Phytotoxicity of sarmentine isolated from long pepper (Piper longum) fruit

Article abstract of DOI:10.1021/jf102087c

Discovery of novel natural herbicides has become crucial to overcome increasing weed resistance and environmental issues. In this article, we describe the finding that a methanol extract of dry long pepper (Piper longum L.) fruits is phytotoxic to lettuce (Lactuca sativa L.) seedlings. The bioassay-guided fractionation and purification of the crude extract led to isolation of sarmentine (1), a known compound, as the active principle. Phytotoxicity of 1 was examined with a variety of seedlings of field crops and weeds. Results indicated that 1 was a contact herbicide and possessed broad-spectrum herbicidal activity. Moreover, a series of sarmentine analogues were then synthesized to study the structure-activity relationship (SAR). SAR studies suggested that phytotoxicity of sarmentine and its analogues was specific due to chemical structures, i.e., the analogues of the acid moiety of 1 were active, but the amine and its analogues were inactive; the ester analogues and amide analogues with a primary amine of 1 were also inactive. In addition, quantification of 1 from different resources of the dry P. longum fruits using liquid chromatography-mass spectrometry showed a wide variation, ranging from almost zero to 0.57%. This study suggests that 1 has potential as an active lead molecule for synthesized herbicides as well as for bioherbicides derived from natural resources.

Full text of DOI:10.1021/jf102087c

9994 J. Agric. Food Chem. 2010, 58, 9994–10000
DOI:10.1021/jf102087c
Phytotoxicity of Sarmentine Isolated from Long Pepper
(Piper longum) Fruit
HUAZHANG
H
UANG,* CHRISTY M. MORGAN, RATNAKAR N. ASOLKAR,
M
ARJA E. KOIVUNEN
,
AND
PAMELA G. MARRONE
Marrone Bio Innovations, 2121 Second Street, Suite B-107, Davis, California 95618
Discovery of novel natural herbicides has become crucial to overcome increasing weed resistance and
environmental issues. In this article, we describe the finding that a methanol extract of dry long pepper
(Piper longum L.) fruits is phytotoxic to lettuce (Lactuca sativa L.) seedlings. The bioassay-guided
fractionation and purification of the crude extract led to isolation of sarmentine (1), a known compound, as
the active principle. Phytotoxicity of 1 was examined with a variety of seedlings of field crops and weeds.
Results indicated that 1 was a contact herbicide and possessed broad-spectrum herbicidal activity.
Moreover, a series of sarmentine analogues were then synthesized to study the structure-activity
relationship (SAR). SAR studies suggested that phytotoxicity of sarmentine and its analogues was
specific due to chemical structures, i.e., the analogues of the acid moiety of 1 were active, but the amine
and its analogues were inactive; the ester analogues and amide analogues with a primary amine of 1
were also inactive. In addition, quantification of 1 from different resources of the dry P. longum fruits using
liquid chromatography-mass spectrometry showed a wide variation, ranging from almost zero to 0.57%.
This study suggests that 1 has potential as an active lead molecule for synthesized herbicides as well as
for bioherbicides derived from natural resources.
KEYWORDS: Piper longum; sarmentine; phytotoxicity
INTRODUCTION
high use rates of the raw materials, exploration of other phyto-
chemicals as bioherbicides or herbicide leads is necessary.
Utilization of synthetic herbicides not only prevents economic
loss in food production but also improves quality of crop
products (1). However, the use of synthetic herbicides may cause
adverse effects on the environment and human health (2,3), and it
has led to increasing resistance among many weed species (4).
Therefore, it is necessary to develop alternative means for weed
management that are ecofriendly, economical and efficacious (5).
Application of natural phytochemicals as weed management
provides an alternative to chemical herbicides (6). In nature, many
natural compounds have allelopathic properties. When released
in air or soil, they kill neighboring weeds or inhibit their germina-
tion and/or growth. Moreover, many of these compounds are
easily biodegradable due to environmental factors such as light,
oxygen, temperature and/or biological metabolic enzymes. These
phytotoxic chemicals include phenolic compounds (e.g., catechin,
ellagic acid, sorgoleone, juglone, ceratiolin, usnic acid), terpenoids
(e.g., 1,8-cineole, geranial, neral, cinmethylin, solstitiolide), quassi-
noids (e.g., ailanthone, chaparrine, ailanthinol B), benzoxazinoids
(e.g., hydroxamic acids), glucoinolates (e.g., glucohirsutin, hirsutin,
arabin), and some amino acids such as meta-tyrosine (7-9).
Some of the phytotoxic compounds such as clove oil (Matratec),
d-limonene (GreenMatch and Nature’s Avenger) and lemongrass
oil (GreenMatchEX) have been commercialized as bioherbicides in
the US for weed management. However, due to the high cost and
Long pepper, Piper longum L. (Piperaceae), is a slender aromatic
climber with perennial woody roots. It grows primarily in tropical
regions. The fruits, commonly known as “pippali” in India and “Bi
Bo” in China, are used as a spice and also as a preservative in
pickles. Whole pepper plants are also used as cattle feed. In tradi-
tional medicinal practice, P. longum fruits have been advocated to
be beneficial in the treatment of diseases and ailments such as
gonorrhea, menstrual pain, tuberculosis, sleeping problems, respira-
tory tract infections, chronic intestinal pain, and certain forms of
arthritis (10-13). Other reported beneficial effects of P. longum
include analgesic and diuretic effects, relaxation of muscle tension,
and alleviation of anxiety (14, 15). In addition, pipernonaline from
P. longum has been found to possess mosquito larvicidal activ-
ity (16). To our knowledge, phytotoxic compounds from Piper
species have never been reported before.
