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In vitro antimicrobial and anticancer activity of Cinnamomum Zeylanicum linn bark extracts

Authors:
  • SHRIMATI INDIRA GANDHI COLLEGE, TIRUCHIRAPPALLI
  • Shrimati Indira Gandhi College, Tiruchirappalli

Abstract and Figures

One of the most common threats is the spread of multidrug resistant pathogens. Cancer is another major problem leads to death. Hence a search for new, plant based, risk-free, superior compounds with novel antimicrobial and anticancer activities is the need of the day. The aim of this study was to evaluate the antimicrobial and anticancer effects of bark of Cinnamomum zeylanicum. The antimicrobial activity of extracts of Cinnamomum zeylanicum bark (aqueous, methanol and chloroform) against bacterial and fungal clinical isolates like Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Aspergillus niger and Candida albicans was determined. The anticancer activity was studied by MTT and AO/EB staining on hepato carcinoma cell lines ((Hep G2 cell line). The result of antimicrobial study showed that methanolic extract had better antibacterial and antifungal activity. The most susceptible bacterial and fungal strains were Bacillus subtilis and Aspergillus niger, respectively. The methanolic extract showed MIC value of 2.5mg/ml for Bacillus subtilis and 5mg/ml for Aspergillus niger. The results of in vitro anticancer studies by MTT assay on Hep G2 cell line in the presence of methanolic extract of Cinnamomum zeylanicum bark showed an IC50 value of 150μg/ ml. The AO/EB staining also showed that the methanolic extract was able to induce apoptotic activity in HepG2 cells after 24 hours of incubation at a concentration 150μg/ ml. This study proved that Cinnamomum zeylanicum bark is a reliable and safer herbal drug that can be used in pharmaceutical preparations for infectious and malignant diseases.
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Full Proceeding Paper
IN VITRO ANTIMICROBIAL AND ANTICANCER ACTIVITY OF CINNAMOMUM ZEYLANICUM LINN
BARK EXTRACTS
B. VARALAKSHMI1*, A. VIJAYA ANAND2, T. KARPAGAM1, J. SUGUNA BAI3, R. MANIKANDAN 4
1Department of Biochemistry, Shrimati Indira Gandhi College, 2Department of Biochemistry, MIET College, 3Department of Biochemistry,
Seethalakshmi Ramaswami College, 4Department of Chemistry, SSK polytechnic college, Tiruchirappalli. Email: chembioooo@yahoo.co.in
Received: 20 Nov 2013, Revised and Accepted: 21 Dec 2013
ABSTRACT
One of the most common threats is the spread of multidrug resistant pathogens. Cancer is another major problem leads to death. Hence a search for
new, plant based, risk-free, superior compounds with novel antimicrobial and anticancer activities is the need of the day. The aim of this study was
to evaluate the antimicrobial and anticancer effects of bark of Cinnamomum zeylanicum. The antimicrobial activity of extracts of Cinnamomum
zeylanicum bark (aqueous, methanol and chloroform) against bacterial and fungal clinical isolates like Bacillus subtilis, Staphylococcus aureus,
Escherichia coli, Aspergillus niger and Candida albicans was determined. The anticancer activity was studied by MTT and AO/EB staining on hepato
carcinoma cell lines ((Hep G2 cell line). The result of antimicrobial study showed that methanolic extract had better antibacterial and antifungal
activity. The most susceptible bacterial and fungal strains were Bacillus subtilis and Aspergillus niger, respectively. The methanolic extract showed
MIC value of 2.5mg/ml for Bacillus subtilis and 5mg/ml for Aspergillus niger. The results of in vitro anticancer studies by MTT assay on Hep G2 cell
line in the presence of methanolic extract of Cinnamomum zeylanicum bark showed an IC50 value of 150μg/ ml. The AO/EB staining also showed that
the methanolic extract was able to induce apoptotic activity in HepG2 cells after 24 hours of incubation at a concentration 150μg/ ml. This study
proved that Cinnamomum zeylanicum bark is a reliable and safer herbal drug that can be used in pharmaceutical preparations for infectious and
malignant diseases.
Keywords: Cinnamomum zeylanicum, Antimicrobial, MIC, Anticancer, MTT, AO/EB staining, Apoptosis.
INTRODUCTION
There has been a progress in the prevention, control and eradication
of microbial diseases by the development of antimicrobials. Due to
the evolution and adaptation of microbes, there is threats of new
diseases and re-emergence of old diseases [1]. Cancer is a life-
threatening disease and leads to high rates of mortality worldwide.
Investigations for finding new plant based antimicrobial and
anticancer compounds are imperative and interesting. There are
many studies on anticancer herb/plant extracts in cell line models
[2-4].
Cinnamomum zeylanicum tree belongs to the family, Lauraceae most
noted for its bark, which provides the world with the commonly
known culinary spice, cinnamon. Cinnamon has medicinal property
and has been used to treat gastrointestinal complaints and other
ailments [5]. Cinnamon possesses antiallergenic, anti-inflammatory,
anti-ulcerogenic, anti-pyretic, antioxidant, anaesthetic activities [6].
Antioxidant studies with Cinnamomum zeylanicum bark showed
better free radical scavenging capacity against a battery of free
radicals [7]. In the present study, Cinnamomum zeylanicum bark was
analyzed for its antimicrobial activity against clinical bacterial and
fungal pathogens. Apoptosis is the mode of cell death induced by
stimuli such as drugs, stress, radiation etc., The study was also
aimed to evaluate the anticancer ability of Cinnamomum zeylanicum
bark extract by inducing apoptosis in human hepato carcinoma cells
(Hep G2 cell lines).
