Research Article
Antibacterial and Anticancer Activity and Untargeted Secondary
Metabolite Profiling of Crude Bacterial Endophyte Extracts from
Crinum macowanii Baker Leaves

Tendani E. Sebola ,1 Nkemdinma C. Uche-Okereafor,1 Lukhanyo Mekuto,2

Maya Mellisa Makatini,3 Ezekiel Green,1 and Vuyo Mavumengwana4

1Department of Biotechnology and Food Technology, Faculty of Science, University of Johannesburg, Doornfontein Campus,
Johannesburg, South Africa
2Department of Chemical Engineering, Faculty of Engineering and the Built Environment, University of Johannesburg,
Doornfontein Campus, Johannesburg, South Africa
3Molecular Sciences Institute, School of Chemistry, University of the Witwatersrand, Johannesburg, South Africa
4DST-NRF Centre of Excellence for Biomedical Tuberculosis Research,
South African Medical Research Council Centre for
Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences,
Stellenbosch University, Tygerberg Campus, Cape Town, South Africa

Correspondence should be addressed to Tendani E. Sebola; 200905353@student.uj.ac.za

Received 15 June 2020; Revised 29 September 2020; Accepted 26 November 2020; Published 10 December 2020

Academic Editor: Karl Drlica

Copyright © 2020 Tendani E. Sebola et al. ,is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.

,is study isolated and identified endophytic bacteria from the leaves ofCrinummacowanii and investigated the potential of the bacterial
endophyte extracts as antibacterial and anticancer agents and their subsequent secondary metabolites. Ethyl acetate extracts from the
endophytes and the leaves (methanol: dichloromethane (1 :1)) were used for antibacterial activity against selected pathogenic bacterial
strains by using the broth microdilution method. ,e anticancer activity against the U87MG glioblastoma and A549 lung carcinoma
cells was determined by theMTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay.
Bacterial endophytes that were successfully isolated fromC.macowanii leaves includeRaoultella ornithinolytica,Acinetobacter guillouiae,
Pseudomonas sp., Pseudomonas palleroniana, Pseudomonas putida, Bacillus safensis, Enterobacter asburiae, Pseudomonas cichorii, and
Arthrobacter pascens. Pseudomonas cichorii exhibited broad antibacterial activity against both Gram-negative and Gram-positive
pathogenic bacteria while Arthrobacter pascens displayed the least MIC of 0.0625mg/mL. Bacillus safensis crude extracts were the only
sample that showed notable cell reduction of 50% against A549 lung carcinoma cells at a concentration of 100μg/mL. Metabolite
profiling of Bacillus safensis, Pseudomonas cichorii, andArthrobacter pascens crude extracts revealed the presence of known antibacterial
and/or anticancer agents such as lycorine (1), angustine (2), crinamidine (3), vasicinol (4), and powelline. It can be concluded that the
crude bacterial endophyte extracts obtained fromC.macowanii leaves can biosynthesize bioactive compounds and can be bioprospected
for medical application into antibacterial and anticancer agents.

1. Introduction

,e emergence of infectious diseases worldwide due to
bacteria and viruses still poses a serious public health
concern, claiming the lives of half a million people a year and
amounting to 25% of the total deaths worldwide [1]. Even

with the discovery and production of new and improved
antibiotics, the resistance of pathogenic microorganisms to
drugs has increased enormously [2]. Ventola [3] indicated
that the causes of antibiotic resistance include overuse,
inappropriate prescribing, and extensive agricultural use.
,ere is, therefore, an imminent need to discover and

Hindawi
International Journal of Microbiology
Volume 2020, Article ID 8839490, 15 pages
https://doi.org/10.1155/2020/8839490

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https://doi.org/10.1155/2020/8839490


develop new drugs to combat antimicrobial resistance [4].
,e World Health Organization (WHO) has declared that
antibiotic resistance is a global public health concern,
claiming that there are at least 700,000 annual deaths
globally [5, 6]. Novel therapies ought to be discovered to
combat antimicrobial resistance [7]. Bacterial infection at-
tributes about 15% of cancers worldwide and, therefore, is a
serious health concern [8].

In South Africa, brain cancer seems to be more prev-
alent in males (0.57%) than females (0.38%) [9]. Gliomas
are common primary central nervous system (CNS) tumors
mostly affecting the brain [10]. Glioblastomas are aggres-
sive cancers with poor prognosis and an average patient
survival of 18 months [11]. In South Africa, lung cancer is
more common in males with 4.91% and females with 2.52%
[9]. Lung cancer has been deemed as one of the most
prevalent cancers in the developed world and has a very
poor survival rate since most patients are diagnosed at a
stage when curative treatment is impossible. It is one of the
hardest cancers to diagnose [12]. ,e WHO has ranked
cancer as the first leading cause of death globally to people
below the age of 70 years, with an estimate of 18.1 million
new cancer cases and 9.6 million deaths from cancer in the
year 2018 [13]. Kumar et al. [14] described that discovery
and development of chemotherapeutic agents are vital in
the treatment of cancer, since currently used therapies are
ineffective and have side effects, and many important
anticancer drugs, such as camptothecin, penochalasin A,
and chaetoglobosin E, have been isolated from endophytes.
,erefore, further investigations on bacterial endophytes
have to be conducted.

Endophytes have been reported from different plant
species and plant parts, in various geographical locations
and diverse environmental conditions [15]. Endophytes
inhabit their host plants; they improve drought tolerance
and produce protective compounds which protect host
plants from biotic and abiotic factors [16]. Furthermore,
Dembitsky [17] reported that endophytes produce bioactive
secondary metabolites, such as alkaloids and lactones that
display antimicrobial and anticancer properties. ,ese
bioactive compounds could be explored further for medical,
agricultural, and pharmaceuticals use [18]. Endophytes in-
habit unique biological niches growing in uncommon en-
vironments, and their isolation and identification are vital
for further exploration [19, 20].

Crinum macowanii Baker is a medicinal plant native to
east, central, and southern Africa [21]. ,e bulbs, leaves,
and roots of C. macowanii possess medicinal properties
and have been used traditionally to treat or manage an-
imal and human diseases [20, 21]. ,e different plant parts
are used for chest pains, diarrhea, tuberculosis, and
stimulate milk production in cattle and, thus, are over-
exploited and overharvested for medicinal purposes [21].
Biological activities of secondary metabolites are attrib-
uted to the components working in a mixture of com-
pounds (synergy) as compared to when in isolation [22];
therefore, metabolite fingerprinting of crude endophytes
extracts is vital in drug discovery. Morare et al. [24] and
Sebola et al. [25] reported on the isolation of bacterial

endophytes from the bulbs and the antibacterial activities
of their crude extracts, leaving bacterial endophytes iso-
lated from the leaves unexplored. Mixtures of natural
extracts are effective in the search for new drugs since they
reduce drug-resistant phenotype and hence this study was
conducted [26].

