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ISOLATION, PURIFICATION AND IN VITRO CYTOTOXICITY ACTIVITIES OF COUMARIN ISOLATED FROM ENDOPHYTIC FUNGI, ALTERNARIA SPECIES OF CROTALARIA PALLIDA

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In the present investigation was aimed to isolate and identification of bioactive compound from Crotalaria pallida endophytic fungal Alternaria species ethanol extract and evaluated for in vitro cytotoxicity assays using onion roots, yeast and greengram germination method. MAE method was used for isolation of potent compound and subjected to purification from HPLC with standard compounds. In vitro cytotoxicity methods were used to know the toxicity level of the isolated compound. MAE method yielded major bioactive compounds along with three coumarin(s) and it was confirmed with all four tests and the 5 th test confirmed as a phenolic agent. Endophytic fungal coumarin inhibited the actively growing onion root meristematic cells by possessing various abnormalities by producing highest percentage of cytotoxicity (18.05%), the death of yeast was observed in extract treated yeasts and the percentage of toxicity was 88.03% and at 400 µL of endophytic coumarin inhibited the greengram germination and there is no germination was observed. The fungal coumarin also cleaved the DNA in yeast and it is a strong evidence of toxicity in DNA level and shown apoptosis activity. For all experiments, standard coumarin was used. The endophytic fungal coumarin results were near to equal of standard. From the obtained results, the endophytic fungi Alternaria species have the ability to produce coumarin and it exhibited as strong in vitro cytotoxicity activity. It can be used as a cancer drug after test against cancer cell lines. Please cite this article in press as Umashankar T et al. Isolation, purification and in vitro cytotoxicity activities of coumarin isolated from endophytic fungi, Alternaria species of Crotalaria pallida. Indo American Journal of Pharm Research.2015:5(02).
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Indo American Journal of Pharmaceutical Research, 2015 ISSN NO: 2231-6876
ISOLATION, PURIFICATION AND IN VITRO CYTOTOXICITY ACTIVITIES OF
COUMARIN ISOLATED FROM ENDOPHYTIC FUNGI, ALTERNARIA SPECIES OF
CROTALARIA PALLIDA
Umashankar T1, Govindappa M1*, YL Ramachandra2 Chandrappa CP1, Padmalatha Rai S3 and
Channabasava R1
1Natural Product Laboratory, Department of Biotechnology, Shridevi Institute of Engineering & Technology, Sira Road, Tumakuru-
572 106, Karnataka, INDIA.
2Department of P.G. Studies and Research in Biotechnology & Bioinformatics, Kuvempu University, Jnana Sahyadri, Shankaraghatta
Shimoga, Karnataka -577 45, INDIA.
3Department of Biotechnology, School of Life Sciences, Manipal University, Manipal-576 104, Karnataka, INDIA.
Corresponding author
Dr M Govindappa
Natural Product Laboratory,
Department of Biotechnology,
Shridevi Institute of Engineering & Technology,
Sira Road, Tumakuru-572 106, Karnataka, INDIA.
+91-7204238327,
+91-816-2212628,
endophytessiet@gmail.com
Copy right © 2015 This is an Open Access article distributed under the terms of the Indo American Journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ARTICLE INFO
ABSTRACT
Article history
Received 24/02/2015
Available online
12/03/2015
Keywords
Alternaria Species,
Coumarin,
HPLC,
Cytotoxicity,
Antiproliferative.
In the present investigation was aimed to isolate and identification of bioactive compound
from Crotalaria pallida endophytic fungal Alternaria species ethanol extract and evaluated
for in vitro cytotoxicity assays using onion roots, yeast and greengram germination method.
MAE method was used for isolation of potent compound and subjected to purification from
HPLC with standard compounds. In vitro cytotoxicity methods were used to know the
toxicity level of the isolated compound. MAE method yielded major bioactive compounds
along with three coumarin(s) and it was confirmed with all four tests and the 5th test
confirmed as a phenolic agent. Endophytic fungal coumarin inhibited the actively growing
onion root meristematic cells by possessing various abnormalities by producing highest
percentage of cytotoxicity (18.05%), the death of yeast was observed in extract treated yeasts
and the percentage of toxicity was 88.03% and at 400 µL of endophytic coumarin inhibited
the greengram germination and there is no germination was observed. The fungal coumarin
also cleaved the DNA in yeast and it is a strong evidence of toxicity in DNA level and shown
apoptosis activity. For all experiments, standard coumarin was used. The endophytic fungal
coumarin results were near to equal of standard. From the obtained results, the endophytic
fungi Alternaria species have the ability to produce coumarin and it exhibited as strong in
vitro cytotoxicity activity. It can be used as a cancer drug after test against cancer cell lines.
