Bioprospecting lovastatin production from a novel producer Cunninghamella blakesleeana

Article abstract of DOI:10.1007/s13205-018-1384-y

Beside anti-cholesterol activity, lovastatin garners worldwide attention for therapeutical application against various diseases especially cancer. A total of 36 filamentous fungi from soil samples were isolated and screened for lovastatin production by yeast growth bioassay method. C9 strain (later identified as Cunninghamella blakesleeana) was screened as potential strain of lovastatin production. Further confirmation of the compound was made using TLC, HPTLC and HPLC in which similar Rf value, densitogram peak and chromatogram peak against the standard lovastatin were observed, respectively. The purified lovastatin subjected for IR analysis showed a lactone ring peak at 1763.63?cm^?1 similar to standard lovastatin. Further structural analysis including NMR and LC–MS of the purified lovastatin reassures the molecular formula and molecular weight similar to standard. In quantitative terms, C. blakesleeana, Aspergillus terreus and Aspergillus flavus produced 1.4?mg?g^?1 DWS, 0.83?mg?g^?1 DWS and 0.3?mg?g^?1 DWS of lovastatin, respectively, (p < 0.0001) without any optimization. Lovastatin showed significant antioxidant property with IC₅₀: 145.9?μg?mL^?1 (140?μL), and the percentage of inhibition is maximum at 199.5?μg/mL which is statistically significant (p < 0.0001).

Full text of DOI:10.1007/s13205-018-1384-y

3
Biotech (2018) 8:359
https://doi.org/10.1007/s13205-018-1384-y
ORIGINAL ARTICLE
Bioprospecting lovastatin production from a novel producer
Cunninghamella blakesleeana
1
1
1
1
Janani Balraj · Karunyadevi Jairaman · Vidhya Kalieswaran · Angayarkanni Jayaraman
Received: 20 October 2017 / Accepted: 30 July 2018
©
Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
Beside anti-cholesterol activity, lovastatin garners worldwide attention for therapeutical application against various diseases
especially cancer. A total of 36 filamentous fungi from soil samples were isolated and screened for lovastatin production by
yeast growth bioassay method. C9 strain (later identified as Cunninghamella blakesleeana) was screened as potential strain
of lovastatin production. Further confirmation of the compound was made using TLC, HPTLC and HPLC in which similar
Rf value, densitogram peak and chromatogram peak against the standard lovastatin were observed, respectively. The purified
−
1
lovastatin subjected for IR analysis showed a lactone ring peak at 1763.63 cm similar to standard lovastatin. Further struc-
tural analysis including NMR and LC–MS of the purified lovastatin reassures the molecular formula and molecular weight
−
1
similar to standard. In quantitative terms, C. blakesleeana, Aspergillus terreus and Aspergillus flavus produced 1.4 mg g
−
1
−1
DWS, 0.83 mg g DWS and 0.3 mg g DWS of lovastatin, respectively, (p<0.0001) without any optimization. Lovastatin
−
1
showed significant antioxidant property with IC : 145.9 µg mL (140 µL), and the percentage of inhibition is maximum
5
0
at 199.5 µg/mL which is statistically significant (p<0.0001).
Keywords Lovastatin · Cunninghamella blakesleeana · Antioxidant · Anticancer
Introduction
2009). Among the statins, lovastatin (C H O ) formerly
24 36 5
known as Mevinolin, Monacolin K and Mevacor was the
first accepted statin by United States Food and Drug Admin-
istration (USFDA) in 1987 as a potent hypercholesterolemia
agent (Manzoni and Rollini 2002; Tobert 2003). Lovastatin,
a secondary metabolite produced during the secondary phase
(idiophase) of fungal growth exists as open β-hydroxyl form
(mevinolinic acid—biologically active) and the synthetic
lactone form (mevinolin—biologically inactive), while the
latter is hydrolyzed to biologically active (open ring) form
through liver enzyme when administered invasive (Gupta
et al. 2007). Apart from its anti-lipidemic activity, it is also
widely used for various functions as anti-inflammatory
agent, apoptotic inducer in cancer cells, restorer of renal
function, suppressor of tumor necrosis factor in coagula-
tion process and protective agent during host cellular dam-
age (Tobert 2003; Zamvil and Steinman 2002). It is also
commonly employed as a potent therapeutic agent in the
treatment of major diseases that involves coronary artery,
peripheral vascular, cerebrovascular, renal disease, multiple
sclerosis, ischemic condition, bone fracture, Alzheimer’s and
to a negligible extent in cancer (Seraman et al. 2010; Kumar
et al. 2000; Tandon et al. 2005).
Statins, a precise effective competitive inhibitor of
3
-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA)
reductase (mevalonate: NADP1 oxidoreductase, EC
.1.1.34) which catalyzes the rate-limiting reduction of
1
HMG-CoA to mevalonate in mevalonate pathway of cho-
lesterol biosynthesis has revolutionized the treatment of
hypercholesterolemia (Endo 2004; Istvan 2003; Bizukojc
and Ledakowicz 2007; Kamath and Janakiraman 2012).
They are the polyketide compounds produced by fungi dur-
ing their secondary metabolism. It is widely used as a lipid-
lowering drugs (lowers LDL cholesterol Levels) to reduce
the risk of cardiovascular events in dyslipidemic patients
(
Endo 2004; Davidson and Robinson 2006; Ludman et al.
