
3.23 s
54.3 q
3.83s
55.6 q
C-7
3.86s
55.7
C-7
OH/NH
OC(O)Me
–
–
–
–
8.45 s (NH)
2.23, 2.15, 2.14, 2.07, 1.98,
–
C-4a, 4b, 8a, 9a
–
7.9 brs (NH)
2.21, 2.17, 2.09 x2, 2.01,
2.0, 1.53, (7x s)
–
–
C-4a, 4b, 8a, 9a
–
22.0, 20.7, 20.5 x3, 20.4,
20.2 (7q)
170.7, 170.1, 169.9, 169.5,
20.9, 20.6, 20.6 x3, 20.5 x2
(7q)
170.4 x2, 170.1, 170.0,
169.4 x2, 169.2
1
–
.94, 1.91 (7x s)
C(O)–Me
–
–
–
–
1
69.4, 169.2 x2
NMR Spectra of 1 (CF3COOD/D2O), and 10 and 11 (in CDCl3) were recorded at 400 ( H) and 100 (13C) MHz, using a Varian spectrometer. The coupling constants (J values, in Hz) are in parentheses. The multiplicities of carbon
1
13
signals were determined by C NMR DEPT experiments. Assignments were made using 2D NMR COSY, HMBC and HMQC experiments.
a
Signals superimposed on each other.
Signals superimposed with solvent.
b

V. Samoylenko et al. / Journal of Ethnopharmacology 127 (2010) 357–367
363
with CHCl –MeCN (98:2) as solvent system to afford acetates 13
determined fluorometrically by SpectraMax M5 fluorescence plate
reader (Molecular Devices, Sunnyvale, CA) with an excitation and
emission wavelength of 320 and 380 nm, respectively, using the
SoftMax Pro program.
3
(
29 mg) and 17 (47 mg).
(
80 (c 0.025, MeOH); CD (in 1,4-dioxane) ꢀmax, nm ([ꢂ] in
−)-Procyanidin B2 acetate (13): Colorless solid; [˛]D23.5
−
mdeg): 233 (−5.14), 245 (73.43), 274 (−12.35), 285 (−10.86);
−1
IR (film) ꢁmax, cm : 2963, 2917, 2855, 1767, 1621, 1596,
208, 1122, 1049, 901; 1H and
13
2.6. In vitro cytotoxicity assay
1
C NMR data were identical
to those reported in the literature (Korver and Wilkins, 1971;
The in vitro cytotoxic activity was determined against four
human cancer cell lines (SK-MEL, KB, BT-549 and HepG2), monkey
kidney fibroblasts (VERO) and pig kidney epithelial cells (LLC-PK11).
All cell lines were obtained from the American Type Culture Col-
lection (ATCC, Rockville, MD). The assay was performed in 96-well
tissue culture-treated microplates. Cells were seeded at a density
of 25,000 cells/well and incubated for 24 h. Samples at different
concentrations were added and plates were again incubated for
48 h. The number of viable cells was determined using Neutral Red
according to a modification of the procedure of Borenfreund et al.
(1990). IC50 values were determined from dose–response curves
of percent growth inhibition against test concentrations. Doxoru-
bicin was used as a positive control, while DMSO was used as the
negative (vehicle) control.
Barrett et al., 1979; Khan et al., 1997); HRESIMS m/z 1016.2859
+
[
(
MNH4]+ (calcd. for C50H50O22N, 1016.2824); 1021.2346 [MNa]
+
calcd. for C50H46O22Na, 1021.2378); 1037.2160 [MK] (calcd. for
C50H46O22K, 1037.2118).
ˇ-d-Fructofuranosyl(2 → 5)fructopyranose octaacetate (17):
Colorless solid; [˛]D2
3.5
−1
+30.4 (c 0.1, CHCl ); IR (film) ꢁmax, cm
:
3
1
2
942, 1746, 1433, 1372, 1222, 1050; H NMR (CDCl , J in Hz) ıH 5.72
3
ꢀ
ꢀ
(
5
1H, d, 4.0, H-3), 5.63 (1H, dd, 2.0, 8.8, H-3 ), 5.44 (1H, d, 2.0, H-4 ),
ꢀ
.15 (1H, m, H-2 ), 5.07 (1H, dd, 4.0, 6.0, H-4), 4.86 (1H, brd, 17.6,
ꢀ
ꢀ
H-6 ), 4.63 (1H, brd, 17.6, H-6 ), 4.55 (1H, d, 9.6, H-1), 4.45 (1H, m,
ꢀ
H-5), 4.35 (1H, dd, 2.8, 12.4. H-1 ), 4.30 (1H, d, 9.6, H-1), 4.22 (1H,
dd, 5.6, 12.4, H-6), 4.10 (1H, dd, 5.2, 12.4, H-1 ), 4.05 (1H, dd, 4.8,
ꢀ
13
1
2.4, H-6), 2.15, 2.10, 2.05, 2.04 (x3), 2.02, 2.01 (8s, CH ); C NMR
3
ꢀ
(
CDCl ) ıC 197.9 (s, C-2 ), 179.9, 170.4, 169.8, 169.6, 169.5, 169.4,
3
1
7
1
2
69.02, 169.0 (8s, C O), 107.9 (s, C-2), 80.6 (d, C-5), 78.4 (d, C-3),
ꢀ
ꢀ
ꢀ
6.1 (d, C-4), 74.2 (d, C-3 ), 68.3 (d, C-4 ), 68.0 (d, C-5 ), 66.6 (t, C-
2
1
.7. Cell based assay for antioxidant activity (Rosenkranz et al.,
992; Scudiero et al., 1988)
ꢀ
ꢀ
), 62.8 (t, C-6 ), 61.7 (t, C-6), 61.5 (t, C-1), 20.7 (x2), 20.6 (x2), 20.5,
+
0.4, 20.3, 20.2 (8q, CH ); HRESIMS m/z 696.2330 [MH O] (calcd.
