Relationship of Circulating High-Density Lipoprotein Cholesterol and Anemia
Relationship of Circulating High-Density Lipoprotein Cholesterol and Anemia
Md. Aminul Haque Khan 1 , Rokshana Rabeya 2 , Muhammad Saiedullah 3 , Rukhsana Parvin 4 , Sohel Ahmed 5 and Md. Rezwanur Rahman 6
1. Department of Biochemistry, Enam Medical College, Dhaka 1340, Bangladesh 2. Biochemistry Laboratory, Enam Medical College Hospital, Dhaka 1340, Bangladesh 3. Department of Applied Laboratory Sciences, Bangladesh University of Health Sciences, Dhaka 1216, Bangladesh 4. Department of Medicine, Enam Medical College & Hospital, Dhaka 1340, Bangladesh 5. Department of Biochemistry & Molecular Biology, Jahangirnagar University, Dhaka 1342, Bangladesh 6. Department of Biochemistry, Delta Medical College, Dhaka 1216, Bangladesh
Received: February 16, 2013 / Accepted: April 19, 2013 / Published: May 30, 2013.
Abstract : Circulating level of low HDLC (high-density lipoprotein cholesterol) represents a common critical risk factor for IHD (ischemic heart disease) and may further aggravate the condition in anemic subjects, as the presence of anemia itself is a threat to cardiovascular consequences. To investigate the relationship of circulating HDLC with anemia, first we determined the levels of total hemoglobin (Hb) in a total of 301 subjects (male, n = 158; female, n = 143) randomly, and then examined the circulating levels of HDLC in fasting condition. Age of the study subjects was 47.9 ± 16.6 (mean ± SD) years. Both the male and female subjects were divided into three groups according to their levels of Hb. The relationship of circulating levels of HDLC with the levels of total Hb was statistically analyzed. In case of the male subjects, we found that the levels of HDLC differed significantly among the three groups with different levels of Hb (P = 0.0233) and decrease in the levels of HDLC correlated significantly with the gradual decrease of total Hb level (r = 0.2504; P = 0.0015). In female subjects, we observed a similar trend of difference among the three groups (P = 0.0685). However, decrease in the levels of HDLC correlated significantly with the gradual decrease of Hb level (r = 0.2199; P = 0.0083). Altogether, this study demonstrates that decrease in the circulating HDLC is related to the gradual decrease of Hb level. This study also indicates that circulating level of HDLC may be influenced by the level of total Hb and reveals the cardiovascular risks in anemia as well.
Key words : High density lipoprotein cholesterol, hemoglobin, anemia.
1. Introduction or those with LVH (left ventricular hypertrophy), respectively [1]. Chronic anemia may increase preload,
Anemia is an important potential nontraditional risk reduce afterload and lead to increased cardiac output factor for CVD (cardiovascular diseases) [1]. There [2]. In the long term, this may result in maladaptive are pathophysiologic reasons why the presence of LVH which in turn is a well recognized risk factor for anemia may lead to adverse cardiovascular CVD outcomes and all-cause mortality [3-5]. Several consequences. In theory, the presence of anemia may studies in various populations have evaluated the also exacerbate cardiac ischemia as a result of importance of anemia as a risk factor for adverse decreased supply or increased demand for oxygen, outcomes. In patients with heart failure, anemia has such as in patients with underlying coronary disease for the most part been found to be associated with an
increased risk for hospitalization and all-cause
Corresponding author: Md. Aminul Haque Khan, MBBS, MCPS, FCGP, FCPS, MD, professor, research field: clinical biochemistry.
mortality [4, 6-9]. In patients with coronary artery E-mail: aminhkhan@yahoo.com.
Relationship of Circulating High-Density Lipoprotein Cholesterol and Anemia
disease, the relationship between anemia and adverse Earlier studies supported an inverse correlation outcomes is not consistent. Some studies have
between circulating HDLC and coronary risk in demonstrated an adverse effect of anemia on CVD
patients with normal or elevated LDLC. In theory, the outcomes while others have demonstrated no adverse
presence of anemia may also exacerbate cardiac effect of anemia on CVD outcomes [10, 11]. In a
ischemia as a result of decreased supply or increased study of atherosclerosis risk in communities, anemia
demand for oxygen, such as in patients with was found associated with CVD outcomes [12]. In
underlying coronary disease or those with LVH, United States, in the NHANES II (Second National
respectively [1]. The combination of anemia and Health and Nutrition Examination Survey) mortality
decrease in HDL may, therefore, be more detrimental. study, after adjustment for traditional CVD risk
In this study, we have assessed the relationship of factors and other covariates, there was no significant
circulating HDLC with anemia.
association between anemia and CHD (coronary heart disease) mortality [13].
2. Materials and Methods
HDL (high-density lipoprotein) enhances
2.1 Study Design
cholesterol transport from peripheral tissues to liver and thus maintains blood cholesterol level at normal
The present research was a cross sectional study. level and reduces the risk of IHD (ischemic heart
2.2 Study Subjects
disease) and other atherosclerotic disorders [14]. Nascent HDL particles released from the liver pick up
Between July 2009 and December 2010, 301 adult cholesterol from peripheral tissues and then come
subjects, both males and females, were randomly back to liver to release cholesterol. In doing so, some
selected from patients attending the out-patient of the HDL particles in liver are assaulted by hepatic
department (OPD) of Enam Medical College Hospital, lipase and turn into lipid-depleted nascent HDL
Savar, Dhaka advised for hematological investigations. particles which along with newly produced nascent
Out of the total subjects, 158 were males and 143 particles repeat the process of cholesterol extraction
were females. Age of the subjects was 47.9 ± 16.6 from peripheral tissues [14]. HDL contains years. Both classes of subjects were further divided paraoxonase, an antioxidant enzyme which shows
into 3 (three) groups based on their total hemoglobin native antioxidant activity to prevent initiation of
(Hb). Subjects with Hb level < 10 g/dL were atherogenic events [14]. Apo A-1 of HDL stabilizes
categorized as Group A, with Hb level from 10-14 PGI 2 (prostacyclin I 2 ) and thus prevents coronary
g/dL as Group B and Hb level > 14 g/dL as Group C. artery constriction [14]. As noted by the National
Following the determination of total Hb, fasting levels NCEP-ATP III (Cholesterol Education Program-Adult
of circulating HDLC were measured. Treatment Panel III) guidelines, low HDLC
2.3 Biochemical Analysis
(high-density lipoprotein cholesterol) represents a key determinant of the Framingham risk score and a
Levels of total Hb were measured by common critical risk factor for IHD [15]. Low HDLC
spectrophotometric method using an automated levels, defined as below 40 mg/dL for men and 50
hematology analyzer, XT-2000i (Sysmex, Japan) and mg/dL for women, remain prevalent [15-17]. HDL cholesterol was estimated directly by Numerous prospective cohort studies support a timed-endpoint method using an automated powerful inverse correlation between circulating
biochemistry analyzer (Synchron CX ® system, HDLC levels and coronary risk [18-25].
Beckman Coulter, USA).
Relationship of Circulating High-Density Lipoprotein Cholesterol and Anemia
2.4 Statistical Analysis
Table 1 Circulating levels of HDLC in different groups of male subjects (n = 158).
Statistical analysis was done using the GraphPad
P value Prism Version 5.04 for Windows. One way analysis of
Total Hb (g/dL)
HDLC (mg/dL) (Mean ± SD)
< 10 (n = 20)
variance (ANOVA) test was applied to compare the
0.0233 levels of HDLC among different hemoglobin groups
10-14 (n = 54)
in males and females. The relationship between the
Table 2 Circulating levels of HDLC in different groups of
levels of total Hb and fasting levels of circulating
female subjects (n = 143).
HDLC was determined by Pearson’s r-test. A P value
Total Hb (g/dL)
HDLC (mg/dL) P (Mean ± SD)
value less than 0.05 was considered as significant.
< 10 (n = 43)
3. Results and Discussion
10-14 (n = 91)
Tables 1 and 2 show the results of ANOVA analyses to compare the levels of circulating HDLC and the
r levels of total Hb among the three different groups of = 0.2504, p = 0.0015 male and female subjects, respectively. We found that
the levels of circulating HDLC differed significantly
/d g m
with the levels of total Hb among the three groups of
male subjects as shown in Table 1 (P = 0.0233). In case
HDL
of female subjects, the difference among the three
groups was close to be significant statistically (P = 0.0685) and we observed a similar trend.
Figs. 1A and 1B show the correlation between the
circulating HDLC and the levels of total Hb in male Hb levels (g/dL)
and female subjects, respectively. We found that
100 B
decrease in the levels of circulating HDLC positively associated with the gradual decrease in the levels of
total Hb in both the male (r = 0.2504) and female (r =
g/ 60
dL
0.2199) subjects. The correlation was found highly
C r = 0.2199 significant in both male (P = 0.0015) and female (P =
D p = 0.0083 0.0083) subjects.
L 40
Circulating low HDLC represents a common critical risk factor for IHD. The presence of anemia
itself is a threat to cardiovascular consequences. Low
HDLC in anemic subjects may be more detrimental. Hb levels (g/dL)
In the present study, the relationship of circulating
Fig. 1 Relationship between HDL cholesterol (HDLC) and Hb levels in male (A) and female (B).
HDLC and anemia has been investigated. Results of
this study clearly demonstrate that circulating levels of male subjects. In case of female subjects, a similar HDLC differed significantly with different levels of
trend was observed and the difference was close to total Hb (P = 0.0233) and decrease in circulating
significance (P = 0.0685). Decrease in the circulating HDLC was positively associated with the gradual
HDLC was positively associated with the gradual decrease of total Hb levels (r = 0.2504; P = 0.0015) in
decrease of total Hb levels in female subjects (r =
Relationship of Circulating High-Density Lipoprotein Cholesterol and Anemia
0.2199; P = 0.0083). These findings reveal that HDL cholesterol levels. But more studies with larger decrease in the circulating HDLC was strongly related
number of subjects are required to come to a decision to the gradual decrease of total Hb levels. This study
whether there is significant association of hemoglobin indicates that circulating levels of HDLC may be
levels with HDL cholesterol levels. influenced by the levels of total Hb. The possible
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May 2013, Vol. 7, No. 5, pp. 464-467
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Development of in-vitro Susceptibility Testing for
Pathogenic Bacteria
Fouad Houssein Kamel 1 , Chiman Hameed Saeed 2 , Ashti M. Amin 2 and Saleem Saaed Qader 2
1. Hawler Polytechnic University, Erbil, Iraq
2. Medical Research Centre, Hawler Medical University, Erbil, Iraq
Received: November 27, 2012 / Accepted: March 02, 2013 / Published: May 30, 2013.
