CORDYCEPS MILITARIS, A NOVEL BIO-LARVICIDE AGAINST DENGUE AND MALARIA VECTORS

Abstract

The invention relates to a novel bio-larvicide Cordyceps militaris, an entomopathogenic fungus. The Cordyceps militaris, acts as a bio-larvicide and is used for controlling the vector of dengue and malaria. Wherein¸ the Cordyceps militaris has 100 % larvae mortality in a concentration range of 0.25% to 1%. The invention also provides process for preparation of bio-larvicide.

Specification

Description:FIELD OF THE INVENTION:
The present invention relates to the field of biotechnology. Particularly, the invention provides a novel bio-larvicide Cordyceps militaris, an entomopathogenic fungus. In particular, Cordyceps militaris, acts as a bio-larvicide and is used for controlling the vector of dengue and malaria. The invention also provides process for preparation of bio-larvicide.

BACKGROUND OF THE INVENTION:
Dengue virus (DENV) infection is one of the most rapidly spreading arboviral diseases of humans in the world. It is a major health problem among those dwelling in tropical and subtropical areas, and it is spreading globally, with approximately 129 countries now thought to be DENV-endemic areas. The incidence of DENV infection continues to increase. An estimated 100-400 million people are infected annually, and 96 million people require medical care. There is currently no specific drug to treat DENV infection. Supportive treatment is the only treatment option for hospitalized DENV-infected patients. Attempts to develop an effective dengue vaccine have been ongoing for decades. Although the availability of a DENV vaccine is necessary for the prevention and control of viral infection, anti-DENV drugs for the treatment of DENV infection are also important for preventing disease progression, for reducing disease severity, and for interrupting the spread of the virus.

Biological resources in and from nature harbor provide large and diverse assortments of bioactive compounds. Natural compounds from herbs and medicinal plants are recognized for their safety and effectiveness, and many of them have been used as traditional medicines by people from many countries around the world. Since these natural compounds often contain a broad spectrum of biological activities, they have the potential to be further purified and developed into drugs for the treatment of many human disease conditions. Cordyceps (ascomycete) fungus has been used in Chinese medicine since ancient times. The pharmacological properties of Cordyceps extract have been studied in both infectious and non-infectious diseases. Cordycepin (3'-deoxyadenosine) is an adenosine derivative, and a major bioactive compound in Cordyceps sinensis and Cordyceps militaris. This compound has been previously reported to have antiviral activity.

Cordyceps species are an entomopathogenic fungi widely used in traditional Chinese medicine. It has been reported that Cordyceps species possessed antioxidant/antiaging, antimicrobial, immunomodulatory, anti-inflammatory and antitumor effects. Cordycepin as the major constituent of Cordyceps militaris, detected from fruiting bodies, and is a derivative of the nucleoside adenosine. This molecule is being produced industrially because of its insecticidal, antibacterial and antitumor effects. Impregnated with the molecules of immense pharmacological potentials (cordycepin, cordycepic acid, adenosine polysaccharides). Among other products isolated from Cordyceps militaris are polysaccharides and ergosterol with a number of biological effects: antioxidant, antiinflammatory, antimetastatic, antitumor, immunomodulatory, steroidogenic, hypoglycaemic and hypolipidaemic. Polysaccharides extracted from fruiting bodies of Cordyceps militaris showed immunostimulating properties and stopped growth of melanoma cells. Some of the prior art discussing the antiviral activity of Cordyceps (ascomycete) fungus have been listed below.

Panya A et al. Cordycepin Inhibits Virus Replication in Dengue Virus-Infected Vero Cells. Molecules (2021);26(11);3118 illustrates Cordycepin (30 -deoxyadenosine), which is a major bioactive compound in Cordyceps (ascomycete) fungus that has been used for centuries in Chinese traditional medicine, was reported to exhibit antiviral activity. However, the anti-larval activity of Cordycepin against mosquito vectors of dengue and malaria is unknown.

Ghosh et al., Plant extracts as potential mosquito larvicides. The Indian Journal of Medical Research (2012); 135(5); 581-598 illustrates application of active toxic agents from plant extracts as an alternative mosquito control strategy. These are non-toxic, easily available at affordable prices, biodegradable and show broad-spectrum target-specific activities against different species of vector mosquitoes. In this article, the current state of knowledge on phytochemical sources and mosquitocidal activity, their mechanism of action on target population, variation of their larvicidal activity according to mosquito species, instar specificity, polarity of solvents used during extraction, nature of active ingredient and promising advances made in biological control of mosquitoes by plant derived secondary metabolites have been reviewed.