3
2
+
for C28H42O19N, 696.2351); C28H38O19; 701.1903 [MNa] ; (calcd.
for C28H38O19Na, 701.1905).
The effect of samples on the generation of ROS in
Myelomonocytic HL-60 cells is determined by the DCFH-DA
ꢀ ꢀ
2 ,7 -dichlorofluorescin diacetate) method. HL-60 cells (ATCC)
(
2.5. Inhibition kinetics assay using recombinant human MAO-A
were cultured in RPMI-1640 medium with 10% fetal bovine serum,
penicillin (50 units/mL) and streptomycin (50 ␮g/mL). 125 ␮L of
and MAO-B
6
the cell suspension (1 × 10 cells/mL) was added to the wells of a
Recombinant human monoamine oxidase A (MAO-A) and
monoamine oxidase B (MAO-B) were obtained from BD Biosciences
Bedford, MA). To investigate the effect of extracts on MAO-A
and MAO-B, the kynuramine deamination assay was adapted for
6-well plates. The inhibition kinetics of MAO-A and MAO-B by
inhibitors/extracts was determined in a range expected to produce
0–90% inhibition. The IC50 values for extracts, pure compounds
9
6-well plate. After treatment with different concentrations (i.e.,
six concentrations of 62.50, 31.25, 15.63, 7.82, 3.91 and 1.95 ␮g/mL
were used) of the test samples for 30 min, cells were exposed
to 100 ng/mL phorbol 12-myristate-13-acetate (PMA, Sigma) for
(
9
3
0 min. DCFH-DA (Molecular Probes, 5 ␮g/mL) was added and
cells were further incubated for 15 min. Levels of fluorescent DCF
(produced by ROS catalyzed oxidation of DCFH) was measured
on a PolarStar Galaxy plate reader with excitation wavelength
at 485 nm and emission at 530 nm. Inhibition of ROS generation
by test samples is determined in terms of % decrease in DCF
production compared to the vehicle control. Vitamin C and trolox
were used as the positive control in each assay. DCFH-DA is a
non-fluorescent probe that diffuses into cells, where cytoplas-
3
and reference standards were determined at five concentrations
of 1, 0.1, 0.01, 0.001 and 0.0001 ␮g/mL for MAO-A, where as for
MAO-B, percent inhibitions of all crude extracts were evaluated at
three concentrations, 100, 10 and 1 ␮g/mL. All experiments were
carried out in duplicate. A fixed substrate concentration and vary-
ing inhibitor concentrations were used to determine the IC50 value
at the point where 50% inhibition of the catalytic activity of the
enzyme occurred. For MAO-A, the substrate concentration of 80 ␮M
kynuramine was chosen because the apparent Km value for sub-
strate binding has been reported previously was approximately
ꢀ
ꢀ
mic esterases hydrolyse the DCFH-DA to 2 ,7 -dichlorofluorescin
DCFH). The ROS generated within HL-60 cells oxidize DCFH to
(
ꢀ
ꢀ
the fluorescent dye 2 ,7 -dichlorofluorescein (DCF). The ability of
the test materials to inhibit exogenous cytoplasmic ROS-catalyzed
oxidation of DCFH in HL-60 cells is measured in comparison to PMA
treated vehicle control. The cytotoxicity of samples to HL-60 cells
4
0 ␮M (Parikh et al., 2002). Since Km is the substrate concentra-
tion at half Vmax, therefore, 2 × Km (2 × 40 = 80 ␮M), was selected for
determining IC values. Similarly, for MAO-B, substrate concentra-
4
5
0
was also determined after incubating the cells (2 × 10 cells/well in
tion of 50 ␮M kynuramine was chosen. The assay was performed
with the addition of inhibitor. Inhibition was calculated as per-
cent of product formation compared to the corresponding control
2
25 ␮L) with test samples for 48 h by XTT method. Briefly, 25 ␮L of
XTT-PMS solution (1 mg/mL XTT solution supplemented by 25 ␮M
of PMS) was added to each well. After incubating for 4 h at 37 C,
absorbance was measured at a dual wavelength of 450–630 nm on
a Bio-Tek plate reader.
