Field of the invention
This invention relates to isolation and identification endophytic fungus from leaves of Nothapodytes nimmoniana and investigation of Camptothecin (CPT): an anticancer plant biomolecule.
Background of the Invention
References which are cited in the present disclosure are not necessarily prior art and therefore their citation does not constitute an admission that such references are prior art in any jurisdiction. All publications, patents and patent applications herein are incorporated by reference to the same extent as if each individual or patent application was specifically and individually indicated to be incorporated by reference.
Several patents have been issued for the endophytic fungus, Camptothecin and bioactive compounds. For example, WO2020044374A1 provides herein Sustainable production of camptothecin in the suspension culture of the endophyte from Nothapodytes nimmoniana, is disclosed. A novel high yielding and sustainable, camptothecin producing endophytes (A. alstroemeriae (NCIM 1408) and A. burnsii (NCIM 1409))and process for producing camptothecin from the said endophytes from Nothapodytes nimmoniana. The endophyte A. burnsii(NCIM 1409) is a high yield and sustainable, camptothecin producing endophyte (fungal strain) forming while colony mycelium which turns black during sporulation and forms aerial hyphae. The strain was able to produce 150-200 µg/g DW biomass or 1.5-3 mg/L of CPT titer. The highest yielding endophyte (A. alstroemeriae (NCIM 1408) demonstrates a high yield of camptothecin up to 300-400 µg/g DW biomass.
Another patent, US8765147B2 belongs to endophytic fungi from higher plants such as a Pteromischum sp. plant, and to extracts and compounds from such fungi that have desirable biological activities, such as antifungal and immunosuppressive activities. The present disclosure further relates to compositions comprising such extracts and compounds, as well as methods of making and using the compositions.
Another patent, US20060134762A1 provides a novel source of microorganism for production of Camptothecin and related camptothecinoids. The invention also discloses its isolation, screening for Camptothecin production, growth, fermentation requirements and chemical analysis of Camptothecin (camptothecinoids).
Another patent, BR102012005283A2 obtaining bioactive secondary compounds of phoma herbarium endophyte with broad spectrum antimicrobial activity. The endophytic fungus phoma herbarium, isolated from the medicinal plant luehea divaricata, is a non-pathogenic fungus from humans, producing effective secondary metabolites compared to a commercial fungicide against the alternaria alternata phytopathogenic fungi, guignardia citricarpa and fusarium solani f. sp. glycines. It has action against the bacteria escherichia coli, micrococcus luteus, salmonella typhi, human pathogens and phytopathogenic bacteria xantomonas asc. phaseoli. The invention assists in the control of plant pathogenic fungi such as cocoa and soybean as well as assisting in the control of human and plant pathogenic bacteria. Moreover, this invention is an alternative to the use of agrochemicals, as it is a natural product, easy to handle, easy to produce in the environmentally friendly environment. The amount of secondary metabolites obtained from the total fermentation of the potato-dextrose medium by the pophy herbarium endophytic fungus is usually around 120mg per liter of fermented medium. From 300 mg of concentrated fungal crude extract (metabolites), 2.6 to 47.1 mg of bioactive metabolites with antimicrobial action are obtained, and in laboratory tests 11 to 188.5 are required. µg / ml metabolites for effective control of plant pathogenic fungi.
Fungus are the most vital groups of eukaryotic organisms for producing various novel metabolites that are used as drugs. Endophytic fungi are symbiotically associated biota of living plant tissues that induce a symptomless disease to their hosts and are not host-specific. Endophytes are the source of bio-pharmacological compounds. Endophytic fungi have proven to be a significant source of novel compounds with fascinating biological activities and a prominent level of biodiversity. Most fungal endophytic research draws attention to screening endophytes for the existence of novel as well as some high-value biomolecules. So far, many of the medicinal plants have been targeted to isolate the bioactive molecules directly. The endophytic fungus colonizes the healthy tissues of the host plant without creating any disease signs.
According to the literature, the endophytic fungus has a strong interest in accumulating natural bioactive compounds or secondary metabolites as a host for medical, agricultural, and industrial purposes.