MATERIALS AND METHODS
The cinnamon bark was purchased from local market,
Tiruchirappalli. The bark was dried, powdered and stored in a
sterile container until use. A voucher specimen was deposited at the
Rapinath Herbarium (BV001) and identified by Dr. John Britto of St.
Joseph’s College, Tiruchirappalli. The solvents and chemicals used
were of analytical grade.
Test Microrganisms
The bacterial and fungal strains used were clinical isolates obtained
from Eumic Analytical Lab and Research Institute, Tiruchirappalli.
The bacterial strains used were Bacillus subtilis, Escherichia coli, and
Staphylococcus aureus. The fungal strains used were Aspergillus
niger and Candida albicans.
Preparation of Plant Extracts and Inoculation Medium
10g of powered bark of Cinnamomum zeylanicum was homogenized
in 100ml of the solvents of varying polarity such as water, methanol
and chloroform. The organic extracts were dried at 60°C kept away
from sunlight. The residue was weighed and dissolved in Dimethyl
Sulfoxide (DMSO; 20mg/50µl) to obtain the desired concentration.
Aqueous extracts were prepared fresh.
The nutrient broth was prepared and sterilized by autoclaving at
121˚C and at 15lbs for 15 minutes. Bacterial and fungal cultures
were sub cultured in liquid medium at 37˚C for 8h and further used
for the test (104-105CFU /ml).
Antibacterial Assay
Kirby bauer Agar Well Diffusion method was used to study the effect
of various bark extracts on the selected bacterial strains [8]. The
sterilized nutrient agar medium was aseptically poured (20ml) into
the sterile petri-plates and allowed to solidify. The bacterial broth
cultures were separately swabbed on petri-plate using a sterile bud.
Then wells were made by well cutter. The organic and aqueous
extracts of bark (20 µl) were added to each well aseptically and were
incubated at 37˚C for 24 hours. The zone of inhibition was measured.
Amphicillin disc was used as a positive control [9]. The experiment
was repeated and the mean of triplicates was calculated.
Antifungal Assay
The antifungal activity of bark extracts against Aspergillus niger and
Candida albicans were assayed by agar well diffusion method similar
to antibacterial assay, except that Rose Bengal Agar medium [10]
was used. Amphotericin B disc was used as standard. The petriplates
were incubated at 37˚C for 48 hrs. The antifungal activity was
assayed by measuring the zone inhibition [9]. The experiment was
repeated in triplicates and mean value was calculated.
Assay of Minimum Inhibition Concentration (MIC)
The MIC was determined with the methanolic extract which showed
the maximum zone of inhibition, according to the NCCLS protocol
[11]. Sterile nutrient broth was used for bacteria and Rose Bengal
broth was used for fungal strains. Serial dilutions of the methanolic
extract of Cinnamomum zeylanicum bark in DMSO was done to
obtain the concentrations ranging from 10, 5, 2.5, 1.25, 0.63, 0.31,
International Journal of Pharmacy and Pharmaceutical Sciences
ISSN- 0975-1491 Vol 6, Suppl 1, 2014
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0.16, 0.08 and 0.04 mg/ml. 100 µl from each tube was then
transferred into 96-well microtitre plates. Each well was then filled
with 100 µl microorganism suspension. Well containing
microorganism suspension without methanolic extract served as
positive control and the well without microorganism suspension
served as negative control. The microtitre plates were incubated at
37ºC for 24 hours for bacterial strains and 37ºC for 48 hours for
fungal strains with intermittent shaking and observed for visible
growth.
Anticancer Drug Screening by MTT Assay
The anticancer assay was performed on Human hepatocellular
carcinoma cells (HepG-2 cell line) supplied by National Centre for
Cell Science (NCCS), Pune, India, with non-toxic dose of the bark
extract and its dilutions. The cells were cultured in standard RPMI-
1640 medium containing 2 mM glutamine supplemented with 12 %
(v/v) heat inactivated fetal bovine serum and antibiotics (100 U/ml
penicillin and 100μg/ml streptomycin) in a humidified atmosphere
of 95% O2 and 5% CO2 at 37°C. Cells were maintained in 25 cm2
flasks and were sub cultured every 3 4 days.
The cell viability was determined by MTT (3-[4, 5- dimethylthiazol-
2-yl]-2, 5-diphenyltetrazolium bromide) Assay. MTT is cleaved by
mitochondrial enzyme dehydrogenase of viable cells, yielding a
measurable purple product formazan. This formazan production is
directly proportional to the viable cell number and inversely
proportional to the degree of cytotoxicity [12].
HepG-2 cells were seeded onto a 96-well plate at a density of 5000
cells/well in 200 μL of medium for 24 hours. Various concentrations
of methanolic extract of Cinnamomum zeylanicum bark (50, 100,
150, 200 µg/ml), was added to the wells and were incubated for 24
and 48 h. A control well without bark extract was maintained to
compare the hundreds percent cell viability. After the incubation
period 100 μl MTT (5 mg/ml in phosphate buffered saline) was
added to each well and incubated for 3 hours in dark. Then MTT was
discarded and 150 μl of DMSO was added to each well. The purple
colour developed was measured at 570 nm with microplate reader
(Bio-Rad). The IC50 value was calculated from the graph.
Percent cell viability was calculated as follows:
% of cell death = 100 - (OD of Test/OD of Control X 100)
Acridine Orange-Ethidium Bromide (AO/EB) Staining
Acridine orange is a vital dye and will stain both live and dead cells.