Please cite this article in press as Umashankar T et al. Isolation, purification and in vitro cytotoxicity activities of coumarin
isolated from endophytic fungi, Alternaria species of Crotalaria pallida. Indo American Journal of Pharm Research.2015:5(02).
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Vol 5, Issue 02, 2015. Umashankar T et al. ISSN NO: 2231-6876
INTRODUCTION
Fungal endophytes are micro-organisms that colonize living, internal tissues of plants without causing any immediate and
overt negative effects [1]. The fungal endophytes have proven to be promising sources of many biologically active natural products
[2]. Crotalaria pallida is a terrestrial, annual, erect herb, up to 150 cm tall. Taproot white or brown and stem grooved, solid,
glabrous. A novel antimicrobial peptide has been exhibited as a strong antimicrobial agent against human pathogenic micro-organisms
[3] and lectin [4]. In the folk and Ayurvedic medicines, C. juncea is used as blood purifier, abortificient, astringent, demulcent, emetic,
purgative and in the treatment of anaemia, impetigo, menorrhagia and psoriasis [5]. Crotalaria species possess anticancer properties
[6]. Presence of coumarin has reported from Crotalaria madhurensis [7] and Crotalaria ramosissima [8].
The antimicrobial, antioxidant, anti-inflammatory, lipoxygenase, xanthine oxidase (XO) and acetylcholinesterase activities
and phenolic contents of different solvent extracts (ethanol, ethyl acetate, chloroform, petroleum ether and water) of Crotalaria
pallida were evaluated using standard in vitro methods and extracts exhibited anti-HIV activity [9]. Umashankar et al. [10] have
reported four fungal endophytes from two different parts and they have shown the presence of coumarin(s) presence. These partially
purified coumarins have been shown as strong antioxidants and antimicrobial activity. The natural coumarin isolated from various
plants is a ubiquitous plant metabolite with strong antioxidant, antimicrobial, antiviral, anti-inflammatory, antihypertensive,
antitubercular, anticonvolusant, multiple sclerosis, antidiabetic and anticancer properties [11].
Further, the present investigation was aimed to identify and characterize the exact coumarin present in leaf fungal endophyte,
Alternaria species and evaluated for in vitro cytotoxicity assays to know their efficacy.
Materials and METHODS
Mass culture of fungal endophyte, Alternaria species and extraction
Endophytic fungi, Alternaria species were isolated from leaf part of Crotalaria pallida. Mass cultured the fungi using potato
dextrose broth for 8 days at room temperature (26+20C). After incubation, the fungal mycelium mat was taken for extraction using
ethanol. Based on the earlier report of Umashankar et al. [10], Microwave Assisted Extraction (MAE) method was used, the
endophytic fungal mat mixed with ethanol was kept for extraction in microwave method at 2 cycles of 5 minutes each at 1000C and
analyzed the percentage of coumarin.
Identification of coumarin in Alternaria species
Identification of coumarin in the extracts
Test 1 3ml of ethanol extract was evaporated to dryness in a vessel and the residue was dissolved in hot distilled water. It was then
cooled and divided into two test portions, one was the reference, second was the test. To the second test tube, 0.5 ml of 10 NH4OH
was added. The occurrence of intense/fluorescence under UV light is a positive test for the presence of coumarins and derivatives. The
experiment was carried out for all the experiments in three replicates [12].
Test 2 5ml of the extract was evaporated to dryness and the residue was dissolved in 2ml of distilled water. The aqueous solution
was divided into two equal parts in test tubes. One part was the reference. To the other test tube, 0.5ml of 10%ammonia solution was
added and the test tubes were observed under UV light indicated. The occurrence of a bluish green fluorescence under UV light
indicated the presence of coumarin derivatives [12].
Test 3 To the concentrated alcoholic extract of drug few drops of alcohol FeCl3 solution was added. Formation of deep green colour,
which turned yellow on addition of conc. HNO3, indicates coumarins presence.
Test 4 The alcoholic extract was mixed with 1N NaOH solution (one ml each). Development of blue green fluorescence formation
indicates presence of coumarins.
Test 5, Detection of phenols
In beakers, 5ml of each previous filtered extract were taken and 1ml of FeCl3 (1%) and 1ml K3(Fe(CN)6) (1%) were added.
The appearance of fresh radish blue color indicated the presence of polyphenols [13].
Isolation, identification and purification of coumarin from Alternaria species
TLC The solvent extract was centrifuged at 6000 rpm for 10 min and then filtered with Whatman No. 1 filter paper and evaporated
at a constant temperature of 620C in hot air oven until a very concentrated extract was obtained. The mobile phase was toluene:
benzene: ethyl acetate (60:20:10) and visualized under UV light.