Endophytes are producing a variety of compounds such as alkaloids, steroids, terpenoids, isocoumarin derivatives, quinones, flavonoids, cytochalasines, furandiones, phenylpropanoids, lignans, peptide with antibiotic, antiviral, volatile antibiotic, anticancer, antioxidant, insecticidal, anti-diabetic, and immunosuppressive properties. The production of active metabolites by endophytes that were previously only known to be produced by their host plants because of putative horizontal genetic transactions between the host and endophytes further supports endophytes as a therapeutic plant alternative. Endophytes with substantial bioactivity are thought to be produced by medicinal plants living in biodiversity hotspots.
Endophytic fungus Stemphylium botryosum and Colletotrichum spaethianum were characterized from the leaves of Nothopodytes nimmoniana by using 18s rRNA sequencing and the BLASTN tool.
A total of two endophytic fungi were isolated and purified from the leaf of plant. Fungi development was discovered after little pieces of fresh leaves were placed on PDA plates at 28±2° C for 7 days then the fungal hyphae were transferred to mycological media. The BLAST analysis of nucleotide sequences was placed in the Gene Bank of NCBI (National Centre for Biotechnology Information, India) with the entry number KC584603.1, which shows 99 percent identity with Stemphylium botryosum sp. and JN940738.1 that show 98% of identity with Colletotrichum spaethianum sp. The ribosomal gene database was accessed (http://ncbi.nim.nih.gov/). however, no investigations on the same fungus species from the N. nimmoniana plant have been reported. Camptothecin (CPT), a cytotoxic quinoline alkaloid, is widely used as anticancer agent in the treatment of lung, uterine, colon and breast cancer by inhibiting DNA topoisomerase I.
The present invention describes the isolation and identification endophytic fungus from leaves of Nothapodytes nimmoniana and investigation of Camptothecin (CPT): an anticancer plant biomolecule.
The primary object of the present invention is to isolate and identify the endophytic fungus from the leaves of Nothapodytes nimmoniana plant.
Another object of the present invention is to isolate endophytic fungi by the pure culture technique and identified by using of 18 S rRNA sequencing.
Another object of the present invention is to use HPLC analysis for knowing the percentage of secondary metabolites Camptothecin (CPT) after growing of endophytic fungus.
Another object of the present invention is to discover fungi development after little pieces of fresh leaves of plant placing on PDA plates at 28±2° C for 7 days.
These and other objects and advantages of the present invention will become readily apparent from the following detailed description.
Summary of Invention
This summary is not a comprehensive overview of the disclosure and does not reflect the main/essential features of the establishment or specify the scope of the establishment. Its sole purpose is to present some of the concepts presented here in a simpler way as a precursor to more detailed explanations presented later.
The present invention relates to isolation and identification endophytic fungus from leaves of Nothapodytes nimmoniana and investigation of Camptothecin (CPT): an anticancer plant biomolecule.
In some embodiments of the present invention, initially the mature leaves were washed carefully with running tap water for 20-30 minutes, followed by Dettol soap solution for 2 min, then washed with liquid Dettol solution for 2 min. After this treatment, leaves were rinsed thoroughly with purified distilled water to remove the traces of soap and disinfectant.
In some embodiments of the present invention, these leaves were transferred to sterile beaker under laminar airflow. Then, explants were treated with 70% ethanol for 2-3 minutes and explants again washed with sterilize distilled water for 6-7 times. Then, washed with freshly prepared 0.1% mercuric chloride (HgCl2) solution in sterilized distilled water for 2-4 min. Finally, leaves are washed 6-7 times with sterilized distilled water for surface sterilized.
In some embodiment of this aspect of the present invention, these surface-sterilized leaves were put on sterilized petri-dish and cut into small pieces and were injured with sterile scalpel blade. Leaf pieces were transferred in the potato dextrose agar (PDA) medium in the aseptically condition. Plates were incubated at 28 ± 2°C for 7 days.
