Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241116EnglishN2019March30HealthcareA Study of Histopathological Spectrum of Non Neoplastic Urothelial Lesions
English0107Nisha AttriEnglish Madan Kumar SolankiEnglishBackground: Specimens from the bladder are commonly encountered by the general surgical pathologist. Emphasis is usually placed on the neoplasms of bladder, particularly if malignant, owing to their bad prognosis. A good command of benign lesions occurring in the bladder, and knowledge of their preneoplastic potential will help pathologists to confidently diagnose malignancy versus its benign mimickers and guide the urologists in choosing the appropriate therapy for the patient.
Aims & Objectives: To present a mixture of benign entities, and discuss their histologic characteristics, hoping to provide a practical review for the general surgical pathologist.
To discuss group of benign disorders that can cause diffuse bladder wall thickening and might be misdiagnosed as malignancy on cystoscopy.
Material and Methods: A retrospective study of 10 years was carried out on bladder biopsies received from Jan 2004 to Dec 2014 at a tertiary care hospital in mumbai. The histopathological records of patients were screened for collection of data like age, sex, clinical history, gross and microscopy. The final diagnosis was given on routine Hematoxylin & Eosin stained sections.
Results: Total 123 cases of non neoplastic bladder lesions were studied, out of which 69 were males and 54 were females with male to female ratio being 1.3:1.The spectrum of non neoplastic pathological lesions included cystitis, nephrogenic adenoma, BCG induced granuloma, malakoplakia and endometriosis. Cystitis was the most common finding. Most common age group affected by the non neoplastic lesions was 41-50 years of age.
Conclusion: A wide variety of benign entities are present in the bladder and are more commonly encountered by the general surgical pathologist. Knowledge of the histologic characteristics of these lesions, their preneoplastic potential, and the possible pitfalls can help the pathologist serve the patient better.
EnglishBladder, Benign, Cystitis, Histopathological spectrum, Non neoplasticINTRODUCTION
Non-neoplastic urinary bladder lesions, are responsible for substantial morbidity throughout the world. The non-neoplastic lesions in particular cystitis comprise an imperative source of symptoms and signs. These diseases are considered more disabling than fatal. Precise diagnosis of urinary bladder lesions requires concurrent data from urology, radiology and surgical pathology. After cystoscopy, histopathology is considered as the primary diagnostic tool for patients who are suspected of having bladder tumours because there is direct light microscopic visualization of the bladder mucosa pathology. Emphasis is usually placed on the neoplasms of bladder, particularly if malignant, owing to their bad prognosis. A good command of benign lesions occurring in the bladder, and knowledge of their preneoplastic potential will help pathologists to confidently diagnose malignancy versus its benign mimickers and guide the urologists in choosing the appropriate therapy for the patient.
AIMS & OBJECTIVES
To present a mixture of benign entities, and discuss their histologic characteristics, hoping to provide a practical review for the general surgical pathologist.
To discuss group of benign disorders that can cause diffuse bladder wall thickening and might be misdiagnosed as malignancy on cystoscopy.
MATERIALS AND METHODS
A retrospective study was done over a period of ten years in the Department of Pathology, T.N.M.C & B.Y.L Nair Ch. Hospital, Mumbai from 1st Jan 2004 to 31st Dec 2014 and included 123 cases. The material for the study was comprised of biopsies from Transurethral resection of bladder tissue (TURBT). The biopsy specimens were received in 10% formalin. Data was collected in a preset performa. The histopathology records of patients were screened for collection of data like age, sex, clinical history, gross, microscopic findings and final diagnosis. All details of the patients were used anonymously and identity of the subject was not revealed anywhere in the study. The entire tissue was processed in all the cases. Routine processing was done starting from fixation, dehydration, embedding and section cutting. Staining was done by Hematoxylin and eosin . Histopathological examination of specimens was carried out and the lesions were classified into various types on light microscopy. Data was analyzed and tabulated for evaluation.
INCLUSION CRITERIA
All the TURBT biopsies received in the Department of Pathology, T.N.M.C & B.Y.L. Nair Ch. Hospital, Mumbai from Jan 2004-Dec 2014.
Adequacy of biopsy sample.
EXCLUSION CRITERIA
Autolysed specimen
Inadequate biopsies.
.All patients with incomplete data regarding clinical profile and histopathological reports were excluded from study.
RESULTS
A total of 123 urinary bladder biopsies were studied in a period 10 years. Hematuria was the most common clinical symptom in 28 cases followed by dysuria in 21 cases, increased frequency in 16 cases and burning micturition in 15 cases.
Age of the patients in our study ranged from 41 years to 80 years with a
peak age incidence of 66 cases in the 4th decade followed
by 35 cases in the 5th decade. Least number of cases were seen
in 7th and 8th decade.
Males were more commonly affected than females. In the present
study there were 69 male patients (56.1%) and 54 females (43.9%)
with M:F ratio of 1.3:1.
Among the 123 cases of non-neoplastic lesions, majority were of papillary/polypoidal cystitis (35.77%). Other conditions in descending order were chronic non specific cystitis (23.58%), tubercular cystitis (17.8%), acute cystitis (10.57%), follicular cystitis (1.63%), acute on chronic cystitis (1.63%), nephrogenic adenoma (1.63%), BCG induced granuloma (1.63%), malackoplakia (0.82%), haemorrhagic cystitis (0.82%) and endometriosis (0.82%).
Average non neoplastic cases were 10-20 per year.
Non neoplastic lesions were most commonly seen in the 4th decade. Amongst the total 123 non neoplastic cases, 53.65% were seen in the 4th to 5th decade.
Out of the 123 non neoplastic cases, 56.1% were males and 43.9% were females.
Out of 123 non neoplastic lesions patients mostly presented with hematuria (38 cases), dysuria (26 cases), frequency (21 cases) and burning micturition (19 cases).
