South African Journal of Botany 139 (2021) 449
457

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South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

Isolation and purification of bioactive metabolites from an endophytic

fungus Penicillium citrinum of Azadirachta indica
Puja Kumaria, Arti Singha, Dheeraj K. Singhc, Vijay K. Sharmab, Jitendra Kumara,
Vijai Kumar Guptad, S. Bhattacharyae, R.N. Kharwara,*
a
Mycopathology and Microbial Technology Laboratory, CAS in Botany, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh 221005, India
b
Agricultural Research Organization (ARO) Rishon LeZion, Israel
c
Department of Botany, Harish Chandra PG College, Varanasi, India
d
Center for Safe and Improved Food & Biorefining and Advanced Materials Research Center, Scotland’s Rural College (SRUC), Kings Buildings, West Mains Road,
Edinburgh EH9 3JG, UK
e
Faculty of Science, Department of Chemistry, Banaras Hindu University, Varanasi, India

A R T I C L E I N F O A B S T R A C T

Article History: Endophytic fungi are the plant symbiont with highly diverse nature and poorly defined ecological impor-
Received 25 September 2020 tance in host fitness. Although there are the reports on the isolation and characterization of fungal endo-
Revised 11 February 2021 phytes from a variety of hosts, there is still no report of Penicillium citrinum from Azadirachta indica. In this
Accepted 15 February 2021
study, an endophytic P. citrinum was isolated from A. indica. The purified fraction of secondary metabolites
Available online 1 April 2021
was characterized by combining TLC, GC-MS, 1H NMR and 13C NMR analyses. The TLC purified fraction was
Edited by by S Gupta identified as milbemycin. The pure fraction did not show any antioxidant activity while crude extract showed
strong antioxidant activity (DPPH inhibition capacity; IC50 = 52.13 mg ml
1). The secondary metabolites dis-
Keywords:
played significant antimicrobial activity against human’s pathogenic bacteria and fungi. The inhibition zones
Azadirachta indica
between 15 and 20 mm were recorded against Gram +ve Staphylococcus aureus, Enterococcus faecalis and
Penicillium citrinum
Gram
ve Aeromonas hydrophila, while maximum inhibition of 29 mm was observed against Trichophyton
TLC
GC-MS mentagrophytes. P. citrinum can be a promising fungus that has broad spectrum antimicrobial activity and
Biological activity may provide future insight towards the production of bioactive compounds.
© 2021 SAAB. Published by Elsevier B.V. All rights reserved.

1. Introduction source of bioactive compounds with antimicrobial, insecticidal and

anticancer activities (Strobel and Daisy, 2003; Yu et al., 2010;
Literally, the term endophyte is used for fungi, bacteria and acti- Kharwar et al., 2011; Han et al., 2013; Verma et al., 2014;
nomycetes, which inhabit inside the healthy tissues like leaf, flower, Kharwar et al., 2012; Goutam et al., 2020; Frooq et al., 2020). A wide
stem, seed and root without causing any apparent symptoms to the range of anticancer compounds, as many of them were host origin,
host plant and have been recovered from all plant species examined have been reported from endophytic Taxomyces andreanae of Taxus
to date (Strobel and Strobel, 2007; Kharwar et al., 2009; wallichiana, Eupenicillium parvum of A. indica and Colletotrichum
Franquelo and Perez- Rodriguez, 2016; Bary, 1866). Earlier Studies gloeosporioides of Piper nigrum (Chitra et al., 2014; Kusari et al., 2012;
have shown that endophytes are beneficial to their host, as they pro- Stierle et al., 1993; Wu et al., 2009). Recently, an antimicrobial and
duce diverse chemicals in their niche, which are utilized by the host antitumor compound “Terrien” has been reported from endophytic
for the adaptations, survival, superiority, enhancing nutrient supply, fungus Aspergillus terreus isolated from Achyranthes aspera
and in overcoming the abiotic and biotic stresses (Johnston- (Goutam et al., 2017). Such alternative potential fungal sources may
Monje and Raizada, 2011). It is reported that medicinal plants with reduce the prices of host mimetic compounds and overexploitation
an ethnobotanical history harbor endophytic fungi, which are a rich of host plants (Verma et al., 2009; Su et al., 2012).
Azadirachta. Indica A. Juss., derived its name from ‘Azad’ meaning
free; ‘Dirakht’ meaning tree; ‘i-Hind’ meaning of the Indian origin,
Abbreviation: TLC, thin layer chromatography; GC-MS, gas chromatography- mass
spectromentry; NMR, nuclear magnetic resonance; DPPH, 2,2-diphenyl-1-picrylhydra-
thus it literally means “the free tree of India”. A. indica is commonly
zyl; PDA, potato dextrose agar; SDA, Sabouraud dextrose agar; MHA, Mueller-Hinton known as “Neem” or “Indian Lilac” and is mainly native to the Indian
agar; BOD, biological oxygen demand; ITS, internal transcribed spacer; PCR, polymer- subcontinent. In the ethnobotanical history of India, neem is being
ase chain reaction; EtOAc, ethyl acetate used as a highly medicinal plant against leprosy, eye disorders,
* Corresponding author.
bloody nose, skin ulcers, and as birth control from many years. In
E-mail address: [email protected] (R.N. Kharwar).