Abstract : A new method developed for in-vitro susceptibility test in medical laboratories consist of micro tubes or gloves containing dehydrated tryptic soya broth, 5% glucose, 0.1% bromothymol blue and one type of antibiotics (ampicillin, tetracycline and chloramphenicol) with critical concentration MIC (minimum inhibitory concentration) for susceptibility. Standard quality control strains of bacterial (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa) suspension were adjusted to 0.5
McFarland turbidity standard (1 × 10 6 cell/mL) were used in inoculation the media and incubated two hours at 37 °C. The MIC of ampicillin against E. coli, S. aureus, and P. aeruginosa were 4, 32, and 256 µg/mL of the media for the bacteria, respectively, while the MIC of tetracycline against bacteria were 512, 512 and 32 µg/mL, respectively, the MIC of chloramphenicol were 512, 32 and 512 µg/mL, respectively. Where, the resistant bacteria to the antibiotics could grow and ferment glucose sugar producing a color change of the media from blue to yellow, while the sensitive bacteria do not grow or show no change in color. This study result compared with common used antibiotic disk method obtaining similar results. This developed method characterized by fast (only two hours) and less cost in comparison to conventional technique. The new micro tube strip is highly stable (more than one year) with more sensitive in detection of variable pathogenic bacteria including standard bacteria strains compared with conventional technique.
Key words : Susceptibility, MIC, antibiotic, medical laboratories technique.
1. Introduction
attained in blood with the normally recommended dose of the antimicrobial agent and implies that an
The clinical presentation of an infectious disease infection caused by this microorganism may be reflects the interaction between the host and the appropriately treated with the antimicrobial agent. The microorganism. This interaction is affected by the host term resistant indicates that the microorganism is immune status and microbial virulence factors. Signs resistant to concentrations of the antimicrobial agent and symptoms vary according to the site and severity that can be attained with normal doses and implies of infection [1]. The diagnosis requires a composite of that an infection caused by this microorganism could information including history, physical examination, not be successfully treated with this antimicrobial radiographic findings, and laboratory data [2].
agent [5, 6].
The responsibility of the microbiology laboratory Many bacteria have unpredictable susceptibilities to includes not only microbial detection and isolation but antimicrobial agents and their susceptibilities can be also the determination of microbial susceptibility to measured in vitro to help the selection of the most antimicrobial agents [3, 4]. The term susceptible appropriate antimicrobial agent. The widely used means that the microorganism is inhibited by a susceptibility testing methods are the disk diffusion concentration of antimicrobial agent that can be and broth agar dilution tests. The MIC (minimum
Corresponding author : Fouad Houssein Kamel, assistant inhibitory concentration) of a particular drug to a professor, research field: microbiology. E-mail: particular organism can be quantitatively measured
fhkamel2013@yahoo.com.
Development of in-vitro Susceptibility Testing for Pathogenic Bacteria
in-vitro through the broth agar or micro/or macro (E. coli, S. aureus and P. aeruginosa) were inoculated dilution test. These testing methods have been
in tubes containing various antimicrobial agents. After standardized and the NCCLS (National Committee of
incubation for 2 h at 37 °C, results were reported (as a Clinica1 Laboratory Standards) provides susceptibility
change in color of the media) by naked eye. test guidelines [6-8]. In this study we tried to develop
2.4 Conventional Antibiotic Discs Method
a new technique for susceptibility test of pathogenic bacteria to the antibiotics.
Inoculums of the test organism were prepared as before. Sterile cotton swabs were impregnated with
2. Materials and Methods
the test and control organisms separately. These swabs
2.1 Measurement of MIC (Minimum Inhibitory were used to inoculate the specified areas of the Concentration)
Petri-dishes with test and control organisms. Antibiotic discs were applied with light pressure on
2.1.1 Stock Solution the agar surface using flamed forceps after the Ampicillin (Aldrich/Sigma) (50 mg/mL), inoculums had dried. The Petri-dishes were incubated Tetracycline (Aldrich/Sigma) (5 mg/mL), at 37 °C for 18-24 h and the results were reported Chloramphenicol (Aldrich/Sigma) (34 mg/mL). (radial width of the zones outside the antibiotic discs)
2.1.2 Agar Dilution
by naked eye [11].
The anti-bacterial agents was measured by using broad spectrum antibiotics (ampicillin, tetracycline
3. Results and Discussion
and chloramphenicol) of different concentrations (0.5, 2, 4, 8, 16, 32, 64, 128, 256 and 512 µg/mL)
Table 1 shows different concentrations of ampicillin and inoculated with standard tested organisms (E.
tested against standard bacterial suspension of E. coli, coli , S. aureus and P. aeruginosa) which were
S. aureus and P. aeruginosa were inoculated in the prepared by emulsifying part of the growth from
media. Tested critical concentrations in sterile each of 5 similar colonies in saline and incubated at
condition for ampicillin were equivalent to MIC break
37 °C for 2 h. point which were 4, 32, 256 µg/mL for tested bacteria, Turbidity of the suspension was adjusted to 0.5
respectively. While, the average number of bacteria
6 6 McFarland standards 6 (BioMerieux, France) were 15.6 × 10 , 12.9 × 10 and 2.1 × 10 bacteria/mL, spectrophotometrically at 600 nm wavelength (1 × 10 6
respectively. The E. coli was more sensitive to cell/mL) [9-11].
ampicillin followed by S. aureus and then P. aeruginosa .
2.2 Preparation of Susceptibility Strip Test Table 2 shows the results of testing the sensitivity
The test strip consists of micro tubes containing of standard pathological bacteria (E. coli, S. aureus dehydrated (lyophilized) media tryptic soya broth,
and P. aeruginosa) for tetracycline using different glucose (5%) and bromothymol blue (0.1%) and
concentrations of the antibiotics. Where it was noted broad-spectrum antibiotics in critical concentration
that the focus MIC to the bacteria were 512, 512, and tested for all antibiotics were equivalent to the MIC
32 µg/mL, respectively, and the average number of break point for susceptibility in sterile condition. The 6 6 bacteria were 16.2 × 10 6 , 16.2 × 10 , and 5.4 × 10 pH was adjusted to 7.4 (Alkaline, blue color).
bacteria per mL, respectively.
P. aeruginosa was seen to be more sensitive to
2.3 Application of Strip Test tetracycline, whereas the bacteria S. aureus and E. coli
Bacterial suspension of particular microorganisms had the same degree of sensitivity.
Development of in-vitro Susceptibility Testing for Pathogenic Bacteria
Table 1 MICs of ampicillin for different concentrations of E. coli, S. aureus and P. aeruginosa. No. of bacteria (1 × 10 6 ) for different concentration of ampicillin (µg/mL)
Isolates
256 512 E. coli 34.2 33.3 15.6 32.7 29.4 6.9 26.7 25.2 24 21
S. aureu s 24.3 16.8 29.4 16.2 15.9 12.9 3.3 18.9 15 21 P. aeruginosa
Table 2 MICs of tetracycline for different concentrations of E. coli, S. aureus and P. aeruginosa. No. of bacteria (1 × 10 6 ) for different concentration of tetracycline (µg/mL)
Isolates
256 512 E. coli 48.3 39.9 37.5 35.1 32.4 29.1 20.1 14.4 19.2 16.2
S. aureu s 20.4 17.7 22.2 33.3 9.6 25.8 20.1 18.6 17.7 16.2 P. aeruginosa
Table 3 MICs of chloramphenicol for different concentrations of E. coli, S. aureus and P. aeruginosa. No. of bacteria (1 × 10 6 ) for different concentration of chloramphenicol (µg/mL)
Isolates
256 512 E. coli 21.9 6.6 3.3 9.9 17.1 14.4 13.2 12.3 6 4.2 S. aureu s
42.3 37.8 25.8 23.7 22.8 21.9 26.4 26.1 24.3 13.8 P. aeruginosa
Table 4 MICs and MBCs of ampicillin, tetracycline and chloramphenicol among E. coli, S. aureus and P. aeruginosa.
Chloramphenicol Isolates MIC, µg/mL
Ampicillin
Tetracycline
MBC, µg/mL
MIC, µg/mL
MBC, µg/mL
MIC, µg/mL MBC, µg/mL
4 S. aureu s
E. coli 4 32 512
16 32 512 P. aeruginosa
Whereas, Table 3 showed sensitivity test of respectively. The MBC of chloramphenicol were 4, different concentrations of chloramphenicol against
512, and 128 µg/mL, respectively, for tested bacteria. the standard pathogenic strains (E. coli, S. aureus and
In this study glucose sugar selected in the P. aeruginosa ). The MIC of the bacteria were 512, 32,
preparation of the new test strip because most types of and 512 µg/mL, respectively. The average number of
bacteria containing the enzyme fermented glucose
6 6 bacteria were 4.2 × 10 6 , 21.9 × 10 and 4.8 × 10 sugar. Bromothymol blue dye was the most suitable bacteria per mL, respectively. It was note that S.
dye used to indicate fermentation process and can note aureus was more sensitive to chloramphenicol,
the color change clearly with the naked eyes as it is in whereas the bacteria E. coli and Pseudomonas
Fig. 1.
aeruginosa had the same degree of sensitivity. The process of freeze drying had important role in In addition to the MIC value, the MBC (minimum
maintaining culture media in the pockets of test strips bactericidal concentration) value of antibiotics was
and control small quantities used in addition to the estimated for all pathogenic strains as it is shown in
ability to keep for a long time at low temperatures Table 4.
(4-6 °C).
Though the values of the MBC of ampicillin were The authors concluded from the results of the study
32, 64, and 512 µg/mL for E. coli, S. aureus and P. provide a great opportunity to work in diagnostic aeruginosa , respectively, but MBC of tetracycline
laboratories, wherever they are located inside or were 128, 16 and 16 µg/mL for standard bacteria,
outside the provinces. The implementation of potential
Development of in-vitro Susceptibility Testing for Pathogenic Bacteria
a- Micro tubes strip before inoculated bacterial growth
b- Micro tubes strip after inoculated bacterial growth
Fig. 1 Antibiotic susceptibility test performing micro tubes strip before (a) and after (b) inoculated bacterial growth. Blue color: sensitive; Yellow: resistance bacteria.
sensitivity testing and providing of best services, as
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increases the speed of the delivery of the required Hauser, D.L. Longo, L.R. Jameson (Eds.), Harrison’s treatment to the patient. Finally, authors recommend
Principles of Internal Medicine, McGraw-Hill, Inc., 2001, the Ministry of Health for the adoption of the way to
pp. 867-882. [8] D.A. Drossman, M. Camilleri, E.A. Mayer, W.E.
ensure the test required in all diagnostic laboratories. Whitehead, AGA technical review on irritable bowel
References yndrome, Gastroenterology 123 (6) (2002) 2108-2131.
[9] U. Ghoshal, P. Ranjan, S.R. Naik, A. Ayyagari, Spectrum [1] E.J.O. Baron, L.R. Peterson, S.M. Finegold, Bailey and
and antibiotic sensitivity of bacteria contaminating the Scott’s Diagnostic Microbiology, 9th ed., CV Mosby, St.
upper gut in patients with malabsorption syndrome from Louis, 1994.
the tropics, BMC, Gastroenterology, 2003. [2] E.W. Koneman, S.D. Allen, P.C. Schreckenberg, W.C.
[10] R. Bauman, Microbiology with diseases by taxonomy, Winn, Color Atlas and Textbook of Diagnostic
Personal International Edition, 2nd ed., 2007, p. 298. Microbiology, 4th ed., JB Lippincott, Philadelphia, 1992.