*
Angayarkanni Jayaraman
angaibiotech@buc.edu.in
Janani Balraj
jananibiotech12@gmail.com
1
Cancer Therapeutics Lab, Department of Microbial
Biotechnology, Bharathiar University, Coimbatore,
Tamil Nadu 641046, India
Vol.:(0
1
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3 Biotech (2018) 8:359
The first anti-cholesterol drug (Triparnol-1958) was
banned due to its side effect and the second drug ML236B
Compacitin) isolated from Penicillium citrinum was also
Microorganism and inoculum preparation
(
Filamentous fungi were isolated using nylon net litter
bag technique from the collected soil samples of Pat-
tanam village (coconut area soil), Coimbatore (sugarcane
soil) and Nilgiris (tea plantation area) which are present
in the southern regions of Tamil Nadu, India (Kondo
et al. 1994). Isolation of desired fungal cultures were
done initially by plating the collected soil samples and
then the individual colonies of micro-fungi were picked
and purified by streaking onto the fresh Potato Dextrose
Agar (PDA) medium. The pure fungal isolates were kept
on PDA medium at 4 °C and re-cultured every 4 weeks.
For preparing a spore suspension, 10 mL of spore suspen-
sion medium (0.9% NaCl, 0.1% Tween80) was added to
a well-sporulated PDA slants of the isolates. The surface
was scrapped with a loop and then the suspension was
collected. The suspension was agitated completely using
a cyclomixer and finally filtered through a glass wool. The
concentration of the spore suspension was measured using
a hemocytometer and the final concentration was adjusted
withdrawn owing to its carcinogenic effect (Kirby 1967;
Endo et al. 1976). Finally, the lovastatin (Mevinolin) was
discovered from Aspergillus terreus which gained popu-
larity due its multiple therapeutic potential advantages
with no side effects. This attracted the interest of many
researchers to identify a better alternate source of fungi
that could produce lovastatin which lead to the identifi-
cation of fungi such as Monascus (M.) ruber, A. flavus,
M. pilosus, M. purpureus, A. fischeri, A. parasiticus, A.
umbrosus, Acremonium chrysogenum, A. flavipes, P. funic-
ulosum, Trichoderma (T) viridae and T. longibrachiatum
(
Endo and Monacloin 1979; Sayyad et al. 2006; Wang
et al. 2003; Valera et al. 2005; Samiee et al. 2003). In
spite of long-term search for about three decades, a com-
mercially successful fungal strain was not yet identified for
efficient production of lovastatin apart from the conven-
tional A. terreus. Solid-state fermentation was employed
for lovastatin production in the present study. There are
several reasons and a vital aspect is higher production.
For instance, Barrios-González and Miranda (2010) stated
that solid-state fermentation (SSF) showed better lovas-
tatin yield than submerged fermentation (Smf). Pandey
et al. (2001) stated that SSF is highly advantageous than
submerged with the reasons such as usage of wide range
of agricultural wastes and thereby reduced overall cost,
better yield and stability of the product and ease of optimi-
zation due to low-cost constituents in the media. In addi-
tion, Suraiya et al. (2018) stated that the products obtained
can be directly consumed after sterilization. However, the
several prevailing patents in lovastatin production deal
with submerged fermentation; Biocon, India ltd, a lead-
ing commercial sector in India, has successfully produced
lovastatin using SSF and promoted the same for commer-
cialization (Suryanarayan 2003).
8
to 1 × 10 spores/mL which was used as inoculums for the
whole study (Alexopoulos et al. 1996).
Cultivation conditions
Each fungal species was cultivated in a solid-state fermen-
tation. The solid substrate (wheat bran) was initially dried
in an oven at 60 °C and accurately weighed to 10 g in Petri
plates (100 mm × 17 mm) followed by moistening with
distilled water and autoclaved for 40 min at 121 °C. After
cooling, the medium was inoculated with 1 mL (10%, v/w)
of spore suspension and the moisture content of the media
was adjusted to 60% by adding sterile distilled water. The
medium was thoroughly mixed and incubated at 28 °C in a
humidity-controlled incubator for 7 days. After incubation,
the Petri dishes were harvested and analyzed for lovastatin
content (Valera et al. 2005).
Hence, the present study aims in identifying and charac-
terizing the alternative source of high-yielding lovastatin
fungal producer through solid-state fermentation, charac-
terizing the purified lovastatin in terms of structural and
functional aspects and feasible primary application.
Extraction of lovastatin
After 8 days of incubation, the solid substrate was dried
at 40 °C for 24 h and gently crushed. Lovastatin extracted
was carried out with 10 mL of ethyl acetate by shaking at
Materials and methods
1
80 rpm for 2 h followed by filtration through Whatman
Chemicals
No. 1 paper. After which, 1 mL of 1% trifluoroacetic acid
was added to 1 mL of extract and finally the solution was
incubated for 10 min (lactonization of hydroxyl acid form
of lovastatin). The resultant extract was used for analytical
studies (Kamath et al. 2015a, b).
Analytical grades of the chemicals with high purity
obtained from Sigma-Aldrich and Merck were used in the
experiments.
1
3

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Page 3 of 19 359
Detection of lovastatin
supernatant was used as test solution for HPTLC analysis.
Test samples (0.6 µL) along with lovastatin standard were
loaded on to the plate coated with silica gel 60 F254 using
Hamilton syringe and run in CAMAG Linomat5 instru-
ment. The sample-loaded plate was kept in TLC twin trough
developing chamber (after saturated with solvent vapor) with
respective mobile phase (chloroform:methanol—9.5:0.5).
The hot air was used to evaporate the solvent from the
developed plate. The plate was kept in photo-documentation
chamber (CAMAG TLC SCANNER 3) and the image was
captured at UV 254 nm. For each TLC run, lovastatin stand-
ard was applied for Rf comparison. The area of the graph
was used to calculate the yield of lovastatin (Lingappa et al.
2004).