Ethidium bromide will stain only cells that have lost membrane
integrity [13].
HepG-2 cells were grown in 6-well plates treated with methanolic
extract of Cinnamomum zeylanicum bark at 150 μg/ml (IC50) for 24
and 48 h. The cells grown without methanolic extract of
Cinnamomum zeylanicum bark served as control. The culture
medium was removed and the cells were washed thrice with PBS,
stained with 10μL of fluorescent dyes i.e. AO/EB (1:1 ratio at
100μg/mL) (Sigma Aldrich, USA) for 5 min. After washing thrice
with PBS, morphological changes were observed under an
epifluorescence microscope (Carl zeiss, Germany) for apoptotic
changes at Ex/Em=510/595 nm for ethidium bromide and at
Ex/Em=500/530 nm for acridine orange.
RESULTS
Antibacterial Activity of Cinnamomum zeylanicum Bark
The bark extracts were evaluated for their antibacterial activity.
Figure 1 and Plate 1 shows the antibacterial function of aqueous,
methanol and chloroform extracts of the bark of Cinnamomum
zeylanicum on the selected bacterial strains which were Bacillus
subtilis Escherichia coli and Staphylococcus aureus.
Fig. 1: Antibacterial effect of Cinnamomum zeylanicum bark extracts
The zone of inhibition for Bacillus subtilis, Escherichia coli and
Staphylococcus aureus were found to be the more in the methanolic
extract which is comparable with the standard drug ampicillin and
followed by aqueous and chloroform extracts.
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Antifungal Activity of Cinnamomum zeylanicum Bark
The antifungal function of the bark extracts were determined
against the fungal clinical isolates like Candida albicans and
Aspergillus niger. The methanolic extract of the bark was found to
cause more inhibition of the growth of Aspergillus niger than
aqueous and chloroform extracts (Figure II and Plate II) and
compared with that of standard drug amphotericin B. All the three
extracts showed moderate inhibition on Candida albicans.
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Fig. 2: Antifungal effect of Cinnamomum zeylanicum bark extracts
Table 1: Minimum Inhibitory Concentration
S. No.
Microorganims
Methanolic extract concentration (mg/ml)
1
Bacillus subtilis
2.5
2
Eschericia coli
>5
3
Staphylococcs aureus
2.5
4
Asperillus niger
5
5
Candida albicans
>5
By collective observation it was revealed that methanolic extract
showed a good antibacterial activity and moderate antifungal
activity. The aqueous and chloroform extracts of the bark showed
the moderate antibacterial and lesser antifungal activity.
Minimum Inhibition Concentration
Table 1 depicted the MIC values for methanolic extract against the
selected microorganisms. The methanolic extract of bark showed a
low MIC of 2.5mg/ml against Bacillus subtilis and Staphylococcus
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aureus but E.coli showed MIC of more than 5mg/ml and hence the
least susceptible bacteria among the three. The methanolic extract of
bark showed the lowest MIC of 5mg/ml against Aspergillus niger but
Candida albicans showed MIC of more than 5mg/ml and hence the
lesser susceptible fungi.
Anticancer Activity by MTT Assay
Significant dose and time-dependent cytotoxicity was observed by
MTT assay. The IC50 values of methanolic extract of Cinnamomum
zeylanicum bark were found to be 200 and 150µg/ml after 24 and 48
hr respectively. At this concentration the extract was toxic to more
than 50% of HepG2 cells. The control tube (without bark extract)
showed 100% viability of cancer cells (Figure III).
Acridine Orange and Ethidium Bromide Staining
Treatment of HepG2 cells with methanolic extract of Cinnamomum
zeylanicum bark for 24 and 48 hours and staining with AO/EB
fluorescent stain exhibited characteristic apoptotic morphology, i.e.,
cell shrinkage, nuclear condensation and fragmentation and the
results were represented in Plate 3.
Fig. 3: MTT Assay
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Under fluorescence microscope the control cells treated for 24 hours
and 48 hours had no morphological changes; nucleus was smoother
and was uniformly bright green together with the cytoplasm (Plate
3A & 3C). The cells treated with 150 μg/ml of methanolic extract of
Cinnamomum zeylanicum bark for 24 h stained green and contained
bright green dots in the nuclei showed the presence of early
apoptotic cells (Plate 3B). HepG2 cells treated with 150 µg/ ml of
methanolic extract of Cinnamomum zeylanicum bark for 48 hours
integrated ethidium bromide and stained orange indicated the
presence of condensed and fragmented nuclei which showed the
presence of late apoptotic cells (Plate 3D). Dead cells were stained
orange (Plate 3B and 3D).
DISCUSSION
Antimicrobial activity
The bacterial strains used were of different taxonomy. Bacillus
subtilis is a Gram positive spore forming rods; Staphylococcus aureus
is a Gram-positive cocci and Escherichia coli is a Gram-negative
enterobacteria. The findings of this study showed that Cinnamomum
zeylanicum bark extracts had inhibited both Gram-positive bacteria
and Gram-negative bacteria indicating broad spectrum inhibitory
effect. Gram positive bacteria were more susceptible than Gram-
negative bacteria by the action of Cinnamomum zeylanicum bark
extracts, demonstrating antibacterial effect which was comparable
with that of the standard drug amphicillin.
There are several reports in the literature indicating the
antibacterial and antifungal activity of the medicinal plants. Many
studies reported the incapability of herbal antimicrobial agents to
inhibit growth of Gram-negative bacteria [14] due to the presence of
complex cell wall structure which decreases the penetration of
bacterial cells by herbal extracts. But in the present study,
Cinnamomum zeylanicum bark extracts moderately inhibited the
growth of E.coli, proving penetrating ability of extracts in to
bacterial cells.
