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ANTIMICROBIAL AND ANTIPROLIFERATIVE, PRO-APOPTOTIC ACTIONS OF KOMBUCHA FERMENTED SOLUTIONS AGAINST COLON AND HEPTAO CANCER CELL LINES

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Kombucha fermented solutions (KFSs) were produced from tea (KTFS), rice (KRFS) and barely (KBFS) after 8 and 10 days. These solutions tested as antimicrobial activity against microorganisms (Listeria monocytogenes, Shigelladysenteriae, Salmonella enteritidis, Staphylococcus aureus, Citrobacter sp., Klebsiella pneumoniae, Esherichia coli and Candida albicans) by agar well diffusion assay. KTFS was higher inhibitory activity than KRFS or KBFS, which gave the largest clear halo-zone diameter against S. enteritidis. Minimum lethal concentration: minimum inhibition concentration ratio wascalculated to known the KFSs action. All tested KFSs were micobicidal effect against S. aureus and C. albicans (≤2) and microbiostatic effect against other pathogenic bacteria (≥4). Objective: The study extends to examine the growth inhibitory effects and Apoptotic abilities of KFSS, on human colon (HCT-116) and liver (HEpG-2) cancer cells. Material and method: using Neutral Red Uptake Assay and the AO/EB dual Staining assays to detect anti proliferative and apoptosis properties. Results: indicated that KTFS had more antitumor activity against different tumor cells than KRFS and KBFS. Although all tested KFSs were found to reduce the cell viability in a concentration manner, but the magnitude of reduction was high in case of KTFS with lower IC50 values, and induces a higher percentage of apoptotic cells after 24 h of exposure of HEpG-2 cell line. These results suggest that the KFSs possess interesting antiproliferative properties and induce apoptosis on the HCT-116 and HEpG-2 cancer cells associated with significant antimicrobial activity. These findings provide additional support for the traditional use of KFSs in the treatment of metabolic diseases and various types of cancer.
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Soheir et al. World Journal of Pharmaceutical and Life Sciences
ANTIMICROBIAL AND ANTIPROLIFERATIVE, PRO-APOPTOTIC ACTIONS OF
KOMBUCHA FERMENTED SOLUTIONS AGAINST COLON AND HEPTAO CANCER
CELL LINES
Khadiga A. Abou-Taleb1, Naglaa M. Ebeed2, Dr. Soheir S. Abd el-salam*3 and Shimaa A. Amin1
1Department of Agric. Microbiology, Fac. of Agric., Ain Shams Univ., Cairo, Egypt.
2Department of Genetics, Faculty of Agric., Ain Shams University, Cairo, Egypt.
3Department of Botany, Faculty of Science, Banha University, Qalubiya, Egypt.
Article Received on 13/08/2017 Article Revised on 04/09/2017 Article Accepted on 25/09/2017
INTRODUCTION
       
fermented different types of acetic acid bacteria and
yeasts in symbiotic association with two portions, a
floating cellulose pellicle layer and the sour liquid broth.
Kombucha tea is a popular beverage across the world for
its refreshing taste and beneficial effects on human
health. The beneficial effects of Kombucha tea are
attributed to the presence of tea polyphenols, gluconic
acid, glucuronic acid, lactic acid, vitamins, amino acids,
antibiotics, and a variety of micronutrients produced
during fermentation (Jayabalan et al, 2007). Kombucha
tea is known to show a remarkable antimicrobial activity
against a broad range of microorganisms. Many
scientific studies have been done on this subject and the
Kombucha broth has demonstrated inhibitory activity
against many pathogenic microorganisms of both Gram
positive and Gram negative origin (Dufresne and
Farnworth, 2000). Kombucha tea has demonstrated the
ability to inhibit the growth of pathogens such as
Helicobacter pylori (the causative organism of peptic
ulcers), Escherichia coli (the causative organism of
common diarrhea), Entamoeba cloacae, Pseudomonas
aeruginosa, Staphylococcus aureus, Staphylococcus
epidermis, Agrobacterium tumefaciens, Bacillus cereus,
Aeromonashydrophila, Salmonella typhimurium,
Salmonella enteritidis, Shigellasonnei,
Leuconostocmonocytogenes, Yersinia enterocolitica,
Campylobacter jejuni, and Candida albicans (Sreeramulu
et al, 2001; Sreeramulu et al, 2000; Dufresne and
Farnworth, 2000). Some studies have demonstrated that
Kombucha tea shows not only antibacterial activity but
Research Article
ISSN 2454-2229
wjpls, 2017, Vol. 3, Issue 8, 120-132
World Journal of Pharmaceutical and Life Sciences
WJPLS
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SJIF Impact Factor: 4.223
*Corresponding Author: Dr. Soheir S. Abd el-salam
Department of Botany, Faculty of Science, Banha University, Qalubiya, Egypt.
ABSTRACT
Introduction: Kombucha fermented solutions (KFSs) were produced from tea (KTFS), rice (KRFS) and barely
(KBFS) after 8 and 10 days. These solutions tested as antimicrobial activity against microorganisms (Listeria
monocytogenes, Shigelladysenteriae, Salmonella enteritidis, Staphylococcus aureus, Citrobacter sp., Klebsiella
pneumoniae, Esherichia coli and Candida albicans) by agar well diffusion assay. KTFS was higher inhibitory
activity than KRFS or KBFS, which gave the largest clear halo-zone diameter against S. enteritidis. Minimum
lethal concentration: minimum inhibition concentration ratio wascalculated to known the KFSs action. All tested
KFSs were micobicidal effect against S. aureus and C. albicans ( and microbiostatic effect against other
pathogenic bacteria (). Objective: The study extends to examine the growth inhibitory effects and Apoptotic
abilities of KFSS, on human colon (HCT-116) and liver (HEpG-2) cancer cells. Material and method: using
Neutral Red Uptake Assay and the AO/EB dual Staining assays to detect anti proliferative and apoptosis
properties. Results: indicated that KTFS had more antitumor activity against different tumor cells than KRFS and
KBFS. Although all tested KFSs were found to reduce the cell viability in a concentration manner, but the
magnitude of reduction was high in case of KTFS with lower IC50 values, and induces a higher percentage of
apoptotic cells after 24 h of exposure of HEpG-2 cell line. These results suggest that the KFSs possess interesting
antiproliferative properties and induce apoptosis on the HCT-116 and HEpG-2 cancer cells associated with
significant antimicrobial activity. These findings provide additional support for the traditional use of KFSs in the
treatment of metabolic diseases and various types of cancer.
KEYWORDS: Kombucha fermented solutions; Minimum inhibitory concentration; Pathogenic microorganisms;
Cytotoxicity, antiproliferative, apoptosis.
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Soheir et al. World Journal of Pharmaceutical and Life Sciences
also antifungal activity (Battikh et al, 2013). There are
several testimonials available in the internet for the
anticancer properties of Kombucha tea from Kombucha
drinkers throughout the world, but there are no scientific
reports to validate the facts. The chemoprevention of
human cancers has long been the subject of research. The
chemopreventive effects of tea polyphenols have been
demonstrated in animal models of cancers of lung, skin,
esophagus, colon, and mammary gland, but the
anticancer property of Kombucha tea was not well
characterized. Carcinogenesis is a multistage process, in
which different genes are prime targets for
chemopreventive agents because they regulate
intracellular, cell-surface or extracellular functions.
