ACCT 3220 Fall 2013 Exercise #3 PART I Presented below is information related to Falco Corp. July   1 Falco Corp. sold to Legler Co. merchandise having a sales price of $10,000 with terms 2/10, net/60. Falco records its sales and receivables net. 5 Accounts receivable of $12,000 (gross) are factored with Rothchild Credit Corp. without recourse at a financing charge of 9%. Cash is received for the proceeds; collections are handled by the finance company. (These accounts were all past the discount period.) Dec. 29 Legler Co. notifies Falco that it is bankrupt and will pay only 10% of its account. Give the entry to write off the uncollectible balance using the allowance method. (Note: First record the increase in the receivable on July 11 when the discount period passed.) Instructions: Prepare all necessary entries in general journal form for Falco Corp. PART II Fiore Corporation factors $250,000 of accounts receivable with Winkler Financing, Inc. on a with recourse basis. Winkler Financing will collect the receivables. The receivables records are transferred to Winkler Financing on August 15, 2014. Winkler Financing assesses a finance charge of 2% of the amount of accounts receivable and also reserves an amount equal to 4% of accounts receivable to cover probable adjustments. Instructions: (a)   What conditions must be met for a transfer of receivables with recourse to be accounted for as a sale? (b)   Assume the conditions from part (a) are met. Prepare the journal entry on August 15, 2014, for Fiore to record the sale of receivables, assuming the recourse obligation has a fair value of $3,000. PART III Inventory information for Part 311 of Seminole Corp. discloses the following information for the month of June. June  1 Balance 300 units @ $10 June 10 Sold 200 units @ $24 11 Purchased 800 units @ $11 15 Sold 500 units @ $25 20 Purchased 500 units @ $13 27 Sold 250 units @ $27 Instructions: (a)   Assuming that the periodic inventory method is used, compute the cost of goods sold and ending inventory under (1) LIFO and (2) FIFO. (b)   Assuming that the perpetual inventory method is used and costs are computed at the time of each withdrawal, what is the value of the ending inventory at LIFO? (c)   Assuming that the perpetual inventory method is used and costs are computed at the time of each withdrawal, what is the gross profit if the inventory is valued at FIFO? (d)   Why is it stated that LIFO usually produces a lower gross profit than FIFO? KOWSAR Int J Endocrinol Metab. 2010;8(2):68-73 Journal home page: www.IJEM.org Effect of garlic on serum adiponectin and interleukin levels in women with metabolic syndrome Faranak Sharifi 1*, Abdolkarim Sheikhi 1, Mahnaz Behdad 1, Nouraddin Mousavinasab 1 1 Metabolic Diseases Research Center, Valieasr Hospital, Zanjan University of Medical Sciences, Zanjan, IR Iran A B S T R A C T Background: Metabolic syndrome is considered to be an inflam.

1. ACCT 3220
Fall 2013
Exercise #3
PART I
Presented below is information related to Falco Corp.
July 1
Falco Corp. sold to Legler Co. merchandise having a sales price
of $10,000 with terms 2/10, net/60. Falco records its sales and
receivables net.
5
Accounts receivable of $12,000 (gross) are factored with
Rothchild Credit Corp. without recourse at a financing charge
of 9%. Cash is received for the proceeds; collections are
handled by the finance company. (These accounts were all past
the discount period.)
Dec. 29
Legler Co. notifies Falco that it is bankrupt and will pay only
10% of its account. Give the entry to write off the uncollectible
balance using the allowance method. (Note: First record the
increase in the receivable on July 11 when the discount period
passed.)

2. Instructions: Prepare all necessary entries in general journal
form for Falco Corp.
PART II
Fiore Corporation factors $250,000 of accounts receivable with
Winkler Financing, Inc. on a with recourse basis. Winkler
Financing will collect the receivables. The receivables records
are transferred to Winkler Financing on August 15, 2014.
Winkler Financing assesses a finance charge of 2% of the
amount of accounts receivable and also reserves an amount
equal to 4% of accounts receivable to cover probable
adjustments.
Instructions:
(a)
What conditions must be met for a transfer of receivables with
recourse to be accounted for as a sale?
(b)
Assume the conditions from part (a) are met. Prepare the journal
entry on August 15, 2014, for Fiore to record the sale of
receivables, assuming the recourse obligation has a fair value of
$3,000.

3. PART III
Inventory information for Part 311 of Seminole Corp. discloses
the following information for the month of June.
June 1
Balance
300 units @ $10
June 10
Sold
200 units @ $24
11
Purchased
800 units @ $11
15
Sold
500 units @ $25
20
Purchased
500 units @ $13
27
Sold
250 units @ $27

4. Instructions:
(a)
Assuming that the periodic inventory method is used, compute
the cost of goods sold and ending inventory under (1) LIFO and
(2) FIFO.
(b)
Assuming that the perpetual inventory method is used and costs
are computed at the time of each withdrawal, what is the value
of the ending inventory at LIFO?
(c)
Assuming that the perpetual inventory method is used and costs
are computed at the time of each withdrawal, what is the gross
profit if the inventory is valued at FIFO?
(d)
Why is it stated that LIFO usually produces a lower gross profit
than FIFO?
KOWSAR
Int J Endocrinol Metab. 2010;8(2):68-73
Journal home page: www.IJEM.org
Effect of garlic on serum adiponectin and interleukin levels in
women with
metabolic syndrome

5. Faranak Sharifi 1*, Abdolkarim Sheikhi 1, Mahnaz Behdad 1,
Nouraddin Mousavinasab 1
1 Metabolic Diseases Research Center, Valieasr Hospital,
Zanjan University of Medical Sciences, Zanjan, IR Iran
A B S T R A C T
Background: Metabolic syndrome is considered to be an
inflammatory situation with
highly production of adipokines. Garlic and its products are
known to induce anti-
inflammatory effects. However, no data are available on the
anti- inflammatory effects
of garlic in subjects with metabolic syndrome.
Objectives: This study was designed to investigate the effects of
a chemically wellchar-
acterized garlic preparation on biomarkers for inflammation,
and lipid metabolism in
subjects with metabolic syndrome.
Patients and Methods: This study was a double-blind,
randomized, placebo-controlled
trial in 40 adult women, aged > 18 year, who were diagnosed to
have metabolic syn-
drome based on ATPIII criteria and 10 normal women. The
cases were randomly as-
signed to 2 parallel treatment groups: garlic tablets (1.8 g/d), or
placebo. Serum adi-
ponectin, interleukin 6, TNF α and lipid profiles were measured
at baseline and after 6
weeks of treatment and anthropometric measurements were
recorded.
Results: Compared to the placebo group, garlic treatment
resulted in significantly low-
er weight and waist circumference in women with metabolic

6. syndrome. No effect on
weight was detected in normal subjects with garlic. None of the
inflammatory biomar-
kers and plasma lipid levels showed significant differences
between the garlic-treated
and the placebo groups.
Conclusions: This study confirms that garlic has no effect on
major plasma lipoproteins
and furthermore has no impact on inflammatory biomarkers in
women with meta-
bolic syndrome.
A R T I C L E I N F O
Article history:
Received: 5 Jul 2010
Revised: 12 Sep 2010
Accepted: 26 Dec 2010
Keywords:
Garlic
Metabolic syndrome
Lipids
TNF alpha
Interleukin 6
Adiponectin
Article Type:
Original Article
Copyright c 2011, Iran Endocrine Society, Published by
Kowsar M.P.Co. All rights reserved.
* Corresponding author at: Faranak Sharifi, Metabolic Diseases
Research
Center, Valieasr Hospital, Zanjan University of Medical