◦
(
enzyme–substrate reaction) without the inhibitors. The reac-
tions were carried out in 0.1 M potassium phosphate buffer at pH
.4. Incubations mixtures contained 5 ␮g/mL of MAO-A (50 ␮L in
7
buffer) and 10 ␮g/mL of MAO-B (50 ␮L in buffer). The inhibitor
was dissolved in DMSO or in buffer (if not dissolved in DMSO).
The total reaction volume was 200 ␮L yielding a final DMSO con-
centration of 1.0% in the reaction mixture. The reaction mixtures
2
.8. Inhibition assay for acetylcholinesterase,
butyrylcholinesterase and catechol-O-methyl transferase enzymes
activity
◦
were pre-incubated for 10 min at 37 C followed by the addition of
Acetylcholinesterase, butyrylcholinesterase and catechol-O-
methyl transferase enzymes assays were carried out as described
previously (Ellman et al., 1961; Learmonth et al., 2004).
MAO-A/MAO-B to initiate the reactions. Reactions were incubated
for 20 min at 37 C and were stopped immediately by the addition
of 75 ␮L of 2N NaOH. The formation of 4-hydroxyquinoline was
◦

3
64
V. Samoylenko et al. / Journal of Ethnopharmacology 127 (2010) 357–367
3
. Results and discussion
firmed by a 2D NMR HMBC experiment (Table 2), which showed key
correlations between H-1 and C-3, C-4a, C-9a, C-10, C-11 and C-14,
ꢀ
The hot aqueous extracts of fresh and dried large branches of
and H-10 and C-1, C-1 (i.e., anomeric carbon), C-9a and C-12, con-
Banisteriopsis caapi demonstrated significant MAO-A inhibitory and
antioxidant activity (Table 1). A bioassay-guided fractionation of
crude aqueous extract of Banisteriopsis caapi by reversed-phase
column chromatography (RPCC) afforded a polar non-alkaloidal
fraction by eluting with MeCN, which showed potent antioxidant
and moderate MAO-B inhibitory activities. This was followed by
firming the formation of a seven-member ring between C-1 and N-2
1
10
11
12
13
14
2
of THH skeleton (– C– C– C– C– C– C– N–), and the linkage
ꢀ
of C-1 of glucose moiety at C-10. HMBC further showed cross-
peaks between N -H (ıN–H 8.45) and C-4a, C-4b, C-8a and C-9a,
9
and between C-7–OMe (ıH-7–OMe 3.84) and C-7 (ı 156.8), confirm-
C
ing the presence of a free indole N–H proton and –OMe group at
C-7. Finally, the configuration of compound 10 was assigned by a
Circular Dichroism spectra and NOESY experiment (vide infra).
Compound 11 was also dextrorotatory {[˛]D +31.1 (c 0.4,
an alkaloidal fraction, eluted with MeCN–H O as solvent, which
2
showed potent MAO-A and moderate MAO-B inhibitory activites.
Subsequent RPCC of polar alkaloidal fraction yielded two new
alkaloidal glycosides 1 and 2, as major markers, while polar non-
alkaloidal fractions afforded the potent antioxidants 8 and 9.
Furthermore, centrifugal preparative TLC (CPTLC) of alkaloidal frac-
tions yielded the tetrahydronorharmine (4), and four known MAO
inhibitors ˇ-CA’s 3 and 5–7.
CHCl )}, and analyzed for C
H
N O by ESIHRMS. The 1H and
37 44 2 18
C NMR (Table 2) showed the presence of seven acetyl groups, and
3
13
its THH carbon skeleton fused with a seven-member ring, via five
oxygenated carbons C-10–C-14, was found to be similar to those
of acetate 10. The 1H NMR spectrum of 11 was generally similar to
those observed for 10 except for significant shieldings of chemical
3.1. Structure elucidation
shift values for protons H-1, H-10 and H-12, and deshieldings of
ꢀ
H-11, H-13 and H-1 , compared to those observed in 10 (ı
5.08,
H-1
Compound 1 was analyzed for C23H30N O by ESIHRMS. The
ıH-10 3.98, ıH-11 5.77, ıH-12 5.32, ıH-13 5.87, ıH-1
ꢀ
4.91 vs. ıH-1 5.98,
4.54, respec-
2
11
UV spectrum of 1 demonstrated absorption bands at ꢀmax 226,
69 and 290 nm, typical of THH chromophore (Hashimoto and
Kawanishi, 1975, 1976), and IR spectrum showed absorption bands
ıH-10 4.32, ıH-11 5.20, ıH-12 5.61, ıH-13 5.72 and ıH-1
ꢀ
2
tively, for 10). A COSY spectrum showed correlations between ıH-10
3.98, ıH-11 5.77, ıH-12 5.32 and ıH-13 5.87, and between ıH-10 3.98
(brd, J = 10.0 Hz) and ıH-1 5.08 (d, J = 10.0 Hz), suggesting a similar
system [–CH–CH(OR)–CH(OAc)–CH(OAc)–CH(OAc)–] as observed
for 10, although the chemical shift values for these protons were
−1
at ꢁmax 3550 and 1740 cm , attributed to hydroxyl and amide
ıC-14 175.3) groups, respectively. The NMR spectrum of 1, recorded
in CF COOD + D O, revealed the presence of three aromatic protons
(
3
2
13
ıH-5 6.90, ıH-6 6.19 and ıH-8 6.35, a methine proton at ıH-1 4.44,
two methylene groups at ıH-3 3.05 and 3.31 (each 1H), and ıH-4
different to those of 10. Likewise, the C NMR also displayed sig-
ꢀ
nificant deshielding of C-10 and C-1 (ı
81.2, ıC-1 101.6 vs.