Acridine orange/ethidium bromide (AO/EB) staining is
used to visualize nuclear changes and apoptotic body
formation that are characteristic of apoptosis. Cells are
viewed under a fluorescence microscope and counted to
quantify apoptosis (Ribble et al, 2005). The ethidium
bromide/acridine orange stain (EB/AO stain) is a
viability stain that detects apoptotic cells. Viability stains
determine the membrane integrity of a cell based on the
uptake or exclusion of a dye from the cell (Park et al,
2000). Ethidium bromide is a dye that is o  
         
   - 

-
     l, 2006). The possible cancer
preventive activity of Kombucha tea has received much
attention in recent years. So, the objective of this study
was to evaluate the antiproliferative activity of
Kombucha fermented solutions from tea (KTFS), rice
(KRFS) and barely (KBFS) against two human cancer
cell lines: human colon carcinoma cells (HCT-116) and
human liver carcinoma (HEpG-2) by using Neutral Red
Uptake Assay (NRU colorimetric Assay) and its
antimicrobial activity against Gram-negative and Gram-
positive pathogenic microorganisms.
MATERIALS AND METHODS
Preparation of Kombucha fermented solutions (KFSs)
After boiling 1L of tap water for 15min, 4g of substrates
(black tea, rice or barely) was added and infused for 15
min then removed by filtration. 70g/L of commercial
sucrose was dissolved into the filtered infusion before it
has cooled. After cooling to room temperature, pour the
solution into a glass jar has been previously sterilized at
121°C for 20min. these solutions were inoculated with
10% of the fermentation broth from the previous
fermentation of black tea obtained under the same
conditions. The glass jars were covered with a clean
piece of cloth and fixed with rubber bands. The
fermentation was carried out under room temperature
(25°C) for 10 days. The harvesting was done after
2, 4, 6, 8 and 10 days of fermentation. Kombucha
tea, rice or barely was centrifuged at 10.000 rpm for 15
min and the supernatant was used as Kombucha
fermented solutions (KFSs).
Pathogenic strains
Seventeen pathogenic bacteria and one yeast strains used
in this investigation were Listeria monocytogenes1,2& 3,
Shigelladysenteriae1&2 and Salmonella enteritidis which
collected from Department of Agric. Microbiology, Fac.
of Agric., Ain Shams University and Staphylococcus
aureus1&2, Citrobacter sp.1&2, Klebsiella
pneumoniae1,2,3&4, Esherichia coli1,2&3and Candida
albicans were obtained from Egyptian hospital. These
strains were maintained on nutrient agar(Difco Manual,
1984) or Sabouraud dextroseagar (BAM, 1998) slants at
4°C for bacteria and yeast, respectively.
Inoculum preparation
From 4 to 5 colonies of a pure tested microbial culture
were collected from agar plate after 24h of incubation
period and subculture into tube containing 4 ml of
Müller-Hinton broththen incubated at 37°C until it
achieves the turbidity of 0.5 MacFarland standard after
24 h incubation according to NCCLS (1998). The
inoculum was standardized by measurement the optical
density using spectrophotometer at 625nm which ranged
from 0.08-0.12. Standardized inoculum has a
concentration of 1-2×108cfu/ml for bacteria and 1-
5×108cfu/ml for yeast and diluted to1:10 in sterile saline
solution to obtain the coveted concentration of
106cfu/ml.
Antimicrobial Activity
The antimicrobial activity of KFSs was tested by agar
well diffusion assay (Mo et al, 2005) against pathogenic
strains. Müller-Hinton agar (MHA)medium was poured
into Petri dishes. Suspensions (100µl) of target strain
previously incubated for 24 h were spread on the plates,
and wells of 7 mm in diameter were made with a sterile
cork borer (ShahidiBonjar, 2004; Atata et al, 2003).
KFSs samples obtained after 2, 4, 6, 8, and 10 days from
fermentation periods were centrifuged at 10,000 rpm for
10 min to remove cell debris and supernatant samples
(100µL) were then transferred into the wells in the agar
plates previously inoculated with the target strain. The
plates were allowed to stand until KFSs samples were
completely absorbed and after then incubated at 37°C for
overnight (Sreeramulu et al, 2001). The growth
inhibition was observed by the naked eye and the
inhibition zone diameter (IZD) was measured using a
ruler after 24 h of incubation. The antimicrobial activity
was evaluated by measuring the growth inhibition zone
surrounding the wells. Acetic acid sample was used as
control at the same concentration as that of fermented
solutions after 8 and 10 days were prepared and sterilized
by filtration and then used for antimicrobial testing, as
described previously. Antibiotic was used as standard.
Each experiment was carried out in triplicate and the
average diameter±standard deviation of the IZD was
recorded.
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Soheir et al. World Journal of Pharmaceutical and Life Sciences
Determination of minimum inhibitory concentration
(MIC) by agar plate dilution method
The agar dilution method was carried out by Clinical and
Laboratory Standards Institute (CLSI). Serial dilutions
(1/2, 1/5, 1/10, 1/25, 1/50, 1/100 and 1/125) of the tested
KFSs were added to the melted MHAmedium at 50°C
and mixed fully to get the desirable final concentrations
of6.800 (control), 3.400, 1.360, 0.680, 0.272, 0.136,
0.068 and 0.054mg/ml for Kombucha tea fermented
solution (KTFS), 5.14, 2.57, 1.03, 0.514, 0.2056, 0.1028,
0.0514 and 0.04112mg/ml for Kombucha rice fermented
solution (KRFS) and 4.60 (control), 2.30, 0.92, 0.46,
0.184, 0.092, 0.046 and 0.0368mg/ml for Kombucha
barely fermented solution (KBFS), respectively (Figure
1). The media containing KFSs were poured immediately
on plates,then leave till solidify. Suspensions (100 µl) of
target strain previously incubated for 24h were spread on
the plates with glass rods. The plates were allowed to
stand until inoculum suspensions were completely
absorbed (pre-diffusion) and after then incubated at 37°C
for 24h. The lowest concentration (highest dilution) of
tested agent preventing appearance of growth was
examine by the naked eye and is described as MIC.
Plates of MHA + inoculation were a positive control and
MHA + KFS were negative control (Mazzola et al,
2003).
Time-kill test
After selected MIC of Kombucha fermented solutions,
  pathogenic microorganisms at
different periods ranged from 1.5-24h.
Determination of the minimum lethal concentration
(MLC)
Minimum bactericidal or fungicidal concentration (MBC
or MFC) was recorded as a lowest KFSs concentration
killing 99.9% of the bacterial or fungal inoculate after
24h incubation at 37°C. The MBCs or MFCs were
determined by selecting plates or dilutions that exhibited
no growth during MIC determination; a portion of agar
from each plate was sub-cultured on nutrient and
Sabouraud dextrose agar media for bacterial or yeast
strains, respectivelythen incubated at 37°C for 24h. The
MLC was determined and 
(Rabe et al, 2002).
Evaluation of KFSsaction
The action ofKFSsas antimicrobial agent on the bacterial
and fungal strains can be described with ratio of MLC:
MIC. The action was microbicidaland microbiostatic
effects when the ratio =1 or 24, respectively(Berche
et al, 1988).
Cytotoxicity and Antiproliferative of KFSs
Cytotoxicity of KFSs were tested against human
carcinoma HCT-116 (colon cancer) and HEpG-2 (liver
cancer) cell lines.
Cell Culture
Human carcinoma cell lines were obtained from
American Type Culture Collection (ATCC; Manassas,
VA, USA). Cells were grown in complete culture
medium (Dulbecco's Modified Eagles Medium (DMEM,
SIGMA, USA) with 10% FBS in 5% CO2 at 37°C under
high humidity. The trypan blue dye exclusion test was
used to assess cell viability (Siddiqui et al, 2008). Only
cells that showed a viability of more than 98% were used
in this study.