Out of 123 non neoplastic cases, majority were of papillary / polypoidal cystitis (35.77%).Other conditions in descending order were chronic non specific cystitis(23.58%), tubercular cystitis(17.8%), acute cystitis(10.57%), follicular cystitis(1.63%), acute on chronic cystitis(1.63%), nephrogenic adenoma (1.63%), BCG induced granuloma (1.63%) , malackoplakia (0.82%), haemorrhagic cystitis (0.82%), and endometriosis (0.82%)
Out of 88 cases of cystitis, 14.6% of the cases showed von Brunn’s nests.
DISCUSSION:
The term 'von Brunn's nests' refers to the presence of nests of transitional cells in the lamina propria, detached from the overlying urothelium. These nests arise by a process of invagination from the overlying urothelium and the term von Brunn's buds can be used when they are attached to the urothelium. It is the detachment from the overlying epithelium that can be problematic, particularly if the nests lie deeper in the lamina propria and are numerous.
Wiener et al and Volmar et al reported the occurrence of von Brunn nests in 89% of normal bladder mucosa. (1) (2). In our study, 14.6% of the cases of cystitis showed von Brunn’s nests. Srikousthubha et al had also shown the association of von Brunn’s nest with cystitis, which is in consonance with our observation (3). (Table-6) (n=18) (FIGURE-1)
Hameed and Humphrey reported cystitis cystica as a group of urothelial disorders featuring cystic cavities in the center of the von Brunn nests and dilated cystic cavities lining the true urothelium while the term cystitis glandularis was attributed to the glandular epithelium with mucosal cells mostly lining the cystic cavity, with cuboidal or columnar epithelium (4). In autopsy cases Harik and O' Toole reported cystitis cystica and glandularis in 60% of the normal bladder mucosa (5). Hameed and Humphrey emphasized that there is no relationship between the appearance of lesion in the bladder and the occurrence of bladder cancer (4).
In contrast to the typical form of cystitis glandularis, during the present study it was found that the cells lining the glands in the intestinal type cases were tall and columnar with abundant mucin. These cells were admixed with goblet cells and the epithelium resembled to that of the intestine. The two types of cystitis glandularis may coexist. In the present study, 3.3% cases of the non neoplastic lesions showed cystitis cystica and cystitis glandularis. (n=4) (FIGURE-2,3 &4)
Cystitis (91.8%) was documented as the most common non neoplastic lesion during the present study. There were cases of papillary polypoidal cystitis, tubercular cystitis, follicular cystitis, haemorrhagic cystitis and chronic non specific cystitis. In a study done in India by Kumar et al, 35% cases of cystitis were reported (6). Vaidya et al, in their study found 58.95% cases of chronic non specific cystitis (7). Srikoustubha et al in their study reported 25 non neoplastic cases and 21 cases (84%) of inflammatory lesions (3). Out of the 21 inflammatory lesions,15 cases (71.4%) were of polypoid chronic non specific cystitis, 3 cases (14.28%) were of tubercular cystitis and 1 case (4.76%) each of follicular cystitis, malackoplakia and squamous metaplasia. (n=113)
Papillary polypoidal cystitis refers to an exophytic , inflammatory lesion that occurs mostly due to the presence of indwelling catheters and is characterised histologically by normal to hyperplastic urothelium overlying a congested, chronically inflamed, and edematous stroma. Although these exophytic lesions grossly mimic a papillary neoplasm, they can be easily distinguished as an inflammatory pseudopolyp on microscopy. It usually arises when inflammation accompanied by edema leads to the papillae or polyp formation. The term papillary cystitis refers to when thin finger-like papillae are present, and polypoid cystitis when the lesions are edematous and broad based. Consistent with the several studies, our study encountered 44 cases (35.77%) of papillary polypoidal cystitis. Most of the cases had history of catheterisation and showed thickened bladder walls on sonography and congested mucosa on cystoscopy. Microscopically, lamina propria showed edema and chronic inflammatory cell infiltrate. (n=44) (FIGURE-5,6)
Another entity, tubercular cystitis was also encountered. The kidneys are the most common site of infection in genitourinary tuberculosis and are infected through hematogenous spread of the bacilli. Bladder lesions are without exeption secondary to renal tuberculosis. The diagnosis is based on culture studies by isolation of the causative organism, but biopsy occasionally may be required. In the present study all the 22 cases (17.8%) of tubercular cystitis showed the characteristic epithelioid cell granulomas, histiocytes, lymphocytes and langhan’s type of giant cells. As many as 5 cases (4.1%) showed caseous necrosis. Vaidya et al, in their study found 0.93% cases of Tubercular cystitis (7). Appropriate stains should reveal the presence of acid-fast bacilli (8). (n=22) (FIGURE-7)
Another entity, follicular cystitis was used to describe the presence of lymphoid follicles with germinal centers in the wall of the urinary bladder (9). The term is a misnomer because it does not necessarily indicate inflammation. However, it does sometimes follow infection, repeated transurethral biopsies, or the instillation of intravesical chemoherapeutic agents or BCG. In the present study it was encountered in 1.63% cases of non neoplasic lesions. (n=2) (FIGURE-8)
Haemorrhagic cystitis which was known to occur from intravesical instillation of chemotherapeutic agents such as thiotepa and mitomycin C was also encountered. These agents induce cytologic atypia (which can be confused with CIS), including cytomegaly, pleomorphism, and hyperchromasia (10). These changes were limited to the superficial cells and mitotic activity was not evident. Microscopically, the bladder was characterized by marked edema and haemorrhage throughout the lamina propria, with ulceration and fibrinopurulent exudate. During the regenerative stage, macrophages and fibroblasts occupied the lamina propria, whereas the overlying epithelium exhibited an increased mitotic rate, increased thickness, and marked atypia (11) (12) . Presently it was diagnosed in 0.82% of the non neoplastic cases examined. (n=1) (FIGURE-9)
Malakoplakia, reported to be a rare chronic inflammatory disease ,was first described by Michaelis and Gutmann in 1902. It is known to have a female predominance with 50 years as an average age of presentation (13). From etiology point of view malakoplakia has been reported to represent a peculiar response to infection which has been attributed to disturbed immune response or abnormal macrophage or lysosomal activity in the host (14) (15). Its occurrence has been reported in the renal pelvis, ureter and urethera (16). In the present study, only one case (0.82%) of malakoplakia was encountered in a 45 years old female, which on microscopy showed Michaelis Gutmann bodies and numerous histiocytes infiltrating the lamina propria. (n=1) (FIGURE-10)
Nephrogenic adenoma , a metaplastic lesion was characterized by aggregates of cuboidal or hobnail cells with clear or eosinophilic cytoplasm and small nuclei without prominent nucleoli (17). These cells lined thin papillary fronds on the surface or formed tubular structures within the lamina propria of the bladder . It was fairly well circumscribed and did not extend into the muscularis propria. It was thought to result from an inflammatory insult or local injury (18). It was originally described in the trigone and given its name because it was thought to arise from mesonephric rests. This process has generally been thought to be metaplastic and the terms 'nephrogenic metaplasia' or adenomatous metaplasia' are preferred by some. Recently Mazal et al. studied cases of nephrogenic adenoma in kidney transplant recipients (19). Based on fluorescent in situ hybridization (FISH) and lectin studies, it was concluded that these lesions were derived from shed tubular cells originating from the transplanted kidney rather than a metaplastic process. Other authors have come to the same conclusion, based on other data, including the expression of PAX2 (20).