https://doi.org/10.1016/j.sajb.2021.02.020
0254-6299/© 2021 SAAB. Published by Elsevier B.V. All rights reserved.
P. Kumari, A. Singh, D.K. Singh et al. South African Journal of Botany 139 (2021) 449
457

some previous studies, several bioactive compounds like ten- mem- 2.4. Molecular identification of endophytic fungus
bered lactones from Phomopsis sp.; javanicin from Chloridium sp., aza-
dirachtin from Eupenicillium parvum, and two new solanapyrone DNA was extracted from a freshly grown fungal culture on PDA
analogues from Nigrospora sp. YB141, have been isolated and charac- plate. The isolation of total genomic DNA was carried out by the
terized (Kusari et al., 2012; Wu et al., 2009, 2008). method described by Sim et al. (2010) with minor modifications. The
Our research work has always been supervised as a part of the ITS and 5.8 s ribosomal genes were amplified with the universal fun-
memoranda to isolate and identify new fungal endophytes from gal primer: ITS 1F (5` TCC GTA GGT GAA CCT GCG G 3`) and ITS 4R (5`
Indian medicinal plants and bioactive compounds by different func- TCC GCT TAT TGA TAT GC 3`) in a thermal cycler (Prima- 96, HIME-
tional assays to alleviate the dearth of highly functionalized antibiot- DIA). All fragments were amplified in total 25 ml reaction mixture
ics (Verma et al., 2014; Kharwar et al., 2012; Goutam et al., 2017; including 2.5 ml 10X PCR Buffer, 0.5 ml of dNTPs, 1ml of each primer
Verma et al., 2009; Sajitha et al., 2013; Sim et al., 2010; (ITS1 and ITS 4), 0.25ml of Taq 1unit, 18.750ml of milli Q (ultra pure)
Kharwar et al., 2020). As Penicillium sp., has been the prime focused water and 1ml of DNA sample. PCR was carried out with a hot start at
fungus in the area of antibiotics after the discovery of penicillin; the 95 °C for 5 min (pre- denaturation) followed by 35 cycles at 94 °C for
endophyte Penicillium citrinum AIB5 isolated from Azadirachta indica 1 min, 52 °C for 1 min (anealing), 72 °C for 1 min (extension) and a
A. Juss., was selected for the screening and purification of potential final extension at 72 °C for 5 min. The amplified product was then
secondary metabolites using the thin layer chromatography (TLC), purified and eluted by Real Genomics Hi Yield Gel/PCR DNA Mini Kit.
gas chromatography mass spectrophotometry (GC
MS), followed by The pure amplified DNA product was sequenced in Genie, Bangalore,
structural elucidation of the pure compound by 1H NMR and 13C Karnataka, India. The strain sequence matched with the reference
NMR. sequences available in the database of National center for Biotechnol-
ogy Information (NCBI) using the basic local alignment search tool
(nBLAST) for its closest match.
2. Material and methods To construct phylogenetic tree, Molecular Evolutionary Genetics
Analytics 7 was used. The optimal tree with the sum of branch
2.1. Sampling length = 14.13467475 is shown. The percentage of replicates trees in
which the associated taxa clustered together in the bootstrap test
For the isolation of a potential fungal endophyte, bark tissue sam- (1000 replicates) is shown next to the branches (Saitou and Nei,
ple was collected from the symptomless, mature and healthy Azadir- 1987; Kumar et al., 2016a).
achta indica growing in the botanical garden of Banaras Hindu
University campus, located in Varanasi, Uttar Pradesh, India (25° 2.5. Fermentation and extraction of secondary metabolites from fungal
16`3`N 82° 59` 19° E) in October 2015. endophyte