In some embodiment of this aspect of the present invention, Fungi were transferred onto fresh potato dextrose agar (PDA) medium following by pure culture technique. A small block of agar medium contained fungus spore was cut out by flame-sterilized scalpel/inoculating needle and placed onto fresh Potato dextrose agar (PDA) medium in the centre of plate and incubated at 28 ± 2°C for 7 days.
These and other aspects of the embodiments herein will be better appreciated and understood when considered in concurrence with the following explanation and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications.
Brief summary of the drawings
Figure 1 Nothapodytes nimmoniana leaves inoculation
Figure 2 Nothapodytes nimmoniana leaves inoculation
Figure 3 Fungus Culturing from leaves of Nothapodytes nimmoniana
Figure 4 Fungus strain isolated from leaves of Nothapodytes nimmoniana: (a) Stemphylium botryosum (S1) and (b) Colletotrichum spaethianum (S2)
Figure 5 Spectral presentation of Nucleotide sequence BLAST analysis of endophytic fungus– Seq210-S1-NS1-NC100519B
Figure 6 Spectral presentation of Nucleotide sequence BLAST analysis of endophytic fungus– Seq210_S1_NS4_NC100519B (Reverse Complement)
Figure 7 Spectral presentation of Nucleotide sequence BLAST analysis of endophytic fungus– Seq210_S1_NS4_NC100519B (Reverse Complement)
Figure 8 Spectral presentation of Nucleotide sequence BLAST analysis of endophytic fungus– Seq210-S2-NS1-NC100519B
Figure 9 Spectral presentation of Nucleotide sequence BLAST analysis of endophytic fungus– Seq210-S2-NS4-NC100519B (Reverse Complement)
Figure 10 Spectral presentation of Nucleotide sequence BLAST analysis of endophytic fungus– Seq210-S2-NS4-NC100519B (Reverse Complement)
Figure 11: HPLC chromatogram of standard Marker Camptothecin (CPT) detection
Figure 12: HPLC chromatogram of standard Marker Camptothecin (CPT) detection
Figure 13: HPLC chromatogram for CPT detection in fungi sample S-2
Brief Summary of the Tables
Table 1: Identification of endophytic fungus strains (NCIM, CSIR-NCL, Pune, India) by the using of BLAST tool program
Table 2: BLAST n hits (top 5) Stemphylium botryosum CBS714.6818SrRNA gene, partial sequence; from type material, LOCUS NG 061146, 1020 bp DNA linear PLN
Table 3: NCBI- BLAST n hits (top5)- Colletotrichum spaethianum CBS 167.49 18S rRNA gene, partial sequence: from type material, Locus NG-062847 1020 bp DNA linear
Table 4: The endophytic fungus and their dry weight (g)
Table 5: CPT content from endophytic fungus of Nothapodytes nimmoniana
These embodiments are described in sufficient detail to enable those skilled in the art to practice the embodiments and all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The present invention relates to isolation and identification endophytic fungus from leaves of Nothapodytes nimmoniana and investigation of Camptothecin (CPT): An Anticancer Plant Biomolecule.
In some embodiments of the present invention, these leaves were transferred to sterile beaker under laminar airflow. Then, explants were treated with 70% ethanol for 2-3 minutes and explants again washed with sterilize distilled water for 6-7 times. Then, washed with freshly prepared 0.1% mercuric chloride (HgCl2) solution in sterilized distilled water for 2-4 min. Finally, leaves are washed 6-7 times with sterilized distilled water for surface sterilized.
In some embodiment of this aspect of the present invention, these surface-sterilized leaves were put on sterilized petri-dish and cut into small pieces and were injured with sterile scalpel blade. Leaf pieces were transferred in the potato dextrose agar (PDA) medium in the aseptically condition. Plates were incubated at 28 ± 2°C for 7 days.
In some embodiment of this aspect of the present invention, Fungi were transferred onto fresh potato dextrose agar (PDA) medium following by pure culture technique. A small block of agar medium contained fungus spore was cut out by flame-sterilized scalpel/inoculating needle and placed onto fresh Potato dextrose agar (PDA) medium in the centre of plate and incubated at 28 ± 2°C for 7 days.
In some embodiments of the present invention, the isolated endophytic fungi were inoculated into a liquid Sabouraud’s broth consisting of 40?g/L dextrose and 10?g/L peptone for enrichment. The endophyte was grown in a 500?mL Erlenmeyer’s flask containing 100?mL of liquid broth (pH 5.6) for a period of 7 days at 220?rpm in an incubator shaker.
In some embodiments of the present invention, Chromosomal DNA of fungi was obtained by spin column kit (HiMedia) India or similar manufacturers. Fungal 18SrRNA gene (1500 bp) was augmented through polymerase chain reaction by using thermal cycler and were purified using exonuclease-I-shrimp alkaline phosphatase (Exo-SAP)