,e main aim of this study was to isolate and identify
bacterial endophytes from Crinum macowanii leaves and to
explore the role of endophytic crude extracts as potential
antibacterial and anticancer therapeutic agents and further
profiling of the secondary metabolite produced by the
isolated endophytes as a means to halt the overharvesting of
Crinum macowanii leaves.

2. Materials and Methods

2.1. Sample Collection. ,e collection of the leaves was done
according to a method described by Sebola et al. [25], where
fresh, healthy C. macowanii leaves showing no apparent
symptoms of disease or herbivore damage were collected
from the Walter Sisulu National Botanical Garden (Roo-
depoort, Gauteng, South Africa, 26°05′10.4″S 27°50′41.5″E).
After collection, the samples were placed in sterile poly-
ethylene bags and transferred to the laboratory at 4°C before
being thoroughly washed with sterile distilled water and
used within hours of harvesting.

2.2. Isolation of Bacterial Endophytes. Bacterial endophytes
were isolated from the leaves of the plant by a method
described by Jasim et al. [27] and Sebola et al. [25] with
minor modifications. Briefly, the leaves were cut into
small pieces of about 10 cm using a sterile pair of scissors.
,e cut leaves were treated with 5% Tween 20 (Sigma-
Aldrich, South Africa) (enough to cover the plant ma-
terial) and vigorously shaken for 5 minutes. Tween 20 was
removed by rinsing several times with sterile distilled
water, followed by disinfection with 50mL of 70% ethanol
for 1 minute. Traces of the ethanol were removed by
rinsing with sterile distilled water 5 times. ,e sample was
then treated with 1% sodium hypochlorite (NaClO) for 10
minutes and again rinsed five times with sterile distilled
water. ,e last rinse was used as a control, and 100 μL of
this was plated on Potato Dextrose agar (PDA) (HiMedia,
USA) and Nutrient Agar (NA) (Oxoid, USA). ,e sample
was then macerated in sterilized phosphate-buffered sa-
line (PBS). ,e macerated sample was serially diluted up
to 10−3 dilution, and each dilution was inoculated (using a
spread plate method) in triplicate on nutrient agar. ,e
NA plates were incubated at 30°C (Inco,erm, Labotec,
Johannesburg, South Africa). Growth was monitored
periodically for 5 days. ,e effectiveness of the sterili-
zation was monitored on the wash control plate, with
growth indicating poor sterilization. Under such cir-
cumstances, the plates for the plant part were discarded
and the sterilization was repeated. Distinct colonies were
selected and subcultured on nutrient agar to obtain pure
isolates. Pure bacterial isolates were preserved in 50%

2 International Journal of Microbiology



glycerol in a ratio of 500 μL glycerol : 500 μL overnight
broth culture and kept at −80°C.

2.3. Morphological Identification of Endophytic Bacteria

2.3.1. Gram Staining. To determine the shape and Gram
stain reaction, a method described by Sandle [28] was used.
Pure colonies were subjected to Gram staining to establish
morphological characteristics such as shape and Gram stain
reaction. Gram stain slides were observed using a compound
bright-field microscope (OLYMPUS CH20BIMF200) with
1000x magnification.

2.4. Molecular Identification

2.4.1. Genomic DNA Extraction, Polymerase Chain Reaction,
and Sequencing. ,e 16S rRNA gene of the bacterial endo-
phyte was amplified according to a method described by
Kuklinsky-Sobral et al. [28]. DNA extraction was done using a
ZR Fungal/Bacterial Kit™ (Zymo Research, catalog NO R2014)
according to themanufacturer’s instructions. Polymerase chain
reaction (PCR) was done to amplify the 16S rRNA gene of each
bacterial endophyte with the primers 16S-27F: 5′-
AGAGTTTGATCMTGGCTCAG-3′ and 16S-1492R: 5′-
CGGTTACCTTGTTACGACTT-3′, using DreamTaq™ DNA
polymerase (,ermo Scientific™). PCR products were gel
extracted (Zymo Research, Zymoclean™ Gel DNA Recovery
Kit) and sequenced in the forward and reverse directions on the
ABI PRISM™ 3500xl Genetic Analyzer. ,e sequencing was
performed at Inqaba Biotechnical Industries (Pty) Ltd., Pre-
toria, South Africa. ,e PCR products were cleaned with
ExoSAP-it™ following the manufacturer’s recommendations.
Purified sequencing products (Zymo Research, ZR-96 DNA
Sequencing Clean-up Kit™) were analyzed using CLC Main
Workbench 7, followed by a BLAST search (NCBI).

2.5. Phylogenetic Analysis. ,e obtained sequences were
screened for chimeras using DECIPHER23 and subjected to
BLASTanalysis using the National Center for Biotechnology
Information (NCBI) database against the 16S rDNA se-
quence database (bacteria and archaea) to identify the closest
bacterial species. Bacterial species with 98–100% similarities
were selected for phylogenetic analysis. Alignments of nu-
cleotide sequences were performed using MUSCLE with
default options. ,e positions containing gaps or missing
nucleotide data were eliminated. Phylogenetic trees were
constructed using a Neighbor-Joining (NJ) method (Saitou
and Nei, 1987) based on the Tamura-Nei model [29]. A total
of 1000 replications were used for bootstrap testing. All
branches with greater than 50% bootstraps were considered
to be significant [31]. All evolutionary analyses were con-
ducted in MEGA 7.0 [31]. ,e 16S rRNA gene sequences of
bacterial isolates identified in the study were deposited in
GenBank (https://www.ncbi.nlm.nih.gov/genbank/) with
the accession numbers as stated in Table 1. ,e assigned
names of the bacterial isolates were based on the BLAST
homology percentages as well as the phylogenetic results.

