molecules

Article
Antioxidant, Antibacterial and Dyeing Potential of Crude
Pigment Extract of Gonatophragmium triuniae and Its
Chemical Characterization
Ajay C. Lagashetti 1,2 , Sanjay K. Singh 1,2, * , Laurent Dufossé 3, * , Pratibha Srivastava 2,4 and Paras N. Singh 1,2

1 National Fungal Culture Collection of India (NFCCI), Biodiversity and Palaeobiology Group,
MACS’ Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India;
lagashettiajay@gmail.com (A.C.L.); pnsingh@aripune.org (P.N.S.)
2 Faculty of Science, Savitribai Phule Pune University, Pune 411007, India
3 CHEMBIOPRO Chimie et Biotechnologie des Produits Naturels, ESIROI Département Agroalimentaire,
Université de la Réunion, F-97490 Sainte-Clotilde, Ile de La Réunion, France
4 Bioprospecting Group, MACS’ Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India;
psrivastava@aripune.org
* Correspondence: sksingh@aripune.org (S.K.S.); laurent.dufosse@univ-reunion.fr (L.D.);
Tel.: +91-20-25325103 (S.K.S.); +33-66-8731906 (L.D.)

Abstract: Filamentous fungi synthesize natural products as an ecological function. In this study, an
interesting indigenous fungus producing orange pigment exogenously was investigated in detail as
it possesses additional attributes along with colouring properties. An interesting fungus was isolated
from a dicot plant, Maytenus rothiana. After a detailed study, the fungal isolate turned out to be a
species of Gonatophragmium belonging to the family Acrospermaceae. Based on the morphological,



cultural, and sequence-based phylogenetic analysis, the identity of this fungus was confirmed as


Gonatophragmium triuniae. Although this fungus grows moderately, it produces good amounts of
Citation: Lagashetti, A.C.; Singh,
pigment on an agar medium. The fermented crude extract isolated from G. triuniae has shown
S.K.; Dufossé, L.; Srivastava, P.; Singh,
antioxidant activity with an IC50 value of 0.99 mg/mL and antibacterial activity against Gram-
P.N. Antioxidant, Antibacterial and
Dyeing Potential of Crude Pigment
positive bacteria (with MIC of 3.91 µg/mL against Bacillus subtilis, and 15.6 µg/mL and 31.25 µg/mL
Extract of Gonatophragmium triuniae for Staphylococcus aureus and Micrococcus luteus, respectively). Dyeing of cotton fabric mordanted
and Its Chemical Characterization. with FeSO4 using crude pigment was found to be satisfactory based on visual observation, sug-
Molecules 2022, 27, 393. https:// gesting its possible use in the textile industry. The orange pigment was purified from the crude
doi.org/10.3390/molecules27020393 extract by preparative HP-TLC. In addition, UV-Vis, FTIR, HRMS and NMR (1 H NMR, 13 C NMR),
Academic Editor: Jacqueline
COSY, and DEPT analyses revealed the orange pigment to be “1,2-dimethoxy-3H-phenoxazin-3-one”
Aparecida Takahashi (C14 H11 NO4 , m/z 257). To our understanding, the present study is the first comprehensive report on
Gonatophragmium triuniae as a potential pigment producer, reporting “1,2-dimethoxy-3H-phenoxazin-
Received: 13 December 2021
3-one” as the main pigment from the crude hexane extract. Moreover, this is the first study reporting
Accepted: 5 January 2022
antioxidant, antibacterial, and dyeing potential of crude extract of G. triuniae, suggesting possible
Published: 8 January 2022
potential applications of pigments and other bioactive secondary metabolites of the G. triuniae in
Publisher’s Note: MDPI stays neutral textile and pharmaceutical industry.
with regard to jurisdictional claims in
published maps and institutional affil- Keywords: Gonatophragmium triuniae; pigments; bioactivity; dyeing; chemical characterization
iations.

1. Introduction
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland. Ascomycetous filamentous fungi are known to produce bio-pigments extensively used
This article is an open access article as colourants, additives, colour intensifiers, antimicrobial, antioxidants, etc., in different
distributed under the terms and industries such as food, beverages, cosmetics, textiles, and pharmaceuticals [1–3]. Natural
conditions of the Creative Commons pigments/colours are gaining increased attention currently because of the adverse effects
Attribution (CC BY) license (https:// of synthetic colours on humans and the environment. Most of the synthetic colours have
creativecommons.org/licenses/by/ been found to be toxic, allergic, and carcinogenic to human beings, and hazardous to the
4.0/). environment [4–6]. This has increased the need for safe, natural, eco-friendly pigments as

Molecules 2022, 27, 393. https://doi.org/10.3390/molecules27020393 https://www.mdpi.com/journal/molecules

Molecules 2022, 27, 393 2 of 23

an alternative to synthetic pigments. Increasing demand for natural pigments necessitates

a greater need to explore colours or pigments from safe, natural sources, especially from
microbes (bacteria, fungi, algae, lichens, and actinomycetes).
Fungi are currently emerging as a better and excellent source of natural pigments.
Many researchers have reported fungi of different taxonomic groups exhibiting the pro-
duction of pigments of diverse chemicals classes such as carotenoids, flavins, ubiquinones,
anthraquinones, quinines, phenazines, etc. [1,7–10]. Due to the additional attributes of
these “mycopigments” such as antimicrobial, anticancer, antioxidant, cytotoxicity, activity,
etc. in addition to colouring property, they are being extensively used for a wide range of
applications in food, textiles, medicines, paints, cosmetics, and electronics [8,9]. Some fun-
gal pigments such as azaphilones, astaxanthin, Arpink Red, riboflavin, β-carotene isolated
from Monascus, Xanthophyllomyces dendrorhous, Penicillium oxalicum, Ashbya gossypii, and
Blakeslea trispora, respectively, are already in the market for their commercial and industrial
applications [11]. Numerous studies have reported the dyeing potential of fungal pigments
and suggested their possible use in the textile industry for dyeing different types of textile
fabrics like cotton, silk, wool, etc. [7,8,12,13].
Literature indicates that extensive studies have been done worldwide on produc-
tion, optimization, and applications of pigments from common ascomycetous fungi like
Monascus, Talaromyces, Aspergillus, Penicillium, Fusarium, etc. [8,9]. Besides these conven-
tional ones, several other genera, such as Epicoccum, Trichoderma, Alternaria, Chaetomium,
etc., are reported to have good pigment production potential [7,14–18]; however, several
genera of filamentous ascomycetes are still unknown and unexplored for their pigment
production potential and exploitations. These unexplored fungi might prove to be a hidden
treasure of novel bio-active pigments having a variety of applications.
Taking this view into account, we have planned the present research in which we
have isolated an uncommon fungus, Gonatophragmium triuniae, from infected leaves of the
Maytenus rothiana, a plant endemic to central Maharashtra (Western Ghats). Interestingly,
it was found that this rare fungus produces a very good extracellular orange pigment
on solid media [potato dextrose agar medium (PDA)] as well as in liquid media [potato
dextrose (PD) broth]. For the characterization of pigment, pure culture of G. triuniae was
subjected to flask level fermentation in PD broth, and pigments were extracted from the
culture filtrate using Hexane and dried. The dried Hexane extract was then examined
for its antimicrobial and antioxidant properties and also assessed for its dyeing potential
on cotton fabric using two mordants (Alum & FeSO4 ). Finally, by preparative thin-layer
chromatography (TLC), we purified an orange pigment from the crude pigment extract and
identified it as “1,2-dimethoxy-3H-phenoxazin-3-one” based on ultra-violet (UV); Fourier
transform infrared (FTIR); high-resolution mass (HRMS) spectroscopy; and 1 H & 13 C NMR,
COSY, and DEPT analysis.
Several natural and synthetic phenoxazines are well known for their bioactivity and
dyeing properties. These phenoxazines were found to exhibit antioxidant, antibacterial,
anti-proliferative, and anti-tumoral activities [19]. 3H-phenoxazin-3-one and its derivatives
exhibiting numerous biological activities (antimicrobial, anticancer, antitumor, antiviral,
antitubercular, anticoccidial, antineoplastic, phytotoxic, and cell growth-stimulating) have
been reported from different microorganisms such as actinomycetes, lichens, and fungi [20].
Phenoxazine class of pigments such as Phenoxazone, Pycnosanguin, Cinnabarine, O-acetyl
cinnabarine, 2-Amino-9-formylphenoxazone-1-carbonic acid, and 9-Hydroxymethyl-2-
methylaminophenoxazone-1-carbonic acid methyl ester have been described from the
fungus Pycnoporus sanguineus [21]. Similarly, Chandrananimycins A-C belonging to class
3H-phenoxazin-3-one, exhibiting antitumor activity against colon cancer (CCL HT29);
breast cancer (LCL H460, CNCL SF268, MACL MCF-7); lung cancer (LXFA 526L, LXFL
529L); melanoma (MEXF 514L); and kidney tumor cells (PRCL PC3M, RXF 631L) has
also been reported from Actinomadura sp. [22,23]. One of the recent studies evaluated the
antibacterial activity of the synthesized derivatives of 3H-phenoxazin-3-one [20]. Based
on the history of Phenoxazin class of pigments, their promising bioactivity, and their
Molecules 2022, 27, 393 3 of 23

dyeing property, we may consider present compound 1,2-dimethoxy-3H-phenoxazin-3-one

isolated from G. triuniae NFCCI 4873 as a good colourant as well as a potential candidate
for application in the textile and pharmaceutical industry.

2. Results and Discussion

2.1. Morphological Identification
Leaf lesions, amphigenous, necrotic spots single or irregular in concentric rings, later
spots unite to form large spots. Margin: greyish-white; center: white. Colonies hypophyl-
lus, velvety, brown. Mycelium superficial. Hyphae branched, septate, pale olivaceous
to subhyaline, smooth-walled, up to 6.5 µm wide. Stroma and hyphopodia are absent.
Conidiophores arising from superficial hyphae, dichotomously branched, multi-septate
(6–8), the width of conidiophore gradually decreasing towards the length; macronema-
tous to mononematous, erect, smooth-walled, highly geniculate, nodose, basal half part
of conidiophore olivaceous brown and subhyaline to light olivaceous towards the apex,
55–145 × 3.22–7 µm. Conidiogenous cells integrated, polyblastic, terminal to intercalary,
swollen towards the apex, variable in size: 10–20 µm long, bearing 10–15 loci, scars thick-
ened and darkened, dentate or plate-like about 1 µm diameter. Conidia solitary, dry,
holoblastic, acropleurogenous, clavate, cylindrical, straight to slightly curved, 0–1 septate,
smooth-walled, subhyaline to light olivaceous, base narrowly truncate, apex obtuse, hilum
thickened and darkened, 6–15 × 2–3.6 µm (Figure 1).
Colonies on Potato Dextrose Agar (PDA), slow-growing, reach 26–29 mm diameter
after 4 weeks of incubation at 25 ◦ C; front view of colony light yellow (4A4), circular,
raised, aerial mycelium slightly cottony, margin smooth. Reverse dark brown (7F8) with
diffusible yellowish orange (5B8) pigment in entire media. Mycelium branched, septate,
smooth-walled, with frequent anastomosis, hyaline, sterile. Colonies on Potato Carrot Agar
(PCA), reach 27–28 mm diameter after 4 weeks at 25 ◦ C; front view of colony grey (6C1),
circular, raised, slightly cottony with margin smooth. Reverse dark brown (6F8) and with
diffusible yellowish orange (5B8) pigment in entire media. Mycelium branched, septate,
smooth-walled, anastomosis, hyaline, sterile (Figure 1).