Herewith we are describing the isolation and structure elucidation
of sarmentine (1), a phytotoxic compound from P. longum, and the
phytotoxic spectrum of this compound against field crops and
weeds. The structure-activity relationship of sarmentine (1) was
preliminarily studied by synthesizing a series of its analogues. Finally,
the percent content of sarmentine (1) in different dry P. longum fruit
samples obtained from different sources was then investigated.
MATERIALS AND METHODS
˚
Chemical. Sepra C18-E (50 μM and 60 A) and silica gel sorbent
*Corresponding author. Fax: 530-750-2808. E-mail: hhuang@
marronebio.com.
(70-230 mesh size) were purchased from Phenomenex (Torrance, CA)
pubs.acs.org/JAFC
Published on Web 08/17/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 18, 2010 9995
and Fisher Scientific (Fair Lawn, NJ), respectively. Polyoxyethylene
(20) sorbitan monolaurate (i.e., Glycosperse O-20 KFG) and sodium
lauryl sulfate (i.e., SLS) were obtained from Lonza Inc. (Allendale, NJ)
and Spectrum Chemical Mfg. Corp. (New Brunswick, NJ), respectively.
Cyclopentylamine, trans-cinnamic acid, ethyl trans-2-cis-4-decadienoate,
hexamethyleneimine, trans-2-decenoic acid, decanoic acid, and 4-di-
methylaminopyridine were purchased from ACROS Organics (Morris
Plains, NJ). 5-Biphenyl-4-ylmethyl-tetrazole-1-carboxylic dimethylamide
(LY2183240) was purchased from Sigma-Aldrich. All other chemicals
were reagent grade.
Fruits of P. longum and Pretreatment. Four samples of dry P.
longum fruits were purchased from Chinese medicinal herb stores. Two of
them were purchased from WAN FUNG Chinese Herb Shop in Rich-
mond, CA, on May 25, 2008, and March 23, 2009, respectively. The other
two samples were purchased from WAH TSUN Chinese Herb Co. in
Sacramento, CA, on June 2, 2008, and March 4, 2009, respectively. The
fruits were completely ground with a coffee grinder (Toastmaster Inc.,
Boonville, MI). The freshly ground powder of the fruits was extracted with
appropriate solvents as described below.
Superfund Analytical Core at Mass Spectrometry Laboratory in the
University of California at Davis.
Synthesis of Sarmentine Analogues. To the ice-cooled carboxylic
acid (3 mmol) solution in dichloromethane (20 mL) were sequentially added
1-ethyl-3-(30-dimethylaminopropyl)carbodiimide (3.3 mmol) and 4-dimethy-
laminopyridine (3 mmol). After 5 min, amine (3.3 mmol) was added to the
reaction solution. The reaction was slowly warmed to room temperature and
continued overnight. The reaction was extracted with ethyl acetate (200 mL),
and the organic phase was dried with anhydrous sodium sulfate. After
evaporation under vacuum, the residue was run through a silica gel column
with an appropriate ratio of ethyl acetate in hexane (i.e., from 1:10 to 1:4 =
ethyl acetate: hexane). The yields of the final products ranged from 85% to
90%. The final products (see Table 3) were characterized with proton NMR,
mass spectrum and melting point analyses. Melting point was measured by
OptiMelt (automated melting point system: SRS, Stanford Research Sys-
tems, Sunnyvale, CA). The ramp was set up at 1 °C/min.
N-Cyclopentyldecanamide (3): white solid, mp 47.3-48.5 °C; 1H
NMR (CDCl3) δ (ppm) 5.35 (br, 1H), 4.22 (m, J=7.00, 1H), 2.12 (t, J=7.20,
2H), 1.98 (m, 2H), 1.59-1.67 (m, 6H), 1.26-1.36 (m, 14H), 0.88 (t, J=7.00,
3H); ESIþ m/z=[M þ H]þ=240.
Weed and Crop Seedlings. All seedlings of weeds and crop plants
were planted in 8 ꢀ 8 ꢀ 7.2 cm or 10 ꢀ 10 ꢀ 9 cm plastic pots. All pots were
stored in a greenhouse with 28 °C temperature and 60% humidity.