7. Sciences, Zanjan,
IR Iran. Tel: +98-9121412528, Fax: +98-2417270815.
E-mail: [email protected]
Background
Diet is a key factor in the development of some human
diseases, including cardiovascular disease. The health-
benefits of fruit and vegetable consumption, because of
the anti-inflammatory properties of their phytochemical
components, have been suggested (1). One such source
is garlic (Allium sativum). The majority of garlic (65%)
is water, and the bulk of the dry weight is composed of
fructose-containing carbohydrates, followed by sulfur
c 2011 Kowsar M.P.Co. All rights reserved.
Please cite this paper as:
Sharifi F, Sheikhi AK, Behdad M, Mousavinasab N. Effect of
garlic on serum adiponectin and interleukin levels in women
with meta-
bolic syndrome. Int J Endocrinol Metab. 2010;8(2):68-73.
compounds, protein, fiber, and free amino acids (2). It
also contains high levels of saponins, phosphorus, po-
tassium, sulfur, zinc, moderate levels of selenium and
Vitamins A and C, and low levels of calcium, magnesium,
sodium, iron, manganese, and B-complex vitamins; gar-
lic also has a high phenolic content (3). Recently interest
in garlic as a preventive factor in cardiovascular diseases
has increased. Some studies showed an effect for garlic
in lowering plasma lipid concentrations and preventing
progressive atherosclerotic changes and consequently
reducing the incidence of cardiovascular events (4-6).
Other studies however do not support these observa-
tions and reported no significant effects on these vari-

8. ables (7-9). Clustering of some cardiovascular risk factors
such as abdominal obesity, dyslipidemias, hypertension
Int J Endocrinol Metab. 2010;8(2):68-73
69Garlic, serum adiponectin and interleukin in metabolic
syndrome Sharifi F et al.
and glucose intolerance has been recognized as metabol-
ic syndrome (10), of which central obesity was suggested
to be the basic component. Obesity is associated with
chronic inflammation due to overproduction of proin-
flammatory cytokines, including tumor necrosis factor
(TNF)-α. In vitro studies show that high concentrations of
garlic decrease cytokine production in endothelial cells,
suggesting anti-inflammatory properties (11, 12). To our
knowledge, no studies have reported the effects of garlic
on inflammatory biomarkers in the metabolic syndrome
in humans.
Objectives
This study was designed to investigate the effects of
garlic on markers of inflammation associated with CVD.
In addition, the effects of garlic on plasma lipid concen-
trations, fasting plasma glucose and body weight were
investigated.
Patients and Methods
Subjects
This is a double-blind, randomized, placebo-controlled
study with 3 parallel groups. A total of 50 women, aged
more than 18 years, including 40 cases with metabolic

9. syndrome (MS) and 10 normal female controls were
completed the study. All the subjects with MS were out-
patients at the Endocrine Clinic of Valieasr general Hos-
pital of Zanjan University of Medical Sciences, and were
diagnosed with metabolic syndrome based on the ATPIII
criteria (13). All the subjects with acute or chronic infec-
tion or inflammatory disorders were excluded. They
were also excluded if they were smokers, pregnant or
used any medication (i.e. aspirin, metformin, steroid or
nonsteroidal anti-inflammatory drugs) that interferes
with the measures of the study or affects insulin sensi-
tivity. All the subjects with known diabetes mellitus and
those with triglyceride levels over 400 mg/dL were also
excluded. Normal subjects without any cardiovascular
risk factors were volunteers who drawn from the same
socioeconomic population. Approval for the study was
obtained from the ethics committee of Zanjan Univer-
sity of Medical Sciences. All participants were notified
about the goals of the study, and informed consent was
obtained.The study was registerd in IRCT, code number:
138706121179N1.
Study design
From a list of 102 adult subjects with known metabolic
syndrome who were outpatients of endocrine clinic,50
people were selected with simple random sampling and
recalled. They were randomly assigned to 1 of the 2 treat-
ment arms by a nurse. The subjects received either the
garlic preparation (daily dose of 1.8 g; two 300 mg garlic
tablets three times per day ) or placebo (2 garlicmatching
placebo tablets morning, noon and evening). An addi-
tional 12 normal volunteer women were serially assigned
to the third arm of the study and received garlic tablets
with the same doses of the cases. The total treatment

10. period was 6 wk with follow-up visits scheduled after
2, 4, and 6 wk. On these visits the study medication was
counted, their medical history was taken and adverse
Parameter Metabolic syndrome
placebo group (No. = 20)
Metabolic syndrome garlic
group (No. = 20)
Normal women on garlic
(No. = 20)
Before After Before After Before After
Age 47.9 ± 3 - 50.5 ± 2.9 - 49.2 ± 3.4 -
BMI a 29.1 ± 0.9 29 ± 0.8 29.6 ± 0.8 29 ± 0.9 26.6 ± 1.4 26.1 ±
1
Waist circumference (cm) 103.3 ± 2.7 103 ± 2.7 103.9 ± 2 102 ±
2 87 ± 4.3 88.1 ± 4
Systolic blood pressure (mm Hg) 122 ± 3.1 119 ± 2.5 123 ± 3
122.5 ± 2.9 j 109 ± 3.1 104 ± 4.2
Diastolic blood pressure (mm Hg) 77 ± 2.1 77.5 ± 1.6 81.5 ± 2.7
81 ± 2.6 75.5 ± 3 72 ± 2.9
FPG b (mmol/l) 6.26 ± 0.58 6.2 ± 0.4 5.7 ± 0.27 5.4 ± 0.2 4.6 ±
0.19 4.46 ± 0.14
Chol c (mmol/l) 5.7 ± 0.3 6.37 ± 0.3 i 5.5 ± 0.3 5.4 ± 0.3 4.5 ±
0.4 4.7 ± 0.4
TG d (mmol/l) 2.5 ± 0.24 2.8 ± 0.25 2.7 ± 0.2 2.75 ± 0.35 1.39

11. ± 0.25 1.14 ± 0.14
HDL e-c (mmol/l) 1.01 ± 0.03 1.09 ± 0.2 i 1.08 ± 0.05 1.07 ±
0.08 1.28 ± 0.09 1.48 ± 0.1
Insulin (pmol/l) 45.8 ± 12.3 38.1 ± 8.3 48.3 ± 11 51.8 ± 15.9 27
± 7.2 29.8 ± 11.8
HOMA index f 1.8 ± 0.48 1.4 ± 0.32 1.7 ± 0.38 1.9 ± 0.7 0.88 ±
0.27 0.85 ± 0.28
Adiponectin (ng/ml) 87.7 ± 15 72.5 ± 14.2 91.9 ± 13.3 70.8 ±
8.8 56.5 ± 9.6 36.2 ± 6 k
IL6 g (pg/ml) 0.85 ± 0.4 0.61 ± 0.19 1.3 ± 0.49 1.13 ± 0.55 0.7
± 0.2 1.3 ± 0.2
TNF α h (pg/ml) 1.66 ± 0.28 2.17 ± 0.4 2.35 ± 0.39 2.18 ± 0.5
1.89 ± 0.3 5.5 ± 0.8 k
a BMI: Body Mass Index, b FPG: Fasting Plasma Glucose, c
Chol: Cholesterol, d TG: Triglycerides, e HDL: High Density
Lipoprotein, f HOMA Index: Homeostasis Model
Assessment Index, g IL6: Interleukin 6, h TNF α: Tumor
Necrosis Factor α, i P < 0.05 for difference between baseline
and after intervention in metabolic syndrome
group, j P<0.05 for difference between baseline and after
intervention in metabolic syndrome garlic group, k P < 0.05 for
difference between baseline and after
intervention in normal women garlic group
Table 1. Characteristics of the study population (Data are shown
as Mean ± SE)