2.2. Molecular Identification and Phylogeny

Mega Blast analysis of ITS sequence of G. triuniae NFCCI 4873 showed 100% identity
with type strain G. triuniae CBS138901; whereas LSU sequence showed 99.74% identity with
G. epiloblii CPC 34889 and 99.61% similarity with G. triuniae CBS 138901. A phylogenetic
tree was constructed based on combined ITS & LSU rDNA sequence data of a total of
19 genetically-related isolates, which shows that our isolate was clustered with G. triuniae
CBS138901 with a very strong bootstrap value of 98.4 (Figure 2). Therefore, based on
combined morphological and molecular phylogenetic analysis, the present isolate was
identified as G. triuniae.
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Figure 1. Gonatophragmium triuniae (NFCCI 4873): (a) Numerous conidiophores in low magnifica‐
Figure 1. Gonatophragmium triuniae (NFCCI 4873): (a) Numerous conidiophores in low magnification;
tion; (b) Single dichotomously branched conidiophore; (c) Conidiophore and conidiogenous cells 
(b) Single dichotomously branched conidiophore; (c) Conidiophore and conidiogenous cells bearing
bearing dark scars (showing arrows); (d) Numerous conidia in high magnification; (e) SEM of co‐
dark scars (showing arrows); (d) Numerous conidia in high magnification; (e) SEM of conidiophores
nidiophores with attached conidia; (f) SEM of conidiophore bearing intercalary and terminal conid‐
with attached conidia; (f) SEM of conidiophore bearing intercalary and terminal conidiogenous cells
iogenous cells with dark scars (showing arrows); (g,h) SEM image of conidia; (i–l) Colonies of G. 
with dark scars (showing arrows); (g,h) SEM image of conidia; (i–l) Colonies of G. triuniae NFCCI
triuniae NFCCI 4873 on PDA and PCA (front and reverse view); (m,n) Hyaline sterile hyphal bundle 
4873 on PDA and PCA (front and reverse view); (m,n) Hyaline sterile hyphal bundle and anastomosis
and anastomosis in‐vitro culture (showing arrows); (o) SEM of sterile hyphal bundles with coiling 
in-vitro culture (showing arrows); (o) SEM of sterile hyphal bundles with coiling (showing arrow).
(showing arrow). 

2.2. Molecular Identification and Phylogeny 
Mega Blast analysis of ITS sequence of G. triuniae NFCCI 4873 showed 100% identity 
with type strain G. triuniae CBS138901; whereas LSU sequence showed 99.74% identity 
with G. epiloblii CPC 34889 and 99.61% similarity with G. triuniae CBS 138901. A phyloge‐
netic tree was constructed based on combined ITS & LSU rDNA sequence data of a total 
of 19 genetically‐related isolates, which shows that our isolate was clustered with G. triu‐
niae CBS138901 with a very strong bootstrap value of 98.4 (Figure 2). Therefore, based on 
combined  morphological  and  molecular  phylogenetic  analysis,  the  present  isolate  was 
identified as G. triuniae. 

 
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Molecules 2022, 27, 393 5 of 23

  rDNA
Figure 2. Phylogenetic tree of G. triuniae NFCCI 4873 based on the combined ITS & LSU
sequence data. Digits on the nodes represent the likelihood bootstrap values.
Figure  2.  Phylogenetic  tree  of  G.  triuniae  NFCCI  4873  based  on  the  combined  ITS  &  LSU  rDNA 
sequence data. Digits on the nodes represent the likelihood bootstrap values. 
2.3. Analysis of Pigment Production on Different Media
After 4 weeks of incubation, G. triuniae NFCCI 4873 produced yellowish orange (5B8)
2.3. Analysis of Pigment Production on Different Media 
pigment on potato dextrose agar (PDA), potato carrot agar (PCA), Sabouraud dextrose agar
(SDA), and Czapek Yeast Extract Agar (CYA), which was completely diffused in media. In
After 4 weeks of incubation, G. triuniae NFCCI 4873 produced yellowish orange (5B8) 
contrast, no pigment production was observed on cornmeal agar (CMA) and Czapek Dox
pigment on potato dextrose agar (PDA), potato carrot agar (PCA), Sabouraud dextrose 
agar (CZA) (Figure 3).
agar  (SDA),  and  Czapek  Yeast  Extract  Agar  (CYA),  which  was  completely  diffused  in 
media. In contrast, no pigment production was observed on cornmeal agar (CMA) and 
Czapek Dox agar (CZA) (Figure 3). 
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Figure 3. Studies on pigment production by G. triuniae (NFCCI 4873) on different media:
Figure 3. Studies on pigment production by G. triuniae (NFCCI 4873) on different media: (a,b) G. 
(a,b) G. triuniae on PDA (front and reverse view); (c,d) G. triuniae on PCA (front and reverse view);
triuniae on PDA (front and reverse view); (c,d) G. triuniae on PCA (front and reverse view); (e,f) G. 
(e,f) G. triuniae on SDA (front and reverse view); (g,h) G. triuniae on CMA (front and reverse view);
triuniae on SDA (front and reverse view); (g,h) G. triuniae on CMA (front and reverse view); (I,j) G. 
(I,j) G. triuniae on CYA (front and reverse view; and (k,l) G. triuniae on CZA (front and reverse view).
triuniae on CYA (front and reverse view; and (k,l) G. triuniae on CZA (front and reverse view). 

2.4. Pigment Production in Liquid Media

2.4. Pigment Production in Liquid Media 
G. triuniae NFCCI 4873 started pigment production earlier in PD broth of Hi-media
G. triuniae NFCCI 4873 started pigment production earlier in PD broth of Hi‐media 
compared to natural PD broth and natural PC broth. However, pigment production
compared to natural PD broth and natural PC broth. However, pigment production in‐
increased in natural PD broth and PC broth after 28 days of incubation compared to the
creased in natural PD broth and PC broth after 28 days of incubation compared to the PD 
PD broth of Hi-media (Figure 4). Scanning of coloured culture filtrate of G. triuniae NFCCI
broth of Hi‐media (Figure 4). Scanning of coloured culture filtrate of G. triuniae NFCCI 
4873 from three different media at a wavelength ranging from 390–760 nm shows that
4873  from  three  different  media  at  a  wavelength  ranging  from  390–760  nm  shows  that 
pigment production was higher in natural PD broth than PD broth (Hi-media) and natural
pigment production was higher in natural PD broth than PD broth (Hi‐media) and natural 
PC broth (Figure 5). This clearly shows that natural potato dextrose broth supports the
PC broth (Figure 5). This clearly shows that natural potato dextrose broth supports the 
pigment production by G. triuniae NFCCI 4873 and suggests it as a good media for optimum
pigment production by G. triuniae NFCCI 4873 and suggests it as a good media for opti‐
pigment production.
mum pigment production. 

 
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Figure 4. Analysis of pigment production by G. triuniae NFCCI 4873 in different liquid media. 
Figure 4. Analysis of pigment production by G. triuniae NFCCI 4873 in different liquid media.
Figure 4. Analysis of pigment production by G. triuniae NFCCI 4873 in different liquid media. 

 
 
Figure 5. UV–Vis spectroscopy scanning (390–760 nm) of culture filtrates of different media. 
Figure 5. UV–Vis spectroscopy scanning (390–760 nm) of culture filtrates of different media. 
Figure 5. UV–Vis spectroscopy scanning (390–760 nm) of culture filtrates of different media.
   
   

 
 
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2.5. Fermentation and Extraction of Pigments 
Upon filtration of 6‐L fermentation broth of G. triuniae NFCCI 4873, approximately 4 
2.5. Fermentation2.5. Fermentation and Extraction of Pigments 
and Extraction of Pigments
L of coloured culture filtrate and 75 g of dry fungal biomass were obtained. Pigments from 
Upon filtration of 6‐L fermentation broth of G. triuniae NFCCI 4873, approximate
Upon filtration of 6-L fermentation broth of G. triuniae NFCCI 4873, approximately 4 L
the coloured culture filtrate were extracted with Hexane, and the concentration of hexane 
of coloured L of coloured culture filtrate and 75 g of dry fungal biomass were obtained. Pigments f
culture filtrate and 75 g of dry fungal biomass were obtained. Pigments from
extract in a rota evaporator under reduced pressure yielded 526.26 mg of crude hexane 
the coloured the coloured culture filtrate were extracted with Hexane, and the concentration of hex
culture filtrate were extracted with Hexane, and the concentration of hexane
extract. This dried crude hexane extract was then used for subsequent analysis, testing, 
extract in a rotaextract in a rota evaporator under reduced pressure yielded 526.26 mg of crude hex
evaporator under reduced pressure yielded 526.26 mg of crude hexane
and purification (Figure 6). 
extract. This dried crude hexane extract was then used for subsequent analysis, test
extract. This dried crude hexane extract was then used for subsequent analysis, testing,
and purificationand purification (Figure 6). 
(Figure 6).

   
Figure 6. Schematic representation of fermentation, extraction, and purification of the compound 
Figure 6. Schematic representation of fermentation, extraction, and purification of the compo
Figure 6. Schematic representation of fermentation, extraction, and purification of the compound
from a pure culture of G. triuniae NFCCI 4873. 
from a pure culture of G. triuniae NFCCI 4873. 
from a pure culture of G. triuniae NFCCI 4873.
2.6. UV–Vis Spectroscopy Analysis of Hexane Extract 
2.6. 2.6. UV–Vis Spectroscopy Analysis of Hexane Extract 
UV–Vis Spectroscopy Analysis of Hexane Extract
Dried hexane extract dissolved in methanol showed maximum absorption at 220 nm 
Dried hexane extract dissolved in methanol showed maximum absorption at 220
Dried hexane extract dissolved in methanol showed maximum absorption at 220 nm
(λmax
(λ max)) upon scanning at a wavelength ranging from 190–760 nm (Figure 7). 
(λmax) upon scanning at a wavelength ranging from 190–760 nm (Figure 7). 
upon scanning at a wavelength ranging from 190–760 nm (Figure 7).

 
 
Figure 7. UV–Vis spectrum of hexane extract dissolved in methanol of G. triuniae NFCCI 4873. 
Figure 7. UV–Vis spectrum of hexane extract dissolved in methanol of G. triuniae NFCCI 4873. 
Figure 7. UV–Vis spectrum of hexane extract dissolved in methanol of G. triuniae NFCCI 4873.

 
 
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2.7. Antagonistic Activity Testing 
2.7. Antagonistic Activity Testing
The dual
The dual  culture 
culture assay 
assay shows 
shows thatthat  G.  triuniae 
G. triuniae NFCCI NFCCI  4873  retarded 
4873 retarded the  growth 
the growth of fungalof 
fungal plant pathogens (Colletotrichum gloeosporioides, Fusarium oxysporum, and Fusarium 
plant pathogens (Colletotrichum gloeosporioides, Fusarium oxysporum, and Fusarium solani).
solani). Among them, G. triuniae showed 30% inhibition of radial growth of F. solani, fol‐
Among them, G. triuniae showed 30% inhibition of radial growth of F. solani, followed by
lowed by 23% inhibition of radial growth of C. gloeosporioides and 16% inhibition of radial 
23% inhibition of radial growth of C. gloeosporioides and 16% inhibition of radial growth of
F.growth  of  F. This
oxysporum. oxysporum. 
indicatesThis 
that indicates 
our isolatethat 
hasour  isolate  has 
the potential to the  potential 
inhibit to  inhibit 
the growth the 
of other
growth of other fungal plant pathogens (Figure 8). 
fungal plant pathogens (Figure 8).