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Identification of coumarin by reversed phase liquid chromatography with gradient elution
The extraction system comprised of a domestic microwave (Trio-triple) manufactured by Samsung CE108MDF-B
(Bangalore, India) equipped with 230v/50Hz with a nominal maximum power of 900W, a reflux unit, 6 power levels, representation
of the analysis of mean values from each level for a particular factor. Agilent technologies High Performance Liquid Chromatograph
1100 series equipped with quaternary solvent delivery pumps, diode array detector, autosampler and integration with Chemstor
Software from Agilent. Stainless steel analytical columns were Inertsil® ODS-3V, C18 (250 x 4.6 mm I. D., 5 μm) (GL Science Inc,
India).
Chemicals and materials
Acetonitrile (ACN, HPLC gradient grade, Rankem RFCL Limited), orthophospharic acid (Analytical grade, Spectrochem)
and methanol (MeOH, HPLC gradient grade, Rankem RFCL Limited), HPLC-grade water (Millipore). Reference standards of
Coumarin >99.0% (1-2 Benzopyrone, Sigma-Aldrich Lot No.#030M1441V),p-coumaric acid >98.0% (Sigma-Aldrich Lot
No.#110M1259V) and 2-Hydroxy cinnamic acid 97.0% (Sigma Aldrich Lot No.#STBB1076V) were obtained from Sigma-Aldrich,
India.
Preparation of standard solution
Accurately weigh and transferred 50 mg each of mg of coumarin , p-coumaric acid and 2-hydroxy cinnamic acid standard
into 100.0 mL volumetric flask, dissolve and dilute to volume with diluent. Further transfer 5.0ml of this solution into 50.0 mL
volumetric flask dilute to volume with diluent.
Reagents preparation
0.05% v/v of orthophospharic acid buffer
Transfer 1.0ml of orthophosphoric acid into 2000ml water. Filter this solution through 0.45µm membrane filter and sonicate to degas.
Mobile phase-A:
0.05% v/v orthophospharic acid buffer.
Mobile phase-B:
Mix 995 mL of Methanol and 5mL of 0.05% v/v orthophospharic acid Buffer. Filter through 0.45µ membrane filter and degas.
Mobile phase-C:
Mix 995 mL of Acetonitrile and 5mL of 0.05% v/v orthophospharic acid Buffer. Filter through 0.45µ membrane filter and degas.
Diluent preparation:
Mix 90:10 ratio of Methanol and water.
Chromatographic conditions
Column : Inertsil® ODS-3V, C18 (250 x 4.6 mm I. D., 5 μm)
Flow : 1.0ml/min
Column Temperature: 25°C
Wavelength : 275nm
Injection volume : 10µl
Run time : 60 minutes
Plant and endophytic fungal material
1.0g of powdered plant material and endophytic Alternaria species fungal mycelial mat (screened through mesh 44) were
transferred in to 25.0ml volumetric flask. Added about 20ml of diluent and sonicate for 120minutes.cooled to room temperature
diluted to volume with diluent. Filtered through Accu-Bond SPE Cartridge (Agilent Technologies, India) discard the first few ml of
the filtrate.
Time
Mobile phase-B (in %)
Mobile phase-C (in %)
0.0
5.0
15.0
10.0
5.0
15.0
20.0
15.0
15.0
30.0
20.0
15.0
35.0
15.0
15.0
40.0
5.0
15.0
50.0
5.0
15.0
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Standard chromatogram
Retention at 18.7 is p-coumaric acid, 30.227 is 2-hydroxy cinnamic acid and 31.231 is coumarin.
Antimitotic activity using Allium cepa root tips
Growing A. cepa root tips
The fresh and healthy bulbs of onion were obtained from the local market of Tumakuru, Karnataka, India. To achieve
sprouting, the bulbs were placed in contact with distilled water and extract in a 25 ml beaker at room temperature (26±2 0C) for 2 days
in the dark individually. The distilled water was changed every 24 h between 9 to 10 h. Bulbs with root length of 2 cm and above
(range= 2.2-3 cm) were selected for A. cepa test [14].
Antimitotic studies
The bulbs that developed uniform roots were used for the experiment. These roots (three roots per concentrations) were
incubated with ethanol extract of Alternaria species and plant extract at various concentrations of 400 and 50 µg/ml. A control was set
with three roots from the same bulb with distilled water and 0.2M PBS (pH 7.2) was used as a medium. Mitotic index were recorded
at 12, 24 and 48 h of incubation and compared with that of control bulbs placed over distilled water and 0.2M PBS and quercetin was
used as standard drug [14].