The synthetic larvicides against mosquito nuisance and transmitted vector borne diseases are expensive, cause environment toxicity and in long term led to development of insecticide resistance during disease transmitting vector species. Moreover, the prior art does not report the antilarval activity of Cordycepin against mosquito vectors of dengue and malaria. Hence, there is a need to develop an affordable water soluble and easily available bio-larvicidal preparation.
OBJECTIVE OF THE INVENTION:
It is a main objective of the present invention is to provide a bio-larvicide from Cordyceps militaris against dengue and malaria vectors, that does not impart toxicity into the solvent.

It is another objective of the present invention is to provide a process for the preparation of bio-larvicide from Cordyceps militaris.

It is another objective of the present invention is to provide a process for preparation of bio-larvicide Cordyceps militaris from bio-foods.

SUMMARY OF THE INVENTION:
In an embodiment, the present invention provides a bio-larvicide for a vector of dengue and malaria, wherein the bio-larvicide is Cordyceps militaris.

In an embodiment of the present invention, there is provided a bio-larvicide, wherein the vector of dengue and malaria is selected from Aedes aegypti mosquito, Anopheles mosquito and culex mosquito.

In an embodiment of the present invention, there is provided a bio-larvicide, wherein the bio-larvicide has 100 % larvae mortality in a concentration range of 0.25% to 1% of the aqueous extract.

In another embodiment, the present invention provides a process for preparation of a bio-larvicide from Cordyceps militaris, the process comprising:
incubating a mother culture Cordyceps militaris in a growth medium under a dark condition at 22? for 30 days;
producing a spawn in a first liquid medium followed by its incubation with continuous shaking at 120 rpm for 10-15 days in dark at 22 ?;
inoculating the spawn culture on a substrate medium containing a substrate in a second liquid medium;
incubating the spawn first under dark conditions for 15 days and then in light (2000-3000 Lux) at 22? to obtain a fruiting bodies of Cordyceps militaris; and
preparing an aqueous extract from fruiting bodies of Cordyceps militaris, wherein the aqueous extract has a concentration in the range of 0.1% to 1.0%.
In an embodiment of the present invention, there is provided a process, wherein the growth medium is selected from potato dextrose agar (PDA), malt extract agar (MEA) and yeast extract agar (YEA).

In an embodiment of the present invention, there is provided a process, wherein the first liquid medium comprises potato dextrose broth (24 gm/L), peptone (5 gm/L), yeast extract (3 gm/L), KH2PO4 (1 gm/L), MgSO4 (0.5 gm/L) and vitamin B1(50 mg/L).

In an embodiment of the present invention, there is provided a process, wherein the substrate is selected from brown rice, wheat and corn.

In an embodiment of the present invention, there is provided a process, wherein the second liquid medium comprises glucose (30 gm/L), peptone (5 gm/L), yeast extract (3 gm /L), KH2PO4 (1 gm/L), tri ammonium citrate (1 gm/L), MgSO4 (0.5 gm/L), vitamin B1 (15 mg/L) and vitamin B12 (10 mg/L).

In an embodiment of the present invention, there is provided a process, wherein the bio-larvicide is used for controlling the vector of dengue and malaria.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

Figure 1 illustrates the schematic representation of various steps to prepare the bio-larvicide for a vector of dengue and malaria, wherein a.) represents Master cultures, b.) represents Mother culture, c.) represents spawn and d.) represents fruiting bodies of Cordyceps militaris.

Figure 2 illustrates the pictorial representation of larvicidal activity of aqueous extract of Cordyceps militaris on larvae of Aedes aegypti mosquito, Anopheles mosquito and Culex mosquito

DETAILED DESCRIPTION OF THE INVENTION:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully interpreted and comprehended. However, any skilled person or artisan will appreciate the extent to which such embodiments could be generalized in practice.

It is further to be understood that all terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting in any manner the scope.

Unless defined otherwise, all technical and scientific expressions used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertains.

In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below which are known in the state of art.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Also, the term 'composition' does not limit the scope of the invention for multiple compositions that can be illustrated for best mode of the invention.

All modifications and substitutions that come within the meaning of the description and the range of their legal equivalents are to be embraced within their scope. A description using the transition "comprising" allows the inclusion of other elements to be within the scope of the invention.