Seedlings including pigweed (Amaranthus retroflexus L.), barnyard grass
(Echinochloa crus-galli L.), bindweed (Convolvulus arvensis L.), crabgrass
(Digitaria sanguinalis L.), dandelion (Taraxacum officinale F.), lambs-
quarter (Chenopodium album L.), annual bluegrass (Poa annua L.), wild
mustard (Brassica kaber L.), black nightshade (Solanum nigrum L.), curly
dock (Rumex crispus L.), horseweed (Conyza canadensis L.), sweet corn
(Zea mays L.) and wheat PR 1404 (Triticum aestivum L.) were planted in
potting soil mixture. Seedlings including rice M-104 (Oryza sativa L.),
sedge (Cyperus difformis L.) and sprangletop (Leptochloa fascicularis
Lam) were planted in mud which was collected adjacent to a rice field
(Woodland, CA). When treated, all seedlings were 15 days old except for
rice (10 days), wheat (20 days), corn (20 days), sprangletop (20 days), sedge
(20 days) and horseweed (70 days).
Bioassay-Guided Fractionation and Isolation. The active com-
pound was isolated by four major steps described as follows: (1) The
methanol extract of freshly ground P. longum fruit powder was screened in
a 96-well plate bioassay with Bibb lettuce (Lactuca sativa L.) seedlings, and
positive hits were obtained (i.e., death at 144 h after treatment). (2) The
methanol extract (0.5 g) was then subjected to separation through a reverse
phase C18 column and was eluted with 20%, 40%, 60%, 80% and 100%
methanol in water. Fractions were dried under vacuum and efficacy was
re-evaluated by 96-well plate bioassay with Bibb lettuce seedlings. The
active fraction, the most hydrophobic fraction, was used to guide the next
stepfor separation. (3) Ethyl acetate extract (17.6 g) was loaded into a flash
column. The column was sequentially eluted with hexane (1 L), hexane/
ethyl acetate (3:1, 1 L), hexane/ethyl acetate (1:1, 1 L), ethyl acetate (1 L)
and acetone (1 L). Based on indication of thin layer chromatography
(TLC), nine fractions were collected. The efficacy of each fraction was
evaluated by foliar spraying of barnyard grass. The concentration of each
fraction was 5 mg/mL with a carrier solution consisting of 4% ethanol and
0.2% glycosperse O-20 KFG. The active fractions (3.4 g) were combined
together and subjected to the next step. (4) A secondary silica column
separation was performed with a combination of hexane and ethyl acetate
(3:1) as an elution solvent to obtain the active ingredient (0.96 g). This
active ingredient (0.96 g) was recrystallized at -20 °C in a mixture of
hexane and ethyl acetate which yielded a crystal compound (0.83 g),
colorless oil at room temperature. Purity was examined by liquid chro-
matography and mass spectrometry (LC/MS). Detailed conditions for
LC/MS are described below in the later section of Materials and Methods.
Structural Analysis. Structural identification of the active compound
was based on data from both nuclear magnetic resonance (NMR) spectra
and high resolution mass spectrometry. NMR spectra including 1H, 13C,
DEPT, COSY, HMQC and HMBC were acquired from a Bruker Avance
600 spectrometer (Bruker BioSpin Corporation, Billerica, MA). Chemical
shift values are given in ppm downfield from an internal standard
(trimethylsilane). Signal multiplicities are represented as singlet (s), doub-
let (d), double doublet (dd), triplet (t), quartet (q), quintet (quint) and
multiplet (m). Exact mass of the active compound was determined by high-
performance liquid chromatography-tandem mass spectroscopy from
N-Cyclopentyldecen-2-amide (4): white solid, mp 71.8-72.5 °C; 1H
NMR (CDCl3) δ (ppm) 6.82 (dt, J1=15.20, J2=7.20, 1H), 5.71 (d, J=
15.20, 1H), 5.33 (br, 1H), 4.27 (m, J=7.00, 1H), 2.15 (m, 2H), 2.10 (m, 2H),
1.67 (m, 2H), 1.60 (m, 2H), 1.40 (m, 4H), 1.28 (m, 8H), 0.88 (t, J=7.00,
3H); ESIþ m/z=[M þ H]þ=238.
N-Cyclopentyl 2E,4Z-decadienamide (5): 1H NMR (CDCl3) δ
(ppm) 7.55 (dd, J1 = 14.80, J2=11.70, 1H), 6.06 (t, J=11.70, 1H), 5.79
(d, J1=14.80, 1H), 5.75 (m, 1H), 5.50 (br, 1H), 4.30 (m, J=7.00, 1H), 2.29
(q, J = 8.20, 2H), 2.01 (m, 2H), 1.68 (m, 2H), 1.61 (m, 2H), 1.40 (m, 2H),
1.28 (m, 6H), 0.88 (t, J=7.00, 3H); ESIþ m/z=[M þ H]þ=236.
N-Cyclopentyl, trans-cinnamamide (6): white solid, mp 144-145
°C; 1H NMR (CDCl3) δ (ppm) 7.62 (d, J = 15.6 Hz, 1H), 7.50 (d, J = 7.0
Hz, 2H), 7.35 (m, 3H), 6.37 (d, J = 15.6, 1H), 5.61 (d, J = 5.0, Hz, 1H,
NH), 4.35 (m, J = 7.0, 1H), 2.06 (m, 2H), 1.71 (m, 2H), 1.64 (m, 2H), 1.46
(m, 2H); ESIþ m/z = [M þ H]þ = 216.
