Online - 2455-3891

Vol 10, Issue 10, 2017 Print - 0974-2441

Research Article

PURIFICATION AND CYTOTOXICITY STUDY OF LOVASTATIN FROM SOIL FUNGI

BALRAJ JANANI1, GANESAN SAIBABA2, GOVINDARAJU ARCHUNAN2, KALIESWARAN VIDHYA1, JAIRAMAN

KARUNYADEVI1, JAYARAMAN ANGAYARKANNI1*
1
Department of Microbial Biotechnology, Bharathiar University, Coimbatore, Tamil Nadu, India. 2Department of Animal Science,
Bharathidasan University, Trichirapalli, Tamil Nadu, India. Email: angaibiotech@[Link]
Received: 18 May 2017, Revised and Accepted: 30 June 2017

ABSTRACT

Objective: The objective of the present study is to evaluate the anticancer potential of lovastatin obtained from fungal source.

Methods: About 15 fungal cultures were isolated from soil samples collected from Bharathiar University, India, and all are identified and characterized
through microscopic characterization. Lovastatin producing capability was confirmed through bioassay against Saccharomyces cerevisiae, and the
ability of selected fungus to produce lovastatin was further confirmed by high-performance liquid chromatography. Maximum lovastatin producing
fungi were further selected for purification (overloaded elution chromatography) and characterization done using inhibition rate (IR). 5-diphenyl
tetrazolium bromide (MTT) assay using A549 cell line was performed for antitumor activity evaluation.

Results: Among the 15 isolates, Aspergillus flavus exhibited the maximum zone of inhibition (1.5 cm) against the test organism through solid-state
fermentation. The resemblance in retention time (RT) of peak shown in chromatograms of standard lovastatin (RT=25.1 minutes) and sample
(RT=25.1 minutes) were similar. This confirmed the presence of lovastatin in the selected fungal isolate (A. flavus). The presence of two functional
groups in lovastatin C=O and O-H was confirmed by IR spectrum 50% of cell lysis was observed in MTT assay.

Conclusion: Lovastatin obtained from soil fungi is capable of producing lovastatin in good proportions. Obtained fungal lovastatin exhibited significant
antitumor activity against A549 cell line. Like other biological derivatives, lovastatin from soil fungi had greater potential in anticancer activity, and
further biosynthetic pathway insights in their production can improve the yield which aid in large scale production.

Keywords: High-performance liquid chromatography, 3-hydroxy-3-methylglutaryl coenzyme A reductase, Lovastatin, Inhibition rate, 5-diphenyl
tetrazolium bromide.

© 2017 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license ([Link]
org/licenses/by/4. 0/) DOI: [Link]

INTRODUCTION effective in the treatment of hypercholesterolemia. Lovastatin and

its semi-synthetic derivatives are very important drugs to treat heart
Fungi were once looked out as a source for the variety of enzymes but disease. Lovastatin was produced by several fungal species including
now they are exploited for its secondary metabolites too. The complex Monascus purpureus, Monascus ruber, Monascus pilosus, Monascus
metabolic pathways in fungal cells favor the production of different pubigerus, Monascus vitreus, [2-4] and Aspergillus terreus [5-11]. The
types of secondary metabolites which have high pharmacological commercial production of lovastatin is mainly through submerged
value. The secondary metabolites include antibiotics, pigments, toxins, fermentation of A. terreus [12,13]. Lovastatin is an intracellular product
antitumor agents, immune modulating agents, receptor antagonists, and is mostly accumulated in mycelia. In submerged fermentation, its
and pesticides. They have a major effect on the health, nutrition, and yield is proportional to the amount of biomass, with the high cell density
economy of our society. They are often unusual structures and their and hence viscosity of the fermentation broth increases and there was a
formation is regulated by nutrition, growth rate, feedback control, difficulty in stirring and oxygen mass transfer; an alternative strategy to
enzyme inactivation, and enzyme induction. produce lovastatin is by solid-state fermentation (SSF) process. Despite
the low substrate cost and low energy consumption, the SSF can offer
Polyketides constitute the largest class of fungal secondary
a good environment for fungi to grow; therefore, high mycelia density
metabolites. They are generally found in soil microorganisms and
and high lovastatin production can be expected [14-20].
there are more than 7000 known polyketides. The large arrays
of structurally diverse polyketide produced by microorganisms Recent evidence showed that statins not only reduce the mortality of
originate from repetitive connections of short-chain fatty acids, for cardiac diseases but also the incidence of cancer up to 28-33% [21].
example, acetate or propionate by pathways very similar to those of Lovastatin had shown great promise beyond their well-documented
fatty acid biosynthesis [1]. Polyketides were a rich source of many lipid lowering effects and they also suppress a variety of leukemic
pharmaceuticals including antibiotics, anticancer drugs, cholesterol cell lines and a wide array of solid tumor cells in vivo, by inhibiting
lowering drugs, immunosuppressants, and other therapeutics. Out of the synthesis of non-sterol isoprenoid compounds [22]. Owing to the
the very important classes of polyketides is “statin” which includes beneficial aspects of lovastatin, an attempt was made in this study to
lovastatin, mevastatin, and pravastatin. screen and characterize lovastatin producing fungi from the soil sample.
Lovastatin, also known as monacolin, is a kind of fungal metabolite, METHODS
serving as a competitive inhibitor of 3-hydroxy-3-methylglutaryl
coenzyme A reductase, the rate-limiting enzyme in cholesterol Microorganism and inoculum preparation
biosynthesis [2]. It can effectively reduce plasma cholesterol levels Filamentous fungi were isolated from soil samples collected from
in various mammalian species including human and hence they are Bharathiar university campus, Coimbatore, India. Isolation of desired
 Janani et al.
Asian J Pharm Clin Res, Vol 10, Issue 10, 2017, 258-262

