Soubhagya K B

TRENDS IN ADVANCED BIOLOGY Trends in Advanced Biology It encompasses 50 research articles presented as part of an International Conference on Advanced Biology organized by iCEIB, University of Kerala. It mainly includes innovative research articles at the intersection of life sciences where integrative and emerging fields of investigations go hand in hand. The articles mainly focus on the themes Stress Physiology, Disease Biology. Environmental Biology and Biodiversity Conservation and envisage the role of advanced techniques such as genomics and proteomics in disease biology, preservation of the diversity of species as well as sustainable utilization of species and ecosystem - Published by iCEIB, University of Kerala, Kariavattom, Thiruvananthapuram. TRENDS IN ADVANCED BIOLOGY Prof. Suhara Beevy S. Dr. Mariamma Cherian Dr. Darsan B. Menon Dr. Anil Kumar T. R. Dr. Mini V.S. Editors iCEIB Published by iCEIB University of Kerala, Kariavattom Thiruvananthapuram Prof. Suhara Beevy S. Dr. Mariamma Cherian Dr. Darsan B. Menon Dr. Anil Kumar T. R. Dr. Mini V.S. TRENDS IN ADVANCED BIOLOGY Editors Published by iCEIB University of Kerala, Kariavattom Thiruvananthapuram Prof. Suhara Beevy S. Dr. Mariamma Cherian Dr. Darsan B. Menon Dr. Anil Kumar T. R. Dr. Mini V.S. Editors Prof. Suhara Beevy S Dr. Mariamma Cherian Dr. Darsan B. Menon Dr. Anil Kumar T.R. Dr. Mini V. S. Cover page design and Layout Dr. Anil Kumar T. R. Copyright @ 2022 University of Kerala. This work is subject to copyright. All rights are reserved. No part of the publication may be reproduced, distributed or transmitted in any form or by any means, including photocopying, recording or mechanical methods without the prior written permission of the publisher, except in case of brief quotations embodied in critical reviews and certain other non-commercial uses permitted by copyright law. For permission requests write to the author, addressed “Attention: Permission” at s.- Published by iCEIB University of Kerala, Kariavattom Thiruvananthapuram - ISBN- Ordering information:- Contents Title Page No. Endospore forming Bacillus spp. as growth promoter in black pepper cuttings A.B. Anju, K. N. Anith, N. Chitra, S. Anu Rajan and V. I. Soumya..................................1 Antioxidant activity of three marine microalgae Nostoc sp., Chaetoceros muelleri and Nannochloropsis oculata M. Sakthipriya, P. Priyadharshini , S. Jeyanthi, P. Santhanam and M. Divya.....................7 Analysis of the lignin removal efficiency of hydrogen peroxide as a pre-treating agent on the wheat straw M.Iyyadurai, P. Merlin sobia, K. Paritosh, V. Vivekanand, M. Krishnaveni, S. Venkatesh..................................................................................................................12 Biodegradation of polycyclic aromatic hydrocarbon by bacteria from marine ecosystem S. Vineetha, Darsan B. Menon, V. S. Mini, M. C. Subhash Peter, S. Suhara Beevy and T. R Anilkumar ..........................................................................21 Golden Oak Mushroom (Shiitake) –A new member in mushroom flora of Kerala C. V. Deepa Rani, Lulu Das, N.P. Lishma...........................................................................36 Virtual screening of plant derived compounds against angiotensin converting enzyme related carboxypeptidase (ACE2) of SARS-COV-2 using molecular docking R. Rathika, S. Venkatesh, M. Krishnaveni.........................................................................................40 Documentation and macronutrients analysis of homestead-based fodder crops in Alappuzha District, Kerala S.R. Dhanya and V. Rajani..................................................................................................50 Impact of drought stress on morpho-physiological traits and membrane damage in the cultivars of sesame (Sesamum indicum L.) M. L. Anchu, S. Jeyaraj and S. Suhara Beevy...............................................................55 In vitro and in silico cytotoxicity evaluation of leaf extracts from Naregamia alata Wight &Arn. K. B Soubhagya, Sruthy Elsa Madhu and BenojMathew...............................................67 Phytoplankton diversity of Vairamkonamchira, a freshwater body of Anchal Panchayat. F. Jensy Roshan and Aparna T. Nair.............................................................................79 Short- and long-term effect of light exposure on photosynthetic and antioxidant machinery of Vigna unguiculata L. Riya Johnson and Jos T