2.2. Isolation of endophytic fungi The culture was grown in 300 ml of potato dextrose broth (PDB)
in two 500 ml flasks at 28 § 2 °C and incubated for 21 days. The cul-
An endophytic fungus was isolated from the sterilized bark tissue ture broths were extracted thrice with an equal volume of ethyl ace-
of A. indica (Meliaceae), following the surface sterilization methodol- tate (EtOAc). The EtOAc extract was concentrated using the rotary
ogy described by Petrini et al. (1993) with slight modifications evaporator (IKA RV 10, IKA, Staufen, Germany) at 40 °C temperature
(Kharwar et al., 2009; Wu et al., 2008; Hood et al., 1989). Within 4 h on 65 rpm, and the extract was then slowly dried under a fume hood.
of the collection, the plant samples were rinsed in running tap water The concentrated metabolites were dissolved in methanol and evalu-
for 10 min to remove the debris and soil particles adhered to it fol- ated for antimicrobial activity. Thin layer chromatography (TLC) was
lowed by washing with double distilled water. The surface treatment performed using a mixture of chloroform, methanol and ethyl acetate
was done by submerging the air dried tissue samples (2 cm) in 70% (9:3:5) as mobile phase and the spots were observed under 254 and
ethanol for 1 min, sodium hypochlorite (4% available chlorine) for 366 nm UV light. The extracted crude was applied (1 mg 10 ml
1) on
2 min and again in 70% ethanol for 15 s followed by thrice washing 60F254 silica gel TLC, pre-coated on aluminum plates (Merck). The
with sterile distilled water. The washed tissue samples were then movement of the analyte was expressed by its retention factor (Rf).
allowed to surface dry in the laminar cabinet. The tissues were then In this preliminary screening, the crude was fractionated into 11
cut into small pieces of 5 £ 5 mm2, and that was then put over the bands, and all the bands were furthered proceeded for antibacterial
streptomycin (150 mg ml
1) amended potato dextrose agar, PDA and antifungal activities.
(HIMEDIA) Petri plates. The plates were then sealed with the parafilm
strip and incubated inside BOD (Calton Super Deluxe, NSW, New 2.6. Study of antimicrobial activity and detection of active metabolites
Delhi) at 28§2 °C temperature and observed for the emergence of of crude extract
fungal mycelia at a regular interval of 2-3 days. The emerging fungal
mycelia were transferred and grown to a fresh PDA plate inside BOD The crude and the fractionated metabolites of endophytic fungus
at 28 § 2 °C temperature. After 5-6 days isolates were preserved in were screened for antibacterial and antifungal activities against six
PDA slants, 20% glycerol at -20 °C and paraffin oil at room tempera- human bacterial pathogens and four fungal pathogens employing
ture. disk diffusion assay. The fraction (F3) of the crude developed into
crystal was dissolved in methanol and 10 ml (1 mg 10 ml
1) solution
was loaded to the sterilized filter paper disc (5 mm diameter). The
2.3. Morphological identification of endophytic fungus antibacterial test was performed against the following Gram negative
and Gram positive bacteria Aeromonas hydrophila IMS/GN11, Shigella
The isolated fungus from A. indica was identified on the basis of boydii IMS/GN17, Salmonella typhi MTCC 3216, Escherichia coli IMS/
their morphological characteristics including conidia and conidio- GN19, Staphylococcus aureus ATCC 25923 and Enterococcus faecalis
phores structures using laboratory manuals (Barnett and IMS/GN7. The lawn of test bacterial cultures was prepared with a cot-
Hunter, 1998). Scanning electron microscopy was also performed ton swab on the surface of solidified Mueller-Hinton agar
using the protocol described by Ezra et al. (2004). For that actively (HIMEDIAÒ ) Petri plates. Sterile paper disc containing the crude
growing fungus was dried, coated with copper by sputtering techni- extract and TLC fractions (1 mg 10 ml
1) were separately placed on
ques and examined under a QUANTA 200 (FEI Company, Netherland). the surface of the Muller-Hinton agar medium already seeded with
450
P. Kumari, A. Singh, D.K. Singh et al. South African Journal of Botany 139 (2021) 449
457