Yeast growth inhibition bioassay
In our study, the method of screening lovastatin by bioas-
say was adopted from Babu et al. (2011) in which Candida
albicans 3102 was used as indicator organisms. In bioassay
method, a clear inhibition zone around the indicator organ-
isms found to be observed and the diameter of the inhibition
zone is proportional to the concentration of lovastatin in the
samples. Yeast growth inhibition bioassay was performed
by seeding 15 mL of glucose yeast peptone agar medium
with 0.25 mL of yeast culture (C. albicans, purchased from
NCIM, Pune). Yeast-inoculated medium was poured into a
1
5 cm diameter of glass Petri dish. After solidifying, 6-mm-
diameter wells were made with the aid of sterile cork borer
and to which 10–70 μL of the extracts were added. Ethyl
acetate and the standard solution of lovastatin (commercial
lovastatin tablet) were used as negative and positive controls,
respectively. The standard was prepared based on Friedrich
et al. (1995) with a slight modification in which the lovas-
tatin tablet obtained from Merck was crushed using mortar
and pestle, and suspended in ethyl acetate followed by soni-
cation and filtration. Antifungal drug (fluconazole) was also
added to the wells. A. flavus and A. terreus [gifted by Indian
Institute of Technology (IIT), New Delhi] were also used as
reference strains. All the plates were incubated at 26 °C for
High‑performance liquid chromatography (HPLC)
The prepared sample extracts and a commercial lovastatin
standard were quantitatively analyzed for the presence of
lovastatin in the fungal extracts. For HPLC, a C18 column
(250 mm_4.6 mm_5 mm internal diameter) with diode array
detector was used. Acetonitrile and water which was acidi-
fied with 1.1% phosphoric acid in the ratio of 70:30 v/v were
used as mobile phase. The eluent flow rate was maintained
at 1.5 mL/min and the detection was carried out at 238 nm
with an injection volume of 20 mL (Samiee et al. 2003).
1
6 h. The concentration of standard lovastatin with respect
Identification and taxonomic positioning of 18S
rRNA gene sequence of C9
to the diameter of zone of inhibition against indicator organ-
isms were observed and used for calculating the presence of
lovastatin in the crude samples.
Based on the screening results, the isolate C9 was selected
for further studies. The isolate C9 was identified to the genus
level by observing its morphological and conidial features in
the culture growth (Alexopoulos et al. 1996). The selected
fungi were identified at species level based on the 18S rRNA
gene nucleotide sequence obtained by PCR amplification
of the ITS region, followed by DNA sequencing, BLAST
analysis and sequence similarity comparison using Gen-
Bank nucleotide databases. The ITS region of 18 s rRNA
fragment (ITS1/ITS4) was successfully amplified using the
fungal universal primers (ITS15′-TCCGTAGGTGAACCT
GCGG-3′ and ITS4 5′-TCCTCCGCTTATTGATATGC-3′).
The DNA sequencing was outsourced to Macrogen Ltd.
The 18S rRNA sequence was analyzed for its similarity
and homology with the existing sequences available in the
data bank of National Center for Biotechnology Information
(NCBI) using the BLAST search tool (http://www.ncbi.nlm.
nih.gov/). The sequences were aligned using the ClustalW
program in BioEdit version 7.0.5.3. A phylogenetic tree was
constructed by neighbor-joining method using PHYLIP soft-
ware version 3.69. Treeview 1.6.6 was used to visualize the
phylogenetic tree along with a bootstrap analysis for carry-
ing out 1000 replicates (Saitou and Nei 1987).
Thin‑layer chromatography (TLC)
Lovastatin in the fungal extracts was analyzed by
TLC performed on silica gel-G (200 meshes) with
chloroform:methanol (90:10 v/v) as the mobile phase. Lov-
astatin (10 mg) was dissolved in 100 mL of ethyl acetate and
is used as a standard solution. For TLC detection, 10 µL of
standard Lovastatin and tenfold-concentrated fungal extracts
were spotted onto TLC plates. Plates were developed three
times with mobile phase and spots were made visible in
iodine chamber. The distances travelled by each spot in base-
line and relative RF values were calculated. By comparing
the standard RF values for the chosen mobile phase, the
lovastatin present in the samples was identified (Nidhiya
et al. 2015; Lingappa et al. 2004).
High‑performance thin‑layer chromatography (HPTLC)
The spot obtained after TLC analysis of the fungal extract
was scrapped and dissolved in 1 mL of ethyl acetate. The
mixture was centrifuged at 3000 rpm for 5 min and the
1
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Page 4 of 19
3 Biotech (2018) 8:359
Purification of lovastatin
by a Nicolet Avatar Model FT-IR spectrophotometer (Naz-
zal et al. 2002).
Conventional purification
Nuclear magnetic resonance spectrometry (NMR)
Unlactonized crude lovastatin extract was concentrated
1
(
(
10 mL) under reduced pressure to get viscous mass
Skoog et al. 1988). The elute was initially purified by
The final structure of the test compound was identified by H
1
3
NMR and C NMR. The purified lovastatin was dissolved
in chloroform for NMR experiments according to Berger
and Baun (2004). The NMR spectrum was recorded using a
Varian Mercury plus 500 MHz.
silica ranging from 60 to 120 mesh column chromatogra-
phy using a dichloromethane:ethyl acetate step gradient
(
100:0–0:100). A volume of 250 mL was collected at each
step and analyzed for the presence of compound by TLC.
The fractions containing lovastatin compound were pooled
and concentrated in vacuum.
Antioxidant assay
Free radical scavenging activity measurement
Purification by preparative HPLC
DPPH (2,2-diphenyl-1-picrylhydrazl) radical was deter-
mined using 1 mL of the sample with 5 mL of 0.1 mM
methanol solution of DPPH followed by a vortex and incu-
bation at 27 °C for 20 min. 0.1 mM methanol solution of
DPPH alone served as the control. For negative control,
4.0 mL methanol and 1.0 mL of DPPH solution were used.