 
Figure 8. Antagonistic activity of G. triuniae NFCCI 4873: (a) C. gloeosporioides on PDA (control),
Figure 8. Antagonistic activity of G. triuniae NFCCI 4873: (a) C. gloeosporioides on PDA (control), (b,c) 
(b,c) G. triuniae against C. gloeosporioides on PDA (front and reverse view), (d) F. oxysporum on PDA
G. triuniae against C. gloeosporioides on PDA (front and reverse view), (d) F. oxysporum on PDA (con‐
trol), (e,f) G. triuniae against F. oxysporum on PDA (front and reverse view), (g) F. solani on PDA 
(control), (e,f) G. triuniae against F. oxysporum on PDA (front and reverse view), (g) F. solani on PDA
(control), and (h,i) G. triuniae against F. oxysporum on PDA (front and reverse view). 
(control), and (h,i) G. triuniae against F. oxysporum on PDA (front and reverse view).

2.8. Antioxidant Activity Testing of G. triuniae NFCCI 4873

2.8. Antioxidant Activity Testing of G. triuniae NFCCI 4873 
From the result of in-vitro antioxidant activity, it was observed that hexane extract
From the result of in‐vitro antioxidant activity, it was observed that hexane extract of 
G. triuniae NFCCI 4873 showed satisfactory dose-dependent DPPH radical scavenging
ofG. triuniae NFCCI 4873 showed satisfactory dose‐dependent DPPH radical scavenging ac‐
activity with an IC
tivity with an IC 50 value of 0.99 mg/mL when tested with standard ascorbic acid
50 value of 0.99 mg/mL when tested with standard ascorbic acid (IC
(IC50
50 value 
value of 0.24 mg/mL). This suggests the promising antioxidant potential of crude hexane
of 0.24 mg/mL). This suggests the promising antioxidant potential of crude hexane extract 
extract of G. triuniae NFCCI 4873 (Figure 9).
of G. triuniae NFCCI 4873 (Figure 9). 

 
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23 
 

 
 
Figure 9. Scavenging effects of hexane extract of G. triuniae NFCCI 4873 on DPPH radical. 
Figure 9. Scavenging effects of hexane extract of G. triuniae NFCCI 4873 on DPPH radical. 
Figure 9. Scavenging effects of hexane extract of G. triuniae NFCCI 4873 on DPPH radical.
2.9. Antimicrobial Activity Testing by Disc Diffusion 
2.9. Antimicrobial Activity Testing by Disc Diffusion
2.9. Antimicrobial Activity Testing by Disc Diffusion 
A crude hexane extract of G. triuniae showed promising antibacterial activity in the 
A crude hexane extract of G. triuniae showed promising antibacterial activity in the
A crude hexane extract of G. triuniae showed promising antibacterial activity in the 
disc diffusion assay (Figure 10). The mean diameters of the inhibition zones of test strains 
disc diffusion assay (Figure 10). The mean diameters of the inhibition zones of test strains
disc diffusion assay (Figure 10). The mean diameters of the inhibition zones of test strains 
for  the  crude  hexane  extract  of  G.  triuniae  NFCCI 4873 are  shown  in  Table 1.  From  the 
for the
for  crude hexane
the  crude  hexane extract of G.
extract  of triuniae NFCCI
G.  triuniae  4873 are shown
NFCCI 4873 are  in Table
shown  in 1. From the
Table 1.  results
From  the 
results of disk diffusion assay, it was observed that crude hexane extract G. triuniae NFCCI 
of disk diffusion assay, it was observed that crude hexane extract G. triuniae NFCCI
results of disk diffusion assay, it was observed that crude hexane extract G. triuniae NFCCI  4873
4873 showed promising antimicrobial activity mainly against Gram‐positive bacteria (B. 
showed promising antimicrobial activity mainly against Gram-positive bacteria (B. subtilis,
4873 showed promising antimicrobial activity mainly against Gram‐positive bacteria (B. 
subtilis, S. aureus, and M. luteus), displaying an average zone diameter of 17.33 mm, 18.67 
S. aureus, and M. luteus), displaying an average zone diameter of 17.33 mm, 18.67 mm,
subtilis, S. aureus, and M. luteus), displaying an average zone diameter of 17.33 mm, 18.67 
mm, and 17.33 mm respectively (Table 1). However, it showed very little activity against 
and 17.33 mm respectively (Table 1). However, it showed very little activity against Gram-
mm, and 17.33 mm respectively (Table 1). However, it showed very little activity against 
Gram‐negative  bacterium  R.  planticola  with  an  zone
average  zone  diameter  of  12.33  mm; 
negative bacterium
Gram‐negative  R. planticola
bacterium  with anwith 
R.  planticola  average diameter
an  average  of 12.33 mm;
zone  diameter  whereas
of  12.33  mm; 
whereas no activity was observed against E. coli and P. aeruginosa. In general, S. aureus 
no activity was observed against E. coli and P. aeruginosa. In general, S. aureus was more
whereas no activity was observed against E. coli and P. aeruginosa. In general, S. aureus 
was more sensitive to the crude hexane extract, followed by B. subtilis and M. luteus, which 
sensitive to the crude hexane extract, followed by B. subtilis and M. luteus, which show
was more sensitive to the crude hexane extract, followed by B. subtilis and M. luteus, which 
show similar sensitivity to the crude hexane extract. The study revealed that crude hexane 
similar sensitivity to the crude hexane extract. The study revealed that crude hexane
show similar sensitivity to the crude hexane extract. The study revealed that crude hexane 
extract of G. triuniae NFCCI 4873 has potential antibacterial compounds that can be used 
extract of G. triuniae NFCCI 4873 has potential antibacterial compounds that can be used in
extract of G. triuniae NFCCI 4873 has potential antibacterial compounds that can be used 
in medicines for their possible applications as an antibiotic.   
medicines for their possible applications as an antibiotic.
in medicines for their possible applications as an antibiotic.   

 
 
Figure 10. Antimicrobial activity of hexane extract of G. triuniae NFCCI 4873 against different test 
Figure 10. Antimicrobial activity of hexane extract of G. triuniae NFCCI 4873 against
Figure 10. Antimicrobial activity of hexane extract of G. triuniae NFCCI 4873 against different test 
bacteria. 
different test bacteria.
bacteria. 
   
   

 
 
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Table 1. The zone of inhibition of test organisms against sample and standards with control. 
Molecules 2022, 27, 393 11 of 23
The Diameter of the Zone of Inhibition in Millimeters 
(Values are Average of Three Readings) 
Samples 
E. coli 
Table 1. B. subtilis  P. aeruginosa 
The zone of inhibition S. aureus 
of test organisms R. planticola 
against sample M. luteus 
and standards with control.
MTCC 739  MTCC 121  MTCC 2453  MTCC 2940  MTCC 530  MTCC 2470 
The Diameter of the Zone of Inhibition in Millimeters
xtract of G. triuniae NFCCI 
‐  17.33 ± 1.53 (Values are
‐  Average18.67 ± 0.58  12.33 ± 0.58 
of Three Readings) 17.33 ± 1.53 
4873  Samples
E. coli MTCC B. subtilis P. aeruginosa S. aureus R. planticola M. luteus
Streptomycin  23.33 ± 2.89 
739 18 ± 0 
MTCC 121 9 ± 0 2453
MTCC ‐ 
MTCC 294019.67 ± 0.58 
MTCC 53038.66 ± 1.15 
MTCC 2470
Ampicillin 
Hexane extract 18.33 ± 2.89  30.67 ± 2.08  ‐  14.33 ± 1.15  13.67 ± 0.58  54 ± 0 
Chloramphenicol 
of G. triuniae 23.33 ± 2.89 
- 29 ± 1.00 
17.33 ± 1.53 ‐  - 26 ± 1.00 
18.67 ± 0.58 19 ± 1.00 
12.33 ± 0.5826.67 ± 1.53 
17.33 ± 1.53
NFCCI 4873
floxacin hydrochloride  43.33 ± 2.89  42 ± 1.00  40 ± 0  34 ± 1.00  30.33 ± 0.58  36.67 ± 1.53 
Streptomycin 23.33 ± 2.89 18 ± 0 9±0 - 19.67 ± 0.58 38.66 ± 1.15
omycin hydrochloride 
Ampicillin ‐ 
18.33 ± 2.89 23.33 ± 0.58 
30.67 ± 2.08 ‐  - 20 ± 1.00 
14.33 ± 1.1518.67 ± 0.58 
13.67 ± 0.5826.67 ± 0.58 
54 ± 0
Milli‐Q water 
Chloramphenicol ‐ 
23.33 ± 2.89 29 ±‐ 1.00 ‐  - ‐  ± 1.00
26 ‐  19 ± 1.00 ‐  26.67 ± 1.53
Ciprofloxacin
Methanol  ‐ 
43.33 ± 2.89 42 ±‐ 1.00 ‐  ± 0
40 ‐  ± 1.00
34 ‐ 30.33 ± 0.58 ‐  36.67 ± 1.53
hydrochloride
Vancomycin
- 23.33 ± 0.58 - 20 ± 1.00 18.67 ± 0.58 26.67 ± 0.58
2.10. MIC and MBC of Crude Pigment 
hydrochloride
Milli-Q water - - - - - -
Methanol
The MIC of the crude hexane extract was found to be 3.91 μg/mL against B. subtilis 
- - - - - -
and 15.6 μg/mL against S. aureus; whereas it was 31.25 μg/mL for M. luteus. The results of 
MIC show that B. subtilis had the lowest MIC, while the highest MIC obtained was for M. 
2.10. MIC and MBC of Crude Pigment
luteus (Figure 11). 
The MIC of the crude hexane extract was found to be 3.91 µg/mL against B. subtilis
The  MBC  of  the  crude  hexane  extract  of  G.  triuniae  NFCCI  4873  was  found  to  be 
and 15.6 µg/mL against S. aureus; whereas it was 31.25 µg/mL for M. luteus. The results
1mg/mL against B. subtilis MTCC 121 and>1 mg/mL against S. aureus MLS 16 MTCC 2940; 
of MIC show that B. subtilis had the lowest MIC, while the highest MIC obtained was for
whereas it was 0.25 mg/mL against M. luteus MTCC 2470. 
M. luteus (Figure 11).

 
Figure 11. Microtitre plate showing MIC of crude pigment of G. triuniae NFCCI 4873. 
Figure 11. Microtitre plate showing MIC of crude pigment of G. triuniae NFCCI 4873.