[11] J.R. Kerr, Antibiotic treatment and susceptibility testing, [3] C.M. Kunin, Detection, Prevention and Management of
Journal of Clinical Pathology 58 (2005) 786-787. Urinary Tract Infections, 4th ed., Lea & Febiger,
[12] E.J.O. Baron, L.R. Peterson, S.M. Finegold, Processing Philadelphia, 1987.
clinical specimens for anaerobic bacteria: Isolation and [4] P.R. Murray, E.J. Baron, M.A. Pfaller, P.C. Tenover, R.H.
identification procedures, in: Anonymous (Ed.), Bailey Yolken, Manual of Clinical Microbiology, 6th ed.,
and Scott’s Diagnostic Microbiology, Mosby, American Society for Microbiology, Washington, DC,
Philadelphia, 1994, pp. 474-503.
1995. [13] V. Sudha, A. Prasad, S. Khare, R. Bhatia, Antimicrobiol [5] J.E. Pennington, Respiratory Infections: Diagnosis and
susceptibility testing in India—A statue survey, Indian J. Management, 3rd ed., Raven Press, New York, 1994, pp.
Med Microbiol. 19 (4) (2001) 222-223.
May 2013, Vol. 7, No. 5, pp. 468-474
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Inhibitory Effect of Alcoholic Extract of Sage Leaves on the Growth of Pathogenic Fungi Causing External Ear and Skin Infections
Maha Akram Al-Rejaboo and Omar Mu’ayad Al-Obaidy Department of Biology, University of Mosul, Mosul 41002, Iraq
Received: March 19, 2013 / Accepted: April 09, 2013 / Published: May 30, 2013.
Abstract: Fifteen pathogenic fungi were isolated from patients with external ear infection like Aspergillus niger and 100 pathogenic fungi were isolated from patients with skin infections like Trichophyton mentagrophytes which was isolated from body skin infections (also four different species of the genus Trichophyton was isolated) as well as another fungus which is Microsporum audouinii was isolated from the head scalp, and Aspergillus ustus which was isolated as normal flora from infected skin inflammation. These fungi are opportunistic that can cause skin infections and inflammation of the external ear which increases the severity and the length of the disease, so the effect of different concentrations of alcoholic extract of sage leaves was tested against these fungi and it was found that the extract had an inhibition effect on growth of the fungus A. ustus by 80.7% at a concentration of 20 mg/mL, while inhibited the fungus A. niger by 77.3% at the same concentration, whereas inhibited the fungus T. mentagrophyte by 53.3% at the same concentration, and the fungus Microsporum audouinii was inhibited at the same concentration by 86.6%, also the extract showed different inhibition values against the other four species of the fungus Trichophyton. The plant sage is considered as good antifungal towards studied fungi of otitis externa and skin diseases and can be used prospectively as antifungal antibiotics against fungi causing external ear infections and skin infections.
Key words : Sage leaves, pathogenic fungi.
1. Introduction the form of bushes, not more than a meter in height and with broad leaves.
Sage (Salvia species) has been used as a herb with It is a desirable plant and almost no home devoid of beneficial healing properties for millennia. The name it and preferred to be used boiled with tea which is itself comes from the Latin word for health (salvare or known as the tea apple, and has a good flavor and his heal). Ancient authors called it elelisphakon. This drink is used after boiling as a medicine for term most likely refers to several species, such as respiratory disease, especially tuberculosis disease. Salvia fruticosa Mill., Salvia officinalis L. and Salvia Willershausen et al. [3] have mentioned that the herb pomifera L. [1]. sage had a significant presence as a feedstock in the Sage is a herb that has many benefits, especially in medical compounds used in medicine and teeth and the process of healing of animal tissues, as well as an oral surgery because of its (astringent) properties and antidote against many kinds of bacteria and fungi. The also had a distinct role in reducing infections and name sage derived from Latin and means “healing”
abscesses in the oral cavity.
was mentioned by Toussaint-Samat [2] that the plant Newall [4] has mentioned during his experiments sage which is popularly known is an aromatic plant in on animals that plant sage had an inhibitory effect on
Corresponding author : Maha Akram Al- Rejaboo , Ph.D., the effectiveness of the cerebral cortex of these assistant professor, research field: mycology. E-mail: Mahaalrejaboo@yahoo.com. animals as well as had a parasympathetic nervous
Inhibitory Effect of Alcoholic Extract of Sage Leaves on the Growth of Pathogenic
Fungi Causing External Ear and Skin Infections
characteristics on the digestive tract of rabbits. randomly from patients and preserved in the middle of It was mentioned by Takeo et al. [5] that studies and
a transport agricultural medium type (Stuart) from tests on this plant in China have resulted in the
Oxoid company until transferred to the lab. They were preparation of medical extract from the extract of this
cultured on the middle of Petri dishes containing SGA herb that has antimicrobial properties, and has the
(Sabroud Glucose Agar) [8] and incubated at 25 ± 2 benefit of calming the nerves, as well as for the
ºC for a week and record the results, and the treatment of pain resulting from intestinal cramps, in
pathogenic fungi were isolated and preserved in which it works on the relaxation of smooth muscle in
slanted medium for further diagnostic tests. the walls of cardiovascular and digestive system.
2.1.1.2 Cutaneous Mycoses
The use of sage extract is of obvious importance in Skin scraping were taken from skin infections form the treatment of pain associated with rheumatic
150 patients with skin diseases after examined disease as well as helps to discourage menstrual pain
clinically by specialists in the Division of and complications and menstrual disorders in women
Dermatology at Al-Zahrawi Educational Hospital and [6].
Al-Salam Educational Hospital in Mosul city, Iraq Basset [7] stated that the plant sage had many
using the method of skimming skin and direct benefits as astringent, antiseptic, aromatic, expelling
examination with KOH (potassium hydroxide) (10%) the wind, producing estrogen, reduce sweating and it
is reinforcing material. The samples were tested microscopically to make Also the same researcher said that sage is useful in
sure of the presence of the fungal elements and the the treatment of asthma, and has antagonistic results were recorded. The positive skin scales were
properties against bacterial and fungal infections skin, cultured on SAG with the addition of the antibiotic as well as for the treatment of mouth sores and gum
(Chloramphenicol) to prevent the growth of bacteria because the grass is astringent and used to treat
and incubated at 28 ± 2 °C for a period of three weeks diarrhea.
with the observation of the results consistently during And according to that, this study was aimed to test
the incubation period, and fungal isolates were the antimicrobial activity of the plant sage against
preserved in slanted media for further diagnostic tests. some kinds of human pathogenic fungi and the
2.1.2 Diagnosis of Fungi
possibility to use its extract as a medical drug against The isolated fungi were diagnosed depending on the
these pathogens. external shape and appearance of the fungal colonies
2. Materials and Methods
whether it is yeasts or molds and the diagnosis was done using the differential media specified for each
2.1 Isolation and Identification of Pathogenic Fungi genus or species, and it was used three basic cultural from Different Sources medias for the diagnosis of the different types of the
2.1.1 Isolation of Fungi genus Aspergillus which they are: Fungi were isolated from two different locations in
(1) Czapek yeast extract agar (CYA); the human body are:
(2) malt extract agar;
2.1.1.1 Inflammation of the External Ear (3) 25% glycerol nitrate agar (GN 25%).
50 samples were collected from patients suffering The diagnosis was done depending on the from inflammations of the external ear from classification keys for the genus Aspergillus [10]. Al-Zahrawi educational hospital in Mosul city. Ear
The different types of the genus Trichopyton and and nose division, and ear swabs were collected
Microsporum were diagnosed using different media
Inhibitory Effect of Alcoholic Extract of Sage Leaves on the Growth of Pathogenic
Fungi Causing External Ear and Skin Infections
and methods such as: infections were isolated and diagnosed microscopically (1) Sabouraud’s dextrose agar with 5% NaCl,
and also by using selective media in which Aspergillus Cornmeal agar, Lactrimel agar, Trichophyton agars
niger was isolated from the external ear infection, while 1-7 [8, 11];
other fungi such as Trichophyton mentagrophytes, T. (2) Hair penetration test was described by Ref. [12]; terrestre , T. verrucosum, T. schoenleinii, T. rubrum and
(3) Urase enzyme test: Microsporum audouinii were isolated from skin This test was used to determine the ability of the
infection also the opportunistic fungus Aspergillus fungus to produce urase enzyme and it is a depended
ustus was isolated. It was found that the highest ratio of method in distinguishing the type Trichophyton
infection of the chronic otitis media happened for mentagrophytes from Trichophyton rubrum in seven
people between 31-50 years old, while the highest ratio days in which T. mentagrophytes produces the
of infection of the fungal skin diseases happened for enzyme while T. rubrum does not [13, 14];
people between 16-30 years old (Figs. 1 and 2) and the (4) Slide-culture technique:
highest ratio of infection for the ear and skin happened The slide-culture technique was used to study the
for people who were not medicinal drug users (Figs. 3 microscopic characteristics of the isolated fungi [15]
and 4), and because of these opportunistic fungi that and the diagnosis clues were used depending in
attack the immunosuppressed persons, this research addition to the previously mentioned source on [8,
was conducted to study the possibility of using plant 16].
sage as antimicrobial to these pathogens.
2.2 Preparation of the Alcoholic Extract of the Plant
The alcoholic extraction was done depending on the -c
td 20 Peo
method described by Al-Nu’aman [17]. The
-p 15
concentrations 5 mg/mL, 10 mg/mL, 15 mg/mL and
,% le 10
20 mg/mL were prepared from the extract with the
medium (SGA) and the lowest inhibitory concentration (MIC) was determined, also the control 0
was used (Petri dish containing medium without any Age (Year)
concentration added), and then the sensitivity of the Fig. 1 Age distribution for patients with chronic otitis
media.
fungal isolates toward this extract was done by inoculation of 6 mm diameter of seven days fungal
colony discs with the use of three replicas for each
45 In fe 40
concentration, then plates were incubated at 28 ± 2 °C
-c 35 td
for 7-14 days depending on the fungus type and then
Peo 30
results were recorded.
-p 25 le ,% 20
3. Results and Discussion 15
It was found that 100 samples from the 150 samples 5
of skin infections were of fungal infections, and from
Age (Year)
the 50 samples of ear infections 15 were of fungal
Fig. 2 Age distribution for patients with fungal skin
infections. Fungi associated with external ear and skin
diseases.
Inhibitory Effect of Alcoholic Extract of Sage Leaves on the Growth of Pathogenic
Fungi Causing External Ear and Skin Infections
76% increasing the extract concentration, in which the ratio
Non-Drug
of inhibition was 46.1% at concentration 5 mg/mL,
Users
67.31% at concentration 10 mg/mL, 71.1% at concentration 15 mg/mL and 80.7% at concentration
20 mg/mL for the fungus Aspergillus ustus (Fig. 5), while the ratio of inhibition on A. niger was 20% at
Drug
concentration 5 mg/mL, 60% at concentration 10 mg/mL, 66.6% at concentration 15 mg/mL and 77.3% at concentration 20 mg/mL (Fig. 6).