N-(Decanoyl)pyrrolidine (7): 1H NMR (CDCl3) δ (ppm) 3.45 (t, J=
6.80, 2H), 3.40 (t, J = 6.80, 2H), 2.24 (t, J = 7.20, 2H), 1.94 (quint, J=
6.80, 2H), 1.84 (quint, J = 6.80, 2H), 1.62 (quint, J = 7.20, 2H), 1.25-1.30
(m, 12H), 0.87 (t, J=7.20, 3H); ESIþ m/z=[M þ H]þ=226.
N-(2-Decenoyl)pyrrolidine (8): 1H NMR (CDCl3) δ (ppm) 6.90 (dt,
J1 = 15.20, J2 = 7.00, 1H), 6.07 (d, J = 15.20, 1H), 3.52 (t, J = 6.30, 2H),
3.50 (t, J = 6.30, 2H), 2.19 (m, 2H), 1.96 (quint, J=7.00, 2H), 1.85 (quint,
J=7.00, 2H), 1.44 (m, 2H), 1.28 (m, 8H), 0.88 (t, J=7.00, 3H); ESIþ
m/z=[M þ H]þ=224.
(2E,4Z-Decadienoyl)pyrrolidine (9): 1H NMR (CDCl3) δ (ppm)
7.62 (dd, J1 = 14.60, J2=11.70, 1H), 6.17 (d, J=14.60, 1H), 6.13 (t, J=
11.70, 1H), 5.78 (m, 1H), 3.55 (t, J=7.00, 2H), 3.52 (t, J=7.00, 2H), 2.30 (q,
J=7.40, 2H), 1.97 (quint, J=7.40, 2H), 1.87 (quint, J=7.40, 2H), 1.40
(quint, J= 7.40, 2H), 1.29 (m, 4H), 0.88 (t, J= 7.00, 3H); ESIþ m/z=
[M þ H]þ = 222.
N-(trans-Cinnamoyl)pyrrolidine (10): white solid, mp 100.6-
101.7 °C; 1H NMR (CDCl3) δ (ppm) 7.70 (d, J=15.5 Hz, 1H), 7.53 (d,
J=7.0 Hz, 2H), 7.36 (m, 3H), 6.74 (d, J=15.5, 1H), 3.63 (t, J=7.0, 2H),
3.60 (t, J = 7.0, 2H), 2.01 (quint, J=7.0, 2H), 1.91 (quint, J=7.0, 2H);
ESIþ m/z = [M þ H]þ = 202.
N-(Decanoyl)piperidine (11): 1H NMR (CDCl3) δ (ppm) 3.55 (t, J=
5.20, 2H), 3.39 (t, J = 5.20, 2H), 2.31 (t, J = 7.60, 2H), 1.58-1.65 (m, 4H),
1.52-1.57 (m, 4H), 1.20-1.30 (m, 12H), 0.87 (t, J=7.20, 3H); ESIþ m/z=
[M þ H]þ=240.
N-(2-Decenoyl)piperidine (12): 1H NMR (CDCl3) δ (ppm) 6.82 (dt,
J1 = 15.20, J2 = 7.00, 1H), 6.23 (d, J = 15.20, 1H), 3.59 (t, J = 6.30, 2H),
3.47 (t, J = 6.30, 2H), 2.17 (m, 2H), 1.64 (quint, J = 5.60, 2H), 1.56 (quint,
J=5.60, 4H), 1.44 (quint, J=7.00, 2H), 1.28 (m, 8H), 0.88 (t, J=7.00,
3H); ESIþ m/z = [M þ H]þ = 238.
(2E,4Z-Decadienoyl)piperidine (13): 1H NMR (CDCl3) δ (ppm)
7.59 (dd, J1 = 14.60, J2 = 11.70, 1H), 6.34 (d, J = 14.60, 1H), 6.13 (t, J =
11.70, 1H), 5.78 (m, 1H), 3.62 (t, J = 7.00, 2H), 3.45 (t, J = 7.00, 2H), 2.31
(q, J = 7.40, 2H), 1.67 (quint, J = 7.40, 4H), 1.57 (quint, J = 7.40, 2H), 1.40
(m, 2H), 1.27 (m, 4H), 0.88 (t, J = 7.00, 3H); ESIþ m/z = [M þ H]þ = 236.
N-(trans-Cinnamoyl)piperidine (14): white solid, mp 118.9-119.9 °C;
1H NMR (CDCl3) δ (ppm) 7.64 (d, J=15.5 Hz, 1H), 7.52 (d, J=7.2 Hz, 2H),

9996 J. Agric. Food Chem., Vol. 58, No. 18, 2010
Huang et al.
7.36 (m, 3H), 6.90 (d, J = 15.5, 1H), 3.67 (s, 2H), 3.59 (s, 2H), 1.68 (m, 2H),
1.62 (m, 4H); ESIþ m/z = [M þ H]þ = 216.
Table 1. 1H and 13C NMR (CDCl3) Data of Sarmentine (1)
N-(Decanoyl)hexamethyleneimine (15): 1H NMR (CDCl3) δ (ppm)
3.52 (t, J=6.00, 2H), 3.42 (t, J=6.00, 2H), 2.30 (t, J=7.80, 2H), 1.66-
1.74 (m, 4H), 1.60-1.66 (m, 2H), 1.50-1.6.0 (m, 4H), 1.20-1.30 (m, 12H),
0.87 (t, J=7.20, 3H); ESIþ m/z=[M þ H]þ=254.