In some embodiments of the present invention, sequence analysis of purified amplicons was done by standard protocol of Sanger in ABI 3500xl genetic analyzer (Life Technologies, USA) as per the manufacturer’s instructions. After that file of Sequencing (ab1) modified by advanced software of CHROMASLITE (version 1.5). These edited sequences were analyzed by Basic Local Alignment Search Tool (BLAST).
A method for the isolation and identification endophytic fungus from leaves of Nothapodytes nimmoniana and investigation of Camptothecin (CPT): an anticancer plant biomolecule consists of steps: -
a) washing the collected mature leaves carefully with running tap water for 20-30 minutes;
b) transferring these leaves to sterile beaker under laminar airflow, and explants were treated with 70% ethanol for 2-3 minutes and Explants again washed with sterilize distilled water for 6-7 times;
c) washing with freshly prepared 0.1% mercuric chloride (HgCl2) solution in sterilized distilled water for 2-4 min followed by washing 6-7 times with sterilized distilled water for surface sterilized;
d) putting these surface-sterilized leaves on sterilized petridish and cutting into small pieces;
e) transferring the leaf pieces in the potato dextrose agar (PDA) medium in the aseptically condition, plates were incubated at 28 ± 2°C for 7 days;
f) transferring the fungi onto fresh potato dextrose agar (PDA) medium following by pure culture technique;
g) cutting out a small block of agar medium contained fungus spore by flame-sterilized scalpel/inoculating needle and placed onto fresh Potato dextrose agar (PDA) medium in the centre of plate and incubated at 28 ± 2°C for 7 days;
h) periodically sub-culturing these fungi into fresh PDA plates until the pure cultures obtained which was moved for identification;
i) inoculating the isolated endophytic fungi into a liquid Sabouraud’s broth consisting of 40?g/L dextrose and 10?g/L peptone for enrichment;
j) growing the endophyte was grown in a 500?mL Erlenmeyer’s flask containing 100?mL of liquid broth (pH 5.6) for a period of 7 days at 220?rpm in an incubator shaker;
k) after incubation, filtering the fungal isolates through a Whatsman filter paper no. 1, and washed the mycelium pellets repetitively with sterilized distilled water and dried in hot air oven at 70°C overnight;
l) washing the Mycelial mass thoroughly with sterilized distilled water and then homogenized by homogenizer in 2 ml methanol;
m) extracting the homogenate from fungal cultures for the CPT (Camptothecin) by the method of Ultra-sound assisted extraction using a sonication probe (Labman pro-250) at 20 kHz at room temperature;
n) doing the extraction by use of equal volume of chloroform: methanol (4:1 v/v) solvent mixture and repeated thrice times;
o) evaporating the prepared extracts to dryness at 37°C for removing of organic solvent and the dried residues were suspended in HPLC grade chloroform: methanol (9:1 v/v) solvent mixture; and
p) subjecting the prepared extracts to high-performance liquid chromatography (HPLC) with standard CPT for the detection of CPT.
The method as claimed in claim 1, wherein two endophytic fungus Stemphylium botryosum and Colletotrichum spaethianum were characterized from the leaf of plant.
The method as claimed in claim 1, wherein endophytic fungus Stemphylium botryosum (S-1) and Endophytic fungus Colletotrichum spaethianum (S-2) helped to produce more amount of CPT.
The method as claimed in claim 1, wherein the content of CPT found in Endophytic fungus Stemphylium botryosum (S-1) and Endophytic fungus Colletotrichum spaethianum (S-2) was 0.008% and 0.011%.
The method as claimed in claim 1, wherein plant Nothapodytes nimmoniana produces good amount of CPT which is use in the treatment of cancer.
EXAMPLE 1
Experimental
Material and Methods
Collection of Plant Material
Plant material Nothapodytes nimmoniana was collected from Mahabaleshwar, Pune (Maharashtra) in the month of February. The plant parts (leaves and fruits) were authenticated by The National Institute of Science Communication and Information Resources, New Delhi, India. Authentication No. NISCAIR/RHMD/Consult/2021/3823-24-2.
