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“Enhancement of Vincristine Under in Vitro Culture of Catharanthus Roseus Supplemented with Alternaria Sesami Endophytic Fungal Extract As Biotic Elicitor”

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“Enhancement of Vincristine Under in Vitro Culture of Catharanthus Roseus Supplemented with Alternaria Sesami Endophytic Fungal Extract As Biotic Elicitor” “Enhancement of Vincristine Under In Vitro Culture of Catharanthus Roseus Supplemented With Alternaria Sesami Endophytic Fungal Extract As Biotic Elicitor” Kanchan Birat Department of Pharmacognosy & Phytochemistry Tariq Omar Siddiqi Department of Botany, School of Chemical and Life Sciences Showkat Rasool Mir School of Pharmaceutical Education & Research Junaid Aslan Department of Pharmacognosy & Phytochemistry Rakhi Bansal Department of Pharmacognosy & Phytochemistry Wasim Khan Department of Pharmacognosy & Phytochemistry Bibhu Prasad Panda ( [email protected] ) Department of Pharmacognosy & Phytochemistry https://orcid.org/0000-0001-5110-2945 Research Article Keywords: Vindoline, Vincristine, Catharanthus roseus, Alternaria sesami, Endophyte Posted Date: July 12th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-308452/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/19 Abstract Vincristine, one of the major vinca alkaloid of Catharanthus roseus(L.) G. Don. (Apocynaceae) was enhanced under in vitro culture of C.roseus using fungal extract of an endophyte Alternaria sesami isolated from the surface-sterilized root cuttings of C.roseus. Vindoline, a precursor molecule of Vincristine was detected for the rst time from the fungal endophyte A.sesami which was used as biotic elicitor to enhance Vincristine content in the C.roseus callus.It was identied using high performance liquid chromatography and mass spectroscopy techniques by matching retention time and mass data with reference molecule. Supplementing heat sterilized A.sesami endophytic fungal culture extract into callus culture medium of C. roseus enhanced the Vincristine content in C. roseus callus by 21.717% after 105 day culture. Introduction Catharanthus roseus (L.) G. Don. (Apocynaceae), known commonly as Madagascar periwinkle or rosy periwinkle, has been a centre of attraction for Vinca alkaloids. Dimeric Vinca alkaloids (vinblastine and vincristine) are anticancer drugs that have been originally extracted from the plant. These indole alkaloids are highly potent anticancer drugs and nd application in various types of cancers. Their biological properties rely on binding to tubulin protein and inhibit microtubule formation and mitotic spindle dynamics thus inhibiting mitosis process.[23] Further, Vinca alkaloids act as angiogenesis inhibitor thus disrupting the intracellular transport and blood ow to tumor.[2]Vindoline, a monomeric Vinca alkaloid constitutes the lower half of dimeric Vinca alkaloid vinblastine.[13] It serves as natural precursor for biosynthesis of vinblastine and vincristine along with catharanthine. Vindoline also acts as synthetic precursor for semi synthetic analogue of Vinca alkaloids such as vindesine (Eldesine®), vinorelbine (Navelbine®) and vinunine (Javlor®). These semi synthetic molecules exhibit superior ecacy in the treatment of breast, bladder and lung cancer.[3] Due to medicinal and pharmaceutical importance and low abundance of natural Vinca alkaloids such as vincristine and vinblastine, C. roseus is the most extensively studied medicinal plant for bioactive secondary metabolites. Advanced strategies over conventional breeding methods for the production of therapeutically important alkaloids have been carried outusing in vitro culture methods like callus culture, cell suspension, hairy root, etc.[7, 14] Although, semi synthetic analogue of Vinca alkaloids (vindesine, vinorelbine and vinunine) are superior to natural Vinca alkaloids (vinblastine and vincristine) but their clinical supply depends on availability of their precursor molecules i.e. monomeric natural Vinca alkaloids (vindoline and catharanthine).[11] Therefore, there is need to explore other sources (non plant) for uninterrupted supply of precursor molecules (monomeric Vinca alkaloids such as vindoline) and enhancement of natural vinca alkaloids in plant. Fungal endophytes are well known as a rich resource of functional molecules and have been reported to produce a plethora of bioactive compounds inside and outside their host plant. Therefore, endophytic fungi are currently explored as alternative source for plant bioactive compounds. Some of the endophytic Page 2/19 fungi are reported to produce taxol, podophyllotoxin, deoxypodophyllotoxin, camptothecin, hypericin, silybin A and B, etc.[18, 19, 20, 21, 24] In the year 1998, Guo et al rst reported Alternaria sp. (a fungal endophyte) producing vinblastine obtained from C. roseus.[8] In the year 2004, Xianzhi et al found an unidentied endophytic fungus from the leaves of C. roseus capable of producing vincristine.