The methanolic extract of Cinnamomum zeylanicum bark was found
to cause significant inhibition of the growth of Aspergillus niger and
Candida albicans compared to aqueous and chloroform extracts and
was comparable with that of standard drug amphotericin B.
Studies by Kil et al., (2009)proved that n-hexane, ethyl acetate, n-
butanol, methanol and water fractions of sorghum (Sorghum bicolor
Moench), the methanol extract elicited the maximum antimicrobial
activity against Escherichia coli, Staphylococcus aureus, Klebsiella
pneumoniae, Salmonella typhimurium, Bacillus subtilis and Candida
albicans [15].
Studies by Liwei et al.,(2004)[16] showed that dimeric, trimeric, and
higher oligomeric proanthocyandins with doubly linked bis-flavan-
3-ol units were present in Cinnamomum zeylanicum bark. In this
study the antimicrobial activity of Cinnamomum zeylanicum may be
due to the presence of these secondary metabolites. The methanolic
extract of the bark of Cinnamomum zeylanicum had strong
antimicrobial activity showed that the active components have been
extracted in methanol.
In the current investigation, the methanolic extract of Cinnamomum
zeylanicum bark exerted low MIC of 2.5mg/ml against Bacillus
subtilis and Staphylococcus aureus but Escherichia coli showed MIC
of more than 5mg/ml and hence the least susceptible bacteria
among all. The methanolic extract of bark showed the lowest MIC of
5mg/ml against Aspergillus niger but Candida albicans showed MIC
of more than 5mg/ml and hence the lesser susceptible fungi.
The Chinese medicinal plants extracted with hot water, methanol
and acetone were evaluated for their antifungal activity, among
which the acetone extracts had the lowest MIC values indicating
their effective antifungal property [17].
Manvi Malwal and Renu Sarin (2010)studied the invitro
antimicrobial efficacy and MIC of root extracts of Murraya koenigii
(Linn) Spreng against four bacterial strains (Escherichia coli,
Staphylococcus aureus, Bacillus subtilis, and Salmonella typhi) and
three fungal strains (Candida albicans, Aspergillus niger and
Trichophyton rubrum) [18]. The most susceptible bacterial and
fungal strains were Staphylococcus aureus and Trichophyton rubrum,
for methanolic of roots showed MIC value of 0.078mg/ml and 0.156
mg/ml respectively.
Our results were congruent with the results of others.
Anticancer activity
MTT is considered to be a reliable assay to determine the extent of
cell viability. In the present study the IC50 values of methanolic
extract of Cinnamomum zeylanicum bark were found to be 200 and
150µg/ml after 24 and 48 hr respectively. The results of the study
also showed that the induction of apoptosis by methanolic extract of
Cinnamomum zeylanicum bark in human hepatoma cancer cells
indicates its anticancer activity.The study by Venkatakrishnan et al.,
(2010) reported that the ethanolic extract of Pleurotus ostreatus
repressed the cell proliferation in a dose-dependent manner in
leukemia cells (HL-60 cell line) [19]. The methanolic extract of Piper
sarmentosum possessed anticarcinogenic properties in HepG2 cells
[20]. Similarly, Zhang and Poporich (2008) estimated, using the MTT
assay, the inhibition of cell proliferation in Hep G2 (liver carcinoma)
cells by soya saponins, which was found to be dose-dependent [21].
Shahrul Hisham et al., (2009), reported on the anticarcinogenic
activity of an ethanolic extract from Piper sarmentosum in HepG2 by
MTT assay [22]. The IC50 value for HepG2 cells was 12.5 μg mL-
1.Treatment with 10, 12 and 14 μg mL-1 of ethanolic extracts caused
typical apoptotic morphological changes in HepG2 cells on staining
with acridine orange and ethidium bromide.
Lingadurai et al., (2011) studied the cytotoxic effect of the leaves of
Bichofia javanica extract on human leukemic cell lines (U937, K562,
and HL60). 10 μg/ml methanolic extract of Bichofia javanica showed
significant cytotoxicity [23].
Our result was congruent with result of others.
CONCLUSION
The present study strongly iterates the antimicrobial and anticancer
capacity of the bark of Cinnamomum zeylanicum and scientifically
validates it for use as a component in medicinal preparations,
especially against pathogenic attack and proliferative diseases.