N-(2-Decenoyl)hexamethleneimine (16): 1HNMR (CDCl3) δ(ppm)
6.91 (dt, J1=15.20, J2=7.00, 1H), 6.21 (d, J=15.20, 1H), 3.57 (t, J=6.00,
2H), 3.49 (t, J=6.00, 2H), 2.17 (m, 2H), 1.73 (m, 4H), 1.56 (m, 4H), 1.45 (m,
2H), 1.28 (m, 8H), 0.88 (t, J=7.00, 3H); ESIþ m/z=[M þ H]þ=252.
(2E,4Z-Decadienoyl)hexamethleneimine (17): 1H NMR (CDCl3)
δ (ppm) 7.64 (dd, J1 = 14.60, J2 = 11.70, 1H), 6.30 (d, J = 14.60, 1H),
6.16 (t, J = 11.70, 1H), 5.78 (m, 1H), 3.60 (t, J = 7.00, 2H), 3.51 (t, J =
7.00, 2H), 2.31 (q, J = 7.40, 2H), 1.76 (m, 4H), 1.57 (m, 4H), 1.40 (m, 2H),
1.30 (m, 4H), 0.88 (t, J = 7.00, 3H); ESIþ m/z = [M þ H]þ = 250.
N-(trans-Cinnamoyl) hexamethleneimine (18): white solid, mp
position
13C
1H (J = Hz)
1
165.4
120.0
142.4
128.8
143.4
33.1
2
6.08 1H d (14.4)
3
7.26 1H dd (14.4, 10.8)
6.16 1H dd (10.8, 9.6)
6.07 1H dt (14.4, 7.2)
2.13 2H quartet (7.2)
1.39 2H quintet (7.2)
1.27 2H m
4
5
6
7
26.3
24.5
8
1
9
22.7
14.2
1.27 2H m
0.87 3H t (7.2)
3.50 2H t (7.2)
122.6-123.3 °C; H NMR (CDCl3) δ (ppm) 7.70 (d, J=15.4 Hz, 1H),
10
10
20
30
40
7.52 (d, J=7.6 Hz, 2H), 7.36 (m, 3H), 6.88 (d, J=15.4, 1H), 3.63 (t, J=
6.0, 2H), 3.61 (t, J=6.0, 2H), 1.76 (m, 4H), 1.59 (m, 4H); ESIþ m/z=
[M þ H]þ = 230.
46.6
31.5
1.95 2H quintet (7.2)
1.85 2H quintet (7.2)
3.52 2H t (7.2)
28.6
46.0
Comparison of the Percent Content of the Sarmentine (1) from
Different Fruit Samples. Freshly ground fruit powder (10 g) of different
samples was soaked in ethyl acetate (50 mL) for 20 h at room temperature.
The solution was filtered by a Whatman qualitative filter paper (No. 1, L
155 mm). The residue and filter paper were washed with ethyl acetate (25
mL). The combined organic phase was dried under vacuum. The weight of
each extract was recorded. The active compound in the extracts was
quantified by LC/MS.
mortality), III (20-40% mortality), IV (40-60% mortality), V (60-80%
mortality) and VI (80-100% mortality).
RESULTS AND DISCUSSION
Structure Elucidation. The active compound was identified on
the basis of the following evidence: High resolution mass data
(TOF MS ESIþ) is 222.5386, indicating that the molecular formula
of the active compound is most likely C14H23NO. The data from
1H and 13C NMR listed in Table 1 further support this mole-
cular formula. The 1H NMR spectrum indicated the presence of
23 protons including four olefinic (-CHd), one methyl (-CH3)
and eight methylene (-CH2-) protons. The 13C NMR and
DEPT-135 spectrum (not shown) confirmed the presence of 14
carbons accounting for one amide carbonyl (N-CO-), four ole-
finic carbons (-CHd), eight methylene carbons (-CH2-) and
Quantification of Sarmentine (1) in the Ethyl Acetate Extract of
Dry Fruits by (LC/MS). Conditions of liquid chromatography and
mass spectrometry are specifically described as follows. Chromatographic
separation was performed at 25 °C on a Thermo high performance liquid
chromatography (HPLC) instrument equipped with Finnigan Surveyor
PDA plus detector, autosampler plus, MS pump and a 4.6 mm ꢀ 100 mm
Luna C18 5 μm column (Phenomene, Torrance, CA). The solvent system
consisted of water (solvent A) and acetonitrile (solvent B). The mobile
phase began at 10% solvent B and was linearly increased to 100% solvent
B over 20 min and then held for 4 min, and finally returned to 10% solvent
B over 3 min and kept for 3 min. The flow rate was 0.5 mL/min. The
injection volume was 10 μL, and the samples were kept at room
temperature in an auto sampler. The active compound was detected by
a positive electrospray ionization mode in a full scan mode (m/z 100-1500
Da) on a LCQ DECA XPplus mass spectrometer (Thermo Electron Corp.,
San Jose, CA). The flow rate of nitrogen gas was fixed at 30 and 15 arb for
the sheath and aux/sweep gas flow rate, respectively. Electrospray ioniza-
tion (ESI) was performed with a spray voltage set at 5000 V and a capillary
voltage at 35.0 V. The capillary temperature was set at 400 °C.