2.11. Dyeing of Cotton Fabric 
The MBC of the crude hexane extract of G. triuniae NFCCI 4873 was found to be
1 mg/mL against B. subtilis MTCC 121 and>1 mg/mL against S. aureus MLS 16 MTCC
Results of the dyeing experiment showed that cotton fabrics mordanted with differ‐
2940; whereas it was 0.25 mg/mL against M. luteus MTCC 2470.
ent mordants (FeSO4 and Alum) show more pigment uptake than un‐mordanted fabric. 
Among the two mordants used, cotton fabrics mordanted with FeSO
2.11. Dyeing of Cotton Fabric 4 have shown more 

pigment  uptake  than  cotton 

Results fabrics 
of the dyeingmordanted 
experimentwith  different 
showed concentrations 
that cotton of  Alum 
fabrics mordanted with dif-
(Figures 12 and 13). This clearly shows that pigments of G. triuniae NFCCI 4873 have po‐
ferent mordants (FeSO4 and Alum) show more pigment uptake than un-mordanted fab-
tential applications in the textile industry for dyeing different textile fabrics. Moreover, 
ric. Among the two mordants used, cotton fabrics mordanted with FeSO4 have shown
more pigment uptake than cotton fabrics mordanted with different concentrations of
previous studies have already reported phenoxazines and their derivatives as promising 
Alum (Figures 12 and 13). This clearly shows that pigments of G. triuniae NFCCI 4873 have
textile dyes. Their intense colours and chemical nature make them excellent vat dyes. Be‐
potential applications in the textile industry for dyeing different textile fabrics. Moreover,
sides this, these dyes act as good colourants for paint, ink, papers, candles, soap, and plas‐
previous studies have already reported phenoxazines and their derivatives as promising
tic materials [24]. This confirms that the main pigment “1,2‐dimethoxy‐3H‐phenoxazin‐3‐
textile dyes. Their intense colours and chemical nature make them excellent vat dyes. Be-
one” isolated and characterized from the present study may have promising dyeing po‐
sides this, these dyes act as good colourants for paint, ink, papers, candles, soap, and plastic
tential in the textile industry. 
materials [24]. This confirms that the main pigment “1,2-dimethoxy-3H-phenoxazin-3-one”
isolated and characterized from the present study may have promising dyeing potential in
the textile industry.
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Figure 12. Dyeing of mordanted and un-mordanted cotton fabrics with crude pigment extract
Figure 12. Dyeing of mordanted and un‐mordanted cotton fabrics with crude pigment extract of G. 
Figure 12. Dyeing of mordanted and un‐mordanted cotton fabrics with crude pigment extract of G. 
triuniae NFCCI 4873. 
of G. triuniae NFCCI 4873.
triuniae NFCCI 4873. 

  
Figure 13. Cotton fabrics dyed with crude pigment extract of G. triuniae NFCCI 4873. 
Figure 13. Cotton fabrics dyed with crude pigment extract of G. triuniae NFCCI 4873. 
Figure 13. Cotton fabrics dyed with crude pigment extract of G. triuniae NFCCI 4873.

2.12. GC-MS Analysis of Crude Hexane Extract

2.12. GC‐MS Analysis of Crude Hexane Extract 
2.12. GC‐MS Analysis of Crude Hexane Extract 
GC-MS analysis of crude hexane extract showed the peaks along with the mass
GC‐MS analysis of crude hexane extract showed the peaks along with the mass of the 
GC‐MS analysis of crude hexane extract showed the peaks along with the mass of the 
of the organic molecules present in the extract. The chromatogram showed 25 peaks
organic molecules present in the extract. The chromatogram showed 25 peaks correspond‐
organic molecules present in the extract. The chromatogram showed 25 peaks correspond‐
corresponding to the molecules (Figure S7). The detailed data of compounds present in
ing to the molecules (Figure S7). The detailed data of compounds present in the extract, 
ing to the molecules (Figure S7). The detailed data of compounds present in the extract, 
the extract, their molecular weight, and respective retention time is presented in Table 2.
their molecular weight, and respective retention time is presented in Table 2. Crude hex‐
their molecular weight, and respective retention time is presented in Table 2. Crude hex‐
Crude
ane  hexanemajorly 
extract 
ane  extract  extract majorly
majorly  showed 
showed showed the presence
the  presence 
the  presence  of fatty
of  fatty 
of  fatty  acids[n‐hexadecanoic 
acids 
acids  [n-hexadecanoic acid 
[n‐hexadecanoic  acid and
acid  and 
and 

  
Molecules 2022, 27, 393 13 of 23

octadecanoic acid] and their derivatives, esters (glycidyl palmitate and dibutyl phthalate),
and alkanes (hexatriacontane and undecane).
Table 2. Compounds identified from hexane extract by GC–MS analysis.

Relative Content
No. Compound Name Retention Time Molecular Weight
(%)
1 Undecane 8.361 156 0.34
2 Unknown 17.817 283 0.21
1,2-benzenedicarboxylic acid,
3 18.243 278 0.40
bis(2-methylpropyl) ester
alpha-D-Mannopyranose, 5TMS
4 18.594 540 0.19
derivative
5 Dibutyl phthalate 18.717 278 0.98
6 n-Hexadecanoic acid 19.116 256 3.56
7 Dibutyl phthalate 19.189 278 0.75
8 Phthalic acid, butyl nonyl ester 19.386 348 0.30
alpha-D-Mannopyranose,
9 19.459 540 0.28
1,2,3,4,6-pentakis-O-(trimethylsilyl)-
10 Hexadecanoic acid, trimethylsilyl ester 19.854 328 1.75
11 9-Octadecenoic acid, methyl ester, (E)- 20.401 296 0.21
12 9-Octadecenoic acid, (E)- 20.801 282 17.62
13 Octadecanoic acid 20.989 284 2.85
14 Unknown 21.380 355 0.25
9-Octadecenoic acid, (E)-, TMS
15 21.422 354 1.37
derivative
16 9,12-Octadecadienoic acid (Z,Z)- 21.550 280 0.47
17 Unknown 21.655 327 1.13
18 Hexatriacontane 21.747 506 2.71
19 Unknown 21.820 383 0.51
20 Glycidyl palmitate 22.149 312 0.53
21 Unknown 22.355 340 0.65
22 Unknown 22.697 355 28.08
23 Unknown 22.854 411 30.35
24 Unknown 23.126 397 2.97
9-octadecenoic acid, 1,2,3-propanetriyl
25 23.990 884 1.56
ester,

2.13. Purification of Pigment

Purification of crude pigment extract of G. triuniae NFCCI 4873 by preparative TLC
yielded 40 mg of purified orange compound (band 2), which was named PNS-1-OR (Figure 14).
This purified pigment was used for further chemical characterization for identification.
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Figure 14. Separation of compounds in the crude pigment of G. triuniae NFCCI 4873 on TLC plate 
Figure 14. Separation of compounds in the crude pigment of G. triuniae NFCCI 4873 on TLC plate
using HP‐TLC. 
using HP-TLC.

2.14. Chemical Characterization of Pure Compound (PNS-1-OR)

2.14. Chemical Characterization of Pure Compound (PNS‐1‐OR) 
HRMS spectrum
HRMS  spectrum  of of 
thethe 
pigment gave agave 
pigment  sodium
a  sodium  + Na]+ at
adduct [Madduct  [M m/z
+ 280Na](C
+  14 H11
at  NO280 
m/z  4 Na)
and molecular ion peak of
(C14H11NO4Na)  and  molecular  C H NO at m/z 258 [M+1], suggesting C
14 11ion  4peak  of  C14H11NO4  at  m/z  258  H NO
14 [M+1], 
11 as its molec-
4 suggesting 
Cular
14H11formula (Figure S2). The IR spectra showed a significant carbonyl (−C=O) peak at
NO4 as its molecular formula (Figure S2). The IR spectra showed a significant car‐
1647 cm− 1 , (−C=N) 2331 cm− 1−1,, (−C=N) 2331 cm
bonyl (−C=O) peak at 1647 cm and aromatic C-H stretching at 2943 cm− 1 (Figure S1).
−1, and aromatic C‐H stretching at 2943 

1,2-dimethoxy-3H-phenoxazin-3-one:
cm−1 (Figure S1).    This compound is orange solid. mp 145−150 ◦ C;
UV (MeOH) λmax (log ε) 220 nm; IR (KBr) This 
1,2‐dimethoxy‐3H‐phenoxazin‐3‐one:  2943, 2331, is 
νmax compound  1647 cm−1solid. 
orange  ; 1 H and 13 C NMR
mp  145−150 
data, see Table 3; HR-MS +
m/z 258 [M+1] (calcd. for C14 H11 NO4 , m/z 257).
°C; UV (MeOH) λ max (log ε) 220 nm; IR (KBr) νmax 2943, 2331, 1647 cm −1;  1H and  13C NMR 

data, see Table 3; HR‐MS m/z 258 [M+1] + (calcd. for C14H11NO4, m/z 257). 
1 H NMR (500 MHz, CDCl ) and 13 C NMR (125 MHz, CDCl ) spectroscopic data for
Table 3. The 3 3
The 1H NMR,  13C NMR details are shown in Table 3 along with the reported data and 
1,2-dimethoxy-3H-phenoxazin-3-one isolated from G. triuniae NFCCI 4873 along with the reported
were found in agreement with the natural product, 1,2‐dimethoxy‐3H‐phenoxazin‐3‐one 
one [25].
(Figure 15) reported from Acrospermum viticola, a leaf spot fungus of Mulberry [25]. 
1,2-dimethoxy-3H-phenoxazin-3-one 1,2-dimethoxy-3H-phenoxazin-3-
Table 3. The 1H NMR (500 MHz, CDCl 3) and 13C NMR (125 MHz, CDCl3) spectroscopic data for 1,2‐
from one Reported from
Position
dimethoxy‐3H‐phenoxazin‐3‐one isolated from G. triuniae NFCCI 4873 along with the reported one 
G. triuniae NFCCI 4873 A. viticola
[25]. 
δC δH (J in Hz) δC δH (J in Hz)
1,2‐dimethoxy‐3H‐phenoxazin‐3‐one from 
1 145.2 C   - 1,2‐dimethoxy‐3H‐phenoxazin‐3‐one Re‐
145.1 C -
Position  G. triuniae NFCCI 4873 
2 145.8 C - ported from A. viticola 
145.9 C -
δC  3 181.8
δH C=O
(J in Hz)  - δC  181.8 C=O δH (J in Hz) -
4 104.6 CH 6.23, s 104.7 CH 6.23, s
1  145.2 C  ‐  145.1 C  ‐ 
4a 147.2 C - 147.3 C -
2  145.8 C  5a 143.4 C‐  - 145.9 C  143.5 C ‐  -
3  181.8 C=O 6 115.9 CH‐  7.33, dd (8.1,181.8 C=O 
1.07) 116.0 CH ‐ 7.33, dd (8.2)
4  104.6 CH 7 132.2 6.23, s 
CH 7.54, td (7.7, 1.37)
104.7 CH  132.2 CH 7.53, td (8.2)
6.23, s 
8 125.3 CH 7.39, td (7.71, 1.37) 125.3 CH 7.39, td (8.2)
4a  147.2 C  ‐  147.3 C  ‐ 
9 130.3 CH 7.92, dd (7.93, 1.53) 130.3 CH 7.92, dd (8.2)
5a  143.4 C  9a 132.6 C‐  143.5 C  132.7 C ‐  -
6  115.9 CH 10a 7.33, dd (8.1, 1.07) 
147.2 C 116.0 CH  147.8 C 7.33, dd (8.2) -
7  1-OMe
132.2 CH  62.2 CH3
7.54, td (7.7, 1.37)  4.12, s 132.2 CH  62.3 CH3 7.53, td (8.2)  4.12, s
8  2-OMe
125.3 CH  61.1 CH 3
7.39, td (7.71, 1.37)  4.14, s 125.3 CH  61.2 CH 3 4.14, s
7.39, td (8.2) 
9  130.3 CH  7.92, dd (7.93, 1.53)  130.3 CH  7.92, dd (8.2) 
The 1 H NMR, 13 C NMR details are shown in Table 3 along with the reported data and
9a  132.6 C    132.7 C  ‐ 
10a  were found in agreement  with the natural product,
147.2 C  1,2-dimethoxy-3H-phenoxazin-3-one
147.8 C  ‐ 
(Figure 15) reported from Acrospermum viticola, a leaf spot fungus of Mulberry [25].
1‐OMe  62.2 CH3  4.12, s  62.3 CH3  4.12, s 
2‐OMe  61.1 CH3  4.14, s  61.2 CH3  4.14, s 