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Ten bacterial species were isolated and monthly variations in their count were recorded from three edible tuna fishes. Phytochemical analysis and antibacterial activity of hexane, chloroform, methanol, and distilled water extracts of twelve common spices, against the isolated bacteria were evaluated. The study indicates that these pathogenic bacteria in all three tuna fish species cause various human health problems upon consumption. ZUSAMMENFASSUNG: Eine Studie zur phytochemischen Analyse und antibakteriellen Vorgängen bei Stämmen pathogener Bakterien isoliert aus kommerziell wichtigen, eßbaren Meeresfischen, Euthynnus affinis (Cantor), Katsuwonus pelamis L. und Auxis Thazard (Lacepede) (Family Scombridae). Zehn Bakterienarten wurden isoliert und monatliche Variationen ihrer Anzahl bei drei essbaren Thunfischen, aufgezeichnet. Phytochemische Analysen und antibakterielle Aktivität von Hexan-, Chloroform-, Methanol-und destillierten Wasserextrakten von zwölf gängigen Gewürzen, wurden mit ihrer Wirkung auf die isolierten Bakterien bewertet. Die Studie zeigt, dass diese in allen drei Thunfischen art vorkommenden pathogenen Bakterien beim Menschen nach Verzehr verschiedene gesundheitliche Probleme verursachen. REZUMAT: Un studiu privind analiza fitochimică și activitatea antibacteriană a condimentelor asupra bacteriilor patogene izolate din pești marini comestibili importanți din punct de vedere comercial, Euthynnus affinis (Cantor), Katsuwonus pelamis L. și Auxis thazard (Lacepede) (Familia Scombridae). Zece specii bacteriene au fost izolate și s-au înregistrat lunar variațiile numărului lor de la trei pești de ton comestibili. Au fost evaluate analizele fitochimice și activitatea antibacteriană a extractelor cu hexan, cloroform, metanol și apă distilată din douăsprezece condimente comune, împotriva bacteriilor izolate. Studiul indică faptul că aceste bacterii patogene din cele trei specii de ton provoacă diverse probleme de sănătate umană în urma consumului. S. Patrata and J. S. R. Aluri-Phyto-chemical study and antibacterial activity of spices on fish bacteria (37 ~ 72) 38
... The demonstrated in vitro anticancer-related activity of Ceylon cinnamon was mainly on cytotoxicity against different carcinoma cell lines (Herdwiani et al. 2016;Abeysekera et al. 2016b). From the different parts of this plant, particularly the bark, leaf and bark essential oils have been tested on breast carcinoma (MCF7, T47D), lung carcinoma (A-549), prostate carcinoma (PC-3), brain carcinoma (glioblastoma, T98G), hepatocarcinoma (HePG2), human cervix carcinoma (SiHa), human skin carcinoma (A431), human neuroblastoma (SK-N-MC), oral carcinoma (KB cells) and lymphoid leukaemia (L1210 cells) cell lines (Abeysekera et al. 2016b;Herdwiani et al. 2016;Varalakshmi et al. 2014;Sudan et al. 2013;Priyarani et al. 2010;Zu et al. 2010). ...
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Cinnamon Botany, Agronomy, Chemistry and Industrial Applications
... Minimal inhibitory concentration of the cinnamon oil was estimated to be 20 μL/mL and it was under the results obtained in the study conducted by Chaudhari et al. 5 and Zainal-Abidin et al. 41 The strength of probiotic was determined taking into consideration the literature available on the safe and effective strength of probiotic strains in children from a dental point of view which ranges from 10 5 to 10 9 CFU/g with 10 9 CFU/g being most effective. 13,[41][42][43][44][45] As per the safety guidelines for probiotics use in children, a healthy child can safely take up to 10 billion per day. 46,47 In the current study, a probiotic blend of 10 billion was used composed of 5 billion CFU/gm L. rhamnosus (TSP-Lrh1) and 5 billion CFU/gm of L. plantarum (TSP-Lp1). ...
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Introduction: Among the various plants studied, cinnamon has emerged as a potential herbal antimicrobial agent. Besides the medicinal plants, recently probiotics have also been recognized to affect cinnamon bark oil Streptococcus mutans (S. mutans) and other harmful oral and gut microflora. Aim and objective: This placebo-controlled study aims to compare the antimicrobial potential of cinnamon bark oil incorporated and probiotic blend [Lactobacillus plantarum (TSP-Lp1), and Lactobacillus rhamnosus (TSP-Lrh1)] incorporated mucoadhesive patch against salivary S. mutans in caries active 7-10-year-old children. Design: It was a double-blinded placebo-controlled study with n = 60. They were randomly allotted into three groups-Group I: Cinnamon patch, group II: probiotic patch, and group III: control patch (placebo) with n = 20 in each group. Materials and methods: The study was carried out in three phases. In the first phase, the minimal inhibitory concentration (MIC) of cinnamon bark oil was determined against S. mutans followed by the formulation of cinnamon and probiotic patches. After a washout period of 2 weeks and a collection of baseline saliva samples, these patches were tested on the subjects from respective groups for 14 days with twice a day placement protocol. On the 15th day, saliva samples were collected and cultured, CFU/mL of the saliva of S. mutans for each subject was recorded and compared with baseline samples. Feedback in the form of a questionnaire was obtained from the patients. Statistical analysis: Descriptive statistics, paired t-test for intragroup comparison, unpaired t-test for intergroup comparison, analysis of variance (ANOVA) for intergroup comparison, and post hoc Scheffe's. Results: The results showed that both cinnamon patch and probiotic patch were comparable to each other in terms of their anti-S. mutans activity. The intragroup comparison of the CFU/mL count showed a highly significant reduction from baseline to post-intervention for both the groups (p = 0.001). Conclusion: Both cinnamon and probiotic blend have a strong antimicrobial property owing to their ability to cause significant reduction in salivary S. mutans and both the patches showed good patient acceptance. How to cite this article: Gandhi HA, Srilatha KT, Deshmukh S, et al. Comparison of Antimicrobial Efficacy of Cinnamon Bark Oil Incorporated and Probiotic Blend Incorporated Mucoadhesive Patch against Salivary Streptococcus mutans in Caries Active 7-10-year-old Children: An In Vivo Study. Int J Clin Pediatr Dent 2020;13(5):543-550.