ꢀ
C-10
2
.51 (2H), and a methoxyl group at ıH 3.23, suggested the presence
ıC-10 72.9, ıC-1 96.7), shielding of C-11 and C-14 (ıC-11 69.5, ıC-14
ꢀ
of a C-1 substituted THH nucleus. It also showed four oxymethine
protons at ıH-10 4.23, ıH-11 4.44, ıH-12 4.09 and ıH-13 4.04, either as
broad singlets or multiplets, which showed 2D NMR COSY corre-
lations for the system [–CH(OH)–CH(OH)–CH(OH)–CH(OH)–]. The
164.4 vs. ıC-11 77.2, ıC-14 170.6, respectively, for 10), compared to
those observed for 10, suggesting compound 11 appeared to be
a diastereoisomer of 10. The gross structure was established by
2
3
HMBC experiments, which revealed J- and J-correlation of pro-
tons and carbons of ring A–C, and glucose moiety involving H-1
and C-3, C-4a, C-9a, C-10, C-11 and C-14, and H-10 and C-1, C-
13
C NMR spectrum showed the presence of a glucose moiety (ıC-1
4.4, ıC-2 71.8, ıC-3 71.7, ıC-4 66.1, ıC-5 72.3 and ı 61.5), and a
ꢀ
9
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
C-6
13
ꢀ
methoxyl group (ıC-7–OMe 54.3). In addition, the C NMR showed
signals for C-1–C-9a of THH base skeleton, four oxymethine car-
bons and a quaternary amide carbonyl, which were assigned by
using 2D NMR HMQC and HMBC experiments of 1. Compound 2
was also analyzed for C23H30N O by ESIHRMS and its UV spec-
1 , C-9a and C-12. The presence of a free indole N–H proton and
C-7–OMe group was also confirmed by HMBC, which showed corre-
lation between N –H (ıN–H 8.45) and C-4a, C-4b, C-8a and C-9a, and
9
between C-7–OMe (ıH-7–OMe 3.84) and C-7 (ıC 156.8), respectively,
as observed for 10.
2
11
trum demonstrated absorption bands at ꢀmax 226, 267 and 294 nm,
as observed for 1. The presence of amide carbonyl and hydroxyl
groups was suggested from the IR spectrum. Compound 2 was
highly insoluble in most of the NMR solvents, like 1, and there-
fore, the spectroscopic analyses were performed on the acetates
for both the compounds.
The absolute stereochemistry of C-1 stereocenter of compounds
10 and 11 was established by Circular Dichroism spectra and NOESY
correlations. The CD spectra of the two compounds displayed a high
amplitude strong positive Cotton effect in the 310–320 nm regions,
which is explicable to C-1(R) chiral center with ˛-orientation of H-1.
These data was in agreement with C-1(R) mitragynine and analogs
(Lee et al., 1967) with strong positive Cotton effect at 270–300 nm
regions, while C-1(S)-speciogynine (Lee et al., 1967) and C-1(S)-
cadambine (Brown and Fraser, 1974) exhibited strong negative
Cotton effect in these regions in their CD spectra. In addition, the
Acetylation of a fraction (L-1) containing mixture of 1 and 2
afforded the corresponding heptaacetates 10 and 11, which were
subsequently separated and purified by CPTLC (see Section 2).
Compound 10 was analyzed for C37H44N O by ESIHRMS, and
2
18
1
13
1
found to be levorotatory; [˛]D −8.7 (c 0.6, CHCl ). Its H and
C
H NMR spectrum showed small coupling constant (J = 4.3 Hz) of
3
NMR spectra displayed signals for seven acetyl groups, a methoxyl
10, compared to large value (J = 10 Hz) for 11, suggesting cis- and
trans-relationships between H-1 and H-10 protons in these two
compounds, respectively. Based on this assumption, the spatial ori-
entation of the relevant protons was confirmed by NOESY spectrum
of 10 and 11 (Fig. 2). Compound 10 showed correlation between H-
1, H-3a, H-11 and H-12, and H-10 and H-3a, indicating that these
protons are ˛-cofacially oriented, thereby C-10-glucosyl, and C-
11 and C-12-acetoxy groups were on the ˇ-face of the molecule.