Puthur....................................................................................84 Etiology of fungi causing leaf rot disease of coconut in Kerala Susha S. Thara; Deepthi S. Nair; Divya S. and Aashitha Joy.........................................95 An induction of antioxidant defense and protease inhibitor activity in Solanum lycopersicum Linn. Pretreated with laminarin against Alternaria solani infection S. P Sowmiyarithika. and N Radhakrishnan...............................................................106 Bioaccumulation of E. coli in Villorita cyprinoides of Ashtamudi Estuary, Ramsar Site -1204 in Kerala, South west coast of India Letty Titus and B.T. Sulekha....................................................................................120 Studies on the endemic and threatened flowering plants of Vagamon hills, Western Ghats, India Anoop P. Balan and A. J. Robi..................................................................................128 Validation of anti-diabetic activity in Plectranthus vettiveroides and identification of lead molecules through in silico method R. P. Remya, S.Sreekumar and C. K.Biju...................................................................136 Role of Bioinformatics in target identification and differentiation of papillary thyroid carcinoma: A study based on GEO dataset Febby Payva and K. S. Santhy..................................................................................148 Evaluation of heavy metal pollution in water, sediment and Meretrix casta, an edible bivalve species of Asramam in Ashtamudi wetland, Kerala, India N. Parvathy, B.T. Sulekha and S. Sheeba...................................................................157 Status and distribution of medicinal plants in the Malamel rock exposure of Edamulakkal Panchayat, Kollam District Anila George, Morvin Mathew Kizhakekara, Nilja Arjun............................................165 Diversity of exopolysaccharide producing bacteria from soil based insect nesting structures N. Sruthi Suresh, N. Chitra, S. Anu Rajan, V. I. Soumya and K. N.Anith......................175 Pro-adipogenic PU.1 AS long noncoding RNA expression changes during adipogenesis B.Surumi. R. Rajalakshmi, G. M. Nair, A. Jayakumaran nairA. Gangaprasad, C. Prabhakumari................................................................................................................183 Study on butterfly diversity in a sacred groovelocated in Alapad village. C. J. Vishnupriya and Praseeja cheruparambath..........................................................194 Establishment of axenic culture of Hyophila involuta (Hook.) A. Jaeger Megha Santhosh, Meenu Mathew, Abraham Mathew.................................................201 Comparative pharmacological study of Piper nigrum L., Piper longum L. and Piper betle L. Sruthy Elsa Madhu, K. B Soubhagya. and Benoj Mathew...........................................207 In vitro and in silico cytotoxicity evaluation of leaf extracts from Naregamia alata Wight &Arn. 1*, 2 K. B Soubhagya1*., Sruthy Elsa Madhu1 & Benoj Mathew2 PG & Research Department of Botany, St. Peter’s College, Kolencherry 1 Department of Biotechnology, Rubber Research Institute of India, *- Abstract Globally cancer is one among the leading causes of death, exerting a massive burden on the society and health systems resulting in a continuous demand for new therapies in treating this life-threatening disease. Plants contain various phytochemicals which are investigated for their medicinal properties due to recent increase of interest in the use of plant-based medications. The integration of experimental and computational approaches has led to the discovery of novel compounds with therapeutic properties. In the present study, petroleum ether and chloroform extract from leaves of Naregamia alata Wight &Arn., an endangered plant species found in Western Ghats of India belonging to the family Meliaceae was used. Mitotic index studies exhibited a considerable reduction in cell division and no chromosomal damage or mitotic anomalies were detected. The cytotoxicity of the leaf extract of N. alata was studied using MTT assay in the MCF-7 breast cancer cell line. The cell lines revealed moderate inhibitory properties when treated with leaf extracts at different concentrations. The in vitro cytotoxicity findings were supported using in silico method by molecular docking studies of 4 among the 34 compounds identified in N. alata against 1TUB receptor using MCule, online drug discovery platform. The compounds