Neutral red uptake (NRU) assay
An NRU assay was performed according to Siddiqui et
al(2010).Post treatment with KFSs, the cells were
washed with phosphate-buffered saline (PBS;0.01 M; pH
7.4). Cells were then incubated for 3h in medium
suppleme The cells were
then subjected to 3 h of incubation. The supernatant was
removed, and the cells were washed with a solution of
0.5% CH2O and 1% CaCl2. Subsequently, a solution of
1% CH3COOH and 50% EtOH was added, and the dye
was extracted. The plates were then read at a wavelength
of 550nm using UVvisible spectrophotometer
multiplate reader (Synergy HT, Bio-Tek, USA).
Ethidium Bromide/Acridine Orange Staining (EB/AO
staining)
Induction of apoptosis by the KFSs were investigated
with AO/EB staining(Ribble et al, 2005). The cells
(2×105) were treated with concentratio  
KFSs and cultured overnight in a humidified CO2
incubator at 37°C. The rest of the adherent cells were
detached with Trypsin-EDTA (1ml) for 2min. The media
and the detached cells from the same sample were pooled
together and centrifuged at 1,000 rpm for 5 min. Cell
pell 
EB/AO dye mix (EB/AO dye mix contained 100
each dye). Stained cell suspensions (10 
visualized using an Olympus (1X70-S1F2) inverted
fluorescence microscope at 100X magnification and
photographed using Nikon D700 camera.
Statistical analysis
Data were expressed as means, standard deviations,
 -test and using IBM® SPSS® Statistics
software (2011). The significance of difference was
considered to incl
RESULTS AND DISCUSSION
Influence of KFSs on pathogenic microorganism
Three types of Kombucha fermented solutions produced
from tea (KTFS), rice (KRFS) and barely (KBFS) were
tested as antimicrobial activity against genus
Staphylococcus, Listeria and Candida (as Gr+veand
Salmonella, Citrobacter, Klebsiella, Escherichia,
andShigella (as Gr-ve).
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Soheir et al. World Journal of Pharmaceutical and Life Sciences
Table 1: Inhibition of pathogenic bacteria and yeast growth by Kombucha fermented solutions (KFSs) produced
from tea, rice and barley during different fermentation times.
Pathogenic bacteria
and yeast
KTFS
KBFS
KRFS
(days)
2
4
6
8
10
2
4
6
8
10
2
4
6
8
10
Staph. aureus 1
++
+
±
-
-
++
++
+
-
-
++
++
+
-
-
Staph. aureus 2
++
+
±
-
-
++
++
+
-
-
++
++
+
-
-
S. enteritidis
++
+
+
-
-
++
+
+
-
-
++
+
±
-
-
Citrobactersp.1
+
+
+
-
-
++
+
+
-
-
++
+
±
-
-
Citrobactersp. 2
+
+
+
-
-
++
+
+
-
-
++
+
±
-
-
K. pneumoniae 1
++
+
+
-
-
++
+
±
-
-
++
+
+
-
-
K. pneumoniae 2
++
+
+
-
-
++
+
+
-
-
++
+
+
-
-
K. pneumoniae 3
++
+
+
-
-
++
+
+
-
-
++
+
+
-
-
K. pneumoniae 4
+
+
+
-
-
++
+
+
-
-
++
++
+
-
-
L.monocytogenes1
+
+
±
-
-
++
++
+
-
-
++
++
+
-
-
L. monocytogenes2
+
+
±
-
-
++
++
+
-
-
++
++
+
-
-
L. monocytogenes3
+
+
±
-
-
++
++
+
-
-
++
++
±
-
-
E. coli 1
+
±
±
-
-
++
+
+
-
-
++
++
±
-
-
E. coli 2
+
±
±
-
-
++
+
+
-
-
++
++
+
-
-
E. coli 3
+
±
±
-
-
++
+
+
-
-
++
++
+
-
-
S. dysenteriae1
+
±
±
-
-
++
+
±
-
-
++
+
+
-
-
S. dysenteriae2
+
+
+
-
-
++
++
+
-
-
++
++
+
-
-
C. albicans
+
+
+
-
-
++
++
+
-
-
++
++
+
-
-
-= No growth, ± = Very Scanty growth, + = Scanty growth, ++ = Moderate growth, KTFS= Kombusha tea fermented
solution, KRFS = Kombusha rice fermented solution, KBFS= Kombusha barley fermented solution.Staph=
Staphylococcus, S. enteritidis= Salmonella enteritidis, K. =Klebsiella, L.= Listeria, S. dysenteriae= Shigelladysenteriae
& C.= Candida.
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Soheir et al. World Journal of Pharmaceutical and Life Sciences
Table 2: Inhibition zone diameter (IZD) of pathogenic bacteria and yeast influence with kombusha fermented solutions after 8-10 days of fermentation period and
compared with acetic acid concentrations and biotin as antibiotic.
Pathogenic bacterial
and yeast
Antibiotic
(mg/ml)
AA conc. (mg/ml) as presented in
Treatments
KTFS
KBFS
KRFS
KTFS
KBFS
KRFS
6.70
5.12
4.34
8
10
8
10
8
10
Staph. aureus 1
29cd±0.08
29cd±0.04
28cde±0.04
20h±0.11
32c±0.13
30cd±0.25
26e±0.11
25ef±0.14
25ef±0.03
23fgh±0.08
Staph. aureus 2
28cde±0.15
29cd±0.13
24f±0.10
21gh±0.09
30d±0.20
28cde±0.35
20h±0.02
21gh±0.17
25ef±0.11
25ef±0.08
S. enteritidis
30cd±0.14
33c±0.04
22fgh±0.13
24f±0.21
38a±0.10
37a±0.11
18i±0.10
19hi±0.09
23fgh±0.10
22fgh±0.02
Citrobactersp.1
30cd±0.18
29cd±0.01
28cde±0.04
20h±0.07
35b±0.11
32c±0.13
25ef±0.08
25ef±0.13
25ef±0.04
23fgh±0.16
Citrobactersp. 2
21gh±0.11
21gh±0.01
21gh±0.01
16j±0.03
21gh±0.03
20h±0.07
20h±0.12
21gh±0.22
19hi±0.07
20h±0.18
K. pneumoniae 1