The mature leaves were washed carefully with running tap water for 20-30 minutes., followed by Dettol soap solution for 2 min, then washed with liquid Dettol solution for 2 min. after this treatment, leaves were rinsed thoroughly with purified distilled water to remove the traces of soap and disinfectant. These leaves were transferred to sterile beaker under laminar airflow. Then, explants were treated with 70% ethanol for 2-3 minutes and Explants again washed with sterilize distilled water for 6-7 times. Then, washed with freshly prepared 0.1% mercuric chloride (HgCl2) solution in sterilized distilled water for 2-4 min. Finally, leaves are washed 6-7 times with sterilized distilled water for surface sterilized. These surface-sterilized leaves were put on sterilized petridish and cut into small pieces and were injured with sterile scalpel blade. Leaf pieces were transferred in the potato dextrose agar (PDA) medium in the aseptically condition, shown in figure no. 1 and figure no.2. Plates were incubated at 28 ± 2°C for 7 days.
Fungi were transferred onto fresh potato dextrose agar (PDA) medium following by pure culture technique. A small block of agar medium contained fungus spore was cut out by flame-sterilized scalpel/inoculating needle and placed onto fresh Potato dextrose agar (PDA) medium in the centre of plate and incubated at 28 ± 2°C for 7 days, shown in figure no. 3.
Purification and Preservation of Endophytic Fungi
These fungi were periodically sub-cultured into fresh PDA plates until the pure cultures obtained. Afterward, pure cultures were moved for identification. For further use, the isolated and purified endophytic fungus were transferred into PDA slants and maintained at 4°C.
The isolated endophytic fungi were inoculated into a liquid Sabouraud’s broth consisting of 40?g/L dextrose and 10?g/L peptone for enrichment. The endophyte was grown in a 500?mL Erlenmeyer’s flask containing 100?mL of liquid broth (pH 5.6) for a period of 7 days at 220?rpm in an incubator shaker, shown in figure no. 3.
Identification of Endophytic Fungi
Molecular Characterization of Fungal Endophytes
PCR Amplification and Sequencing of 18S rRNA gene
Chromosomal DNA of fungi was obtained by spin column kit (HiMedia) India or similar manufacturers. Fungal 18SrRNA gene (1500 bp) was augmented through polymerase chain reaction by using thermal cycler and were purified using exonuclease-I-shrimp alkaline phosphatase (Exo-SAP).
Sequence analysis of purified amplicons were done by standard protocol of Sanger in ABI 3500xl genetic analyzer (Life Technologies, USA) as per the manufacturer’s instructions. After that file of Sequencing (ab1) modified by advanced software of CHROMASLITE (version 1.5). These edited sequences were analyzed by Basic Local Alignment Search Tool (BLAST) and identified with comparing neighboring culture sequence provided from the National Centre for Biotechnology Information (NCBI) database that recognized regions of local similarity between sequences. The BLAST program defined as term of comparatively analysis of nucleotide or protein sequences to sequence of databases and then computes the statistical significance of matches. The basis of BLAST program concludes relationships of evolutionary and functional between sequences along with identification of members related to gene families. There are two steps of the BLASTN program:
(i) The BLASTN program is Preliminary search that helps to get potentially close related type strain sequences.
(ii) The pairwise alignment to analyze the sequence similarity values between the query sequence and the sequences identified in step(i).
Therefore, individual isolate is conveyed with the first five-ten hits observed in the alleged database. Further, alignment of multiple sequence and phylogenetic analysis is therefore recommended for accurate species prediction and evolutionary relationship.
The PCR conditions used were initial denaturation at 95° C for 2-3 min, annealing at 50-60° C for 30sec, extension at 72 ° C for 2 min, denaturation at 95° C for 30 s, and a final extension at 72° C for 7 min followed by 30 cycles. The PCR products were purified using Exonuclease I – Shrimp Alkaline Phosphatase (Exo-SAP).
Biomass production of endophytic fungus
In this study,each fungus mycelium isolated and grown in liquid Sabouraud broth media containing peptone 1.0 and dextrose 4.0 %. Agar –plugs containing mycelium were used as inoculums. The Erlenmeyer flask each containing 100 ml Sabouraud broth with mycelium inoculums were kept for 3-4 days on a incubator shaker with 120 rpm at 28°C. After incubation, the fungal isolates were filtered through a Whatsman filter paper no. 1. The mycelium pellets were washed repetitively with sterilized distilled water and dried in hot air oven at 70°C overnight. The dry weight of the fungus was calculated by using the following formula:
Dry weight of fungus = (weight of filter paper + fungus mycelium) - (weight of filter paper)
Extraction of Mycelia for detection of Camptothecin (CPT)