For the fungus Trichophyton mentagrophytes, the ratio of inhibition was 6.6% at concentration 5 mg/mL,
20% at concentration 10 mg/mL, 26.6% at
Fig. 3 Distribution of otitis media infected people according to their usage of medicinal drug.
concentration 15 mg/mL and 53.3% at concentration
20 mg/mL. For the other species of Trichophyton, the ratio of inhibition was as follow for the concentrations
5 mg/mL, 10 mg/mL, 15 mg/mL, and 20 mg/mL,
Non-Drug Users
respectively.
T. terrestre 25.3%, 40%, 57.3% and 60%, for T. verrucosum 41.3%, 68%, 96% and 97.3%, for T. schoenleinii 25.3%, 44%, 60% and 62.6%, and for T. rubrum 17.3%, 36%, 48% and 60% at concentrations
Drug
5 mg/mL, 10 mg/mL, 15 mg/mL and 20 mg/mL,
Users
respectively.
It was found from Table 2 that T. verrucosum was
the most sensitive to the extract that showed the
Fig. 4 Distribution of fungal skin diseases infected people
highest inhibition ratio 97.3% at concentration 20
according to their usage of medicinal drug.
mg/mL of the extract.
Table 1 shows the average of fungal colonies Finally, for the fungus Microsporum audouinii the diameter treated with different concentrations of
ratio of inhibition was 53% at concentration 5 mg/mL, alcoholic extract of plant sage leaves. It was noticed
75% at concentration 10 mg/mL, 83.3% at from Table 2 that the percentage of alcoholic extract
concentration 15 mg/mL, and 86.6% at concentration inhibition on studied fungi was increased by
20 mg/mL, as shown in Fig. 7.
Table 1 The effect of plant sage leaves extract on the average of fungal colonies diameter (cm).
Concentrations, mg/mL
5.2 1 1.5 1.7 2.8 Aspergillus ustus 7.5 1.7 2.5 3 6 A. niger 7.5 3.5 5.5 6 7 Trichophyton mentagrophytes
7.5 3 3.2 4.5 5.6 T. terrestre 7.5 2.0 0.3 2.4 4.4 T. verrucosum 7.5 2.8 3 4.2 5.6 T. schoenleeinii
7.5 3 3.9 4.8 6.2 T. rubrum 6 0.8 1 1.5 2.8 Microsporum audouinii
Inhibitory Effect of Alcoholic Extract of Sage Leaves on the Growth of Pathogenic
Fungi Causing External Ear and Skin Infections
Table 2 The percentage ratio of plant sage leaf alcoholic extract effect on the fungal colonies. The ratio of inhibition (%)
Concentrations (mg/mL)
Fungi
Control
20 15 10 5 0 80.7 71.1 67.3 46.1 Aspergillus ustus
0 77.3 66.6 60 20 A. niger 0 53.3 26.6 20 6.6 Trichophyton mentagrophytes 0 60 57.3 40 25.3 T. terrestre 0 97.3 96 68 41.3 T. verrucosum 0 62.6 60 44 25.3 T. schoenleeinii 0 60 48 36 17.3 T. rubrum 0 86.6 83.3 75 53.3 Microsporum audouinii
fungi, and that indicates the increase in inhibition with the increase in concentrations.
Table 3 shows the effect of the antifungal ketoconazole on the average of colonies growth diameter of studied fungi, while Table 4 shows the percentage ratio of effect of this antibiotic on fungal colonies.
Standard A. ustus For the fungus Aspergillus ustus, the ratio of
Fig. 5 Effect of plant sage extract on growth of A. ustus.
inhibition with antibiotic was 100% for all used concentrations, also the same results was obtained for
the other fungi (Trichophyton mentagrophytes and Microsporum audouinii ), whereas, for the fungus A. niger the ratio of inhibition was 20% at concentration
0.1 mg/mL, 29.3% at concentration 0.5 mg/mL, 60% at concentration 1 mg/mL, 73% at concentration 1.5 mg/mL, also the other species of the genus
Standard A. niger Trichophyton showed different inhibition ratios at
Fig. 6 Effect of plant sage extract on growth of A. niger.
different concentrations as in Tables 3 and 4.
It was noticed from the above results that the inhibition of the antibiotic ketoconazole was higher than alcoholic extract of plant sage leaves, and this may be because the alcoholic extract contain (as shown from results) active antifungal compounds but in low concentrations that will not give 100% inhibition as compared with ketoconazole.
Standard Microsporum audouinii Xiao [6] mentioned that plant sage contain chemical
Fig. 7 Effect of plant sage extract on growth of M.
compounds especially dihydrotanshinone in which
audouinii .
this compound stops the hyphal growth of the It was noticed from results that the highest
following fungi: Trichophyton rubrum, T. tonsulans inhibition ratio for the alcoholic extract of plant sage
var. sulfureum, Epidermophyton flocossum and was at concentration 20 mg/mL for all mentioned
Sabouranditis canis .
Inhibitory Effect of Alcoholic Extract of Sage Leaves on the Growth of Pathogenic
Fungi Causing External Ear and Skin Infections
Table 3 The effect of ketoconazole on the average of fungal colonies diameter (cm).
Concentrations (mg/mL)
Control
Fungi
1.5 1 0.5 0.1 5.2 0 0 0 0 Aspergillus ustus
5.3 6 A. niger
7.5 0 0 0 0 Trichophyton mentagrophytes 7.5 0.4 0.7 0.8 2 T. terrestre 7.5 0.6 0.9 1.2 1.4 T. verrucosum 7.5 0.7 0.9 1.1 1.4 T. schoenleinii 7.5 3.1 3.5 3.9 4 T. rubrum
6 0 0 0 0 Microsporum aaudouinii
0: indicates no growth.
Table 4 The percentage ratio of the effect of ketoconazole on fungal colonies growth. The ratio of inhibition, %
Concentrations, mg/mL
0 100 100 100 100 Aspergillus ustus
0 72 60 29.3 20 A. niger
0 100 100 100 100 Trichophyton mentagrophytes
0 94.6 90.6 89.3 73.3 T. terrestre
0 92 88 84 81.3 T .verrucosum
0 90.6 88 85.3 81.3 T .schoenleinii 0 58.6 53.3 48 46.6 T. rubrum
0 100 100 100 100 Microsporum audouinii
Farag et al. [18] and Dragan et al. [19] had found that
agreement with our results.
the alcoholic extract of plant sage taken from stems or It was mentioned by Mishra and Dubey [21] that leaves of the plant had inhibitory effect on different
alcoholic extract of plant sage had an inhibitory effect kinds of fungi and bacteria such as: Pseudomonas
against bacteria and fungi, and concluded that the aerugenosa , Salmonella enteritidis, Staphylococcus
concentration 2% inhibits the growth and activities of aureus , Bacillus subtilis, Fusobactrium nucleatum,
the fungi specially Aspergills parasiticus in a ratio of Peptostreptococcus anaerobius ,
87.6% and prevents the formation of the fungal toxin gingivalis , Treponema denticola, Treponema vencenti,
Porphyromonas
(aflattoxin) type B and G in a ratio of 96%. Sarcina lutea , Sacchromyces cervisiae, Candida
4. Conclusion
albican and Aspergillus niger. Also it was indicated by Dulger and Hacioglu [20] that the alcoholic extract
From all above and from the obtained results of the of plant sage had an inhibitory effect against different
research, it was concluded that the alcoholic extract of types of fungi especially: Candida spp., Aspergillus
plant sage leaves had an inhibitory effect against nige r, A. flavus, Alternaria spp., Fusarium oxysporum,
different kinds of pathogenic fungi such as Aspergillus Pencillium frequenta and Cryptococcus spp., which
niger associated with external ear infection, was in agreement with the results. They also had
Trichopyton mentagrophytes and Aspergillus ustus concluded that the inhibitory effect of plant sage
associated with human skin infection, that is extract depend on its concentration and the MIC
recommended to use this extract as a natural antibiotic (minimum inhibitory concentration) for fungi range
against the previously mentioned pathogenic microbes, from 3.12 mg/mL to 25 mg/mL and that was in
also it is recommended to test this extract against the
Inhibitory Effect of Alcoholic Extract of Sage Leaves on the Growth of Pathogenic
Fungi Causing External Ear and Skin Infections
other different kinds of pathogenic fungi and bacteria. [11] D. Ellis, The University of Adelaide, (MycologyOnline), 2013, http://www.mycology.adelaide.edu.au/Fungal_
References
descriptions. [12] Y.M. Clayton, G. Midgley, Identification of Agents of
[1] D. Rivera, C. Obón, F. Cano, The botany, history and Superficial Mycoses in Medical Mycology: A Practical traditional uses of threelobed sage (Salvia fruticosa
Approach, Information Press Ltd, Oxford England, 1989. Miller) (Labiatae), Economic Botany 48 (1994) 190-195.
[13] E.J. Baron, L.R. Peterson, S.M. Finegold, Diagnostic [2] M. Toussaint-Samat, A History of Food, Oxford
Microbiology, Mosby, 1994, pp. 168-760. University Press, London, 1996.
[14] E.S. Beneke, A.L. Rogers, Medical Mycology Manual, [3] B. Willershausen, I. Gruber, G. Hamm, The influence of
3rd ed., Burgess Publishing Company, London, 1970, p. herbal ingredient on the plaque index and bleeding
tendency of the gingival, The Journal of Clinical [15] E.W. Koneman, G.D. Roberts, S.E. Wnght, Practical Dentistry 2 (3) (1991) 75-78.
Laboratory Mycology, 2nd ed., William and Wilkins, [4] C.A. Newall, L.A. Anderson, J.D. Phillipson, A Guide for
USA, 1979, p. 163.
Health Care Professionals, Oxford University Press, [16] K.J. Kwon-Chung, J.E. Bennett, Medical Mycology, Lea London, 1996.
and Febiger, Philadelphia, London, 1992, p. 866. [5] S. Takeo, K. Tanonaka, K. Hirari, K. Kawaguchi, M.
[17] A.Y. Al-Nu’aman, The molecular effect of some Ogawa, A. Yagi, et al., Beneficial effect from the root of
climbing plants on the growth and metabolism of some saliva on post hypoxic recovery of cardiac contractile
gram positive and negative germs, Ph.D. Thesis, College force, Biochemical Pharmacology 40 (5) (1990)
of Science, University of Mosul, Iraq, 1998. (in Arabic) 1137-1143.
[18] R.S. Farag, Z.Y. Daw, S.H. Abo Raya, Influence of some [6] P.G. Xiao, Excerpts of the Chinese pharmacopeia in
spice essential oils on Aspergillus para siticus growth and herpes, species and medical plants, Recent Advances in
production of aflatoxin in synthetic medium, Journal of Botany, Holticultres and Pharmacology, Vol. 4, Oryx
Food Science 54 (1) (1989) 74-76. press, Arizona, 1989, pp. 42-114.
[19] T. Dragan, V. Novica, A. Anas, Chemical constitutes and [7] A.M.A. Basset, 90 Healing Herbs (Arabic Book),
antimicrobial activity of ethanol extract obtain from the National Center of Researches, Iraq, 1992.
flower leaf and stem of Salvia officinalis, Journal Serb. [8] G.S. De Hoog, J. Guarro, Atlas of Clinical Fungi, 2nd ed.,
Chemic Soc. 68 (11) (2003) 17-24. Universtal Rovirai virgili, Spain, 1995, p. 720.