19hi±0.07
19hi±0.05
18i±0.08
14jk±0.12
13jkl±0.05
10l±0.11
12k±0.02
10l±0.07
11kl±0.11
10l±0.23
K. pneumoniae 2
24f±0.18
20h±0.11
20h±0.04
15j±0.04
10l±0.07
8m±0.08
8m±0.07
9lm±0.12
9lm±0.11
9lm±0.17
K. pneumoniae 3
23fgh±0.14
24f±0.08
22fgh±0.03
18i±0.16
23fgh±0.03
23fgh±0.09
20h±0.13
17i±0.18
18i±0.04
16j±0.08
K. pneumoniae 4
28cde±0.19
26e±0.02
24f±0.07
21gh±0.04
24f±0.13
24f±0.18
20h±0.08
21gh±0.20
18i±0.07
15j±0.13
L. monocytogenes1
21gh±0.07
27e±0.06
20h±0.02
23fgh±0.03
25ef±0.06
25ef±0.16
12k±0.06
13k±0.11
19hi±0.13
19i±0.24
L. monocytogenes2
26e±0.08
25ef±0.05
21gh±0.13
17i±0.01
20h±0.02
18i±0.07
15j±0.03
16j±0.10
18i±0.03
19hi±0.17
L. monocytogenes3
26e±0.18
26e±0.03
22fgh±0.08
21gh±0.07
20h±0.09
20h±0.12
18i±0.04
18i±0.13
20h±0.12
19i±0.25
E. coli 1
13k±0.11
17i±0.01
18i±0.03
16j±0.12
24f±0.11
22fgh±0.14
20h±0.03
21gh±0.21
17i±0.08
17ij±0.22
E. coli 2
17i±0.14
27e±0.01
25ef±0.05
11l±0.08
28cde±0.04
25ef±0.07
23fgh±0.08
21gh±0.05
10l±0.02
8m±0.03
E. coli 3
19hi±0.05
25f±0.23
21gh±0.13
14jk±0.11
28cde±0.03
26e±0.08
14jk±0.03
15j±0.12
15j±0.13
12k±0.24
S. dysenteriae1
20h±0.19
28cde±0.09
20fgh±0.02
17i±0.09
20h±0.11
19i±0.20
15j±0.03
13k±0.25
17i±0.08
15j±0.03
S. dysenteriae2
12kl±0.14
15j±0.01
15j±0.03
21gh±0.12
12kl±0.11
13jkl±0.22
12kl±0.03
10l±0.18
20h±0.13
20h±0.06
C.albicans
17i±0.15
26e±0.07
14jk±0.03
12kl±0.14
10l±0.13
8m±0.25
11l±0.03
10l±0.11
9lm±0.04
9lm±0.16
Antibiotic=hibiotic (mg/ml); AA= acetic acid, Conc.= concentration, 8 and 10= fermented solution period; KTFS= Kombusha tea fermented solution, KRFS = Kombusha
rice fermented solution, KBFS= Kombusha barley fermented solution. ± Standard deviation (SD). Values in the same column (followed by letters with aliphatic series)
sharing the same letters do not differ significantly whereas the values followed letters in different alphabetic series are significantly different according to Duncan (1975) at
5% level.Staph= Staphylococcus, S. enteritidis= Salmonella enteritidis, K.=Klebsiella, L.= Listeria, S. dysenteriae= Shigelladysenteriae& C.= Candida.
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125
Soheir et al. World Journal of Pharmaceutical and Life Sciences
Table 3: Minimum inhibition concentration (MIC) of Kombucha fermented solutions produced after 8 days’ fermentation period on pathogenic bacteria and yeast.
Pathogenic
bacteria
and yeast
MIC (Dilution tested of Kombusha)
KTFS
KBFS
KRFS
1/2
1/5
1/10
1/25
1/50
1/100
1/125
Cont.
1/2
1/5
1/10
1/25
1/50
1/100
1/125
Cont.
1/2
1/5
1/10
1/25
1/50
1/100
1/125
Cont.
Staph. aureus 1
-
-
-
-
-
-
+
-
-
-
-
-
-
+
++
-
-
-
-
-
-
-
+
-
Staph. aureus 2
-
-
-
-
-
-
+
-
-
-
-
-
-
+
++
-
-
-
-
-
-
-
+
-
S. enteritidis
-
-
-
-
-
-
+
-
-
-
-
-
-
++
++
-
-
-
-
-
-
-
++
-
Citrobactersp.1
-
-
-
-
-
-
+
-
-
-
-
-
-
+
++
-
-
-
-
-
-
-
+
-
Citrobactersp. 2
-
-
-
-
-
-
+
-
-
-
-
-
-
+
++
-
-
-
-
-
-
-
+
-
K. pneumoniae 1
-
-
-
-
±
+
+
-
-
-
-
-
+
+
++
-
-
-
-
-
+
+
++
-
K. pneumoniae 2
-
-
-
-
±
+
+
-
-
-
-
-
±
+
++
-
-
-
-
-
+
+
++
-
K. pneumoniae 3
-
-
-
-
±
+
+
-
-
-
-
-
±
+
++
-
-
-
-
-
+
+
++
-
K. pneumoniae 4
-
-
-
-
±
+
++
-
-
-
-
-
±
±
++
-
-
-
-
-
+
+
++
-
L. monocytogenes1
-
-
-
-
-
-
++
-
-
-
-
-
-
++
++
-
-
-
-
-
-
-
++
-
L. monocytogenes2
-
-
-
-
-
-
++
-
-
-
-
-
-
++
++
-
-
-
-
-
-
-
++
-
L. monocytogenes3
-
-
-
-
-
-
++
-
-
-
-
-
-
++
++
-
-
-
-
-
-
-
++
-
E. coli 1
-
-
-
-
±
++
++
-
-
-
-
-
±
±
+
-
-
-
-
-
±
++
++
-
E. coli 2
-
-
-
-
±
++
++
-
-
-
-
-
±
±
+
-
-
-
-
-
±
++
++
-
E. coli 3
-
-
-
-
±
++
++
-
-
-
-
-
±
±
+
-
-
-
-
-
±
++
++
-
S. dysenteriae1
-
-
-
-
±
++
++
-
-
-
-
-
±
+
+
-
-
-
-
-
±
++
++
-
S. dysenteriae2
-
-
-
-
±
++
+++
-
-
-
-
-
+
+
++
-
-
-
-
-
±
++
+++
-
C. albicans
-
-
-
+
++
++
++
-
-
-
-
+
+
++
++
-
-
-
-
+
++
++
++
-
Spectrum of activity
(%)
18/1
8
18/1
8
18/1
8
17/1
8
8/18
8/18
18/18
18/
18
18/1
8
18/1
8
17/1
8
8/18
18/18
18/1
8
18/1
8
18/1
8
17/1
8
8/18
8/18
18/18
100
100
100
94
44.4
44.4
100
10
0
100
100
94
44.4
100
100
100
100
94
44.4
44.4
100
-= No growth, ± = Very Scanty growth, + = Scanty growth, ++ = Moderate growth, Cont.= Control (100%) Kombusha fermented solutions before diluted, KTFS= Kombusha
tea fermented solution, KRFS = Kombusha rice fermented solution, KBFS= Kombucha barley fermented solution.Staph= Staphylococcus, S. enteritidis= Salmonella
enteritidis, K.=Klebsiella, L.= Listeria, S. dysenteriae= Shigelladysenteriae& C.= Candida.
Table 4: Time-kill test of Kombucha fermented solutions on different pathogenic microorganisms after exposure of different periods during 24 h.