Microscopic investigations
The root tip cells were fixed with stained examined using a compound microscope. The treated roots were rinsed with
distilled water and cut into segments about 1-2 cm length from the tips and fixed in ethanol: glacial acetic acid (3:1), hydrolyzed for 5
min with 1N HCl at 70 0C and stained with 2 % acetocaramine for 1 h. Stained root tips were excised and squashed on a clean glass
slide with a drop of 45 % acetic acid and examined under microscope. In all the slides, 100-300 cells were counted to determine the
number of cells in interphase and dividing phase. The mitotic index was calculated by using the formula:
Mitotic Index = Number of dividing cells/ Total number of cells X 100
Changes in chromosome morphology were photographed under (100 X) a light microscope with a Sony photographic camera.
Evaluation of cytotoxic effect of coumarin on seed germination of Vigna radiata
Inexpensive cytotoxic assay of p-coumaric acid on sprouting seeds has been carried out using green-gram (V. radiata) seeds
by the method [15], seeds were purchased from a grocery store of good quality. For germination assays, The seeds were surface
sterilized with 0.1% mercuric chloride solution for 2 minutes and washed thoroughly with tap water and then with distilled water for
30 minutes, seeds were placed in solutions of lectin from endophytic fungi, Alternaria sp. and plant extract at two concentrations (200
and 400 µg/ml) taken in 24-well plates and dry seeds of equal weight were added one each to microtitre wells and the plate was closed
with the lid and left at room temperature for 24 h for imbibed with water. Similarly, standard anticancer agent coumarin was also
prepared at various concentrations (50 and 400 µg/ml) and control plates were prepared using water. At the end of the test period (24
h), the seeds were weighed after drying them on a dry tissue paper. For morphological studies, the time of sprouting was extended to
either 48 or 72 h, the length of the radicals was measured in cm at the end of 48, 72 h and growth inhibitory effect of coumarin was
identified and photographs were taken.
Antiproliferative activity
Antiproliferative study was evaluated by yeast (S. cerevisiae) model previously reported [16].
Preparation of yeast inoculum
The yeast was inoculated with sterilized potato dextrose broth and incubated at 37 0C for 24 h, referred as seeded broth. The
seeded broth diluted with sterilized distilled water, in order to get 25.4 x 104 cells (average).
Cell viability count
For cell viability count solution containing 2.5 ml of potato dextrose broth and 0.5 ml of yeast inoculum was prepared in
separate test tubes. To each test tube 1 ml lectin extract from the endophytic fungi and plant extract was added at different
concentration (400 and 50 µg/ml) respectively. To control test tube 1ml of PBS was added without extracts. All tubes were incubated
at 37 0C for 12 h. Similarly for positive control standard drug quercetin was used. In the above cell suspension, 0.1% methylene blue
dye was added in all tubes and cells were observed under the microscope. The number of viable cells, which does not stain and look
transparent with oval shape while dead cells get stained and appeared blue in color were counted in 16 chambers of hemocytometer
and percentage of cell viability was calculated using the formula [14].
% viable cells =
No. of Viable Cells Counted
X 100
Total Cells Counted
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RESULTS AND DISCUSSION
In MAE method, the Alternaria species was yielded highest amount (3.688 mg/g)) of coumarin whereas, the plant Crotalaria
pallida flower extract had shown 3.881 mg/g. To obtain of coumarin and related compounds from plants or endophytic fungal species
from MAE is one of the best methods in present day [10, 12]. We have tested the Alternaria species extract to know coumarins and
their derivatives and it was shown positive test for tested coumarins (coumarin, O-coumari acid and hydroxy cinnamic acid) and
phenol test was also positive (Table 1). Coumarins are phenolic substances made up of fused benzene and α-pyrone rings [17]. All the
coumarin(s) tests were strong evidence for presence of coumarin(s) and its derivatives.
Table 1. Identification of coumarin and phenol from leaf endophyte, Alternaria species.
Samples
Test 1
Test 2
Test 3
Test 4
Test 5
Plant
+
+
+
+
+
Alternaria sp.
+
+
+
+
+
Data based on three replicates of each experiment, +: presence
The above three coumarin(s) were visualized under UV light in TLC experiment and was compared with standard
coumarin(s). The present interested compound, coumarin was compared with standard coumarin and it was confirmed (Fig 1). The
results are confirmed with the earlier findings [10].
Fig 1. TLC of plant, endophytic extract and standards, A) Alternaria sp. B) p-Coumaric acid, C) 2-Hydroxy coumaric acid and
D) Coumarin
In HPLC, the ethanol extract of Alternaria species was yielded three important coumarin(s) along with other phytochemicals
and were identified with standard compounds based on retention time. The crude extract of Alternaria species was yielded low amount
of all the three coumarins (Fig 2). All the coumarin(s) were identified based on retention time of the compounds compared with
standard and retention time of p-coumaric acid is 18.7, 30.227 is 2-hydroxy cinnamic acid and 31.231 is coumarin (Fig 3).
