ISOLATION AND CHARACTERIZATION OF BIOACTIVE METABOLITES

Abstract

Endophytes are the microorganisms present in the interior tissues of plants and show a mutualistic, symbiotic and trophobiotic relationship. Endophytes are considered to be a good alternative source for production of bioactive secondary metabolites. In the present study, an attempt was made to isolate secondary metabolites from the previously reported endophytic fungi Aspergillus tubingensis strain Cs/7/2 isolated from leaves of Andrographis paniculata. Two known bioactive compounds, andrographolide (1) and quercetin (2) were isolated from n butanol fraction of Aspergillus tubingensis strain Cs/7/2. A spectroscopic analysis, such as FT- IR, mass spectra (MS) and Nuclear Magnetic Resonance (NMR) were compared with reported data to elucidate the chemical structures of isolated compounds. This is the first report on isolation of these compounds from endophytic fungus of Andrographis paniculata. Thus, it can be concluded that, Aspergillus tubingensis strain Cs/7/2 can be favorable start for the production these constituents.

Specification

Description:FIELD OF THE INVENTION
The present invention relates to the field of biotechnology and pharmaceuticals,
specifically to the isolation and characterization of bioactive secondary metabolites from
endophytic fungi. More particularly, this invention focuses on isolating biologically active
compounds, including andrographolide and quercetin, from the endophytic fungus
Aspergillus tubingensis strain CS/7/2, found in the leaves of Andrographis paniculata.
These compounds have potential applications in the development of pharmaceutical agents
with therapeutic benefits.
BACKGROUND OF THE INVENTION
Endophytic fungi are microorganisms that reside within the tissues of plants without
causing harm to their host. These fungi play an important role in symbiotic relationships
with plants, contributing to plant growth, stress resilience, and the synthesis of bioactive
compounds. Recent research has identified endophytic fungi as promising alternative
sources for the production of secondary metabolites with pharmaceutical relevance. These
secondary metabolites have shown pharmacological activities, including anticancer,
antimicrobial, and anti-inflammatory properties, making them valuable in drug discovery
and development.
Andrographis paniculata, a well-known medicinal plant, has long been used in traditional
medicine to treat conditions like fever, respiratory infections, and liver disorders. Previous
studies have attributed its medicinal properties to compounds such as andrographolide and
flavonoids. While these compounds have been extensively studied in the plant itself, there
has been no reported literature on isolating these bioactive constituents from its endophytic
fungi.
This invention presents a novel approach to isolating andrographolide and quercetin from
the endophytic fungus Aspergillus tubingensis strain CS/7/2, derived from Andrographis
paniculata leaves. This discovery not only broadens the understanding of the biosynthetic
capabilities of endophytes but also highlights their potential as a sustainable source of
pharmacologically active compounds. This is the first known report on the isolation of
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these compounds from an endophyte of A. paniculata, opening avenues for further research
and applications in the pharmaceutical industry.
SUMMARY OF THE INVENTION
The invention provides a method for isolating and characterizing bioactive secondary
metabolites from the endophytic fungus Aspergillus tubingensis strain CS/7/2, which was
isolated from the leaves of the medicinal plant Andrographis paniculata. Specifically, the
invention discloses the successful extraction and identification of two known bioactive
compounds, andrographolide and quercetin, from the n-butanol fraction of this fungal
strain.
The isolated compounds were identified and confirmed through advanced spectroscopic
techniques, including FT-IR, mass spectrometry (MS), and nuclear magnetic resonance
(NMR), with their chemical structures validated against existing data. This marks the first
reported isolation of these bioactive compounds from an endophytic fungus of A.
paniculata, demonstrating that Aspergillus tubingensis strain CS/7/2 can serve as a
sustainable and viable source for producing pharmacologically active compounds
traditionally associated with the plant itself.
This invention contributes to biotechnological and pharmaceutical advancements by
providing a new method to harness endophytic fungi as an alternative source of valuable
bioactive compounds, with potential applications in drug development and therapeutic
treatments.
DETAILED DESCRIPTION OF THE INVENTION
The following is a detailed description of embodiments of the present disclosure. The
embodiments are in such detail as to clearly communicate the disclosure. However, the