However several features of the tubular component of nephrogenic adenoma may pose particular diagnostic difficulty and merit emphasis. Tiny tubules containing mucin and apparently lined by a single cell with a compressed nucleus may simulate signet cells. The haphazard distribution of the tubules may simulate the appearance of invasive adenocarcinoma. Oliva and Young reviewed the microscopic appearances of 80 cases with unusual nephrogenic adenoma of the urinary tract . Only 2 cases (1.63%) were documented in the present study. (n=2) (FIGURE-11)
Urothelial carcinoma in situ (CIS) and high risk papillary carcinomas were commonly treated with intravesical installations of BCG which induced an intense inflammatory reaction (21). It was characterized by the presence of non caseating epithelioid cell granulomas and multinucleated giant cells. The granulomas were situated in the superficial third of the lamina propria and were associated with an intense lymphocytic infiltrate. In the present study only 1.63% of the non neoplastic lesions were documented as BCG induced granulomas. (n=2)
Genitourinary involvement by endometriosis was seen in 1% to 2% of cases. More than 200 cases of vesical endometriosis have been described, making bladder the most common site of involvement within the urinary tract (22) . It affected women between the second and fifth decades of life, but rarely seen in postmenopausal women receiving exogenous estrogen . Interestingly, rare cases of vesical endometriosis have been reported in men with prostate carcinoma who were receiving exogenous estrogen therapy (23) (24). The occurrence in men most likely represented activation of müllerian rests by exogenous estrogens.
Clinically, patients presented with urgency, frequency, suprapubic pain, and rarely hematuria. A mass was frequently apparent either by palpation or cystoscopic examination (25) . Microscopically, the lesion resembled endometriosis elsewhere; endometrium-like glands seen in association with endometrial stromal cells and recent or old hemorrhage. Only 1 case of endometriosis was encountered during this study. (n=1) (FIGURE-12)
CONCLUSION: A wide variety of benign entities present in the bladder are more commonly encountered by the general surgical pathologist. Knowledge of the histologic characteristics of these lesions, their preneoplastic potential, and the possible pitfalls can help the pathologist serve the patient better.
ACKNOWLEDGEMENTS; Special thanks to Dr. Vinaya B.Shah for her expert guidance and to the Head, Department of Pathology, T.N.M.C & B.Y.L. Nair Hospital, Mumbai for providing with all necessary facilities. Authors also acknowledge the immense help received from the scholars whose articles are cited and included in references. The authors are grateful to editors and publishers of all those articles , journals and books from where the literature for this article has been reviewed and discussed.
CONFLICTING INTERESTS; None
FUNDING; None
Englishhttp://ijcrr.com/abstract.php?article_id=2588http://ijcrr.com/article_html.php?did=25881. Wiener DP, Koss LG, Sablay B, Freed SZ. The prevalence and significance of Brunn’s nests, cystitis cystica and squamous metaplasia in normal bladders. J Urol. 1979 Sep;122(3):317–21.
2. Volmar KE, Chan TY, De Marzo AM, Epstein JI. Florid von Brunn nests mimicking urothelial carcinoma: a morphologic and immunohistochemical comparison to the nested variant of urothelial carcinoma. Am J Surg Pathol. 2003 Sep;27(9):1243–52.
3. Srikousthubha, Sukesh, C V R, Hingle S. Profile of lesions in cystoscopic bladder biopsies: a histopathological study. J Clin Diagn Res. 2013 Aug;7(8):1609–12.
4. Hameed O, Humphrey PA. Pseudoneoplastic mimics of prostate and bladder carcinomas. Arch Pathol Lab Med. 2010 Mar;134(3):427–43.
5. Harik LR, O’Toole KM. Nonneoplastic Lesions of the Prostate and Bladder. Arch Pathol Lab Med. 2012 Jul;136(7):721–34.
6. Kumar M, Yelikar BR. Spectrum of Lesions in Cystoscopic Bladder Biopsies -A Histopathological Study. Ai Ameen J Med Sci. 2012;5(2):1–3.
7. Vaidya S, Lakhey M, K C S, Hirachand S. Urothelial tumours of the urinary bladder: a histopathological study of cystoscopic biopsies. JNMA J Nepal Med Assoc. 2013;52(191):475-8.
8. Lage JM, Bauer WC, Kelley DR, Ratliff TL, Catalona WJ. Histological parameters and pitfalls in the interpretation of bladder biopsies in bacillus Calmette-Guerin treatment of superficial bladder cancer. J Urol. 1986 May;135(5):916–9.
9. Sarma KP. On the nature of cystitis follicularis. J Urol. 1970 Nov;104(5):709–14.
10. Murphy WM, Soloway MS, Lin CJ. Morphologic effects of thio-TEPA on mammalian urothelium. Changes in abnormal cells. Acta Cytol. 1978;22(6):550–4.