... The demonstrated in vitro anticancer-related activity of Ceylon cinnamon was mainly on cytotoxicity against different carcinoma cell lines (Herdwiani et al. 2016;Abeysekera et al. 2016b). From the different parts of this plant, particularly the bark, leaf and bark essential oils have been tested on breast carcinoma (MCF7, T47D), lung carcinoma (A-549), prostate carcinoma (PC-3), brain carcinoma (glioblastoma, T98G), hepatocarcinoma (HePG2), human cervix carcinoma (SiHa), human skin carcinoma (A431), human neuroblastoma (SK-N-MC), oral carcinoma (KB cells) and lymphoid leukaemia (L1210 cells) cell lines (Abeysekera et al. 2016b;Herdwiani et al. 2016;Varalakshmi et al. 2014;Sudan et al. 2013;Priyarani et al. 2010;Zu et al. 2010). ...
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Sri Lanka is the market leader of Ceylon cinnamon with a 90% global market share. Cinnamon is one of the leading foreign exchange earners from among agricultural exports of Sri Lanka. The cinnamon extent of cultivation, production, and yield has only marginally increased over the past few decades despite high potential in the global market. However, the major competitive product cassia produced and exported predominantly by Indonesia, China, and Vietnam has contributed to the erosion of Ceylon cinnamon market share, notwithstanding warning from leading health agencies about its negative impact on health due to high content of coumarin. Ceylon cinnamon has the potential to become Sri Lanka’s number one foreign exchange earner from the agriculture sector by building on its competitive edge as the main true cinnamon supplier. Product and process innovation with a focus on compliance with food safety and quality requirements remains the most feasible and practical option to exploit the competitive edge of Ceylon cinnamon in the global marketplace. Marketing strategies should focus on product diversification, value addition, and brand recognition.
... In this regard, Cinnamomum verum J. Presl, commonly known as Ceylon cinnamon an evergreen tree with aromatic barks and leaves are known to contain phytochemicals such as cinnamaldehyde, cinnamic acid, and eugenol that could exert anticancer effects. [6][7][8] This herb is widely cultivated and is easily available throughout the world. It is not costly and can be easily procured. ...
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Aim and objective: The present study was conducted to assess the in vitro anticancer effects of Cinnamomum verum J. Presl extract and its active constituents, such as cinnamaldehyde, 4 hydroxycinnamic acid, and eugenol on oral squamous cell carcinoma cell line. Materials and methods: Aqueous, ethanolic, and hydroalcoholic extracts of C. verum J. Presl (bark) were prepared using standardized protocols. Cinnamaldehyde, 4 hydroxycinnamic acid, and eugenol were quantified in the extracts. Total saponins, tannins, and polyphenols were quantified in the selected extracts. A commercially available SCC-25 cell line was cultured according to standard protocol. The anticancer effects of the extract, active compounds, and standard cisplatin were assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity, acridine orange/ethidium bromide staining, DNA, fragmentation assay, cell cycle analysis by flow cytometry, and JC-1 staining (5,5',6,6'-tetrachloro1,1',3,3'tetraethylbenzimidazolylcarbocyanine iodide). Results: The hydroalcoholic extracts demonstrated a higher quantity of the active ingredients cinnamaldehyde, 4 hydroxycinnamic acid, and eugenol. The selected extract and active compounds demonstrated anticancer effects via apoptosis induction and S-phase arrest. Apoptosis induction was exerted by the extract via alteration in mitochondrial membrane potential. Conclusion: Cinnamomum verum J. Presl and its active compounds exhibited in vitro anticancer effects on oral squamous cell carcinoma. Further studies in animal models have to be carried out to assess toxicity and in vivo effects. Clinical significance: The anticancer properties of Cinnamomum verum J. Presl could be explored further for prevention and management of oral squamous cell carcinoma.
... Curcuma longa (Turmeric) is quite well-known as an anti-cancer herb [23][24][25]. Cinnamomum zeylenicum (Cinnamon bark) is able to induce apoptosis in cancer cell-lines [26,27]. Bacopa monnieri (Water hyssop) is possessing some cytotoxic effects [28,29]. ...
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UNANI MEDICINE AND CANCER Christer Sundqvist. Prepublished article, 2020 Petrafoundation, Helsinki, Finland https://www.petrafoundation.com/en/foundation Unani medicine is an alternative medical system originating in ancient Greece almost 2500 years back. It is now practiced primarily in India. Herbal remedies, dietary practices and alternative therapies characterize Unani medicine. Let us study what it can offer for a cancer patient.
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Cell biology, plant-based extracts, structural chemistry, and laboratory in vitro or in vivo experiments are the principal aspects or interfaces that can contribute to discovering new possibilities in cancer therapy and to developing improved chemotherapeutics. Forestry residues can be used for their wealthy resource in polyphenols and other phytoconstituents known for anticancer properties. This review is designed to bring together information on the in vitro or in vivo anticancer potential of woody vascular plants especially the bark extracts (BE) and biosynthesized metallic nanoparticles (BMN) using bark extracts. Type of extracts, main phytoconstituents found in extracts responsible for the anticancer activity, and targeted cancerous cell lines were followed. The literature data were collected via Clarivate Analytics, Science Direct, PubMed, and Google Academic (2011–2021). The search terms were: bark extracts, metallic nanoparticles, silver nanoparticles, gold nanoparticles, anticancer, cytotoxic activity, antiproliferative effect, and antimetastatic potential in vitro and in vivo. All of the search terms listed above were used in different combinations. The literature data highlight the efficaciousness of the BE and BMN as anticancer agents in in vitro experiments and showed the mechanism of action and their advantage of nontoxicity on normal cells. In vitro testing has shown promising results of the BE and BMN effect on different cancer cell lines. In vivo testing is lacking and more data is necessary for drug development on animal models.