2.6. Extraction of Crude Extracts from C. macowanii Leaves.
,e extraction of secondary metabolites from the leaves was
carried out using the method previously described by Yadav
and Agarwala [32] and Sebola et al. [25].C. macowanii leaves
were washed, cut into small pieces, and air-dried at room
temperature. ,e dried plant material was blended into a
fine powder using a commercial blender. 150 g of the pre-
pared plant material was mixed with 2 L of a 50 : 50
methanol : dichloromethane solution. ,is was allowed to
shake for 3 days on a platform shaker (Amerex Gyromax,
Temecula, CA, USA) at 200°rcf. ,e solution was filtered
through Whatman No. 1 filter paper; the filtrate was
evaporated on a rotatory evaporator and allowed to air dry in
a desiccator.

2.7. Extraction of Crude Extracts from Bacterial Endophytes.
,e extraction of crude extracts from each isolated endo-
phytic bacterium was carried out using the method previ-
ously described by Sebola et al. [25]. Briefly, LB broth (1 L)
was prepared in 2 L of broth and was measured into a 4 L
Erlenmeyer flask leaving room for aeration and autoclaved at
121°C for 15min. Each 4 L flask was inoculated with one of
the endophytic bacteria as listed in Table 1, shaken at 200°rcf,
and incubated at 30°C, an ideal temperature for the growth
of the endophytes [33]. After 7 days of cultivation, sterile
XAD-7- HP resin (20 g/L) (Sigma, Johannesburg, South
Africa, BCBR6696V) was added to the culture for 2 h, shaken
at 200 rcf. ,e resin was filtered through cheesecloth and
washed three times with 300mL of acetone for each wash.
,e acetone soluble fraction was concentrated using a rotary
evaporator, and a dark yellowish viscous extract was ob-
tained, which was transferred into a measuring cylinder.
Depending on the volume, ethyl acetate was added in a ratio
of 1 :1 (v/v). ,e mixture was vigorously shaken for about
10min, decanted into a separating funnel, and allowed to
separate and each phase is collected in a conical flask. ,is
process was repeated until the dark yellowish viscous liquid
obtained after removing the acetone became a very light-
yellow liquid. ,e ethyl acetate fraction was evaporated
using a rotary evaporator, and the brown extract obtained
was stored in an amber bottle in a cool dry place until the
analysis was done. ,e light-yellow liquid was evaporated,
and no reasonable extract or further analysis was done on
this substance. ,e brown crude endophyte extracts were
used for antibacterial and anticancer assays and metabolite
fingerprinting.

2.8. Antibacterial Analysis of Crinummacowanii Bulbs Crude
Extracts and Endophytic Bacterial Crude Secondary Metab-
olite Extracts. ,e evaluation of the antimicrobial activity of
the crude secondary metabolite extract was performed using
the minimum inhibition concentration (MIC) method as
previously described by Sebola et al., Sebola et al., and
Andrews, [25, 34, 35]. Eleven pathogenic bacterial species,
namely, Bacillus cereus (ATCC10876), Bacillus subtilis
(ATCC19659), Streptococcus epidermidis (ATCC14990),
Staphylococcus aureus (ATCC25923), M. smegmatis
(ATCC21293), Mycobacterium marinum (ATCC927),

International Journal of Microbiology 3

https://www.ncbi.nlm.nih.gov/genbank/


Enterobacter aerogenes (ATTC13048), Escherichia coli
(ATCC10536), Klebsiella pneumonia (ATCC10031), Proteus
vulgaris (ATCC 33420), and Proteus aeruginosa
(ATCC10145) were used. ,e antibiotic Streptomycin was
used as the positive control and was prepared by weighing
0.032mg in 1mL of sterile distilled water while 0.1% DMSO
was used as a negative control.

2.8.1. Sample Preparation. ,e crude leave extract and crude
endophytic extracts were weighed separately into empty
autoclaved McCartney bottles to ensure sterility. A minimal
amount of dimethyl sulfoxide (DMSO) (0.1%) was used to
dissolve the crude extracts, and Mueller-Hinton (MH) broth
was added to bring the volume of the dissolved crude extract
to a concentration of 32mg/mL as the stock solution.

2.8.2. Microtiter Plate Assay. Serial dilutions were carried
out using the MH broth from 16mg/mL down to 0.031mg/
mL, which was the lowest inhibition observed. ,e exper-
iment was carried out in five repeats using a 96-well
microtiter plate. ,e outer wells of the plate were filled with
sterile distilled water (sdH2O). ,e inoculum (100 μL) was
added to each well that did not contain the sdH2O. ,e
diluted crude extract samples (100 μL) were added in five
wells horizontally, and the concentrations decreased in a
vertical order from 16mg/mL down to 0.031mg/mL. ,e
plates were covered and incubated overnight at 37°C. After
incubation, 10 μL of 0.02% (w/v) Resazurin sodium salt dye
solution was added to the wells, and the resulting solution
was incubated for another two hours. On reduction, resa-
zurin changes color from blue to pink to clear as oxygen
becomes limited within the medium, indicating metabolism
and the viability of bacterial cells, as well as no effect of the
crude extracts on the bacteria. Any well with a known
concentration showing a slight color change was used as
MIC. ,e wells were visually inspected for color changes.

2.9. Anticancer Assays. ,e evaluation of the anticancer
activity of the crude secondary metabolite extract was per-
formed using the MTS (3-(4, 5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
assay as previously described by Sebola et al. [25]. A stock

solution of 200 μg/mL of all crude extracts (leave crude ex-
tracts and endophytic crude extract) was prepared in 0.1%
DMSO and sonicated. Serial dilutions were done according to
[36, 37]. Briefly, dilutions were carried out using growth
media from 100 μg/mL to 3.13 μg/mL. MTS (3-(4, 5-di-
methylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sul-
fophenyl)-2H-tetrazolium) in vitro cancer cytotoxicity assay
was carried out to determine a change in cell viability through
the use of a color change. ,e MTS compound (yellow) is
metabolized by viable cells to form a dark purple-colored
compound, while dead cells turn the color of the MTS
compound pink. ,e samples were run in duplicate across
three plates (n� 6), and the average values obtained were
reported. ,e U87MG (glioblastoma) cells and A549 (lung
carcinoma) cells were grown using normal tissue culture
techniques using Dulbecco’s Modified Eagle Medium (Merk,
Johannesburg, SA) supplemented with 15% fetal bovine se-
rum (FBS) (Merck, Johannesburg, SA).,e cells (1× 105 cells/
ml) were incubated in 96-well plates at 37°C overnight, with
the subsequent addition of the crude bulb extracts and crude
endophytic extracts, in concentrations of 100 μg/mL, 50.0 μg/
mL, 25.0 μg/mL, 12.5 μg/mL, 6.25 μg/mL, 3.125 μg/mL, and
0 μg/mL.,e cells were left to incubate for 4 days, whereupon
MTS (5 μL) (Promega, Madison, WI, USA) was added to the
cells. ,e absorbance values were measured at 490 nm after
1 hr, 2 hr, and 4hr incubation periods. Cell viability was then
calculated using the formula

%Cell viability �
Ea − Ba( 􏼁

Ca − Ba( 􏼁
× 100, (1)

where Ea is the absorbance of the extract, Ba is the absor-
bance of the blank, and Ca is the absorbance of the control
[39]. ,e positive control used for all conducted tests was
auranofin, as it is able to inhibit thioredoxin reductase as
well as the ubiquitin–proteasome system (UPS) by targeting
proteasome-associated deubiquitinase, thus inducing lung
cancer cell apoptosis by selenocystine [39–41].