studied were selected based on their highest percentage of occurrence in the plant viz. γ-Himachalene (11.2%), βCaryophyllene (10.8%), β-Sesquiphellandrene (10.7%) and Caryophyllene oxide (9.78%). Among the 4 phytochemicals screened Sesquiphellandrene and β-Caryophyllene exhibited the highest negative docking score (-7.7 and -7.4 respectively) which shows their higher affinity to the target, 1TUB receptor. This depicts that these compounds present in Naregamia alata can act as potential drug in cancer treatment. Keywords: Cancer, cytotoxicity study, in vitro, in silico, 1TUB receptor 67 1. Introduction Naregamia alata Wight &Arn., a shrub belonging to the family Meliaceae is endemic to peninsular India and is found throughout Kerala (Fig. 1). The family consists of 51 genera and 575 species distributed in the tropics and subtropics of which 19 genera and 70 species are found in India. The genus Naregamia consists of two species of which one occurs in India in the states of Maharashtra, Andhra Pradesh, Karnataka, Tamil Nadu and Kerala (Nambiar et al., 1995). Fig. 1. Naregamia alata Wight &Arn. It is an herbal medicine used for the treatment of various ailments like asthama, bronchitis, eczema, pruritus, scabies, jaundice, anaemia, arthritis, biliousness, phlegm vomiting. It has alexiteric, antibacterial, depurative, antipyretic, emetic, and antioxidant properties and its methanolic extract also showed hepato-protective activity (Prashasti et al., 2020). N. alata is believed to be a highly potential plant against cancer owing to the presence of various phytochemicals in it. However, no documented study has been conducted in this line. Here, we aimed our study on N. alata leaves, which is less explored. Furthermore, in silico studies is used to determine the drug-likeness properties of the phytochemical compounds present in N. alata using the Mcule webserver, conducting molecular docking simulations which is useful in supporting the in vitro cytotoxicity studies of the compounds and to identify their binding sites on the tubulin receptor (PDB ID: 1TUB). Highly dynamic mitotic-spindle microtubules are among the most successful targets for anticancer therapy. Microtubules are composed of alpha- and beta-tubulin subunits assembled 68 into linear protofilaments. Among the two components, β-tubulin has been a common drug target for various diseases including cancer (Majcher et al., 2018). Microtubule-targeted drugs such as paclitaxel and Vinca alkaloids, were formerly considered to work by increasing or decreasing the cellular microtubule mass. Even though these causes might have a role in their chemotherapeutic actions, we currently know that at lower concentrations, microtubuletargeted drugs can suppress microtubule dynamics without altering microtubule mass; which leads to mitotic block and apoptosis (Jordan, 2014). This study using the leaf extract of N. alata is carried out with the following objectives: (i)To examine the genotoxic effect in the roots of Allium cepa (ii) To determine the cytotoxic activity of the extracts of . alata using MTT assay in the MCF 7 breast cancer cell line and (iii) Molecular Docking of phytochemicals present in of N. alata against 1TUB protein. 2. Materials and Methods 2.1. Collection and authentication of plant material The plants were collected from Ernakulam district of Kerala during the months of November and December. The plant specimens were taxonomically identified as N. alata. 2.2. Method of extraction Fresh leaf samples of N. alata were collected. The collected leaf samples were washed under running tap water, shade dried and powdered using blender. Extract was prepared in petroleum ether and chloroform using dried leaf powder in Soxhlet apparatus. The extracts were dried and diluted with fresh ethanol at concentrations of 1 mg/1mL (stock solutions). 2.3. Cytotoxic analysis 2.3.1. Mitotic index and chromosomal aberrations The leaf extract of N. alata in chloroform was prepared for cytotoxicity study by grinding clean fresh leaves using mortar and pestle. The extract obtained was filtered using Whatman No.1 filter paper. In this study, the root meristems of Allium cepa (onion) is used for the study of the effects of extract on the genetic material. The dry outer scales were removed from the onion and stem of the bulbs were scraped to expose the root primordia. The bulbs were placed in 10 ml vials. The basal plate of the bulb was held in contact with distilled water. Water was replaced every 24 h. to avoid contamination. After rooting, roots about 3 cm long were treated with the test compound for different durations (1, 24, 48 h.) and similarly distilled water controls were maintained. The treated roots were fixed in freshly prepared acetic acid: ethanol mixture (1:3). 