12. Int J Endocrinol Metab. 2010;8(2):68-73
70 Garlic, serum adiponectin and interleukin in metabolic
syndromeSharifi F et al.
events were recorded. The participants were asked not
to change their diet or physical activity during the study
and to report any changes. The 300 mg tablets of garlic
were manufactured (Amin pharmaceutical co., Isfahan,
Iran) and matching placebo tablets were produced un-
der standard manufacturing practice standards (Tehran
University of Medical Sciences, Tehran, Iran). All study
medication was labeled, and dispensed by the Valieasr
Hospital endocrine clinic.
Measurements
Body weight was measured to the nearest 0.1 kg with a
balanced beam scale, while wearing light clothing, and
height was measured with a stadiometer to the nearest
0.5 cm. BMI was calculated based on the weight/height²
formula. Waist circumference between the lowest rib and
the iliac crest, at the level of umbilicus, was measured in
duplicate to the nearest millimeter using flexible tape.
Blood pressure was measured with the subject seated
using a random zero sphygmo-manometer. Systolic (Ko-
rotkoff phase I) and diastolic (Korotkoff phase V) blood
pressure was measured twice on the left upper arm and
the average of the two measurements was used for analy-
sis.In all the women, blood samples were collected be-
tween 8.00-9.00 AM, after at least 12 hours of fasting. The
basal levels of adiponectin, insulin, interleukin 6 (IL6),
tumor necrotizing factor α (TNF α) and plasma glucose
were measured, and a lipid profile was also obtained. The
Homeostasis Model Assessment Index (HOMA Index) was
used to determine the level of insulin resistance and was

13. calculated according to the following equation:
[Insulin (μU/mL)] [FPG (mmol/L)] /22.5. Insulin resis-
tance was diagnosed in cases with a HOMA index of more
than 2.1, (14).
Insulin levels were measured via an electrochemilu-
minescence immunoassay (ECLIA) using commercially
available kits (Roche, German), and adiponectin was
measured using human adiponectin ELISA kits (Bioven-
dor, Germany) with a limit of detection 7 ng/ml. Intra and
inter-assay CV for the assay were 7 and 8 % in the lower
limit and 6.4% and 7.3% for upper limit concentrations
respectively. TNF-α was measured with the use of ELISA
kits (Bender medsystem), with a sensitivity of 5pg/ml
and inter and intra-assay CVs of 8.1 and 7.7% respectively.
IL 6 was measured by ELISA kits (Bender medsystem). The
sensitivity of the kit was 0.92 pg/mL. Inter and intra-assay
CV for the kits were 5.2% and 3.4% respectively.
Statistical methods
Data are presented as Mean ± SE. Between group com-
parisons of changes in lipid and adipokines parameters
were done using one-way ANOVA. Mann-Whitney test was
used for those variables without normal distribution.
Within group assessments of changes in the parameters
were analyzed by paired the t-test and Kruskal-Wallis Test.
Significance of differences was evaluated using the SPSS
version 11.5 and defined at a 0.05 level of confidence.
Results
At the end of the study, of 50 subjects with MS, five in
the garlic group and six in the placebo arm did not com-

14. plete the study and were excluded. Of 12 normal subjects,
who entered in the study, 10 completed it. All the exclud-
ed subjects were followed by telephone. No complication
with the medication was reported by the excluded sub-
jects. One additional subject with MS entered later to the
placebo arm of the study and finally the study popula-
tion consisted of 50 subjects, 20 subjects with metabolic
syndrome in the garlic group, 20 subjects with meta-
bolic syndrome in the placebo group, and 10 normal
subjects in the garlic group. No significant differences
were observed in the baseline characteristics among the
3 groups. The adverse events reported in this study were
mild and data showed a good compliance for the medica-
tions. Clinical parameters and markers of inflammation
at the 6 week assessment of women with metabolic syn-
drome before and after intervention are shown in Table1.
Compared with placebo group, garlic treatment re-
sulted in significantly lower weight (P = 0.04) and waist
circumference (P < 0.001) in women with metabolic
syndrome. No effect on weight (71.4 ± 3.8 Kg before treat-
ment vs. 71.8 ± 4 Kg after treatment, p = 0.4) and waist
circumference (87.8 ± 4.3 cm before treatment vs. 88± 4.4
cm after treatment, p = 0.4) was detected in normal sub-
jects with garlic. None of the inflammatory biomarkers
and plasma lipid levels showed significant differences
between the garlic-treated and placebo groups with met-
abolic syndrome (Table2).
While significant rise was seen in serum adiponectin
concentration in normal subjects with garlic (56.5 ± 9.6
ng/ml after treatment vs. 36.2 ± 9.6 ng/ml before treat-
ment, p = 0.024), no significant changes in other inflam-
matory biomarkers or lipid concentrations were ob-
served in normal subjects using garlic.

15. Discussion
This double blind placebo-controlled study demon-
strated no effect of a 6-wk treatment with garlic tablets
on plasma lipid concentrations of women with metabol-
ic syndrome. Although a significant statistical reduction
in weight and waist circumference was seen with garlic,
the reduction was not clinically significant. The main
finding of this study was that garlic has no detectable ef-
fects on TNF-α, interleukin 6 and adiponectin concentra-
tions in women with metabolic syndrome, which makes
it unlikely that garlic exerts a beneficial effect on car-
diovascular disease prevention by anti-inflammatory or
lipid-lowering mechanisms. For a long time a preventive
effect on atherogenesis has been considered for garlic
because its lipid lowering properties. It has been shown
that garlic inhibits enzymes involved in lipid synthesis,
decreases platelet aggregation, and prevents lipid per-
Int J Endocrinol Metab. 2010;8(2):68-73
71Garlic, serum adiponectin and interleukin in metabolic
syndrome Sharifi F et al.
Parameter
(Mean ± SE a)
Average treatment effect
(97.5% CI)
Garlic compared with placebo
Metabolic syndrome
Placebo group (No. = 20)

16. Metabolic syndrome
Garlic group (No. = 20)
% Change (97.5% CI of difference) p-value
Waist circumference (cm) (-1.2)-(0.6) (-2.4)-(-0.7) (-1.2)-(-0.1)
0.036
Systolic blood pressure (mmHg) (-5.4)-(1.4) (-3.7)-(2.7) (1.3) -
(1.7) 0.5
Diastolic blood pressure (mmHg) (-3)-(5.8) (-5.5)- (5.7) (-2.5)-
(-0.1) 0.7
FPG b (mmol/l) (-3.3)-(11.9) (-12.3) - (6.6) (-9)- (-5.3) 0.2
Chol c (mmol/l) (3.6)-(21.6) (-11.7) - (20.4) (-15.3)-(-0.8) 0.3
TG d (mmol/l) (-4.8)-(31.6) (-29)- (41) (-24.2)-(9.4) 0.6
HDL e (mmol/l) (0.12)-(16.8) (-14)-(17) (-14.1)-(0.2) 0.4
Insulin (pmol/l) (-22)-(208) (-17.6)-(63) 4.4-(-145) 0.2
Adiponectin (ng/ml) (-39.4)-(39.2) (-38)-(44.7) (1.4)-(5.5) 0.9
IL6 f (pg/ml) (-105.4)-(527) (-74.8)-(213) (30.6)-(-314) 0.3
TNF α g (pg/ml) (-49)-(168) (-55.7)-(100) (-6.7)-(-68) 0.5
a SE: Standard Error of mean, b FPG: Fasting Plasma Glucose,
c Chol: Cholesterol, d TG: Triglycerides, e HDL: High Density
Lipoprotein, f IL6: Interleukin 6, g TNF α:
Tumor Necrosis Factor α
Table 2. Average treatment effects of placebo and garlic on