Fungal mycelium was filtered to separate the mycelia and broth. Mycelial mass was washed thoroughly with sterilized distilled water and then homogenized by homogenizer (Remi Electro tech, RPM 8000) in 2 ml methanol. The homogenate was extracted from fungal cultures for the CPT (Camptothecin) by the method of Ultra-sound assisted extraction using a sonication probe (Labman pro-250) at 20 kHz at room temperature. The extraction was done using equal volume of chloroform: methanol (4:1 v/v) solvent mixture and repeated thrice times. The prepared extracts were evaporated to dryness at 37°C for remove of organic solvent and the dried residues were suspended in HPLC grade chloroform: methanol (9:1 v/v) solvent mixture. The prepared extracts were subjected to high-performance liquid chromatography (HPLC) with standard CPT for the detection of CPT.
HPLC analysis
CPT detection was characterized from mycelium by the using of HPLC analysis. Test solutions were directly analyzed by HPLC (UELC- Shimadzu). The column used was C18 (4.6 × 150mm) and particle size 5 µ. Acetonitrile: H2O (70:30) solvent system flow rate 1ml/min. The CPT content in the samples was evaluated with the help of photodiode detector. The chromatograms were retrieved at the absorption maxima of standard CPT (256 nm). Quantification of CPT was done by determining the calibration curve using a range of concentrations of standard CPT.
Preparation of standard solution of marker compounds Camptothecin (CPT)
The Stock solution of Camptothecin was prepared by dissolving 5 mg of accurately weighed CPT in chloroform-methanol mixture (3:1) and making the volume upto 5 mL with methanol. From this stock solution, standard solutions of 10 µg/mL to 50 µg/ml were prepared by transferring aliquots (0.1 to 0.5 mL) of stock solution to 10 mL volumetric flasks and adjusting the volume with methanol.
Preparation of sample solutions of fungi
The dried extracts of fungus (10 mg) were transferred to volumetric flask and made volume upto 10 mL with methanol and centrifuged at 4000 rpm for 10 min. supernatant are transferred to vials to furnish the final concentration of 100 µg/ml.
EXAMPLE 2
Results and Discussion
Isolation of endophytic fungi from leaves of Nothapodytes nimmoniana
The plant part of Nothapodytes nimmoniana was chosen leaves for isolation of endophytes. The small pieces of fresh leaves were placed on PDA plates at 28±2° C for 7 days, and then fungus growth was found. These fungal hyphae were transferred on mycological media for pure culture, two endophytic fungi were obtained to be Stemphylium botryosum (Seq210_S1) and Colletotrichum spaethianum (Seq210_S2), mentioned in the table no.1. The nucleotide sequence analysis by NS sequencing using BLAST was deposited in the NCBI GenBank with an accession number is KC584603.1 that was shown 99% of identity with Stemphylium botryosum sp. andJN940738.1 that was shown 98% of identity with Colletotrichum spaethianum sp, shown in table no. 2 and 3 respectively.
Sequences were used for analysis of Seq210 S1 NS1 NS4 (RC) NC100519B (GCATTATACCGTGAAA and TACGAGAAATCAAAG) shown in figure no.5, Seq210 S1 NS1 NC100519B (GCATTATACCGTGAAA and TGTCAGAGGTGAAATTC), Seq210 S1 NS4 NC100519B (Reverse Complement) (TTCATGATAACTTTA and TACGAGAAATCAAAG) shown in figure no.6 and figure no. 7, Seq210 S2 NS4 NS4(RC)_NC100519B (TTATACAGCGAAACT and ACGAGAAATCAAAGT), Seq210 S2 NS1 NC100519B (TTATACAGCGAAACT and CGAAAAGCATTTGCC) shown in figure no.8, Seq210 S2 NS4 NC100519B (Reverse Complement) (CGAATCGCATGGCC and ACGAGAAATCAAAGT) shown in figure no. 9 and figure no. 10.
Table 1: Identification of endophytic fungus strains (NCIM, CSIR-NCL, Pune, India) by the using of BLAST tool program
Sr. No. Strain Objects Primer NCBI BLAST Type Comments
1. Seq 210-S1 -NC100519 B Identification of 18S rRNA gene ++ Strain 18S rRNA NS1-NS4 (1021bp) Genomic region (NG-061146.1) Stemphylium botryosum Identities: 1011/1019 (99%)
Genomic region (KC584603.1) Stemphylium botryosum Identities: 1011/1019(99%) Strain shown closest homology with Stemphylium sp.(closer to botryosum)
2. Seq210-S2 -NC100519 B Identification of 18S rRNA gene ++ Strain 18S rRNA NS1-NS4 (1021bp) Genomic region (NG-062847.1) Colletotrichum spaethianum Identities: 1001/1022(98%) Genomic region (JN940738.1) Colletotrichum spaethianum Identities: 1001/1022(98%) Strain shown closest homology with Colletotrichum sp.(closer to spaethianum)
NC= Nucleotide Code, NS= Nucleotide Sequence, bp= base pair
Table 2: BLAST n hits (top 5) Stemphylium botryosum CBS714.6818SrRNA gene, partial sequence; from type material, LOCUS NG 061146, 1020 bp DNA linear PLN.
Description Max Score
Total
Score
Query
Cover
Percent
Identification
Accession
Stemphylium botryosum CBS
714.6818SrRNA gene, partial sequence; from type material