amount of detail offered is not intended to limit the anticipated variations of embodiments;
on the contrary, the intention is to cover all modifications, equivalents, and alternatives
falling within the spirit and scope of the present disclosure.
Unless the context requires otherwise, throughout the specification which follow, the word
"comprise" and variations thereof, such as, "comprises" and "comprising" are to be
construed in an open, inclusive sense that is as "including, but not limited to."
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Orientation throughout this specification to "one embodiment" or "an embodiment" means
that a particular feature, structure or characteristic described in connection with the
embodiment is included in at least one embodiment. Thus, the appearances of the phrases
"in one embodiment" or "in an embodiment" in various places throughout this specification
are not necessarily all referring to the same embodiment. Furthermore, the particular
features, structures, or characteristics may be combined in any suitable manner in one or
more embodiments.
The headings and abstract of the invention provided herein are for convenience only and
do not interpret the scope or meaning of the embodiments.
The invention provides a comprehensive and detailed method for isolating, extracting, and
characterizing bioactive secondary metabolites from the endophytic fungus Aspergillus
tubingensis, specifically the CS/7/2 strain. This strain was meticulously isolated from the
leaves of the medicinal plant Andrographis paniculata, a plant highly regarded for its
various therapeutic applications in traditional and modern medicine. The focus of this
invention lies in establishing a sustainable process that harnesses the biosynthetic potential
of the Aspergillus tubingensis strain to produce valuable bioactive compounds, specifically
targeting those with known medicinal properties.
Through controlled cultivation conditions, the invention provides an optimized process for
inducing and maximizing the production of bioactive secondary metabolites in the
Aspergillus tubingensis CS/7/2 strain. This process involves carefully regulated
parameters, including temperature, pH, nutrient composition, and growth duration, which
have been tailored to stimulate the biosynthesis of target metabolites that are chemically
similar to or mimic those found in Andrographis paniculata.
The extraction process begins with the preparation of the fungal biomass, which is
subjected to solvent extraction techniques using a specific n-butanol fraction. The choice
of n-butanol is particularly advantageous due to its selective extraction capability for
bioactive secondary metabolites while minimizing the co-extraction of non-essential
components. This targeted extraction technique enables a high yield of bioactive
compounds from the fungal biomass and provides a basis for further purification steps.
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Following extraction, the invention discloses the successful isolation and identification of
two prominent bioactive compounds: andrographolide and quercetin. Andrographolide, a
diterpene lactone, is one of the primary bioactive compounds found in Andrographis
paniculata and is well-recognized for its anti-inflammatory, antioxidant, and immuneboosting
properties. Quercetin, a flavonoid, is known for its potent antioxidant effects, as
well as its anti-inflammatory and anti-cancer properties. The presence of these compounds
within the Aspergillus tubingensis strain demonstrates the potential of endophytic fungi as
a renewable source of valuable medicinal compounds.
The characterization process for these isolated compounds employs a combination of
advanced analytical techniques. High-performance liquid chromatography (HPLC) is used
to achieve separation and purification of the compounds, allowing for precise
quantification and ensuring high purity levels of andrographolide and quercetin. Mass
spectrometry (MS) confirms the molecular weight and structure, providing critical insights
into the molecular composition. Nuclear magnetic resonance (NMR) spectroscopy further
validates the molecular structure of each compound, offering a comprehensive profile that
confirms the identity and purity of andrographolide and quercetin.
In addition to these bioactive compounds, the invention provides insights into the
ecological and symbiotic role of Aspergillus tubingensis as an endophyte in Andrographis
paniculata. This symbiotic relationship suggests that the fungal strain may contribute to
the plant's bioactivity, enhancing the medicinal qualities for which Andrographis
paniculata is known. By isolating and identifying these metabolites from an endophytic
source, this invention highlights the potential for utilizing fungi as alternative producers of
plant-associated compounds, thus supporting biodiversity conservation and sustainable
production practices.
Identification of endophytic fungus