2.10. LC-QTOF-MS Analysis

2.10.1. Instrumentation. Secondary metabolites of the crude
leave extracts and crude endophytes extracts were identified
using an LC-QTOF system with a Dionex UltiMate 3000

Table 1: Endophytic bacteria isolated from C. macowanii leaves.

Sample code Assigned bacterial name GenBank accession number Similarity (%) Gram reaction
Colony

morphology (pigmentation,
texture, form)

TES 02B Raoultella ornithinolytica MF943227 99 −Rod Cream white, moist, circular
TES 02C Acinetobacter guillouiae MF943228 100 −Rod Cream white, dry, circular
TES 05A Pseudomonas sp. MF943233 99 −Rods White, dry, filamentous
TES 05B Pseudomonas palleroniana MF943234 100 −Rods Pale yellow, viscid, filamentous
TES 06A Pseudomonas putida MF943235 99 −Rods Milky white, moist, circular
TES 07A Bacillus safensis MF943236 100 +Rod Milky white, viscoid, circular
TES 07B Bacillus safensis MF943237 100 +Rod White, dry, circular
TES 10A Enterobacter asburiae MF943239 99 −Rod Pale yellow, moist, filamentous
TES 14A Pseudomonas cichorii MF943243 99 −Rods Yellow, moist, circular
TES 15A Arthrobacter pascens MF943244 99 +Rods Cream white, viscid, circular

4 International Journal of Microbiology



UHPLC (,ermo Scientific, Darmstadt, Germany) coupled
to a Compact™ QTOF (Bruker Daltonics, Bremen, Ger-
many) that uses an electrospray ionization (ESI) interface,
following a modified method by Hoffman et al., Changwa
et al., Want, Tapfuma et al., and Tapfuma et al. [42–46]. ,e
instrument parameters used in this study are listed in Ta-
ble 2. Instrument operation control and acquisition was
done using HyStar software version 2.1 (,ermo Scientific,
Darmstadt, Germany). ,e analytical run was set at 40
minutes.

,e gradient flow used for the mobile phase is listed in
Table 3.

2.10.2. Data Processing. Spectral data processing was per-
formed on Bruker Compass DataAnalysis software version
4.3 (Bruker Daltonics, BremHub, California, USA). MetFrag
web tool version 2.1 software (Git Hub, California, USA) was
used to compare fragment patterns of fragmented ions with
those from compound databases, namely, PubChem,
ChemSpider, and KEGG [48]. Additional databases that
were used include METLIN (Scripps Research, California,
USA) and KNapSAcK (Kanaya Laboratory, Japan) [49].
Blank containing methanol for plant extracts and NB for
bacteria endophytes were also analyzed in the same con-
ditions. Comparison of the two base chromatograms (from
the crude extracts and endophyte extracts and the controls)
allowed for filtering out impurities from the growth medium
[45, 46].

3. Results and Discussion

3.1. Molecular and Morphological Identification of Bacterial
Endophytes from C. macowanii leaves. Endophytes inhabit
unique biological niches growing in unusual environments.
,eir isolation and identification are vital for further ex-
ploration [49, 50]. In this study, a total of 9 bacterial en-
dophytes were isolated and characterized from the leaves of
C. macowanii. as seen in Table 1.

Seven Gram-negative bacteria were observed. Pheno-
type diversity of the endophyte contributes to the Gram
reaction and colony morphology of the endophytes [51]
and hence the diverse endosphere, while the growth rate
and size of the host plant also have an effect on the diverse
endophytic community [52]. ,e endophytic community
of plants is influenced by the age of the host plant, geo-
graphic location, and abiotic factors such as temperature
[52, 53]. ,is would explain the diversity of the bacterial
endophytes isolated.

Bacterial genera including Pseudomonas, Bacillus, and
Burkholderia have been isolated as endophytes from leaves
of medicinal plants [54]. ,ese bacteria genera predominate
in medicinal plants, as they assist the host plant in mineral
nutrient composition and protection against abiotic and
biotic stresses [55]. ,e BLAST search conducted of the
results of the 16S rDNA gene sequence revealed that the
isolated endophytes belong to bacterial genera, such as
Raoultella, Acinetobacter, Pseudomonas, Bacillus, Enter-
obacter, and Arthrobacter, as seen in Figure 1. ,is supports
our observed results.

Pseudomonas are common bacteria associated with
plants and have been isolated from a number of plant species
and tissues. ,ey display a positive effect on host plant
growth such as reducing drought stress and producing plant
hormones such as 1-aminocyclopropane-1-carboxylic acid
(ACC) and Indole-3-acetic acid (IAA) and acting as bio-
control agents [26, 49, 56, 57]. Our isolate TES05A showed
94% similarity with Pseudomonas sp. WB4.4-99. Endophytic
isolate TES14A showed 83% similarity with Pseudomonas
cichorii Pc-Gd-1. Endophytic Pseudomonas cichorii has been
isolated from potato cultivar [58]. Isolate TES06A displayed
a 99% similarity with Pseudomonas putida PF45. Endophytic
Pseudomonas putida have been isolated from mango or-
chard [59]. Isolate TES05B showed 99% similarity with
Pseudomonas palleroniana IHB B 7234. Endophyte Pseu-
domonas palleroniana has been isolated from bananas and is
reported to fix free nitrogen, solubilize phosphates, and
produce siderophores in vitro [60].