fungal cultures were initially isolated by plating collected soil samples tightly into the 60 cm × 3.5 cm size column chromatography without
on potato dextrose agar (PDA) and characterized by following standard air bubbles) using solvents n-hexane and petroleum ether mixture
microbial methods such as morphological and microscopic properties. (1:1 v/v). White powder was obtained by subsequent evaporation of
Individual colonies of microfungi were picked and purified by streaking solvents [4].
onto fresh PDA medium. The pure fungal isolates were kept on PDA
medium at 4°C and recultured every 4 weeks. For preparing a spore Infrared spectrometry
suspension, towel sporulated PDA slants of isolates, 10 ml spores A pinch of KBr pellet was oven-dried and transferred into a mortar. The
suspension medium (0.9% NaCl, 0.1% Tween80) was added. The surface purified sample at about 0.1-2% was added, mixed, and ground to a
was scrapped with a loop and suspension was collected. The suspension fine powder. The mid-range inhibition rate (IR) spectra, 4000-400 cm−1
was agitated thoroughly using cyclomixer to suspend the spore and were recorded on a Nicolet Avatar Model fourier transform infrared
filtered through a glasswool. The concentration of the spore suspension spectrophotometer [28].
was measured using an hemocytometer and adjusted to 1 × 108 spores/ml
which was used as inoculum throughout the study [23]. Cytotoxicity assay
Cytotoxicity evaluation of secondary metabolites was performed using
Media and cultivation conditions 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
To screen the potent lovastatin producing fungal strain, lovastatin assay as described by Ghasemi-Mobarakeh et al. (2008) [29]. A549 cells
production was carried out using isolated fungal strains through SSF. were trypsinized, and 5000 cells were seeded in a flat-bottomed 96-well
In the fungal fermentation, solid substrate (wheat bran; particle size polystyrene-coated plate and were incubated for 24 hrs. Different
0.3-0.5 mm) was dried in an oven at 60ºC, accurately weighed to 8 g concentrations of commercial lovastatin and lovastatin from fungal
in Petri plates (100 mm x 17 mm), appropriately moistened with extract (purified fraction) were prepared in Dulbecco’s modified Eagle’s
0.8 ml (10% v/w) of spore suspension. The medium (glucose [20 g/l], medium with 5% fetal bovine serum. The viability of the cells treated
lactose [20 g/l], Na-glutamate [12.5 g/l], KH2PO4 [5 g/l], K2HPO4 [5 g/l], with different concentrations of commercial lovastatin and lovastatin
FeSO4.7H2O [0.2 g/l], MnSO4.4H20 [0.1 g/l], ZnSO4.7H2O [0.2 g/l], from fungal extract was estimated after 48 hrs of incubation. Optical
MgSO4.7H2O [0.1 g/l], CaCl2.2H2O [20 mg/l], CuCl2.2H2O [5/mg/lit], density was read at 570 nm using 96-well Multiskan™ GO microplate
H3BO3 [11 mg/l], (NH4)6Mo7O24.4H2O [5 mg/l], pH adjusted to 6.5) spectrophotometer (Thermo Scientific, USA). The cells were given with
was thoroughly mixed and incubated at 30°C, in a humidity-controlled 2, 4, and 6 µg/ml of lovastatin and cells without any treatment were used
incubator for 7 days. After incubation, the fungal cultures were as a control. The experiment was done in triplicates (mean±standard
harvested and analyzed for presence of lovastatin [17,24]. deviation).

