Isolation and characterization of polyphenol type-A polymers from cinnamon with insulin-like biological activity.
ABSTRACT The causes and control of type 2 diabetes mellitus are not clear, but there is strong evidence that dietary factors are involved in its regulation and prevention. We have shown that extracts from cinnamon enhance the activity of insulin. The objective of this study was to isolate and characterize insulin-enhancing complexes from cinnamon that may be involved in the alleviation or possible prevention and control of glucose intolerance and diabetes. Water-soluble polyphenol polymers from cinnamon that increase insulin-dependent in vitro glucose metabolism roughly 20-fold and display antioxidant activity were isolated and characterized by nuclear magnetic resonance and mass spectroscopy. The polymers were composed of monomeric units with a molecular mass of 288. Two trimers with a molecular mass of 864 and a tetramer with a mass of 1152 were isolated. Their protonated molecular masses indicated that they are A type doubly linked procyanidin oligomers of the catechins and/or epicatechins. These polyphenolic polymers found in cinnamon may function as antioxidants, potentiate insulin action, and may be beneficial in the control of glucose intolerance and diabetes.
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ABSTRACT: Cinnamomum cassia (cinnamon) proanthocyanidins (PACs) are believed to have anti-hyperglycemic potential via stimulation of insulin sensitivity. The present study investigates the carbohydrate hydrolyzing enzyme inhibition of cinnamon PACs. Five grams of cinnamon bark powder were extracted in 100 mL acetone solution (CAE) (acetone: water: hydrochloric acid, 70:29.9:0.01) for 2 h at room temperature and in 100 mL deionized water for 30 min at 90 °C (CWE). PACs were purified from CAE using LH-20 (CAE-PAC) to be further evaluated. PAC contents were evaluated by 4-Dimethylaminocinnamaldehyde (DMAC) assay and yielded 795, 177 and 123 mg/g, for CAE-PAC, CAE and CWE respectively. The total phenolic contents of CAE and CWE were determined to be 