17. clinical parameters and markers of inflammation at the 6-week
assessment of women with meta-
bolic syndrome
oxidation of oxidized erythrocytes and LDL (15, 16). How-
ever, most clinical trials have indicated no reduction in
total cholesterol with garlic and its effect on blood pres-
sure and oxidative-stress reduction is controversial (17).
The different results obtained in clinical trials may be
due to usage of different garlic preparations with differ-
ent dosage, selection of subjects, and duration of trials.
Different garlic preparations have different properties.
Some toxicity has been reported with raw garlic. Garlic
powder must be prepared at a suitable temperature and
is associated with some toxicity. No major studies on the
efficacy of garlic oils have been reported (18). Aged garlic
extracts (AGE) have increased sulfur compounds with the
loss of allicin. There is also substantial variability in the
contents of garlic preparations, with inadequate defini-
tions of the biologically active and available constituents
and their dissolution properties making it difficult to
confirm the garlic in these trials. There is some evidence
that higher garlic doses are associated with more signifi-
cant effects. In the present study, we chose a relatively
high dose of the garlic tablets (1.8 g/d, approximately
equivalent to 9 mg allicin/d) to be able to detect the po-
tentially beneficial effects of garlic. Measurement of gar-
lic components plasma concentrations would have been
helpful, but, unfortunately, these were not measured in
our study. Our data are in agreement with many of the
recent findings that did not observe any effects of garlic
on plasma lipids (7, 8). To our knowledge, this is the first
report for the effects of garlic on lipid concentrations
in subjects with metabolic syndrome. Although a clini-
cally significant rise in HDL-cholesterol was observed
in normal women with garlic, this difference was not

18. statistically significant. This result may be due to small
sample size of normal women in our study. No signifi-
cant changes in total cholesterol, triglyceride and low-
density lipoprotein cholesterol (LDL-C) were observed in
normal women using garlic. Central obesity and conse-
quent insulin resistance are major factors of metabolic
syndrome and confer an increased risk of atherosclero-
sis and cardiovascular disease (CVD). The possible role of
inflammatory cytokines originating from adipose tissue
in this process is still being elucidated. A growing body
of evidence suggests that inflammatory processes play
an important role in the pathophysiology of atheroscle-
rosis (19-21). Substances like interleukin (IL)-6 and tumor
necrosis factor (TNF)-α, are indicators of oxidative stress
and may play a role in promoting adverse vascular out-
comes in the metabolic syndrome (9). Low levels of adi-
ponectin and increased leptin also have been reported in
this syndrome. Adiponectin is inversely associated with
insulin resistance and inhibits inflammation. IL-6 is a cy-
tokine that increases triglyceride formation and insulin
resistance. Levels of IL-6 are linked to insulin resistance,
adiposity (22), and the metabolic syndrome (23). TNF-α
is produced mostly by fatty tissue, but also by macro-
phages and endothelial cells. Its levels correlate with the
amount of adiposity and have been linked to the increase
in insulin resistance seen in obesity (24, 25). We chose
adiponectin, IL-6 and TNF- α as primary outcomes of this
study because of their possible effect on atherogenesis in
the metabolic syndrome. Some in vitro studies indicated
a reduced cytokine production in endothelial cells with
high concentrations of garlic suggesting that it has anti-
inflammatory properties (15, 26, 27).
Among limited number of clinical trials performed with
garlic and different preparations of garlic, most evalu-

19. ated the effect of garlic on glucose control, lipid profiles,
hypertension and platelet aggregation (1, 7, 28, 29); only
two clinical trials have been conducted to evaluate the ef-
fect of garlic on inflammatory cytokines, and both have
shown that garlic has no effect on these markers (30, 31).
Of these, one study was performed in overweight smok-
Int J Endocrinol Metab. 2010;8(2):68-73
72 Garlic, serum adiponectin and interleukin in metabolic
syndromeSharifi F et al.
er subjects, and the other in men with coronary artery
disease; our results were in agreement with these two
studies and revealed no significant changes in IL-6, TNF-α
and adiponectin after a 6-week duration of treatment
with garlic tablets, in women with metabolic syndrome.
A rise in adiponectin concentration in normal women
after garlic administration in this study may suggest a
different effects of garlic in different health conditions,
suggesting a preventive role for cardiovascular disease
with garlic in healthy people. However considering the
small sample size of normal subjects and lack of normal
people on placebos in this study, any results in the nor-
mal group should be interpreted cautiously.
In conclusion, our data demonstrated that a 6-week
treatment with a high-dose, garlic tablets had no anti-
inflammatory or lipid-lowering effect in women with
metabolic syndrome. This suggests that short-time ad-
ministration of garlic has no significant effect on the
inflammatory processes associated with atherosclero-
sis in a high risk population. Furthermore, increases in
adiponectin levels in normal women with garlic showed

20. that garlic tablets may have some anti-inflammatory ef-
fects in healthy people, and may prevent cardiovascu-
lar events in this group. More investigations with larger
sample sizes and longer duration of interventions are
recommended.
Financial support
This study was supported by Zanjan University of Medi-
cal Sciences and was approved as the residency thesis of
Dr. Behdad.
Conflict of interest
None declared.
Acknowledgement
We are grateful to Dr. Hamidi, Mr. Rahimi and to the
laboratory staff for their assistance in this research.
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26. DOI 10.1007/s00217-006-0295-z
O R I G I N A L PA P E R
V. Ani · M. C. Varadaraj · K. Akhilender Naidu
Antioxidant and antibacterial activities of polyphenolic
compounds
from bitter cumin (Cuminum nigrum L.)
Received: 16 November 2005 / Revised: 8 February 2006 /
Accepted: 13 February 2006 / Published online: 9 March 2006
C© Springer-Verlag 2006
Abstract Cumin is one of the commonly used spices in
food preparations. It is also used in traditional ayurvedic
medicine as a stimulant, carminative and astringent. Ear-
lier we have reported that bitter cumin (Cuminum nigrum
L.) possess the most potent antioxidant activity among
cumin varieties—cumin, black cumin and bitter cumin. In
this study, we have further characterized the polyphenolic
compounds of bitter cumin and also their antioxidant and
antibacterial activity using different model systems. The
major polyphenolic compounds of cumin seeds were ex-
tracted with 70% methanol, 70% acetone, water, separated
by HPLC and their structures were elucidated by LC-MS.
The profile of phenolic acids/flavonols in bitter cumin were
found to be gallic acid, protocatechuic acid, caffeic acid,
ellagic acid, ferulic acid, quercetin and kaempferol. The
antioxidant activity of the cumin extract was tested on 1,1-
diphenyl-2-picryl hydrazyl (DPPH) free radical scaveng-
ing, soybean lipoxygenase-dependent lipid peroxidation,
rat liver microsomal lipid peroxidation and superoxide an-
ion (O2−) scavenging. The bitter cumin extract exhibited
high antioxidant activity with IC50 values of 14.0 ± 0.5 µg,
28.0 ± 3.0 µg, 110 ± 14.0 µg and 125.4 ± 8.7 µg of the ex-
tract, respectively for DPPH free radical scavenging, soy-

