Bioactive potential of endophytic Myrothecium sp. isolate M1-CA-102, associated with Calophyllum apetalum

Figures & data

Table 1. Isolates of Myrothecium spp. obtained from Calophyllum apetalum and Garcinia Morella.

Sl. no.	Isolate code	Source	Season	Locations	Accession number
Calophyllum apetalum
1	M1-CA-102	Bark	Summer	Shimoga	JX862206
2	M2-CA-203	Twig	Rainy	Shimoga	JX908884
3	M3-CA-213	Twig	Rainy	Shimoga	JX908885
4	M4-CA-222	Bark	Rainy	Shimoga	JX908886
5	M5-CA-158	Bark	Summer	Mysore	JX908887
6	M6-CA-301	Twig	Winter	Kajre	JX984612
7	M7-CA-302	Twig	Winter	Kajre	JX984613
8	M8-CA-305	Twig	Winter	Kajre	JX984614
9	M9-CA-360	Twig	Winter	Mysore	JX984615
Garcinia morella
10	M10-GM-111	Twig	Summer	Nagara Fort	JX984616
11	M11-GM-123	Twig	Summer	Nagara Fort	JX984617
12	M12-GM-130	Twig	Summer	Madikeri	JX984618
13	M13-GM-345	Bark	Winter	Nagara Fort	JX984619
14	M14-GM-367	Twig	Winter	Shimoga	JX984620
15	M15-GM-368	Twig	Winter	Madikeri	JX984621
16	M16-GM-369	Twig	Winter	Madikeri	JX984622

Figure 1. TLC profiles of the crude Myrothecium (M1-CA-102) extract. (a) TLC plate visualized under UV light and (b) TLC plate after development in iodine.

Table 2. Percent scavenging activity of DPPH and ABTS radicals by extracts of 16 Myrothecium isolates.

Sl. no.	Isolate code	% DPPH radical scavenging activity	% ABTS radical scavenging activity	Total phenol content (mg/mL)
1	M1-CA-102	71.24 ± 2.02	99.45 ± 3.55	154 ± 2.45
2	M2-CA-203	23.87 ± 1.89	39.22 ± 3.75	23 ± 3.35
3	M3-CA-213	35.9 ± 2.22	35.35 ± 2.45	28 ± 3.27
4	M4-CA-222	15.2 ± 3.09	22.3 ± 3.64	17 ± 3.49
5	M5-CA-158	52.09 ± 3.45	68 ± 2.85	55 ± 2.65
6	M6-CA-301	11.05 ± 3.23	21.89 ± 2.48	13 ± 1.99
7	M7-CA-302	40.21 ± 2.67	33.66 ± 2.53	31 ± 1.58
8	M8-CA-305	16.25 ± 1.22	18 ± 3.33	10 ± 2.66
9	M9-CA-360	10.26 ± 2.36	15.03 ± 3.85	8 ± 2.95
10	M10-GM-111	21.5 ± 2.87	28.55 ± 3.05	17 ± 3.88
11	M11-GM-123	22.36 ± 3.56	26.79 ± 3.66	20 ± 2.76
12	M12-GM-130	35.45 ± 1.65	38.75 ± 2.85	27 ± 3.22
13	M13-GM-345	21.22 ± 1.90	27.5 ± 2.58	20 ± 3.82
14	M14-GM-367	36.85 ± 2.85	41.44 ± 3.57	38 ± 3.63
15	M15-GM-368	19.32 ± 2.69	23.7 ± 3.23	17 ± 2.87
16	M16-GM-369	33.62 ± 2.55	38.29 ± 2.55	32 ± 2.90
BHT (+control)	–	85.25 ± 3.05	100 ± 2.95	–

Bold values signify that amongst the 16 isolates screened the isolate M1-CA-102 has given the highest antioxidant activity and has been taken up for further studies.

Each result is expressed as mean ± SD (n = 3).

Total phenolic content is expressed in mg gallic acid equivalent (GAE)/100 g dry weight (DW).

Table 3. Antioxidant activity of the crude extract of Myrothecium sp. M1-CA-102 isolated from twigs of Calophyllum apetalum and its TLC fractions.

Sl. no.	Test samples	DPPH (IC50) (μg/mL)	ABTS (IC50) (μg/mL)
1	M1-CA-102 (crude)	10 ± 1.25	6 ± 2.32
2	M-I (TLC fraction)	45 ± 2.09	43 ± 1.46
3	M-II (TLC fraction)	45 ± 2.5	40 ± 1.37
4	M-flu (TLC fraction)	40 ± 1.95	21 ± 2.22
5	Ascorbic acid (+ control)	32.30 ± 1.86	75.2 ± 2.83
6	Butylated hydroxytoluene (+ control)	14.46 ± 1.57	19.5 ± 2.51
7	Quercetin (+ control)	6.1 ± 2.08	7.03 ± 1.64

Each result is expressed as mean ± SD (n = 3).

Table 4. Antimicrobial activity of the crude extract of Myrothecium sp. M1-CA-102 and the TLC fractions.