In addition, NOESY spectra also showed cross-peaks between H-
and a glucose moiety (Table 2). The 1H NMR spectrum of 10, in
CDCl , demonstrated multiplicities for all the protons, previously
3
not observed for 1 in CF COOD + D O, and structure 1 was fully sub-
3
2
stantiated by 2D NMR COSY, HMQC, HMBC and NOESY experiments
on 1-Ac (10). The presence of tetra-O-acetyl-ˇ-d-glucose moiety
(
[
Baddeley et al., 2005) was suggested from its C and 1H NMR
1
3
ꢀ
ꢀ
ıH-12 96.7, 71.4, 72.1, 68.6, 72.8, 62.2; C-1 –C-6 , respectively; ı
ꢀ
H-1
4
.54 (d, J = 4.8 Hz)]. A 2D NMR COSY spectrum showed correlations
ꢀ
ꢀ
ꢀ
between four acetoxy protons at ıH-10 4.32, ıH-11 5.20, ıH-12 5.61
and ıH-13 5.72, together with the correlation between ıH-10 4.32
1 and H-1 and H-5 , and H-5 and H-11, suggesting they are in
close spatial proximity among themselves. A close comparison of
the NOESY spectra of 11 with those of 10 revealed clear differences
for the stereocenters C-10 and C-13, which was earlier indicated
by the differences in 1H NMR chemical shift values for H-10–H-13
(
brd, J = 4.3 Hz) and ı
5.98 (d, J = 4.3 Hz), suggesting C-10 of the
H-1
1
10
11
12
13
system [– CH- CH(OR)- CH(OAc)– CH(OAc)– CH(OAc)–] was
connected with C-1 of THH base skeleton. The structure was con-

V. Samoylenko et al. / Journal of Ethnopharmacology 127 (2010) 357–367
365
gel/solvent: CHCl –MeOH (2:8)] and not reactive to Dragendorff’s
3
reagant. The 1H and C NMR spectra of 4 was generally similar to
those observed for THH (5), except for the differences associated
with the presence of a methylene group at C-1 (ıH-1 4.15, 2H, m; ıC
13
4
1
1.1, t), instead of a methine substituted with a methyl group (ıH-1
H, m; ıC 50.9, d; ıH-10 1.69, d, J = 6.4 Hz; ıC 18.1) of 5. In addition,
a close comparison between the NMR spectra of 4 and other THH
analogs (Faizi and Naz, 2002), led to the conclusion that 4 contained
a methylene group instead of the methine group at C-1 of THH, sug-
gesting 4 was a C-1 nor-derivative of THH (5). Complete assignment
of the spectral data was carried out using 2D NMR experiments, due
to unavailability of spectral data in the literatures. The HMBC exper-
iment showed three bond correlations between H -1, C-3, C-4a and
2
two bond correlations between H -1 and C-9a. It also showed cor-
2
relations between H-5, C-6 and C-7, and the H7–OMe group showed
cross-peak with C-7, suggesting the substitution of the OMe was at
C-7. Based on the foregoing data compound 4 was established as
THNH.
During the course of the investigation, three sugars, ˇ-d-
fructofuranosyl (2 → 5) fructopyranose (d-difructose) (14), sucrose
and ˇ-d-glucose were isolated as major primary metabolites.
Compound 14 was isolated as gum and homogenous on TLC [Rf
13
0
.35, silica gel/solvent: CHCl –MeOH (3:7)]. Its C NMR spec-
3
trum was generally similar to those observed for fructan-type
disaccharide with two fructofuranosyl units (Timmermans et
al., 2001), which displayed 12 major carbon signals consisting
four triplets, six doublets and two singlets, the latter assigned
ꢀ
to anomeric carbons at C-2 and C-2 (ıC-2 101.7, ıC-2
ꢀ
97.8 in
13
CD OD). Comparison of the C NMR data of 14 with those of ˇ-d-
3
fructofuranosyl(2 → 2)ˇ-d-fructofuranose, isolated from Morinda
citrifolia (Lin, 2004), suggested the presence of a non-symmetrical
dimmer for 14, instead of a symmetrical dimmer, which reported
only six 13C NMR signals due to symmetrical nature of the molecule.
On acetylation fraction (H-1) containing 14 as a major compound
afforded the corresponding octaacetate 17 by using CPTLC (see Sec-
tion 2). Compound 17 was analyzed for C28H38O19 by positive ion
1
13
ESIHRMS, and found to be dextrorotatory. Its H and C NMR spec-
tra (in CDCl ) displayed signals for eight acetyl groups, in addition
3
to 12 carbon signals consisting four triplets, six doublets and two
singlets, the latter assigned to one anomeric carbon (ıC-2 107.9)
and a carbonyl (ıC-2
ꢀ
197.9), suggesting one of the anomeric cen-
1
ter of 14 was opened during acetylation. The H NMR spectrum
of 17 demonstrated multiplicities for all the protons, previously
not observed for 14, and structure 14 was fully substantiated by
2D NMR COSY, HMQC, HMBC and NOESY experiments on 14-Ac
Fig. 2. 2D NMR NOESY correlations of 10 and 11.