TES02C displayed a sequence identity of 100% to Aci-
netobacter guillouiae OTU-b62. Different Acinetobacter
species have been isolated from potato cultivars [58].
TES15A isolates revealed 100% to Arthrobacter pascensH19.
Arthrobacter spp. has been isolated from ethnomedicinal
plants in Southern India [61]. Bacillus endophytes have been
isolated from sunflower, potatoes, and cotton and assist host
plants in phosphate solubilization and auxin production
[56]. Our endophytic isolates (TES07A and TES07B) dis-
played 100% similarity to Bacillus safensis TMV13-3. Ba-
cillus spp. Isolates TES02B had a similarity of 62% to
Raoultella terrigena m 5. Endophytic Raoultella ornithino-
lytica has been isolated from mountain-cultivated ginseng
plants [62]. A 64% similarity was observed between our
isolate TES10A and Enterobacter asburiae E6 – 2. Endo-
phytic Enterobacter asburiae has been isolated from date
palm and promotes plant growth [63].

To the best of our knowledge, this is the first report on
the isolation of Arthrobacter pascens and Enterobacter
asburiae from C. macowanii.

3.2. Antibacterial Evaluation of Crude Bacteria Endophyte
Extracts from the Leaves. ,e lowest MIC (0.0625mg/mL)
was observed from Arthrobacter pascens crude extract and
crude extracts against B. subtilis, respectively. ,e crude
extracts of most of the endophytes showedMIC values below
1.00mg/mL. ,e leave crude extracts displayed noteworthy
activity against both Gram-positive and Gram-negative
bacteria as seen in Table 4.

C. macowanii leaves have been used traditionally to
cleanse the blood, treat coughs, kidney, and bladder diseases
in humans and animals, and also t treat coughs and diarrhea
[20]. ,e results obtained in this study indicate the inhi-
bition of B. subtilis, M. smegmatis, and P. vulgaris at
0.500mg/mL, and S. aureus, E. coli, and K. pneumonia were
inhibited at 0.250mg/mL and S. epidermidis and
P. aeruginosa at 1.00mg/mL and 0.125mg/mL, and
respectively.

,ese data provide scientific justification for the eth-
nomedicinal uses of the leaves, as the inhibited bacteria are

International Journal of Microbiology 5



the main causative agents of the ailments the leaves are used
to treat. S. aureus, E. coli, and K. pneumoniae were inhibited
by a final concentration of 250 μg·mL−1 by alkaloids such as
crinine, cherylline, crinamidine, 3-O-acetylhamayne, and
bulbispermine which have been isolated from C. macowanii
leaves [64]. To the best of our knowledge, this study is the
first to report on the extraction of crude extract from leaves
of C. macowanii and its antibacterial activity.

,e cultivation of plants to obtain bioactive compounds
has led to drawbacks, such as overharvesting of plants to

obtain bioactive compounds, different environmental con-
ditions tend to produce low yields, and total synthesis and
semisynthesis are challenging due to complex structures
[65]. A number of endophytic microorganisms have pro-
duced anticancer, antimicrobial, antidiabetic, insecticidal,
and immunosuppressive compounds [66]. Plants growing in
a variety of places possibly harbor endophytes with novel
natural products [66, 67].

Raoultella ornithinolytica crude extract had MIC values
ranging from 0.250 to 16mg/mL, with the most significant

100

100

98

100

62

58
64

61

93

99

99
44

97

83
94

0.10

MF943233 Pseudomonas sp. strain TES05A
MF943243 Pseudomonas cichorii strain TES14A
AM934698 Pseudomonas sp. WB4.4-99
KU923370 Pseudomonas cichorii strain Pc-Gd-1
MF943234 Pseudomonas palleroniana strain TES05B
KJ767367 Pseudomonas palleroniana strain IHB B 7234
MF943235 Pseudomonas putida strain TES06A
MF838696 Pseudomonas putida strain PF45

MF943227 Acinetobacter guillouiae strain TES02C
KJ147068 Acinetobacter guillouiae isolate OTU-b62

MF943244 Arthrobacter pascens strain TES15A
KC934811 Arthrobacter pascens strain H19

MF943236 Bacillus safensis strain TES07A
MF943237 Bacillus safensis strain TES07B
KY882055 Bacillus safensis strain TMV13-3

MF943227 Raoultella ornithinolytica strain TES02B
AY292873 Raoultella terrigena isolate m 5
MF943239 Enterobacter asburiae strain TES10A
KY938112 Enterobacter asburiae strain E6-2

GQ203544 Hypocrea lixii strain SWFC8926

Figure 1: Neighbor-joining phylogenetic tree of 16S rRNA gene sequences of endophytes isolated from C. macowanii leaves showing the
relationship with other similar species selected from GenBank.

Table 2: Parameters of the LC-QTOF-MS/MS system.

Specification Setting

Column Raptor ARC-18 column with dimensions of 2.7 μm (particle size), 2.1mm (internal diameter), 100mm
(length) and 90 Å (pore size) (Restek, Bellefonte, USA

Injection volume 5 μL
Operating Reverse phase ultra-high-performance liquid chromatography (RP-UHPLC)
Capillary voltage at 4.5 kV
End plate offset −500V
Dry heater nebulizer gas
pressure 1.8 bar

Scan 50 to 1300m/z

Table 3: Gradient flow profiles of the mobile phase.

Time (min) Flow (mL/min) Solvent A (0.1% formic acid in H2O (v/v)) Solvent B (0.1% formic acid in acetonitrile (v/v))
0–2 300 95 5
2–30 300 5 95
30–40 300 95 5

6 International Journal of Microbiology



Ta
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4:

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International Journal of Microbiology 7



inhibition observed for K. pneumonia, E. coli, and
M. marinum at concentrations of 0.500mg/mL, and
P. aeruginosa was inhibited at concentrations of 0.250mg/
mL. Microcin genes have been reported to be present on
Raoultella ornithinolytica [62] and microcins are antibac-
terial peptides produced by Enterobacteria [68]. ,is could
explain the observed results. Acinetobacter guillouiae crude
extract showed MIC values of between 0.500 and 16mg/mL.
,e crude extract showed activity against M. marinum at
0.500mg/mL.

Bacillus safensis crude extract showed MIC values of
between 0.125 and >16mg/mL. ,e crude extract showed
activity against B. subtilis, M. marinum, and E. coli at
concentrations of 0.125mg/mL, 0.500mg/mL, and
0.250mg/mL, respectively. Crude endophytic extracts of
Bacillus safensis isolated from Ophioglossum reticulatum
L. displayed antibacterial activity against Staphylococcus
aureus and Escherichia coli [69]. ,is is in agreement with
the obtained results.