[20] B. Dulger, N. Hacioglu, Antifungal activity of endemic [9] C.W. Emmons, C.H. Binford, J.P. Utz, K.J. Chung,
Salvia in Turkey, Tropical J. of Pharmaceutical Research Medical Mycology, 3rd ed., Lea and Febiger,
7 (3) (2008) 1051-1054.
Philadelphia, 1977, pp. 117-167. [21] A.K. Mishra, N.K. Dubey, Fungotoxicity of essential oils [10] J.I. Pitt, A.D. Hocking, Fungi and Food Spoilage, 2nd ed.,
of Amomum subulatum against Aspergillus flavous, Academic Press, Sydney, 1997, p. 593.
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May 2013, Vol. 7, No. 5, pp. 475-482
Journal of Life Sciences, ISSN 1934-7391, USA PUBLISHING
DAVID
External Gastric Balloon in Obesity Treatment
Mesut Basak 1 , H. Erdem Gozden 2 , Gulay Turan 2 , Hayrettin Mutlu 3 and Emine Pakir 3
1. Department of Social Work, University of Yalova, Yalova 77100, Turkey 2. Department of Internal Medicine, Umraniye Education and Resarch Hospital of Ministiry of Health, Istanbul 34400, Turkey 3. Department of Internal Medicine, University of Fatih, Istanbul 34844, Turkey
Received: October 06, 2012 / Accepted: March 01, 2013 / Published: May 30, 2013.
Abstract: O besity is an important and chronic disease. It is known that obesity is closely related with many chronic disorders. Thus, well knowledge about effects of obesity and its alternative treatments is important for determining ideal treatment of obesity and its
complications. In this study the authors tried to use a different method that is called EGB (External Gastric Balloon) for obesity treatment which is completely non-invasive. 91 patients were observed, including 47 of study group and 44 of control group. In the study group patients used a corset for 2.51 ± 1.07 months without hypocaloric diet and exercise. And during this time average weight loss was 6.08 kg. On the other hand, the control group was planned to take 1400-2400 kcal/day diet (approximately 1695.45 ± 256.95 kcal/day). Patients in the control group diet for a period 4.48 ± 2.17 months. Average weight loss was 6.06 kg. Patients in the study group weight loss with corset usage time between positive and statistically significant relationship at the level of 38.6% was observed (P < 0.05). The higher weight loss can be seen with long corset using period. A statistically significant relationship between duration of the diet in the control group patients and weight loss was not seen (P > 0.05).
Key words : Obesity, external gastric balloon, satiety corset.
1. Introduction hypothalamic center include neural afferents, hormones and metabolites. Vagal inputs are
Obesity is a major public health problem all over particularly important, bringing information from the world. The prevalence of obesity is increasing viscera, such as gut distension. Hormonal signals rapidly in developing countries. Obesity is spreading include leptin, insulin, cortisol and gut peptides. fast that its quickly turning into a pandemic. Obesity Among the letter are ghrelin, which is made in the increases mortality and morbidity and leads to huge stomach and stimulate feeding, and peptide YY (PYY) economic losses. In USA, $100 billion annually is and cholecystokinin, which are made in the small spent for the treatment of obesity. In USA, there are intestine and signal to the brain through direct action 100 million obese and overweight adults. 19 years or on hypothalamic control centers and/or via the vagus older, 31% of men, 35% of women and 20-25% of nerve. Especially, leptin is an hypophageic signal children are obese or overweight [1]. protien that reduces oral dietary intake [3]. If leptin There are different ways to determine whether binds to hypotalamic reseptors, it stimulates anorexic people are overweight or obese. The most commonly peptides and it reduces dietary intake [4]. used method is BMI and waist circumference [2].
Appetite is influenced by many factors that are
2. External Gastric Balloon
integrated by the brain, most importantly within the In very old times, people in war had connected a hypothalamus. Signals that impinge on the piece of stone on their stomach not to feel hunger and
they really managed to do this. The authors thought Corresponding author: Mesut Basak, Ph.D., professor, research field: internal medicine. E-mail: that the people who applied this method that make
drmesutbasak@gmail.com.
External Gastric Balloon in Obesity Treatment
people feel no hunger will feel satiated them at the same time, and moved from this basic idea.
The basic principle of intragastric balloon method is, to reduce the blank space in stomach with placing the balloon into the stomach and also to create the feeling of early saturation with a small amount of eating. The aim of “gastric clamp method” means to put handcuffs in stomach, is to reduce gastric volume by dividing the stomach into two spaces. Thus, person’s early
satisfaction is provided again.
Fig. 2 Tail of corset.
In external gastric balloon method, an elastic belt with the width of 28 cm and length that varies
according to the person’s body diameter that passes over their epigastriums, is used (Fig. 1).
Inside of this satiety belt, at the part of epigastrium,
a ball-shaped balloon with a changing diameter (12 cm/13 cm) according to the corset size that is used by people, is placed (Figs. 2 and 3). The balloon’s valve part for inflation is placed facing to the outside of the corset into the inner surface of the corset in a pocket
Fig. 3 Head of corset.
(Fig. 4). The valve is removed from the inside surface to the others through a hole and it is inflated from the valve till it becomes like a stone (Fig. 5).
This balloon can be taken out from the pocket of the corset anytime you need to wash out or for anything else, by its portable adhesive system (Fig. 6).
The belt is fastened around the body while the upper-medium edge of the balloon is fixed as it sits on epigastrium, just 1 cm below the xiphoid. And the
other arm of the corset is sticked onto the balloon part
Fig. 4 Valve of corset.
Fig. 1 View of corset. Fig. 5 The balloon’s valve part for inflation with pump.
External Gastric Balloon in Obesity Treatment
Fig. 6 Balloon.
Fig. 7 Support bands.
of corset, through the adhesive property. When the user wears the corset and fastens the corset arms with each other, the balloon provides pressure on the stomach of the user and reduces the volume of the stomach. A flexible band with a certain length of 36 cm and width of 9 cm is sticked on corset through the adhesive parts on center and edges focusing on the middle part will be on the balloon just to prevent the balloon from being seen aesthetically from the outside and could press on epigasrium better. A second band’s
upper edge placed just below the first one’s lower Fig. 8 Frontal view of corset.
edge to prevent corset folding above and to diminish risk of balloon’s moving up (Fig. 7). People wear this corset when they get up in the morning and throughout the night.
The method of EGB is completely non-invasive (Figs. 8 and 9). This method intends to press on the outside of the stomach using a balloon, and to reduce the space of with diminishing the volume of stomach. When people start eating, they feel full, eating half of what they normally would eat.
Consequently, it makes people feel staiated faster
Fig. 9 Lateral view of corset.
with less eating and satisfies a person enough
3. Materials and Methods
between meals that they have no urge for junk food. Thus people have a decreased appetite during the day
The present study was planned with 60 patients of thanks to usage of “Satiety Balloon (SB)” and begin
study group and 60 patients of control group in order to eat less. Satiety of the person applying balloon
to compare with two groups. However, 13 patients in makes a reduced desire to eat excessively.
the study group and 16 patients of control group could Individual, whose will of excessive eating is taken
not be followed-up due to inappropriate situation of off his hands, becomes disciplined and has a new
patients.
lifestyle. This study was conducted on the patients who
External Gastric Balloon in Obesity Treatment
appealed to Internal Medicine Clinic in Umraniye VARITEKS Company was used. Training and Research Hospital, Istanbul, Turkey. In
While examining the conclusions of the study this study the authors observed 91 patients, including
NCSS (Number Cruncher Statistical System) 2007 &
47 of study groups and 44 of control groups. At first, PASS 2008 Statistical Software (Utah, USA) program patients were asked about their symptoms (gastric and
has been used and the datas were analysed in addition gastroesophageal reflux complaints), history, medical
to Descriptive statistical methods used to evaluate the resume, family history, eating behaviors, eating
study data (mean, standard deviation and frequency) frequency and quantity, frequency and quantity of
Besides the comparison of quantitative data showing between-meals and subsequently physical examination
normal distribution, Student t-test comparisons
between the two groups, non-normally distributed body fat (%), HbA1c levels) was made. In addition,
(waist circumference (cm), weight (kg), BMI (kg/m 2 ),
parameters between two groups, Mann Whitney U test laboratory tests (CBC, glucose, urea, creatinin, AST,
was used for comparisons. Within group comparisons ALT, total cholestrol, LDL, HDL, TG, Na, K, Ca,
of normally distributed parameters, paired sample t TSH, fT4, insulin and cortisol levels), radiologic tests
test was used. Parameters in the evaluation of the (chest X-ray and abdominal ultrasound) and ECGs
relationship between Spearman’s correlation analysis were examined, too. Patients were called for was used, significance P < 0.05 level were evaluated. examining at the end of 1st, 2nd, 3rd, 4th, 5th, 6th
When an international patent application was months and inquired for gastrointestinal complaints
maken, PCT institution remarked that they have preceding going through physical examination (waist
already received two patents like the application
identified previously. The first one of these patents fat (%), HbA1c levels) again in each control. At the
circumference (cm), weight (kg), BMI (kg/m 2 ), body
has been taken in 1985 although its patent information last ones, as new, laboratory tests (CBC, glucose, urea,
could not be found on google search engine or from creatinin, AST, ALT, total cholestrol, LDL, HDL TG,
any international patent links (PCT: DE 35 00 078 Na, K, Ca, TSH, fT4, insulin and cortisol levels) were
A1). Besides this, the authors have failed to reach a evaluated again.
scientific publication related to this product made by The control group was planned to take 1400-2400
recipients of this patient, any of our literature review. kcal/day diet (approximately 1695.45 ± 256.95) by
The second patent is understood to be taken on the means of Schofield formulation which was used while
2009 even though there has no scientific publications this diet calculated, considering weight and sex. These
found (PCT: ES 1 067 680 U). The national patent patients were observed for 2-10 months and called for
date is 18 November 2008. Thus it can be understood calculating BMI and body fat ratios, body weight and
that the authors took the domestic patient before the waist circumferences at the beginning and 3rd, 6th,
international patient date of the above-mentioned 9th months.
second patient. Although the first patient with German The brand of weighing instrument we used in our
descent is earlier than ours, in the research on the study is “In body 520”. National patent of EGB
internet the authors could not found a patient or any application was taken by Destek Patent Company on
publication related to product of this patient. The our behalf. Besides they have applied for the
authors think that it means it is impossible for us to be international patent (PCT) for us, and the procedure is
effected by this patient or product. still going on. Upon this request, the satiety corset was
4. Results
produced and developed by VARITEKS Company on our behalf. In this study the satiety corset produced by
This study is based on 91 patients, including 47 of
External Gastric Balloon in Obesity Treatment
study group and 44 of control group. Patients using were 86.40 kg and 80.34 kg, respectively. corset in the study group periods ranged from 1 month
In the study group patients used a corset for 2.51 ± to 6 months and the average corset using time were
1.07 months without hypocaloric diet and egzercise.
