Indian J Microbiol (July–Sept 2011) 51(3):338–344
`DOI 10.1007/s12088-011-0096-3
`
`O R I G I N A L A R T I C L E
`
`Effect of Mangrove Tea Extract from Ceriops decandra (Griff.)
`Ding Hou. on Salivary Bacterial Flora of DMBA Induced Hamster
`Buccal Pouch Carcinoma
`
`Natarajan Sithranga Boopathy •
`Kathiresan Kandasamy • Manivannan Subramanian •
`Jeon You-Jin
`
`Received: 22 May 2009 / Accepted: 20 September 2009 / Published online: 27 February 2011
`Ó Association of Microbiologists of India 2011
`
`Abstract The objective of this study was to investigate
`the effects of mangrove tea on salivary bacterial flora in
`DMBA induced hamster buccal pouch carcinoma. Tea
`from mangrove plant Ceriops decandra was administered
`against DMBA induced buccal pouch carcinoma in ham-
`ster rats. The chemical constitutions and quality of man-
`grove tea is similar with the commercial tea Camellia
`sinensis. The Hamster rats were painted thrice a week with
`DMBA in their right buccal pouch, and also administrated
`orally with 1.25% of Ceriops tea extract, on alternate days
`of the DMBA treatment. Appropriate control animals were
`maintained. After 14 weeks of treatment, bacterial species
`in saliva were enumerated, tumor incidences were analyzed
`using histopathological section and tumor volume in the
`animals was quantified using water-displaced method. The
`decreased counts of beneficial bacteria and increased
`counts of harmful bacteria were associated with increased
`volume of tumors. The present study concluded that the tea
`extract from C. decandra prevents the oral cancer inci-
`dences and maintain the good health conditions of the
`animals.
`Keywords Beneficial bacteria Harmful bacteria
`Oral cancer Ceriops decandra Mangrove tea DMBA
`
`N. Sithranga Boopathy (&) K. Kandasamy M. Subramanian
`CAS in Marine Biology, Annamalai University, Parangipettai,
`Cuddalore 608 502, Tamilnadu, India
`e-mail: nsboopathy@gmail.com
`
`J. You-Jin
`School of Biomedical Sciences, Jeju National University, Jeju,
`South Korea
`
`123
`
`Introduction
`
`Oral cancer is the sixth most common malignancy world-
`wide [1] and is particularly prevalent in developing coun-
`tries, such as in Southeast Asia, where up to 40% of all
`malignancies are located within the oral cavity [2]. More
`than 90% of cancers in mouth are squamous cell carcino-
`mas (SCCs), originating from the oral mucosa [3]. With an
`average of all stage, 5-year survival rate for oral cancer of
`less than 50%, the annual mortality figures are comparable
`to those of carcinoma of the cervix and malignant mela-
`noma [4, 5]. Bacteria are known to associate with cancer
`tissues. Nagy et al. [6] have demonstrated a difference in
`the microflora associated with the surface of tumors in
`comparison to control sites. Patients with oral cancer tend
`to possess significantly low concentration of beneficial
`bacteria in their saliva. This is of particular interest because
`of its potential application as a diagnostic tool to predict
`oral cancer [7]. Tea that contains many antioxidants is a
`pleasant and safe drink that is enjoyed by people across the
`globe. Tea leaves are manufactured as black, green, or
`oolong. Black tea represents *78% of total consumed tea
`in the world, whereas green tea accounts for *20% of tea
`consumed. The concept of ‘‘use of tea for promotion of
`human health and prevention and cure of diseases’’ has
`become a subject of intense research in the last decade. The
`health benefits of tea are ranging from a lower risk of
`certain cancers to weight
`loss, and protection against
`infections, like bacterial and viral, to chronic debilitating
`diseases, including cancer, coronary heart disease, stroke,
`and osteoporosis. [8]. the mangrove plant Ceriops decan-
`dra has traditionally [9, 10] and scientifically rich in
`medicinal values [11–13]. Our research team attempted to
`extract black tea from a mangrove species Ceriops dec-
`andra which contains large amount of theaflavin (giving
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`Indian J Microbiol (July–Sept 2011) 51(3):338–344
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`339
`
`flavour) and theambrugin (neurostimulant) [13]. These
`compounds are produced when polyphenols (present in
`cytoplasms) are allowed to ferment by polyphenol oxidase
`(present in cell vacuoles). The tea had no toxicity in mice,
`and had a better quality than commercial teas, as evident by
`sensory evaluation tests performed with our centre people.