Fig 2. Ethanol extract of Alternaria species showing different phytochemicals along with three different coumarin(s) in HPLC.
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Fig 3. The standard coumarin, p-coumaric acid and hydroxy cinnamic acid peaks in HPLC.
For further, we had purified the compound, coumarin based on retention time (Fig 4) and was repeated many times to collect
the same at higher concentration. Similar results were noticed by Martino et al. [18] and Govindappa et al. [9] using Melilotus
officinalis and Crotalaria pallida. The purified endophytic fungal coumarin was run in the HPLC and confirmed with standard. The
coumarin isolated from Alternaria species in UV spectra was similar to standard (Fig 5-6).
Fig 4. Purified coumarin from Alternaria species showing 18.705 retention time.
Fig 5. Spectra of standard coumarin in UV.
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Fig 6. Spectra of isolated coumarin from Alternaria species.
By repeating HPLC method, we have collected the coumarin at high amounts for in vitro cytotoxicity studies. The ethanol
extract of Alternaria species coumarin was found to be potent in function in antimitotic, antiproliferative and growth inhibition of
green gram assays. The lower concentration of 200 µl was more significant in reducing onion root cell division and effective in
reduction in index value after 48h of treatment. We have found that Alternaria species ethanol extract was induced the antimitotic
activity at various stages of cell cycle viz., chromosomal, nucleolar and cellular abnormalities, Cell shrinkage and arrest of cellular
multiplication at interphase, B) Abnormal distribution of chromosomes at metaphase, C) Lagging chromosomes and chromosomal
bridge at anaphase and D) Chromosomal condense and cell shrinkage at anaphase. Chromosomal fragmentation, dislocation,
abnormality in movement was observed in various stages of cell division (Fig 7) and was compared with normal mitotic phases (Fig
8). The antimitotic index value decreased with treatment of Alternaria species couamrin (18.03) was compared with standard
coumarin (12.14). The results are more or similar to standard. The untreated control was showed 99.01 mg/ml (Table 2). Similar
results were observed with earlier reports of Sadananda et al. [19], Channabasava and Govindappa [20] and Govindappa et al. [14]
and Umashankar et al. [10] by using endophytic fungal extracts. The isolated coumarin had shown potential antimitogenic activity by
inducing structural changes in chromosome.
Fig 7. Normal mitotic phases, A) Interphase, B) Metaphase, C) Anaphase & D) Telophase.
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Fig 8. Chromosomal, Nucleolar and Cellular abnormalities due to Alternaria species coumarin treatment, A) Cell shrinkage
and arrest of cellular multiplication at interphase, B) Abnormal distribution of chromosomes at metaphase, C) Lagging
chromosomes and chromosomal bridge at anaphase and D) Chromosomal condense and cell shrinkage at anaphase.
Table 2. Antimitotic activitiy of coumarin isolated from Alternaria species extract in Allium cepa root tips.
Sample
Concentration
% of cytotoxicity
Control
Distilled water
99.01
Alternaria species coumarin
400 mg/ml
18.05
Coumarin (Std)
1mg/ml
12.14
Data based on three replicates of each experiment.
The Alternaria species coumarin was evaluated against yeast in antiproliferative activity and it showed potent in inhibition of
yeast cell growth. The number of dead cells was counted in coumarin treated sample and calculated using given formula. The purified
coumarin of Alternaria species inhibited the growth of yeast cells above 76.03%, whereas standard showed 88.6% (Table 3). This
indicates the purified coumarin have shown similar activity and it leads to death of yeast by inducing toxicity and death cells with
debris was observed with treated cells (Fig 9). Even at low concentration of endophytic fungal coumarin induced the highest
percentage of cytotoxicity compared with standard and distilled water control.
Table 3. In vitro antiproliferative activity of Alternaria species coumarin extract in yeast cells.
Sample
Concentration
% of cytotoxicity
Control
Distilled water
0.0
Sample A (Alternaria sp.)
10mg/ml
76.03
Coumarin (Std)
1mg/ml
88.6
Data based on three replicates of each experiment.
Fig 9. Different extracts treated yeast cells. A. control and B. Alternaria species coumarin extract treated yeast cells (stained
cells are dead cells).