69 Hematoxylin squash method is used (Marimuthu, 1960). Heidenhains stain solution was prepared by dissolving 0.5 g of hematoxylin in a mixture of 95% ethanol and distilled water. Root samples were washed thoroughly in distilled water and hydrolyzed in 1 N HCl at room temperature for about 15 min. Roots were mordanted using 4% ferric ammonium sulphate for 15– 20 min and after washing in distilled water they were stained in 0.5% hematoxylin for 10–20 min and finally washed and squashed in a drop of 45% acetic acid on a clean slide and sealed with either DPX or Dunlop Rubber Solution. Mitotic indices were recorded from treated and control samples by analyzing a minimum of 2000 nuclei involving 6 root tips from 3 bulbs. The frequency of division was calculated. The formula adopted in this method was n x 100/N, where ‘‘n” is the number of dividing cells and ‘N’ is the total number of cells scanned. The frequency of chromosomal aberrations in control and treated root tips were determined and classified (Buckton et al., 1973). Around, 100 dividing cells from each bulb were considered for scanning mitotic anomalies like break, anaphase bridge, lagging chromosome, abortive anaphase, multipolar spindles etc. 2.3.2. MTT assay MTT assay is used to measure cytotoxicity (loss of viable cells). This assay is based on the metabolic reduction of the soluble MTT salt (3-(4,5-dimethylthiazol-2-yl) -2,5 diphenyl tetrazolium bromide) that reflects the normal function of mitochondria dehydrogenase activity and cell viability, into an insoluble, colored formazan product, which was measured spectrophotometrically (Sadeghi-Aliabadi et al., 2014). MTT assay (Mosmon, 1983) is based on the capability of live but not dead cells to reduce a yellow tetrazolium dye to a purple formazan product. MCF-7 Cells were maintained in DMEM medium, supplemented with 10% Fetal Bovine Serum, at 370C in humidified atmosphere with 5% CO2. The cells were plated in 96 well flat bottom tissue culture plates at a density of approximately 1.2 x 104 cells/well and allowed to attach overnight at 370C. The medium was then discarded, and cells were incubated with different concentrations of the extract for 24 h. Once the incubation is over, medium was discarded, and 100 ml fresh medium was added with 10 ml of MTT (5 mg/ml). After 4 h., the medium was discarded and 100 ml of DMSO was added to dissolve the formazan crystals. Absorbance was read at 570 nm in a microtitre plate reader. Cell survival was calculated by the following formula: Viability % = (Test OD / Control OD) x 100 Cytotoxicity % = 100 - Viability% 70 2.4. Molecular docking simulations All computational analyses were performed on Microsoft Windows 11 Home platform in ASUS Vivobook 11th Gen Intel(R) Core (TM) i3-1115G4. Docking was performed to study the binding efficacy of the phytochemicals and the target. The more negative binding energy values obtained were considered as the binding affinity value of the ligands for each docking experiments. These values and their interaction with the tubulin protein were tabulated. 2.4.1. Tubulin structure The structure of Tubulin (PDB ID: 1TUB) was downloaded from RCSB Protein Bank, PDB (Fig. 2). It is a 98.58 KDa protein. The alpha- beta tubulin heterodimer is the structural subunit of microtubules, which are cytoskeletal elements that are essential for intracellular transport and cell division in all eukaryotes Fig. 2. Structure of Tubulin heterodimer (PDB ID: 1TUB) with chains coloured green and yellow indicating α and β tubulins respectively 2.4.2. Binding-site preparation The prediction of binding site was done with respect to the available literature and then the data was verified using CAVER WEB, using which identified tunnels, their properties, energy profiles and trajectories for ligands’ passages were calculated and visualized. 2.4.3. Screening for phytochemicals Based on previous literature, 34 phytochemical compounds present in N. alata were identified, from which four were selected for our study based on their highest percentage of occurrence in the plant viz. γ-Himachalene (11.2%), β-Caryophyllene (10.8%), βSesquiphellandrene (10.7%) and Caryophyllene oxide (9.78%). 