152 and 134 mg/g respectively. All extracts were adjusted to the same PAC content (180, 90, 45 and 20 μg) and the inhibitory activity against rat α-glucosidase was determined. The CAE-PAC fraction had very low rat α-glucosidase inhibitory activity, CAE had the highest (IC50 0.474 mg/mL total phenolic (TP) basis) followed by CWE (IC50 0.697 mg/mL TP basis). The specific maltase and sucrase inhibitory activities were determined and CAE (IC50 0.38 and 0.10 mg/mL TP basis) had higher inhibition than CWE (IC50 0.74 and 0.37 mg/mL TP basis). Results suggest that the observed bioactivity is not PAC dependent and that CAE has a higher anti-hyperglycemic potential than CWE via inhibition of carbohydrate hydrolyzing enzymes.Plant Foods for Human Nutrition 04/2014; · 2.36 Impact Factor
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ABSTRACT: Water soluble cinnamon extract has been shown to increase insulin sensitivity and modulate macrophage activation, a desirable trait for the management of obesity or atherosclerosis. Our present study investigated whether cinnamon water extract (CWE) may influence the differentiation of monocytes into macrophages and the activity of macrophage scavenger receptors, commonly observed in atherosclerotic lesions. We investigated the effect of CWE on the expression of various surface markers and the uptake of acetylated low density lipoprotein (LDL) in phorbol-12-myristate-13-acetate (PMA)-stimulated THP-1 cells. The protein levels of PMA or macrophage-colony stimulating factor (M-CSF)-stimulated type 1 macrophage scavenger receptor (SRA) were analyzed. Finally, the role of extracellar signal-related kinase (ERK) 1/2 in SRA synthesis and the effect of CWE on PMA-stimulated ERK1/2 were determined. CWE inhibited the differentiation of monocyte by decreasing the expression of CD11b, CD36 and SRA and the uptake of acetyl LDL. CWE suppressed the upregulation of SRA by M-CSF and modulated ERK1/2 activity, which was required for PMA-induced SRA synthesis. Our results demonstrate that CWE was able to interfere with monocyte differentiation and macrophage scavenger activity, indicating its potential in preventing the development of atherosclerotic lesions.BMC Complementary and Alternative Medicine 03/2014; 14(1):90. · 2.08 Impact Factor