27. bean lipoxygenase-dependent lipid peroxidation, rat liver
microsomal lipid peroxidation and superoxide anion scav-
enging. Further, the extract offered a significant protection
against DNA damage induced by hydroxyl radicals. Among
a spectrum of food-borne pathogenic and spoilage bacteria
tested, the cumin extract significantly inhibited the growth
of Bacillus subtilis, Bacillus cereus and Staphylococcus
aureus. Thus, bitter cumin with an array of polyphenolic
V. Ani · K. A. Naidu (�)
Biochemistry and Nutrition, Central Food Technological
Research Institute,
Mysore 570 020, India
e-mail: [email protected]
Fax: +91-821-2517233
M. C. Varadaraj
Human Resource Development, Central Food Technological
Research Institute,
Mysore 570 020, India
compounds possesses potent antioxidant and antibacterial
activities.
Keywords Bitter cumin . Phenolic acids . DPPH
radicals . Superoxide anion scavenging . Lipid
peroxidation . DNA damage . Antibacterial activity
Introduction
A number of physiological processes in human body lead to
the generation of a series of oxygen-centered free radicals
and other reactive oxygen species (ROS) as by-products.
ROS play a positive role in energy production, phagocy-
tosis, regulation of cell growth, intercellular signaling and
synthesis of biologically important compounds. However,

28. overproduction of ROS is also harmful to the body because
the oxidation induced by ROS can result in cell membrane
disintegration, membrane protein damage and DNA muta-
tion, which can further initiate or propagate the develop-
ment of many diseases [1, 2]. Antioxidants are compounds
that can delay, inhibit, or prevent the oxidation of oxi-
dizable matters by scavenging free radicals and diminish
oxidative stress. Plants contain a wide variety of antiox-
idant phytochemicals or bioactive molecules, which can
neutralize the free radicals and thus retard the progress of
many chronic diseases associated with oxidative stress and
ROS. The intake of natural antioxidants has been associ-
ated with reduced risk of cancer, cardiovascular disease,
diabetes and diseases associated with ageing. Studies on
dietary free radical scavenging molecules have attracted
the attention to characterize phenolic compounds and other
naturally occurring phytochemicals as antioxidants.
Spices and condiments have become an integral part of
human diet to impart flavour, taste and colour to the food.
Spices are also considered as nutraceuticals in view of their
nutritional, medicinal and therapeutical properties. Cumin
is one of the most popular spices used in food preparations
in India and South East Asia. Cumin (Cuminum cyminum)
and black cumin (Nigella sativa) are widely as spice
condiment in vegetarian and non-vegetarian preparations
110
along with other spices in India and Arabia. Bitter cumin
(Cuminum nigrum L.) locally known as ‘Shahi jeera’ or
‘Kashmiri’ cumin belongs to the family Apiaceae and
grows mainly in Central Asia and India. It is used in spice
mix or garam masala, pickles, wheat and rice dishes. It is

29. bitter in taste compared to other two varieties of cumin viz.,
normal cumin and black cumin. In traditional ayurvedic
medicine, it is used as a stimulant, carminative, astringent
and useful in dyspepsia and diarrhea [3]. Earlier we
reported a comparative study on the antioxidant potency of
the three cumin varieties and showed that bitter cumin pos-
sess high antioxidant activity compared to other two cumin
varieties [4]. In this study, we report isolation and charac-
terization of bioactive polyphenolic compounds from bitter
cumin and their antioxidant and antimicrobial properties.
Materials and methods
Materials
C. nigrum seeds were obtained from the local market, iden-
tified and authenticated at the Department of Botany, Agri-
culture University, Bangalore. Diphenyl-picryl hydrazyl
(DPPH), linoleic acid, soybean type IV lipoxygenase,
Tween 80, Tris, calf thymus DNA, reference standards of
phenolic acids viz., gallic acid, protocatechuic acid, caffeic
acid, ellagic acid, ferulic acid, quercetin and kaempferol,
thiobarbituric acid were purchased from Sigma Chemical
Co., MO, USA. Nictinamide adenine dinucleotide-reduced
(NADH) and phenazine methosuphate (PMS) were pur-
chased from Hi Media, Mumbai, India. Nitroblue tetra-
zolium (NBT) was purchased from Sisco Research Labo-
ratories, Mumbai, India. pUC18 DNA was purchased from
Bangalore Genei, Bangalore, India. All other chemicals
and solvents used were analytical grade.
Extraction of polyphenolic compounds
from C. nigrum seeds
Bitter cumin seeds were powdered in a mixer grinder. The
powder was defatted with hexane in a soxhlet’s apparatus

30. for 6 h. Five grams of defatted cumin powder was extracted
with 100 ml of 70% aqueous methanol and 70% aqueous
acetone in 1:1 ratio by stirring for 2 h. The residue was ex-
tracted thrice with above solvents. The combined extracts
were concentrated under vacuum in a rotavapour and sub-
jected to hydrolysis with 2N HCl to facilitate the breakage
of glycosides. It was then phase-separated with hexane to
remove any traces of fatty acids and subsequently with
ethyl acetate to extract polyphenolic compounds. The ethyl
acetate phase was concentrated under vacuum. The residue
was weighed and kept at 4 ◦C until use.
Estimation of total phenolic compounds
The total phenolic content of the above extract was esti-
mated by Folin–Ciocalteau method [5]. In brief, the extract
Table 1 Phenolic compounds identified in C. nigrum seeds
Polyphenolic compounds Concentration (µg/g dry weight)
Gallic acid 173.50
Protocatechuic acid 130.50
Caffeic acid 500.90
Ellagic acid 150.10
Ferulic acid 375.50
Quercetin 154.60
Kaempferol 94.70
was dissolved in methanol and an aliquot of this solution
was added to 2 ml of 2% Na2CO3 and after 2 min 100 µl
of Folin-reagent (diluted 1:1) was added. After 30 min, the
absorbance was measured at 750 nm using Shimadzu UV-
Visible Spectrophotometer-1601. The total phenol content
was expressed as gallic acid equivalents (GAE) per mg of
extract calculated from standard graph of gallic acid.

31. Estimation of polyphenolic compounds from bitter
by HPLC and LC-MS
The hydrolyzed cumin extract was dissolved in methanol
and subjected to HPLC for the qualitative and quantitative
analysis of phenolic contents. The HPLC system Shimadzu
LC-10 A (Japan) was equipped with dual pump LC-10AT
binary system, UV detector SPD-10A, Phenomenex Luna
RP, C18 column (i.d. 4.6 mm × 250 mm) and data was
integrated by Shimadzu Class VP series software. The fol-
lowing gradient programme was employed (A) acetic acid
(1%) and (B) acetonitrile; 18% B at 0 min, 32% at 15.0 min
and finally to 50% at 40.0 min. The amount of phenolic
compounds (µg/g dry weight) were calculated by compar-
ison of peak areas (254 nm) of the samples with that of
standards (Fig. 1 and Table 1). The HPLC retention times
0
20
40
60
80
100
120
0 2.5 5 10 15 20 30 40 60 100 140 200
Bitter cumin extract (µg)

32. R
em
ai
n
in
g
D
P
P
H
(
%
)
BHT
C.nigrum
BHA
Fig. 1 C. nigrum seed extract BHA and BHT-scavenged DPPH
free
radicals. Values mean ± SEM of three individual experiments
111
Table 2 HPLC and MS characteristics of C. nigrum seeds