11. Philips FS, Sternberg SS, Cronin AP, Vidal PM. Cyclophosphamide and urinary bladder toxicity. Cancer Res. 1961 Dec;21:1577–89.
12. Beyer-Boon ME, de Voogt HJ, Schaberg A. The effects of cyclophosphamide treatment on the epithelium and stroma of the urinary bladder. Eur J Cancer.1978 Oct;14(10):1029-35.
13. Michaelis L GC. Einschlusse in blastentumoren. Z Klin Med. 1902;47:208–15.
14. Damjanov I, Katz SM. Malakoplakia. Pathol Annu. 1981;16(Pt 2):103–26.
15. Stanton MJ, Maxted W. Malacoplakia: a study of the literature and current concepts of pathogenesis, diagnosis and treatment. J Urol. 1981 Feb;125(2):139–46.
16. Sharma TC, Kagan HN, Sheils JP. Malacoplakia of the male urethra. J Urol. 1981 Jun;125(6):885–6.
17. Bhagavan BS, Tiamson EM, Wenk RE, Berger BW, Hamamoto G, Eggleston JC. Nephrogenic adenoma of the urinary bladder and urethra. Hum Pathol. 1981 Oct;12(10):907–16.
18. Stilmant MM, Siroky MB. Nephrogenic adenoma associated with intravesical bacillus Calmette-Guerin treatment: a report of 2 cases. J Urol. 1986 Feb;135(2):359–61.
19. Mazal PR, Schaufler R, Altenhuber-Müller R, Haitel A, Watschinger B, Kratzik C, et al. Derivation of Nephrogenic Adenomas from Renal Tubular Cells in Kidney-Transplant Recipients. N Engl J Med. 2002 Aug 29;347(9):653–9.
20. Rahemtullah A and Olivia E 2006. Nephrogenic Adenoma: An update on an innocuous but troublesome entity. Ad Anat Pathol. 2006;13:247–55.
21. Young RH, Bostwick DG. Florid cystitis glandularis of intestinal type with mucin extravasation: a mimic of adenocarcinoma. Am J Surg Pathol. 1996 Dec;20(12):1462–8.
22. Melamed M.R. VNG and GH. The urinary sediment cytology in a case of malakoplakia. Acta Cytol. 1962;6:471–4.
23. Lewin KJ, Fair WR, Steigbigel RT, Winberg CD, Droller MJ. Clinical and laboratory studies into the pathogenesis of malacoplakia. J Clin Pathol. 1976 Apr;29(4):354–63.
24. Nieh PT, Althausen AF. Malacoplakia of the ureter. J Urol. 1979 Nov;122(5):701–2.
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241116EnglishN2019March30Life SciencesSeasonal and Geographical Variations in Antimicrobial Activity of Selected Mangroves from Krishna Estuary
English0815Vijaya Kumar KovvadaEnglish Rosaiah GorrepatiEnglish Babu KakumanuEnglish Tirupathi Swamy NattalaEnglish Rajesh ButtiEnglishObjective: To determine the seasonal and geographical impact on plant secondary metabolite production and antimicrobial activity.
Methods: Three mangrove plants viz., Ceriops tagal, Bruguiera cylindrica and Lumnitzera racemosa commonly found in Gilakaladindi and Malakayalanka regions of Krishna Estuary, Andhra Pradesh, India were selected for the present study. The study was done during 2014-15 summer, rainy and winter in both the stations. Leaf samples were extracted with methanol and subjected to preliminary phytochemical screening. The methanolic leaf extracts were tested against bacterial pathogens Bacillus cereus, B. subtilis, Staphylococcus aureus, Escherichia coli and a fungal pathogen Candida albicans.
Results: The methanolic extracts of leaves collected during rainy season showed maximum zone of inhibition at both the stations Gilakaladindi and Malakayalanka. Among the studied plants leaf methanolic extract of C.tagal was recorded maximum antibacterial activity against B. subtilis and B. cereus (18±0.05 mm), followed by Bruguiera cylindrica both at Gilakaladindi and Malakayalanka respectively. But the extracts of L.racemosa showed less inhibition activity (7±0.00 mm) in all the seasons at both the stations.
Conclusion: The study demonstrated that mangroves produce bio-active compounds optimally in rainy season that are effective against microbial proliferation which may further help in synthesis of natural antibiotics in place of commercial antibiotics to heal infectious diseases.
EnglishGilakaladindi, Malakayalanka, Phytochemicals, RainyINTRODUCTION
Mangroves are a specific group of plant communities, habituated to adverse environments includes high salt content, relative humidity with fluctuated water currents.[1] These contrary conditions consequently results in reduced photosynthetic rate and thereby reduced growth.[2,3] Of all the stressors soil and water salinity inflict more on plant development especially in semiarid and arid regions.[4] In order to cope up with these unfavorable conditions, mangrove switch on to different modifications at morphological, physiological, biochemical and molecular levels.[5] Production of secondary metabolites is one of such physiological change which tends the plant to protect themselves against harmful microorganisms along with grazing threat.[6] Bryant et al.[7] have hypothesized that when plants are stressed, an exchange occurs between carbon to biomass production or formation of defensive secondary compounds.
The local geo-climate, seasonal changes, external factors such as light, temperature and humidity may affect composition of secondary metabolites. The synthesis of secondary metabolites in response to environmental factors is nothing but an adoptable strategy leading to tolerance to abiotic stress.[8,9] On the other side the mangrove plant extracts have proven activity against human, animal and plant pathogens and have been used in folklore medicine since ages.[10,11,12] Till now more than 200 bio-active compounds have been isolated from true mangroves of tropical and subtropical population.[13] As said, seasonal changes influence much on production of secondary metabolites which is less explored and badly overlooked. On the other side right season for the collection of leaf material to extract maximum secondary metabolites is inevitable in order to get much benefit. Krishna estuary is one such neglected area with less exploration specifically on seasonal changes. The Krishna deltaic region is in subtropical humid climate with hot summers and moderate winters, which get annual rainfall due to south west monsoon.[14,15] Gilakaladindi and Malakayalanka are the prominent biodiverse mangrove reserves in Krishna estuary on East Coast of India located at latitudes 16° 8?56.1624??, 15°58?34.5354 and longitudes 81°9?46.029??, 81°5?53.3616?? respectively. Coupled with their richness and diversity mangroves of Gilakaladindi and Malakayalanka have also physiological peculiarities as a result of environmental conditions to which they belong. Each species of mangroves has a particular range of tolerance to environmental factors.[16, 17]
The present study aims at identify the seasonal and geo-climatic changes on secondary metabolite production in terms of antimicrobial activity on selected mangrove species Lumnitzera racemosa, Ceriops tagal and Bruguiera cylindrica.