 
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OCH 3
9 1
8 9a N 10a OCH 3
2

7 3
5a O 4a O
6 4
 
Figure 15. Natural product 1,2-dimethoxy-3H-phenoxazin-3-one.
Figure 15. Natural product 1,2‐dimethoxy‐3H‐phenoxazin‐3‐one. 
The pigment is isolated as an orange solid: yield (40 mg, 20%), mp 148–150 ◦ C. The
1H The pigment is isolated as an orange solid: yield (40 mg, 20%), mp 148–150 °C. The 
NMR shows the distribution of protons signals between 1.0 and 8.0 ppm. Analysis of
1H NMR shows the distribution of protons signals between 1.0 and 8.0 ppm. Analysis of 
the11 H NMR (Figure S3) and 2D-COSY spectra (Figure S4) revealed a sequence of 11 total
the  H NMR (Figure S3) and 2D‐COSY spectra (Figure S4) revealed a sequence of 11 total 
hydrogens at δ 4.12 (s, 3H, OCH3 ), 4.14 (s, 3H, OCH3 ), 6.23 (s, 1H, 4-H), 7.33 (dd, 1H, J = 8.1,
hydrogens at δ 4.12 (s, 3H, OCH3), 4.14 (s, 3H, OCH3), 6.23 (s, 1H, 4‐H), 7.33 (dd, 1H, J = 
1.07 Hz, 6-H), 7.39 (td, 1H, J = 7.71, 1.37 Hz, 8-H), 7.54 (td, 1H, J = 7.7, 1.37 Hz, 7-H), and
8.1, 1.07 Hz, 6‐H), 7.39 (td, 1H, J = 7.71, 1.37 Hz, 8‐H), 7.54 (td, 1H, J = 7.7, 1.37 Hz, 7‐H), 
7.92 (dd, 1H, J = 7.9, 1.53 Hz, 9-H). The 1 H NMR of 1a showed two singlets at δ 4.11 (s, 3H,
and 7.92 (dd, 1H, J = 7.9, 1.53 Hz, 9‐H). The 
OCH3 ) and δ 4.14 (s, 3H, OCH3 ), revealing two
1H NMR of 1a showed two singlets at δ 4.11 
methoxy groups at positions one and two.
(s, 3H, OCH 3) and δ 4.14 (s, 3H, OCH3), revealing two methoxy groups at positions one 
A singlet was observed at δ 6.23 (s, 1H, C-4) next to the carbonyl group at position four.
and two. A singlet was observed at δ 6.23 (s, 1H, C‐4) next to the carbonyl group at posi‐
The characteristic signals for aromatic protons were observed at δ 7.33 as a doublet for (dd,
tion four. The characteristic signals for aromatic protons were observed at δ 7.33 as a dou‐
J = 8.09 Hz, 1H, 6-H), triplet at δ 7.39 (td, J = 7.71Hz, 1H, 8-H), triplet at δ 7.54 (td, J = 7.70,
blet for (dd, J = 8.09 Hz, 1H, 6‐H), triplet at δ 7.39 (td, J = 7.71Hz, 1H, 8‐H), triplet at δ 7.54 
1H, 7-H), and doublet at δ 7.92 (dd, J = 7.93 Hz, 1H, 9-H). The 1 H COSY spectrum showed
(td, J = 7.70, 1H, 7‐H), and doublet at δ 7.92 (dd, J = 7.93 Hz, 1H, 9‐H). The 
the correlation of a 6-H proton at δ 7.33 with a 7-H proton at δ 7.54 only. The
1H COSY spec‐
8-H proton
trum showed the correlation of a 6‐H proton at δ 7.33 with a 7‐H proton at δ 7.54 only. The 
at δ 7.39 showed a correlation with 7-H and 9-H at δ 7.54 and 7.92, respectively. The 7-H
8‐H proton at δ 7.39 showed a correlation with 7‐H and 9‐H at δ 7.54 and 7.92, respectively. 
proton at δ 7.54 displayed a correlation with 6-H and 8-H at δ 7.33 and 7.39, respectively.
The 7‐H proton at δ 7.54 displayed a correlation with 6‐H and 8‐H at δ 7.33 and 7.39, re‐
The 9-H proton at 7.92 displayed a correlation with 8-H at δ 7.39.
spectively. The 9‐H proton at 7.92 displayed a correlation with 8‐H at δ 7.39. 
The 13 C spectrum of PNS-1-OR was observed between 60 ppm and 200 ppm. The
The 13C spectrum of PNS‐1‐OR was observed between 60 ppm and 200 ppm. The 
13 C NMR spectra (Figure S5) showed 14 carbons at δC 61.1 (OCH3 ), 62.2 (OCH3 ), 104.6 13C 

NMR spectra (Figure S5) showed 14 carbons at δ  61.1 (OCH ), 62.2 (OCH ), 104.6 (C‐4), 

(C-4), 115.9 (C-6), 125.3 (C-8), 130.3 (C-9), 132.2 (C-7), 132.6 (C-9a), 143.4 (C-5a), 145.2 (C-1),
C 3 3

115.9 (C‐6), 125.3 (C‐8), 130.3 (C‐9), 132.2 (C‐7), 132.6 (C‐9a), 143.4 (C‐5a), 145.2 (C‐1), 145.8 
145.8 (C-2), 147.2 (C-4a, 10a), and 181.8 (C-3). The DEPT 135 analysis (Figure S6) showed
(C‐2), 147.2 (C‐4a, 10a), and 181.8 (C‐3). The DEPT 135 analysis (Figure S6) showed the 
the carbons that are attached to hydrogens. Therefore, peaks observed at δ 61.22 and
carbons that are attached to hydrogens. Therefore, peaks observed at δ 61.22 and 62.26 
62.26 belong to two methoxy carbon. The peak detected at δ 104.65 corresponds to C-4.
belong  to  two  methoxy 
The displayed peaks at δcarbon. 
115.98,The  peak 130.38,
125.375, detected 
andat 132.21
δ  104.65  corresponds 
belong to  C‐4. 
to C-6, C-8, C-9, The 
and
displayed peaks at δ 115.98, 125.375, 130.38, and 132.21 belong to C‐6, C‐8, C‐9, and C‐7, 
C-7, respectively.
respectively. 
The elemental analysis of CHN for the formula C14 H11 NO4 was obtained as C 64.97,
The elemental analysis of CHN for the formula C
H 4.29, and N 5.44%, which was found in agreement with 14H11NO
the4 was obtained as C 64.97, 
calculated values C 65.37,
H 4.31, and N 5.44%. It was confirmed that the isolated pigment is 1,2-dimethoxy-3H-
H 4.29, and N 5.44%, which was found in agreement with the calculated values C 65.37, 
phenoxazine-3-one.
H  4.31,  and  N  5.44%.  It  was  confirmed  that  the  isolated  pigment  is  1,2‐dimethoxy‐3H‐
phenoxazine‐3‐one. 
3. Materials and Methods
3.1. Collection and Isolation of Fungus
3. Materials and Methods 
Infected leaves of Maytenus rothiana were collected in sterile paper bags from the West-
3.1. Collection and Isolation of Fungus 
ern Ghat region (Mahabaleshwar), Maharashtra, India. Collected samples were transported
Infected  leaves  of  Maytenus  rothiana  were  collected  in  sterile  paper  bags  from  the 
to the laboratory and stored in a refrigerator at 4 ◦ C till their processing. Collected leaves
Western  Ghat  region  (Mahabaleshwar),  Maharashtra,  India.  Collected  samples  were 
(infected with fungus) were used to isolate fungus. The lower leaf surface was found to be
transported to the laboratory and stored in a refrigerator at 4 °C till their processing. Col‐
colonized by fungus. For the in-vitro culture of fungus, spore mass was lifted with the help
lected leaves (infected with fungus) were used to isolate fungus. The lower leaf surface 
of a fine needle from the infected leaf surface and suspended in 1 mL sterile distilled water
was found to be colonized by fungus. For the in‐vitro culture of fungus, spore mass was 
incorporated with Tween 20. Then, with a micropipette’s help, 200 µL of spore suspension
lifted with the help of a fine needle from the infected leaf surface and suspended in 1 mL 
was spread on a 2% Neutral agar plate using a spreader, and the plate was incubated at
sterile distilled water incorporated with Tween 20. Then, with a micropipette’s help, 200 
25 ◦ C overnight. On the next day, the plate was observed under the CX-21 compound
μL of spore suspension was spread on a 2% Neutral agar plate using a spreader, and the 
microscope, and germinated single spores with agar block were picked up with the help of
plate was incubated at 25 °C overnight. On the next day, the plate was observed under 
a sterile needle and transferred on sterile potato dextrose agar (PDA) plates. Plates were
the  CX‐21  compound 
incubated at 25 ◦ C formicroscope, 
7 days. Theand 
puregerminated  single  spores 
growing colonies with  agar 
were further block  were 
sub-cultured on
picked up with the help of a sterile needle and transferred on sterile potato dextrose agar 
fresh PDA plates and slants. Slants were stored in a refrigerator at 4 ◦ C till further use.
(PDA) plates. Plates were incubated at 25 °C for 7 days. The pure growing colonies were 

 
Molecules 2022, 27, 393 16 of 23

3.2. Morphological Identification and Deposition of Fungal Culture

Necrotic lesions on the infected leaves were marked. Scrape mount slides were
prepared in lactophenol cotton blue and observed under a microscope; based on the
literature, the fungus was identified as Gonatophragmium sp. Similarly, slides were prepared
in lactophenol cotton blue mount from axenic culture. Microphotographs were taken using
Carl Zeiss AXIO-10 microscope, and scanning electron microscopic (SEM) images were
taken using images ZEISS EVO MA 15 Scanning electron microscope at 20 KV.
A pure culture of G. triuniae was inoculated on potato dextrose agar (PDA) and potato
carrot agar (PCA) to study cultural and microscopic characters. Plates were incubated at
25 ◦ C for 14 days, and cultural and microscopic characters were noted upon completion
of incubation. Slide culture [26,27] and grass leaf technique [28] were used to get the
sporulation, but no sporulation was observed.
Live and pure fungal culture of G. triuniae were deposited in the National Fungal
Culture Collection of India (NFCCI), Agharkar Research Institute, Pune, under the accession
number NFCCI 4873. Voucher culture G. triuniae NFCCI 4873 was deposited in Ajrekar
Mycological Herbarium (AMH), Agharkar Research Institute, Pune, with accession number
AMH 10289.