test bacterium in a Petri plate. The paper disc impregnated with 10 ml 2.8.4. Identification of compounds
methanol was used as a negative control. The plates were incubated Interpretation of the obtained mass spectrum of GC
MS was
for 24 h at 35 § 2 °C and then were analyzed for antibacterial activity made using the database of National Institute of Standards and Tech-
by observing the zone of inhibition. The fractions were also tested nology (NIST) having more than 62,000 patterns. The obtained spec-
against four fungal pathogens including Candida albicans, Trichophy- trum was compared with the spectrum of the known/standard
ton mentagrophytes, T. rubrum and Microsporum gypseum on Sabour- component present in the library. The name, molecular weight, and
aud dextrose agar plates. The plates were incubated at 25 § 2 °C for structure of the compounds of the rest materials were ascertained.
3-4 days, and the plates were analyzed for antifungal activity by
observing the zone of inhibition. Each test was done in triplicates, 3. Results
and the mean of the diameter of the inhibition zones with standard
deviation has been recorded. 3.1. Isolation and identification of endophytic fungus P. citrinum AIB5

2.7. DPPH free radical scavenging activity Endophytic fungus isolated from the surface sterilized bark tissue
of A. indica, was initially identified as Penicillium sp. based on their
The crude extract was screened for antioxidant assay using 2,2- morphological features including colony and spore characteristics
diphenyl-1- picryhydrazyl (DPPH) (Wu et al., 2009). Methanolic using light and electron microscope shown in Fig. S1 and Fig. 1. A
extract was made at 1 mg 10 ml
1 concentration and mixed with long chain of conidia with varied shapes at each sterigmata were
1000 ml DPPH (Sigma) solution and incubated in a dark room for observed with 2-4 branches of mutulae and phialides. The spores
30 min at normal room temperature. Only 10 ml methanol with were round and bead- like (1.6
2.1 mm in diameter) either in a chain
DPPH was used as negative control. Similar concentration of ascorbic or free with fragile attachment shown in Fig. 1. An ITS- 5.8S rDNA
acid was used as a positive control. The absorbance was recorded at analysis followed by a BLAST search tool revealed that the closet rela-
517 nm and the percentage (%I) inhibition of free radicals was evalu- tive to this fungus was Penicillium citrinum 3
4
3
5
1 KY069209
ated for both the crude metabolite of P. citrinum AIB5 along with L- (Fig. 2) and with the help of plate morphology, microscopic structures
ascorbic acid as positive control using formula: and molecular approaches confirmed the identification as Penicillium

 citrinum AIB5 (Code) and the sequence was further deposited to the
% I ¼ Ablank
Atest sample =Ablank  100 GenBank of NCBI with accession number MF686385.
P. citrinum AIB5 was found to be a fast- growing fungus produced
where Ablank: Absorbance of negative control
15
17 mm colony in 7
10 days on PDA plate. The fungus displayed
A test sample: Absorbance of methanolic extract
gray to green mycelia with adequate sporulation. The reverse side of
IC50 was calculated for P. citrinum AIB5 by plotting straight line
the colony appeared compact and yellow in color displayed in Fig. S1.
equation