Pathogenic bacteria
and yeast
Time of Exposure (h)
KTFS
KBFS
KRFS
1.5
3
6
9
12
15
24
1.5
3
6
9
12
15
24
1.5
3
6
9
12
15
24
Staph. aureus 1
+
+
+
-
-
-
-
+
+
+
-
-
-
-
+
+
+
-
-
-
-
Staph. aureus 2
+
+
+
-
-
-
-
+
+
+
-
-
-
-
+
+
+
-
-
-
-
S. enteritidis
+
+
+
-
-
-
-
+
+
+
-
-
-
-
+
+
+
-
-
-
-
Citrobactersp.1
+
+
+
+
-
-
-
+
+
+
+
-
-
-
+
+
+
+
-
-
-
Citrobactersp. 2
+
+
+
+
-
-
-
+
+
+
+
-
-
-
+
+
+
+
-
-
-
K. pneumoniae 1
+
+
+
+
-
-
-
+
+
+
+
-
-
-
+
+
+
+
-
-
-
K. pneumoniae 2
+
+
+
+
-
-
-
+
+
+
+
-
-
-
+
+
+
+
-
-
-
K. pneumoniae 3
+
+
+
+
-
-
-
+
+
+
+
-
-
-
+
+
+
+
-
-
-
K. pneumoniae 4
+
+
+
+
-
-
-
+
+
+
+
-
-
-
+
+
+
+
-
-
-
L. monocytogenes1
+
+
+
+
-
-
-
+
+
+
+
-
-
-
+
+
+
+
-
-
-
L. monocytogenes2
+
+
+
+
-
-
-
+
+
+
+
-
-
-
+
+
+
+
-
-
-
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126
Soheir et al. World Journal of Pharmaceutical and Life Sciences
L. monocytogenes3
+
+
+
+
-
-
-
+
+
+
+
-
-
-
+
+
+
+
-
-
-
E. coli 1
+
+
-
-
-
-
-
+
+
-
-
-
-
-
+
+
-
-
-
-
-
E. coli 2
+
+
-
-
-
-
-
+
+
-
-
-
-
-
+
+
-
-
-
-
-
E. coli 3
+
+
-
-
-
-
-
+
+
-
-
-
-
-
+
+
-
-
-
-
-
S. dysenteriae1
+
+
-
-
-
-
-
+
+
-
-
-
-
-
+
+
-
-
-
-
-
S. dysenteriae2
+
+
-
-
-
-
-
+
+
-
-
-
-
-
+
+
-
-
-
-
-
C. albicans
+
+
+
+
+
-
-
+
+
+
+
+
-
-
+
+
+
+
+
-
-
-= No growth, + = growth, KTFS= Kombusha tea fermented solution, KRFS = Kombusha rice fermented solution, KBFS= Kombusha barley fermented solution.Staph=
Staphylococcus, S. enteritidis= Salmonella enteritidis, K.=Klebsiella, L.= Listeria, S. dysenteriae= Shigelladysenteriae& C.= Candida.
Table 5: Minimum lethal concentration (MLC) of Kombucha fermented solutions on various Gram positive and negative microorganisms.
Pathogenic bacteria and
yeast
Dilution tested of Kombusha
KTFS
KBFS
KRFS
1/2
1/5
1/10
1/25
1/50
1/100
1/2
1/5
1/10
1/25
1/50
1/100
1/2
1/5
1/10
1/25
1/50
1/100
Gr+ve
Staph. aureus 1
-
-
-
-
-
-
-
-
-
-
+
+
-
-
-
-
-
-
Staph. aureus 2
-
-
-
-
-
-
-
-
-
-
+
+
-
-
-
-
-
-
L. monocytogenes1
-
-
-
+
+
+
-
-
-
+
+
+
-
-
-
+
+
+
L. monocytogenes2
-
-
-
+
+
+
-
-
-
+
+
+
-
-
-
+
+
+
L. monocytogenes3
-
-
-
+
+
+
-
-
-
+
+
+
-
-
-
+
+
+
C. albicans
-
-
+
+
+
+
-
+
+
+
+
+
-
+
+
+
+
+
Gr-ve
S. enteritidis
-
-
+
+
+
+
-
+
+
+
+
+
-
-
+
+
+
+
Citrobactersp.1
-
-
+
+
+
+
-
+
+
+
+
+
-
-
+
+
+
+
Citrobactersp. 2
-
-
+
+
+
+
-
+
+
+
+
+
-
-
+
+
+
+
K.pneumoniae 1
-
-
+
+
+
+
-
+
+
+
+
+
-
-
+
+
+
+
K. pneumoniae 2
-
-
+
+
+
+
-
+
+
+
+
+
-
-
+
+
+
+
K.pneumoniae 3
-
-
+
+
+
+
-
+
+
+
+
+
-
-
+
+
+
+
K.pneumoniae 4
-
-
+
+
+
+
-
+
+
+
+
+
-
-
+
+
+
+
E. coli 1
-
-
+
+
+
+
-
-
+
+
+
+
-
+
+
+
+
+
E. coli 2
-
-
+
+
+
+
-
-
+
+
+
+
-
+
+
+
+
+
E. coli 3
-
-
+
+
+
+
-
-
+
+
+
+
-
+
+
+
+
+
S. dysenteriae1
-
-
+
+
+
+
-
-
+
+
+
+
-
+
+
+
+
+
S. dysenteriae2
-
-
+
+
+
+
-
-
+
+
+
+
-
+
+
+
+
+
-= No growth, + = Growth. Staph= Staphylococcus, S. enteritidis = Salmonella enteritidis, K.=Klebsiella, L.= Listeria, S. dysenteriae= Shigelladysenteriae& C.= Candida.
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127
Soheir et al. World Journal of Pharmaceutical and Life Sciences
Table 6: Bacterio/fungi-static (-) and Bacter/fung-icidal (+) effects of Kombucha fermented solutions.
Pathogenic bacteria
and yeast
KTFS
KBFS
KRFS
MIC
(mg/ml)
MLC
(mg/ml)
MLC/
MIC
Effect
MIC
(mg/ml)
MLC
(mg/ml)
MLC/
MIC
Effect
MIC
(mg/ml)
MLC
(mg/ml)
MLC/
MIC
Effect
Staph. aureus 1
0.068
0.068
1
+
0.1028
0.2056
2
+
0.046
0.046
1
+
Staph. aureus 2
0.068
0.068
1
+
0.1028
0.2056
2
+
0.046
0.046
1
+
S. enteritidis
0.068
1.36
20
-
0.1028
2.57
25
-
0.046
0.92
20
-
Citrobactersp.1
0.068
1.36
20
-
0.1028
2.57
25
-
0.046
0.92
20
-
Citrobactersp. 2
0.068
1.36
20
-
0.1028
2.57
25
-
0.046
0.92
20
-
K. pneumoniae 1
0.272
1.36
5
-
0.2056
2.57
12.5
-
0.184
0.92
5
-
K. pneumoniae 2
0.272
1.36
5
-
0.2056
2.57
12.5
-
0.184
0.92
5
-
K. pneumoniae 3
0.272
1.36
5
-
0.2056
2.57
12.5
-
0.184
0.92
5
-
K. pneumoniae 4
0.272
1.36
5
-
0.2056
2.57
12.5
-
0.184
0.92
5
-
L. monocytogenes1
0.068
0.68
10
-
0.1028
0.514
5
-
0.046
0.46
10
-
L. monocytogenes2
0.068
0.68
10
-
0.1028
0.514
5
-
0.046
0.46
10
-
L. monocytogenes3
0.068
0.68
10
-
0.1028
0.514
5
-
0.046
0.46
10
-
E. coli 1
0.272
1.36
5
-
0.2056
1.028
5
-
0.184
2.3
12.5
-
E. coli 2
0.272
1.36
5
-
0.2056
1.028
5
-
0.184
2.3
12.5
-
E. coli 3
0.272
1.36
5
-
0.2056
1.028
5
-
0.184
2.3
12.5
-
S. dysenteriae1
0.272
1.36
5
-
0.2056
1.028
5
-
0.184
2.3
12.5
-
S. dysenteriae2
0.272
1.36
5
-
0.2056
1.028
5
-
0.184
2.3
12.5
-
C. albicans
0.68
1.36
2
+
0.514
1.028
2
+
0.46
0.92
2
+
     -   Staph= Staphylococcus, S. enteritidis = Salmonella enteritidis, K.=Klebsiella, L.= Listeria, S. dysenteriae=
Shigelladysenteriae& C.= Candida.