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ABSTRACT: 17 / 1 / 93 ﭘﺬﻳﺮش ﺗﺎرﻳﺦ : 24 / 3 / 93 1 ﻣﻴﺮﻓﻴﻀﻲ ﻣﺎﻧﻲ * 2 ﺗﻮرزﻧﻲ زاده ﻣﻬﺪي زﻫﺮا 3 ﻣﻴﺮﻓﻴﻀﻲ زﻫﺮا ﺳﻴﺪه 4 آﺑﺎدي ﺟﻌﻔﺮ اﺻﻐﺮي ﻣﺤﻤﺪ 5 رﺿﻮاﻧﻲ ﺣﻤﻴﺪرﺿﺎ 6 ﺷﻮﻗﻲ ﻣﻬﻨﺎز 1 -،ﻣﺎﻣﺎﻳﻲ داﻧﺸﻜﺪه ،ﻣﺎﻣﺎﻳﻲ ﮔﺮوه اﺳﺘﺎدﻳﺎر اﻳﺮان ،ﻛﺮج ،ﻛﺮج واﺣﺪ ،اﺳﻼﻣﻲ آزاد داﻧﺸﮕﺎه 2 -،ﻣﺎﻣﺎﻳﻲ ﭘﺮﺳﺘﺎري داﻧﺸﻜﺪه ،ﻣﺎﻣﺎﻳﻲ ﮔﺮوه ﻣﺮﺑﻲ ،ﻛﺮج ،اﻟﺒﺮز ﭘﺰﺷﻜﻲ ﻋﻠﻮم داﻧﺸﮕﺎه اﻳﺮان 3 -ﻣﺮﻛﺰ ،روﻣﺎﺗﻮﻟﻮژي ،داﺧﻠﻲ ﮔﺮوه داﻧﺸﻴﺎر ﺑﻴﻤﺎري ﺗﺤﻘﻴﻘﺎت ﻋﻠﻮم داﻧﺸﮕﺎه ،روﻣﺎﺗﻴﺴﻤﻲ ﻫﺎي اﻳﺮان ،ﻣﺸﻬﺪ ،ﻣﺸﻬﺪ ﭘﺰﺷﻜﻲ 4 -ﺗﺤﻘﻴﻘﺎت ﻣﺮﻛﺰ ،آﻣﺎرزﻳﺴﺘﻲ اﺳﺘﺎدﻳﺎر ﻣﺼﺪوﻣﻴﺖ از وﭘﻴﺸﮕﻴﺮي اﭘﻴﺪﻣﻴﻮﻟﻮژي داﻧﺸﻜﺪه ،ﻫﺎ ،ﺗﺒﺮﻳﺰ ﭘﺰﺷﻜﻲ ﻋﻠﻮم داﻧﺸﮕﺎه ،وﺗﻐﺬﻳﻪ ﺑﻬﺪاﺷﺖ اﻳﺮان ،ﺗﺒﺮﻳﺰ 5 -دﻛﺘﺮ ﺣﺮﻓﻪ اي اﻳﺮان ،ﻛﺮج ،داروﺳﺎزي اي 6 -،اﺳﻼﻣﻲ آزاد داﻧﺸﮕﺎه ،ﭘﺮﺳﺘﺎري ﮔﺮوه ﻣﺮﺑﻲ اﻳﺮان ،ﻛﺮج ،ﻛﺮج واﺣﺪ ﺧﻼﺻﻪ ﻣﻘﺪﻣﻪ اﻳﻦ ﻣﻄﺎﻟﻌﻪ ﺑﻴﻤﺎران در ﺧﻮن ﭼﺮﺑﻲ و ﻗﻨﺪ ﻛﻨﺘﺮل ﻫﺎي ﺷﺎﺧﺺ ﺑﺮ دارﭼﻴﻦ ﺧﻮراﻛﻲ ﻣﻜﻤﻞ ﺗﺎﺛﻴﺮ ﺑﺮرﺳﻲ ﻫﺪف ﺑﺎ ﮔﺮدﻳﺪ اﻧﺠﺎم دو ﻧﻮع دﻳﺎﺑﺘﻲ . روش ﻛﺎر اﻳﻦ ﻛﺎرآزﻣﺎﻳﻲ ﺑﺎﻟﻴﻨﻲ ﺑﺮ 75 ﺑﻴﻤﺎر دﻳﺎﺑﺖ ﻧﻮع دو) 30 ﻧﻔﺮ دارﭼﻴﻦ ﻛﻨﻨﺪه درﻳﺎﻓﺖ و 45 داروﻧﻤﺎ ﻛﻨﻨﺪه درﻳﺎﻓﺖ ﻧﻔﺮ (ﻫﺎي ﺳﺎل در ﻛﺮج ﺷﻬﺮ در 1389 -1391 ﺷﺪ اﻧﺠﺎم . ،ﮔﺮوه دو ﻛﭙﺴﻮل روز ﻧﻮد ﻳﺎ دارﭼﻴﻦ ﺣﺎوي ﺧﻮراﻛﻲ ﻫﺎي داروﻧﻤﺎ) ﻧﺸﺎﺳﺘﻪ) (500 ﻧﺎﻫﺎر و ﺻﺒﺤﺎﻧﻪ از ﺑﻌﺪ روز در دوﺑﺎر ،ﮔﺮم ﻣﻴﻠﻲ (ﻧﻤﻮدﻧﺪ ﻣﺼﺮف را . ﺧﻮن ﻧﻤﻮﻧﻪ ورﻳﺪي ﮔﻴﺮي اﻧﺪازه ﺟﻬﺖ ﺳﺎﻋﺘﻪ دو ﺧﻮن ﻗﻨﺪ ،ﻧﺎﺷﺘﺎ ﺧﻮن ﻗﻨﺪ (BS2hpp) ، ﮔﻠﻴﻜﻮزﻳﻠﻪ ﻫﻤﻮﮔﻠﻮﺑﻴﻦ (HbA1c) اﻧﺴﻮﻟﻴﻦ ، ﻣﻄﺎﻟﻌﻪ ﭘﺎﻳﺎن و آﻏﺎز در آن اﺟﺰاي و ﺗﺎم ﻛﻠﺴﺘﺮول ،ﮔﻠﻴﺴﻴﺮﻳﺪ ﺗﺮي ،ﺳﺮم ﺷﺪ ﮔﺮﻓﺘﻪ . اﺳﺘﻔﺎده ﺑﺎ ﻧﻴﺰ اﻧﺴﻮﻟﻴﻨﻲ ﻣﻘﺎوﻣﺖ اﺳﻜﻮر از HOMA ﮔﺮدﻳﺪ ﻣﺤﺎﺳﺒﻪ . اﻓﺰاز ﻧﺮم و آﻧﻮا ،ﺗﻲ ،اﺳﻜﻮر ﻛﺎي ﻫﺎي آزﻣﻮن SPSS ﺗﺤﻠﻴﻞ و ﺗﺠﺰﻳﻪ ﺟﻬﺖ ﺷﺪ اﺳﺘﻔﺎده ﻫﺎ داده . ﻧﺘﺎﻳﺞ ﻣﺪاﺧﻠﻪ اﻧﺘﻬﺎي در ﻛﺎﻫﺶ داري ﻣﻌﻨﻲ در ﺷﺎﺧﺺ ﺗﻮده ﮔﺮوه ﺑﻪ ﻧﺴﺒﺖ دارﭼﻴﻦ ﻛﻨﻨﺪه درﻳﺎﻓﺖ ﮔﺮوه در ﺑﺪﻧﻲ ﺷﺪ دﻳﺪه داروﻧﻤﺎ) 01 / 0 p= (ﻣﺘﻐﻴﺮﻫﺎي ﺳﺎﻳﺮ در ﺗﻔﺎوت اﻳﻦ اﻣﺎ ﻧﺒﻮد دار ﻣﻌﻨﻲ ﺑﺮرﺳﻲ ﻣﻮرد . ﺑﻴﻤﺎران در ﭼﻨﻴﻦ ﻫﻢ ﻣﻴﺎﻧﮕﻴﻦ دارﭼﻴﻦ ﻛﻨﻨﺪه درﻳﺎﻓﺖ FBG ، BS2hpp ، HbA1c اﺳﻜﻮر ،ﺳﺮم اﻧﺴﻮﻟﻴﻦ ، HOMA ﺗﻮده ﺷﺎﺧﺺ و داﺷﺖ ﻣﺪاﺧﻠﻪ از ﻗﺒﻞ ﺑﻪ ﻧﺴﺒﺖ داري ﻣﻌﻨﻲ اﺧﺘﻼف ﺑﺪﻧﻲ) 05 / 0 p< ﻣﻮارد ﺗﻤﺎﻣﻲ ﺑﺮاي (.Medical Journal of Mashhad University of Medical Sciences. 06/2014; 57:533-541.