The active compound standard was obtained by repeated crystal-
lization in laboratory until only one peak at 1 mg injection level was
shown under a wavelength of 210 nm with two different mobile phases. A
series of standard concentrations (125, 62.5, 31.25, 15.6, 7.8, 3.9, 1.95, and
0.976 ng/mL) was made in ethanol. Three independent samples (2.5 μg/mL
in ethanol) for each extract were made. Mass spectra were run in a SIM
mode with a mass range of 221.0-224.0 and retention time of 16.94 min.
The limits of detection (LOD) of the active compound were determined by
running decreasing amounts of standard solution until the ratio of the
signal of the active compound over the background was greater than or
equal to 3. The concentration of sarmentine in fruit samples was presented
by an average of three independent samples with a standard deviation.
Evaluation of Herbicidal Activity. Herbicidal activity of the active
compound and synthesized compounds was evaluated by foliar spraying.
Carrier solution contained 2% ethanol, 0.2% glycosperse O-20 and 0.1%
sodium lauryl sulfate. Freshly prepared solution with the evaluated
compound at a concentration of 5 mg/mL was used. In the spectrum
study, spraying volume was dependent on the foliar surface area, ranging
from approximately 1 to 3 mL/pot. One or two pots of plants were treated
for each. Number of pots treated was dependent on both foliar area and
availability. In the structure-activity relationship study, one pot of
barnyard grass was used per treatment and the spraying volume of each
compound was 3 mL of 5 mg/mL. Phytotoxicity was evaluated 3 days after
spraying. Efficacy of phytotoxicity was graded as I (no effect), II (<20%
1
one methyl carbon (-CH3). From the H-1H COSY spectrum
(not shown), two spin systems were constructed. One contained
four consecutive methylenes (-CH2CH2CH2CH2-), and the
other contained four conjugated olefin protons further connected
to CH2CH2CH2CH3. Moreover, data from HMBC (not shown)
indicated that the above-mentioned two spin systems were con-
nected through an amide to give the planar structure of sarmentine
(1). Finally, the stereochemistry of the two double bonds were
assigned as trans based on the coupling constants (J = 14.4 Hz).
The search in the literature (SciFinder) indicated that the com-
pound 1 is a known compound called sarmentine, which was first
isolated from dry P. sarmentosum fruit powder (17) and also from
P. nigrum (18). However, this is the first report of isolation from P.
longum and of exhibiting phytotoxicity.
Herbicidal Spectrum of Sarmentine (1). The phytotoxicity of
sarmentine (1) depended on its concentration and plant species.
The optimal concentration of sarmentine (5 mg/mL) for excellent
control of barnyard grass (Figure 1) was chosen for herbicidal
spectrum study. Due to the hydrophobic property of sarmentine
(1), a carrier solution containing 0.2% glycosperse O-20, 2%
ethanol and 0.1% sodium lauryl sulfate was used. This carrier
solution contains a high concentration of surfactants, and sar-
mentine (1) can be suspended in this solution for 15 min. To
mitigate the poor suspension, the sarmentine (1) solution was
prepared immediately prior to foliar application. The carrier
solution alone did not show any phytotoxicity toward tested
plants when results were recorded.
Sarmentine (1) displayed phytotoxicity against a variety of
plants including crop plants and weeds (Table 2). Phytotoxicity of

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J. Agric. Food Chem., Vol. 58, No. 18, 2010 9997
Figure 1. Concentration dependence of sarmentine phytotoxicity on barnyard grass. Pots from left to right were treated with the carrier solution, 5.0, 2.5, and
1.25 mg/mL of sarmentine, respectively. The carrier solution consisted of 2% ethanol and 0.2% glycosperse O-20 KFG. Each treatment was 3 mL/pot.
Table 2. Phytotoxicity Spectrum of Sarmentine (1)
no.
plant name
efficacya
no.
plant name
efficacya
1
2
3
4
5
6
7
8
pigweed (Amaranthus retroflexus L.)
barnyard grass (Echinochloa crus-galli L.)
bindweed (Convolvulus arvensis L.)
crabgrass (Digitaria sanguinalis L.)
horseweed (Conyza canadensis L.)
sedge (Cyperus difformis L.)
VI
VI
VI
VI
II
9
lambsquarter (Chenopodium album L.)
annual bluegrass (Poa annua L.)
wild mustard (Brassica kaber L.)
black nightshade (Solanum nigrum L.)
curly dock (Rumex crispus L.)