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128
Soheir et al. World Journal of Pharmaceutical and Life Sciences
Table 7: In vitro growth inhibitory activityIC50
values (μg/mL) of the KFSs againstHCT-116 (human
colon carcinoma cells) and HEpG-2G-2 (liver HEpG-
2atocellular cells).
Sample
*IC50 (µg/ml)
HCT-116
HEpG-2G-2
Kombucha tea (KTFS)
46.3
46.7
Kombucha Rice (KRFS)
162.19
115.8
Kombucha barley (KBFS)
161.3
266.0
*IC50 (µg/ml) = 50% inhibition of cell growth.
Figure 1: Illustrative scheme of successive dilutions
method.
Figure 2: The percentage distribution of Kombucha
sensing pathogenic microorganisms isolates into three
categories that express as diameter of zone inhibition
(mm). KTFS= Kombusha tea fermented solution,
KRFS = Kombusha rice fermented solution, KBFS=
Kombusha barley fermented solution.
Figure 3: Cell viability from NRU cytotoxicity assay
after 48h exposure of human colon carcinoma cells
(HCT-116) and human liver carcinoma (HEpG-2G-2)
cells exposed to KTFS, KRFS and KBFS.
Figure 4: In vitro cytotoxicity IC50 value of KTFS,
KRFS and KBFS exposed to HCT-116 and HEpG-
2G-2 human cells line as determined by Neutral Red
Uptake Assay.
Figure 5: Fluorescent microphotograph of HEpG-2
cells treated with 45μg/mlKFSs, stained with AO/EB.
(a) control cells show a uniform green fluorescence
and (B, C, and D) cells treated with KFSs appeared
green with bright green nuclei indicating nuclear
fragmentation and early apoptotic cells; also, cells
show late apoptotic cells by the orange red
appearance due to the incorporation of both ethidium
bromide and acridine orange (bold arrow) and
(regular arrows) indicate early and late apoptotic
cells respectively. (Magnification 400X).
Data in Table 1 showed that all tested pathogenic
bacteria and yeast were resistant to all tested KFSs
during fermentation times ranged from 2:6 days, which
gave growth density on disc-agar diffusion plates ranged
from moderate (++) to very scanty (±) growth. At
fermentation times ranged from 8-10 days, all tested
KFSs were inhibited the growth of all tested pathogenic
strains (which gave inhibition zone). So, KTFS, KRFS
and KBFS during 8:10 days of fermentation period were
selected as antimicrobial activity for further studies. In
addition to, theseveral Kombucha teas inhibited the
pathogenic bacteria in human and shrimp belong to
species of Salmonella enteritidis, Pseudomonas
aeruginosa, Vibriocholeraand V. vulnificus and the IZD
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Soheir et al. World Journal of Pharmaceutical and Life Sciences
increased gradually with increase fermentation period of
Kombucha from 0-14 days (Talawat et al, 2006).
Moreover, both Kombucha green and black teas gave
activities against all the Gr+veand Gr-ve bacteria include;
S. epidermidis, S. aureus, M.luteus, E. coli, S.
typhimuriumand L.monocytogenes(Deghrigue et al,
2013; Battikh et al, 2012).
Results in Table 2 clearly show that tested 3 types of
KFSswere high inhibition activities for tested pathogenic
microorganisms as compared with hibiotic and acetic
acid. The IZD was ranged from 8 to 38, 25 or 26 mm of
KTFS, KRFS or KBFS during 8-10 days fermentation
periods, respectively. Whereas acetic acid (as chemical
killer) and hibiotic (as antibiotic) were achieved the
antimicrobial activities ranged from 15-33mm and 12-
30mm, respectively. Also, results represented that KTFS
was preferred than KRFS or KBFS, which gave the
highest antimicrobial activity against of
S.enteritidis(38mm) followed by Citrobactersp.1 (35
mm), S. aureus1 (32mm) and S. aureus2 (30 mm).
While, KRFS gave the maximum activity against of
S.aureus1, Citrobactersp.1 and S. aureus2 being 25 mm
of IZD. In case of KBFS, the highest IZD was observed
with S.aureus 1 (26 mm), then Citrobactersp.1 (25mm)
and E. coli 2 (23mm).
No significant difference between KFSsproduced after 8
days until 10 days, so it was selected the 8 days for
reduce the time and cost of Kombucha production. The
tested pathogenic microorganisms were classified into 3
categories (high, moderate and weak) according to IZD
(sensibility of KTFS, KRFS and KBFS) ranged from 25-
38 mm, from 16-24 mm and from 8-15 mm, respectively
(Figure 2). In first category (high IZD), the number of
pathogenic bacteria with high sensitivity to KTFS, KRFS
and KBFS being 7, 3 and 2 represented about 39, 17 and
11% of all tested pathogenic organisms, respectively.
While in second category consisted of a high number of
these tested organisms were recorded a moderate
sensitivity to all tested KFSs. These results are
accordance with Jayabalan et al (2014) they observed
that Kombucha with high antimicrobial efficiency
against pathogenic microorganisms of both Gram-
positive and Gram-negative origin. Acetic acid and
catechins are known to inhibit a number of Gr+&-ve
microorganisms (Sreeramulu et al, 2000). Moreover, the
IZD of green fermented tea was ranged from 12-22mm,
while it varied from 10.5-19mm for black fermented
tea(Battikh et al, 2012). So, it was found that Kombucha
exhibited its strongest antimicrobial effect against S.
epidermidis, M. luteus, L. monocytogenes and P.
aeruginosa (IZD  
be stated that the KFSs produced after 8 days were
selected for further investigation. Also, it was observed
that the 4-pathogenic bacteria S. enteritidis,
Citrobactersp.1, S. aureus1 and S. aureus2 were high
susceptibility to KTFS more than acetic acid and hibiotic
due to Kombucha tea is largely attributable to the
presence of organic acids, particularly acetic acid, large
proteins, catechins and antibiotics (Jayabalan et al, 2007;
, 2000). In addition,
the fermented tea with antibacterial feature, it might due
firstly to organic acids and ethanol formation and
secondly to polyphenolic compounds within the tea
(Talawat et al, 2006). Also, the metabolites produced by
the bacteria and/or yeasts during the fermentation of
Kombucha tea are responsible for its antimicrobial
activity (Sreeramulu et al, 2001).
Evaluation of MIC of tested KFSs
Results in Table 3 demonstrated that the antimicrobial
spectra activity of tested KTFS, KRFS and KBFS at
dilution ranged from 1/2- 1/10 were 100% and dilutions
of 1/25, 1/50 and 1/100 were exhibited activity with
94.0%, 44.4% and 44.4% against the tested pathogenic
microorganisms, respectively, except KBFS without
spectrum activity at 1/100 dilution. Also, data recorded
that the MIC values ranged from 0.0544~3.4 mg/ml,
0.04112~2.57 mg/ml and 00.0368~2.3mg/ml for tested
KTFS, KRFS and KBFS, respectively. Both KTFS and
KRFS at 1/100 dilution (with MIC 0.068 & 0.046mg/ml,
respectively) and KBFS at 1/50 dilution (with MIC
0.1028mg/ml) had a bacteriostatic effect on genus of
Staphylococcus, Salmonella, Citrobacter and Listeria.