In the present invention, the anti-larval potential of Cordycepin, a major bioactive component of Cordyceps militaris at very low concentration have been disclosed. Also, the fungal larvicide has no possible toxic effects. Cordyceps militaris also known as caterpillar fungus have the larvicidal activity against the vector of dengue and malaria such as Aedes aegypti mosquito, Anopheles mosquito and culex mosquito. The fungus forms 1-8 centimetres high, club-shaped and orange/red fruiting bodies, which grow out of dead underground pupae. The club is covered with the stroma, into which the actual fruit bodies, the perithecia, are inserted. The surface appears roughly punctured. The inner fungal tissue is whitish to pale orange.

In an aspect, the present invention provides a bio-larvicide for a vector of dengue and malaria, wherein the bio-larvicide is Cordyceps militaris.

In an embodiment of the present invention, there is provided a bio-larvicide, wherein the vector of dengue and malaria is selected from Aedes aegypti mosquito, Anopheles mosquito and Culex mosquito.

In an embodiment of the present invention, there is provided a bio-larvicide, wherein the bio-larvicide has 100 % larvae mortality in a concentration range of 0.25% to 1% of the aqueous extract.

In another aspect, the present invention provides a process for preparation of a bio-larvicide from Cordyceps militaris, the process comprising:
incubating a mother culture Cordyceps militaris in a growth medium under a dark condition at 22? for 30 days;
producing a spawn in a first liquid medium followed by its incubation with continuous shaking at 120 rpm for 10-15 days in dark at 22 ?;
inoculating the spawn culture on a substrate medium containing a substrate in a second liquid medium;
incubating the spawn first under dark conditions for 15 days and then in light (2000-3000 Lux) at 22? to obtain a fruiting bodies of Cordyceps militaris; and
preparing an aqueous extract from the fruiting bodies of Cordyceps militaris, wherein the aqueous extract has a concentration in the range of 0.1% to 1.0%.

In an embodiment of the present invention, there is provided a process, wherein the growth medium is selected from potato dextrose agar (PDA), malt extract agar (MEA) and yeast extract agar (YEA).

In an embodiment of the present invention, there is provided a process, wherein the first liquid medium comprises potato dextrose broth (24 gm/L), peptone (5 gm/L), yeast extract (3 gm/L), KH2PO4 (1 gm/L), MgSO4 (0.5 gm/L) and vitamin B1 (50 mg/L).

In an embodiment of the present invention, there is provided a process, wherein the substrate is selected from brown rice, wheat and corn.
In an embodiment of the present invention, there is provided a process, wherein the second liquid medium comprises glucose (30 gm/L), peptone (5 gm/L), yeast extract (3 gm /L), KH2PO4 (1 gm/L), tri ammonium citrate (1 gm/L), MgSO4 (0.5 gm/L), vitamin B1 (15 mg/L) and vitamin B12 (10 mg/L).

In an embodiment of the present invention, there is provided a process, wherein the bio-larvicide is used for controlling the vector of dengue and malaria.

EXAMPLES:
Having described the basic aspects of the present invention, the following non-limiting examples illustrate specific embodiments thereof. Those skilled in the art will appreciate that many modifications may be made in the invention without changing the essence of invention.

Example 1. Preparation of fruiting bodies of Cordyceps militaris.
Master cultures of Cordyceps militaris C-1164 were provided by JAM BIOFOODS, Bahadurgarh, Haryana, India. Master cultures were further multiplied on potato dextrose agar (PDA) medium in test tubes under dark at 22?. After incubation of mother cultures for 30 days. Spawn was produced in liquid medium containing potato dextrose broth (24 gm/L), peptone (5 gm/L), yeast extract (3 gm/L), KH2PO4 (1 gm/L), MgSO4 (0.5 gm/L) and vitamin B1 (50 mg/L) followed by its incubation with continuous shaking at 120 rpm for 10-15 days in dark at 22?. Spawn cultures were inoculated on substrate medium containing Brown Rice 25 gm + 50 ml liquid medium in each culture bottle. The composition of liquid medium was glucose (30 gm/L), peptone (5 gm/L), yeast extract (3 gm /L), KH2PO4 (1 gm/L), tri ammonium citrate (1 gm/L), MgSO4 (0.5 gm/L), vitamin B1 (15 mg/L) and vitamin B12 (10mg/L). The fruiting bodies were developed by incubating the spawn first under dark conditions for 15 days and then were transfer in light (2000-3000 Lux) at 22?. Fruiting bodies of 3-8 centimeters were developed in 45 days (Figure 1).