VI
VI
VI
VI
VI
VI
VI
I
10
11
12
13
14
15
16
III
VI
VI
sweet corn (Zea mays L.)
wheat (PR 1404) (Triticum aestivum L.)
rice (M-104) (Oryza sativa L)
sprangletop (Leptochloa fascicularis Lam.)
dandelion (Taraxacum officinale F.)
a Efficacy of phytotoxicity was graded as I (no effect), II (<20% mortality), III (20-40% mortality), IV (40-60% mortality), V (60-80% mortality) and VI (80-100% mortality).
sarmentine (1) was dependent on the plant species, ranging from
zero to 100% control. No visible phytotoxicity (after 10 days) was
shown on rice plants toward sarmentine (1). Slight phytotoxicity
of sarmentine (1) on sedge and horseweed was shown. However,
high phytotoxicity of sarmentine (1) was observed on pigweed,
barnyard grass, bindweed, crabgrass, sprangletop, dandelion,
lambsquarter, annual bluegrass, wild mustard, black nightshade,
curly dock, sweet corn and wheat. Phytotoxicity may be related to
age of the plants. For example, horseweed (70 days old) was much
older than other weeds, and less phytotoxicity was shown.
Structure-Activity Relationship (SAR). To quickly understand
which part of the sarmentine (1) molecule plays a crucial role on
the phytotoxicity, a series of sarmentine analogues were synthe-
sized and evaluated on barnyard grass. The SAR study (Table 3)
suggested that neither the long unsaturated fatty acid nor pyrro-
lidine of sarmentine (1) alone is crucial for phytotoxicity, but the
amide bond with a secondary amine seemed to be necessary. This
conclusion was supported by the following experimental results.
Phytotoxicity remained the same when the acid moiety of
sarmentine (1) was replaced by structurally similar fatty acids
such as 2E,4Z-decadienoic acid (i.e., geometric isomer of 2E,4E-
decadienoic acid) with two double bonds (9), 2E-decenoic acid
with one double bond (8) and decanoic acid without any double
bonds (7), and even structurally different acid such as trans-
cinnamic acid (10). This suggested that the acid moiety of
sarmentine (1) can vary when the amine is pyrrolidine. Similarly,
when the acid moiety remained the same, the amine could vary.
For example, phytotoxicity remained the same with decanoic acid
when the amine was changed from a five-membered ring (7) to a
six- or seven-membered ring (11 and 15, respectively). However,
phytotoxicity dropped dramatically when the amide bond with a
secondary amine was changed into an ester bond (e.g., 2) and an
amide bond with a primary amine (e.g., 3-6). In addition, results
from the SAR study (Table 3) also indicate that the amine
moiety of sarmentine, pyrrolidine (23), and its analogues such as
cyclopentylamine (22), hexamethyleneimine (25) and piperidine
(24) were nontoxic to barnyard grass; but the analogues of the
acid moiety of sarmentine such as decanoic acid (20), 2E-decenoic
acid (21) and trans-cinnamic acid (19) were very active. To obtain
a better SAR, the length of the carbon chain in the acid moiety,
disubstituted amines (nonring system) and changing carbonyl
group into other groups such as phosphate or sulfone should be
further investigated.
Symptoms of Phytotoxicity. Sarmentine (1) is phytotoxic by
contact. When plants were exposed to sarmentine, phytotoxic
symptoms included bent stems, closed-up leaves, or slight black
tiny spots (scorching) on the leaves which then became bigger and
bigger until they covered the whole leaves. These symptoms could
be clearly observed within half an hour to 2 h after spraying. Most
phytotoxicity occurred within 7 h after spraying. These observed
symptoms were very similar to that of middle-chain fatty acids
such as decanoic acid (20) (Figure 2).
Our results suggest that sarmentine (1) and its analogues most
possibly possess the same mode of action as the middle-chain
fatty acids such as decanoic acid (20) and 2E-decenoic acid (21),
which disrupt the cell membrane and then initiate peroxidation
driven by radicals (19,20). Phytotoxicity of sarmentine (1) and its
analogues can be a direct and indirect (or hydrolytic) action. The
indirect mechanism involves the acid moiety of sarmentine and its
analogues acting after the active ingredients were hydrolyzed
plant amide hydrolases (21). For example, 2E,4E-decadieneoic
acid, one of the hydrolyzed metabolites of sarmentine (1), is
structurally similar to decanoic acid (20) and 2E-decenoic acid
(21). It seems reasonable to assume that phytotoxicity of sarmen-
tine (1) results from the formation of 2E,4E-decadieneoic acid.
However, this possible hydrolytic mechanism may be totally
excluded. First of all, esters are typically hydrolyzed by serine
proteases with similar or much higher rates when compared with
structurally similar amides (22-24). Therefore, trans-2-cis-4-
decadienoate (2) should be at least as active as its amide analogues

9998 J. Agric. Food Chem., Vol. 58, No. 18, 2010
Huang et al.
Table 3. Phytotoxicity of Sarmentine (1) and Its Analogues on Barnyard Grass
* Efficacy of phytotoxicity was graded as I (no effect), II (<20% mortality), III (20-40% mortality), IV (40-60% mortality), V (60-80% mortality) and VI (80-100% mortality).