MATERIALS AND METHODS
Collection of Plant Material
Leaf samples were collected during summer (March to May) rainy (July to September) coinciding with South west monsoons and in winter season (October to February). Two different sites were chosen and the distance between the sites was about 25 kms. Healthy and fresh leaves were collected from selected three mangroves i.e. Lumnitzera racemosa, Ceriops tagal and Bruguiera cylindrica of Krishna estuary. The collected leaves were washed with tap water and double distilled water until dust is removed from surface of the leaves. The leaves are shade dried at room temperature. Dry leaf material was powdered with the help of mechanical grinder and sieved.
Extraction
The leaf powders were extracted with 80% methanol using soxhlet apparatus for about 18 hours. The crude extracts obtained were evaporated and concentrated by using vacuum rotary evaporator (Buchi Labortech Ag, model I, R-215) under reduced pressure. The dried extracts were preserved at 4°C until further use.
Antimicrobial screening
The antibacterial activity of the obtained leaf extracts was carried out by agar well diffusion method. Nutrient agar (NA) was used for culturing the test bacteria. Nutreint agar medium was sterilized at 15 lbs pressure (121 °C) for 15 min, later cooled and inoculated with 0.1 ml of test bacterial suspension. The inoculated medium was poured into petri plates under aseptic conditions. After solidification, wells of about 5 mm diameter were made with sterilized cork borer [18]. Solvent extract (50 μl, 100 μl and 150 μl) was added to each well and the addition of solvent alone served as control. The inoculated plates were incubated at 30 °C and the diameter of the inhibition zone was measured after 24 h on bacterial pathogens such as Staphylococcus aureus (MTCC 3160), Bacillus subtilis (ATCC 6633), Bacillus cereus (MTCC 430) Escherichia coli (MTCC 43) and fungal pathogen Candida albicans (ATCC 10231).
Phytochemical screening
Shade dried plant material was extracted with 80% methanol and were tested for qualitative phytochemical screening. [19,20]
Alkaloids
The methanolic extract was evaporated to dryness and the residue is dissolved in 1% HCl. To this solution Mayer’s reagent was added. Appearance of any precipitate or turbidity indicates the presence of alkaloids.
Saponins
The plant extract is evaporated to dryness. Tap-water was added and shaken vigorously. Formation of persistent foam of about 2 cm is taken as a positive reaction.
Terpenoids and steroids
A 50% H2SO4 is added along the sides of the test tube containing a mixture of methanolic HCl and acetic anhydride. If there is any change in color, from green to blue-green (sometimes via red or blue) indicates the presence of terpenoids and steroids.
Tannin
The methanolic extract is evaporated to dryness and the residue was dissolved in water and tested with 1% gelatin solution and 1% gelatin salt solution (1 g) gelatin dissolved in 10 g of NaCl (w/w) to separate volumes. The appearance of white precipitate will be regarded as a positive reaction.
Anthocyanin
To the plant extract was added equal volume of methanolic HCl. Appearance of red or purple color indicates the presence of anthocyanidins.
Phenolic compounds
The formation of intense color in the extract, on adding 1-2 drops of 1% ferric chloride to the extract is considered as a positive reaction test.
Flavonoids
Few ml of methanolic extract is added with conc. HCl and Mg powder. The presence of flavonoids can be identified by the development of pink or magenta or red colored foam.
Coumarin
To the methanolic extract, a few drops of alcoholic sodium hydroxide were added. Formation of yellow color indicated the presence of coumarins.
Quinones
To the 1 ml of methanolic extract, 1 ml of conc. H2SO4 was added. Formation of red color shows the presence of Quinone’s.
Resins
Plant extracts were treated with acetone. To this, small amount of water was added and shaken. The appearance of turbidity indicates the presence of resins.
Test for glycosides
To the methanolic extract a little amount of anthrone was added. To this a few drops of conc. H2SO4 was added and warmed gently over water bath. The presence of glycosides was identified by dark green color formation.
Soil and Water analysis
Field data like temperature, salinity and pH were recorded during morning to noon. Water salinity was estimated with the help of hand refractometer and pH was measured using Elico pH meter (model L.C-120). Data was collected on five plants of each species growing in different locations in both the sites. Soils are air-dried, crushed with a wooden mallet, passed through a 2 mm sieve and were analyzed for inorganic ions using the standard procedure.[21] Saturation extracts of soils are analyzed for electrical conductivity using an electrical conductivity meter. Soil pH was determined on air-dried samples using a 1: 2.5 soil to 1 M KCl ratio. Exchangeable Na+ Ca2+, Mg2+, K+, P, Zn2+, Cu2+ and Mg2+ present soil and water samples were analyzed. [22]
RESULTS
The methanolic leaf extracts were screened for various phytochemical constituents and represented in Table 1. The Phytochemical analysis of the methanol extracts revealed the presence of alkaloids, flavanoids, phenols, resins, saponins and tannins in all the studied plants of both Gilakaladindi and Malakayalanka estuaries in all the seasons (Table 1). But the existence of anthocyanins, quinines, glycosides and terpenoids were not reported in L. racemosa in all the seasons at both the stations. Along with the L. racemosa the presence of anthocyanins and quinines not observed in B. cylindrica in all the seasons. The existence of flavonoids also not recorded in B. cylindrica in all the seasons at both the stations. On the other side the presence of coumarins and glycosides not observed in C. tagal in both the stations during all the seasons. These variations in presence or absence of phytochemicals among three selected mangroves may correspond to different bioactive constituents and may be responsible for variations in antimicrobial potency among the species.