1836
1836
99%
99.21%
NG-061146.1

Stemphylium botryosum strain CBS
714.6818S ribosomal RNA gene, partial sequence

1836
1836
99%
99.21% KC584603.1

Alternaria iridiaustralis CBS11848618S rRNA gene, partial
sequence; from type material

1831
1831
99%
99.12%
NG-063035.1

Alternaria eichhorniae CBS 489.9218S rRNA gene, partial sequence;
from type material

1831
1831
99%
99.12%
NG-063034.1

Alternaria burnsii CBS 107.38 18S rRNA gene, partial sequence; from
type material

1831
1831
99%
99.12%
NG-063033.1

Table 3: NCBI- BLAST n hits (top5)- Colletotrichum spaethianum CBS 167.49 18S rRNA gene, partial sequence: from type material, Locus NG-062847 1020 bp DNA linear.
Description Max
Score
Total
Score
Query Cover
Percent
Identification
Accession
Colletotrichum spaethianum CBS
167.49 18S rRNA gene, partial sequence; from type material

1766
1766
99%
97.95%
NG-062847.1

Colletotrichum spaethianum strain CBS167.4918S ribosomal RNA
(SSU) gene, partial sequence

1766
1766
99%
97.95%
JN940738.1

Colletotrichum brevisporum strain LC060018S ribosomal RNA(SSU)
gene, partial sequence

1762
1762
100%
97.85%
JN940357.1

Colletotrichum truncatum CBS
151.35 18S rRNA gene, partial sequence; from type material

1760
1760
99%
97.85%
NG-062848.1

Colletotrichum dematium CBS
125.25 18S rRNA gene, partial sequence; from type material

1760
1760
99%
97.85%
NG-062846.1

Dry weight of endophytic fungi
The endophytic fungus isolated from Leaf samples Stemphylium botryosum (Seq210-S1) and Colletotrichum spaethianum (Seq210-S2) contained 0.293g and 0.157g, shown in table no. 4.
Table 4: The endophytic fungus and their dry weight (g.)
Plant Sample Endophytic fungus Dry weight
Leaf1 Stemphylium botryosum (Seq210-S1) 0.293g
Leaf2 Colletotrichum spaethianum (Seq210-S2) 0.157g
Determination of CPT by HPLC
Camptothecin standard was run in HPLC and the chromatograms shown in figure. The single peak of camptothecin was obtained at the retention time of 4.344 min. Endophytic fungus S-1 and S-2 were shown single peak at the retention of 5.822min and6.066 min respectively.
The camptothecin content recorded from Endophytic fungus S-1 and S-2 were 82.71 µg/g and 113.85 µg/g dry weight of samples. In the present study, the content of CPT found in Endophytic fungus Stemphylium botryosum (S-1) and Endophytic fungus Colletotrichum spaethianum (S-2) was 0.008% and 0.011% respectively, shown in table no.5.
Table 5: CPT content from endophytic fungus of Nothapodytes nimmoniana
Endophytic fungus Amount of camptothecin present in dry weight of sample (µg/g) Camptothecin content in percentage (%)
Stemphylium botryosum (Seq210-S1) 82.71 0.008
Colletotrichum spaethianum (Seq210-S2) 113.85 0.011
Conclusion:
The selected fungus identified as Stemphylium botryosum (Seq210_S1) and Colletotrichum spaethianum (Seq210_S2) analyzed by Basic Local Alignment Search Tool (BLAST) with closest culture sequence retrieved from the National Centre for Biotechnology Information (NCBI). The endophytic fungus isolated from Leaf samples Stemphylium botryosum (Seq210_S1) and Colletotrichum spaethianum (Seq210_S2) contained 0.293g and 0.157g. Endophytic fungi often produce secondary metabolites in increase amount which may be effective. Endophytic fungus S-1 and S-2 were shown single peak at the retention of 5.822 min and 6.066 min comparatively camptothecin marker at the retention time of 4.344 min respectively. In the present study, the content of CPT found in Endophytic fungus Stemphylium botryosum (S-1) and Endophytic fungus Colletotrichum spaethianum (S-2) was 0.008% and 0.011% respectively.