`The present study was to determine the effect of mangrove
`tea on the bacterial flora which associated with saliva of
`DMBA induced hamster buccal pouch carcinoma.
`
`housed at six per polypropylene cage and provided with
`standard pellet diet (Mysore Snack Feed Ltd., Mysore,
`India) and water ad libitum. The animals were maintained
`at a temperature of 28 ± 2°C with an alternating 12-h
`light/12-h dark cycle. The animals were maintained as per
`the norms provided by the Bioethic Committee of An-
`namalai University (IAEC/CPCSEA 270 Dated 01.
`07.2005).
`
`Materials and Methods
`
`Collection and Preparation of Plant Material
`
`Leaves of the mangrove plant species, Ceriops decandra
`were collected from the forest of Pichavaram (11° 270 N;
`79° 470 E) situated in south east coast of India. The spec-
`imen was identified and its holotype (No. R90) has been
`deposited in the herbarium of the Centre of Advanced
`Study in Marine Biology, Annamalai University, Parangi-
`pettai, Tamil Nadu, India. The leaf sample was washed
`with tap water to remove epiphytes and other external
`matter.
`
`Preparation of Black Tea Extract
`
`Black tea was extracted from the leaves of Ceriops dec-
`andra, adopting the method of [14]. Fresh leaves were
`spread on a trough and allowed to dry using a warming
`blender. A known weight of the macerated sample was
`placed in a piece of cloth and distilled water was contin-
`uously trickled over it for 1 h. The fermented sample was
`dried in a hot air oven at 95°C for 30 min. The tea extract
`was freshly prepared every day using 1.25 g of Ceriops tea
`powder in 100 ml boiled water.
`
`Chemicals Used for the Study
`
`(DMBA) was
`Carcinogen Dimethylbenz[a]anthracene
`purchased from Sigma Chemical Company, USA and used
`for this study. All other reagents used were of analytical
`grade.
`
`Experimental Animals Used
`
`The Syrian hamster cheek pouch epithelium has been a
`valuable model in studies of chemical carcinogens inter-
`actions with oral tissues [15, 16]. Therefore, male Syrian
`hamsters were used as experimental animals with an age of
`8–10 weeks weighing 85–90 g obtained from the National
`Institute of Nutrition Hyderabad, India. The animals were
`
`Experimental Design
`
`The animals were randomized into experimental, control
`groups, and divided into four groups of six animals each.
`Animals in group I was treated as untreated control. The
`animals in group II were painted with a 0.5% solution of
`DMBA in liquid paraffin on the right buccal pouch using a
`no. 4 brush three times a week for 14 weeks. Each appli-
`cation treated approximately 0.4 mg of DMBA. Group III
`animals were painted with DMBA as in group II; In
`addition, the animals were administered with 1.25% of
`freshly prepared mangrove tea extract twice per day. Group
`IV animals received the same dose of mangrove tea extract
`alone. The experiment was terminated at the end of 14th
`week and animals were sacrificed by cervical dislocation
`after an over night fast and the fresh tissue were used for
`estimations.
`
`Isolation and Identification of Bacteria from Saliva
`
`Saliva samples were collected from the buccal region of the
`animal using sterile Whatman filter paper discs (with a
`diameter of 5 mm). The sample was serially diluted and
`plated on culture media deMan Rogosa Sharpe (MRS),
`streptococcus selection agar (SSA) and modified milk agar
`and incubated at 37°C for 36 h. All the determinations
`were carried out in triplicate. The counts are expressed as
`colony forming unit (CFU) per ml of the sample. Identifi-
`cation was done by following the keys of Bergy’s manual
`of determinative bacteriology [17, 18].
`
`Histopathological Observations
`
`The specimens were maintained in 10% formalin solution
`for processing. Embedded in paraffin, the specimens were
`sectioned and examined under a microscope at 409 mag-
`nification, after staining with hematoxylin and eosin.
`
`Statistical Analysis
`
`One-way ANOVA and Duncan multiple range tests were
`used to compare mean values at 0.05 probabilities.
`
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`340
`
`Results
`
`Cancer Incidences
`
`Tea from mangrove plant Ceriops decandra effectively
`prevented the DMBA induced carcinogenesis. The DMBA
`treated animals showed 100% squamous cell carcinoma
`where as the tea extract treated animals showed hyperplasia
`alone. We did not observed any cancer incidences in both
`untreated control and tea extract alone treated animals.