Isolation and Characterization of Polyphenol Type-A Polymers
fromCinnamon with Insulin-like Biological Activity
RICHARD A. ANDERSON,*,†C. LEIGH BROADHURST,‡MARILYN M. POLANSKY,†
WALTER F. SCHMIDT,‡ALAM KHAN,†,§VINCENT P. FLANAGAN,⊥
NORBERTA W. SCHOENE,†AND DONALD J. GRAVES|
Nutrient Requirements and Functions Laboratory, Beltsville Human Nutrition Research Center, USDA,
ARS, Bldg 307C, Rm 223, Beltsville, Maryland 20705-2350, Nuclear Magnetic Resonance
Laboratory, ARS, USDA, Beltsville, Maryland 20705-2350, Food Composition Laboratory,
Beltsville Human Nutrition Research Center, USDA, ARS, Beltsville, Maryland 20705-2350, and
Department of Molecular Cellular and Developmental Biology, University of California,
Santa Barbara, California 93106-9610
The causes and control of type 2 diabetes mellitus are not clear, but there is strong evidence that
dietary factors are involved in its regulation and prevention. We have shown that extracts from
cinnamon enhance the activity of insulin. The objective of this study was to isolate and characterize
insulin-enhancing complexes from cinnamon that may be involved in the alleviation or possible
prevention and control of glucose intolerance and diabetes. Water-soluble polyphenol polymers from
cinnamon that increase insulin-dependent in vitro glucose metabolism roughly 20-fold and display
antioxidant activity were isolated and characterized by nuclear magnetic resonance and mass
spectroscopy. The polymers were composed of monomeric units with a molecular mass of 288. Two
trimers with a molecular mass of 864 and a tetramer with a mass of 1152 were isolated. Their
protonated molecular masses indicated that they are A type doubly linked procyanidin oligomers of
the catechins and/or epicatechins. These polyphenolic polymers found in cinnamon may function as
antioxidants, potentiate insulin action, and may be beneficial in the control of glucose intolerance
KEYWORDS: Glucose; insulin; diabetes; cinnamon; polyphenols; spice
Despite extensive diabetes research, the prevention and
control of type 2 diabetes mellitus (type 2 DM) remain unclear.
Diet has been shown to play a definite role in the onset of type
2 DM, and the diets commonly consumed in the United States
and other westernized countries appear to increase the incidence
of diabetes (1). The high refined sugar and high fat content of
US diets are likely to be partly responsible, but the low intake
of traditional herbs, spices, and other plant products may also
be involved. The recommended use of plants in the treatment
of diabetes dates back to approximately 1550 BCE (2). For the
majority of the world population, drug treatment for diabetes
is not feasible and alternative treatments need to be evaluated.
Plants are important not only for the control of type 2 DM but
also for its prevention, especially for people with elevated levels
of blood glucose and glucose intolerance who have a greater
risk of developing diabetes.
Common spices such as cinnamon, cloves, and bay leaves
display insulin potentiating activity in vitro (3). It was thought
that these spices might also have high chromium (Cr) concen-
trations, because biologically active forms of Cr potentiate
insulin activity (4). However, there are no correlations between
total Cr concentrations and insulin potentiating activity in these
plant products (3). Only a small portion of the total Cr in
biological systems is associated with insulin potentiating activity.
In addition to improving cellular glucose metabolism, cin-
namon may provide additional benefits for persons with diabetes
through its antioxidant activity. Cinnamon, in addition to cloves,
cumin, curcumin, many mint family plants, and others that are
usually high in flavonoids, which are potent antioxidants, may
be synergistic with vitamins and trace elements (5-8). Dried
ground and fresh spices were found to be highly effective at
preventing lipid peroxidation of cooked ground fish. The order
of effectiveness for dried spices was cloves > cinnamon >
cumin g black pepper g fennel ) fenugreek (9). Specific
antioxidant phytochemicals that have been identified in cin-
namon include epicatechin, camphene, eugenol, gamma-ter-
pinene, phenol, salicylic acid, and tannins (8).
* To whom correspondence should be addressed. Tel.: 301/504-8091.
Fax: 301/504-9062. E-mail: firstname.lastname@example.org.
†Nutrient Requirements and Functions Laboratory, Beltsville Human
Nutrition Research Center.
‡Nuclear Magnetic Resonance Laboratory.
§Present address: Department of Nutrition, NWFP Agricultural Uni-
versity, Peshawar, Pakistan.
⊥Food Composition Laboratory, Beltsville Human Nutrition Research
|University of California.
J. Agric. Food Chem . 2004, 52, 65−7065
10.1021/jf034916b CCC: $27.50©2004 Am erican Chem ical Society
Published on Web 12/03/2003
From an aqueous extract of commercial cinnamon, we have
identified polyphenolic polymers that increase glucose metabo-
lism roughly 20-fold in vitro in the epididymal fat cell assay
(10). These appear to be rather unique, because other cinnamon
or similar compounds display little or no biological activity.
Additionally, approximately 50 plant extracts have also been
investigated in this assay, and none have shown activity equal
to that of cinnamon (11).