2.51 ± 1.07 months. Patients in the control group diet And during this time average weight loss was 6.08 kg. periods were ranging from 1 month to 10 months to an
On the other hand the control group was planned to average of 4.48 ± 2.17 months. Waist circumference
take 1400-2400 kcal/day diet (approximately 1695.45 in the study group of patients varies between 81 cm
± 256.95 kcal/day). Patients in the control group diet and 137 cm with an average of 108.51 ± 14.01 cm.
period was 4.48 ± 2.17 months. Average weight loss Corset circles varies between 125 cm to 76 cm an
was 6.06 kg. Patients in the study group weight loss average of 100.52 ± 11.52 cm. Patients in the control
with corset usage time between positive and group were applied to 2400 kcal diet calorie levels
statistically significant relationship at the level of ranging from 1400 kcal to an average of 1695.45 ±
38.6% was observed (P < 0.05). The higher weight 256.95 kcal.
loss can be seen with long corset using period. A Patients body fat varied between 23% and 55%,
statistically significant relationship between duration average body fat varies were 41.28 ± 7.68%.
of the diet in the control group patients and weight Hemoglobin levels of patients varied between 9 g/dL
loss was not seen (P > 0.05).
and 16 g/dL and average of 12.75 ± 1.35 g/dL. The mean body mass index of patients in the study Between 28% and 47% of the cases hemotocrit levels
group was statistically significantly higher than were changed, and averaged 38.69 ± 3.89%. patients in the control group in an advanced level (P < Sedimentation levels of the patients were changing
between 2 mm/h and 47 mm/h and the mean was Body fat ratios of the patients in the study group
17.07 ± 11.92 mm/h, the median was 12 mm/h. were higher than the control group patients, although CRP levels of patients varied between 0.2 mg/dL to
these differences weren’t statistically significant but
9.9 mg/dL with an average of 2.41 ± 1.74 mg/dL,
close to significance (P > 0.05).
median 0.7 mg/dL. HbA1c levels of the cases varied The first measurement of HDL level and last between 5.1% and 6.9%, an average of 5.68 ± 0.42%.
increased measurement of HDL level was statistically Patients’ cortisol levels were changing between 5.6
significant in the patients of working group (P < 0.05). mcg/dL and 46.6 mcg/dL in an average of 14.24 ±
According to the first measurement of the patients
7.54 mcg/dL, median was 13.8 mcg/dL. Insulin levels in the last measurement, Blood Sugar Tests, ALT, of the patients varied between 3.8 mclU/mL and 61
AST, total cholesterol, LDL, triglycerides, sodium, mclU/mL with an average of 12.73 mcIU/mL ± 9.20,
potassium, calcium, TSH and fT4 statistically median 11 mclU/mL.
significant difference in the average is not available The first measuring weight of patients in the study
(P > 0.05).
group was significantly higher than patients in the “Do you have a complaint after using the corset?” control group (P < 0.05). The last weight of patients in
answers to the question of the 47 cases, none of the study group was significantly higher than patients in
findings of the stomach complaint has been found. the control group at the end of the study (P < 0.01).
“Do you have a complaint like gastroesophageal First mean weight measurement in the study group
reflux after the corset?” answers to the question of the was 96.39 kg in the beginning of the study and the last
47 cases of 4.3% in the study group (n = 2) mean weight measurement was 90.25 kg at the end of
gastroesophageal reflux to the complaint, the findings the study. In the control group these measurement
were encountered. “After the use of corset any skin
External Gastric Balloon in Obesity Treatment
reaction or dermatitis were looking for?” answered to group found 2.8% decrease [6]. In our study, at the the question of the 47 cases and 4.3% (n = 2) that the
end of the 10 weeks BMI invariably fall at a rate of complaints stated. “Do you have a reduction in the
9.6% was found. In a study based on long term effects frequency and amount of eating after using the
of orlistat in Norway, it was indicated that there has corset?” answer to the question of the 47 cases by
no effect on frequency of the dietary intake in obese answering all these questions is yes. “Do you have a
patients and it was negatively affecting the effect of reduction in the frequency and amount of between
the drug [7]. In our study, the eating habits of all meals?” answers to the question of 47 cases who
patients improved, the frequency of dietary intake and answered yes to this question is all waist appetie were decreased. In RIO (rimonobant in obesity) circumference levels last seen was measured 8.76 cm
European Study overweighted and obese patients were decrease in the average according to the first
given hypocaloric diet (600 kcal less then daily measurement of the waist circumference in working
needed) and given 20 mg/day rimonobant, a group of the cases and that was significant (P < 0.01).
cannabinoid reseptor antagonist. At the end of the one The advanced decline in average 3.48 kg/m 2 at metric
year the study showed rimonobant’s effect on weight measurements in BMI according to the level seen in
loss was average 6.6 kg [8]. On the other hand in our recent measurements was statistically significant (P <
study average corset using duration was 2.5 month 0.05).
and our patient’s weight loss was 6.08 kg. A meta-analysis of all published randomised controlled
5. Discussion
trials to assess the efficacy and safety of rimonabant,
Obesity prevalence is significantly increasing which made by Christens et al. [9], patients given worldwide. As a result of use of thecnology, in
rimonabant 20 mg per a day who are overweight and developed countries, a physical activity of individuals
obese, average weight loss was 4.7 kg in one year [9]. are decreasing and because of this obesity is becoming
This study showed that in 2.5 months corset user more common. Obesity is an important risk factor for
patients’ weight loss was 6.08 kg. Another study with cardiometabolic diseases, including diabetes mellitus,
rimonabant, Despres et al., randomly assigned 1,036 hypertension, dyslipidemia, and coronary hearth overweight and obese patients with untreated disease and should be treated.
dislipidemia, a double blind therapy with either To prevent all these complications, for the treatment
placebo or rimonabant at a dose of 5 mg or 20 mg of obesity, today’s low calorie diet lose weight
daily for 12 months in addition to a hypocaloric diet. treatment, drug treatment, exercise, eating behavior
Rimonabant at a dose of 20 mg was associated with disorder therapy and surgical treatments are being
6.7 ± 0.5 kg weight loss [10]. In our study average tested. Especially as drug therapy with orlistat, in
corset using duration was 2.5 month and our patient’s China, overweight and obese 228 patients are tracked
weight loss was 6.08 kg. Another comparative study, with addition of low calorie diet for 24 weeks. At the
hypocaloric diet added with 20 mg rimonabant and 5 end of the study maximum weight loss was 5.0 ± 3.7
mg rimonabant treatment in obese patients, at the end kg [5]. In our study, avarage duration of corset using
of the one year, at a dose of 20 mg rimonabant group time was 2.5 ± 1.07 month and avarage weight loss
weight loss was 4.9 kg while at a dose of 5 mg was (6.08 ± 5.11) 6.08 kg. Another overwieght and
rimonabant group weight loss was 1.3 kg, both of obese 30 patients with coronary artery disease and
these results were staticially significant according to hypercholesterolemia given 3 × 120 mg orlistat for 12
placebo group [11]. In our study, the average corset weeks and 12 weeks later BMI rate in the orlistat
using duration was 2.5 months and without any
External Gastric Balloon in Obesity Treatment
hipocaloric diet weight loss was 6.08 kg. As decrease at 3.48 kg/m 2 and 6.08 kg weight loss was anti-obesity treatment and long-term use of indicated in 2.51 ± 1.07 months according to the first sibutramine, serotonin and noradrenaline reuptake
BMI and weight, and also in this study we didn’t inhibitors, was made with double-blind, randomized,
advice any hypocaloric diet or excersize with our
corset application. In Italy 2,515 patients to whom patients took sibutramine, 155 patients took placebo
multicenter study, BMI 35 ± 3.2 kg/m 2 with 154
intragastric balloon application applied were observed treatment. In addition to this treatment made retrospectively for the weight loss and comorbidity. hipocaloric diet regulation. The weight loss was 8.2
At this study the patients who took balloon application kg in sibutramine group after 6 months, weight loss
were observed with 1000 kcal diet and medical was 3.9 kg in the placebo group were found [12]. In
therapy for 6 months and the intragastric balloon was our study, the average corset using duration was 2.5
taken away at the end of 6 months. After that months and without any hipocaloric diet weight loss
approximately 4.9 ± 12.7 kg/m 2 decrease was was 6.08 kg.
ascertained in BMI. In 2 patients (0.08%) had got 172 patients to whom an invasive technique called
gastric dilatation, 5 patients (0.19%) got gastric “laporoscopic gastric band application” made were
perforation (4 of these had a history of gastric surgery observed between 2003 and 2009. The ones whose
before) and 2 patients had died, while 2 patients got BMI were 38.5 kg/m 2 at the begining became 35.9 healed with laporoscopic surgery. 32 patients (1.27%)
2 kg/m 2 three months later with a fall of 2.6 kg/m [13]. had eusophagitis, 5 patients had (0.2%) gastric ulcer In our study BMI levels were found 3.48 kg/m 2 [16]. Although intragastric balloon application is an decreased according to the last measurements for the
effective procedur on healing comorbid illnesses duration of 2.5 months. In a retrospective study in
because of overweight and weight loss, in our study America on the reliability of the laporoscopic gastric
we had 3.48 kg/m 2 decrease in BMI in 2.5 months band application, 3,000 patients were examined in
while in this study they got 4.9 kg/m 2 decrease in BMI, general and band related complications; and in 6 months, in spite of diet. Another point that we
death-reoperation rate was looked at: Band slipped at want to underline is that we had neither a death nor a
a rate of 4.5%; port related problems were reported complication Genco et al. [16] had in their study. In 3.3%; band erosion was 0.2% (7 patients) and 11
our study the efficacy of EGB on metabolic and patients (0.4%) needed to be reoperated. Three of ten
cardiovascular risc factors is not analysed in people died in the first 30 days after the surgery; and 2
details.
of these 3 were found to be associated with the surgery and 7 of 10 people were lost for reasons
6. Conclusion
unrelated to surgery [14]. As a result, although very This new method in obesity treatment is non low mortality rate, in our study we encountered no
invasive and does not require any diet. We planned complications that will result in mortality. Another
new studies about longer usage of Satiaty Corset. method in the treatement of obesity called And at these studies efficacy on metabolic syndrome
“intragastric balloon application” was used in a study and risk factors are planned to be explored. In that was conducted on 303 patients in Italy in 1998
additon to this plan, we will also try to determine and reported at the end of 4 months 13.9 kg decrease
how the metabolites like “Leptin, Ghrelin, EGF
(endothelial growth factor), Neuropeptide Y, patients were given diets as 1000 kcal per day beside
in weight and 4.8 kg/m 2 decrease in BMI. In this study
Adiponectin” will be affected by the usage of Satiety the application [15]. In our study the last BMI got a
Balloon.
External Gastric Balloon in Obesity Treatment
References
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370 (2007) 1706-1713.
www.emedicine.com/endocrinology/obesity (update May [10] J.P. Despres, A. Golay, L. Sjöström, Effects of 21, 2009, p. 1).
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patients with dyslipidemia, The New England Journal of internal medicine, in: Biology of Obesity, 17th ed.,
Medicine 353 (2005) 2121-2134. Chapter 74, The McGraw-Hill Companies, New York,
[11] C. Curioni, C. Andre, Rimonabant for overweight or 2008, Volume 2, pp. 462-464.