The DNA fragmentation was observed in Alternaria species coumarin treated yeast cells after 48h in antiproliferative
activity. Due to toxic effect of coumarin, the yeast DNA was broken. It indicates that inhibited the DNA, further DNA replication it
may be due to activating topoisomerase or direct cleavage of DNA (Fig 10). The result was confirmed the findings of Govindappa et
al. [2014] and Channabasava and Govindappa [20]. Most of the anticancer drugs of natural products and their synthetic compounds
have activity to cleave the DNA and cause DNA damage. The natural products may suppress the DNA replication and they induced
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apoptosis. The advantage of the use of yeast as in vitro model for cytotoxic assays is that the complete genome comprises only 6250
defined genes and most importantly, many genes that are altered in human tumours have homologs and it is thought to provide a
valuable resource to achieve a greater understanding about human cancer and hopefully give insights into new approaches for therapy.
Fig 10. DNA fragmentation assay, A) Untreated lane and B) Endophytic extract treated lane.
The growth inhibitory effects of two different concentrations of coumarin extract exhibited remarkable and significant
reduction in the length of the radical in all the treated seeds compared to control. Figure 11 depicts that the germination of green gram
seeds in water, coumarin and standard coumarin treated. The development of sprouting was observed in treated with water (control)
and lower concentration of endophytic fungal coumarin green gram seeds after 48 h and their dry seed weight were 56-64, 56-64, 56-
64 and 56-64 in distilled water treated control, standard (2.0 mg/ml), endophytic coumarin (400 µl/ ml) and endophytic coumarin (200
µl/ ml) respectively. No sprouting was observed in Alternaria species coumarin and standard treated green gram had shown 2% (Fig
11). It confirms that the inhibition of root development may be due to toxicity of the compound, coumarin and it is dose dependent.
After treatment, incubation periods of 24 and 72h, the average weight of dry seeds, average seed weight and average shoot length were
measured. Endophytic fungal coumarin was shown less average seed weight and no growth of seeds compared to standard coumarin
(Table 4). Similar results were reported by Sadananda et al. [19], they inhibited the green gram germination/sprouting by using
endophytic fungal lectins and Umashankar et al. [10] have inhibited the green gram germination by using endophytic p-coumaric acid.
This result indicates that the coumarin isolated from endophytic fungi, Alternaria species have similar effects when compared with
standard coumarin.
Fig 11. Green gram seed treatment with different concentrations of endophytic extracts and standard coumarin, A) Control
(Distilled water), B) Coumarin 2.0 mg/ml (Std), C) Alternaria species coumarin (400 µl/ml) and D) Alternaria species coumarin
(200 µl/ml).
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Table 4. Observation of 24 h treated green gram seeds with different concentrations of Alternaria species coumarin extract
after 72 h of further incubation (24 to 72 h).
Observation
A
Control
(Distilled water)
B (Std)
(2.0 mg/ml)
C
(400 µl/ml)
D
(200 µl/ml)
Weight of dry seeds
(mg /seed) before imbibitions
56-64
56-64
56-64
56-64
Average seed weight after
incubation (24 +72 h) (mg/seed)
228-256
81-96
84-90
88-111
Average shoot length of
seed in mm (24 +72 h)
24-36
0-2
-
0-6
A) Control (Distilled water), B) Coumarin 1.0 mg/ml (standard), C) Alternaria species coumarin (400µl/ml) and D) Alternaria species
coumarin (200µl/ml)
CONCLUSION
Based on the results, the leaf endophytic fungi, Alternaria species of Crotalaria pallida have yielded all the suspected three
different coumarin(s) in MAE method and also other coumarin identification methods. The purified coumarin from fungal extract
showed strong antimitotic activity through various mechanisms in onion actively growing root cells and antiproliferative activity by
cause of yeast cells. The endophytic fungal coumarin had also showed inhibition of green gram germination by act as a toxic. This is
the first report in nationally and internationally isolating coumarin from endophytic fungal species and its in vitro cytotoxicity assays.
It can be used as a cancer drug after testing against cancer cell lines.
Conflict of interest
We declared that we have no any conflict of interest.
ACKNOWLEDGEMENTS
We thank, Dr MR Hulinaykar, Managing Trustee, Sri Shridevi Charitable Trust (R.) and Dr SM Shashidhara, Principal,
SIET, Tumkur, India for encouragement and Dr S Lokesh, DOS in Applied Botany and Biotechnology, University of Mysore,
Mysore, India for assistance in identifying endophytic fungi.
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... Presently there are more than 1300 coumarins have been identified in plants, bacteria, and fungi [1][2][3]. Coumarins have their specific fingerprints as antiviral [4], antimicrobial [5], antioxidant, anti-inflammatory [6,7], antiadipogenic [8], cytotoxic [9], apoptosis [10], antiprolilferative [11], antitubercular [12] and cytotoxicity [13] agent(s). Due to these wide range of pharmacological values, coumarins and its derivatives has got more importance in synthesis and production. ...