3.2. Characterization of secondary metabolites of P. citrinum

2.8. Characterization of compound
During fermentation, the color of broth medium was changeable
2.8.1. UV
Vis spectroscopic analysis in color from yellow (0
7 days) to orange (8
15 days) then to red
A solution was prepared by dissolving 1 mg compound in 4 ml of (15
21 days). The crude secondary metabolites were extracted using
MeOH. Thereafter 3 ml of the solution was taken in a quartz cuvette triple extraction method in ethyl acetate solvent from P. citrinum
and observed for wavelength scanning ranging between 300 and AIB5 grown for 21 days at 28§2 °C given in Fig. S2.
400 nm with distilled MeOH as a reference. The absorption band of
the compound was observed at 329 nm (SPECORDÒ 210 PLUS). 3.2.1. Antimicrobial and antioxidant acitivity of P. citrinum
Broad spectrum antibacterial activity was noticed against human
pathogenic bacteria including Aeromonas hydrophila IMS/GN11, E.
2.8.2. NMR spectroscopy coli IMS/GN19 (Gram
ve) and S. aureus ATCC 25923, E. faecalis IMS/
The 1H and 13C- NMR spectra were recorded on a JNM-ECZ500R/ GN7 (Gram +ve). S. boydii IMS/GN17 and S. typhi MTCC 3216 (Gram
S1 Spectrometer, with an operating field strength of 500 MHz. The 1H -ve) were not inhibited by the crude extract. The crude extract was
and 13C chemical shifts were measured using Chloroform:d (CDCl3). also found active against two human pathogenic fungi including T.
mentagrophytes and C. albicans, whereas inactive against T. rubrum
2.8.3. GC
MS analysis and M. gypseum given in Table 1, 2.
GC
MS analysis of the crude extract and pure compound were Crude extract showed significant DPPH inhibition activity. IC50 of
performed on a TRACE 1300, TSQ DUO (Thermo Scientific) and for extract was recorded as 52.13 mg ml
1 in comparison to positive con-
NIST MS search 2.2 and Gas Chromatograph interfaced to a mass trol ascorbic acid as 34.01 mg ml
1 shown in Fig. S3. Pure fraction (F3)
spectrometer (GC
MS) equipped with an elite-I, fused silica capillary which has shown antimicrobial activity did not show the antioxidant
column (size,1D x 1MDF), composed of 100% dimethyl polysiloxane. activity.
For GC
MS detection, an electron ionization system with the ionizing
energy of 70 eV was used. Helium gas (99.999%) was used as the car- 3.2.2. Characterization of fraction F3 using GC
MS and 2D NMR
rier gas at a constant flow rate 1 ml min
1. Injection volume 1 ml was In the process of compound purification steps, 11 fractions were
employed (split ratio 10:00); injector temperature 250 °C; Ion source separated through TLC plate using 9:3:5 ratio of chloroform: metha-
temperature 23 °C. The oven temperature was programmed from nol: ethyl acetate, and again the 11 bands were screened against
100 °C (isothermal for 2.00 min), with an increase of 250 °C for above mentioned pathogens (Fig. S2). Out of 11, fraction third (F3)
5.00 min hold, ending with 11.00 min isothermal at 300 °C. Mass had exhibited antibacterial and antifungal activity, which was
spectra were taken at 0.00 kV; an interval of 3 min and end time was selected for further characterization of the compound. Activity of this
35.49 min. Total GC- MS running time was 38.49 min. The relative% compound was registered against 6 pathogenic bacteria and 4 human
amount of each component was calculated by comparing its average pathogenic fungi given in Tables 1, 2. The zone of inhibition of the
peak area to the total area. The turbo mass software was used to ana- purified band F3 was higher than that of the crude extract shown in
lyze mass spectra and chromatograms. Fig. 6.
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P. Kumari, A. Singh, D.K. Singh et al. South African Journal of Botany 139 (2021) 449
457

Fig. 1. Microscopic images (a) biverticilliate conidiopores at 100X resolution; SEM images: (b) pair of conidiophores cross linked each other at 30mm (c) Showing beads like struc-
tures of conidia sticked with each other, looks like a chain of white pearl, and (d) size of those beads like conidia in round shape as 1.6, 1.9 and 1.9, respectively.

Table 1 the macrolide group (antibacterial and antifungal) and these groups,
Inhibition zone (mm) of antibacterial activity against A. hydrophila, E. coli, S. aureus, E. belong to the polyketide class of natural products. So the purified mil-
faecalis, S. boydii and S. typhi. Where, (-: no activity); Cr: Crude extract; F3: Pure fraction.
bemycin may be the reason behind the enhanced antimicrobial activ-
Compounds Human bacterial pathogens ity; its activity could have been suppressed in crude extract due to
the presence of many more compounds.
S. aureus E. faecalis A. hydrophila S. boydii S. typhi

Cr 21§1.732 12.66§ 1.527 13.3 § 2.080

F3 24 § 1.000 13.6 § 2.080 22 § 1.00

Table 2
Inhibition zone (mm) of antifungal activity against T. mentagrophytes, C. albicans, T.
rubrum, M. gypseum. Where, (-: no activity); Cr: Crude extract; F3: Pure fraction.

Compounds Human fungal pathogens

T. mentagrophyte C. albicans T. rubrum M. gypseum

Cr 12.33 § 0.577 10.66§ 0.577

F3 29.66 § 1.154 11.66§ 1.520

In GC
MS, milbemycin B, 5- demothoxy-5-one-6, 28-anhydro-

25-ethyl-4-methyl-13‑chloro-oxime was detected on RT: 8.34
8.67
shown in Fig. 3, and it was also confirmed with 2D NMR spectroscopy
shown in Figs. 4 and 5.
1
H NMR spectra showed the characters signal using chloroform: d
(CDCl3, ppm) for OH (8.22), CH (4.75, 5.28), CH2 (2.00
3.73) and CH3
(0.81
1.50). In 13C NMR spectra it shown the signals using the same
Fig. 2. 18S rRNA based neighbor-joining phylogenetic tree from ITS sequences show-
solvent deuterated chloroform for CH3 (11.5 - 30.3), CH2 (48.9
ing the closest relatives of P. citrinum AIB5 (accession no. MF685385) from NCBI Gen-
63.4), CH (109.7
117.2), CN (146.6), CO (169.0
198.6) and COO Bank data base and the realtion between Penicillium species. Bootstraps values (100
(207.2), respectively given in Figs. 4 and 5. As milbemycin belongs to replicates) are shown next to the branches.