Enterobacter asburiae crude extract showed MIC values
of between 0.125 and 16mg/mL. ,e crude extract showed
activity againstM. smegmatis and E. coli at concentrations of
0.500mg/mL and M. marinum and K. pneumonia at con-
centrations of 0.125mg/mL. Endophytic crude extracts of
Enterobacter asburiae displayed antibacterial activity against
K. pneumoniae, E. coli, S. aureus, and B. cereus. Enterobacter
strains [70] have been reported to produce antibacterial
lipopeptides with a broad activity [71]. ,is supports the
obtained results.

Arthrobacter pascens crude extract showed MIC values
of between 0.0625 and >16mg/mL. ,e most active inhi-
bition was against B. subtilis at 0.0625mg/mL. ,e crude
extract showed activity against S. aureus and E. aerogenes at
concentrations of 1.00mg/mL. Arthrobacilin, an antibac-
terial compound produced by Arthrobacter spp., showed
inhibition against S. aureus [72, 73]. ,is could explain the
antibacterial activity observed.

Pseudomonas sp. crude extract showed MIC values of
between 0.0625 and >16mg/mL. ,e most active inhibition
was against M. marinum at 0.0625mg/mL. Mupirocin
produced by Pseudomonas strains has been reported to
possess antibacterial activity [74]. Pseudomonas palleroniana
crude extract showed MIC values of between 0.250 and
>16mg/mL. ,e most active inhibition was against
P. vulgaris at 0.250mg/mL. ,e crude extract showed ac-
tivity against E. aerogenes and P. aeruginosa at concentra-
tions of 1.00mg/mL and M. marinum and K. pneumonia at
concentrations of 0.500mg/mL. Endophytic crude extracts
from Pseudomonas palleroniana were reported to inhibit
Escherichia coli and Staphylococcus aureus [50]. Pyoluteorin
produced by Pseudomonas palleroniana strains has been
reported to contain antibacterial activity [75]. Pseudomonas
putida crude extract showedMIC values of between 1.00 and
>16mg/mL. ,e most active inhibition was against
P. aeruginosa at 1.00mg/mL. Antibiotics pyoluteorin,
phenazine-1-carboxamide, and phenazine-1-carboxylic acid
were produced by Pseudomonas putida strains [75]. ,is
could explain the antibacterial activity observed. Pseudo-
monas cichorii crude extract showed MIC values of between

0.125 and 16mg/mL. ,e crude extract showed activity
against E. coli and P. vulgaris at concentrations of 1.00mg/
mL, M. smegmatis and P. aeruginosa at concentrations of
0.125mg/mL, B. subtilis at 0.250mg/mL, and K. pneumonia
at 0.500mg/mL. To the best of our knowledge, this is the first
report on the antibacterial activity of crude endophyte ex-
tracts from Pseudomonas cichorii.

Cos et al. [76] state that a concentration of <0.1mg/mL for
a crude sample is the ideal concentration for anti-infective
bioassays whereas [77, 78] recommend that crude samples
with a concentration of 1.00mg/mL and ≤100 μg/ml
(0.100mg/ml) and ≤625 μg/ml are considered to be very
significant and moderately significant and therefore note-
worthy for minimal inhibitory concentration. Stringent
endpoints for anti-infective bioassays ought to be set to
prevent false results and confusion, taking into consideration
the sensitivity of extracts and test microorganisms, extraction
methods, and solvents used [66, 67].

It was observed from the results that crude endophyte
extract from Pseudomonas sp. and Arthrobacter pascens
obtained from C. macowanii leaves had noteworthy anti-
bacterial activity against the pathogenic bacteria used in this
study and can be used as antibacterial agents against and
M. marinum and B. subtilis infections, respectively.

3.3. Anticancer Evaluation of Crude Bacteria Endophyte Ex-
tracts from the Leaves against Resistance Cancer Cell Lines.
Secondary metabolites produced by endophytic microor-
ganisms’ act as anticancer agents and display significant
potential in medical and veterinary treatments [79, 80].
Anticancer agents paclitaxel and podophyllotoxin have been
isolated from endophytic microorganisms [81].

3.3.1. Anticancer Evaluation of Crude Bacteria Endophyte
Extracts from the Leaves against A549 Lung Carcinoma Cells.
Pseudomonas putida and Bacillus safensis crude extracts
showed a 47% and 50% cell reduction, respectively, against
lung carcinoma cells at a concentration of 100 μg/mL as seen
in Figure 2.

3.3.2. Anticancer Evaluation of Crude Bacteria Endophyte
Extracts from the Leaves against UMG87 Glioblastoma Cells.
Acinetobacter guillouiae crude extracts showed a 42% re-
duction of UMG87 glioblastoma cells at a concentration of
6.25 μg/mL and Arthrobacter pascens crude extracts dis-
played cell reduction of 37% at a concentration of 12.5 μg/ml
as seen in Figure 3.

To the best of our knowledge, this is the first report on
the anticancer activity of C. macowanii leave crude extracts.
No noteworthy activities were observed from the leaves’
crude samples against both cell lines used in this study.
Bioactive compounds such as lycorine, pretazettine, crin-
amine, augustine, and galanthamine are noted to appear in
C. macowanii leaves and have been reported to possess
anticancer activity [82–84].

To the best of our knowledge, this is the first report on
the anticancer activity of crude endophytes extracts from

8 International Journal of Microbiology



Pseudomonas palleroniana, Bacillus safensis, Enterobacter
asburiae, Arthrobacter pascens, and Pseudomonas cichorii.

Acinetobacter guillouiae crude endophyte extract was the
only tested sample that exhibited anticancer against UMG87
glioblastoma cells, with a 31% cell reduction at 100 μg/mL
and 53% cell reduction at 3.13 μg/mL posing as a possible
anticancer agent against brain cancer.

Bacillus safensis crude extracts displayed noteworthy
activity against A549 lung carcinoma cells with 50% cell
reduction at 100 μg/mL. Crude extracts of Bacillus safensis
isolated from sea sponges had anticancer activity against
HepG2 (hepatocellular carcinoma), HCT (colon

carcinoma), and MCF 7 (breast carcinoma) [85]. ,is could
explain the observed results, and crude endophyte extracts
from Bacillus safensis can be used as an anticancer agent
against lung cancer.