Antimicrobial activity
Antibacterial and antifungal activities of selected mangrove plants were studied and analyzed. The present study revealed the significant influence of seasons on potential antimicrobial activity of plant extracts. All the test organisms responded varied to different plant extracts at different concentrations of crude extracts i.e 50 μl, 100 μl, 150 μl and maximum inhibitory action was obtained at 150 μl.
Summer season
The leaf methanolic extracts of Gilakaladindi showed significant inhibiton activity. The plant extracts of mid-summer reported antimicrobial activity ranged from 7-11 mm with a mean average of 9.06 mm at 50 μl concentration. At 100 μl plant extract concentration the sensitivity of test organisms ranged from 8-13 mm with an average mean of 10.26 mm. On the other side at 150 μl the maximum inhibition was ranged from 8-15 mm with a mean average 11.00 mm. The extracts of C. tagal showed potential antimicrobial activity against E. coli (11.00±1.25 mm), S. aureus (13.00±1.00 mm) and B. cereus (15.00±0.25 mm) at 50 μl, 100 μl and 150 μl concentrations respectively (Figure 1). In case of Malakayalanka antimicrobial activity ranged between 7-10 mm with a mean average of 8.26 mm at 50 μl concentration. At 100 μl plant extract concentration microbial activity ranged from 7-12 mm with an average mean of 9.00 mm. At 150 μl the maximum zone of inhibition was ranged from 8-14 mm with a mean average 9.66 mm. At 50 μl concentration, leaf extract of C. tagal recorded maximum antimicrobial activity with S. aureus, E. coli and C. albican (10.00±1.102). Test organisms B. subtilis and E. coli showed highest sensitivity (12.00±0.958) when medium diffused with 100 μl leaf methanolic extracts of C. tagal. When all the test organism treated with 150 μl methanolic leaf extracts C. tagal extracts found to be more potential against B. subtilis with a zone of inhibition of 14.00±0.085 (Figure 2). The results of the present study indicated that the methanolic leaf extracts of summer season of Gilakaladindi showed more inhibiton activity when compared to Malakayalanka.
Rainy season
Rainy season showed highest positive antimicrobial activity and antifungal activity in all the plant species during all the seasons in both Gilkaladindi and Malakayalanka stations. The methanolic extracts of peak rainy season of Gilakaladindi, reported a varied antimicrobial activity 9-13 mm with a average mean of 11.13 mm at 50 μl concentration whereas at 100 μl concentration the inhibitory activity differed between 12-16 mm with an average mean of 14.13 mm. At 150 μl concentration of plant extract the maximum sensitivity is occurred between 14-18 mm with a mean average 15.6 mm. Samples collected during rainy season showed maximum inhibitory activity against all the test organisms. At 50 μl and 100 μl concentrations, leaf methanolic extracts of C. tagal showed high microbial activity 13±0.00 and 16±0.45 against B. subtilis and S. aureus respectively. Increased concentration of methanolic leaf extracts i.e 150 μl especially C. tagal extracts potentially increased the inhibitory activity (18.00±0.00) in B. subtilis and B. cereus (Figure 1) (Plate 1 A, B). Samples collected from Malakayalanka are not potentially bioactive when compared with Gilakaladindi samples in combating harmful microorganisms (Figure 2). In case of Malakayalanka antimicrobial activity varied from 8-12 mm with an average mean of 10.46 mm at 50 μl concentration. At 100 μl concentration inhibitory activity ranged from 9-15 mm with an average mean of 12.13 mm. The potential inhibitory activity at 150 μl concentration ranged from 9-15 mm with a mean average 12.8 mm. B. Cereus (12.00±0.12 mm) strains found to be more sensitive against C. tagal and B. cylindrica at 50 μl concentration. Methanolic leaf extract of C. tagal recorded maximum antimicrobial activity with B. subtilis and E. coli (15.00±0.04) at 100 μl concentration. In case of 150 μl concentration leaf extracts of C. tagal recorded maximum antibacterial activity (15.00±0.00) on S. aureus (Plate 1C), and against fungal organism C. albican (14.00±0.02) (Figure 2) (Plate 1D). Results revealed that the leaf methanolic extracts of rainy season are more sensitive against Bacillus subtilis and Bacillus cereus at Gilakadindi and S. aureus and C. albican at Malakayalanka stations.
Winter season
In winter season all the extracts recorded lowest antibacterial and antifungal activity when compared to summer and rainy seasons. In case of Gilakaladindi antimicrobial activity differed between 6-9 mm with a mean average of 7.53 mm at 50 μl concentration. Methanolic leaf extracts 100 μl concentration recorded antimicrobial activity ranged from 8-10 mm with an average mean of 8.6 mm. At 150 μl concentration of leaf extract antimicrobial sensitivity was ranged from 8-12 mm with a mean average of 9.46 mm. Of all the plants highest microbial activity was reported with C. tagal leaf methanolic extracts in all three concentrations. At 50 μl maximum inhibitory activity (9.00±1.24 mm) was observed against B. subtilis, S.aureus, C. albicans whereas at 100 μl concentration strains B. subtilis, C. albicans recorded more inhibition (10.00±0.110). In case of 150 μl concentration, leaf extract of C. tagal recorded maximum antimicrobial activity with B. subtilis (12.00±0.00) (figure 1). Plant extracts collected from Malakayalanka showed relatively less activity when compared to Gilakaladindi. At 50 μl concentration the mean antimicrobial activity was ranged from 6-8 mm, with a mean of 7.20 mm. At 100 μl, 150 μl concentrations antimicrobial activity varied between 6-9 mm, 7-9 mm and with a mean average of 7.93 mm and 8.40 mm respectively. Except E. coli all the strains showed maximum sensitivity (9.00±0.11) towards C. tagal extracts when compared to other two plant extracts.