452
P. Kumari, A. Singh, D.K. Singh et al. South African Journal of Botany 139 (2021) 449
457

Fig. 3. Mass spectrum of authentic and fungal milbemycin and 1st hit result showing 51% probability.

4. Discussion bark tissue of A. indica in search of potential fungus which could pro-
duce bioactive natural products. In recent years, the investigation of
In the current study, the endophytic fungus P. citrinum was novel natural compounds with diverse biological activities from fun-
observed to have broad spectrum antibacterial activity against both gal endophyte has got remarkable boost due to their agricultural and
Gram (+ve) and Gram (-ve) human pathogenic bacteria, antifungal pharmaceutical exploitation.
activity against two human pathogenic dermatophytes and signifi- In a study, P. citrinum had been reported to show antibacterial
cant antioxidant activity. Many Penicillium species except the P. citri- activity against several human pathogenic bacteria including S.
num have already been reported as endophytes from many medicinal aureus, Bacillus subtilis, E. coli, S. boydii, Vibrio cholera. Further, study
plants including Plectranthus amoinicus, Adenocalymma alliaceum, reported the presence of active molecule as citrinin compound which
Pinus wallichiana (Tiwari and Ganesen, 2020; Kharwar et al., 2011; is responsible for antibacterial activity (Mazumder et al., 2002). In the
Quadri et al., 2014). But in this study, P. citrinum has been isolated as current study, P. citrinum AIB5 has shown significant antibacterial
an endophyte for the first time from the healthy and symptomless activity against S. aureus, E. faecalis, A. hydrophila and antifungal
453
P. Kumari, A. Singh, D.K. Singh et al. South African Journal of Botany 139 (2021) 449
457

Fig. 4. 1H NMR spectra of the fraction F3 seperated on thin layer chromatography of P. citrinum AIB 5 in deutrated chloroform: d (CDCL3, ppm).

activity against T. mentagrophyte and C. albicans. Penicillium species is increased in fraction F3 in comparison to the crude extract as shown
an ocean of compounds in the medical field. As many reports have Figs. 6 and S2, which indicate the compound of fraction F3 is the
revealed the anti-cancer compounds from Penicillium species, asper- active molecules behind the antibacterial in the crude extract. The
phenamate produced by P. buchwaldii and P. spathulatum pure fraction F3 was characterized and identified through GC
MS
(Frisvad et al., 2013). Six new tetramic acid derivatives penicillenols having milbemycin with (51% probability) and its structure was eluci-
A1, A2, B1, B2, C1, C2 together with citrinin, phenol A acid, phenol A dated based on data of 2D NMR (Figs. 4 and 5). Milbemycin belongs to
and dihydrocitrinin were identified from Penicillium sp. an endo- macrolide group (antibacterial and antifungal), and this group
phytic fungus associated to Aegiceras corniculatum (Lin et al., 2008). belongs to the polyketide class of natural products. Milbemycin an
Some novel antibacterial compounds like arisugacins J, G, C and terri- antibiotic compound was first reported from Streptomyces hygrosco-
trems, comazophilones A-F, isocyclocitrinols; antifungal like penicis- picus sub-sp. Aureola rimosus (Hood et al., 1989). Milbemycin D has
teroid A, terretrione A; both antibacterial and antifungal compounds anthelmintic activity against dog parasites (Taliguchi et al., 1980).
such as adametizine A and cillifuranone have been produced by dif- This is the fact that biologically originated crude extract possess a
ferent Penicillium species (Li et al., 2014; Gao et al., 2011a; mixture of bioactive compounds from which separation, characteri-
Amagata et al., 2003; Liu et al., 2015; Wiese et al., 2011; Sasaki et al., zation and identification of pure active molecules are always a big
2005). Similarly, some antimicrobial compounds have also been challenge .
reported from P. citrinum such as citrinin, citrinadin A-B, perinadine In addition to milbemycin, this fungus may still have more poten-
A, scalusamide A-C; penicitrinol G-H, 2,11-dihydoxy-1-methyoxycar- tial to produce other novel natural products by using epigenetic mod-
bonyl-9-xarboxylxanthone and chrysophenol (Sun et al., 2014). ulation, which is very important for the production of cryptic
In the present study, EtOAc was used as crude extracting solvent metabolites (Chaves et al., 2012; Kumar et al., 2016a; Sharma et al.,
which is most common organic solvent used for fungal secondary 2017). This work encourages for isolating and screening of some
metabolite extraction (Nawaz et al., 2020). Extracted fungal crude more bioactive compounds from endophytic Penicillium species. The
was separated in to 11 fractions in the thin layer chromatography compound can be exploited in future with a variety of applications in
(9:3:5 ratio of chloroform: methanol: ethyl acetate) showing the agriculture and pharmaceutical industries because of its considerably
presence of different polarity of compounds as shown in Fig. S2. good yield of a secondary metabolite from fungal mycelium (Xu et al.,
We found that the crude and TLC fraction F3 showed antibacterial 2007). Endophytic fungi isolated from medicinal plants can be used
and antifungal activity against the tested human pathogens (Tables 1 for the production of some rare and new highly active compounds
and 2). We have also observed that size of zone of inhibition with the prospects to reduce the resistivity of the pathogens.
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P. Kumari, A. Singh, D.K. Singh et al. South African Journal of Botany 139 (2021) 449
457