We Claims:

1. A method for the isolation and identification endophytic fungus from leaves of Nothapodytes nimmoniana and investigation of Camptothecin (CPT): an anticancer plant biomolecule consists of steps: -
a) washing the collected mature leaves carefully with running tap water for 20-30 minutes;
b) transferring these leaves to sterile beaker under laminar airflow, and explants were treated with 70% ethanol for 2-3 minutes and Explants again washed with sterilize distilled water for 6-7 times;
c) washing with freshly prepared 0.1% mercuric chloride (HgCl2) solution in sterilized distilled water for 2-4 min followed by washing 6-7 times with sterilized distilled water for surface sterilized;
d) putting these surface-sterilized leaves on sterilized petridish and cutting into small pieces;
e) transferring the leaf pieces in the potato dextrose agar (PDA) medium in the aseptically condition, plates were incubated at 28 ± 2°C for 7 days;
f) transferring the fungi onto fresh potato dextrose agar (PDA) medium following by pure culture technique;
g) cutting out a small block of agar medium contained fungus spore by flame-sterilized scalpel/inoculating needle and placed onto fresh Potato dextrose agar (PDA) medium in the centre of plate and incubated at 28 ± 2°C for 7 days;
h) periodically sub-culturing these fungi into fresh PDA plates until the pure cultures obtained which was moved for identification;
i) inoculating the isolated endophytic fungi into a liquid Sabouraud’s broth consisting of 40?g/L dextrose and 10?g/L peptone for enrichment;
j) growing the endophyte was grown in a 500?mL Erlenmeyer’s flask containing 100?mL of liquid broth (pH 5.6) for a period of 7 days at 220?rpm in an incubator shaker;
k) after incubation, filtering the fungal isolates through a Whatsman filter paper no. 1, and washed the mycelium pellets repetitively with sterilized distilled water and dried in hot air oven at 70°C overnight;
l) washing the Mycelial mass thoroughly with sterilized distilled water and then homogenized by homogenizer in 2 ml methanol;
m) extracting the homogenate from fungal cultures for the CPT (Camptothecin) by the method of Ultra-sound assisted extraction using a sonication probe (Labman pro-250) at 20 kHz at room temperature;
n) doing the extraction by use of equal volume of chloroform: methanol (4:1 v/v) solvent mixture and repeated thrice times;
o) evaporating the prepared extracts to dryness at 37°C for removing of organic solvent and the dried residues were suspended in HPLC grade chloroform: methanol (9:1 v/v) solvent mixture; and
p) subjecting the prepared extracts to high-performance liquid chromatography (HPLC) with standard CPT for the detection of CPT.
2. The method as claimed in claim 1, wherein two endophytic fungus Stemphylium botryosum and Colletotrichum spaethianum were characterized from the leaf of plant.
3. The method as claimed in claim 1, wherein endophytic fungus Stemphylium botryosum (S-1) and Endophytic fungus Colletotrichum spaethianum (S-2) helped to produce more amount of CPT.
4. The method as claimed in claim 1, wherein the content of CPT found in Endophytic fungus Stemphylium botryosum (S-1) and Endophytic fungus Colletotrichum spaethianum (S-2) was 0.008% and 0.011%.
5. The method as claimed in claim 1, wherein plant Nothapodytes nimmoniana produces good amount of CPT which is use in the treatment of cancer.