`
`Enumeration of Bacteria in Saliva
`
`Bacterial counts in the hamsters induced with oral carcinoma
`are given in the Table 1. Animals in control group exhibited
`high counts of lactobacilli (42 ± 11 9 103 CFU ml-1) fol-
`lowed by streptococci (29 ± 17 9 103 CFU ml-1) and bif-
`idobacteria (22 ± 17 9 103 CFU ml-1). The tea extract
`alone treated animals were also rich in lactobacilli and bifi-
`dobacterial counts (53 ± 18 9 103, 31 ± 15 9 103 CFU ml-1
`respectively) and reduced counts of streptococci (25 ± 13 9
`103 CFU ml-1). The DMBA treated groups of animals
`exhibited low counts of lactobacilli (25 ± 10 9 103 CFU
`ml-1) and bifidobacteria (07 ± 1.0 9 103 CFU ml-1) how-
`ever,
`the animals showed high counts of streptococci
`(47 ± 17 9 103 CFU ml-1). Whereas the tea extract in the
`DMBA-treated animals showed increased counts of lacto-
`bacilli and bifidobacteria (37 ± 10 9 103; 17 ± 1.1 9
`103 CFU ml-1 respectively) and decreased counts of strep-
`tococci (32 ± 15 9 103 CFU ml-1). Counts of lactobacilli
`and bifidobacteria were negatively correlated with tumor
`size, whereas streptococcus was positively correlated to
`tumor size (Figs. 1, 2, 3). The ratio between beneficial and
`harmful bacteria of 0.68 coincided with the incidence of
`tumor, whereas there was no tumor incidence when the ratio
`was equal or greater than 1.6 (Table 5).
`
`Bacterial Species in Saliva
`
`Bacterial species were identified in saliva based on mor-
`phological and biochemical characteristics as shown in
`
`Indian J Microbiol (July–Sept 2011) 51(3):338–344
`
`Table 2. There were five species of lactobacilli (Lactoba-
`cillus acidophilus, L.
`lactis, L.
`jensenii, L. casei and
`L. brevis), two species of bifidobacteria (Bifidobcterium
`bifidium and B. longum) and two species of streptococci
`(Streptococcus mutan and S. mitis) identified. The charac-
`teristics of the bacterial species are given in Tables 2, 3, 4.
`
`Histological Observations
`
`The tumour formation is evident by histological observa-
`tions. There was well developed squamous cell carcinoma,
`along with well-defined epithelial and keratin pearls in the
`connective tissue with cellular pleomorphism (Fig. 4).
`However, the animals treated with DMBA ? mangrove tea
`extract, exhibited only hyperplasia. The histological studies
`proved the anti-cancer effect of the mangrove tea extract at
`the dose of 1.25% twice per day. No pathological obser-
`vations were noted either in control animals or the animals
`treated with mangrove tea extract alone. Fig. 4a, d show
`the histological structure of mucosa extracted from control
`and mangrove tea extract alone treated animals which are
`showing normal and intact epithelium. Fig. 4b exhibits the
`abnormal epithelium intruded into the connective tissue
`leading to the formation of spherical pearl’ like structures
`in the mucosa extracted from DMBA–treated animals.
`Fig. 4c exhibits only multi
`layer of epithelial cells
`(hyperplasia) in the mucosa of animal treated with both
`DMBA ? mangrove tea extract. Here there is no abnormal
`intrusion of epithelium and formation of pearl
`like
`structures.