Extraction of lovastatin from the culture media RESULTS

Fermented material was dried at 40°C for 24 hrs, crushed to powder, Isolation of fungal cultures
and 2 g of the powdered material was extracted by 100 ml of methanol- The aim of this study was to find a better filamentous fungus for
water mixture (1:1 v/v) at pH 7.7 in 250 ml Erlenmeyer flask, keeping lovastatin production compared to already available fungi. Nine pure
the flask at 30°C in the rotary shaker at 200 rpm for 2 hrs. After 2 hrs, cultures of fungus were isolated from the soil sample collected in
the mixture was centrifuged at 10,000 rpm for 10 minutes, and the the campus of Bharathiar University by serial dilution technique on
supernatant was filtered through 0.45 µm membrane filter [17]. sabourad dextrose agar. They were characterized using standard
microbiological methods such as morphological and microscopic
Lovastatin detection characteristics. Characterized and identified fungal cultures (1)
The bioassay method for screening the lovastatin producing fungi was Aspergillus sp.1, (2) Fusarium sp., (3) Aspergillus sp.2, (4) Aspergillus
adopted from Kumar et al. (2000) [25] and Vilches et al. (2005) [26] sp.3, (5) Aspergillus niger, (6) Aspergillus fumigatus, (7) Aspergillus
using Saccharomyces cerevisiae as indicator organisms, and the ability oryzae, (8) Aspergillus flavus, and (9) Rhizopus sp. were maintained as
of lovastatin to inhibit fungal growth was tested. Yeast growth inhibition pure cultures on PDA slants at 4°C. The isolates were further analyzed
was performed by seeding 15 ml of glucose yeast peptone agar medium for their potential lovastatin production.
with 0.25 ml of yeast culture (S. cerevisiae). Yeast inoculated medium
was poured into a 15 cm diameter glass Petri dish. After solidifying, SSF and downstream processing of lovastatin
6 mm diameter wells were made with aid of sterile cork borer. Two All the nine fungal strains were grown under SSF to screen their
different volumes of the crude extracts (25 and 50 μL) were taken potential for lovastatin production. At the end of 7 days of fermentation,
and transferred to these wells. Methanol–water mixture (1:1 v/v) and organic phase containing lovastatin was extracted through solvent-
standard solution of lovastatin were used as negative and positive solvent extraction. Then, crude extract was concentrated by drying at
controls, respectively. Plates were incubated at 26°C for 16 hrs and room temperature for further analysis.
afterward inhibition zone were measured [27].
Screening of isolates for lovastatin production through bioassay
Estimation of lovastatin method
Lovastatin in the clear extract was estimated by high-performance The results of bioassay revealed that out of nine, six isolates fungal
liquid chromatography (HPLC) system (Agilent1100series) using cultures showed zone of inhibition for yeast S. cerevisiae. Based on
C-18 (250 mm length × 4.6 mm ID). A mixture of 0.02M phosphate the diameter of inhibition zone (cm) obtained with two different
buffer (pH7.7) and acetonitrile in a ratio of 65:35 (v/v) was used as concentrations of standard and fungal extracts, it has been observed
mobile phase. The 20 µl sample was injected, the eluent flow rate was that maximum lovastatin production was found with A. flavus. The
1.0 ml/minutes, and the detection wavelength was 238 nm. The identity other fungal isolates with decreasing order of their lovastatin producing
of the compound was confirmed with an authentic lovastatin (Sigma- potential are Aspergillus sp.1, Aspergillus sp.2, Aspergillus sp.3,
Aldrich) reference standard [17]. A. fumigatus, and A. oryzae (Fig. 1). A clear inhibition zone around the
indicator organisms was the basis for the selection of suitable fungal
Purification of lovastatin strains for production of lovastatin.
In the fermentation broth, lovastatin is present mostly in its hydroxyacid
form and mevinolinic acid. The isolation of mevinolinic acid from the Confirmation and quantification of lovastatin by HPLC
fermentation broth was done by solvent extraction using methanol: To ensure whether the maximum zone of inhibition exhibited by
Water mixture (1:1 v/v) at pH 7.7. The extract is concentrated by vacuum isolate (A. flavus) on bioassay plates were due to lovastatin activity,
distillation at 106°C for 2 hr. The concentrated extract was subjected to the crude extract were subjected to HPLC for quantitatively confirming
column chromatography (Silica 60-120 mesh was activated and packed the amount of lovastatin in the fungal extract. Analysis of lovastatin