Crude endophytic extract of Enterobacter asburiae,
Pseudomonas sp., Arthrobacter pascens, and Pseudomonas
palleroniana displayed no noteworthy activity against
UMG87 glioblastoma cells and A549 lung carcinoma cells.
,e extracts can be tested on other cancer cell lines to
determine their activity. Pseudomonas sp. are known to
produce anticancer compounds and have been reported to
have activity against a number of human cancer cell lines

A549 lung carcinoma cells

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 Positive
control

100µg/ml
50.0µg/ml
25.0µg/ml

12.5µg/ml
6.25µg/ml
3.13µg/ml

Crude extracts from C. macowanii leaves and endophytes from the leaves

0
20
40
60
80

100
120
140

C
el

l v
ia

bi
lli

ty
 (%

)

Figure 2: Cytotoxic activity of endophytic-derived secondary metabolites and crude extracts on A549 lung carcinoma cells tested at
different concentrations ranging from 100 to 3.13 μg/mL.,e positive control used was auranofin. T1�C. macowanii leaves, T2�Raoultella
ornithinolytica, T3�Acinetobacter guillouiae, T4� Pseudomonas sp., T5�Pseudomonas palleroniana, T6�Pseudomonas putida,
T7�Bacillus safensis, T8� Enterobacter asburiae, T9� Pseudomonas cichorii, T10�Arthrobacter pascens.

0

50

100

150

200

250

300
U87MG glioblastoma cells

T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 Positive
control

100µg/ml
50.0µg/ml
25.0µg/ml

12.5µg/ml
6.25µg/ml
3.13µg/ml

Crude extracts from C. macowanii leaves and endophytes from the leaves

C
el

l v
ia

bi
lli

ty
 (%

)

Figure 3: Cytotoxic activity of endophytic-derived secondary metabolites and crude extracts on UMG87 glioblastoma cells tested at
different concentrations ranging from 100 to 3.13 μg/mL.,e positive control used was auranofin. T1�C. macowanii leaves, T2�Raoultella
ornithinolytica, T3�Acinetobacter guillouiae, T4� Pseudomonas sp., T5�Pseudomonas palleroniana, T6�Pseudomonas putida,
T7�Bacillus safensis, T8� Enterobacter asburiae, T9� Pseudomonas cichorii, T10�Arthrobacter pascens.

International Journal of Microbiology 9



[86, 87]. Pseudomonas sp. and Pseudomonas cichorii dis-
played no noteworthy activity against UMG87 glioblastoma
cells and A549 lung carcinoma cells. Pseudomonas paller-
oniana crude extracts displayed 36% cell reduction at 100 μg/
mL against A549 lung carcinoma cells.

Crude endophyte extracts from Pseudomonas putida
displayed 47% cell reduction at 100 μg/mL against A549 lung
carcinoma cells. P. putida TJ151 is able to produce fluo-
rouracil which is a bioactive aromatic compound and it is an
anticancer drug [59]. L-methioninase, an enzyme produced
by Pseudomonas putida, has shown anticancer activity
against leukemia cell lines, liver HepG2, breast MCF-7, lung
A549, prostate PC3, and colon HCT116 [88, 89]. Methio-
ninase from P. putida and 5-fluorouracil work synergistically
to inhibit tumor growth and hence the activity observed
[90].

Crude endophyte extracts from Raoultella ornithinoly-
tica displayed 43% cell reduction at 100 μg/mL against A549
lung carcinoma cells. Protein complex from
R. ornithinolytica has shown anticancer activity against HeLa
cell line, human endometrioid ovarian cancer line (TOV
112D ATCC CRL-11731), and the human breast adeno-
carcinoma line (T47D ECACC 85102201) resulting in cy-
topathic effect and reduction in the cell number [91, 92].

Crude endophyte extracts of Raoultella ornithinolytica,
Pseudomonas palleroniana, Pseudomonas putida, and Ba-
cillus safensis can be further purified and tested for their
anticancer activity against other types of the cancer cell line.

3.4. Liquid ChromatographyQuadrupole Time-of-FlightMass
Spectrometry LC-Q-TOF-MS Analysis. ,e isolation and
characterization of bioactive secondary metabolites help in
distinguishing between new and already known bioactive
secondary metabolites which help in the development and
discovery of new drug leads [93]. Lycorine and powelline
were some of the identified secondary metabolites present in
the crude extract of C. macowanii leaves, Bacillus safensis,
Pseudomonas cichorii, and Arthrobacter pascens. ,ese are
indicated in Table 5.

Not much work has been done to the leaves, and to the
best of our knowledge, this is the first report of the iden-
tification of secondary metabolites from leaves of
C. macowanii and their endophytes using LC-Q-TOF-MS.
Endophytes are able to produce similar secondary metab-
olites as the host plants by exchanging fragments of their
genomic DNA with the host plant [103, 104]. ,ese sec-
ondary metabolites perform distinct functions such as an-
tibacterial and anticancer activity as performed in this study
[105]. Melicopicine is an acridone alkaloid isolated from
leaves of Teclea and Zanthoxylum species [94]. An acridone
alkaloid melicopicine has been isolated from Melicope
fareana [106] and has anthelmintic and antibacterial ac-
tivities [107]. ,is would explain the noteworthy antibac-
terial activity of the crude leave extract observed in this
study.

Lycorine is an alkaloid previously isolated from
C. macowanii [108]. Leaves of C. macowanii contain more
lycorine as compared to the bulbs and other plant parts such

as roots and flowers [109]. Crude endophyte extras of Ba-
cillus safensis, Pseudomonas cichorii, and Arthrobacter pas-
cens displayed the presence of lycorine, and this is not
surprising as endophytes can metabolize secondary me-
tabolites from the host plant [104]. Lycorine has been re-
ported to possess antibacterial activity and cytotoxic and
antitumor activities [95]. ,is would explain the observed
antibacterial and/or anticancer activity of the crude endo-
phyte extracts.

Angustine is an alkaloid previously isolated from plants
of the Rubiaceae and Loganiaceae family [110]. To the best of
our knowledge, angustine is being identified for the first time
in C. macowanii leaves extracts and its isolated endophytes.

Aulicine and 3-O-methyl-epimacowine are crinine-type
alkaloids and have been isolated from Hippeastrum aulicum
Herb. and Hippeastrum calyptratum Herb. [110, 111].
Evidente and Kornienko [97] reported on the anticancer
properties of these crinine-type alkaloids. ,is would justify
the anti-lung cancer activity of crude Bacillus safensis en-
dophyte extracts. Different cancer cell lines can be used to
determine the anticancer activity of crude Pseudomonas
cichorii endophyte extracts.