(e.g., 9, 13 and 17), which is not supported by our experimental
results (Table 3). In addition, when LY2183240 [a nonselective
and highly potent inhibitor for human serine hydrolases such
as fatty acid amide hydrolase (IC50 =13 nM) and MAG-lipase
(IC50=5.3 nM) (25)] at 32.5 and 325 μM was separately coapp-
lied with N-(decanoyl) pyrrolidine (7), the phytotoxicity of 7 was
not affected at all (data are not shown here). Of course, the fact
that this inhibitor may not inhibit serine hydrolases (e.g., the

Article
J. Agric. Food Chem., Vol. 58, No. 18, 2010 9999
Figure 2. Comparison of phytotoxic symptoms and time course of phytotoxicity of sarmentine and decanoic acid (21). Mustard and barnyard grass are
displayed in the back and front row, respectively. Plants in the left, middle and right columns were treated with carrier solution, sarmentine and decanoic acid,
respectively. The carrier solution consisted of 2% ethanol, 0.2% glycosperse O-20 KFG and 0.1% sodium lauryl sulfate.
Table 4. The Concentration of Sarmentine (1) in Different Samples of Dry P. longum Fruits
% of sarmentine in
ethyl acetate extract
extract wt (g) from 10 g of
ground dry fruit powder
% content of sarmentine
in dry fruit
sample
resources
purchase time
1
2
3
4
WAN FUNG
WAN FUNG
WAH TSUN
WAH TSUN
May 25, 2008
March, 23, 2009
June 2, 2008
March 4, 2009
12.66 ( 0.45
1.83 ( 0.10
0.01 ( 0.001
0.008 ( 0.001
0.45 ( 0.03
0.51 ( 0.03
0.55 ( 0.02
0.66 ( 0.03
0.5697 ( 0.0380
0.0933 ( 0.0055
0.00055 ( 0.00002
0.00053 ( 0.00002
amide hydrolases, lipases) in barnyard grass may explain the
negative results of this experiment. Finally, it is impossible for
serine hydrolases to metabolize sarmentine (1) and its analogues
so quickly that the time course of phytotoxic symptoms of
sarmentine and its analogues matches very well with those of
middle chain fatty acids (Figure 2). The direct action mechanism
with sarmentine (1) and its analogues directly disrupting cell
membrane and then initiating the peroxidation reactions is
supported by the following observed experimental phenomenon
(Figure 2): the appearance and progression of phytotoxic symptoms
of sarmentine (1) and its analogues was similar to that of decanoic
acid (20). Using this mode of action mechanism it is, however,
difficult to explain why esters (e.g., 2) and amides with a primary
amine (e.g., 3-6) did not display the same phytotoxicity. Therefore,
more biochemical and molecular biology data are needed to
elucidate the mode of action of sarmentine and its analogues.
Quantification of Sarmentine in Different P. longum Fruit
Samples. In order to successfully use long pepper fruit as raw mate-
rial for a commercial bioherbicide, the variability of sarmentine
content in dry long pepper fruits obtained from different sources
was investigated. Due to the fact that freshly dried fruits are not
readily available, dry fruits from herb stores were investigated
instead. For quantitative analysis, the LC/MS method was devel-
oped and the standard curve of sarmentine (Y = 83324X - 30784,
R2 = 0.9998; X and Y stand for the concentration of sarmentine
and the peak area, respectively) was obtained. For the method, the
limit of detection for sarmentine was 12 pg per injection (or 1.2
ng/mL). Based on this external standard curve, the concentration of
sarmentine in the extracts of four different P. longum fruit samples
varied dramatically, ranging from 0.0005% to 0.57% (Table 4).
This high variability may result from the age and/or origin of the
dry fruits, which parameters were not available for the test samples.
The first sample we obtained in this study contained the highest
content of sarmentine. However, this may give us an indication that
freshly dried material should be used to study phytochemicals. In
addition, from this study, it seems that the quantity of sarmentine in
the ground powder of dry P. longum fruits can be evaluated by phy-
sical appearance because the higher the concentration of sarmen-
tine, the oilier the ground powder is. For example, no oil was visible
for the ground powder of the two samples from WAH TSUN, but
oily powder was seen for the ground powder of those from WAN
FUNG, especially the sample obtained on May 25, 2008.
Potential Use of Sarmentine and Its Analogues. Biologicaleffects
of sarmentine have been investigated in the past. Sarmentine has
been found to be an in vivo skin antioxidant protecting photoaged
skin (26) and to display antiplatelet aggregation activity (27),
antiplasmodial and antimycobacterial activities (28) and antituber-
culosis activity (29). In addition, sarmentine is used to solubilize
hydrophobic compounds in cosmetics and pharmaceuticals (PCT
Publication No: WO/2008/065451 (30)). In this study, sarmentine
has shown potential for use as a bioherbicide and/or a lead molecule
for synthetic herbicide. However, chemical instability of sarmentine
under environmental (e.g., light, oxygen) and biological conditions
(e.g., P450s, hydrolases) is currently limiting the commercial use
of sarmentine. Structural modification of sarmentine as demon-
strated in this study will probably help solve instability problem,

10000 J. Agric. Food Chem., Vol. 58, No. 18, 2010
Huang et al.
and lead to more commercial application and use of this known
natural compound.
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ACKNOWLEDGMENT
We thank Drs. Jun Yang, Junyan Liu and Ms. Shirley Gee of the
University of California at Davis for assistance in measuring the
high resolution mass of sarmentine and providing some reagents.
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Received for review June 1, 2010. Revised manuscript received August 2,
2010. Accepted August 3, 2010.