Meanwhile, Klebsiella, Eschericha and Shigellagenus
(Gr-ve) were inhibited at 1/25 dilution (with MIC 0.272,
0.2056&0.184mg/ml) of KTFS, KBFS and KRFS,
respectively. While, Candida was inhibited at low
dilution (1/10) with high concentrations of all KFSs
(0.68, 0.514 and 0.46 mg/ml of KTFS, KBFS and KRFS,
respectively) and stimulated in high dilutions. Similarly,
MIC of both Kombucha green and black teas were 150,
228, 280&   E. coli,P. aeruginosa, S.
aureus and S. typhimurium, respectively (Deghrigue et
al, 2013).The tested pathogenic microorganisms were
incubated with the selected MIC of KFSs at different
periods ranged from 1.5-24 h incubation period.
Results in Table 4 represented that the first exposure
periods of each KFSs (1.5 and 3h) were no bacteriostatic
effect on all pathogenic bacteria and yeast. The tested
KFSswere bacteriostatic effect after 6 h exposure period
for both genera of Escherichia and Shigella, 9h for both
genus of Staphylococcus and Salmonella and 12 h for
each genus of Citrobacter, Klebsiella and Listeria. While
Candida albicans took a long time in inhibition was
reached to 15 h of exposure period. Generally, it could
be noticed that the tested pathogenic microorganisms
were inhibited with minimum concentration of KFSs
ranged from 0.68:0.068, 0.514:0.0514 and
0.46:0.046mg/ml during 6-15 h of exposure for KTFS,
KBFS and KRFS, respectively.
MLC of KFSS
Results recorded in Table 5 demonstrated that all tested
KFSS had inhibitor effect on all tested microorganisms.
Both strains of S. aureus were high sensitivity to KFSS
athigh dilutions (with low concentrations) and no growth
appeared when re-subculture on agar medium. The MLC
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Soheir et al. World Journal of Pharmaceutical and Life Sciences
was 0.068 and 0.046mg/ml (at dilution of 1/100) of
KTFS and KRFS and 0.2056mg/ml (at dilution of 1/25)
of KBFS. Whereas, in L. monocytogenes1- 3, the 100%
kill were appeared with MLC ranged from 0.46-
0.68mg/ml at 1/10 dilution of all tested KFSS. However,
in case of C. albicans (Gr+ve), and other Gr-ve bacteria (S.
enteritidis, Citrobactersp.1&2, K. pneumonia 1-4, E.
coli1-3 and S. dysenteriae1&2), the 100% kill were
achieved at low dilution ranged from 1/2-1/5 (at high
concentrations)of all tested KFSS, which MLC recorded
1.36mg/ml of KTFS, ranged from 1.028mg/ml (against
genera Escherichia and Shigella) to 2.57mg/ml (against
genera Candida, Salmonella, Citrobacter and Klebsiella)
of KBFS and ranged from 0.92mg/ml (against genera
Salmonella, Citrobacter and Klebsiella) to
2.3mg/ml(against genera Candida, Escherichia and
Shigella) of KRFS. So, these genera were stimulated
when cultivated on agar medium without substance at
high dilution with low concentrations.
Mode action of KFSS
Results represented in Table 6 clearly showed that action
effect of KFSS against pathogenic strains. The actin
effect was calculated as the ratio of MLC: MIC. It was
found that all tested KFSS had bacter/funal-icidal effect
against both strains of S. aureus1&2and C. albicans
(Gr+ve) which the ratio MLC: MIC was ranged from 1:2.
While KFSS were bacteriostatic effects against other
tested strains. Kombuchafrom lemon balm and black teas
had higher bactericidal effects towardP. mirabilis,S.
aureus and Bacillus sp.    
Greenwalt et al, 1998).
Evaluation of cytotoxicity and antiproliferative
activity against tumor cell lines
Anti-proliferation and induction of apoptosis by the
tested KFSs were studied using NRU and EB/AO
staining assays against HCT-116 and HEpG-2over a
concentration range (30-60
Results presented in Table 7 and Figure 3 displayed a
marked decrease in metabolic activity in cell viability in
a concentration dependent manner exposed to different
KFSS. The results clarify the different response in
cytotoxicitywith different KFSS where, KTFS found to
be more cytotoxic than KRFS and KBFS either on HCT-
116 or HEpG-2cell lines. Maximum reduction in cell
viability at 60µg/mlwas found to be 25% for HEpG-
2exposed to KTFS. Regarding to the cytotoxicity of
KRFS and KBFS, both beverages exhibited nearly
similar inhibitory effects on HCT-116 tumor cells but
lower than KTFS. Meanwhile, treatment of HEpG-
2tumor cells exhibited, KBFS was less active than KTFS
and KRFS. Comparing the cytotoxic efficacy of KTFS in
HCT-116 colon cancer the result showed that the highest
cytotoxic is observed with IC50 46.3µg/ml Meanwhile,
KRFS and KBFS exhibited nearly similar inhibitory
effects less than KTFS with IC50 values of 162.19 and
161.3µg/ml, respectively. In liver cancer HEpG-2, the
higher cytotoxic activity was observed with KTFS with
IC50 value, 46.7µg/ml while the lowest cytotoxic activity
was observed with KBFS (with IC50 value 266µg/ml).
Moreover, KRFS exhibited moderate inhibitory effects
with IC50 value 115.8µg/ml. Data from doseresponse
curve indicated that, the concentrations 30, 45 and
  S could produce highest inhibitory
effects against HCT-116; with value 30.7, 47.5 and 67%,
respectively. Meanwhile, KRFS produced inhibitory
values; 21, 26.61 and 27.4%, respectively. While the
effect of the same concentrations of KBFS produced
lower inhibitory effects; 4.25, 10.4 and 14.63%,
respectively (Table 7 and figure 4).Determination of the
antitumor activities of KFSs against the different tumor
cells indicated that KTFS has more antitumor activity
than KRFS and KBFS. Although all tested KFSs were
found to reduce the cell viability in a concentration
manner, but the magnitude of reduction was high in case
of KTFS. Many researchers have investigated the
antiproliferative properties of KFSs because it might be
contained several compounds were identified as
polyphenols, organic acids and vitamins which reduces
stomach cancer (Cushnie and Lamb, 2005; Taguri et al,
2004; Bauer-Petrovska and Petrushevska-Tozi, 2000)
and D-saccharic acid-1, 4-lactone, which inhibits the
activity of glucuronidase, an enzyme indirectly related
with cancers (Wang et al, 2010). The antitumor
properties of polyphenols which might act as cancer-
blocking agents (Russo, 2007). In addition, KTFS
decreased the survival of prostate cancer cells by down
regulating the expression of angiogenesis stimulators
(Srihari et al, 2013).
Fluorescence microscopic analysis of cell death by
EB/AO staining
Treatment of KFSS led to induction of apoptosis, where,
KTFS exhibited apoptotic signal appears to be
stronger than KRFS and KBFS against HEpG-2 cell line
(Figure 5).KTFS showed increased percentages of late
apoptotic cells than KRFS and KBFSwhich had stained
orange attributed to incorporation of both ethidium
bromide and acridine orange (Figure 5B-C). From these
result, it can conclude that the KFSS fermented solution
has cytocidal or cytostatic phenotype.Apoptotic cells
were increased after treatment with KFSS compared to
control, suggesting that apoptosis could contribute to kill
cancers cells via apoptosis. The figures show the
potential of the KFSS in inducing apoptosis in the HEpG-
2cells by demonstrating the morphological changes
characteristic to apoptosis such as cell shrinkage, nuclear
and cytoplasmic condensation and formation of apoptotic
bodies as compared to the negative control. Live cells
displayed a uniformly green fluorescence which had
regular, round-shaped nuclei in control (Figure 5A). The
early apoptotic cells were observed at a concentration of
45         
bright green dots in their nuclei corresponding to nuclear
fragmentation (Figure 5; bold arrow). The chemo
preventive action of KTFS might be due to its ability to
induce apoptosis. The AO/EB and the NRU assays
showed similar effects on viability with different KFSs.