259
 Janani et al.
Asian J Pharm Clin Res, Vol 10, Issue 10, 2017, 258-262

in HPLC confirmed its identity with that of authentic lovastatin. The Structure analysis
resemblance in retention time (RT) of peak shown in chromatograms of The purified lovastatin obtained after column chromatography process
standard lovastatin (RT=25.1 minutes) and sample (RT=25.1 minutes) was confirmed by IR spectrum. IR spectrum was recorded on Shimadzu
were similar (Fig. 2a and b). This confirmed the presence of lovastatin. spectrometer, and the absorption frequencies are expressed in
Lovastatin quantification was carried out on A. flavus extract (Table 1). reciprocal (cm−1). IR spectrum showed stretching at 1633 and 1643 cm−1
These analytical procedures confirmed the production of lovastatin in and indicating the presence of two C=O groups. A broadband between
the selected fungal isolate (A. flavus). 3369 and 3498 cm−1 showed the presence of O-H group. The presence
of two functional groups in lovastatin C=O and O-H was confirmed by
Purification of lovastatin IR spectrum (Fig. 3).
To use lovastatin as a drug at commercial level, measures have to be
taken to ensure that the final product is not only in its purest form but Cytotoxicity assay
also shows no loss of activity on purification and is safe for human The purified lovastatin showed cytotoxicity against A549 cells lines
consumption. Lovastatin extracted from A. flavus on the 7th day of solid in a dose-dependent manner (Fig. 4). The effective concentration for
fermentation was subjected to column chromatography. The extract 50% cell death (half maximal inhibitory concentration inhibitory
is concentrated by vacuum distillation at 106°C for 2 hrs and then concentration) was found to be 30±5 µg/ml. This result indicated that
poured into the packed activated silica, and the fractions were eluted by lovastatin from fungal extract (A. flavus) is a strong cytotoxic agent
solvents benzene and acetonitrile in the ratio 1:1. Ninety fractions were which partially explains its antitumor activity than authentic lovastatin.
collected (flow rate 1 ml/minute) and all collected fractions were dried
using vacuum evaporator. Up to 60 fractions, yellow colored eluent was DISCUSSION
obtained, which were then followed by watery colored eluent. These
Lovastatin is widely used as an anticholesterolemic drug, and
watery colored eluents were checked for their purity of lovastatin by
researchers have screened several microbes for lovastatin production
HPLC analysis. The fractions from 61 to 66 showed the presence of
from various niches [30]. Soil is the exceptionally rich source for
lovastatin. All the 6 fractions were analyzed by HPLC, and the fraction
potential drug producing organisms, especially for fungi. Therefore,
64 showed the presence of pure lovastatin and the experiment was
in the present study, attempts were made to isolate potential of
conducted thrice to standardize the purification process.
lovastatin producing fungal strains from soil sample collected
from campus of Bharathiar University, Coimbatore, India, which is
described as the culture of choice for the production of lovastatin [5].
The identified fungal isolates belonged to different classes such as
Aspergillus sp.1, Fusarium sp., Aspergillus sp.2, Aspergillus sp.3, A. niger,
A. fumigatus, A. oryzae, A. flavus, and Rhizopus sp. In this, majority of

Fig. 1: Screening of lovastatin producing fungal isolates through

bioassay

Fig. 3: Inhibition rate spectrum of purified lovastatin

b
Fig. 2: (a) High-performance liquid chromatography
chromatogram of standard, (b) high-performance liquid
chromatography chromatogram of lovastatin production in Fig. 4: Cytotoxicity assay after 48 hrs (mean±standard deviation).
Aspergillus flavus Sample 1: Lovastatin from fungal extract. Sample 2: Commercial
lovastatin