MATERIALS AND METHODS:
Three verified samples of cinnamon were tested, including Korintji
lauraceae; Microbial Identification Index (MIDI) class; Korintji cassia;
botanical class, Cinnamomum burmannii (Nees) Blume; a cinnamon
of Chinese origin, MIDI class Tung Hing, botanical class, C. cassia
Blume (Lauraceae); and one of Vietnamese origin, botanical class C.
loureirii Nees, (Lauraceae); which were obtained as a generous gift
from Dr. Carolyn Fisher, McCormick & Company Incorporated,
Baltimore, MD. Samples were verified using a database that utilizes a
fingerprint matching of the volatile oil components. Other ground
commercial cinnamons tested were C. Verum (Ceylon cinnamon or
“canela”, bulk bark) C. loureinii (Vietnamese cinnamon) and C.
burmannii (Korintje cinnamon from Sumatra) (Penzey’s House of
Spices, Muskego, WI) as well as a C. cassia specimen in bulk bark
from China. The cinnamon used for the purification and characterization
was primarily C. burmanni.
Insulin enhancing biological activity was measured using the
epididymal fat cell assay (10, 11). Briefly, 0.43 µCi [U-14C]-glucose,
72 µg glucose, and adipocytes were incubated with insulin and/or
aqueous extracts of cinnamon or its components in a final reaction
volume of 2 mL of Krebs-Ringer phosphate buffer, pH 7.1. Quantitation
of14CO2release by the cells was done using benzethonium hydroxide
(Sigma-Aldrich, St. Louis, MO) as a trapping agent, which is a
replacement for hyamine hydroxide. Similar results were obtained by
trapping14CO2and measuring14C incorporation into lipids using Dole’s
solution (800 mL of 2-propanol, 200 mL of heptane, and 20 mL of 1
N sulfuric acid). The insulin activity ratio was calculated by dividing
the basal cpm of14CO2released by the cells into those of the activity
due to aqueous extracts of cinnamon or its components.
For the purification of the active components of cinnamon, 5 g of
cinnamon was suspended in 100 mL of 0.1 N acetic acid and autoclaved
for 15 min at 15 psi. The supernatant was removed, 4 volumes of
absolute ethanol were added to the supernatant, and the sample was
stored at 4 °C overnight. The sample was filtered using Whatman #40
filter paper. The sample was added to an LH-20 column (5 × 15 cm)
(Pharmacia LKB Biotechnology, Piscataway, NJ) equilibrated with
absolute ethanol and washed with 600 mL of absolute ethanol. The
sample was then eluted with 50% acetonitrile and water with a final
concentration of 0.1N acetic acid. Fractions with insulin-enhancing
activity were collected and concentrated by rotoevaporation and purified
using high performance liquid chromatography (HPLC). Samples were
injected onto a 7.8 × 300 mm, 7 µL, SymmetryPrep C18, column
(Waters Corp., Milford, MA), equilibrated with 90% 0.05 N acetic acid
and 10% acetonitrile at a flow rate of 4 mL/min using a Waters HPLC
chromatography system with Millennium 2100 software and a Waters
996 ultraviolet absorbance detector.
The production of reactive oxygen species was determined using
whole blood samples from rats (12). The molecular probe, 2′7′-
dichlorodihydrofluorescein diacetate, 10 µM, was loaded into the
platelets for 10 min, followed by a 5 min exposure to insulin potentiating
polyphenolic polymers or vehicle control. Collagen at 20 µg/mL was
added and platelets in the samples were monitored for increases in
fluorescence by flow cytometry at 2 min intervals from 0 to 12 min.
1H and13C attached proton test (APT) experiments (13, 14) were
conducted on a Bruker QE Plus 300 NMR spectrometer (Billerica, MA)
operating at 300 MHz for1H and 75 MHz for13C. APT experiments
used a spectral width of 20 000 Hz with 64 000 scans and 16 K data
points zero filled to 32K data points. APT experiments determine the
number of molecular sites that are CH2groups (and/or protonless carbon
atoms) from those that are CH and CH3 groups. Fractions collected
during HPLC analysis to be used for1H NMR analysis were first
concentrated by evaporation of the solvent mobile phase to about 0.3
mL, 0.5 mL of D2O was added, it was again evaporated to 0.3 mL,
and then made to 0.8 mL with D2O. The sample was not evaporated to
dryness, due to difficulty in resolubilizing the compounds once dry.