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Cinnamomum zeylanicum Blume or Ceylon cinnamon is a native medicinal and aromatic plant in Sri Lanka, which has been used extensively in treatment of various diseases in traditional systems of medicine in Asia. It is also a household remedy for primary care in aches, pains and certain other diseases. The claimed health benefits have been extensively tested using modern scientific methods to validate the health properties. It has been used clinically for treatment and management of diseases and in health foods, nutraceutical and cosmeceutical industries. The pharmacological properties tested and reported from C. zeylanicum bark, leaf and essential oils are discussed with special reference to antioxidant, anti-diabetic, antilipidemic, anticancer, anti-inflammatory, antiarthritic, immunomodulatory, antibiotic, antinociceptive, analgesic, cardioprotective, antihypertensive, gastroprotective, skin protection, skin whitening and anti-ageing properties, including clinical studies and toxicology.
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Antioxidant activity, anti-aging effects and cytotoxicity activity of cinnamon essential oils from Cinnamomum zeylanicum were investigated in this study. The antioxidant activities of the cinnamon essential oil at the concentrations of 125, 250, 500, and 1000 µg/mL were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2ʹ-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS). The inhibitory activities against collagenase, elastase and tyrosinase were evaluated for anti-aging effects. The antioxidant activity determined by DPPH and ABTS assays varied from 4.91 - 28.74% and 4.96 - 50.17%, respectively. In addition, cinnamon essential oil at all concentrations tested (100, 200, 500, and 1000 µg/mL) inhibited tyrosinase activity by 61.68 - 93.12 %, collagenase activity by 2.83 - 30.28 % and elastase activity by 4.37 - 33.92 %. The cytotoxicity activity determined by the diphenyltetrazolium (MTT) assay revealed that the cinnamon essential oil at the concentration less than 100 µg/mL did not exhibit cytotoxicity activity on human fibroblast cells while the percentage of cell viability decreased when exposed to this oil at the concentration higher than 150 µg/mL. These results demonstrated that the cinnamon essential oil has antioxidant, tyrosinase inhibitory, collagenase inhibitory, and elastase inhibitory activities. In addition, cinnamon essential oil at each effective concentration did not show any toxicity when tested on normal human fibroblast cell. Therefore, this essential oil could be a potential candidate for cosmetic and pharmaceutical products.
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Piper sarmentosum, locally known as kaduk is belonging to the family of Piperaceae. It is our interest to evaluate their effect on human hepatoma cell line (HepG2) for the potential of anticarcinogenic activity. The anticarcinogenic activity of an ethanolic extract from Piper sarmentosum in HepG2 and non-malignant Chang's liver cell lines has been previously determined using (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) (MTT) assays, where the IC50 value was used as a parameter for cytotoxicity. The ethanolic extract that showed anticarcinogenic properties in HepG2 cells had an IC50 of 12.5 mug mL-1, while IC50 values in the non-malignant Chang's liver cell line were greater than 30 mug mL-1. Apoptotic morphological changes in HepG2 cells were observed using an inverted microscope and showed chromatin condensation, cell shrinkage and apoptotic bodies following May-Grunwald-Giemsa's staining. The percentage of apoptotic cells in the overall population (apoptotic index) showed a continuously significant increase (p < 0.05) in 12.5 mug mL-1 ethanolic extract-treated cells at 24, 48 and 72 hours compared to controls (untreated cells). Following acridine orange and ethidium bromide staining, treatment with 10, 12 and 14 mug mL-1 of ethanolic extracts caused typical apoptotic morphological changes in HepG2 cells. Molecular analysis of DNA fragmentation was used to examine intrinsic apoptosis induced by the ethanolic extracts. These results showed a typical intrinsic apoptotic characterisation, which included fragmentation of nuclear DNA in ethanolic extract-treated HepG2 cells. However, the non-malignant Chang's liver cell line produced no DNA fragmentation. In addition, the DNA genome was similarly intact for both the untreated non-malignant Chang's liver and HepG2 cell lines. Therefore, our results suggest that the ethanolic extract from P. sarmentosum induced anticarcinogenic activity through an intrinsic apoptosis pathway in HepG2 cells in vitro.
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Streptococcus mutans is considered one of the most important cariogenic species of the human oral microbial flora. Total 150 samples were collected from Himachal institute of Dental sciences hospital Paonta sahib to check the prevalence of S. mutans in dental plaque. Total recovered isolates were 126 out of which 90 Streptococcus mutans were isolated. So the prevalence of recovered isolates was 60%. In the present investigation, the evaluation of current efficacy of 10 commercially available antibacterial drugs in India was carried out against S. mutans by Kirby-Bauer disc diffusion method. Of the 10 antibacterial drugs evaluated against the recovered isolate amoxicillin, ciprofloxacin and penicillin G were highly effective in terms of maximum diameter of growth inhibition zones followed by chloramphenicol. Five drugs namely, ofloxacin, tetracycline, erythromycin, chloramphenicol and gentamycin were found to be moderately effective against the three strains of S. mutans. Metronidazole and rifampicin were not effective against the bacteria as they did not show any inhibitory activity. Extracts of Eugenia jambolana showed antibacterial activities against Streptococcus mutans and showed the maximum zone of inhibition.