33. Retention time (min) [M-H]− Fragmented ion Corresponding
fragment Identified compound
6.37 169 125 M-COO Gallic acid
8.41 153 109 M-COO− Protocatechuic acid
13.47 179 135 M-COO− Caffeic acid
17.66 300.8 170 M-125 Ellagic acid
125 Trihydroxybenzene fragment
21.24 193 178 M-O− Ferulic acid
149 M-COO−
29.21 301.1 151 M-free phenol at 2 position and a portion of the
benzopyranone ring moiety
Quercetin
37.31 285 133 M-151 Kaempferol
151 Free phenol at position 2 and a portion of the
benzopyranone part
of the individual peaks of polyphenolic compounds are
shown in Table 2.
An API 200 triple quadrupole mass spectrometer (Ap-
plied Biosystems) was used for determining the mass of the
polyphenolic compounds. Analysis were performed on a
Turbo ions spray source in negative mode by using settings
nebuliser gas 16 (N2) (arbitrary units), focusing potential
− 400 V, entrance potential − 10, declustering potential
(DP) 25–60 and collision energy (CE) 15–35. Full scan
acquisition was performed scanning from m/z 150–700 u
at a cycle time of 2 s. MS product ions were produced
by collision-associated dissociation (CAD) of the selected

34. precursor ions in collision cell. In all the experiments, both
the quadrupoles (Q1 and Q3) were operated at unit resolu-
tion. Product ion scan of selected molecules were carried
out in order to confirm the structure of the compounds.
Determination of antioxidant activity
Measurement of DPPH radical scavenging activity
The DPPH free radical scavenging test was carried out
as described elsewhere [6]. Different dilutions of the
extract of bitter cumin were incubated with 1 ml of DPPH
solution (50 × 10−5 M). The decrease in absorbance due
to scavenging DPPH radicals by bitter cumin extract
was determined at 517 nm using a Shimadzu UV-Visible
Spectrophotometer-1601 (Shimadzu, Kyoto, Japan). BHT
were used as BHA standard. The percentage of remaining
DPPH after 5 min was calculated for individual experi-
ments and the concentration at which 50% of the initial
and remaining DPPH concentrations were calculated from
standard DPPH graph.
Measurement of superoxide anion scavenging activity
The superoxide scavenging ability of the bitter cumin ex-
tract was assessed by a modified method as described else-
where [7]. Superoxide anions were generated in samples
that contained 100 µl each of 1.0 mM NBT, 3.0 mM NADH
and 0.3 mM PMS and the final volume was adjusted to 1 ml
with 0.1 M phosphate buffer (pH 7.8) at ambient tempera-
ture. The reaction mixture (NBT and NADH) was incubated
with or without cumin extract at ambient temperature for
2 min and the reaction was started by adding PMS. The
absorbance at 560 nm was measured against blank sam-
ples for 3 min. Decrease in absorbance in the presence of

35. cumin extracts indicated superoxide anion scavenging ac-
tivity. The percent inhibition was calculated by using the
following formula.
Superoxide scavenging activity (%)
= Control OD − Sample OD
Control OD
× 100
Measurement of rat liver microsomes lipid
peroxidation activity
Rat liver was homogenized in 0.25 M Tris–HCl–Sucrose–
EDTA buffer at pH 7.4. The homogenate was centrifuged
at 7649 × g for 30 min to remove unbroken cells and cell
debris. The supernatant was centrifuged at 100,000 × g in
a refrigerated ultracentrifuge (Beckman L7-65 Ultracen-
trifuge, USA) for 1 h to isolate the microsomal pellet,
which is dissolved, into 2 ml of 125 mM KCl. The protein
content of microsomes was estimated by Lowry method
[8]. One milligram of microsomal protein was incubated
with 0.2 mM FeSO4 and 0.2 mM ascorbic acid with or
without cumin extract in a final volume of 1 ml at 37 ◦C
for 1 h. Malondialdehyde (MDA) formed in the incubation
mixture was reacted with thiobarbituric acid and thiobarbi-
turic acid reactive substances (TBARS) were measured at
535 nm spectrophotometrically [9]. The antioxidant activ-
ity was expressed as decrease in oxidation of microsomal
lipids measured as n moles of MDA formed per minute per
milligram protein using a molar extinction coefficient of
1.56 × 105 M/cm.
Lipoxygenase-dependent lipid peroxidation activity

36. Soybean lipoxygenase-dependent lipid peroxidation was
measured as a decrease in absorbance of lipid hydroper-
112
oxide formation at 234 nm spectrophotometrically [10].
Briefly, the reaction mixture in a final volume of 1 ml con-
tained 200 µM linoleic acid and 5 nM soybean lipoxy-
genase in 50 mM Tris buffer, pH 7.4. The absorption
due to the formation lipid hydroperoxide was monitored
at 234 nm in a Shimadzu UV-Visible spectrophotome-
ter. Different concentrations of bitter cumin extract were
incubated for 2 min with soybean lipoxygenase prior to
initiation of reaction with linoleic acid. The decrease in
lipid hydroperoxide formation in the presence of cumin
extract was calculated using an extinction coefficient
of 25 mM/cm. The enzyme activity was expressed as
moles of hydroperoxide formed per minute per nM of
enzyme.
Antioxidant activity against oxidative damage to DNA
Hydroxyl radicals generated by Fenton reaction was used
to induce oxidative damage to DNA. The reaction mixture
(9 µl) contained 3 µg of calf thymus DNA in 20.0 mM
phosphate buffer saline (pH 7.4) and different concentra-
tions of bitter cumin extract (0.5, 1.0, 1.5 and 2.0 µg) were
added and preincubated with DNA for 15 min at ambient
temperature. The oxidation was induced by treating DNA
with 1.0 mM FeSO4 and 10.0 mM ascorbic acid and incu-
bated them for 1 h at 37 ◦C. Similarly 1.0 µg of pUC18
DNA was incubated with different concentrations of cumin
extract for 30 min at ambient temperature and 2.0 µl each
of 100.0 µM FeSO4, 600.0 µM ascorbic acid and 60.0 mM

37. H2O2 were added. The final volume was adjusted to 20.0 µl
with 20.0 mM phosphate buffer (pH 7.4) and incubated for
1 h at 37 ◦C. The reaction was terminated by the addition of
loading buffer (xylene cyanol, 0.25%; bromophenol blue,
0.25% and glycerol 30%) and the mixture was subjected to
gel electrophoresis in 1% agarose/TAE buffer run at 60 V
for 3 h. DNA was visualized and photographed by a digital
imaging system (Hero Lab, GMBH, Germany).
Determination of antibacterial activity
Antibacterial activity of cumin extract was tested against
food-borne pathogenic and spoilage bacteria viz., Bacillus
subtilis, Bacillus cereus, Enterobacter spp., Escherichia
coli, Listeria monocytogenes, Staphylococcus aureus and
Yersinia enterocolitica by agar diffusion method. Plate
count agar plates were prepared using 1% brain heart infu-
sion broth inoculum of individual bacterial cultures. Five
equidistant wells of 5.0 mm each were made in the solidified
agar medium using sterile stainless steel cork borer. Dif-
ferent concentrations of cumin aqueous methanol–acetone
extract were added to the wells and the control well re-
ceived the same volume of methanol. Initially, the plates
were kept at 6 ◦C for 3 h and then incubated for 22 h at
37 ◦C. The inhibition zones formed around the well were
measured to an accuracy of 0.1 mm and the activity was
calculated as a mean of triplicate for each of the indicated
bacterial species.
Statistical analysis
All the experimental data are presented as mean ± SEM of
three individual samples. Data are presented as percentage
of free radical scavenging/inhibition lipid peroxidation on
different concentration of cumin extracts. IC50 (the concen-
tration required to inhibit 50% of enzyme activity/scavenge