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Soheir et al. World Journal of Pharmaceutical and Life Sciences
According to obtained results it can concluded that
KTFS has strong antiproliferative properties which
increase with concentration. The KTFS exhibits best
antiproliferative activity than the KRFS and KBFS
extract with lower IC50 values, and induces a higher
percentage of apoptotic cells after 24h of exposure.
Scientific studies have claimed that Kombucha has
anticancer effects as well (Dufresne and Farnworth,
2000) due to the presence of tea polyphenols and the
secondary metabolites produced during the fermentation
process (Jayabalan et al, 2014; Jayabalan et al, 2011).
CONCLUSIONS
Kombucha fermented solutions (KFSS) had microbicidal
impact against S. aureusand C. albicans and
microbiostatic effect against other pathogenic bacteria.
Also, it have capable of reducing cancer cell proliferation
andinduced apoptotic cell death and may have a
promising role to play in the development of new
anticancer drugs in the future.
CONFLICTS OF INTEREST
All authors certify that this manuscript has not been
published in whole or in part nor is it being considered
for publication elsewhere. The authors have no conflicts
of interest to declare.
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... [24,25] The anticancer mechanisms of green tea polyphenols include inhibition of gene mutation, inhibition of cancer cell proliferation, induction of cancer apoptosis, and termination of metastasis. [24,26,27] In the traditional medicine, the anticancer activity of kombucha had reported with no scientific knowledge background. Recent studies as Jaya Balan group investigations, take a new overview for anticancer specification of kombucha especially in liver and renal carcinoma. ...
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Background: Colorectal cancer is the third leading to death type of cancer in the world. The therapeutic guideline varied between different methods. As the main therapeutic guideline is chemotherapy, recent studies had shown utilization of natural products in combination with conventional medication, elevate the efficiency of chemotherapeutic methods. Kombucha is a traditional beverage obtained from the fermentation of green tea as a rich source of flavonoid medicinal plant. This study aimed to evaluate the natural potential of combination therapy of this natural product with doxorubicin as a chemotherapeutic agent. Materials and methods: The study was performed as in vitro evaluation of biological activity of kombucha on HCT-116 cell line (human colon cancer cell line). The cytotoxic effect of different kombucha beverages (fermented green tea) in comparison with green tea extract was evaluated by dimethylthiazolyl tetrazolium bromide (MTT) assay. In the next step, anticancer activity of doxorubicin as a general guideline chemotherapeutic agent in combination with kombucha was evaluated by cell cycle analysis and apoptosis assay flow cytometry. Apoptotic genes expression pattern was determined using real-time polymerase chain reaction. The experiments were designed in three independent replications and statistically analyzed using SPSS software. Results: The results show that kombucha compared with the green tea extract caused more (1.2 fold) early apoptosis induction and G0/G1 phase arrest. Moreover, kombucha increased the expression levels of p21, p53, and B-cell leukemia/lymphoma 2 (Bcl-2)-associated X protein genes (2, 2.5, and 1.5 fold, respectively) while it decreased Bcl-2 gene expression level (5-8 fold) compared with doxorubicin alone. Combination of kombucha with doxorubicin shows 2-fold increased G0/G1 phase compared with the doxorubicin treatment. Conclusion: This result indicated that kombucha caused boosted anticancer activity of doxorubicin agent. These findings suggest that kombucha may be has an assistor and useful role in colorectal cancer treatment align with chemotherapy.
... Kombucha produced on red and green tea and prepared using the technique proposed by Abou-Taleb et al., (2017). Four grams of the substrate (red and green tea) soaked in 1 L of boiling tap water for 15 min, then filtered into a sterilized glass jar using filter paper Whatman No. 1. ...
... Some of the metabolites are ethanol, lactic acid, acetic acid, gluconic acid, and glucuronic acid (Jayabalan et al., 2014;Mukadam et al., 2016), which are thought to play a vital role in providing health benefits. It has been reported that kombucha has a hepatoprotective effect (Kabiri et al., 2014;Watawana et al., 2015), antiproliferative effect against cancer cells, and antimicrobial effect against Escherichia coli, Salmonella enterica, Micrococcus luteus, and Staphylococcus epidermidis (Abou-Taleb et al., 2017), while also has hypoglycemic and antilipidemic (Zubaidah et al., 2018), and provides a better healing process to stomach ulcer than omeprazole and black tea (Kaewkod et al., 2019). ...
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Context: Kombucha from tea is reported to be beneficial for health. Moreover, it can be used as a hepatoprotective, antiproliferative, antimicrobial, antidiabetic, and antilipidemic agent and is capable of healing stomach ulcers. But kombucha from other herbal has not been studied, including kombucha from the mangrove (Rhizophora mucronata) fruit. Aims: To evaluate the characteristics and the antidiabetic potential of kombucha herbal tea from R. mucronata fruit based on in vitro, chemistry, and physical analysis. Methods: This study was conducted by an experimental method using R. mucronata herbal tea as a kombucha drink with different sugar concentrations (10, 20, 30%) and fermentation time (7, 14, 21 days) with three replications on each experiment. The analyzed parameters were the inhibition of the α-glucosidase enzyme for antidiabetic activity, total phenolics, total acids, and organoleptic characteristics. Results: The sugar concentration and fermentation time significantly affected the characteristics of the produced kombucha in inhibiting α-glucosidase. The optimum treatment in inhibition was at 10% sugar concentration and 14 days of fermentation time with IC50 of 33.95 ppm. The kombucha from R. mucronata fruit had a pH of 3.11 and contained a total phenolics of 19,679.82 mg GAE/100g, 0.52% of total acids, and was quite preferred by panelists. Conclusions: Kombucha herbal tea of R. mucronata fruit has the potential as an antidiabetic drink with a lower IC50 value than acarbose drug and commercial kombucha tea.
... Kombucha produced on tea (KT), rice (KR), and barley (KB) substrates. It prepared using the technique proposed by Abou-Taleb et al. (2017). Four grams of the substrate (black tea, rice, or barley) soaked in 1 L of boiling tap water for 15 min, then filtered into a sterilized glass jar (length: 12 cm and radius: 3 cm) using filter paper Whatman No. 1. ...
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Three types of Kombucha fermented tea, rice, and barley were produced in order to study their changes of biological and chemical parameters. About a 2-fold increase in mushroom dry weight after 8 days of growth was observed with Kombucha tea. The optical density of the Kombucha extracts gave the same trend. Moreover, Kombucha tea recorded the highest specific growth rate and lowest doubling time. The total acidity, ethanol content, and total protein content were incremented with the fermentation process to reach the peak after 6–8 days as in descending order: tea > barley ≥ rice. The scavenging abilities of DPPH were in descending order, tea (89.69%) > barley (76.19%) > rice (36.04%). The total phenolic compounds of Kombucha tea (88.8 ppm) have the same trend, which more than 3-fold of Kombucha rice (26.11 ppm). Results revealed that the Kombucha tea preservation method using heat treatment (at 76 and 100 °C for 10 min) was not successful, resulting in a decrease in antioxidant amounts to 71.0 and 53.42% and total phenolic compounds to 40.22 and 45.69 ppm, respectively.
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