(
17). The HMBC experiment showed key 2J- and 3J-correlations
between C-2 and H -1, H-3 and H-4, and C-5 and H-3 and H -6 for
2
2
ꢀ
the fructofuranose unit. It also showed correlations between C-2
between the two compounds. A NOESY experiment of 11 (Fig. 2)
showed correlation between H-1, and H-11 and H-13, as well as
H-11 and H-12, suggesting these protons are cis and placed at the
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
and H -1 , H-3 and H-4 , and C-5 and H-4 , and H -6 , attributable
2
2
to acetylated acyclic fructose unit of 17. Based on the foregoing
data compound 14 was established as ˇ-d-fructofuranosyl-(2 → 5)-
fructopyranose.
˛
-face of the molecule, since H-1 had ˛-disposition due to C-1(R)
absolute configuration, like 10, determined by CD spectra. There-
fore, the acetyl groups at C-11, C-12 and C-13 were placed on
the ˇ-face of the molecule. The placement of C-10 glucosyl moi-
ety at the ˛-face of the molecule was evident from the large trans
coupling (J1,10 = 10 Hz) between H-1 and H-10, as well as NOESY
The structures of harmol (3), THH (5), harmaline (6) (Coune
et al., 1980; Atta-ur-Rehman and Tayyaba, 1972), harmine (7),
(−)-2R,3R-epicatechin (8) (Balde et al., 1991; Sun et al., 2006)
and (−)-2R,3R-epicatechin-4ˇ,8-(−)-2R,3R-epicatechin (procyani-
din B2) (9) (Barrett et al., 1979; Korver and Wilkins, 1971; Khan et
ꢀ
correlations between H-10 and H-1 , suggesting their close spa-
1
tial relationships in 11. Analyses of the molecular models further
al., 1997) were determined by physical and spectroscopic data ( H
13
confirmed the above observations. In addition, NOESY spectra also
and C NMR, 2D NMR experiments, and MS) as well as by direct
ꢀ
ꢀ
showed cross-peaks between the protons H-1 , H-5 , and H-11, and
comparison, except for 9, with their respective authentic samples.
ꢀ
1
13
H-6 a and H-11, suggesting their close spatial proximity. Collec-
The H and C NMR spectra of harmaline (6) in CD OD exhibited an
3
tively, these data permitted the assignment of configuration for
compounds 10 and 11, and unequivocally confirmed the structures
of banistenoside A (1) and banistenoside B (2).
equilibrium between imine-enamine tautomeric forms (Coune et
al., 1980; Atta-ur-Rehman and Tayyaba, 1972). The imine was pre-
dominant in DMSO-d6 (aprotic solvent), while both the tautomers
Compound 4 was isolated as gum and analyzed for C12H14N O
were detected in CD OD/trifluroacetic acid (1 drop) as mixtures
2
3
by ESIHRMS. This compound was homogenous on TLC [Rf 0.40, silica
in the NMR spectra. The structures of 8 and 9 were confirmed

3
66
V. Samoylenko et al. / Journal of Ethnopharmacology 127 (2010) 357–367
by chemical derivatization to their corresponding peracetate 12
and 13. Compounds 9 and 13 adopt two conformations in solu-
and mammalian VERO and kidney cell lines (at 10 ␮g/mL), and were
found to be inactive.
◦
tion, in which the interflavan dihedral angles are roughly ± 90 .
The preferred conformation of the pyran ring is a half-chair with
the 3,4-dihydroxyphenyl substituents in pseudo-equatorial posi-
tions (Khan et al., 1997). The absolute configuration of 8 and 9 was
confirmed by optical rotation and Circular Dichroism experiments,
which is consistent with the CD spectra of reference (−)-2R,3R-
epicatechin (for 8) and literature data (for 9) (Korver and Wilkins,
3.3. HPLC analyses and quantification
The isolated markers (1–9) were employed for chemical pro-
filing of Banisteriopsis caapi Da Vine, thereby markers 1–9 were
consistently assigned and quantified from aqueous extracts using
a reverse phase C-18 column by HPLC according to published
methods for ˇ-carboline alkaloids (Abourashed et al., 2003) (using
harmine, harmaline, harmol and harmane as standards) and green
tea catechins/proanthocyanidines (Khokhar et al., 1997) (using cat-
echin and epicatechin as standards). HPLC analysis revealed that
most of the dominant chemical and bioactive markers were present
in high concentrations in dried bark of large branch [1 (0.71%), 2
1
971; Barrett et al., 1979).