Nutrient analysis
Both in Gilakaladindi and in Malakayalanka field stations the available soil and water nutrients were measured and tabulated. As compared with Malakayalanka the soil samples of Gilakaladindi contained less nitrogen (S:81 kg/a; R:81 kg/a and W:80 kg/a), more phosphorus (S:1.99 kg/a; R:1.99 kg/a and W:1.98 kg/a), zinc (S:1.015 ppm; R:1.019 ppm and W:1.015 ppm), iron (S:13.79 ppm; R:13.77 ppm and W:13.78 ppm) and copper (S:6.70 ppm; R:6.54 ppm and W:6.65 ppm). The availability of manganese (S:24.0 ppm; R:24.48 ppm and W:23.50 ppm) and sulphur (S:67 ppm; R:68 ppm and W:67 ppm) were found to be more in Gilakaladindi in all the seasons. The availability of all these components were increased during rainy season when compared to summer and winter seasons (Table 2 and Table 3). Electrical conductivity is an important parameter to evaluate the water quality. Any change in the concentration of chlorides and sulphates may influence the changes in the conductivity. In present study significant variation in electrical conductivity was observed in all the seasons (Table 2 and Table 3). The electrical conductivity values were found to be recorded more during summer in both Gilakaladindi (5.75 m.mmhos/cm) and Malakayalanka (7.58 m.mmhos/cm) stations. Among all the components the concentration of CO-3 was increased slightly (5.7 m.e.q/Lts) during winter season in Malakayalanka region. The availability of chlorides, sulphates, sodium, potassium were slightly decreased during rainy seasons both in Gilakaladindi and Malakayalanka regions (Table 2 and Table 3).
DISCUSSION
In mangrove ecosystem nutrients are considered as the significant components that influence growth and development of the plants.[23] and their availability is majorly based upon season, tidal forces and influx of fresh water. In present study also the availability of nutrients found to be increased during rainy season (Table 2). This increased nutrient availability during rainy season is may be due to influx of not only fresh water but also due to organic minerals carried by the rain water. Moreover the availability of nutrients observed to be more at Gilakaladindi as compared with Malakayalanka is because of high tidal currents at Gialakaladindi when compared to Malakayalanka. Frequent changes in water sources from ocean to fresh water or vice-versa may have significant effect on growth and production of bioactive compounds and result in seasonal variations in microbial activity.[24,25] Seasonal variations in protein, polyphenol and tannin concentration in leaves of Rhizophoraceae was reported and the organic constituents proteins, polyphenol and tannin concentration were generally highest in rainy seasons, intermediate in summer and lowest in winter of mangrove leaves.[26] Earlier studies indicated that anti bacterial activity of the leaves of Ceriops tagal showed 9.78±0.32 mm zone of inhibition against Bacillus cereus and 9.25 ±0.84 mm against Staphylococcus aureus[27,28]. Bruguiera cylindica showed zone of inhibition of 6.8 ±0.84mm against Pseudomonas aeruginosa.[29,30] Highest antibacterial activity of aqueous and methanolic extracts of leaves and shoots of Lumnitzera racemosa and Ceriopsis decandra was previously reported.[31,32] In the present study also rainy season samples showed highest overall mean zone of inhibition of 13.62 mm and 11.79 mm. The high concentration of organic constituents in leaf extracts could be the reason for pronounced microbial activity in rainy season. Excoecaria agallocha leaf extracts of rainy season collected from mumbai coast reported highest antimicrobial and anti fungal activity in Klebisella pneumonia, Aspergillus flavus and Phytopthora infestans.[33] In present study also irrespective of location, samples collected during rainy season showed more activity compared to summer and winter samples. In fact samples collected from Gilakaladindi station showed comparatively more antimicrobial activity over samples of Malakayalanka.
CONCLUSION
The present study concluded that the seasonal and geographical variations were found to be significant in production secondary metabolites. Of all the seasons rainy season is relatively more preferable for sample collection followed by summer. As the methanolic leaf extracts of rainy season showed highest inhibitory activity on bacterial and fungal growth at both sampling sites. Among the three mangrove species investigated leaf extracts of Ceriops tagal showed more inhibitory activity followed by Lumnitzera reacemosa and Bruguiera cylindrica. Further research in isolation and quantification of secondary metabolites from the mangrove leaf samples of rainy season would provide information in developing natural antibiotics with therapeutic potential and pharmacological efficacy.
ACKNOWLEDGEMENT
Author acknowledges the immense help received from the scholars whose articles are cited and included in references of this manuscript. The author is also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
Conflict of Interest: Authors have no conflict of interest
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241116EnglishN2019March30Life SciencesAlcohol Dehydrogenase (ADh) Enzyme is a Potent Biochemical Marker for Submergence Tolerance in Rice (Oryza sativa L.) During Seedling Stage of Growth
English1621Biswajit PradhanEnglish Moushree SarkarEnglish Sabyasachi KundagramiEnglishAim: The incorporation of sub 1 gene in high yielding rice mega genotypes indicate that higher amount of Adh enzyme synthesis is controlled by the genetic factors. Adh enzyme will be the one of the vital factor for underwater respiration process of survival. Hence, rice genotypes with high amount of alcohol dehydrogenase enzyme synthesis during anoxic condition in compare with tolerant checks could be good promise for early introduction to the submergence prone area.
Methodology: Fresh and disease free seeds of fifty diverse rice genotypes including tolerant and susceptible checks were taken and germinated. 0.1 gm cut five days old rice seedlings of various submergence dose of day 1, 2, 3, 4 and 5 along with control were evaluated separately in three replications for Adh enzyme estimation by standard protocols with spectrophotometer.
Results: The peak time of Adh enzyme synthesis was during 72 hours to 96 hour of submergence period. Among paddy genotypes Mahananda along with Sabita and Purnendu performed well in compare with tolerant check paddy genotypes.
Conclusion: As it is controlled by genetic factors, so estimation of Adh enzyme activity under submerged stage could be the potent biochemical marker for the screening for paddy genotypes for seedling stage of crop growth during flash flooding condition.