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Article No.: CD006162, DOI: 10.1002/14651858. 18th ed., Appleton & Lange Publishing Company, [12] V. Di Francesco, T. Sacco, M. Zamboni, L. Bissoli, E. Connecticut, 1997, p. 223. Zoico, G. Mazzali, et al., Weight loss and quality of life [4] Robbins Basic Pathology, 8th ed., Saunders/Elsevier Inc., improvement in obese subjects treated with sibutramine: Philadelphia, 2007, p. 960.
[5] H.J. Zhu, H. Pan, F.Y. Gong, X.F. Lu, Y.D. Peng, Z.M. A double-blind randomized multicenter study, Ann. Nutr. Liu, et al., A comparison of the efficacy and safety of
Metab. 51 (2007) 75-81.
domestic orlistat and imported orlistat in Chinese [13] X.R. Ji, D.L. Chen, X.G. Hu, J.S. Wu, C.W. Ke, K. Yin, overweight and obese patients, Zhonghua Nei Ke Za Zhi
et al., Laparoscopic adjustable gastric banding in the 10 (2009) 825-829.
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Journal of Gastrointestinal Surgery 12 (2009) 551-553. H. Chu, et al., The effects of diet and orlistat on body
[14] A.M. Carelli, H.A. Youn, M.S. Kurian, C.J. Ren, G.A. weight and lipid profiles in high risk Chinese patients
Fielding, Safety of the laparoscopic adjustable gastric with coronary artery disease, obesity and
band: 7-year data from a U.S. center of excellence, in: hypercholesterolemia, Ir. J. Med. Sci. 6 (2009) 173-178.
The 2009 Annual Meeting of the Society of American [7] M. Svendsen, M. Helgeland, S. Tonstad, The long-term
Gastrointestinal and Endoscopic Surgeons (SAGES) 4 influence of orlistat on dietary intake in obese subjects
(2009) 22-25.
with components of metabolic syndrome, Journal of [15] S.B. Doldi, G. Micheleto, M.N. Perrini, R. Rapetti, Human Nutrition & Dietetics 2 (2009) 55-63.
Intragastric baloon: Another option for treatment of [8] L.F. Van Gaal, A.M. Rissanen, A.J. Scheen, O. Ziegler, S.
obesity and morbid obesity, Hepato-Gastroenterology 51 Rössner, Effectts of the cannabinoid-1 receptor blocker
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rimonobant on weight reduction and cardiovascular risk [16] A. Genco, T. Bruni, S.B. Doldi, P. Forestieri, M. Marino, factors in overweight patients, Lancet 4 (2005)
L. Busetto, et al., BioEnterics Intragastric Balloon: The 1389-1397.
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May 2013, Vol. 7, No. 5, pp. 483-490
Journal of Life Sciences, ISSN 1934-7391, USA
DAVID PUBLISHING
Chromatographic Analysis of Thiophenes in Calli and Suspension Cultures of Tagets spp.
Hussein S. Taha 1 , Hamida A. Osman 2 , Mahmoud M.M.A. Youssef 2 , Abdel Monem Y. El-Gindi 3 , Hoda H.
2 Ameen 2 and Asmahan M.S. Lashein 1. Department of Plant Biotechnology, National Research Centre, Dokki, Cairo, Egypt
2. Department of Plant Pathology, National Research Centre, Dokki, Cairo, Egypt 3. Department of Zoology & Agricultural Nematology, Faculty of Agriculture, Cairo University, Cairo, Egypt
Received: December 28, 2012 / Accepted: February 18, 2013 / Published: May 30, 2013.
Abstract : The thiophene isomer fractions were investigated by TLC (thin layer chromatography) and GLC (gas liquid chromatography). The TLC analysis revealed that, 5-(4-acetoxy-1-butynyl)-2,2-bithenyl (BBTOA C , Rf 0.78 I);
5-(3-buten-1-ynyl)-2,2-bithineyl (BBT, Rf 0.39 II) and 2,2,5,2- -terthienyl ( -T, Rf 0.32 III) were detected in extracts of leaf, stem and root calli or suspension cultures of Tagetes erecta and T. patula as well as leaf cell culture of T. patula which treated with DL-tryptophan under light or dark conditions. The GLC quantitative analysis of thiophenes in subjected revealed that the percentage distribution of the major thiophenes in all the analysed samples were determined as follows: BBT II > -T III > BBTOAc I. Another thiophene was determined in relatively small amounts and found to be 5-(3-penten-1-ynyl)-2,2-bithienyl (PBT IV). Other thiophenes were quantitated in trace amounts with unidentified or unknown chemical structure from the investigated extracts.
Key words : Thiophenes, callus, suspension, TLC, GLC, Tagetes erecta, T. patula.
1. Introduction Seeds of T. laxa, T. terniflora, T. minuta and T. campanulata germinated on free growth regulators of
Tagetes species (marigolds) contain biocidal MS [5] medium and the 2 nd to 3 rd week old seedlings compounds of the thiophene group as non-polar were analyzed by HPLC for the presence of products of secondary metabolism. Thiophenes thiophenes [6]. All seedlings were found to contains represent a variety, of biocidal action against 5-(4-hydroxy-1-butynyl)-2-2-bithenyl and 5-(4- nematelminths, bacteria, fungi, virus, insects and algae acetoxy-l-butynl)-2-2-bithenyl. T. campanulata and T. [1], the five main thiophenes accumulated in tagetes laxa seedlings also contained 5-(3-buten tissue are -T, BBT, BBTOH (hydroxyl butenenyl 1-ynyl)-2,2-bithenyl and alpha-terthienyl. Effects of
biothiophene), BBTOA C , BBT (OAc) 2 (diacetoxy
sucrose, light and the number of subcultures on butenenyl bithiophene) [2, 3]. Transformed roots of T. growth and thiophene formation in cell suspension erecta following infection of stems of sterile plantlets and root cultures of T. argentina were studied [7]. The with Agrobacterium rhizogenes strain TR105, four thiophenes studied were BET, BBTOA C , detected thiophenes were 5-(4-hydroxy-1-butenyl) BBTOH and alpha-T, which is the most abundant 2,2-bithienyl,5-(4-acetoxy-1-butenyl),2,2-bithienyl,1,5 were reported in Tagetes cultures. Moreover four -(3-buten-1-ynyl)2,2-bithineyl and 2,2,5,2- - thiophene compounds 5-(pent-1-ol)-2,2-bithienyl; terthienyl [4]. The thiophene pattern was the same in stigma-4,22-dien-3-beta-ol and 5-(4-acetoxy-1- normal root cultures and roots of the intact plant. butenyl)-2,2-bithienyl were detected in Tagetes erecta,
Corresponding author: Hussein S. Taha, Ph.D., research T. patula and T. minuta [8]. Arroo et al. [9] used the field: plant biotechnology. E-mail: hussein.taha2@yahoo.com.
484 Chromatographic Analysis of Thiophenes in Calli and Suspension Cultures of Tagets spp.
incorporation of [S] sulphur in thiophenes by T. patula ULTRA” gas chromatograph equipped with FID using roots as a model to study the regulation of secondary
a Thermo TR-5MS (5% phenyl polysil phenylene metabolism with a limited supply of substrate. They
siloxane) [30 m × 0.25 mm ID × 0.25 µm film] concluded that the rate of thiophene synthesis was
column. The carrier gas was nitrogen (flow rate 35 regulated by a control mechanism that reacts to the
m/min). The injector temperature was 235 °C and the availability of sulphate to the roots.
split ratio was 1:40. The column temperature was This study aiming to qualitative and quantitative
programmed from 50 (3 min isothermal) to 180 °C (10 detection and determination of thiophenes in the leaf
min isothermal) at 5 °C/min. The FID (flame and root calli or suspension cultures compared to
ionization detector) temperature was 250 °C. intact plant extracts of Tagetes sp. using TLC and
3. Results
GLC techniques.
2. Materials and Methods 3.1 Qualitative Detection of Thiophenes Using TLC
Aseptically grown T. erecta and T. patula seedlings The thiophene isomers were separated by TLC after 30 days from germinated seeds on basal
using silica gel plates. The thiophene fractions MS-medium were used as sources of different
5-(4-acetoxy-1-butynyl)-2,2-bithienyl (BBTOA C ), explants i.e. leaf, stem and root. Three sections of 3-4
was detected at Rf 0.78; mm in diameter of each plant part were excised and
5-(3-buten-1-ynyl)-2,2,-bithienyl (BBT) was detected cultured in 150 mL of glass jars containing 25 mL of
at Rf 0.39 and 2,2,5,2- α-Terthienyl (α-T) were MS-medium supplemented with 7.0 mg/L of NAA
detected at Rf 0.32, respectively. The isomers were and 10.0 mg/L of BAP [1].
detected in leaf, stem and root calli and cell suspension extracts of T. erecta and T. patula. This
2.1 Establishment of Tagetes Cell Suspension data is presented in Tables 1 and 2 and Figs. 1 and 2. Establishment of T. erecta and T. patula cell
Leaf cell culture of T. patula treated with suspension was carried out according to the described
DL-tryptophan at the concentrations of 5, 10, 15 and method [10].
20 mM under light or dark conditions is shown in Table 3 and Fig. 3. The isomers appeared as stained
2.2 Thin Layer Chromatography (TLC) bright blue spots on a black background when the
The methanolic extracts from leaf and root calli or plates were sprayed with 5% sulphuric acid. The suspension cultures and intact plant of T. erecta and T.
obtained results from TLC experiments revealed that patula as well as thiophenes as standard authentic
all fractions contain thiophene isomers in pure form. were analyzed using TLC. TLC was performed on
3.2 Quantitative Determination of Thiophenes Using pre-coated silica gel plates 60 F254 (20 cm × 20 cm,
Gas Liquid Chromatography (GLC)
0.25 mm, Merck, Germany). Routine procedure for analytical chromatography of thiophenes using TLC
Data presented in Table 4 and Fig. 4 show the effect was developed using: Hexane:Dioxane (3:1 v/v) as
of culture type (callus or suspension) as well as solvent system according to the described method
addition of tryptophan as an enhancer compound at 15 [11].
mM on the accumulation rate of thiophenes from leaf and root of T. erecta and T. patula incubated under
2.3 Gas Liquid Chromatography (GLC) light (16/8 h) or dark conditions. The main
Gas liquid chromatography was performed with components were BBT, α-T and BBTOA C . PBT and 4 “GAS LIQUID CHROMATOGRAPHY TRACE GC
unidentified thiophenes were detected only in traces.
Chromatographic Analysis of Thiophenes in Calli and Suspension Cultures of Tagets spp. 485
Table 1 Rate flow (Rf) of standard thiophenes compared to extracts of leaf, stem and root calli cultured under light or dark conditions compared to intact plant of T. erecta and T. patula.
T. patula Standard
T. erecta
Thiophenes (Rf)
Type of calli
Type of calli
Stem Root Light condition T1 (0.78)
Intact plant Leaf
Stem
Root
Intact plant Leaf
+ + T2 (0.39)
+ + T3 (0.32)
+ + Dark condition T1 (0.78)
+ + T2 (0.39)
+ + T3 (0.32)
+ + T1 indicated to 5-(4-acetoxy-1-butynyl)-2,2-bithienyl (BBTOA C ); T2 indicated to 5-(3-buten-1-ynyl)-2,2-bithienyl (BBT);
T3 indicated to 2,2,5,2- α-Terthienyl (α-T); + = Positive spot; Calli extract: 80% methanolic extract; Rf = Rate of flow.