38. 50% of free radicals) value was calculated from the dose-
response curves. Antibacterial effect was measured in terms
of zone of inhibition to an accuracy of 0.1 mm and the effect
was calculated as a mean of triplicate tests.
Results and discussion
Spices occupy an important place in our food as taste
and aroma enhancers since ancient times. A variety of
molecules derived from spice possess bioactive properties.
Of these phenolic compounds constitute the largest propor-
tion of known natural antioxidants [11]. There is an increas-
ing interest in natural antioxidant molecules from food to
prevent the deleterious effects of free radicals in biological
systems and also prevent the deterioration of foods due to
oxidation of lipids and microbial spoilage. Cumin is one of
the commonly used spice condiment in both vegetarian and
non-vegetarian food preparations in Asia and Arabia. Our
earlier study showed that bitter cumin was most potent an-
tioxidant among the three cumin varieties [4]. In this study,
we have further characterized the polyphenolic compounds
and also evaluated the antioxidant and antibacterial activity
of bitter cumin.
The polyphenolic compounds from bitter cumin seeds
were extracted with aqueous 70% methanol, 70% acetone
and ethyl acetate to facilitate extraction of low- and high-
molecular weight polyphenols. Thus, with the above sol-
vent system we could extract a number of phenolic acids
and flavonols from bitter cumin (Table 1). The total phenol
content of this extract was estimated to be 551.8 ± 0.82 µg
GAE/mg of extract.
Many phenolic compounds are normally present as gly-
cosides or aglycones in plants. The cumin extract was sub-
jected to acid hydrolysis (2N HCl) to break the glycoside

39. linkages and the polyphenolic compounds in hydrolysate
were separated, identified and quantified by LC-MS. The
identification of the individual phenolic compounds was
achieved by comparing retention time and the peak area
of cumin extract compounds with that of standards). Inter-
estingly, bitter cumin contained a number of polyphenolic
compounds including gallic acid, protocatechuic acid,
caffeic acid, ellagic acid, ferulic acid and also flavonols
such as quercetin and kaempferol (Table 1). The MS char-
acteristics of identified polyphenolic compounds are given
in Table 2. Caffeic and ferulic acids were found to be most
abundant among phenolic acids present in bitter cumin
(Table 1). This is the first report showing the presence of
an array of polyphenolic compounds in bitter cumin seeds.
There are many methods to assess the antioxidant activ-
ity, but each method has its own limitations [12]. Hence,
we tested the effect of bitter cumin extract on different
113
antioxidant assays including DPPH free radical scavenging,
superoxide anion radical scavenging, rat liver microsomal
lipid peroxidation, soybean lipoxygenase-dependent lipid
peroxidation and oxidative damage to DNA to understand
its antioxidant potential. Further, we have also tested its
effect on selected food-borne pathogenic and spoilage
bacteria to assess the antibacterial activity of bitter cumin.
The DPPH radical scavenging is a sensitive antioxidant
assay and is independent of substrate polarity [13].
DPPH is a stable free radical that can accept an electron
or hydrogen radical to become a stable diamagnetic
molecule. Cumin extract exhibited a dose-dependent

40. scavenging of DPPH radicals and 14.0 ± 0.5 µg of extract
was sufficient to scavenge 50% of DPPH radicals/ml. The
radical scavenging effect of bitter cumin was found to
be 2.6 times more potent than the standard BHT (IC50
36.4 ± 1.2 µg/ml) (Fig. 1) but, less potent than BHA (IC50
8.25 ± 0.35 µg). This suggests that bitter cumin is a good
free radical scavenger or hydrogen donor and contributes
significantly to the antioxidant capacity of bitter cumin.
Superoxide anion is a reduced form of molecular oxygen
and plays an important role in the formation of other reac-
tive oxygen species such as hydrogen peroxide, hydroxyl
radical or singlet oxygen [14]. The bitter cumin extract
was found to be an effective scavenger of superoxide anion
radicals in a dose-dependent manner with an IC50 value of
125.4 ± 8.7 µg (Fig. 2) and thus can prevent the formation
of ROS.
Lipid oxidation of fats and fatty foods not only brings
about chemical spoilage in foods, but also produces free
radicals such as peroxy radicals, which are purportedly as-
sociated with carcinogenesis, autogenesis and ageing [15,
16]. Membrane lipids are particularly susceptible to ox-
idation because of their high polyunsaturated fatty acid
content but also of their association in the cell membrane.
Lipid peroxidation is a free-radical chain reaction and the
0
20
40
60
80

41. 100
120
0 25 50 100 200 400
Bitter cumin extract (µg)
p
er
ce
n
ta
g
e
sc
av
en
g
in
g
o
f
su
p
er

42. o
x
id
e
an
io
n
Fig. 2 C. nigrum seed extract scavenged superoxide anion
radicals.
Values mean ± SEM of three individual experiments
0
0
200
400
600
800
1000
50 100 150 200 250 350300 400
Bitter cumin extract (µg)
n
m
o

43. le
s
o
f
M
D
A
f
o
rm
ed
/m
g
o
f
p
ro
te
in
Fig. 3 C. nigrum seeds extract inhibited the peroxidation of rat
liver microsomal membrane lipids. Values mean ± SEM of three
individual experiments
reactive oxygen species can accelerate lipid oxidation [17].
Hydroxyl radicals are the most reactive free radicals capa-
ble of reacting with lipids, polypeptides, proteins and DNA

44. [18]. Bitter cumin extract significantly inhibited hydroxyl
radical induced oxidation of rat microsomal lipids with an
IC50 value of 110.0 ± 14.0 µg (Fig. 3). The polyphenols
present in bitter cumin extract might react with hydroxyl
radical by donating hydrogen atom and convert them to
more stable no radical products and thus prevent the mi-
crosomal lipid peroxidation (Table 3).
The autoxidation of lipids as well as the enzymatic oxi-
dation of fats, oils and fat-containing foods during storage
and processing are responsible for rancidity and deteri-
oration of food quality. Soybean lipoxygenase-dependent
lipid peroxidation is an enzymatic lipid peroxidation as-
say used to determine the antioxidant activity of test
compounds. Cumin extract showed significant inhibition
of lipoxygenase-dependent lipid peroxidation and the in-
hibitory effect is found to be dose-dependent with an IC50
value of 28.0 ± 3.0 µg (Fig. 4). However, bitter cumin is
found to be less potent compared to synthetic antioxidant
BHA. Plant phenols and flavonoids are known to inhibit
lipid peroxidation by quenching lipid peroxy radicals and
reduce or chelate iron in lipoxygenase enzyme and thus
prevent initiation of lipid peroxidation reaction [19–21].
Similarly, the polyphenolic compounds such as quercetin,
caffeic acid, ferulic acid, ellagic acid present in bitter cumin
being free radical scavengers could react with peroxy rad-
ical before the fatty acid reacted with peroxy radicals and
thus inhibited lipid oxidation.
DNA is susceptible to oxidative damage and hydroxyl
radicals oxidize guanosine or thymine to 8-hydroxyl-2-
deoxyguanosine and thymine glycol which change DNA
and lead to mutagenesis and carcinogenesis [22]. In this
study, hydroxyl radicals generated by Fenton reaction were
found to induce DNA strand breaks in calf thymus DNA
and uncoiling of supercoiled DNA. Bitter cumin extract at