EnglishAlcohol dehydrogenase enzyme, Rice, Submergence, Biochemical markerINTRODUCTION
Under flash flooding, few characters were identified as playing a key role in submergence tolerance in rice, the most critical are: maintenance of slow elongation of stem, maintenance of high carbohydrate concentration, retention of high chlorophyll percentages, optimum rates of alcoholic fermentation and energy conservation by maintaining low elongation growth rates during submergence (Jackson and Ram, 2003). Submergence tolerance is a metabolic adaptation in response to anaerobiosis that enables cell to maintain their integrity so that the plant survives hypoxia/anoxia without major damage (Sarkar et al., 2006). Rapid increase of water due to various reasons is very much detrimental for crop establishment where direct sowing is practices (Kawano et al., 2009). Tolerance to submergence stress during various stage of crop growth is also important aspect of submergence breeding (Ito et al., 1999; Mohanty et al., 2000). Tolerance to submergence stress during seedling stage of crop growth is very important for quick regeneration and initiation of new leaves after submergence (Setter et al., 1997). Alcoholic fermentation is the alternative metabolic process that plants seems to get adapted under submerged or oxygen free atmosphere (Green Way and Setter, 1996). Under anaerobic conditions aerobic respiration shift to a less efficient anaerobic fermentation to provide energy for survival (Green Way and Setter, 1996). Alcohol dehydrogenase (Adh) involves in ethanol fermentation pathway that is responsible for the reduction of toxic acetylaldehyde to ethanol, resulting in continuous regeneration of nicotinamide adenine dinucleotide (NAD) in the cytoplasm (Chung and Ferl, 1999). Hence, induction of Adh can enhance survival of plants under flooded conditions (Johnson et al., 1994). Changes in enzyme activity levels have been noted within a day under hypoxic conditions and may occur more quickly under anoxic conditions (Keeley and Franz, 1979). Variation of Adh activity was increased from aerial to submerged condition (Chan and Burton, 1992). Adh activity under submerged condition reaches to an optimum level which provide sufficient energy level for better survival. Estimation of Adh activity could be used as a tool to differentiate tolerant and susceptible level.
So an attempt was done to estimate the alcohol dehydrogenase enzyme activity in paddy genotypes which could provide an aid for bio chemical marker for submergence tolerance. Hence an attempt was made to study the alcohol dehydrogenase enzyme activity of fifty diverse paddy genotypes for tolerance under complete submergence condition during seedling stage of growth.
MATERIALS AND METHODS
Estimation of alcohol dehydrogenase enzyme: The Adh enzyme estimation was done by the modification of process of Tong, W.F. et al. 1989. The fresh and disease free seeds of fifty diverse rice genotypes were taken and germinated. Five days old rice seedlings was transferred to the test tube flooded with deionized water along with control. 0.1 gm cutted shoots of five days old rice seedlings of submergence dose of day 1, 2, 3, 4 and 5 along with control in separate set up with three replications were homogenated in a pre chilled mortar with 1.0 ml of 10 mM Tris-Hcl buffer solution (pH-7.6), containing 0.5mM zinc chloride, 0.5g polyvinylpyrolidone. The homogenate was centrifuged at 5600 x g for 20 mins, at -40C and the supernatant serve as a crude extract for assay of Adh activities. Adh activity was determined by modifying the method of Bonnichsen and Brink (1955). The assay mixture contained 0.1 M glycine- NaOH buffer (pH-9.0), 75mM ethanol and 0.26 mM NAD+. After the addition of enzyme solution to the mixture the initial rate of NAD+ reduction was measure at 340nm with spectrophotometer. One unit of Adh activity was defined as the amount which catalysed 1.0µ mole of NAD+ per min. The calculation of Adh activity was based on manuals of Worthington and Worthington (2011), Units/mL = (A340/min x Cuvette volume x Enzyme dilution) / (6.22 x Sample volume).
This experiment was carried out under laboratory condition at the Dept. of Genetics and Plant Breeding, Calcutta University during 2015-2016.
RESULTS
Table 1 represents an account of estimated value of alcohol dehydrogenase enzyme units/gm/min of fifty diverse rice genotypes of five days aerial condition and five successive days of submergence during seedling stage of growth. The mean value ranges from 42.59 – 98.41 Adh units/gm/min. It was IR64 sub1 showed maximum activities of Adh enzyme. In compare with tolerant checks, rice genotypes like Mahananda, Sabita and Purnendu performed well. From chart 1, 2 and 3, it was revealed that gradually alcohol dehydrogenase synthesis took place during submergence period than the aerial condition. During seedling stage of rice genotypes, the enzyme synthesis reached its peak around 3-4 days of submergence. After that the synthesis of Adh enzyme falls rapidly during 5th day of submergence.
DISCUSSION
The interesting feature is that rice genotypes like Mahananda, Sabita and Purnendu showed Adh units above from the FR43B tolerant check genotypes and below the other tolerant check genotypes. But these rice genotypes performed appreciably well in compare with all tolerant check genotypes. Among three well performed rice genotypes Mahananda is best followed by Sabita and Purnendu. The exhaustion of reserve food materials of the nascent rice seedlings was unable to maintain the Adh activity so long period. It may be the age which determines the reserve food matters and Adh activity during extreme anaerobiosis stress condition. During early seedling stage of growth these genotypes may be considered to be a good promise for cultivation of paddy for submerged prone areas where direct sowing is practiced.
CONCLUSION
Flash flood and heavy rain during just grown rice seedling stage specially for direct seed sowing condition is very much fatal to the crop. As the rapid increase of water causes total inundation of the nascent rice seedlings, survival during seedling stage is very much crucial for regeneration after submergence. The well established sub 1 incorporated rice mega varieties like IR64 sub 1 and Swarna sub 1 showed commendable Adh enzyme activity in the experiment. In compare with the sub 1 introgressed rice varieties, genotype like Mahananda, Sabita and Purnendu performed at par during seedling stage of growth. So these rice genotypes could be a good promise for early selection to be introduced into the submerged prone area where flash flood may occur. So screening of Adh activity could a good tool for differentiating submergence tolerant and susceptible rice genotypes during seedling stage of growth.
ACKNOWLEDGEMENTS
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
SOURCE OF FUNDING: Nil
CONFLICT OF INTEREST: Nil
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