260
 Janani et al.
Asian J Pharm Clin Res, Vol 10, Issue 10, 2017, 258-262

Table 1: Production of lovastatin in A. flavus (crude extract) based column chromatography with benzene and acetonitrile used
as eluents. In organic solvent extraction of lovastatin by Pansuriya
Sample Area Height Lovastatin conc. in mg/g and Singhal (2010) [52] initially, various solvents were screened such
Standard 439303 25053 1 as acetonitrile, methanol, ethyl acetate, butyl acetate, toluene, and
A. flavus 234875 8542 0.33±0.02 chloroform for maximum recovery of lovastatin. Acetonitrile gave
Mean of three replicates, A. flavus: Aspergillus flavus
maximum extraction of both hydroxyl acid and lactone lovastatin which
was similar to the results of our present study. The purified lovastatin
obtained after column chromatography process was characterized
the fungal isolates obtained were Aspergillus sp. strains [31-33]. All by IR spectrum. The presence of two functional groups in lovastatin
these species are known to produce vast array of the primary and C=O and O-H was confirmed by IR spectrum. The IR spectral data of
secondary metabolites [34]. In recent days, SSF appears to be one lovastatin was found to be in concurrence with earlier reported spectral
of the best choices for any metabolite production using cheaper raw data [53,54]. Although statin has been extensively researched on its
materials [7,35,36]. The solid substrates used in SSF are generally property of treating hypercholesterol, several reports suggest that
insoluble in water and play a dual role by supplying of nutrients and cancer incidence of cancer is also reduced by 28-33% [21]. Cytotoxicity
providing anchorage to the cells [37]. Szakacs et al. (1998) reported is one of the properties of antitumor agents [55]. Hence in this study,
that production of lovastatin through SSF is superior than submerged MTT assay results indicate that lovastatin from fungal extract is a strong
fermentation. They observed the highest production of lovastatin cytotoxic agent which partially explains its antitumor activity than
using solid substrates such as wheat bran amended with the nutrient commercial lovastatin. Our results are also in accordance with myeloma
solution. Wheat bran has been previously reported as a best suitable researchers who had used chemotherapeutic agents in combination
carbon source for lovastatin production using A. flavus and A. terreus with statins [56] and other dose-dependent studies [57]. Hence, it can
[17,38]. Hence, in the present study, wheat bran was used as the solid be suggested for therapeutic use as a cancer chemopreventive agent.
substrate for both the fungal sporulation and lovastatin production
by isolated fungal cultures. The fungal cultures were grown under CONCLUSION
SSF conditions to screen their potential for lovastatin production. The
lovastatin yield increased rapidly from day 3 to day 5, which explained An attempt is made in this study to isolate and screen the lovastatin
that although lovastatin is a secondary metabolite and its accumulation producing fungi from soil source. The present study shows among the
in mycelia seems growth related. The maximum lovastatin yield was isolated strains, A. flavus yield more lovastatin than the other isolates
achieved on day 7 after then the yield was almost unchanged. From the based on the bioassay result. Analysis of lovastatin in HPLC confirmed
above results, it is obvious that the SSF can be ended at day 7. Because its identity with that of authentic lovastatin and it is characterized by
lovastatin is an intracellular product and its accumulation is almost IR spectrum. The characterized lovastatin was analyzed for antitumor
simultaneously with cell growth [39]. activity by MTT assay using A549 cell line. Therefore, it could be
concluded that compared to A. terreus which is commercially exploited
The culture extracts prepared in methanol: Water mixture was for lovastatin production, the levels of lovastatin isolated from A. flavus
screened for lovastatin production by employing bioassay against are comparatively lower. Based on these findings, the future work is
S. cerevisiae. Similar approaches have also been reported using Candida aimed at biosynthesis of lovastatin by this fungus could be manipulated