`
`Discussions
`
`The results of present study indicated that the tea extract
`from Ceriops decandra effectively prevented the DMBA
`induced carcinogenesis. The available salivary bacterial
`species of the test animals clearly indicated the health
`status of the host animals. The predominant types of bac-
`teria isolated from the saliva, tongue, dorsum and buccal
`mucosa of rates were Streptococcus spp., Lactobacillus
`
`Table 1 Effect of tea extract of C. decandra on salivary bacterial counts and tumour volume of experimental animals
`
`Treatment
`
`Control
`
`DMBA
`
`DMBA? Tea extract
`
`Tea extracts alone (1.25%)
`
`Lactobacillus
`(9103 CFU ml-1)
`
`Bifidobacteria
`(9103 CFU ml-1)
`
`Streptococci
`(9103 CFUml-1)
`
`42 ± 11a
`25 ± 10b
`36 ± 9c
`53 ± 18d
`
`22 ± 05a
`07 ± 1.0b
`16 ± 1.1c
`31 ± 18d
`
`29 ± 17a
`47 ± 17b
`29 ± 11a
`25 ± 13a
`
`Tumour
`volume (ml)
`
`0.00a
`2.45 ± 0.2b
`0.00a
`0.00a
`
`Values are mean ± standard error from three replicates in each group of animals maintained with six each
`Values not sharing a common superscript are differ significantly at P [ 0.05
`
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`341
`
`Fig. 1 Correlation between
`Lactobacilli (CFU 9 103.ml-1)
`count and tumour volume
`
`Fig. 2 Correlation between
`Bifidiobacteria (CFU 9
`103.ml-1) count and tumour
`volume
`
`spp., and Bifidobacteria [19]. Therefore these three types of
`bacteria were studied in the present investigation. Lactic
`acid bacteria and bifidobacteria are two well-known groups
`of beneficial bacteria which constitute an integral part of
`the health condition. They impart nutritional and thera-
`peutic benefits to their host. The vitamins and enzymes
`produced by the lactic acid bacteria contribute to host
`metabolism. The antimicrobial substances produced by
`these bacteria control
`the proliferation of undesired
`
`pathogens. Lactic acid bacteria produce a soluble com-
`pound which may interact directly with oral tumor cells in
`culture and inhibit their growth [20]. Singh et al. [21] have
`observed that Bifidobacterim longum exerts a strong anti-
`tumor activity against colon cancer. Data from epidemio-
`logical and experimental studies indicate that ingestion of
`lactobacilli and bifidobacteria and their fermented products
`reduce the risk of certain types of cancer and inhibit tumor
`growth [22, 23]. In the present work,
`the counts of
`
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`342
`
`Fig. 3 Correlation between
`Streptococcus (CFU 9
`103.ml-1) count and tumour
`volume.
`
`Indian J Microbiol (July–Sept 2011) 51(3):338–344
`
`Table 2 Biochemical reactions of the species of genus Lactobacillus
`
`Biochemical
`test
`
`Species of lactobacilli
`
`L.
`acidophilus
`
`L.
`lactis
`
`L.
`casei
`
`L.
`jensenii
`
`L.
`brevis
`
`Fructose
`
`Galactose
`
`?
`
`?
`
`Glucose (acid) ?
`
`Glucose (gas)
`
`Gluconate
`
`Lactose
`
`Maltose
`
`Mannitol
`
`Melizitose
`
`Melibiose
`
`-
`
`-
`
`?
`
`?
`
`-
`
`-
`
`-
`
`?
`
`?
`
`?
`
`-
`
`-
`
`?
`
`-
`
`-
`
`-
`
`-
`
`?
`
`?
`
`?
`
`-
`
`?
`
`-
`
`d
`
`?
`
`?
`
`-
`
`?
`
`?
`
`?
`
`-
`
`-
`
`-
`
`?
`
`-
`
`-
`
`-
`
`?
`
`?
`
`?
`
`?
`
`?
`
`?
`
`?
`
`?
`
`-
`
`?
`
`development of illness such as cancer is associated with
`significant shifts in the number of gram negative bacteria
`detectable in oral samples [24]. Nagy et al. [6] have
`demonstrated a difference in the micro-flora associated
`with the surface of tumors in comparison to control.
`Patients with oral cancer tend to possess significantly high
`concentration of certain bacteria in their saliva. Many
`bacterial species have been similarly tested for their car-
`cinogenic potential in monkeys, rats, hamsters, rodents and
`mice. The most carcinogenic bacteria are streptococci
`including Streptococcus mutans, S. sobrinus, S. cricetus,
`and S. rattus; and other carcinogenic bacterial species
`include Actinomyces naeslundii, (formerly A. viscosus),
`S. salivarius, S. sanguis, and Entrococcus faecalis [15].
`The mutants of streptococcus (S. cricetus) are known to
`cause ‘‘caries’’ [25]. The present study recorded high
`counts of streptococci in oral system of tumor bearing
`Hamsters (Table 1). The contribution of probiotic bacteria,
`such as lactobacilli and bifidobacteria are mainly in the
`control of pathogenic microbes, that cause various diseases
`through production of antibacterial protein namely bacte-
`riocin [26, 27] and anti-cancer substances [28]. The dietary
`supplements of lactobacilli are reportedly decreased the
`induction of experimental colon cancer [29]. They stim-
`ulate and modulate the mucosal
`immune system by
`reducing the production of pro-inflammatory cytokines
`through actions on NFjB pathways, increasing produc-
`tion of anti-inflammatory cytokines such as IL-10 and
`host defense peptides such as b-defensin 2, enhancing
`IgA defenses and influencing dendritic cell maturation.