3.2. Biological activity
The hot aqueous extracts of fresh and dried large branches of
Banisteriopsis caapi and isolated compounds 1–9 were evaluated
for MAO inhibitory and antioxidant activities, as well as cyto-
toxicity against selected human cancer and mammalian (VERO)
cells. Assays for MAO-A and MAO-B inhibitions were carried out
fluorometrically at 460 nm emission wavelength using recombi-
nant human brain MAO-A and B enzymes, which showed strong
inhibitory activities against MAO-A (IC50 ∼0.01–0.4 ␮g/mL), and
weak activities against MAO-B (i.e., ∼25% inhibition at 5 ␮g/mL) for
most of the extracts. During the measurement of MAO-B activity,
strong fluorescence interferences at 460 nm emission wavelength
were observed at a concentrations of >5 ␮g/mL, which interfered
with IC50 determinations. To resolve fluorescence interference,
a modified standardized protocol with an emission wavelength
of 380 nm was employed (Parikh et al., 2002). Using this proto-
col, percent inhibitions were determined at 1, 10 and 100 ␮g/mL
for MAO-B, while the MAO-A inhibitions remained closely sim-
ilar to those measured at 460 nm. The extracts of fresh and
dried samples of bark/debarked large branch demonstrated potent
MAO-A inhibition (IC50 0.02–0.05 ␮g/mL) and modest MAO-B
inhibitory activities (IC50 ∼100 ␮g/mL) (Table 1). MAO-A activ-
ity of extracts of fresh/dried stem or bark/debarked large branch
was found to be 2500-fold more potent than MAO-B. The activ-
ity of most potent MAO-A inhibitors harmine (7) or harmaline
(1.93%), 5 (0.10%), 7 (0.67%), 8 (0.43%) and 9 (0.16%)]. Analysis of reg-
ular/commercial Banisteriopsis caapi dried stems showed a similar
qualitative HPLC pattern, but relatively low- content of dominant
markers 1 (0.15, 0.17%), 2 (0.07, 0.15%), 7 (0.04, 0.13%) and 9 (0.03,
0
.38%) in samples BCEx-1 and BCEx-4, respectively, which led to
decreased MAO and antioxidant potency in these samples. How-
ever, two other regular/commercial samples (BCEx-2 and BCEx-3)
were found to be inactive and devoid of markers 1–9.
4. Conclusion
This appears to be the first report of banistenoside A (1)
and banistenoside B (2), containing an unique “azepino[1,2-
a]tetrahydro-ˇ-carboline” carbon framework, either from natural
or synthetic sources. Presumably
1 and 2 appear to be
biogenetically analogous to mitragynine-type alkaloid dihydro-
cadambine (Brown and Fraser, 1974) with the unusual N-4–C-18
bond (i.e., using mitragynine carbon numbering system) in a
seven-membered ring. Therefore, the biogenetic pathway for 1(R)-
banistenoside A and B can be envisioned from the precursor of
indole alkaloids 3˛-H strictosidine via 3˛-H epoxystrictosidine, fol-
lowed by trioxoaglycone (Szabó, 2008), which undergoes extensive
decrboxylation, cyclyzation, oxidation and glycoxylation to 1 and
2. Interestingly, a synthetic azepino-tetrahydro-ˇ-carboline [CAS
610782-53-1] related to the compound 1 or 2 demonstrated potent
˛2-adrenoceptor activity (Din Belle et al., 2003), therefore, fur-
ther biological work on 1 and 2 is warranted. In addition, THNH
(4) and ˇ-d-fructofuranosyl-(2 → 5)-fructopyranose (14) were iso-
lated for the first time from a natural source, and (−)-epicatechin
(8) and (−)-procyanidin B2 (9) for the first time from the genus
Banisteriopsis. During the course of the investigation, 6-methoxy-
THH (pinoline) or its corresponding C-5 oxygenated tryptamine
analogs, which have potential hallucinogenic or behavioral prop-
erties (Rothchild, 2005; Pahkla et al., 1996), were neither identified
nor isolated from this cultivar, or regular/commercial samples. It
is also noteworthy that all the isolated ˇ-carbolines 1–7 are oxy-
genated at C-7 position, suggesting they are biogenetically derived
from corresponding C-6 oxygenated tryptamine precursor.
(
(
6) was found to be >10,000-fold more potent than MAO-B
IC50 2.0 nM/2.5 nM vs. 20 ␮M/25 ␮M), which was proportion-
ately consistent with those observed for the crude extract. The
activity of 3 and 5 was found to be much weaker than 6 or
7
(
against MAO-A, while compounds 1, 2 and 4 were inactive
Table 1).