Table 2 Rate flow (Rf) of standard thiophenes compared to extracts of leaf, stem and root suspensions cultured under light or dark conditions compared to intact plant of T. erecta and T. patula.
T. patula Standard
T. erecta
Thiophenes (Rf)
Type of suspension
Type of suspension
Intact plant
Intact plant
Stem Root Light condition T1 (0.78)
+ + Dark condition T1 (0.78)
+ + T2 (0.39)
+ + T3 (0.32)
+ + T1 indicated to 5-(4-acetoxy-1-butynyl)-2,2-bithienyl (BBTOA C ); T2 indicated to 5-(3-buten-1-ynyl)-2,2-bithienyl (BBT);
T3 indicated to 2,2,5,2- α-Terthienyl (α-T); + = Positive spot; Cell suspension extract: 80 % methanolic extract; Rf = Rate of flow.
Fig. 1 TLC of leaf, stem and root suspensions extracts of T. Fig. 2 TLC of leaf, stem and root suspensions extracts of T. patula .
erecta .
Column (1) = Methanolic extract of T. patula intact plant; Column (1) = Methanolic extract of T. erecta intact plant; Column (2) = Methanolic extract of leaf suspension of T.
Column (2) = Methanolic extract of leaf suspension of T. erecta patula under dark conditions; Column (3) = Methanolic extract
under dark conditions; Column (3) = Methanolic extract of of stem suspension of T. patula under dark conditions; Column
stem suspension of T. erecta under dark conditions; Column (4) (4) = Methanolic extract of root suspension of T. patula under
= Methanolic extract of root suspension of T. erecta under dark dark conditions; Column (5) = Standard thiophene; Column (6)
conditions; Column (5) = Standard thiophene; Column (6) = = Methanolic extract of leaf suspension of T. patula under light
Methanolic extract of leaf suspension of T. erecta under light conditions; Column (7) = Methanolic extract of stem
conditions; Column (7) = Methanolic extract of stem suspension of T. patula under light conditions; Column (8) =
suspension of T. erecta under light conditions; Column (8) = Methanolic extract of root suspension of T. patula under light
Methanolic extract of root suspension of T. erecta under light conditions; Column (9) = Standard thiophene.
conditions; Column (9) = Standard thiophene.
Chromatographic Analysis of Thiophenes in Calli and Suspension Cultures of Tagets spp.
Table 3 Rate flow (Rf) of standard thiophenes compared to extract of leaf suspension of T. patula cultured on MS-medium supplemented with DL-tryptophan as enhancer compound at 5, 10, 15 and 20 mM and intact plant and incubated under light or dark conditions.
Dark Standard
Light
Thiophenes (Rf)
DL-tryptophan (mM)
DL-tryptophan (mM)
Intact plant
Intact plant
+ + + T1 indicated to 5-(4-acetoxy-1-butynyl)-2,2-bithienyl (BBTOA C ); T2 indicated to 5-(3-buten-1-ynyl)-2,2-bithienyl (BBT); T3 indicated to 2, 2, 5, 2- α-Terthienyl (α-T); + = Positive spot; Cell suspension extract: 80 % methanolic extract; Rf = Rate of flow.
1 2 3 4 5 6 7 8 9 10 patula treated with 5 mM of DL tryptophan under light conditions; Column (3) = Methanolic extract of leaf suspension of T. patula treated with 10 mM of DL tryptophan under light conditions; Column (4) = Methanolic extract of leaf suspension of T. patula treated with 15 mM of DL tryptophan under light conditions; Column (5) = Methanolic extract of leaf suspension of T. patula treated with 20 mM of DL tryptophan under light conditions; Column (6) = Standard thiophene; Column (7) = Methanolic extract of leaf suspension of T. patula treated with 5 mM of DL tryptophan under dark conditions; Column (8) = Methanolic extract of leaf suspension of T. patula treated with 10mM of DL tryptophan under dark conditions; Column (9) Methanolic
Fig. 3 TLC of leaf suspension extracts of T. patula treated
extract of leaf suspension of T. patula treated with 15 mM of
with DL-tryptophan.
DL tryptophan under dark conditions; Column (10) = Column (1) = Methanolic extract of T. patula intact plant;
Methanolic extract of leaf suspension of T. patula treated with Column (2) = Methanolic extract of leaf suspension of T.
20 mM of DL tryptophan under dark conditions.
Table 4 Different retention times of standard thiophenes and their relative production percent in root, leaf calli and leaf suspension cultured on MS medium supplemented with tryptophan compared with intact plants of T. erecta and T. patula and incubated under light or dark conditions (Total thiophenes = 100%).
Thiophene relative percentage to total
Unknown Treatments
BBTOA C PBT
0.529 0.513 1.042 T. patula leaf callus (Dark)
T. erecta root callus (Dark)
n.d. 0.417 1.188 T. erecta leaf suspension (Light) 26.112
n.d. n.d. n.d. T. erecta leaf suspension (Dark) 24.971
n.d. n.d. n.d. T. patula root suspension (Light) 27.584
n.d. n.d. n.d. T. patula root suspension (Dark) 27.452
2.149 n.d.
n.d. n.d. n.d. T. patula leaf suspension (Light)
n.d. n.d. n.d. T. patula leaf suspension (Dark)
(15 mM DL-tryptophan)
(15 mM DL-tryptophan)
n.d. n.d. n.d. T. erecta intact plant
n.d. n.d. n.d. T. patula intact plant
n.d. n.d. n.d. R t : Retention time of standard thiophenes, min; α-T = 2,2,5,2-terthienyl; BBT = 5-(3-buten-1-ynyl)-2,2-bithienyl; BBTOA C = 5-(4-acetoxy-1-butynyl)-2,2-bithienyl; PBT= 5-(3-penten-1-ynyl)-2,2-bithienyl; n.d. = non detected;
Chromatographic Analysis of Thiophenes in Calli and Suspension Cultures of Tagets spp. 487
Fig. 4 GLC analysis of thiophene compounds (R t value) from: (a) T. erecta root callus (Dark); (b) T. patula leaf callus (Dark); (c) T. erecta leaf suspension (Light); (d) T. erecta leaf suspension (Dark); (e) T. patula root suspension (Light); (f) T. patula leaf suspension (Light); (g) T. patula leaf suspension + tryptophan (Light); (h) T. patula leaf suspension + tryptophan (Dark); (i) T. patula intact plant; (j) T. erecta intact plant.
Chromatographic Analysis of Thiophenes in Calli and Suspension Cultures of Tagets spp.
Table 5 Distribution patterns of thiophene compounds in root and leaf calli and suspension cultures of T. erecta and T.
patula incubated under light or dark conditions compared with intact plants.
Thiophene compounds (%) of intact plant
α-T relative to
Treatments
BBT relative to
BBTOA C relative to
PBT relative to
intact plant of :
intact plant of:
intact plant of:
intact plant of :
T. patula T. erecta T. patula T. erecta root callus (Dark)
T. erecta
T. patula T. erecta
T. patula T. erecta
18 - T. patula leaf callus (Dark)
- 25 T. erecta leaf suspension Light)
84 - T. erecta leaf suspension (Dark)
- - T. patula root suspension (Light) -
- 38 T. patula root suspension (Dark) -
- 25 T. patula leaf suspension (Light)
(15 mM DL-tryptophan)
- 134 T. patula leaf suspension (Dark) -
- 67 α-T = 2,2,5,2-terthienyl; BBT = 5-(3-buten-1-ynyl)-2,2-bithienyl, BBTOA C = 5-(4-acetoxy-1-butynyl)-2,2-bithienyl; PBT = 5-(3-penten-1-ynyl)-2,2-bithienyl.
15 mM DL-tryptophan under light conditions which detected by (GLC) in T. erecta root callus and T.
Other three unknown thiophene compounds were
gave the highest level of 134% compared with the patula leaf callus under dark conditions.
intact plants. The percentage increases in ( α-T) were The rates of ( α-T) accumulation were 28.116%,
(115%, 114%, 107% and 109%) in T. patula root 27.584% and 27.452% of total thiophenes, from root
suspension under light or dark conditions, T. patula calli of T. erecta under dark condition; root leaf suspension enhanced with 15 mM DL-tryptophan suspension of T. patula under light and dark
under light or dark conditions.
conditions; respectively. Also, the rates of BBT Retention time (Rt) range: BBTOA C I= accumulation were 45.732%, 44.223% and 43.931%
31.995-32.945; BBT II = 31.505-32.438; α-T III = of total thiophenes from leaf suspension of T. erecta
28.287-29.035; PBT IV = 30.188-30.290. under dark condition; leaf suspension of T. patula
4. Discussion
treated with 15 mM of tryptophan under dark condition and leaf suspension of T. erecta under light
The present results of the TLC analysis revealed condition, respectively. Moreover, the accumulation
that, 5-(4-acetoxy-1-butynyl)-2,2-bithienyl (BBTOA C , rates of BBTOA C were 21.259%, 21.218% and
R f = 0.78 I); 5-(3-buten-1-ynyl)-2,2-bithienyl (BBT, R f 20.626% of total thiophenes from leaf suspension of T.
= 0.39 II) and α-Terthienyl (α-T, R f = 0.32 III) were patula treated with 15 mM of tryptophan under dark
detected in extracts of leaf, stem and root calli and cell condition, leaf suspension of T. erecta under dark or
suspension from T. erecta and T. patula as well as leaf light conditions; respectively.
cell culture of T. patula treated with DL-tryptophan Data in Table 5 indicated the distribution pattern of
under light or dark conditions. These results revealed thiophenes in various in vitro cultures as compared to
that all fractions contain the three thiophene T. erecta and T. patula intact plants. Generally, non
compounds I, II, and III in pure form as evidenced by
their specific R f values compared with authentic concentrations of the different extracts calculated as
significant changes in thiophene ( α-T, BBT, BBTOA C )
compounds. This method proved to be simple and percentage of intact total plant contents. However
reliable in isolation of highly purified forms of the PBT was dramatically decreased in most treatments
thiophene compounds at lower cost. except in T. patula leaf suspension supplemented with
In this regard it has been reported that, compounds I
Chromatographic Analysis of Thiophenes in Calli and Suspension Cultures of Tagets spp. 489
and III were thiophene derivatives with nematicidal plants were screened by TLC and analyzed by GC-MS. activity [12]. Moreover, thiophenes in calli and
Three thiophene fractions were obtained containing suspension cultures derived from (leaf and root)
BBT, 5-(3-buten-1-ynyl)-2,2-bithienyl; BBTOA C , explants of T. patula and T. erecta were identified
5-(4-acetoxy-1-butynyl)-2,2-bithienyl and BBTOH, through GLC study. The results of GLC quantitative
5-(4-hydroxy-1-butynyl)2,2-bithienyl. analysis of thiophenes in extracts of root, leaf calli and