13
Fig. 5. C NMR spectra of the fraction F3 purified on thin layer chromatography of P. citrinum AIB5 in denatured chloroform: d (CDCl3, ppm).

Halliwell 1994). Search of new antioxidant molecules is increasingly

required. The present study reports the significant antioxidant activ-
ity of the crude extracted from endophytic fungus P. citrinum. Previ-
ously, P. citrinum has also been reported to possess antioxidant
activity as reported elsewhere (Singh Arora et al., 2012;
Samanthi et al., 2015). In this study, the antioxidant activity of P.citri-
num AIB5 (52.13 mg ml
1) was found very close to the tested positive
control (ascorbic acid-34.01 mg ml
1) showing it’s potential to be
characterized as antioxidant molecule Fig. S3. In recent past, many
endophytic fungi crude have been reported to produce antioxidant
molecules including P. capitatum, P. chinese, M. suaveolens
(Huang et al., 2007). Several antioxidant compounds have been
reported from P. citrinum like 2,3,4- Trimethyl- 5, 7- dihydroxy- 2,3-
dihydrobenofuran and interestingly, two antioxidant active polyketi-
des were also reported from the same fungus associated with lichens
(Chen et al., 2002; Samanthi et al., 2015; Chandra and Arora, 2011;
Sajitha et al., 2013). However, in this study, principal compound
Fig. 6. Antibacterial activity of crude extract (Cr) and fraction 3 (F3) (separated on TLC)
responsible for antioxidant activity is unknown.
against A. hydrophila. Inhibition zone of fraction 3 was increased when separated from
the crude extract (Cr). Where, 3: F3 fraction; Cr: crude extract; C: control (methanol). 5. Conclusion

In this study, P. citrinum as an endophyte recovered from the

Excess generation of free radicals in our body causes oxidative healthy bark tissue of A. indica is the first report with promising bio-
damages to the biomolecules present in our body and eventually logical potential and its crude extract and partially purified com-
they contribute to cancer, Alzheimer`s disease, aging and other neu- pound were tested for antimicrobial and antioxidant activities. The
rodegenerative disorders (Finkel and Hollbrook, 2000; crude extract of P. citrinum showed appreciable potential against
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457