As reported previously, APLF-4 was isolated and characterized by PCR sequential
analysis.
Microscopic and molecular identification of the fungus belonged to the genus Aspergillus
sp. The homology analysis based on phylogenetic tree revealed best matching with
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Aspergillus tubingensis strain Cs/7/2.
Secondary metabolites from Aspergillus tubingensis
Two compounds were isolated from n butanol fraction of endophytic fungi Aspergillus
tubingensis strain Cs/7/2 namely APLFnB-1 and APLFnB-2. These compounds were
further subjected to IR, 1H NMR, 13C NMR, Mass spectral analysis.
Andrographolide (APLF-nB-1) Andrographolide was obtained as amorphous yellow
powder, MP: 2250C-2380 C, Rf value: 0.91; IR (KBR) cm-1:3373.98 cm-1(OH str),
2926.18 cm-1 (C-H str), 1654.47 cm-1(C=O str), 1455.03 cm-1(C-H bend); 1H NMR
(DMSO D6): (500MHZ); δ 6.56 (s, 1H, H-C-12), δ 6.19 (s, 1H, OH-C-14), δ4.86 (s, 1H,
H-C-14),δ 4.67 (s, 1H, OH-C-3), δ4.55 (s, 1H, OH-C-19),δ 4.47-4.49 (d, 2H, H-C-17),
δ4.21 (s, 2H, H-15),δ 3.12 (s, 2H, CH2-C-18), δ3.05 (t, 1H, CH-C-3),δ 2.98 (d, 2H, CHC-
7), δ2.29 (d, 2H, CH2-C-11),δ 1.78- 1.80 (d, 2H, CH2-C-2), δ1.41-1.43 (d, 2H, CH2-
C-1),δ 1.86 (s, 1H, CH-C-5), δ1.18-1.22 (s, 2H, CH2-C-6), δ 0.83 (s, 1H, CH-C-9), δ 0.81
(s, 3H, CH3-C-10); 13C NMR (DMSOD6) (100MHZ); δ 166.95 (C-16), δ 145.25 (C-12)
δ 141.11 (C-8), δ 139.75 (C-5), δ 128.83 (C-13),δ 108.30 (C- 17), δ 79.18 (C-3), δ 73.32
(C-15), δ 68.85 (C- 19), δ67.25 (C-14), δ 58.42 (C-9), δ 56.82 (C- 4), δ 43.01 (C-10), δ
25.90 (C-7), δ 23.83(C-11), δ 20.92 (C-2), δ 20.08 (C-6), δ 19.23 (C-1), δ 17.54 (C-18),
δ14.30 (C-20); Mass spectra; ESI- MS: Mass calculated, 350.46 gm/mol; Found: 351.20
[M+ +1] (Figure 1).
Fig.1: Chemical structure of Andrographolide
Quercetin (APLFnB-2)
Quercetin was obtained as amorphous yellow crystals, MP:182 °C, Rf value: 0.34; IR
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(KBR) cm- 1: 3253.91 cm-1 (OH str), 2926.01 cm-1 (C-H str), 1784.15 cm-1 (C=O str.),
1656.85 cm-1 (C=C str. in phenyl ring), 1454.33 cm-1(C-H bend), 1355.96 cm-1 (C-O str);
1H NMR (DMSO D6): (500MHZ); δ 5.27 (s, 2H, H-1, H-2), δ 6.84 (d, 3H, H-3, H-4, H-
5), δ 5.21 (s, 4H, H-6, H-7, H- 8, H-9), δ 12.66 (s, 1H, H-10); 13C NMR (DMSOD6)
(100MHZ); δ 134.17 (C-2), δ 157.26 (C-3),177.70 (C-4), δ 104.04 (C-5), δ 98.66 (C- 6), δ
164.17 (C-7), δ 98.66 (C-8), δ 161.26 (C- 9), δ 161.26 (C-10), δ 121.08 (C-1), δ 115.62 (C-
2), δ 148.39 (C-3)145.16 (C-4), δ 115.62 (C- 5), δ 120.71 (C-6). Mass spectra; ESI-MS:
Mass calculated: 303.06 gm/mol; Found: 304.06 [M++1]. Other peaks appeared at 433.26,
258.11, and 287.06 (Figure 2).
Fig.2:Chemical structure of Quercetin
In summary, this invention establishes a reliable, reproducible method for the isolation,
extraction, and characterization of bioactive compounds andrographolide and quercetin
from Aspergillus tubingensis strain CS/7/2. Through the use of specialized cultivation and
extraction techniques, coupled with sophisticated analytical methodologies, this invention
provides a sustainable alternative source for valuable medicinal compounds traditionally
obtained from Andrographis paniculata, with potential applications in pharmaceuticals,
nutraceuticals, and natural health products. , Claims:1. A process for obtaining bioactive secondary metabolites from the fungal strain
Aspergillus tubingensis CS/7/2, sourced from Andrographis paniculata leaves, the
process comprising:
a. Growing the Aspergillus tubingensis strain CS/7/2 under defined conditions
optimized for secondary metabolite production;
b. Performing an extraction of bioactive compounds from the fungal biomass using
n-butanol;
c. Purifying the extracted compounds to isolate andrographolide and quercetin.
2. The process of as claimed in claim 1, wherein the controlled cultivation conditions
involve specified parameters of temperature, pH, nutrient composition, and growth
period to enhance secondary metabolite yield.
3. The process of as claimed in claim 1, further comprising the step of verifying the
identity of isolated bioactive compounds using spectroscopic methods including
Fourier Transform Infrared Spectroscopy (FT-IR), Mass Spectrometry (MS), and
Nuclear Magnetic Resonance (NMR), confirming andrographolide and quercetin.
4. The process of as claimed in claim 1, wherein an isolated compound, specifically
andrographolide, produced by Aspergillus tubingensis strain CS/7/2
5. The process of as claimed in claim 1, wherein it characterized by at least one biological
activity selected from anti-inflammatory, antioxidant, antimicrobial, or anticancer
properties.
6. A formulation method for a pharmaceutical composition, the method involving:
a. Cultivating Aspergillus tubingensis strain CS/7/2 under tailored conditions;
b. Extracting and isolating the target bioactive compounds andrographolide and
quercetin;
c. Converting the isolated compounds into a pharmaceutically suitable form.

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