3D and 2D structures were downloaded from PUBCHEM database in Simplified Molecular Input Line Entry System (SMILES) format for further screening procedures (Table 71 1). Drug like compounds were screened and was fed into SWISS-ADME server for further screening based on the pharmacokinetics, drug-likeliness, and medicinal chemistry friendliness of small molecules. In addition, the physicochemical properties and the draggability of the selected phytochemicals were also predicted with SWISS-ADME server (Kothandan et al., 2021). 2.4.4. Multiple ligand docking For the present study, mcule.com online drug discovery platform was employed to carry out docking simulations. It encompasses more than 122 million chemical compounds. For screening default options were used to select our final hits. Molecular docking was carried out on 1-click docking in lead optimization. The downloaded structure of phytochemical in SMILES (Simplified Molecular Input Line Entry System) format and the structure of Tubulin protein (PDB ID: 1TUB) was uploaded along with the binding site centres was uploaded to mcule. The docking between phytochemicals presents in Naregamia alata against Tubulin protein (PDB ID: 1TUB) is done which gives the most suitable binding pose ie., the highest negative docking score. 3. Results and Discussion 3.1. Mitotic index The mitotic index values were obtained by treating the root meristems of onion with fresh leaf extracts prepared in chloroform at 3 different time periods along with the corresponding control. The mitotic index of the root meristem treated with fresh leaf extracts show values 43.21, 37.01 and 24.62 in 1, 24 and 48 h. treatment respectively (Table 2). The treated cells showed a substantial reduction when compared to control. The divisional frequencies were found to decrease at one h. treatment which implies that the fresh leaf extract has an immediate toxic effect on the dividing cells. 3.2. Chromosomal aberrations and mitotic anomalies Leaf extract of N. alata in petroleum ether and chloroform were employed to assess their induction of chromosomal damage and mitotic anomalies at various time periods of exposure in onion root meristem. No chromosomal damage or mitotic anomalies were observed in the onion root meristem exposed to fresh leaf extract of N. alata. 72 Table 1. List of phytochemicals studied during the present study S. No. Phytochemicals studied Pubchem ID 1 γ-Himachalene 577062 2 β-Caryophyllene - 3 βSesquiphellandrene - 4 Caryophyllene oxide - 2D structure 3D structure of of treatment scored division (N) (n) 1 1hr 12,368 4612 43.21 4346 459 273 267 2 24h. 12,433 5345 37.09 4070 233 189 120 3 48h. 12,163 2995 24.62 2910 33 35 27 4 Control 12,246 6385 52.13 5880 263 147 95 cells cells in (n/NX100) Telophase No Anaphase Total No. Total no. of Metaphase Duration Prophase Sl. Mitotic index Table 2. Mitotic index 73 3.3. MTT assay Screening of petroleum ether and chloroform extract of N. alata for anticancer activity against MCF-/7 cell lines revealed the inhibitory potential of the extract in all three concentrations viz. 50, 100, 150 µg. The percentage of cancer cell inhibition profile was found to be concentration dependent (Table 3). Fifty µg concentration brought about 8.28% inhibition followed by 100 mg with 21.52% inhibition and 150 mg with 23.9% inhibition. The cell viability of MCF-7 cell lines significantly reduced after exposure to chloroform extract of N. alata leaf in a dose dependent manner at 150 mg. Table 3. MTT Assay Sl. No. Sample Concentration (µg) Percentage viability Percentage toxicity 1 Control 0 100 0 2 Petroleum ether 50 91.71 8.28 100 78.47 21.52 150 76.00 23.99 50 76.53 23.46 100 73.38 26.61 150 56.35 43.64 3 Chloroform 3.4. Molecular docking simulations To further investigate the ability to inhibit tubulin aggregation by the phytochemicals present in N. alata in cancer cell growth assays, binding energies between the new compounds and β tubulin, one of the subunits of microtubules in the cytoskeleton structure of every eukaryotic cell, were calculated using docking simulations. Among the 4 phytochemicals screened Sesquiphellandrene and β-Caryophyllene exhibited the highest negative docking score, -7.7 and -7.4 respectively (Table 4.) which shows their higher affinity to the target, 1TUB receptor. In the present study, the crude leaf extract showed that it possesses an immediate toxic effect on the developing cells as it reduces the mitotic index even at one h. exposure when compared to control. The treatment also showed a duration dependent mitotoxic effect. Mitotoxicity study has not been reported in leaf