In addition, strong antioxidant activity (Rosenkranz et al.,
992; Scudiero et al., 1988) for inhibition of cellular Reactive
1
Oxygen Species (ROS) generation by phorbol-12-myristate-13-
acetate (PMA) was observed for large branches (IC50 0.4–3.0 ␮g/mL)
(
Table 1). (−)-Epicatechin (8) and (−)-procyanidin B2 (9) demon-
strated potent antioxidant activity; both found to be more potent
than vitamin C (IC50 < 0.13 and 0.57 ␮g/mL vs. 1.34 ␮g/mL), while
8
was also more effective than trolox (IC50 0.14 ␮g/mL). Both the
compounds also showed MAO-B inhibitory activity (IC50 65 and
3
8
5 ␮M, respectively) and weak MAO-A inhibitions (IC50 51.7 and
.5 ␮M for 8 and 9, respectively). However, epicatechin and cat-
echin had previously been reported as MAO-B inhibitors (IC50
8.9 ␮M) (Hou et al., 2005). Evaluation of regular sample of Banis-
teriopsis caapi dried stem (BCEx-1 and BCEx-4) showed consistent
MAO-A inhibitory and antioxidant activity, but was relatively less
potent than bark of large branch of Da Vine for MAO-A inhibitory
Inhibition of MAO-B activity by ˇ-carbolines harmine (7) and
harmaline (6), in addition to potent MAO-A inhibition responsible
for antidepressant activity, provide protection against neurodegen-
eration, and has a potential therapeutic value for the treatment
of Parkinson’s diseases. In addition, oxidative stress induced by
ROS has been strongly associated with the pathogenesis of neu-
rodegenerative disorders, including Parkinson’s and Alzheimer’s
disease (Barnham et al., 2004). Therefore, the presence of two
potent antioxidants, (−)-epicatechin (8) and (−)-procyanidin B2
(9), have significant added value for the protection of neuronal cells
damage by oxidative free radicals. Their selective MAO-B inhibitory
activity (Hou et al., 2005) benefits them even more. Usefulness of
5
(
1
IC50 0.32–0.39 ␮g/mL vs. 0.02–0.03 ␮g/mL) and antioxidant (IC50
.0–7.8 ␮g/mL vs. 1.9–2.0 ␮g/mL) activity. Finally, extracts from
different parts of large branch, together with regular/commercial
samples, were tested against acetylcholinesterase (AChE), butyl-
cholinesterase (BuChE) and catechol-O-methyl transferase (COMT)
enzymes (100 ␮g/mL), and cytotoxic activities (Borenfreund et al.,
1
990) against human breast, melanoma, skin and prostate cancer,

V. Samoylenko et al. / Journal of Ethnopharmacology 127 (2010) 357–367
367
compounds 8 and 9 was reported for amyloid diseases (Castillo
et al., 2002, 2004), as well as protection against PC12 cells from
Aˇ-induced neurotoxicity by compound 8 (Cho et al., 2008; Heo
and Lee, 2005). In addition, proanthocyanidin dimers, including 9,
were recently reported for treating amyloid, ˛-synuclein or NAC
fibrillogenesis in a mammalian subject (Castillo et al., 2004). Col-
lectively, these results give additional basis to the existing claim of
Banisteriopsis caapi stem extract for the treatment of Parkinsonism,
including other neurodegenerative disorders.
Hashimoto, Y., Kawanishi, K., 1975. New organic bases from amazonian Banisteriop-
sis caapi. Phytochemistry 14, 1633–1635.
Hashimoto, Y., Kawanishi, K., 1976. New alkaloids from Banisteriopsis caapi. Phyto-
chemistry 15, 1559–1560.
Heo, H.J., Lee, C.Y., 2005. Epicatechin and catechin in cocoa inhibit amyloid ˇ-protein
induced apoatosis. Journal of Agricultural and Food Chemistry 53, 1445–1448.
Hochstein, F.A., Paradies, A.M., 1957. Alkaloids of Banisteria caapi and Prestonia ama-
zonicum. Journal of the American Chemical Society 79, 5735.
Hou, W.C., Lin, R.D., Chen, C.T., Lee, M.H., 2005. Monoamine oxidase B (MAO-B)
inhibition by active principles from Uncaria rhynchophylla. Journal of Ethnophar-
macology 100, 216–220.
Kawanishi, K., Saiki, K., Tomita, H., Tachibana, Y., Farnsworth, N.R., Bohike, M., 1998.
Chemical components of the Brazilian shamanistic drink “Ayahuaska”. Advances
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Acknowledgement
Khan, M.L., Haslam, E., Williamson, M.P., 1997. Structure and conformation of the
procyanidin B2 dimer. Magnetic Resonance in Chemistry 35, 854–858.
The authors sincerely thank Dr. Larry A. Walker, NCNPR, Univer-
sity of Mississippi and Professor Keith Tipton, University of Dublin,
Ireland, for their valuable advise, suggestions and comments on the
project; Mr. Raymond F. Baker, Harold Lyon Arboretum, University
of Hawaii, for his kind collection and supply of fresh plant mate-
rials from Oahu, HI, and Mr. Frank Wigger and Dr. Bharati Avula,
NCNPR, UM, for NMR support and Mass spectra, respectively. This
work was supported by the National Center for Complementary and
Alternative Medicine (NCCAM), National Institute of Health, Grant
No. 5R21AT003409-02.
Khokhar, S., Venema, D., Hollman, P.C., Dekker, M., Jongen, W., 1997.
HPLC method for the determination of tea catechins. Cancer Letters 114,
71–172.
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