3.3. Molecular Identification and Phylogeny

For the identification and authentication of fungal culture up to species level, molecular
characterization was done. The fungal genomic DNA was isolated following the standard
protocol [29]. Then by polymerase chain reaction (PCR), the ITS (internal transcribed spacer)
and LSU (large subunit) regions of rDNA were amplified from the extracted genomic DNA
using the primers ITS-4 & ITS-5 [30] and LR-0R & LR-7 [31], respectively. FavorPrepTM PCR
Purification Kit (Favorgen, Biotech Corporation, Taiwan) was used to purify PCR products.
Purified PCR products were subjected for sequencing by the BigDye Terminator v3.1 Cycle
Sequencing Kit (Applied Biosystems, Waltham, MA, USA) and ABI Avant 3100 automated
DNA sequencer (Applied Biosystems, USA). The manually edited sequences of ITS and
LSU regions of rDNA of our fungal isolate were deposited in the nucleotide sequence
database of NCBI (Gene Bank Accession Numbers: ITS- MW193329 and LSU- MW144438).
The ITS and LSU rDNA sequences of our fungal isolate were subjected to Mega
BLASTn sequence homology searches. Based on the BLASTn search results, geneti-
cally related species, including genus Gonatophragmium, Acrospermum, Pseudovirgaria, and
Dyfrolomyces, were chosen to construct the phylogenetic tree (Table 4). Phylogenetic anal-
ysis of G. triuniae NFCCI 4873 was performed based on a combined ITS and LSU rDNA
sequence data of a total of 19 fungal cultures. The Eremomyces bilateralis CBS 781.70 was
chosen as an out-group. With the help of the MUSCLE algorithm, multiple sequence
alignment was performed in MEGA 7 [32]. Phylogenetic tree of G. triuniae NFCCI 4873 was
constructed based on combined data of ITS & LSU rDNA sequences in IQ-TREE multicore
version 1.6.11 [33] using the Maximum Likelihood method with best-fit model TN+F+G4.
Selection of the best-fit model was done using the ModelFinder employed in IQ-TREE.

3.4. Analysis of Pigment Production on Different Media

A pure culture of G. triuniae NFCCI 4873 was inoculated on different media such as
potato dextrose agar (PDA), potato carrot agar (PCA), Sabouraud dextrose agar (SDA),
Czapek Dox agar (CZA), cornmeal agar (CMA), and Czapek Yeast Extract Agar (CYA) in
duplicates and incubated at 25 ◦ C for 28 days to assess the pigment production potential.
After incubation, the colour of the pigment diffused in media was recorded using the
Methuen handbook of colour [34].

3.5. Analysis of Pigment Production in Liquid Media

The culture of G. triuniae was also tested for its pigment production ability in different
liquid media such as potato dextrose broth (PDB, Hi-media), natural potato dextrose
broth (n-PDB), and natural potato carrot broth (n-PCB). Four agar blocks of a pure culture
Molecules 2022, 27, 393 17 of 23

of G. triuniae (6 mm diameter) from 20-days-old PDA culture plate were inoculated in

a 250 mL Erlenmeyer flask containing 100 mL media (each media in the separate flask),
and flasks were incubated at 25 ◦ C with 150 rpm. All tests were performed in duplicates.
All flasks were observed intermittently after every week from the date of inoculation for
pigment production, and observations were noted down. After 4 weeks of incubation,
culture broths were filtered, and absorption spectrum analyses of the coloured filtrates
were performed using a UV–VIS spectrophotometer (Shimadzu UV-2450). The absorbance
of coloured culture filtrates was recorded in the visible light range from 390–760 nm with a
10 mm optical pathlength and 0.1 nm resolution.

Table 4. GenBank accession numbers of taxa used for phylogenetic analysis.

GenBank Accession No.

Sr. No. Fungal Culture Strain
ITS LSU
Gonatophragmium
1 NFCCI 4873 MW193329 MW144438
triuniae
Gonatophragmium
2 CBS 138901 NR_137932 NG_058117
triuniae
Gonatophragmium
3 CBS 122271 MH863183 MH874728
epilobii
4 Acrospermum sp. SGSF153 MK335823 MK754265
Acrospermum
5 MFLU 17-2849 - NG_064506
longisporium
Acrospermum
6 M152 - EU940085
gramineum
Acrospermum
7 M151 - EU940084
compressum
8 Pseudovirgaria grisea CPC 19130 JF957607 JF957612
Pseudovirgaria
9 CPC 10702 EU041765 EU041822
hyperparasitica
Pseudovirgaria
10 CPC 10704 EU041766 EU041823
hyperparasitica
Pseudovirgaria
11 CPC 10753 EU041767 EU041824
hyperparasitica
12 Pseudovirgaria grisea CPC 19126 JF957605 JF957610
13 Pseudovirgaria grisea CPC 19128 JF957606 JF957611
14 Dyfrolomyces sinensis MFLU 17-0777 - NG_064507
15 Dyfrolomyces sinensis MFLUCC 17-1344 - MG836699
Dyfrolomyces
16 NTOU3636 - KC692156
tiomanensis
Dyfrolomyces
17 JK 5456A - GU479799
rhizophorae
Dyfrolomyces
18 MFLUCC 15-0635 - KX925435
thamplaensis
19 Eremomyces bilateralis CBS 781.70 NR_145364 NG_059206

3.6. Fermentation and Extraction of Pigments

G. triuniae culture was subjected to flask scale fermentation in a total of 6 L (four flasks
containing 1.5 L of media) of natural potato dextrose broth (PD broth). Each flask was
inoculated with 20–25 mycelial disks (6 mm diameter) of G. triuniae from 3-weeks-old PDA
culture plate using a cork borer and incubated at 25 ◦ C with 100 rpm for 4–6 weeks. After
incubation, the coloured culture broth was filtered through pre-weighed blotting paper, and
culture filtrate was collected in a separate flask. Later, the pigments from the culture filtrates
were extracted thrice with an equal volume of Hexane. With the help of a separating funnel,
the Hexane part was separated from the culture filtrate. The separated hexane part was
evaporated to dryness under reduced pressure in a rota evaporator (Heidolph, Schwabach,
Germany). The resulting concentrated hexane extract was used for further experiments.
Molecules 2022, 27, 393 18 of 23

Finally, biomass collected in a pre-weighed blotting paper was dried at 105 ◦ C for 12–15 h
and weighed to measure the yield of biomass concentration [35].

3.7. UV–VIS Spectroscopy Analysis of Hexane Extract

UV-Visible spectroscopic analyses of the crude hexane extracts were performed using a
UV-VIS spectrophotometer (Shimadzu UV-2450). The crude hexane extract was evaporated
to dryness and then dissolved in methanol solvent, and absorbance was recorded in the
range of 190–760 nm with a 0.1 nm resolution and 10 mm optical path length.

3.8. Antagonistic Activity of G. triuniae NFCCI 4873

Antagonistic activity of G. triuniae against three fungal pathogens (Colletotrichum
gloeosporioides, Fusarium oxysporum & Fusarium solani) was evaluated by dual culture tech-
nique [36]. Mycelial disks of 6 mm diameter were excised from the edge of actively growing
culture of G. triuniae and fungal pathogens and inoculated on opposite ends of PDA plates
equidistant from the periphery (each pathogen separately with test culture). For control,
PDA plates were inoculated with a pathogen without test culture. Plates were then incu-
bated at 25 ◦ C for 14 days. Experiments were performed in duplicates. After completion of
incubation, the radial growth of each fungal pathogen on PDA plates was measured, and
percentage inhibition of radial growth (PIRG) of fungal pathogens was calculated relative
to the control plate using the following formula:

PIRG = [(R1 − R2 )/R1 ] × 100

where R1 is the radial growth of the fungal pathogen in the control plate, and R2 is the
radial growth of the pathogen in the presence of test culture (G. triuniae) [37].

3.9. In-vitro Antioxidant Activity of Crude Pigment

In-vitro antioxidant activity of hexane extracts was tested using DPPH radical scaveng-
ing method [38]. Different concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) of the extract
and standard solution (Ascorbic acid, Sigma, USA) were used, and for that, dilutions were
prepared in methanol. For the assay, 10 µL of extract or standard solution was added to
200 µL of 0.1 mM DPPH in methanol solution in a 96-well microtitre plate (Thermofisher,
Waltham, MA, USA). All reactions were performed in triplicates. The plate was then incu-
bated at 37 ◦ C for 30 min in the dark. After incubation, the absorbance of the solution in
each well was measured at 490 nm using a Synergy HT Multi-detection microplate reader
[BioTek, Winooski, VT, USA]. The percentage of radical scavenging activity of the hexane
extract was calculated by the following formula:

DPPH radical scavenging activity (%) = [(OD control − OD sample)/OD control] × 100

where OD means optical density or absorbance value. The IC50 value (concentration of
sample required to scavenge 50% of free radicals) of hexane extract was determined.

3.10. Antimicrobial Activity of Crude Hexane Extract

Crude pigment sample was screened against a panel of test organisms, including
Escherichia coli (MTCC 739), Bacillus subtilis (MTCC 121), Staphylococcus aureus MLS 16
(MTCC 2940), Pseudomonas aeruginosa (MTCC 2453), Raoultella planticola (MTCC 530), and
Micrococcus luteus (MTCC 2470). The test strains were procured from Microbial Type
Culture Collection (MTCC), CSIR-Institute of Microbial Technology (IMTECH), Chandigarh,
India. Ciprofloxacin hydrochloride (1 mg/mL), vancomycin hydrochloride (1 mg/mL),
chloramphenicol (1 mg/mL), streptomycin (1 mg/mL), and ampicillin (1 mg/mL) against
bacteria, were used as positive controls.
Molecules 2022, 27, 393 19 of 23

3.11. Antimicrobial Activity by Disc Diffusion Method

The antimicrobial activity of the hexane extract of G. triuniae was tested using the disk
diffusion method. The standardized microbial inoculum of the test strain was prepared in
a saline solution (~106 –108 CFU/mL) from the 24 h old culture plates. The culture media
used was Muller-Hinton agar (MHA). A direct colony suspension method was used for
the inoculum preparation in which well-isolated colonies from 24 h old culture plates
were selected and suspended in sterile saline. Then saline suspensions of the test cultures
were adjusted to achieve turbidity equivalent to a 0.5 McFarland standard. This was done
by adjusting the turbidity of the cell suspensions between 0.08 and 0.12 AU in a UV-Vis
Spectrophotometer (Shimadzu UV-2450) at 625 nm as recommended by CLSI guidelines.
This results in a suspension with approximately 1–2 × 108 colony-forming units (CFU)/mL
for Escherichia coli ATCC® a 25922. The resulted suspension was added in sterile molten
Muller-Hinton agar (0.5 mL/100 mL of media). The final concentration of the cells in the
media was 0.5–1.0 × 108 CFU/mL. Then media was poured in Petri plates and allowed to
solidify [39].
Sterile Whatman filter paper discs impregnated with known amounts of the test sam-
ple (1mg/mL of dried hexane extract dissolved in methanol) were placed on the surface
of an agar plate that was inoculated with a standardized suspension of microorganisms
that were to be tested. Standard antibiotics like ampicillin (1 mg/mL), ciprofloxacin hy-
drochloride (1 mg/mL), streptomycin (1 mg/mL), vancomycin hydrochloride (1 mg/mL),
and chloramphenicol (1 mg/mL) were used as positive controls. Paper discs impregnated
with only methanol and sterile water were used as negative controls. Plates were left at
room temperature for 1–2 h for the diffusion of samples and then incubated at 37 ◦ C for
24 h. After completion of incubation, all plates were observed for the zone of inhibition.
The diameters of the zone of inhibition were measured in millimeters, including the disk
diameter (6 mm). All experiments were performed in triplicates.