albicans and Neurospora crassa as test organisms [27,40-42] as a less to yield greater quantities so that the fungus can serve as a potential
expensive and alternative method for lovastatin assay. These yeasts source for industrial exploitation.
form constricted colonies on agar and served as a relatively convenient
method as compared to using N. crassa that shows profuse sporulation. ACKNOWLEDGMENT
The mechanism of antiyeast inhibition could be attributed to (i)
The corresponding author thank Department of Science and Technology
mycosin contact directly with the sterol in the cell membrane causing
(SR/SO/HS-0131/2008; dt: 28/10/2009) for the financial support.
leakage of cell membrane and the loss of intracellular component,
Dr. Govindaraju Archunan acknowledges with thanks to University
(ii) mycosin attached with ribonucleic acid and inhibition of protein
Grants Commission, New Delhi for the award of UGC-BSR faculty fellow.
synthesis, and (iii) mycosin inhibited ergosterol synthesis causing
The instrumentation facility availed from UGC-SAP-II and DST-PURSE
increase of membrane permeability and membrane damage [43]. From
programmes, Bharathidasan University is gratefully acknowledged.
results obtained during the present study, six out of nine isolates have
exhibited the inhibition zone on bioassay plate. Of these, one isolate has
REFERENCES
exhibited maximum inhibition zone of 1.5 cm in 50 µl concentration
and hence it was considered as good lovastatin producer. To ensure 1. Sivasithamparam K, Ghisalberti EL. Secondary metabolism in
whether the zones of inhibition exhibited by A. flavus in bioassay Trichoderma and Gliocladium. In: Trichoderma and Gliocladium
studies were due to lovastatin activity, the crude extract was subjected Basic Biology Taxonomy and Genetics. Vol. 1. London, UK: Taylor &
to HPLC for further confirmation and estimation of lovastatin. Analysis Francis Group; 1998. p. 139-91.
of lovastatin in HPLC is carried out in the form of β-hydroxyacid form. 2. Endo A. Monacolin K. A new hypocholesterolemic agent produced by
a Monascus species. J Antibiot (Tokyo) 1979;32(8):852-4.
Because the β-hydroxyacid form elutes earlier in the chromatographic
3. Negishi S, Huang ZC, Hasumi K, Murakawa S. Productivity of
column [44,45]. And also, the fermentation liquor chiefly contains monacolin K (mevinolin) in the genus Monascus. J Ferment Eng
lovastatin in the form of β-hydroxyacid. The chromatogram reveals 1986;64:509-11.
that RT of standard lovastatin and sample was 25.1 minutes and 4. Manzoni M, Rollini M, Bergomi S, Cavazzoni V. Production and
25.1 minutes, respectively. The concentration of crude extract of purification of statins from Aspergillus terreus strains. Biotechnol
lovastatin was found to be 0.33 mg/g. Therefore, on the basis of results Technol 1998;12(7):529-32.
observed on the bioassay plates and HPLC, it was considered that the 5. Alberts AW, Chen J, Kuron G, Hunt V, Huff J, Hoffman C,
strain A. flavus as a potential lovastatin producing candidate for further et al. Mevinolin: A highly potent competitive inhibitor of
studies. In accordance with our results, A. flavus was considered as the hydroxymethylglutaryl-coenzyme a reeducates and a cholesterol-
best producer of lovastatin in addition to A. terreus and other fungi lowering agent. Proc Natl Acad Sci U S A 1980;77(7):3957-61.
6. Novak N, Gerdin S, Berovic M. Increased lovastatin formation by
[5,46-48]. Similar results have been reported by other investigators for
Aspergillus terreus using repeated fed-batch process. Biotechnol Lett
different fungal species [49-51]. The purity of the lovastatin extracted 1997;19(10):947-8.
could be enhanced through column chromatography, which employs 7. Szakács G, Morovján G, Tengerdy RP. Production of lovastatin by a
silica gel as the stationary phase and an eluent system comprising wild strain of Aspergillus terreus. Biotechnol Lett 1998;20(4):411-5.
benzene and acetonitrile. Similar to our results, Nidhiya et al. (2012) 8. Rodriguez Porcel EM, Casas Lopez JL, Sanchez Perez JA, Chisti Y.
reported that purification of lovastatin was carried using silica gel- Enhanced production of lovastatin in a bubble column by Aspergillus