The absorbance of sample was measured at 517 nm using
methanol to set the blank (Iwashima et al. 2005). The ability
of the sample to scavenge DPPH radical was calculated by
the following formula:
The concentrated sample was subjected to preparative
HPLC equipped with an octadecylsilane column (Delta-
pak C18) equipped with an UV detector. The column was
eluted with acetonitrile and water which was acidified with
1
.1% phosphoric acid (70:30 v/v) at a flow rate of 20 mL/
min. The UV-absorbing peak at 238 nm with a retention
time of 13.687 min was collected and concentrated. The
purity was determined by analytical HPLC using a Nova-
Pak C18 column eluted with acetonitrile and water which
was acidified with 1.1% of phosphoric acid (70:30 v/v).
The purified sample was concentrated by rotary evapora-
tor and stored at − 20 °C for further study (Samiee et al.
DPPH radical scavenging activity (%)
− −Abssampleꢂꢃ
ꢀ
ꢁ
Abscontrol
ꢁAbs
=
ꢂ
× 100, Abs = absorbance.
control
2
003).
Reducing power
Characterization of purified compound
Total reducing power was determined as described by
Oyaizu (1986) by adding 0.1 mL of sample solution at
different concentrations with 2.5 mL of phosphate buffer
Liquid chromatography–mass spectrometry (LC–MS)
analysis
−
1
(0.2 mol L , pH 6.6) and 2.5 mL of potassium ferricyanide
(
1%). The mixture was incubated at 50 °C for 20 min.
The purified sample was subjected to LC–MS analysis
according to the protocol of Beer et al. (2012) at Sophisti-
cated Analytical Instrument Facility in IIT-Bombay, India.
LC–MS analyses were conducted on Q-TOF LC–MS Agilent
Technologies Q-TOF B.05.01 (B5125.1) system equipped
with a binary pump, in-line degasser, auto-sampler and col-
umn oven. LC–MS spectra data were compared with the
literature to identify the peaks of bioactive compound.
2.5 mL of trichloroacetic acid (TCA 10%) was added to
the mixture and centrifuged at 3000 g for 10 min. The
supernatant (5 mL) was mixed with 1 mL of ferric chloride
(0.1%) and the absorbance was measured at 700 nm in a
spectrophotometer. Increased absorbance of the reaction
mixture indicates its activity.
Iron chelating activity
The iron chelating activity was measured by the decrease in
absorbance at 562 nm of the iron (II)—ferrozine complex
Infrared spectrometry
(
Yamaguchi et al. 2000). The reaction mixture contained
A pinch of KBr pellet was oven dried and transferred into
a mortar. The purified sample at about 0.1–2% was added,
mixed and ground to a fine powder. The mid-range of IR
spectra ranging from about 4000 to 400 cm−1 was recorded
0.5 mL of ferric chloride (0.6 mM) and 900 µL of methanol.
The mixture was shaken and left at room temperature for
10 min. To this, 0.1 mL of ferrozine (5 mM) in methanol was
added, then mixed and left for 5 min to complex the residual
1
3

3
Biotech (2018) 8:359
Page 5 of 19 359
2
+
Fe . The control sample contained iron and ferrozine only.
The absorbance of the resulting solution was measured at
standard lovastatin and crude extract of lovastatin of 36
fungal strains along with fluconazole and reference strains
extract were loaded into the well to assess the zone of
inhibition against C. albicans. The graph in Fig. 1a showed
that the C3 and C9 strain crude extracts were much effec-
tive against C. albicans than all other strains when com-
pared to controls and reference strains. The standard plot
of zone of inhibitions against different concentrations of
standard lovastatin and C9 strain crude extract is also
presented in Fig. 1b, c, respectively. This graph showed
that C9 strain crude extract activity is similar to that of
standard lovastatin. The statistical insignificance was
observed when the effect of C9 lovastatin crude extract
was compared with standard lovastatin, fluconazole, A.
flavus and A. terreus (p > 0.0001). This showed that C9
strain crude extract activity is similar to that of lovastatin,
fluconazole, extracts of A. flavus and A. terreus. This also
demonstrated that extract was compared with all the other
strains (p< 0.0001) as represented in Table 2. Moreover, a
statistical significant difference was documented between
standard lovastatin and C3 strain which was lower at 25 µL
and 50 µL except 100 µL. Hence from the above observa-
tions, C9 strain and its crude extract were selected for
further evaluation.
5
62 nm. The ability of the sample to chelate ferrous ion
was calculated relative to the control (consisting of iron and
ferrozine only) using the formula:
ꢀ
ꢁA
− Asample ∕Acontrol
ꢃ × 100,
ꢂ
Chelating effect (%) =
A = absorbance.
control
Statistical analysis
All the experimental samples were done in triplicates to
validate the test results. Statistical analyses were performed
using the latest SPSS 10 version. The mean and standard
deviation values of reading were obtained from the test rep-
licate. The efficiency of test sample was compared between
the commercially available standard and lovastatin sample
by employing independent sample t test and paired sample
t test. A ‘p’ value of less than 0.0001 was considered as
statistically significant.
Results
3
6 fungal strains were isolated from the collected soil sam-
ple and the information related to its source is represented
in Table 1. All the 36 fungal strains that were grown under
solid-state fermentation conditions for the production of lov-
astatin were screened by bioassay method against C. albi-
cans indicator.