3.12. Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal

Concentration (MBC)
3.12.1. Preparation of Pigment Stock
Forty milligrams of crude pigment was dissolved in 1 mL of DMSO in a 2 mL Eppen-
dorf tube, and a tube was mixed well. Then this 1 mL solution was then diluted 10 times in
sterile Muller–Hinton broth (MHB), and the resultant solution was stored in the refrigerator
at 4 ◦ C until further use.

3.12.2. Preparation of Standardized Inoculum

The standard inoculum (0.5 McFarland) of Gram-positive bacteria (Bacillus subtilis,
Staphylococcus aureus MLS 16, and Micrococcus luteus) was prepared by the direct colony
suspension method as recommended by CLSI guidelines in which the OD625 value was
adjusted to the equivalent of 108 CFU/mL in a Shimadzu UV-2450 UV–VIS spectropho-
tometer [39].

3.12.3. MIC and MBC Experiment

The minimum inhibitory concentration (MIC) of the crude pigment extract was as-
sessed using the standard method [39]. The MIC of the crude pigment extract was deter-
mined by the broth microdilution method in a 96-well plate as per the CLSI recommended
protocol. All the wells of the microtitre plate from columns 2–11 were added with 50 µL of
sterile Muller–Hinton broth (MHB). The last (12th ) column was added with 100 µL of MHB
as sterility control. One hundred microlitres of pigment solution (2 mg/mL) was added
to the first column of the 96-well microtitre plate. Then, using a micropipette, a two-fold
serial dilution was performed by transferring 50 µL of the pigment solution from the first
well to the succeeding well and up to the 10th well, and the final 50 µL of the solution
was discarded. Standardized inoculums of bacteria were diluted 1:150 times in sterile MH
broth to get 106 CFU/mL concentrations of bacteria. Then, 50 µL of microbial suspension
Molecules 2022, 27, 393 20 of 23

(106 CFUs/mL) was added in each well from well 2–11; while in rows D and H, from well
1–10, there was no addition of bacterial suspension, which were treated as pigment control.
Plates were incubated at 37 ◦ C for 20 h. Each test was performed in triplicates.
After incubation at 37 ◦ C for 20 h, 30 µL of 0.01% resazurin was added to each well,
and then plates were further incubated for 2 h at 37 ◦ C for the change of colour. The
well-containing lowest concentration of pigment showing no colour change was considered
as the MIC value.
After 20 h of incubation at 37 ◦ C, 10 µL solution from each well (2–11) of the 96-well
microtitre plate was plated on Muller–Hinton agar plate, and plates were incubated at
37 ◦ C for 24 h. After completion of incubation, plates were observed for the growth of
bacteria. The lowest concentrations that completely kill the bacteria and do not show
growth on the MHA plate were considered minimum bactericidal concentration (MBC).

3.13. Dyeing of Cotton with a Crude Pigment of G. triuniae NFCCI 4873

3.13.1. Textile Fabric
Raw cotton fabric was collected from the local market, Pune. Cotton fabric was then
cut into 10 × 10 cm pieces (each weighing 1 gm) and used for the dyeing experiment.

3.13.2. Scouring
Twelve cotton fabric pieces of 10 × 10 cm (12 g) were pre-soaked in milli-Q water and
then cooked in 2.5 L of Milli-Q water containing 20 mL of non-ionic detergent (Triton-X-100)
for 1 h at 70 ◦ C in a water bath to remove oil and dirt. Scoured cotton fabrics were then
rinsed thoroughly with running water and air-dried [40].

3.13.3. Mordanting
Scoured cotton fabric pieces were mordanted by the pre-mordanting technique [40,41].
Cotton fabric pieces (10 × 10 cm) were mordanted with different concentrations (5%, 10%,
and 15% w/w of fabric) of Alum and FeSO4 for 45 min at 70 ◦ C with a 1:20 material to liquid
ratio (MLR). After completing mordanting, fabric pieces were rinsed in running water and
finally allowed to air dry.

3.13.4. Preparation of Dye Bath

One hundred milligrams of dried crude pigment of G. triuniae NFCCI 4873 was re-
dissolved in 500 mL of Milli-Q water using a magnetic stirrer. The resultant coloured
solution was used as a dye bath for dyeing cotton fabric.

3.13.5. Dyeing
Unmordanted and pre-mordanted cotton fabric pieces were dyed with a crude pigment
solution of G. triuniae NFCCI 4873. Cotton fabric pieces (10 × 10 cm) were dyed at a material
to liquor ratio (MLR) of 1:50 at 70 ◦ C for 45 min in a water bath. The pH of the dye bath
was not controlled. Dyed fabric pieces were then treated with 1% acetic acid and washed
thoroughly in running water. The dyed fabric pieces were then rinsed with cold water and
dried overnight in the shed [40].

3.14. GC–MS Analysis

The dried hexane extract was subjected to derivatization using N-Methyl-N-(trimethylsilyl)
trifluoroacetamide [MSTFA] as a silylating agent. For derivatization, about 5 mg of sample
was dissolved in 100 µL of pyridine in a glass vial followed by the addition of 100 µL of
MSTFA. The solution was mixed well, heated at 60 ◦ C for 20 min, and cooled to room
temperature. After derivatization, the sample was subjected to gas chromatography-mass
spectrometric analysis using GCMS-TQ8030 (Shimadzu, Nakagyo-ku, Kyoto, Japan). A
fused silica column [RTX-5MS (30 m × 0.25 mm × 0.25 µm)] was used. The temperature of
the column was programmed from 50 to 280 ◦ C at a rate of 10 ◦ C/min. The injection port
temperature was set at 280 ◦ C, and a split ratio of 1:40 was used for the analysis. Helium
Molecules 2022, 27, 393 21 of 23

was used as the carrier gas at a flow rate of 1.0 mL/min. Electron ionization source of 70 eV
and a mass range of m/z 35–800 U was used for MS detection. The resultant MS peaks
in the GC-MS chromatogram were identified by comparing and matching the mass and
mass fragmentation pattern with the reference mass and mass fragmentation pattern in the
NIST05 MS library.

3.15. Purification of a Compound by HP-TLC

Two-hundred milligrams of crude pigment extract of G. triuniae NFCCI 4873 was
dissolved in 4 mL of acetone, and compounds present in crude extract were separated
on silica plates using HP-TLC (CAMAAG). Hexane:ethyl Acetate (40:60) was used as the
mobile phase; thin aluminium silica plates (Merck) were used as the stationary phase. After
separating compounds through HP-TLC, the orange band separated on plates was cut,
and the compound attached to silica was extracted with acetone. Acetone extracts were
collected in a round bottom flask and dried using a rota evaporator under reduced pressure
using Heidolph rota evaporator. Finally, the weight of the dried, purified pigment was
measured and recorded.

3.16. Chemical Characterization of Purified Orange Compound (PNS-1-OR)

The high-resolution mass spectrum (HRMS) of the pure orange pigment labeled
as PNS-1-OR was recorded on a Bruker IMPACT HD. FTIR spectrum of pigment was
recorded on a Shimadzu-IRAffinity-1 FTIR spectrophotometer in the frequency range
4000–400 cm−1 . Pure compound was subjected to 1 H (500 MHz) and 13 C (125 MHz) NMR
in Bruker 500 MHz NMR instrument using CDCl3 as a solvent for dissolving sample and
TMS as internal standard. Chemical shifts (δ-values) are given in parts per million (ppm),
and the coupling constants (J-values) are given in hertz (Hz).

4. Conclusions
Many fungi of different taxonomic groups producing a wide variety of pigments
of different colours and chemical classes have been reported by researchers across the
world. Among them, some fungal pigments find their application in different industries
possessing promising colouring properties. The present study also reports one of the
unconventional fungi, i.e., G. triuniae, showing very good pigment production potential.
Moreover, this is the first experimental work reporting pigments and other secondary
metabolites from the fungus G. triuniae. Based on the results of the antibacterial activity of
the crude pigment extract, we conclude that the crude pigment extract shows the presence
of antibiotic compounds, exhibiting antibacterial activity against Gram-positive bacteria.
In addition to this, the DPPH radical scavenging activity of the crude pigment extract
confirmed the presence of antioxidant compounds in the crude pigment extract. Such
bioactivities (antibacterial and antioxidant) of the crude pigment extract have elevated
the G. triuniae as a promising source of bioactive compounds for their possible use in the
medicine and pharmaceutical industry. Besides this, the dyeing property of the crude
pigment extract revealed the potential use of pigments of G. triuniae in the textile industry
for dyeing different types of fabrics.
The purification of crude pigment extract of G. triuniae finally yielded into a major
orange-colored phenoxazine class pigment, which was characterized and identified as
1,2-dimethoxy-3H-phenoxazin-3-one (C14 H11 NO4 , M.W. 257), based on UV-Vis, FTIR,
HRMS, and NMR analysis. Although this pigment was already described from fungus
A. viticola, this is the first study reporting the phenoxazine class of pigment from fungus
G. triuniae. Considering the previous studies describing dyeing potential and bioactivity
of phenoxazines, we may finally conclude that the orange pigment “1,2-dimethoxy-3H-
phenoxazin-3-one” is a promising colourant and possible bioactive compound of G. triuniae,
having future applications in the textile and pharmaceutical industry.
Molecules 2022, 27, 393 22 of 23

Supplementary Materials: FTIR (Figure S1), HR-MS (Figure S2), 1 H NMR (Figure S3), COSY (Figure S4),
13 C NMR (Figure S5), and DEPT-135 (Figure S6) spectra for compound 1,2-dimethoxy-3H-phenoxazin-

3-one and GC-MS chromatogram (Figure S7) of hexane extract (PDF).