... Detection of phenols: In beakers, 5 ml of each previous filtered extract were taken and 1ml of FeCl 3 (1%) and 1 ml K 3 (Fe(CN) 6 ) (1%) were added. The appearance of fresh radish blue color indicated the presence of polyphenols [24] Isolation, purification of coumarin from Alternaria species-1 from high performance liquid chromatography (HPLC) Identification, isolation and purification of coumarin were done using Alternaria species-1ethanol extract based on our earlier report [13]. ...
... Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells. Experimental designs were done at 24 and 48h based on procedure of Umashankar et al. [13]. ...
... Presently there are more than 1300 coumarins have been identified in plants, bacteria, and fungi [1][2][3]. Coumarins have their specific fingerprints as antiviral [4], antimicrobial [5], antioxidant, anti-inflammatory [6,7], antiadipogenic [8], cytotoxic [9], apoptosis [10], antiprolilferative [11], antitubercular [12] and cytotoxicity [13] agent(s). Due to these wide range of pharmacological values, coumarins and its derivatives has got more importance in synthesis and production. ...
... Detection of phenols: In beakers, 5 ml of each previous filtered extract were taken and 1ml of FeCl 3 (1%) and 1 ml K 3 (Fe(CN) 6 ) (1%) were added. The appearance of fresh radish blue color indicated the presence of polyphenols [24] Isolation, purification of coumarin from Alternaria species-1 from high performance liquid chromatography (HPLC) Identification, isolation and purification of coumarin were done using Alternaria species-1ethanol extract based on our earlier report [13]. ...
... Since reduction of MTT can only occur in metabolically active cells, the level of activity is a measure of the viability of the cells. Experimental designs were done at 24 and 48h based on procedure of Umashankar et al. [13]. ...
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Endophyte, Alternaria species-1 isolated from leaf part of Crotalaria pallida was selected in isolation, identification and purification of coumarin. HPLC, UV-spectrophotometry, FTIR, NMR and XRD studies were carried out on characterization of endophytic fungal coumarin. In vitro apoptotic activity was carried out against HeLa cervical cancer cell lines. The results of MTT studies, acridine staining activity revealed the coumarin as an effective in inducing apoptotic activity in HeLa cells. The coumarin significantly inhibited the proliferation of HeLa cells and it’s a concentration and time dependent manner. Significant elevation of caspase 3 and 9 and activity was observed in coumarin treated HeLa cells but no elevation or activity of caspase 7, 8, 10 was not observed. Coumarin has shown 0.156 μg/ml of significant IC50 value against viable of HeLa cell lines. Thus, coumarin exerts apoptotic activity by caspase dependent apoptosis by enhancing the caspase 3 and 9 and it degraded the DNA (avoided the further replication). This is the first report in internationally that coumarin isolated from endophyte, Alternaria species-1, purified and characterized and its role was identified in apoptotic activity.
... Isolation and purification of coumarins was done with endophytic fungi, Penicillium species BCt isolated from bark tissue of Calophyllum tomentosum. Wight and was performed as described in previous studies (Umashankar et al., 2015a;Umashankar et al., 2015b). Briefly, the endophytic fungal coumarins extracts were prepared by methanol in Microwave Assisted Extraction (MAE) method for 2 cycles of 5 min each at 100 °C. ...
... Four different qualitative methods were used to identify coumarins in the endophytic fungal extracts as described previously by Umashankar et al. (Umashankar et al., 2015b). ...
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The present investigation was aimed to know the coumarins in the methanol extract of endophytic fungi, Penicillium species BCt isolated from Calophyllum tomentosum bark tissues using qualitative and GC-MS analysis. The endophytic extract was evaluated for anti-HIV activity on three replicating enzymes in vitro and in silico. The methanol extract of Penicillium species confirmed the presence of coumarins in four qualitative methods and yielded four different types of coumarins in GC-MS. In GC-MS analysis, totally seven different phytochemicals were identified based on retention time and compared with available library data. The four coumarins are coumarin (2H-1-benzopyran-2-one), coumaric acid (3-benzofuran-carboxylic acid), hynecromone (coumarin 4), 4-hydroxy-9-(3-methyl-2-butyl) furo (3,2-g) chloronen-7-one) and other three are common phytochemicals. The HIV-1 RT (98) was strongly inhibited by the endophytic fungal extract compared to integrase (118) and protease (158) in vitro analysis. Highest inhibition of integrase was observed with coumarilic acid (-17.62) when attached to Glu-35, Asn-38, Ser-39 amino acids. The protease was inhibited strongly by hymecromone (-16.39) when attached to amino acids of Val-77, Glu-34, Pro-79, Gly-78. The inhibition of RT was observed with coumarilic acid by attaching to Ala-445, Arg-567, Asp-456, Glu-478, Ser-499, Asn-474 (-23.54) significantly. Based on above results, the endophytic fungal coumarins have the ability to inhibit the three replicating enzymes of HIV-1 significantly. The in-silico results are evidence for how coumarins inhibiting the HIV replicating proteins by binding at specific amino acids. The results will help to understand how and where phytochemicals bind to target proteins to inhibit their action and it may help to identification of drugs to treat HIV. To validate our results, the in vivo research is needed.