Rapid confirmation of C9 statin by TLC, HPTLC
and HPLC method
The C9 crude extract showed a Rf value of 0.53 by TLC
(Fig. 2) and their Rf value was compared against the authen-
tic lovastatin Rf value of 0.52 which confirms that the C9
crude extract as a lovastatin compound. HPTLC profile of
the commercial standard lovastatin and C9-extracted crude
lovastatin extract was carried out by measuring the absorb-
ance at 254 nm. The peak height observed in densitogram of
the standard lovastatin showed a correlation with C9 crude
extract as shown in Fig. 3a, b which confirms once again
Screening of lovastatin producers by bioassay
method
The screening of potential lovastatin-producing fungal
strain was carried out by measuring the zone of inhi-
bition against indicators. 25 µL, 50 µL, and 100 µL of
Table 1 Details of fungal
S. no.
Name of sampling area
Source isolation
No. of strains
obtained from
soil
strain isolated from different
agricultural sites of southern
region in Tamil Nadu
1
2
3
Pattanam village, Coimbatore
Pattanam village, Coimbatore
Nilgiris tea plantation area
Coconut soil (C)
Sugarcane soil (S)
Tea soil (T)
14
10
12
36
Total no. of strains
For our conventions, organisms or strains were designated with consecutive number based on the source of
sample collection
1
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359
Page 6 of 19
3 Biotech (2018) 8:359
Fig. 1 Inhibition zones formed at varying concentrations of fungal
extracts in significant strains compared against standards and refer-
ences against C. albicans. a Yeast growth bioassay of all compared
strains at different concentrations. L1 (stan), Standard lovastatin; AF
effect of standard lovastatin at its different concentration in producing
the zone of inhibition against C. albicans. c The zone of inhibition
produced by lovastatin crude extract isolated from C9 strain at dif-
ferent concentrations which is very similar to the effect of standard
lovastatin
(
Flucan), flucanazole; R1 (A. flav), A. flavus; R2; A. terreus. b The
that C9 crude extract as lovastatin compound. The objective
of HPLC analysis of any drug is to confirm the identity of a
drug and provide quantitative results. The HPLC chromato-
gram standard peak was detected at a retention time (RT) of
Morphological characterization of selected C9 strain
The selected C9 strain was characterized using standard
microbiological methods such as morphological properties
(shape, margins evaluation and growth rate) and microscopic
properties (conidial head, conidiophores, vesicles and
conidia). The organism was observed to have conidial
features as shown in the Fig. 5a–c.
1
3.486 and 13.687 min for standard lovastatin and C9 strain
crude sample, respectively, which resembled each other as
represented in Fig. 4a, b. Hence, the presence of lovastatin
is confirmed in a confounding way.
1
3

3
Biotech (2018) 8:359
Page 7 of 19 359
Table 2 Details of fungal
isolates studied for lovastatin
production through yeast growth
inhibition bioassay
S. no. Name of fungal extract
Diameter of zone of
inhibition against C.
albicans (cm)
C9 crude lovastatin statistical sig-
nificance (p<0.0001) ‘p’ value
compared with all
2
5 µL 50 µL 100 µL 25 µL
50 µL
100 µL
1
2
3
4
5
6
7
8
9
Lovastatin (standard)
Fluconazole (Antifungal durg)
A. flavus (Reference compound 1)
A. terreus (Reference compound 2) 0.1
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
C12
C13
C14
S1
S2
S3
S10
T1
T2
T3
T4
T5
T7
1.3
0.6
–
1.5
0.9
0.5
0.9
–
–
0.7
–
–
–
0.3
–
1.2
–
–
–
–
–
0.6
–
0.3
0.44
–
–
0.2
–
1.7
1.3
1.0
1.3
0.5
1.2
1.2
0.68
1.2
1.4
0.7
1.1
1.5
1.2
0.9
1.1
1.1
1
1.2
1.1
0.8
1.3
–
0.3
0.4
0.15
1.0
0.5
1.0
0.5
0.000128 0.000913 0.004025
0.004025 0.000913 0.004025
–
0.000034 0.008538
0.111034 0.000913 0.004025
–
–
0.5
–
–
–
–
–
0.8
–
–
–
–
–
0.2
–
–
0.3
–
–
–
–
–
–
0.000913
0.000913
0.000913 0.000128 0.000913
–
–
–
–
–
–
0.000062
0.000913
0.004025
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
0.000013 0.00002
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.000128
–
0.000913
0.000034
0.000128
0.000128
0.000128
0.000062 0.000062 0.000913
20
21
22
23
24
25
26
27
28
29
–
–
–
0.000128
0.000013 0.000034
0.000128 0.000017 0.004025
–
–
–
–
0.000013
0.000013 0.000913
– 0.000011
–
–
0.22
–
–
0.5
0.1
0.6
0.3
0.000071 0.000034 0.000128
0.000034 0.000128
T10
T11
0.000062 0.000128
3
0
–
0.000013 0.000128
Values in bold indicate significance
Phylogenetic tree analysis—C9 strain
Purification and quantification of lovastatin
Alignment and cladistic analysis of homologous nucleotide
sequences had shown that the sequence belonged to
Cunninghamella blakesleeana UBOCC-A-101341
The purification of lovastatin was done using conventional
method followed by preparative HPLC method (Fig. 7a, b).
The peak observed between standard and sample lovastatin
was similar. Subsequently, lovastatin was quantified
using spectrophotometer at 238 nm. The standard plot of
lovastatin was used to evaluate the concentration of the
purified lovastatin as shown in the Fig. 8a, b, respectively.
C. blakesleeana (C9) strain had shown higher concentration
than all other strains (p < 0.0001). The concentration of
lovastatin by C. blakesleeana was found to be 1.4 mg g−
(
KF225029.1) strain with a bootstrap value of 100%.
Of the species included in the phylogenetic analysis, C.
bertholletiae strain E15 was found to be closely related to
C. blakesleeana UBOCC-A-101341 (KF225029.1). From
the Fig. 6, it was observed that C. polymorpha CBS 779.68
and C. echinulata strain lSa species formed a well-supported
clade with 88% bootstrap support.
1
1
3

359
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3 Biotech (2018) 8:359
−
1
DWS followed by A. terreus with 0.83 4 mg g DWS. Both
the data reflect that definitely C. blakesleeana is a better
strain than A. terreus.