To detect potential structural peaks, which could otherwise be obscured
by a large HDO peak, presaturation (15) was used with each of the
sample peaks with a spectral width of 3000 Hz with typically 512 scans
and 8192 data points per scan. The time required for presaturation using
a continuous wave decoupler was varied with the amount of HDO
present, typically 1.2 s per scan.
Electrospray ionization (ESI) and atmospheric pressure chemical
ionization (APCI) mass spectrometry (MS) analyses were performed
on an LCQ classic ion trap instrument (ThermoElectron Co., San Jose,
CA). Trapped fractions were analyzed by infusion at a flow rate of
5-10 µl/min in the positive ion mode under full scan (m/z 200-2000
Da) and multiple collision induced dissociations (MSn) conditions.
Typical ESI operating conditions were spray voltage 3.5 kV and
capillary at 200 °C. Sheath gas was set at 50% for infusion and 80%
for online HPLC. The MSncollision gas was helium with a collision
energy of 24-30% of the 5 V end cap maximum tickling voltage. APCI
was operated at 4.5 kV and 400 °C with sheath gas at 80%.
The total ethanol extract was analyzed on an Agilent 1100 HPLC
(Wilmington, DE), containing a 150 × 4.6 mm 5 µ Luna C-18 column
(Phenomenex, Torrance, CA) operated at 25 °C and initial flow of 0.175
mL/min with a solvent system consisting of acetonitrile/methanol/water
(26:14:60, containing 0.1% formic acid).
RESULTS AND DISCUSSION
The insulinlike biological activity of the cinnamon fraction
is shown in Figure 1. Data are for the insulin-dependent
breakdown of radiolabeled glucose to carbon dioxide. Similar
results were obtained when glucose incorporation into fat was
measured. When no exogenous insulin was added (control),
there was still a maximal amount of insulin-dependent activity
at the highest level of cinnamon tested (Figure 1). Maximal
activity was similar at both the highest levels of cinnamon and
the highest level of insulin. The activities of the different species
of cinnamon tested were not significantly different.
On the basis of the strong insulin-enhancing biological activity
of the cinnamon fraction, we measured the bioactivity of a
number of compounds derived from cinnamon and other related
compounds (Table 1). None of the compounds tested displayed
any insulinlike or insulin-enhancing activity under our assay
Figure 1. Effects of cinnam ononinsulinactivity. Solid black bar is the
control withnoaddedcinnam onfraction. Secondgray barateachlevel
ofinsulindenotes purifiedcinnam onfraction(7m g/m L) diluted1:20, open
bardenotes 1:10 dilution, horizontal linedbardenotes 1:5, andhatched
baris adilutionof1:2. 25µlwas addedtothe2m L assaym ixture. CPM
denotes counts per m inute.
66 J. Agric. Food Chem ., Vol. 52, No. 1, 2004Anderson et al.
It then became critical to isolate and characterize the insulin-
potentiating compounds (see Materials and Methods). The major
peaks eluting at roughly 14, 19, 26, and 34 min (Figure 2) were
polyphenol polymers with an absorption maximum at 279 nm.
Insulin potentiating activity of these peaks was similar.
Infusion into the MS of the trapped preparative HPLC
fractions from the SymmetryPrep C-18 column showed that they
consisted of oligomers ranging in detected masses from 576 to
1728 Da. The chromatographic peaks at Rt) 14, 19, 26, and
34 min contained a trimer, tetramer, trimer, and mixture of
oligomers, respectively. The trimers and tetramer had molecular
masses of 864 and 1152 Da, respectively. The positive ion ESI
mass spectra of the trapped fractions showed most of the charge
residing on the protonated molecular ion (M + H) with the other
ion abundances at less than 25% of the M + H value. Their
protonated molecular masses indicated that they were A-type
doubly linked procyanidin oligomers of the catechins/epicat-
echins (Figure 3). In both the ESI and APCI mass spectra, the
presence of an ion at m/z 287 appears to be indicative of the
doubly linked A-type catechin/epicatechin oligomers. Doubly
linked type-A catechin/epicatechin oligomers contain C4fC8
carbon and C2fO7 ether bonds between the terminal (T) and
middle (M) units of the trimer. Further support for oligomer
structure comes from the MSnexperiments conducted on the
trimer (M + H, m/z 865), in which losses of similar masses
from each monomer, in addition to the catechin losses m/z 286,
288, and 290 Da, were observed (Figure 4). Single linked
oligomers of the catechins also display the same characteristic
HPLC-APCI-MS ion profiling of the SPE purified ethanol
extract for (M + H, m/z 865) also showed the presence of minor
amounts of additional A-type doubly linked trimers associated
with different monomer linkages and/or stereochemistry of the
aliphatic hydroxyl groups. Similar ion profiles were observed
for other oligomers present in the cinnamon extract. The SPE
HPLC-APCI-MS also confirmed the presence of catechin,
epicatechin, and procyanidins B1 and B2 based upon their Rt
and mass spectra compared to that of authentic material.
