Effects of water-soluble extracts of the fresh mature leaves of Aristolochia bracteolata were studied on common freshwater carp Oreochromis niloticus and observed the Histopathological effect on different tissues, including liver, kidney, and intestine in the laboratory. First, select the two fishes for the experiment. Then, each fish was placed in a glass aquarium containing 10 litres of dechlorinated tap water and named treated and controlled. The 1.5 ml leaf extract exposed fish in the first aquarium; after that, the to and fro movement increased. At the end of the experiment, the mortality of fish occurred. Next, take the tissue sections of organs from both the fishes and treat and stain with haematoxylin and eosin. The stained slides were observed under a compound microscope at 45X and recorded in different observations. Finally, we concluded that the exposure of aqueous leaf extract of Aristolochia bracteolata L. resulted in apparent toxic effects on the kidneys of common carp Oreochromis niloticus.
Aristolochia bracteolateis poisonous and harmful to human beings and other animals. It is commonly known as birthwort, pipevine, or Dutchman's pipe. Aristolochia is widely occurring in the most diverse climates; some species like A. utriformis and A. westlandii are threatened with extinction. Aristolochia is a genus of evergreen and deciduous lianas (woody vines) and herbaceous perennials. In 1993, a series of end-stage renal disease cases were reported from Belgium associated with a weight-loss treatment; Stephania tetrandra is, an herbal preparation, was suspected of being substituted with Aristolochia fangchi. Aristolochia is both a potent carcinogen and a kidney toxin. Epidemiological and laboratory studies have identified Aristolochia as a dangerous kidney toxin; Aristolochia has been shown to be associated with more than 100 cases of kidney failure . Aristolochic acid was linked to aristolochic acid-associated urothelial cancer in a Taiwanese study in 2012 . In 2013, two studies reported that aristolochic acid is a potent carcinogen. Whole-genome and exome analysis of individuals with known exposure to aristolochic acid revealed a higher somatic mutation rate in DNA . Fishes, often called "rich food for poor people," provide essential nourishment, and exceptionally high biological value. The Nile tilapia, Oreochromis niloticus, is the most important freshwater fish in the ujani dam, Bhigwan. These fish species have great economic importance, where the Nile tilapia constitutes about 32% of the total country catch. In this concern, tilapiaspecies are the most common fish in the Bhima River. These tilapia species are well adapted to survie in fresh and brackish water.
II. MATERIAL AND METHOD
A. Collection and Preparation of Sample
The Mature leaves of Aristolochia bracteolata were collected near the Airport of Baramati, Pune, and identified in the Department of Zoology, Vidya Pratishthan's Art, Science and Commerce College, Baramati (Fig.1). The leaves were thoroughly washed and used for extraction. First, aqueous leaf extract was prepared by dissolving 50 gm of leaves in 100 ml of distilled water using the Soxhlets apparatus. After extraction, the extracted sample is kept at room temperature for 24 hours in the laboratory. After 24 hours, the filtered mixture and extract were used immediately in the experiment.
B. Selection of Model Organism for Experimentation
The freshwater fish Oreochromis niloticus (Tilapia) were selected for the experiment. The Oreochromis niloticus was chosen for the study because of its easy availability, hardy nature, rapid growth rate, and tolerance to varied environmental salinity . Fishes were collected from Ujani Dam at Bhigwan, District: Pune. After the collection, it was acclimatised in a glass aquarium (10 litres) for one week. During acclimatisation, the fishes were fed with standard food pellets and were exposed to the natural day and night cycle. The acclimatisation was done at room temperature.
C. Determination of 24hr Sub- Lethal Toxicity Testing
The two fish were selected for the experimentation, and each fish was placed in a glass aquarium containing 10 litres of dechlorinated tap water. The twenty-four hours before the commencement of toxicity testing, stopped feeding the fish. The 1.5 ml extract of Aristolochia bracteolata L. was exposed in the first aquarium, and another set served as the control. The aquarium was observed for 24 h for clinical signs like skin pigmentation, swimming pattern, response to stimuli, and mortality. The 24 hr death was recorded in the test tank. One group of the fish did not receive Aristolochia bracteolata L. and served as the control group.
D. Histological Analysis
After the experimentation, the fishes proceeded for histological analysis. For histological examinations, organs like liver, kidney, intestine, and stomach were collected from the fish (Control and experimental). Collected organs were gently rinsed with physiological saline solution (0.9% NaCl) and immersed in a fixative composed of glacial acetic acid, formaldehyde, and ethanol (1:3:7). The fixed tissues were passed through the alcohol series 50 to 100% for dehydration and finally cleared in alcohol. Tissues were then embedded in paraffin wax (58°C). After embedding, the blocks were subjected to sectioning at 5μ in thickness in a rotary microtome. The tissue section slides were stained with Haematoxylin and Eosin stains. These slides were treated with xylene and mounted in DPX, observed under a research microscope attached to a digital camera, and photographed.
III. RESULT AND DISCUSSION
A. Major Chemical Constitutes of Aristolochia bracteolate
The Aristolochia bracteolate has been investigated for chemical constituents that have been characterised (Table 1). The 15 different secondary metabolites are commonly found in Aristolochia. Itincludes aristolochic acids and esters, aristolactams, aporphines, protoberberines, isoquinolines, benzylisoquinolines, amides, flavonoids, lignans, biphenyl ethers, coumarins, tetralones, terpanoids, benzenoids, steroids, and others respectively. However, most chemical constituents are pharmaceutically important, showing some clinical significance .
Table No 1: Major Chemical Constituents of Aristolochia bracteolata
The effect of water-soluble extracts of the fresh mature leaves of Aristolochia bracteolata was studied on common freshwater carp Oreochromis niloticus and observed mortality in set 1 (treated with extract) at 24 hr. Untreated control fish showed no mortality. After that, both the fishes were dissected and preceded for histological study.
Histology study of fish tissues:
There were different histological changes recorded in tissues of freshwater carp Oreochromis niloticus.
Untreated:In control fish, the histological structure of tissue appeared in normal shape and size. However, the liver tissue showed irregular lobules separated by the hepatocytes and bile ducts. Observed the polygonal cells with a central spherical nucleus and a densely stained nucleolus in the liver tissue.
Treated: The plant extract treated fish liver had slightly vacuolated cells showing evidence of fatty degeneration. In addition, necrosis was observed in some portions of the liver tissue, probably resulting from the excessive work required by the fish to get rid of the toxicant from its body during the process of detoxification. Cells may also have led to necrosis.
We thank Principal Dr. Bharat Shinde, Vidya Pratishthan's Art's, Science and Commerce College, Baramati, Pune and Dr. B.B. Sharma, Kelkar Education Trust, Mumbai, Principal Prof. (Dr.) Preeta Nilesh, KET's V.G. Vaze College of Arts, Science and Commerce (Autonomous), Mumbai, Dr. S.S. Barve, Dy. Director, Scientific Research Center, Mumbai, for the encouragement and providing facilities.
In the present study, exposure of aqueous leaf extract of Aristolochia bracteolata L. to common freshwater Oreochromis niloticus, resulted in apparent toxic effects on their kidney, which was more severe at 24 hr. Several plants have phytotoxic compounds, which have pesticide properties. Several researchers have reported this kind of study. The extract of Aristolochia bracteolata L. can poison fishes leading to morphological alterations in their tissues and organs.
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