`Modulation of cell proliferation and apoptosis through
`cell
`responses to,
`for example, microbially produced
`short chain fatty acids [30]. The present study expected
`the DMBA induced oral cancer could change the chem-
`istry of the mouth, allowing the bacteria to flourish. It
`may be due to the cellular leakage of cancer tissues. The
`
`Raffinose
`
`Ribose
`
`Xylose
`
`Esculin
`
`Oxidase
`
`Catalase
`
`Gelatin
`liquified
`
`Nitrate
`reduction
`
`Casein
`hydrolysis
`
`Indole
`
`H2S
`
`-
`
`-
`
`-
`
`?
`
`-
`
`-
`
`?
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`?
`
`-
`
`-
`
`-
`
`-
`
`-
`
`?
`
`-
`
`?
`
`-
`
`-
`
`?
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`?
`
`-
`
`-
`
`-
`
`-
`
`-
`
`-
`
`d
`
`d
`
`-
`
`-
`
`?
`
`-
`
`-
`
`-
`
`-
`
`? Positive,- Negative, d Variable
`
`lactobacilli and bifidobacteria were high in normal and tea
`extract
`treated animals when compared to streptococci
`which were low in number (Table 1).
`Pathogenic forms of oral cavity bacteria have recently
`been known to associate with cancer
`tissues. The
`
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`343
`
`Table 3 Carbohydrate reactions of the species of genus Bifidobacterium
`
`Species
`
`Sugars
`
`Arabinose
`
`Xylose
`
`Ribose
`
`Glucose
`
`Cellulose
`
`Lactose
`
`Mannitol
`
`Melibiose
`
`Starch
`
`Bifidobacterium bifidium
`
`Bifidobacterium longum
`
`-
`
`?
`
`-
`
`?
`
`-
`
`?
`
`-
`
`-
`
`?
`
`-
`
`?
`
`?
`
`-
`
`-
`
`-
`
`?
`
`-
`
`-
`
`Table 4 Some important tests used to differentiate Streptococcus
`species
`
`Species Growth
`
`Hae Raf Suc Inu
`
`10°C 45°C 2%
`NaCl
`
`6.5%
`NaCl
`
`S. mitis -
`S.
`mutans
`
`-
`
`?
`
`-
`
`?
`
`?
`
`-
`
`-
`
`pH
`9.5
`
`-
`
`-
`
`D
`
`D
`
`? ? ?
`
`- - -
`
`Hae Haemolysis, Raf Raffinose, Suc Sucrose, Inu Inulin
`
`types of bacteria present in the saliva are depending upon
`the conditions of the saliva. The alkaline condition of the
`saliva was observed in DMBA treated tumor bearing
`
`animals, whereas acetic and near neutral pH was
`observed in DMBA ? tea treated animals. So the lac-
`tobacillus and bifidiobacteria cannot survive in the tumor
`bearing animals as they grow in acetic condition. It is
`inferred that
`the reduction in the beneficial bacterial
`counts increased the pathogenic bacterial counts in buccal
`pouch of Hamsters, and this situation made the animal
`weak and susceptible to incidence of tumor (Tables 1 and
`5). The changes in bacterial flora are not
`the direct
`effects of DMBA treatment as proved by anti microbial
`method using disk diffusion method. DMBA showed no
`antimicrobial activity (data not shown). Therefore the
`DMBA effects on bacterial flora are indirect perhaps
`through metabolic changes induced in the test animals.
`
`Fig. 4 Histological changes in
`the mucus tissue of Hamster
`buccal pouch. Untreated control
`(A), treated with DMBA
`(B) treated with DMBA ?
`mangrove tea (C) and mangrove
`tea alone (D) (magnification
`409)
`
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`1:1–11
`
`Table 5 Ratio between beneficial (lactobacilli, bifidobacteria) and
`harmful (streptococcus) bacteria and incidence of tumour volume in
`buccal pouch of Hamsters
`
`Ratio between
`beneficial and harmful
`bacteria(CFU 9103.ml-1)
`
`Tumor
`volume
`(ml)
`
`Treatment
`
`Control
`
`DMBA
`
`1.93 ± 0.15a
`0.68 ± 0.05b
`1.60 ± 0.13a
`
`DMBA? Tea extract
`(1.25%)
`Tea extracts alone (1.25%) 3.14 ± 0.28a
`
`0.00a
`2.45 ± 0.2b
`0.00a
`
`0.00a
`
`Values not sharing a common superscript are differ significantly at
`P [ 0.05
`
`Acknowledgments The authors are thankful to the authorities of
`Annamalai University for having provided with facilities.
`
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