An APT experiment was used to differentiate the relative
abundance of CH, CH2, and CH3 groups. The APT results
confirmed that an aromatic region of CH groups and an aliphatic
region of CH groups, but only one methylene (CH2) group, were
present. The presence of only one (CH2) group for a given
molecular weight simplified structural elucidation.
The MS result of the peak with a protonated mass of 865 Da
fragmented under positive ESI-MSnconditions into multiple
losses of similar masses, consistent with a trimer. Each major
fragment on further fragmentation exhibited similar mass losses.
The trimer mass formula equals C45H36O18, which comprises
The NMR results confirmed the mass spectrometric data. The
only CH2group is at site 4 in the base unit (B) of the trimer.
Each of the three components in the trimer is a close structural
analogue of the other. The coupling pattern and chemical shifts
Table 1. Com pounds FoundinCinnam onandRelatedCom pounds
cinnam icacidm ethylester
2-m ethoxy-cinnam aldehyde
aCom pounds were dissolved at 1 m g/m L in 0.1 N am m oniumhydroxide or
water.Ifsam pleswerenotsoluble, dim ethysulfoxidewasusedtoim provesolubility.
25 µL of sam ples was assayed directly and at a 10-fold dilution. None of these
com pounds displayed insulin enhancing activity.
Figure 2. HPLC separationof biologically active cinnam onextract.
Figure 3. Structure of doubly linkedprocyanidintype-A polym ers found
in cinnam on that enhance insulin activity.
Figure 4. Positive ESI-MSndissociation schem e for the doubly linked
A-type procyanidin protonated trim er (M + H, m /z 865) isolated from
Cinnam on Increases Insulin ActivityJ. Agric. Food Chem ., Vol. 52, No. 1, 200467
are consistent with the structure as drawn. Eight of the nine
sites numbered 2, 3, and 4 are chiral centers. The NMR evidence
is that a mixture of stereoisomers are present, and because the
stereoisomers have not been separated into enantiomers, as-
signing the chiral centers as R or S would be beyond the scope
of this study. If the three components were exactly equivalent,
NMR would not be able to distinguish it from the structure of
Each component of the trimer, terminal (T), middle (M) and
base unit (B) has minor differences in structure: For the trimer,
the M unit would consist of only a single catechin ,and in the
case of the tetramer, the middle unit would consist of two
catechins. The M and B components with and 8-4 linkage are
structurally identical to proanthocyanidin B-1 dimers (17). The
T, M, and B components have 7, 7, and 8 structurally equivalent
protons, respectively (Table 2). The NMR spectrum (Figure
5) shows that the chemical shift and coupling pattern are
consistent with the NMR data for the M and B portion of the
trimer. The H2′, H5′, and H6 aromatic peaks are similar in each
of the trimers. Consistent with the chemical structure presented,
protons for H6 are singlets in M and B; in T both H8 and H6
are present as doublets with a coupling of 3 Hz. The chemical
shift values for the aliphatic groups integrate for the nine
aliphatic nonexchangeable protons. The aliphatic chemical shifts
for the aliphatic protons in T are consistent with a 2,7 as well
as 4,8 cross link to M. Two magnetically non equivalent peaks
routinely occur for diastereoisomers. Hydrolysis of the 2,7-
linkage from T and M during MS analysis would explain why
the three monomer components had equal molecular weights.
Although the structure is drawn with component M above the
plane of the T component, an equally valid structure would be
with the M component below the plane of the T component
(and the B component below the M component).
Because polyphenols often display antioxidant activity, we
determined the activity of the insulin-enhancing fractions
(Figure 6). There was an inhibition of activity of the production
of reactive oxygen species in collagen-stimulated platelets from
rats for fractions eluting at 14 and 19 min. The data are means
of three separate flow cytometric determinations of fluorescence
of the two fractions to reduce the production of the oxidative
signals in activated platelets in whole blood samples.
Cinnamon is one of the most frequently consumed spices and
is both safe and relatively inexpensive. Furthermore, during
aqueous extraction, the overwhelming majority of the lipid-
soluble components of cinnamon bark would remain in the
insoluble fraction. In the NMR spectrum of the partially purified
extract, much less than 10% of the total organic material in
solution was identified as cinncassiols (hydroxyl-substituted
diterpenoids), saturated hydrocarbons, or other unidentified
compounds (data not shown). The lipid soluble fraction of
cinnamon contains the phytochemicals most likely to be toxic
at higher doses, or with chronic cinnamon ingestion (8, 15, 18).