[25] These results show that endophytic fungi existing in C. roseus could be a potential source for production of vindoline (the precursor molecules for biosynthesis of dimeric Vinca alkaloids). In this work, we have reported for the rst time the production of a monomeric natural Vinca alkaloid, vindoline from a fungal endophyte, isolated from Catharanthus roseus and enhancement of vincristine content in C. roseus callus by treating with sterilized cellular extract of endophytic fungus producing vindoline. The fungus was identied as Alternaria sesami (Pleosporaceae) using genomic techniques. Materials And Methods Isolation, maintenance and Fermentation of the Fungal Endophyte: A young and healthy plant material of Catharanthus roseus (L.) G. Don (EC 120837) was obtained from herbal garden of Jamia Hamdard University, New Delhi, India. The leaves, stem and root of the plant were cut in to small pieces (approximately 2mm size) with the help of a surgical blade, and washed in reverse osmosis water using cetrimide. Afterwards, the plant segments were surface sterilized (using optimised method) by adding plant material in 70% ethanol for 1minute, rinsed twice with autoclaved water, and then the plant material was added to 0.1% mercuric chloride for 90 seconds, nally washed thrice with sterile water. The effectiveness of surface sterilization was determined by observing no bacterial growth in the rinsed water on potato dextrose agar (PDA) media after 7days of incubation at 28˚C.The segments were transferred onto petri plates containing PDA media (Himedia, Mumbai, India) supplemented with antibiotic mixture of streptomycin sulphate 250 mg/L and penicillin G 250 mg/L in order to grow endophytic fungus without any contamination. Plates were sealed with paralm and incubated at 28oC until the fungal colonies started oozing out of the explants.All of the isolated endophytic fungal cultures were maintained in PDA slant and sub-cultured in every 30 day interval. Isolated endophytes were cultured in liquid medium (20g dextrose, 1g yeast extract, 3g potassium dihydrogen sulphate, 1.5g magnesium sulphate.7H2O, pH 6.0) in 250mL Erlenmeyer asks at 28oC, 110 rpm in a shaker incubator. After 6 days, the culture (fungal biomass) were separated and analysed for monomeric/dimeric Vinca alkaloids by qualitative analysis using high performance liquid chromatography (HPLC) and mass spectrometry separately. Extraction & analysis of vinca alkaloids: Various endophytes isolated from the explants were analysed for the presence of the secondary metabolites through qualitative analysis mode using high performance liquid chromatography (HPLC) technique. The fungal biomass was obtained by ltration process using Whatman No 1 lter paper and dried at 50oC for 4 hr in a hot air oven. To the dried fungal biomass, methanol was added (25 ml per 5 g of dried biomass) and sonicated (Pulse-30Khz, Gap-10 seconds, amplitude-100%) for 10 min using probe sonicator (VCX 130, Vibra Cell, Sonics, USA). After the sonication Page 3/19 process, supernatant was collected by centrifugation (1537g for 5min), evaporated to dryness in a vacuum evaporator at 60°C. For HPLC-UV analysis, the dried extract was dissolved in equal volume of methanol and ltered through 0.45 µm membrane. Elution was carried out by the mobile phase consisting of methanol and water with 0.1% triethylamine at a ow rate of 1ml/min under gradient mode (55% v/v methanol at 0 min, 65% v/v methanol at 5 min, 70% v/v methanol at 15 min, 80% v/v methanol at 18 min, 90% v/v methanol at 35 min, 55%v/v methanol at 40 min).[10, 27] Detection was carried at 260nm. The mass analysis was carried out on UPLC MS/MS analyser of “WATERS” and data were acquired using MassLynx4.1 software. Source temperature was set at 80°C and the gas ow at 500l/hr. Identication of the endophytic fungal strain: The vindoline producing endophytic fungus was identied and characterized by using molecular biology techniques as per the manufacturer protocol (Bhat Biotech, Bangalore, India). Fungal genomic DNA was extracted from fungus using genomic DNA extraction kit from fresh culture of MPBL 105 (desired endophytic fungus). Amplication of the nuclear internal transcribed spacers (ITS) region was performed using the universal primers ITS1 (5’- TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’). The amplication was carried out in a Master cycler® Thermocycler (Eppendorf, Germany) using the following program. Polymerase chain reaction (PCR) was performed with initial denaturation of 94oC for 2 minutes followed by 40 cycles of denaturation at 94oC for 1 min, annealing at 55.5oC for 1 min and extension at 72oC for 30 sec. Final extension was carried out at 72oC for 10 min. Amplied PCR products were separated in 1% agarose gel containing ethidium bromide in 0.5 µg/ml concentration (for detection of bands under UV) in Tris-Borate –EDTA (TBE) buffer. The
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