Crinine-type alkaloid crinamidine has been isolated
from different plants of the Amaryllidaceae family [113]
and also from the bulbs, flowering stalks, leaves, and roots
of Crinum macowanii [21]. Crinamidine is found in
whole plant parts of the Crinum species [114]. Powelline
is an alkaloid reported to occur in C. macowanii [115] and
Ndhlala et al. [115] reported its occurrence from the
bulbs. Both these crinine-type alkaloids have been re-
ported to possess antibacterial, antitumor, and anticancer
activity [20, 98, 100]. ,is is not alarming as endophytes
can metabolize secondary compounds from the host [105]
and display a number of biological activities as the host
plant.

Vasicinol is a quinazoline alkaloid from Adhatoda
zeylanicaMedic. [117]. Crude endophyte extracts of Bacillus
safensis, Pseudomonas cichorii, and Arthrobacter pascens
displayed the presence of this alkaloid; this is not surprising
as some quinazoline alkaloids are produced by microbes
[118]. Vasicinol can be tested on other resistant pathogenic
bacteria to combat antimicrobial resistance, as its antibac-
terial activity has been reported by Jain et al. [99].

Brefeldin A is a fungal metabolite produced by species of
the Ascomycetes [119], and to the best of our knowledge, it is
being reported for the first time in bacterial endophytes.
Brefeldin A has been reported to possess anticancer activity
[101]; different cancer cell lines can be used to determine its
anticancer activity with different cell lines.

From the results obtained, varying retention times were
observed between the leaves and bacterial endophytes
samples, even though the same chromatography conditions
were used. Factors such as the affinity of the compounds to
the extraction solvents used [120], the change in polarity of
the sample being analyzed, and fragments that make up the
secondary metabolites detected [121], and the formation of
secondary metabolites complexes with extraction solvents
used to influence the different retention times observed, as
they create a sample matrix [122].

10 International Journal of Microbiology



,e identified alkaloids lycorine, crinamidine, and powel-
line are true alkaloids of the Amaryllidaceae family [120, 124],
and this supports the obtained results as Crinum macowanii
belongs to this plant family. ,e availability of these alkaloids
leads to overuse and overharvesting of C. macowanii to obtain
these bioactive compounds [65, 125]. ,e bioprospecting of
endophyte isolatedmetabolites could help save the environment
since endophytes have been reported to contain similar bio-
active compounds as the host plant [66, 126] and in some doing
medicinal plants are being conserved and revenue is generated
by the bioprospecting of metabolites from endophytes [127].

4. Conclusions

,e study revealed the presence and cohabitating of endophytic
bacteria from leaves of C. macowanii, and this has informed us
of the microbial community of C. macowanii. Crude endo-
phyte extracts displayed notable inhibitory activities against
both Gram-positive and Gram-negative bacterial species.
Crude extracts endophytes (Pseudomonas putida and Bacillus
safensis) exhibited promising anticancer activity against lung
cancer. ,e identified secondary metabolites from the endo-
phytes have reported biological activities, and this data raises
the possibility that the overharvesting of C. macowanii for its
medicinal properties will be halted.,is is a promising lead for
drug discovery and bioprospecting. Further extraction of
secondary metabolites from endophytes is still needed.

Data Availability

All the data are provided in full in the results section of this
paper apart from the DNA sequences of the bacterial en-
dophytes available at https://www.ncbi.nlm.nih.gov/
genbank, and accession numbers for each endophyte can
be found in Table 1 of the manuscript.

Conflicts of Interest

,e authors declare that they have no conflicts of interest.

Acknowledgments

Tendani Edith Sebola was awarded a scholarship from the
CSIR DST-Interbursary Support (IBS) bursary and Uni-
versity of Johannesburg Merit Bursary for postgraduate
students. ,e authors would like to thank Walter Sisulu
Botanical Garden for sampling and identification of the
plant material and the mass spec team Mr. Eric Morifi, Mr.
,apelo Mbele, and Ms. Refilwe Moepya at the University of
the Witwatersrand for assisting with the mass spectrometry
experiments. ,e National Research Foundation (NRF) of
South Africa under Grant no. TTK150713125714 funded the
collection, analysis, and interpretation for the isolation and
identification of bacterial endophytes. ,e National Re-
search Foundation (NRF) of South Africa under Grant no.
114384 funded the analysis and interpretation of the anti-
bacterial studies.,eDepartment of Science and Technology
through the Artificial Wetland Research (AWARE) project
funded the analysis and interpretation of the anticancer
studies.

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Table 5: LC-Q-TOF-MS analysis of crude extracts of C. macowanii leaves and their bacterial endophytes.

Compound name Rt (min/sec) m/z Molecular
formula Reported biological activity Sample

Melicopicine 5.64 330.1351 C18H19N1O5 Antiplasmodial activity [94] T1

Lycorine 1.92; 13.76; 36.75;
7.23 288.1236 C16H17N1O4

Antibacterial cytotoxic and antitumor
activities [95]

T1, T7, T9,
T10

Angustine 10.35; 19.23; 25.98;
11.07 314.1388 C20H15N3O1 Antiproliferative activity [96] T1, T7, T9,

T10
3-O-methyl
epimacowine 5.63 288.1586 C17H21NO3 Anticancer [97] T7

Crinamidine 17.57; 15.50; 15.44;
6.32 318.1348 C17H19N1O5 Antibacterial [21] anticancer activity [98] T1, T7, T9,

T10

Vasicinol 4.18; 21.69; 21.51;
8.91 205.0977 C11H12N2O2 Antibacterial activity [99] T1, T7, T9,

T10
Aulicine 1.99 320.1870 C18H25NO4 Anticancer activity [97] T9

Powelline 19.14, 14.05, 17.98,
7.43 302.1389 C17H19N1O4 Antitumor [100] antibacterial [21] T1, T7, T9,

T10
Brefeldin A 11.52 281.1702 C16H24O4 Anticancer [101] and antifungal [102] T10
Note: T1�C. macowanii leaves, T7�Bacillus safensis, T9�Pseudomonas cichorii, T10�Arthrobacter pascens.

International Journal of Microbiology 11

https://www.ncbi.nlm.nih.gov/genbank
https://www.ncbi.nlm.nih.gov/genbank


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