The cinnamon “essential oil”, containing mainly terpenes,
Table 2: Chem ical Shift Assignm ent for Three Trim er Com ponents
proton T chem icalshiftsMchem icalshifts Bchem icalshifts
Figure 5. NMR spectrumof purifiedwater soluble cinnam onfraction.
68J. Agric. Food Chem ., Vol. 52, No. 1, 2004Anderson et al.
aldehydes, and eugenol is not present or present at very low
levels in the water soluble insulin potentiating fractions;
therefore, these fractions are not likely to have significant
toxicity at physiological doses. The insulin-enhancing biological
activity of the polymers isolated from cinnamon is rather unique
as none of the cinnamon or cinnamonlike compounds we tested
displayed any biological activity in the insulin potentiation assay
The polyphenolic polymers have antioxidant effects, which
may provide synergistic benefits for the treatment of diabetes.
Botanical antioxidants may play a role in helping maintain the
integrity of cell membranes by preventing polyunsaturated fatty
acid peroxidation. The lipid composition of muscle membrane
phospholipids reflects the combined influences of diet and
desaturase/elongase activity, and in turn, the phospholipid
composition affects the binding and action of insulin. In general,
the more unsaturated the membrane, the better glucose is
utilized. The more saturated the membrane, the more deleterious
the effects on insulin efficiency (19).
Lipid peroxidation and damage by reactive oxygen metabo-
lites are also major problems in terms of diabetic complications.
Clinical studies have shown that supplementation of both
nutrient and phytochemical antioxidants can reduce or slow the
progression of various complications of diabetes (20-22).
Catechin procyanidins have been reported in a number of
products (23-25). Most of the procyanidins reported are the
single linked B-type procyanidins. We report here that the major
procyanidin oligomers in the ethanolic and aqueous extracts of
cinnamon are doubly linked type-A. These polymers are
consistent with the oligomeric procyanidins reported to be
present in aqueous fractions from Cinnamonium cassia (24) and
are likely doubly linked A-type mers (25) composed of repeating
mass units of 288.
These studies demonstrate that water-soluble polymeric
compounds isolated from cinnamon have insulin-enhancing
biological activity in the in vitro assay measuring the insulin-
dependent effects on glucose metabolism and also function as
antioxidants. These same compounds have been shown to inhibit
phosphotyrosine phosphatase in the insulin-receptor domain and
to activate insulin receptor kinase (26) and function as a mimetic
for insulin in 3T3-L1 adipocytes (27).
These results suggest that compounds present in cinnamon
may have beneficial effects on glucose, insulin and blood lipids
and may prove to be beneficial in the treatment of diabetes.
More than 100 million people worldwide die each year from
diabetes, and for many, drugs or other forms of treatment are
unavailable. It may be possible that many of these people could
benefit from readily available natural products such as cinnamon
that could have profound effects on their overall health. During
the completion of this work, we completed a human study
involving subjects with type 2 diabetes consuming cinnamon
(28). Subjects consumed 1, 3, or 6 g of cinnamon per day for
40 days with 3 placebo groups corresponding to the three groups
that consumed different numbers of capsules containing cin-
namon. There were significant decreases in fasting serum
glucose (18-29%), triglycerides (23-30%), total cholesterol
(12-26%), and LDL cholesterol (7-27%) after 40 days. Values
after 20 days were intermediate. After 40 days, subjects stopped
taking cinnamon from days 40-60. Values after 60 days were
returning to prestudy levels but were largely lower than initial
values. There were no significant changes in the three placebo
groups. The responses to 1, 3, and 6 g of cinnamon per day
were similar, suggesting that even lower levels of cinnamon
may elicit improvements. The magnitude of these effects due
to taking a common spice illustrate the importance of naturally
occurring insulin-enhancing complexes in the prevention and
alleviation of glucose intolerance and diabetes. Benefits in
insulin sensitivity are also likely to lead to decreased incidence
of cardiovascular diseases, which are more than double in people
The technical assistance of J. Pavlovich to D. J. G at the
University of California at Santa Barbara is appreciated.
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Figure 6. Inhibitionofproductionofreactive oxygenspecies inplatelets
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m in, and (1) fraction eluting at 19 m in. (see Figure 2).
Cinnam on Increases Insulin ActivityJ. Agric. Food Chem ., Vol. 52, No. 1, 2004 69