Characterization of purified compound
Infrared spectrometry (IR)
Infrared spectrum of the standard lovastatin and purified
lovastatin is shown in Fig. 9a, b, respectively. The
characteristic peaks of lovastatin were represented at
−
1
−1
3
541 cm (alcohol O–H stretching vibration), 3003.27 cm
−
1
(
olefinic C–H stretching vibration), 2335.87 cm (methyl
−
1
and methylene C–H asymmetric stretching), 1629.90 cm
−1
(
lactone and ester carbonyl stretch), 1375 cm (methyl and
Fig. 2 TLC chromatogram of standard lovastatin (left) and C9-pro-
−1
methylene-bending vibration), 1033.88 cm (ester C–O–C
duced lovastatin crude extract (right)
−1
symmetric bend), 819.77 cm (trisubstituted olefinic C–H)
Fig. 3 HPTLC profile of standard lovastatin compared over the C9-produced lovastation. a HPTLC densitogram of standard lovastatin. b
HPTLC densitogram of lovastatin extracted from C9 strain
1
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Biotech (2018) 8:359
Page 9 of 19 359
Fig. 4 Comparison of HPLC chromatogram of standard lovastatin over for rapid confirmation of the compound. a HPLC chromatogram of
standard lovastatin. b HPLC chromatogram of C9-derived crude extract sample
−
1
1
and 750.33 cm (meta-disturbed benzene-strong) which
confirmed that purified extract of C. blakesleeana is lovastatin.
The observed characteristic peak represented each
chemical group present in a compound between the standard
and purified C. blakesleeana extract which almost remained
same with a small difference in numerical value. Moreover,
terreus. The characteristic peaks of H-NMR shift difference
in the neighborhood of the stereo center C-1; H-1 experienced
a downfield shift of δ 4.63, whereas H-4 experienced the
higher field compared to the H-4 which is at δ 5.80. Further,
1
H-NMR spectrum of the isolated compound suggests that
the presence of characteristic three olefinic protons at 5.80
(1H, q, J=6.00, 3.6 Hz), 6.00 (1H, d, J=9.6 Hz) and 5.52
(1H, brs) corresponding to H-4, H-5 and H-6, respectively.
Four methyl group signals at δ 0.90 (6H, d, J=7.6 Hz), 1.08
(3H, d, J=7.2 Hz), 1.12 (3H, d, J=6.8 Hz) and 0.88 (3H, t,
J=7.6 Hz) were due to H-3/CH , H-7/CH , H-2″/CH and
−1
the presence of lactone ring peak at 1629.90 cm confirmed
that the sample compound was lovastatin (Patel and Patel
2
007). In the present study, it is observed that the lactone
−
1
−1
ring peaks at 1763.63 cm and 1762.35 cm for standard
and C. blakesleeana extract sample correspondingly. This
confirmed that the purified test sample was lovastatin.
3
3
3
H-3″/CH , respectively, three ‘O’ attached methine groups at
3
δ 4.63 (1H, m), 5.39 (1H, m) and 4.37 (1H, brs) were attributed
13
Nuclear magnetic resonance spectrometry (NMR)
and liquid chromatography–mass spectrometry (LC–MS)
analysis
to the position of H-1, H-5′ and H-3′, respectively. The C-
NMR spectrum indicated the presence of four methyls as δ
22.81, 13.85, 16.21 and 11.70, six methylenes at δ 32.69,
3
8.59, 36.17, 32.94, 24.25 and 26.79, three OCH were at
2
1
13
The C. blakesleeana lovastatin data obtained from H and C
NMR were in accordance with the standard data reported in A.
δ 67.86, 62.83, 76.35 due to C-1, C-3′ and C-5′ and three
methine carbons which were assigned to the characteristic of
1
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3 Biotech (2018) 8:359
Fig. 5 Morphological characterization of C9 strain. a C9 strain on PDA plate. b Scanning electron microscopic micrograph of C9 strain. c
Microscopic image of C9 strain showing conidial features
Fig. 6 Phylogenetic tree using
the neighbor-joining method of
C9 strain inferred from analysis
of the ITS region
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Biotech (2018) 8:359
Page 11 of 19 359
Fig. 7 Preparative HPLC profile. a HPLC chromatogram of standard lovastatin. b HPLC chromatogram of purified lovastatin from Cunningha-
mella blakesleeana
the C-4, C-5 and C-6, respectively. Two ester carbonyl carbons
at δ 170.30 and 176.80 are due to C-1′ and C-1″, respectively.
Finally all the spectral data match with lovastatin which is
to be 8-(2-(4-hydroxy-6-oxotetrahydro-2H-pyran-2-yl)
ethyl)-3,7-dimethyl-1,2,3,7,8,8a-hexahydronaphthalen-1-yl
Antioxidant assay
Free radical—scavenging activity measurement
The hydroxyl radical scavenging activity of the purified
1
13
2
-methylbutanoate, a known compound. The H and
C
extracts of C. blakesleeana was dose dependent. At a
−
1
NMR spectra of fungal sample yielded a molecular formula
C H O as shown in Fig. 10a, b. The LC–MS spectral peak
concentration of 199.5 µg mL (200 µL), the scavenging
activity of purified lovastatin was optimum and it is almost
nearly equal to that of the standard lovastatin as shown
in the Fig. 12a. The IC of the purified lovastatin was
24
36
5
of lovastatin produced by C. blakesleeana is represented in
Fig. 11 which ultimately confirmed the compound.
5
0
1
3

359
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3 Biotech (2018) 8:359
Fig. 8 Quantification of purified lovastatin using the standard lovastatin plot. a Standard lovastatin calibration curve at different concentrations
−
1
(
µg mL ). b Estimation of lovastatin from yielding strains
−
1
at 145.9 µg mL (140 µL) demonstrating the inhibitory
activity and it was not higher than the standard.