261
 Janani et al.
Asian J Pharm Clin Res, Vol 10, Issue 10, 2017, 258-262

terreus using a two-stage feeding strategy. J Chem Technol Biotechnol 34. Keller NP, Turner G, Bennett JW. Fungal secondary metabolism-from
2007;82(1):58-64. biochemistry to genomics. Nat Rev Microbiol 2005;3(12):937-47.
9. Bizukojc M, Ledakowicz S. A macro kinetic modeling of the biosynthesis 35. Prabhakar A, Krishnaiah K, Janaun J, Bono A. An overview of
of lovastatin by Aspergillus terreus. J Biotechnol 2007;130(4):422-35. engineering aspects of solid state fermentation. Malays J Microbiol
10. Jia Z, Zhang X, Zhao Y, Cao X. Enhancement of lovastatin production 2005;1(2):10-6.
by supplementing polyketide antibiotics to the submerged culture of 36. Barrios-González J, Baños JG, Covarrubias AA, Garay-Arroyo A.
Aspergillus terreus. Appl Biochem Biotechnol 2010;160(7):2014-25. Lovastatin biosynthetic genes of Aspergillus terreus are expressed
11. Upendra RS, Khandelwal P. Physical mutagenesis based strain differentially in solid-state and in liquid submerged fermentation. Appl
improvement of Aspergillus sp. For enhanced production of lovastatin. Microbiol Biotechnol 2008;79(2):179-86.
Int J Pharm Pharmsci 2016;8(7):163-7. 37. Latha DP, Hemalatha KP. Production of lovastatin by Aspergillus
12. Lopez JC, Pérez JS, Sevilla JF, Fernández FA, Grima EM, Chisti Y. flavipes NCIM 1209 using rice husk under solid state fermentation. Int
Production of lovastatin by Aspergillus terreus: Effects of the C: J Adv Res Eng Appl Sci 2015;4(9):12-23.
N ratio and the principal nutrients on growth and metabolite production. 38. Patil RH, Krishnan P, Maheshwari VL. Production of lovastatin by wild
Enzyme Microb Technol 2003;33(2):270-7. strains of Aspergillus terreus. Nat Prod Commun 2011;6(2):183-6.
13. Radha KV, Lakshmanan D. A review: Lovastatin production and 39. Wei PL, Xu ZN, Cen PL. Lovastatin production by Aspergillus terreus
applications. Asian J Pharm Clin Res 2013;6(3):21-6. in solid-state fermentation. J Zhejiang Univ Sci A 2007;8(9):1521-6.
14. Balakrishnan K, Pandey A. Production of biologically active 40. Baranova NA, Kreiner VG, Egorov NS. The growth of Rhodotorula
secondary metabolites in solid state fermentation. J Sci Ind Res rubra yeasts and their synthesis of ergo sterol on media with lovastatin.
1996;55(5-6):365-72. Antibiot Khimioter 1996;41(11):3-6.
15. Robinson T, Singh D, Nigam P. Solid-state fermentation: A promising 41. Nigam VK. Screening of different fungi for production of lovastatin.
microbial technology for secondary metabolite production. Appl AJBPS 2015;5(44):24.
Microbiol Biotechnol 2001;55(3):284-9. 42. Lakshmanan D, Radha KV. An effective quantitative estimation of
16. Hölker U, Lenz J. Solid-state fermentation. are there any lovastatin from Pleurotus ostreatus using UV and HPLC. Int J Pharm
biotechnological advantages? Curr Opin Microbiol 2005;8(3):301-6. Pharmsci 2012;4(4):462-4.
17. Valera HR, Gomes J, Lakshmi S, Gururaja R, Suryanarayan S, 43. Ganiswara SG, Rianto S, Frans DS, Purwantyastuti. Farmakoligidan
Kumar D. Lovastatin production by solid state fermentation using Tearpi. 5th ed., Vol. 10. Jakarta: Bagain Farmakologi Kedokteram, UI;
Aspergillus flavipes. Enzyme Microb Technol 2005;37(5):521-6. 1985. p. 643.
18. Xu BJ, Wang QJ, Jia XQ, Sung CK. Enhanced lovastatin production by 44. Friedrich J, Žužek M, Benčina M, Cimerman A, Štrancar A, Radež I.
solid state fermentation of Monascus ruber. Biotechnol Bioprocess Eng High-performance liquid chromatographic analysis of mevinolin
2005;10(1):78-84. as mevinolinic acid in fermentation broths. J Chromatogr A
19. Barrios-González J, Mejía A. Production of secondary metabolites by 1995;704(2):363-7.
solid-state fermentation. Biotechnol Annu Rev 1996;2:85-121. 45. Morovján G, Szakács G, Fekete J. Monitoring of selected
20. Thomas L, Larroche C, Pandey A. Current developments in solid-state metabolites and biotransformation products from fermentation
fermentation. Biochem Eng J 2013;15(81):146-61. broths by high-performance liquid chromatography. J Chromatogr A
21. Blais L, Desgagné A, LeLorier J. 3-Hydroxy-3-methylglutaryl 1997;763(1-2):165-72.