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In vitro antimicrobial efficacy of root extracts of Murraya koenigii (Linn.) Spreng. was assessed by disc diffusion method against four bacterial strains (Bacillus subtilis, Escherichia coli, Staphylococcus aureus and Salmonella typhi) and three fungal strains (Aspergillus niger, Candida albicans and Trichophyton rubrum). Minimum inhibitory concentration was also determined. The most susceptible bacterial and fungal strains were S. aureus and T. rubrum, respectively. The root extracts in organic solvents (hexane, methanol and chloroform) showed good antimicrobial activity. However, aqueous extracts could not exhibit any activity. Results of the present investigation indicate that root of M. koenigii possess antimicrobial properties and hence can be exploited for future natural plant based antimicrobial agents.
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Here, we report the antioxidant and antimicrobial activities of sorghum (Sorghum bicolor Moench) extracts prepared from 25 cultivars from South Korea. Four cultivars of sorghum were extracted with methanol, then further fractioned with n-hexane, ethyl acetate, n-butanol, and water. The RC50 (the concentration of antioxidant required to achieve absorbance equal to 50% that of a control containing no antioxidants) value of the DPPH method and reducing power showed higher efficiency in the BuOH layer of all selected cultivars except Neulsusu. The various fractions were then examined for antimicrobial activity by a serial two fold dilution assays using the paper disc method. The methanol extracts showed higher levels of antimicrobial activity than the other fractions. Our results indicate that sorghum extracts could be used as a source of antioxidant and antimicrobial ingredients in the food industry.
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Chinese medicinal plant extracts were screened against some fungal strains, such as Aspergillus niger, Botrytis cinerea, Fusarium moniliforme, Glomerella cingulata, and Phyllosticta caricae. Plants were extracted with hot water, 80% methanol or acetone. Aliquots of the extracts at variable concentrations were then incubated with different fungal strains, and the minimum inhibitory concentrations (MICs) of each plant extract determined. In this study, the methanol extracts of Cinnamomum cassia had MIC values of 13.3 mg ml−1, when tested against F. moniliforme and P. caricae. The acetone extracts of C. cassia had MIC values of 8.3 mg ml−1 and 10 mg ml−1 respectively, when tested against B. cinerea and G. cingulata. The hot water extracts of C. cassia inhibited significantly the growth of A. niger, B. cinerea, F. moniliforme, and P. caricae with MIC values at 10, 11.7, 5, and 6.7 mg ml−1 respectively. The acetone extracts of Curcuma longa inhibited effectively P. caricae with the MIC value at 6.7 mg ml−1. To determine the stability, various plant extracts were stored at 4 and 25 °C over a period of one month and their effects on fungal growth examined. Results show that the acetone extracts of Cu. longa and Coptidis rhizoma maintained their activity against fungal strains when stored at 4 °C, but not at 25 °C. The methanol extracts of C. cassia lost a great portion of inhibitory activities but not all, after stored at 4 °C and 25 °C for one month. The effect of various combinations of these extracts on antimicrobial activity has also been examined. The combinations of herb extracts showed higher inhibitory effect towards tested fungi than that of individual extract. Results from these findings suggest that these herbal extracts may be used as natural antifungal agents to inhibit growth of foodborne pathogen.
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Leaves of Bichofia javanica (BJ) have been traditionally used for many ailments including cancer. In the present study, antileukemic activity of the leaf extract was evaluated on human leukemic cell lines. Human leukemic cell lines U937, K562, and HL60 were purchased from National Facility for Animal Tissue and Cell Culture, Pune, India. The cells were routinely maintained in RPMI 1640 medium supplemented with 10% heat inactivated fetal calf serum. Cultures were maintained at 37ºC in a humidified atmosphere containing 5% CO(2) in air. The methanol extract of BJ (MEBJ) was dissolved in PBS and used at the concentrations of 5, 10, and 15 μg/ml for cell viability and cytotoxicity studies (MTT assay). Cell counts were made in quadruplicate samples at the interval of 24, 48, and 72 h and cytarabine (20 μg/ml) served as standard drug. The apoptotic pathway of cytotoxicity was assessed by DNA agarose gel electrophoresis technique and confirmed by fluorescence and confocal microscopic methods at the concentration of 10 μg/ml. MEBJ showed significant cytotoxicity (P<0.001) in leukemic cell lines in the in-vitro cell proliferation assay. IC(50) of MEBJ was very low (3.5 μg/ml) at 72 h in the HL60 cell line. The apoptotic pathway of cytotoxicity was observed at 10 μg/ml of MEBJ by the fragmented DNA pattern in the apoptosis assay, chromatin condensation, and apoptotic body formation as revealed in the fluorescence and confocal microscopic studies. The present findings support the ethno-medicinal use of BJ for cancer by mediating through the apoptosis pathway.
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Cinnamon (Cinnamomum verum) has been widely used in spices, flavoring agents, and preservatives. Cinnamon polyphenol extract (CPE) may be important in the alleviation of chronic diseases, but the molecular evidence is not substantial. Tristetraprolin (TTP) family proteins have anti-inflammatory effects through the destabilization of pro-inflammatory mRNAs. TTP expression is reduced in fats of obese people with metabolic syndrome and brains of suicide victims. This study used quantitative real-time PCR to explore the effects of CPE on the regulation of TTP, VEGF, and related gene expression in mouse 3T3-L1 adipocytes. CPE (100 μg/mL) increased TTP mRNA levels by up to 10-fold, and this stimulation was sustained over 16 h. The levels of VEGF mRNA, a putative target of TTP, were decreased 40-50% by CPE. It also affected the expression of other genes coding for ZFP36L1 and ZFP36L3 (TTP homologues), GM-CSF, COX2, IL6, APP, G-CSF, and PAI1. This study demonstrated that CPE rapidly induces TTP mRNA and reduces VEGF mRNA and affects the expression of a number of other genes in the cultured adipocytes.