	Zone of inhibition (mm)^a/(minimum inhibitory concentration [μg/mL])^b
Microorganisms	Antibacterial activity						Antifungal activity
Test samples	Klebsiella pneumoniae	Staphylococcus aureus	Escherichia coli	Salmonella typhi	Shigella flexneri	Bacillus subtilis	Candida albicans
M1-CA-102 (crude)	23 ± 1.25 (35)	17 ± 1.22 (88)	17 ± 2.32 (82)	18 ± 1.5 (64)	23 ± 2.07 (40)	20 ± 1.5 (57)	21 ± 1.22 (54)
M-I (TLC fraction)	12 ± 2.09 (143)	11 ± 1.85 (156)	13 ± 2.01 (137)	9 ± 1.0 (185)	12 ± 2.2 (155)	11 ± 2.02 (160)	7 ± 1.5 (225)
M-II (TLC fraction)	6 ± 2.22 (186)	7 ± 2.65 (181)	10 ± 2.0 (165)	7 ± 1.25 (200)	11 ± 1.45 (152)	9 ± 2.0 (170)	0 (515)
M-flu (TLC fraction)	19 ± 1.5 (91)	15 ± 2.05 (110)	12 ± 1.07 (138)	12 ± 1.23 (145)	17 ± 1.25 (105)	18 ± 1.56 (82)	11 ± 2.04 (167)
Chloramphenicol^c	25 ± 2.1 (13.5)	27 ± 2.08 (22)	21 ± 1.9 (21)	23 ± 2.05 (27)	25 ± 1.9 (32)	27 ± 1.25 (47)	–
Nystatin^d	–	–	–	–	–	–	23 ± 2.12 (21.5)

^aAll determinations were done in triplicates, standard deviation for 95% confidence.

^bThe values are mean of three determinations, the ranges of which are <5% of the mean in all case.

^cPositive control for antibacterial activity.

^dPositive control for antifungal activity.

Figure 2. Bacillus subtilis (a) and Candida albicans (b) growth, survival, and death curves on exposure to the crude extract of Myrothecium (M1-CA-102). Each point represents the log of the mean ± SD CFU/mL.

Table 5. Anti-inflammatory activity of Myrothecium extract and its TLC fractions.

	15-Lipoxygenase activity			Human cyclooxygensae-2 activity
Test samples	Final concentration of methanolic extract (μg/mL)	% inhibition	IC50 (μg/mL)	Final concentration of methanolic extract (μg/mL)	% inhibition	IC50 (μg/mL)
M1-CA-102 (crude)	25	22.74		25	79.55
	50	42.57	58	50	94.10	8
	100	90.20		100	100
M-I (TLC fraction)	5	11.22		5	6.03
	10	22.89	25	25	15.11	50
	25	45.16		50	33.45
M-II (TLC fraction)	5	9.31		5	6.27
	10	17.89	57	25	11.9	68
	25	26.16		50	23.42
M-flu (TLC fraction)	5	27.98		5	22.04
	10	49.44	10	25	58.43	21
	25	81.57		50	79.66
Quercetin ( + control)	25	35.72		25	81.29
	50	68.55	36	50	98.65	5
	100	100		100	100

Figure 3. Endophyte extracts assessed on plasmid pBR322 DNA treated by Fenton’s reagent. Lane 1: pBR322 (native plasmid DNA); Lane 2: pBR322 DNA + Fenton’s reagent; Lane 3: pBR322 + Myrothecium extract (50 μg) + Fenton’s reagent; Lane 4: pBR322 + TLC fraction M-II (50 μg) + Fenton’s reagent; Lane 5: pBR322 + TLC fraction M-I (50 μg) + Fenton’s reagent; Lane 6: pBR322 + TLC fraction M-flu (50 μg) + Fenton’s reagent.

Figure 4. Acridine orange – ethidium bromide staining of HeLa cells observed under fluorescent microscope. (A) Control (untreated cells), (B) cells treated with doxorubicin, and (C) cells treated with the crude extract of Myrothecium (M1-CA-102).

Table 6. Cytotoxicity of crude extract of Myrothecium sp. M1-CA-102 and its TLC fractions against HeLa cervix cancer cell lines.

Test samples	Concentration (μg/mL)	% inhibition of HeLa cells	IC50 (μg/mL)
M1-CA-102 (crude)	25	35.47
	50	68.90	37 ± 1.25
	100	98.54
M-I (TLC fraction)	15	25.26
	25	70.54	21 ± 1.2
	50	92.47
M-II (TLC fraction)	15	20.71
	25	42.35	36 ± 1.22
	50	60.12
M-flu (TLC fraction)	15	22.15
	25	47.92	27 ± 1.09
	50	71.15
Doxorubicin (+ control)	15	48.30
	25	64.57	16 ± 1.53
	50	99.05

Figure 5. Analytical HPLC chromatograms of standards and crude endophyte extracts separated on a semi-preparative RP-HPLC column. (a) HPLC chromatogram of Myrothecium extract. (b) HPLC chromatogram of TLC fraction M-I from Myrothecium. (c) HPLC chromatogram of TLC fraction M-II from Myrothecium. (d) HPLC chromatogram of TLC fraction M-flu from Myrothecium. (e) HPLC chromatogram of a mixture of gallic acid, quercetin, and phloroglucinol-R showing specific peak at retention time 3.044, 5.730, and 9.609 min, respectively.

Figure 6. Mass spectra of the TLC fractions of Myrothecium (M1-CA-102). (a) MS of TLC fraction M-I. (b) MS of TLC fraction M-II. (c) MS of TLC fraction M-flu.