45. 114
Table 3 Antioxidant activity
of extract of C. nigrum seed in
different model systems
Correlation coefficient between
total phenol content and
antioxidant activity
Antioxidant assay Free radicals IC50 value (µg)
DPPH free radical scavenging DPPH 14.0 ± 0.5 0.94
Soybean lipoxygenase-dependent
lipid peroxidation
LOO 28.0 ± 3.0 0.92
Microsomal lipid peroxidation OH 110.0 ± 14.0 0.98
Superoxide anion scavenging O2− 125.4 ± 8.7 0.94Note. Values
are mean ± SEMof three experiments
0.5–2.0 µg offered complete protection to DNA damage
induced by hydroxyl radicals in calf thymus DNA and also
reduced uncoiling or open circular form in pUC18 DNA
(Figs. 5 and 6). Thus, the hydroxyl radical quenching abil-
ity of polyphenolic compounds of bitter cumin could be
responsible for the protection against oxidative damage to
DNA.
Spices and their essential oils are reported to possess
antimicrobial activity [23, 24]. The antibacterial effect of
bitter cumin extract was tested against a number of food-

46. borne pathogenic and spoilage bacteria by agar diffusion
method. As shown in Table 4, the bacterial species namely
B. subtilis, B. cereus and S. aureus were found to be highly
sensitive and showed significant inhibition of the growth in
the presence of bitter cumin extract (Table 3). Enterobac-
ter spp. and L. monocytogenes were moderately inhibited,
while E. coli and Y. enterocolitica were resistant to bitter
cumin extract. Thus, Gram-positive bacteria were found
to be more sensitive to bitter cumin extracts than Gram-
negative bacteria. The antibacterial activity of flavonoids
was attributed to inhibition of synthesis of DNA and RNA
and other related macromolecules [25, 26]. Further, pheno-
lic compounds with more than three 3-OH were found to
possess antibacterial activity [27]. Thus, antibacterial ac-
Fig. 4 C. nigrum seeds extract and BHA inhibited soybean
lipoxygenase-dependent oxidation of linoleic acid. Values mean
±
SEM of three individual experiments
Table 4 Antibacterial activity of the extract of C. nigrum seeds
Organism Inhibition
B. subtilis + +
B. cereus + +
Enterobacter sp +
E. coli −
Listeria monocytogens +
S. aureus + +
Y. enterocolitica −
Note. − , Low/no inhibition (<5 mm); + , moderate inhibition
(5–
20 mm); + + , High inhibition (>20 mm)
tivity of bitter cumin could be attributed to the polyphenolic

47. compounds present in the bitter cumin extract.
In conclusion, our studies have demonstrated for the first
time the presence of a mixture of bioactive polyphenolic
compounds such as caffeic acid, ferulic acid, protocate-
chuic acid, ellagic acid, quercetin and kampeferol in bitter
cumin seeds. Bitter cumin seed extract also exhibited
significant antioxidant activity at microgram quantities as
quencher of DPPH radicals, lipid peroxy radicals, hydroxyl
radicals and superoxide anion radicals in different antioxi-
dant systems. Further, bitter cumin extract also showed an-
tibacterial activity by suppressing the growth of pathogenic
bacteria namely B. cereus and S. aureus. Thus, bitter
cumin with a mixture of polyphenolic compounds possess
Fig. 5 Protective effect of the extract of C. nigrum seeds on
oxidative
damage to Calf Thymus DNA. Lane 1: Native Calf Thymus
DNA.
Lane 2: DNA + 1.0 mM FeSO4 + 10.0 mM ascorbic acid. Lane
3:
DNA + 1.0 mM FeSO4 + 10.0 mM ascorbic acid + 0.5 µg cumin
seeds extract. Lane 4: DNA + 1.0 mM FeSO4 + 10.0 mM
ascorbic
acid + 1.0 µg cumin seeds extract. Lane 5: DNA + 1.0 mM
FeSO4
+ 10.0 mM ascorbic acid + 1.5 µg cumin seeds extract. Lane 6:
DNA + 1.0 mM FeSO4 + 10.0 mM ascorbic acid + 2.0 µg cumin
seeds extract
115
Fig. 6. Protective effect of the extract of C. nigrum seeds on
oxida-

48. tive damage to pUC18 DNA. Lane 1: pUC18 DNA. Lane 2:
pUC18
DNA + 10.0 mM FeSO4 + 60.0 mM ascorbic acid + 6.0 mM
H2O2. Lane 3: pUC18 DNA + 10.0 mM FeSO4 + 60.0 mM
ascor-
bic acid + 6.0 mM H2O2 + 0.5 µg of Cumin seeds extract. Lane
4: pUC18 DNA + 10.0 mM FeSO4 + 60.0 mM ascorbic acid +
6.0 mM H2O2 + 1.0 µg of cumin seeds extract. Lane 5: pUC18
DNA + 10.0 µM FeSO4 + 60.0 µM ascorbic acid + 6.0 mM
H2O2 + 2.5 µg of cumin seeds extract. Lane 6: pUC18 DNA +
10.0 mM FeSO4 + 60.0 mM ascorbic acid + 6.0 mM H2O2 +
2.5 µg of BHA (S, supercoiled DNA; N, nicked DNA)
potential antioxidant and antibacterial activities and this
spice can be further exploited for nutraceutical properties.
Acknowledgements Authors are thankful to Dr. V. Prakash,
Direc-
tor and Dr. S.G. Bhat, Head of the Department of Biochemistry
and
Nutrition, Central Food Technological Research Institute,
Mysore
for their constant encouragement and support. Ani. V is
thankful to
the Council of Scientific and Industrial Research (CSIR), New
Delhi,
for the award of Junior and Senior Research Fellowship. This
work
was partly supported by a project awarded to KAN by
Department
of Science and Technology (DST), New Delhi, India.
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51. 26:s2231–s2234
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FCS 690A
Guidelines for Oral Presentation
1. Familiarize yourself with the variety and location of nutrition
and nutrition-related journals. Some useful websites to locate
peer-reviewed journals are:
http://nutrition.org
http://sciencedirect.com
http://scholar.google.com/
http://www.ncbi.nlm.nih.gov/pubmed/
http://library.csun.edu/
2. Allow time to select your 2 articles. As you read, be certain
that you understand the journal articles and specialized
terminology used by the author. Please remember to email the 2
articles to the instructor for approval.
3. PowerPoint® presentations should take a classical,
professional approach to
organization. Most scientific presentations have five elements

57. that comprise the content of the talk:
1) Introduction
· Introduce your topic in broad, general terms.
· Supply background information.
2) Rationale
· State the purpose of the study (goals and objectives).
· Specific statement of hypothesis to be addressed.
3) Methods
· Present how the research question(s) were addressed.
4) Results
· Present what the experiments found.
· Use pictures, charts, tables, graphs, and figures (visual aids).
5) Discussion/Conclusions
· Discuss the implications of the results.
· Discuss the overall importance of the study or possible future
research.
· Do you feel the food item(s) or phytochemical(s) will provide
health benefits? Should it be consumed as a supplement or as
whole food?
· Discuss how the Human Ecological Theory (HET) affects
consumption of this phytochemical. In other words, how does
the environment impact our food intake patterns and

58. consumption of the specific phytochemical(s)?
4. The PowerPoint® presentation will be approximately 20
minutes in length.
Here’s a link which discusses the HET:
http://www.csun.edu/~whw2380/542/Human%20Ecological%20
Theory.htm.
Figure 1: Human Ecological Theory (HET) images.
Figure 2: Relationship of theory to research.