Iron chelating activity
The iron chelating assay was used to evaluate the ability of
purified lovastatin to disrupt the complex formation. In our
study, it was observed that the percentage of inhibition is
Reducing power
−
1
−1
Reducing power reflects the antioxidant activity significantly.
The reducing capacity of the purified extract was observed
maximum at 176.8 µg mL (180 µL) and 199.5 µg mL
(200 µL) which is statistically significant (p<0.0001). At
other doses also, it is statistically significant (p < 0.05)
in case of both test sample (C. blakesleeana extract) and
standard (Fig. 13).
−
1
maximum at 117.4 µg mL (120 µL) and also optimum at
−
1
−1
1
45.95 µg mL (140 µL) and 166.95 µg mL (160 µL). The
reducing power of the purified extracts is also represented
in Fig. 12b.
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Biotech (2018) 8:359
Page 13 of 19 359
Fig. 9 IR analysis. a IR spectrum of standard lovsatatin. b IR specrtum of purified lovsatatin of Cunninghamella blakesleeana
Discussion
commercial production of lovastatin (Lai et al. 2007;
Pecyna and Bizukojc 2011; Subazini and Kumar 2011).
However, various compounds have been produced from
various fungal species, trying a compound production from
a species that are never reported earlier always been crucial
and chances of getting an efficient activity could be high.
In such perspective, C. blakesleeana showed magnificent
innate ability of producing lovastatin which is on par with
its conventional fungal producers. The purified Lovastatin
showed significant zone of clearance and the reasons
could be attributed to competitive inhibition of HMG-CoA
receptor which in turn prevents ergosterol biosynthesis
followed by cell cycle arrest and accompanied by various
To date, there are plenty of reports that dealt with
the production of pharmaceutical drugs such as
antibiotics, anticancer, anti-cholesterol, anti-diabetic,
immunosuppressant, anti-anxiety and antiviral from
fungal origin and it also acts as obvious potent producers
of those useful compounds. At present, organisms that are
familiarly reported to produce lovastatin as a secondary
metabolite include A. terreus, Monascus sp., A. niger,
A. flavus, P. purpurogenum, Pleurotus sp., T. viride and
Penicillium sp., whereas the strains such as Penicillium
sp., M. ruber, and A. terreus are commonly used for the
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3 Biotech (2018) 8:359
Fig. 10 NMR elucidation. a H NMR spectrum. b 13C NMR spectrum of Cunninghamella blakesleeana lovastatin
1
eventual intracellular activities holistically contributing to
cell death. The confirmatory screening techniques including
TLC, HPTLC and HPLC all showed similar agreement with
standard lovastatin. The proximity of the retention time
concluded A. terreus as best producer. Hence, on finding
the best lovastatin producers among the studied organisms
such as A. terreus, A. flavus and C. blakesleeana, it was
evident that C. blakesleeana found to be the predominant
−
1
(
standard: 13.486; lovastatin from C. blakesleeana: 13.687)
producer of lovastatin with a yield of 1.4 mg g DWS.
C. blakesleeana produced a good amount of lovastatin
without optimization unlike other earlier reported strains
which were optimized for attaining considerable production
(Table 3). In addition, it was observed that C. blakesleeana
produced 12% and 23% of lovastatin more than that of A.
terreus and A. flavus, respectively, as shown in the Fig. 8b.
Hence, the present study had lucidly established the fact
that the unreported lovastatin producer C. blakesleeana
can be further explored for commercial production after
conducting extensive studies such as optimization of
medium constituents and other process parameters that
between the standard lovastatin and lovastatin obtained
from C. blakesleeana showed that the lovastatin is no longer
distinguishable than the commercially available lovastatin.
So far, a large number of microbes were screened for the
production of lovastatin in which A. terreus was identified
to be the best lovastatin-producing fungus (production
−
1
level up to 55 mg L ) among other producers (Samiee
et al. 2003). Shindia (1997) had reported the synthesis of
lovastatin by A. oryzae, A. terreus, Doratomyces stemonitis,
Paecilomyces variotii, P. citrinum, P. chrysogenum,
Scopulariopsis brevicaulis and T. viridae, etc. and
1
3

3
Biotech (2018) 8:359
Page 15 of 19 359
Fig. 11 LC–MS spectra of the purified lovastatin
may influence the production of lovastatin than the present
innate capacity of the lovastatin production. Even though
C. blakesleeana is known to be reported for bromhexine
metabolite production (Dube and Kumar 2017), lovastatin
productions have not been reported earlier. Thus, the present
study makes a sincere note and light on C. blakesleeana
ability as lovastatin producer and prospecting area to
explore further production augmentation and modification
of lovastatin.
and matches with the reported lovastatin. Researchers
have also quantified lovastatin with their hydroxy acid
metabolites in rat and mouse plasma and even in humans
using LC/MS/MS (Vlčková et al. 2011; Wu et al. 1997). The
information derived from the above spectroscopic analysis
is also in accordance with our report. This shows that the C.
blakesleeana is producing lovastatin in a form without any
detectable structural differences than that of the commercial
producers of lovastatin.