... The extract exhibited arrests of cells at interphase and prophase with large nucleoli, chromosomal stickiness, bridges and fragments, displacement and clumping of chromosomes were observed. These results were confirmed by other research studies published in literature [12,13,33]. A potent antiproliferative activity was noticed for Penicillium species extract by inhibiting the growth of yeast cells. ...
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The present research was aimed to identify the metabolites in the methanolic extract of endophytic fungi, Penicillium species of Tabebuia argentea and evaluated the extract, in order to emphasize their in vitro cytotoxic activities. The Penicillium species extract was evaluated for cytotoxicity and antiproliferative activity and we identified the phytochemicals present in the extract by a GC-MS method. The actively growing onion root cells were inhibited by arresting the cells at various stages of cell cycles. The growth of yeast was also inhibited by inducing necrosis. 18 different phytochemicals were identified in the extract of Penicillium species by GC-MS and few of these compounds have significance in anticancer activity. Hence, the endophyte, Penicillium species contains very potent phytochemicals depicting a strong cytotoxic activity. Further research is needed to extract the active molecules and to verify their potential anticancer activity. © 2017, Romanian Society for Pharmaceutical Sciences. All rights reserved.
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The present work was aimed to identify phytochemicals in C.uredinicola methanol extract from qualitative, TLC and GC-MS method and evaluated for antioxidant, anti-HIV, anti-diabetes, anti-cholinesterase activity in vitro and insilico . The C.uredinicola extract showed flavonoids, tannins, alkaloids, glycosides, phenols, terpenoids, and coumarins presence in qualitative method. From GC-MS analysis, identified seven different phytochemicals and out of seven, four (coumarin, coumarilic acid, hymecromone, alloisoimperatorin) are coumarins. The C.uredinicola extract have shown significant antioxidant activity in DPPH (73) and FRAP (1359) method. The HIV-1 RT (83.81 + 2.14), gp 120 (80.24 + 2.31), integrase (79.43 + 3.14) and protease (77.63+2.14), DPPIV, β-glucosidase and acetyl cholinesterase activity was significantly reduced by the extract. The 2-diphenylmethyleneamino methyl ester had shown significant interaction with oxidant and HIV-1 proteins whereas alloisoimperatorin have interacted with diabetes and cholinesterase proteins followed by hymecromone with high binding energy. These three phytochemicals are non-carcinogens, non-toxic, readily degradable and have drug likeliness properties. The C.uredinicola phytochemicals are responsible for management of diabetes, HIV-1 and Alzheimer. Further in vivo work is needed to justify our research.
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Background Many biologically important bioactive compounds were identified in Calophyllum tomentosum extract. Hence, we want to identify different endophytic fungal species from different parts of the plants and their bioactive compounds. Objective Identification and characterization of bioactive compounds in extracts of endophytic fungal species of Calophyllum tomentosum. Methods Different parts were used to assess the different endophytic fungi. Two different solvents were used extract and qualitative, TLC and GC-MS were employed to identify the bioactive compounds. The bioactive compounds producing endophytes were identified by using18S rRNA. Results Totally 13 different fungal species were identified from different parts of Calophyllum tomentosum. The C. tomentosum leaf part had shown seven different fungal endophytes followed by bark (four) and stem (two). The methanol extract of Fusarium species (stem), Cladosporium species (bark) and hexane extract of Alternaria species (bark) have shown more amounts of coumarins in qualitative methods and TLC. Based on the above results, for further GC-MS and molecular identification studies, we have selected three endophytes. The GC-MS analysis, the methanol extract of Fusarium species (stem) shown psoralen, furocoumarin (imperatonin or ammidin), Cladosporium species (bark) shows coumarin (2H-1-benzopyran-2-one), coumaric acid (3-benzofurancarboxylic acid), hynecromone (coumarin 4), 4-hydroxy-9-(3-methyl-2-butyl)furo(3,2-g)chloronen-7-one and hexane extract of Alternaria species (bark) have shown coumaric acid (3-benzofurancarboxylic acid). The three fungal endophytes were identified as Fusarium equiseti, Cladosporium uredinicola and Alternaria alternata from 18S rRNA analysis. Conclusions Based on above results the three endophytic fungal species confirms the presence of coumarins and they can use for production of coumarins instead of plant.
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