Author Contributions: Conceptualization, A.C.L., S.K.S.; Formal analysis, A.C.L., P.S.; Methodology,
A.C.L.; Supervision, S.K.S., L.D. and P.N.S.; Writing—original draft, A.C.L.; Writing—review &
editing, S.K.S., L.D., P.S. and P.N.S. All authors have read and agreed to the published version of
the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: We thank Prashant Dhakephalkar, MACS-Agharkar Research Institute, Pune, for
providing the necessary facilities and encouragement to carry out the research work. A.C. Lagashetti
acknowledges CSIR (Council of Scientific and Industrial Research), New Delhi, for granting Senior
Research Fellowship (SRF), and S.P. Pune University, Pune, for granting permission to register for the
Ph.D. degree. We acknowledge technical support and help by S.B. Gaikwad, N.S. Gaikwad, and S.
Bagale. Laurent Dufossé deeply thanks the Conseil Régional de La Réunion, Réunion island, Indian
Ocean, for continuous financial support of research activities dedicated to microbial pigments.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Rao, M.P.; Xiao, M.; Li, W.J. Fungal and Bacterial Pigments: Secondary Metabolites with Wide Applications. Front. Microbiol. 2017,
8, 1113.
2. Mansour, R. Natural Dyes and Pigments: Extraction and Applications. In Handbook of Renewable Materials for Coloration & Finishing;
Scrivener Publishing LLC: Beverly, MA, USA, 2018; Volume 9, pp. 75–102.
3. Ranaweera, S.J.; Ampemohotti, A.A.L.T.; Arachchige, U.S. Advantages, and Considerations for the Applications of Natural Food
Pigments in the Food Industry. J. Res. Technol. Eng. 2020, 1, 8–18.
4. Arora, S. Textile Dyes: Its Impact on Environment and Its Treatment. J. Bioremediation Biodegrad. 2014, 5, 1000e146. [CrossRef]
5. Ardila-Leal, L.D.; Poutou-Piñales, R.A.; Pedroza-Rodríguez, A.M.; Quevedo-Hidalgo, B.E. A Brief History of Colour, The
Environmental Impact of Synthetic Dyes and Removal by Using Laccases. Molecules 2021, 26, 3813. [CrossRef] [PubMed]
6. Manzoor, J.; Sharma, M. Impact of Textile Dyes on Human Health and Environment. In Impact of Textile Dyes on Public Health and
the Environment; IGI Global: Hershey, PA, USA, 2020; pp. 162–169.
7. Caro, Y.; Venkatachalam, M.; Lebeau, J.; Fouillaud, M.; Dufossé, L. Pigments and Colorants from Filamentous Fungi. In Fungal
Metabolites; Merillon, J.-M., Ramawat, K.G., Eds.; Springer International Publishing: Cham, Switzerland, 2017; pp. 499–568.
8. Lagashetti, A.C.; Dufossé, L.; Singh, S.K.; Singh, P.N. Fungal Pigments and Their Prospects in Different Industries. Microorganisms
2019, 7, 604. [CrossRef]
9. Ramesh, C.; Vinithkumar, N.V.; Kirubagaran, R.; Venil, C.K.; Dufossé, L. Multifaceted Applications of Microbial Pigments: Current
Knowledge, Challenges and Future Directions for Public Health Implications. Microorganisms 2019, 7, 186. [CrossRef] [PubMed]
10. Khan, A.A.; Alshabi, A.M.; Alqahtani, Y.S.; Alqahtani, A.M.; Bennur, R.S.; Shaikh, I.A.; Muddapur, U.M.; Shakeel Iqubal, S.M.;
Mohammed, T.; Dawoud, A.; et al. Extraction and Identification of Fungal Pigment from Penicillium europium Using Different
Spectral Studies. J. King Saud Univ.–Sci. 2021, 33, 101437. [CrossRef]
11. Dufossé, L. Microbial Production of Food Grade Pigments. Food Technol. Biotechnol. 2006, 44, 313–323.
12. Kumar, A.; Vishwakarma, H.S.; Singh, J.; Dwivedi, S.; Kumar, M. Microbial Pigments: Production and Their Applications in
Various Industries. Int. J. Pharm. Chem. Biol. Sci. 2015, 5, 203–212.
13. Sajid, S.; Akbar, N. Applications of Fungal Pigments in Biotechnology. Pure Appl. Biol. 2018, 7, 922–930. [CrossRef]
14. Mapari, S.A.; Meyer, A.S.; Thrane, U. Evaluation of Epicoccum nigrum for Growth, Morphology, and Production of Natural
Colorants in Liquid Media and on a Solid Rice Medium. Biotechnol. Lett. 2008, 30, 2183–2190. [CrossRef]
15. Sibero, M.T.; Triningsih, D.W.; Radjasa, O.K.; Sabdono, A.; Trianto, A. Evaluation of Antimicrobial Activity and Identification
of Yellow Pigmented Marine Sponge-Associated Fungi from Teluk Awur, Jepara, Central Java. Indones. J. Biotechnol. 2016, 21,
1–11. [CrossRef]
16. Heo, Y.M.; Kim, K.; Kwon, S.L.; Na, J.; Lee, H.; Jang, S.; Kim, C.H.; Jung, J.; Kim, J.J. Investigation of Filamentous Fungi Producing
Safe, Functional Water-Soluble Pigments. Mycobiology 2018, 46, 269–277. [CrossRef]
17. Wang, W.; Liao, Y.; Chen, R.; Hou, Y.; Ke, W.; Zhang, B.; Gao, M.; Shao, Z.; Chen, J.; Li, F. Chlorinated Azaphilone Pigments with
Anti-microbial and Cytotoxic Activities Isolated from the Deep Sea-Derived Fungus Chaetomium sp. NA–S01–R1. Mar. Drugs
2018, 16, 61. [CrossRef] [PubMed]
Molecules 2022, 27, 393 23 of 23

18. Kalra, R.; Conlan, X.A.; Goel, M. Fungi as A Potential Source of Pigments: Harnessing Filamentous Fungi. Front. Chem. 2020,
8, 369. [CrossRef] [PubMed]
19. Polak, J.; Wlizło, K.; Pogni, R.; Petricci, E.; Graz,
˛ M.; Szałapata, K.; Osińska-Jaroszuk, M.; Kapral-Piotrowska, J.; Pawlikowska-
Pawl˛ega, B.; Jarosz-Wilkołazka, A. Structure and Bioactive Properties of Novel Textile Dyes Synthesised by Fungal Laccase. Int. J.
Mol. Sci. 2020, 21, 2052. [CrossRef] [PubMed]
20. Sudhakar, C.; Reddy, M.K.; Rajyalakshmi, K.; Raju, K.R. Antibacterial Activity of Substituted 2-Bromo-1, 4-Dimethoxy-3H-
Phenoxazin-3-Ones. J. Chem. Pharm. Res. 2016, 8, 571–574.
21. Achenbach, H.; Blümm, E. Investigation of the Pigments of Pycnoporus sanguineus-Pycnosanguin and New Phenoxazin-3-Ones.
Arch. Pharm. 1991, 324, 3–6. [CrossRef]
22. Maskey, R.P.; Li, F.; Qin, S.; Fiebig, H.H.; Laatsch, H. Chandrananimycins A approximately C: Production of Novel Anticancer
Antibiotics from A Marine Actinomadura sp. Isolate M048 by Variation of Medium Composition and Growth Conditions. J. Antibiot.
2003, 56, 622–629. [CrossRef]
23. Mondal, A.; Bose, S.; Banerjee, S.; Patra, J.K.; Malik, J.; Mandal, S.K.; Kilpatrick, K.L.; Das, G.; Kerry, R.G.; Fimognari, C.;
et al. Marine Cyanobacteria and Microalgae Metabolites—A Rich Source of Potential Anticancer Drugs. Mar. Drugs 2020,
18, 476. [CrossRef]
24. Onoabedje, E.A.; Okoro, U.C.; Knight, D.W. Rapid Access to New Angular Phenothiazine and Phenoxazine Dyes. J. Heterocycl.
Chem. 2017, 54, 206–214. [CrossRef]
25. Kinjo, J.E.; Yokomizo, K.; Awata, Y.; Shibata, M.; Nohara, T.; Teramine, T.; Takahashi, K. Structures of Phytotoxins, AV–Toxins C,
D, and E, Produced by Zonate Leaf Spot Fungus of Mulberry. Tetrahedron Lett. 1987, 28, 3697–3698. [CrossRef]
26. Riddell, R.W. Permanent Stained Mycological Preparations Obtained by Slide Culture. Mycologia 1950, 42, 265–270. [CrossRef]
27. Senanayake, I.C.; Rathnayaka, A.R.; Marasinghe, D.S.; Calabon, M.S.; Gentekaki, E.; Lee, H.B.; Hurdeal, V.G.; Pem, D.; Dis-
sanayake, L.S.; Wijesinghe, S.N.; et al. Morphological Approaches in Studying Fungi: Collection, Examination, Isolation,
Sporulation, and Preservation. Mycosphere 2020, 11, 2678–2754. [CrossRef]
28. Srinivasan, M.C.; Chidambaram, P.; Mathur, S.B.; Neergaard, P. A Simple Method for Inducing Sporulation in Seed–Borne Fungi.
Trans. Br. Mycol. Soc. 1971, 56, 31–35. [CrossRef]
29. Aamir, S.; Sutar, S.; Singh, S.K.; Baghela, A. A Rapid and Efficient Method of Fungal Genomic DNA Extraction, Suitable for PCR
Based Molecular Methods. Plant Pathol. Quar. 2015, 5, 74–81. [CrossRef]
30. White, T.J.; Bruns, T.D.; Lee, S.; Taylor, J. Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics.
In PCR Protocols, A Guide to Methods and Applications; Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., Eds.; Academic Press:
San Diego, CA, USA, 1990; pp. 315–322.
31. Vilgalys, R.; Hester, M. Rapid Genetic Identification and Mapping of Enzymatically Amplified Ribosomal DNA from Several
Cryptococcus Species. J. Bacteriol. 1990, 172, 4238–4246. [CrossRef]
32. Kumar, S.; Stecher, G.; Tamura, K. MEGA7, Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol. Biol.
Evol. 2016, 33, 1870–1874. [CrossRef]
33. Nguyen, L.T.; Schmidt, H.A.; Von Haeseler, A.; Minh, B.Q. IQ–TREE: A Fast and Effective Stochastic Algorithm for Estimating
Maximum-Likelihood Phylogenies. Mol. Biol. Evol. 2015, 32, 268–274. [CrossRef]
34. Kornerup, A.; Wanscher, J.H. Methuen Handbook of Colour, 3rd ed.; Eyre Methuen Ltd.: London, UK, 1978; pp. 1–252.
35. Velmurugan, P.; Kim, M.J.; Park, J.S.; Karthikeyan, K.; Lakshmanaperumalsamy, P.; Lee, K.J.; Park, Y.J.; Oh, B.T. Dyeing of Cotton
Yarn with Five Water Soluble Fungal Pigments Obtained from Five Fungi. Fibers Polym. 2010, 11, 598–605. [CrossRef]
36. Morton, D.J.; Stroube, W.H. Antagonistic and Stimulatory Effects of Soil Microorganisms Upon Sclerotium rolfsii. Phytopathology
1955, 45, 417–420.
37. Rabha, A.J.; Naglot, A.; Sharma, G.D.; Gogoi, H.K.; Veer, V. In Vitro Evaluation of Antagonism of Endophytic Colletotrichum
gloeosporioides Against Potent Fungal Pathogens of Camellia sinensis. Indian J. Microbiol. 2014, 54, 302–309. [CrossRef] [PubMed]
38. Jinesh, V.K.; Jaishree, V.; Badami, S.; Shyam, W. Comparative Evaluation of Antioxidant Properties of Edible and Non–Edible
Leaves of Anethum graveolens Linn. Indian J. Nat. Prod. Resour. 2010, 1, 168–173.
39. Balouiri, M.; Sadiki, M.; Ibnsouda, S.K. Methods for In Vitro Evaluating Anti-microbial Activity: A Review. J. Pharm. Anal. 2016,
6, 71–79. [CrossRef] [PubMed]
40. Devi, S.; Karuppan, P. Reddish Brown Pigments from Alternaria alternata for Textile Dyeing and Printing. Indian J. Fibre Text. Res.
2015, 40, 315–319.
41. Janani, L.; Hillary, L.; Phillips, K. Mordanting Methods for Dyeing Cotton Fabrics with Dye from Albizia coriaria Plant Species. Int.
J. Sci. Res. Publ. 2014, 4, 1–6.