The constituent and structural details of the purified
lovastatin were obtained through IR, NMR and LCMS
which all showed similar agreement with the standard
lovastatin. For instance, in IR analysis, the lactone ring
Oxidative stress has been associated with the development
of many diseases such as cardiovascular disease and cancer
which cause higher mortality worldwide. Certain earlier
reports have shown that the lovastatin possesses anticancer
and antioxidant activity (reduces oxidative stress) (Singh
et al. 1997; Kumar et al. 2011). Bhargavi et al. (2016)
have shown that the lovastatin isolated from A. terreus
−
1
−1
peak observed at 1763.63 cm and 1762.35 cm for
standard and C. blakesleeana, respectively. Similarly, the
spectral data obtained through NMR had fine accordance
1
3

359
Page 16 of 19
3 Biotech (2018) 8:359
Fig. 12 Antioxidant and reducing power assessment of purified lovastatin. a Assessment of radical scavenging activity using DPPH. b Reducing
power evaluation of the lovastatin extract
(
KM017963) was effective to accelerate hydroxyl radical
cancer cells generate uncontrolled formation of hydroxyl free
radicals which result in death of healthy cells, tissues, DNA
modifications, membrane peroxidation, enzyme inactivation
and protein oxidations (Mohan Kumari et al. 2011). Hence,
lovastatin was found to be effective than the standard
in accelerating scavenging activity of hydroxyl radical.
Hence, the current study had the significant reflection of an
antioxidant activity and reducing power ability of the purified
lovastatin compound synthesized by C. blakesleeana. Thus
the reported species C. blakesleeana produces lovastatin with
good quantity and similar structural and functional features
on par with earlier reported lovastatin.
−
1
scavenging activity (54.06%) at an IC50 of 3601 µg mL
compared to its positive control (catechin) which showed
−1
8
6.86% at an IC50 value of 350.5 µg mL . The same paper
had reported that lovastatin showed a moderate reduction
in total reduced glutathione (41.58%) compared to its
positive control (ascorbic acid) which showed 68.85% at
−
1
an IC50 value of 19.46 µg mL . Our study had observed
−1
IC50 value of purified lovastatin at 145.9 µg mL (140 µL)
demonstrating its potent inhibitory activity. This difference
could be because of the controls (standard lovastatin) used
for the study. Hydroxyl radicals formed through ascorbic
acid–iron EDTA complex are effectively disturbed by the
lovastatin of C. blakesleeana which reflected the increased
scavenging activity in terms of decreased absorbance as
observed in the present study. It is a well-known fact that
In a commercial perspective, the economy behind the
production can only be analyzed. For instance, wheat bran
acts as an excellent cheaper medium possessing protein, fat
and crude fiber, celluloses, pentoses, vitamins, minerals, and
1
3

3
Biotech (2018) 8:359
Page 17 of 19 359
Fig. 13 Assessment of iron chelating ability of standard lovastatin and purified lovastatin
Table 3 Lovastatin production from various fungal species using solid-state fermentation
S. no.
Reports
Strain
Lovastatin production
Yield after
optimiza-
tion
−
1
(
mg g DWS)
1
2
3
4
Mouafi et al. 2016
Aspergillus fumigatus
3.353
3.6
1.3
0.402
0.27
0.26
1.4
0.83
0.3
Yes
Yes
Yes
Yes
Yes
Yes
No
Kamath et al. (2015a)
Kamath et al. (2015b)
Dikshit and Tallapragada (2015)
Aspergillus terreus (KM017963)
Aspergillus terreus (KM017963)
Monascus sanguineus
Monascus purpureus
Both of the above co-culture
Cunninghamella blakesleeana KP780148.1
Aspergillus terreus
5
Our current report
No
No
Aspergillus flavus
phytochemicals and such richness in constituents made it
as a suitable substrate for commercialization (Kamath et al.
and purification which were comprehensively reviewed
by Mulder et al. (2015). Since the present study aims in
investigating the possible lovastatin production ability of C.
blakesleeana KP780148.1 strain, our future work involves a
detailed insight on exploring optimized higher production in
commercial aspect. Hence, compared with the existing pat-
ent is quite early for this C. blakesleeana KP780148.1 strain
which is newly reported to have lovastatin ability.
2
015a, b; Jahromi et al. 2012; Chanakya et al. 2011; Arranz
and Calixto 2010; Jaivel and Marimuthu 2010; Sramkova
et al. 2009; Sun et al. 2008). Wei et al. (2007) stated that
either wheat or rice bran in SSF gives maximum lovasta-
tin production in a cost-effective way. Regarding patents,
there are plenty of existing patents on lovastatin production
1
3

359
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3 Biotech (2018) 8:359
Friedrich J, Žužek M, Benčina M, Cimerman A, Štrancar A, Radež
I (1995) High-performance liquid chromatographic analysis of
Conclusion
mevinolin as mevinolinic acid in fermentation broths.
togr A 704:363–367
‎
J Chroma-
This is the first report that documented C. blakesleeana
having gene bank accession number KP780148.1 as a
lovastatin producer. This study suggested that the C.
blakesleeana strain may be a better alternative source to the
conventional sources such as A. terreus and A. flavus in the
production of lovastatin. However, the commercialization
of lovastatin production can be feasible after subjecting
to further studies on production optimization. Since the
characterized lovastatin demonstrated significant antioxidant
property, future perspectives are towards improving the yield
of lovastatin followed by its further necessary applications
for establishing its strong anticancer potentials.
Gupta K, Mishra PK, Srivastava P (2007) A correlative evaluation of
morphology and rheology of Aspergillus terreus during lovastatin
fermentation. Biotechnol Bioprocess Eng 12:140–146
Istvan E (2003) Statin inhibition of HMG-CoA reductase: A 3-dimen-
sional view. Atheroscler 4:3–8
Iwashima M, Mori J, Ting X, Matsunaga T, Hayashi K, Shinoda D,
Saito H, Sankawa U, Hayashi T (2005) Antioxidant and antiviral
activities of plastoquinones from the brown alga Sargassum mic-
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plastoquinones. Biol Pharm Bull 28:374–377
Jahromi MF, Liang JB, Ho YW, Mohamed R, Goh YM, Shokryazdan
P (2012) Lovastatin production by Aspergillus terreus using agro-
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