extract of N. alata. However, related 74 observation of mitotoxicity on other medicinal plant extracts have been reported in Psidium guajava and Allophylus edulis in somatic cells of Allium cepa (Rosangela et al., 2003). The extracts brought about a significant decrease in mitotic index in a dose-independent way. The genotoxicity of N. alata leaf extracts were analysed by exposing root meristems of onion to the plant extract at different concentrations and durations. The results showed that the test compound is not toxic to the genetic material and do not induce any chromosomal aberration. The MTT assay is a sensitive, quantitative and reliable colorimetric assay that measure cell viability. The assay is based on the capacity of the cellular mitochondrial dehydrogenase enzyme in living cells to reduce the yellow water-soluble substrate 3-(4,5-dimethylthiazol2yl)-2,5-diphenyl tetrazolium bromide (MTT) into a dark blue/purple formazan product which is insoluble in water. The crude extract with IC 50 of less than 30 mg/ml is considered to be cytotoxic by the American cancer institute (Itharat et al., 2004). The results of the present study suggest that chloroform and petroleum ether leaf extracts of N. alata possess moderate anticancer activity as the IC 50 values achieved were 207.974 mg/ml and 158.176 mg/ml for petroleum ether and chloroform respectively. It is observed that both petroleum ether extract and chloroform extract cause cell growth inhibition in dose-dependent manner. Constituents like tannins, glycosides and flavonoids present in the plant may be responsible for such activity. There is no previous report on the anticancer activity of N. alata. Molecular docking was performed to evaluate binding affinity of phytochemicals to the target protein. The four structures of phytochemicals present in N. alata as described above were docked into the β tubulin and examined their binding affinity. Binding Affinity is the capability of a specific ligand (small molecule) and the strength by which a compound interacts and binds to a target molecule's active sites. This data is used to study and compare the binding affinity of different ligands with their corresponding receptor molecule. Lower the binding energy, greater the affinity of a ligand towards the receptor molecule. Thus, a compound with a higher negative value can be chosen as a viable drug candidate. Sesquiphellandrene and β- Caryophyllene exhibited the highest negative docking scores which depicts their higher affinity to the target, 1TUB receptor. 75 Table 4. Docking results of phytochemicals of N. alata (with white colour) against tubulin heterodimer (chains coloured green and yellow indicating α and β tubulins respectively) Sl. No. Phytochemicals studied 1 γ-Himachalene Docked structure Docking score -5.9 2 β-Caryophyllene -7.4 s3 β-Sesquiphellandrene -7.7 4 Caryophyllene oxide -4.8 76 Docking studies revealed that the phytochemicals in N. alata showed anti-cancer activity and could act by binding tubulin at the dimer interface. The binding of the compounds to tubulin would prevent tubulin polymerization or disassociation and presumably result in apoptosis. This depicts that these compounds present in N. alata can act as potential drug in cancer treatment. 5. Conclusion To conclude, we have demonstrated that the N. alata petroleum ether extract of leaf possesses moderate cytotoxicity whereas the chloroform extract exhibited strong cytotoxic effect on MCF-7 cells at 150 mg concentration. It is observed that both petroleum ether extract and chloroform extract cause cell growth inhibition in dose-dependent manner. The leaf extracts in chloroform induced inhibition of mitotic index and did not induce chromosomal anomalies in the A. cepa root meristems. The chemical constituents present in the extract responsible for cytotoxic activities need to be investigated further. There is no previous record on the cytotoxicity of N. alata. The present data also suggests that the extract is more toxic to cancer cells than normal cells like A. cepa and it do not have any genotoxic effect as it did not cause any mitotic anomalies. We also performed molecular docking simulations to help elucidate the anticancer activities of the phytochemical compounds present in N. alata. In the docking simulations, we employed the crystal structure of the tubulin (PDB ID: 1TUB) as the drug target. The binding affinity values attained because of the docking studies were recorded. 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