coenzyme a reeducates inhibitors and the risk of cancer: A nested case- 46. Javed S, Meraj M, Mahmood S, Hameed A, Naz F, Hassan S, et al.
control study. Arch Intern Med 2000;160(15):2363-8. Biosynthesis of lovastatin using agro-industrial wastes as carrier
22. Lewis KA, Holstein SA, Hohl RJ. Lovastatin alters the isoprenoid substrates. Trop J Pharm Res 2017;16(2):263-9.
biosynthetic pathway in acute myelogenous leukemia cells in vivo. 47. Mangunwardoyo W, Rafliyanti Y, Kusmana D. Bioprospection of
Leuk Res 2005;29(5):527-33. lovastatin in Aspergillus spp. from university of Indonesia culture
23. Alexopoulos CJ, Mims CN, Blackwell M. Introductory Mycology. collection (UICC). World Appl Sci J 2012;16(2):183-8.
New York, USA: John Willey & Sons Inc., Google Scholar; 1996. 48. Osman ME, Khattab OH, Zaghlol GM, El-Hameed RA. Screening for
24. Hajjaj H, Niederberger P, Duboc P. Lovastatin biosynthesis by the production of cholesterol lowering drugs (lovastatin) by some fungi.
Aspergillus terreus in a chemically defined medium. Appl Environ Aust J Basic Appl Sci 2011;5(6):698-703.
Microbiol 2001;67(6):2596-602. 49. Samiee SM, Moazami N, Haghighi S, Aziz-Mohseni F, Mirdamadi S,
25. Kumar MS, Kumar PM, Sarnaik HM, Sadhukhan AK. A rapid technique Bakhtiari MR. Screening of lovastatin production by filamentous fungi.
for screening of lovastatin-producing strains of Aspergillus terreus Iran Biomed J 2003;7(1):29-33.
by agar plug and Neurospora crassa bioassay. J Microbiol Methods 50. Gunde-Cimerman N, Friedrich J, Cimerman A, Benički N. Screening
2000;40(1):99-104. fungi for the production of an inhibitor of HMG CoA reductase:
26. Vilches-Ferrón MA, Casas-López JL, Sánchez-Pérez JA, Production of mevinolin by the fungi of the genus Pleurotus. FEMS
Fernández-Sevilla JM, Chisti Y. Rapid screening of Aspergillus terreus Microbiol Lett 1993;111(2-3):203-6.
mutants for overproduction of lovastatin. World J Microb Biotechnol 51. Nidhiya K, Sathya E, Nitya M. Extraction and purification of lovastatin
2005;21(2):123-5. from non-aflatoxigenic strains of Aspergillus flavus. Int J Biol Pharm
27. Babu RH, Rupa A, Radha S, Prasad NB, Narasimha G. Screening of Res 2012;4:916-21.
lovastatin producing fungi by yeast growth inhibition assay method. 52. Pansuriya RC, Singhal RS. Response surface methodology for
J Pharm Res 2011;4(9):2967-8. optimization of production of lovastatin by solid state fermentation.
28. Nazzal S, Smalyukh II, Lavrentovich OD, Khan MA. Preparation Braz J Microbiol 2010;41(1):164-72.
and in vitro characterization of a eutectic based semisolid self-Nano 53. Alarcón J, Aguila S, Arancibia-Avila P, Fuentes O, Zamorano-Ponce E,
emulsified drug delivery system (SNEDDS) of ubiquinone: Mechanism Hernández M. Production and purification of statins from Pleurotus
and progress of emulsion formation. Int J Pharm 2002;235:247-65. ostreatus (Basidiomycetes) strains. Z Naturforsch C 2003;58(1-2):62-4.
29. Ghasemi-Mobarakeh L, Morshed M, Karbalaie K, Fesharaki M, 54. Patel RP, Patel MM. Preparation and evaluation of inclusion complex
Nasr-Esfahani MH, Baharvand H. Electrospun poly (ε-caprolactone) of the lipid lowering drug lovastatin with?-cyclodextrin. Dhaka Univ J
nanofiber mat as extracellular matrix. Yakhteh 2008;10(3):179-84. Pharm Sci 2007;6(1):25-36.
30. Seenivasan A, Subhagar S, Aravindan R, Viruthagiri T. Microbial 55. Suffness M, Pezzuto JM. Assays for cytotoxicity and antitumor activity.
production and biomedical applications of lovastatin. Indian J Pharm Methods Plant Biochem 1991;6:71-133.
Sci 2008;70(6):701-9. 56. Schmidmaier R, Simsek M, Baumann P, Emmerich B, Meinhardt G.
31. Raper KB, Fennell DI. The Genus Aspergillus. Baltimore USA: Synergistic antimyeloma effects of zoledronate and simvastatin.
Williams & Williams Company, Baltimore Google Scholar; 1965. Anticancer Drugs 2006;17(6):621-9.
32. Klich MA. Identification of Common Aspergillus Species. Utrecht, 57. Wong WW, Tan MM, Xia Z, Dimitroulakos J, Minden MD, Penn LZ.
Netherlands: Centraalbureau Voor Schimmelcultures; 2002. Cerivastatin triggers tumor-specific apoptosis with higher efficacy than
33. Gilman J. A manual of soil fungi. Soil Sci 1957;84(2):183. lovastatin. Clin Cancer Res 2001;7(7):2067-75.

262