human pathogenic bacteria and fungi. We found that partially puri- Farooq, S., Qayum, A., Nalli, Y., Lauro, G., Chini, M.G., Bifulco, G., Chaubey, A., Singh, S.K.,
fied TLC F3 showed better antibacterial activity compared to the Riyaz-Ul-Hassan, S., Ali, A., 2020. Discovery of a secalonic acid derivative from
Aspergillus aculeatus, an endophyte of rosa damascena mill., triggers apoptosis in
crude extract, and a milbemycin compound is characterized from the MDA-MB-231 triple negative breast cancer Cells. ACS Omega. https://doi.org/
same fraction. To our knowledge, A. indica is one of the best potential 10.1021/acsomega.0c02505.
source of endophytic fungus that encourage for the exploitation of Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress and the biology of ageing.
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more endophytic fungi and their cryptic secondary metabolites. For Frisvad, J.C., Houbraken, J., Popma, S., Samson, R.A., 2013. Two new Penicillium species
further studies, media standardization coupled with epigenetic mod- Penicillium buchwaldii and Penicillium spathulatum, producing the anticancer com-
ulations will be carried out for better antimicrobial and antioxidant pound asperphenamate. FEMS Microbiology Letters 339 (2), 77–92.
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Declaration of Competing Interest phytic fungi from Tripterygium wilfordii and their insecticidal activities. African
Journal of Microbiology Research 7 (9), 771–776.
Hood, J.D., Banks, R.M., Brewer, M.D., Fish, J.P., Manger, B.R., Poulton, M.E., 1989. A
Authors declare that they have no conflict of interests or personal novel series of milbemycin antibiotics from Streptomyces strain E225. The Journal
relationships that could have appeared to influence the work of Antibiotics 42 (11), 1593–1598.
Huang, W.Y., Cai, Y.Z., Xing, J., Corke, H., Sun, M., 2007. A potential antioxidant
reported in this paper.
resource: endophytic fungi from medicinal plants. Economic botany 61 (1), 14–30.
Johnston-Monje, D., Raizada, M.N., 2011. Conservation and diversity of seed associated
Acknowledgments endophytes in Zea across boundaries of evolution, ethnography and ecology. PLOS
One 6 (6), e20396. https://doi.org/10.1371/journal.pone.0020396.
Kharwar, R.N., Sharma, V.K., Mishra, A., Kumar, J., Singh, D.K., Verma, S.K.,
The authors are thankful to the Head and Coordinator, CAS and Gond, S.K., Kumar, A., Kaushik, N., Revuru, B., Kusari, S., 2020. Harnessing the
DST-FIST in Botany, Institute of Science, BHU, Varanasi, India, for pro- phytotherapeutic treasure troves of the ancient medicinal plant Azadirachta
viding essential research facilities. Authors appreciably acknowledge indica (Neem) and associated endophytic microorganisms. Planta Medica 86
(13
14), 906–940.
the help of ISLS, BHU, Varanasi, India for technical support. We wish Kharwar, R.N., Verma, V.C., Kumar, A., Gond, S.K., Harper, J.K., Hess, W.M.,
to thank Prof. Gopal Nath, Institute of Medical Sciences, BHU, and Lobkovosky, E., Ma, C., Ren, Y., Strobel, G.A., 2009. Javanicin, an antibacterial naph-
Prof R. Chand, Institute of Agriculture (IAS), BHU, Varanasi, India for thaquinone from an endophytic fungus of neem, Chloridium sp. Current Microbiol-
ogy 58 (3), 233–238.
help in the antimicrobial assay. Special thanks to Prof O. N. Srivastava, Kharwar, R.N., Mishra, A., Gond, S.K., Stierle, A., Stierle, D., 2011. Anticancer compounds
for the Scanning Electron Microscopy (SEM), Nanoscience and Tech- derived from fungal endophytes: their importance and future challenges. Natural
nology Center, BHU. Financial support by DST, New Delhi for the proj- Product Reports 28 (7), 1208–1228.
Kharwar, R.N., Maurya, A.L., Verma, V.C., Kumar, A., Gond, S.K., Mishra, A., 2012. Diver-
ect (SB/SMEQ-121/2014), for part of the work acknowledged. sity and antimicrobial activity of endophytic fungal community isolated from
medicinal plant Cinnamomum camphora. Proceedings of the National Academy of
Supplementary materials Sciences, India Section B: Biological Science 82 (4), 557–565.
Kumar, J., Sharma, V.K., Singh, D.K., Mishra, A., Gond, S.K., Verma, S.K., Kumar, A.,
Kharwar, R.N., 2016a. Epigenetic activation of antibacterial property of an endo-
Supplementary material associated with this article can be found phytic Streptomyces coelicolor strain AZRA 37 and identification of the induced pro-
in the online version at doi:10.1016/j.sajb.2021.02.020. tein using MALDI TOF MS/MS. PLOS One 11 (2), e0147876. https://doi.org/10.1371/
journal.pone.0147876.
Kusari, S., Verma, V.C., Lamshoeft, M., Spiteller, M., 2012. An endophytic fungus from
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