Example 2. Extracts preparation of Cordyceps militaris for larvicidal activities.
Aqueous extracts of fresh fruiting bodies of Cordyceps militaris were prepared in the range of 0.1%, 0.25%, 0.5%, 0.75% and 1.0% and first stage larvae of aedes, anopheles and culex mosquito were transferred in different extracts. To feed the larvae during the experiment 0.1% glucose was added to each extract. Control of each experiment was taken. Larval mortality was observed in the interval of eight hours (Figure 2) (Table 1, Table 2 and Table 3).

Larvicidal activity of Cordyceps militaris.

Table 1. Larvicidal activity of aqueous extract of Cordyceps militaris on 1st stage larvae of Aedes aegypti mosquito.

Aqueous
extract
(%) Larval mortality (%) in aqueous extract of Standard Fruiting body
8 hours 16 hours 24 hours 32 hours 40 hours 48 hours
Control 0.0 0.0 0.0 0.0 0.0 0.0
0.1 0.0 0.0 20 40 80 80
0.25 40 80 80 100 100 100
0.5 60 100 100 100 100 100
0.75 60 100 100 100 100 100
1.0 80 100 100 100 100 100


Table 2. Larvicidal activity of aqueous extracts of Cordyceps militaris on 1st stage larvae of Anopheles mosquito.

Aqueous extract (%) Larval mortality (%) in aqueous extract of Standard Fruiting body
8 hours 16 hours 24 hours 32 hours 40 hours 48 hours
Control 0.0 0.0 0.0 0.0 0.0 0.0
0.1 20 40 40 40 60 80
0.25 20 40 40 60 60 80
0.5 20 60 100 100 100 100
0.75 40 80 100 100 100 100
1.0 40 80 100 100 100 100


Table 3. Larvicidal activity of aqueous extract of Cordyceps militaris on 1st stage larvae of culex mosquito.

Aqueous extract
(%) Larval mortality (%) in aqueous extract of Fruiting body
8 hours 16 hours 24 hours 32 hours 40 hours 48 hours
Control 0.0 0.0 0.0 0.0 0.0 0.0
0.1 0.0 0.0 0.0 0.0 0.0 0.0
0.25 20 20 40 80 100 100
0.5 20 40 80 80 100 100
0.75 20 40 80 100 100 100
1.0 20 40 80 100 100 100

Advantages of the present invention:
• The formulation targets mosquito larvae specifically, reducing the population of adult mosquitoes and consequently lowering the transmission of diseases such as malaria, dengue fever, and Zika virus.
• Being derived from a natural source, it is generally safer for humans and domestic animals, reducing health risks associated with chemical treatments.
• This larvicide can be integrated into broader pest management strategies, combining biological control with other methods for more comprehensive and effective mosquito control.
• By controlling mosquito population more effectively and sustainably, the formulation contributes to reduction in mosquito-borne diseases, and thus leads to improved public health outcomes.
• Cordyceps militaris can be cultivated under controlled conditions, making the production of the Bio-larvicide more sustainable from natural resources.
, Claims:1. A bio-larvicide for a vector of dengue and malaria, wherein the bio-larvicide is Cordyceps militaris.

2. The bio-larvicide as claimed in claim 1, wherein the vector of dengue and malaria is selected from aedes Aegypti mosquito, Anopheles mosquito and Culex mosquito.

3. The bio-larvicide as claimed in claim 1, wherein the bio-larvicide has 100 % larvae mortality in a concentration range of 0.25% to 1% of the aqueous extract.

4. A process for preparation of a bio-larvicide from Cordyceps militaris, the process comprising:
incubating a mother culture of Cordyceps militaris in a growth medium under a dark condition at 22? for 30 days;
producing a spawn in a first liquid medium followed by its incubation with continuous shaking at 120 rpm for 10-15 days in dark at 22 ?;
inoculating the spawn culture on a substrate medium containing a substrate in a second liquid medium;
incubating the spawn first under dark conditions for 15 days and then in light (2000-3000 Lux) at 22? to obtain a fruiting bodies of Cordyceps militaris; and
preparing an aqueous extract from fruiting bodies of Cordyceps militaris, wherein the aqueous extract has a concentration in the range of 0.1% to 1.0%.

5. The process as claimed in claim 4, wherein the growth medium is selected from potato dextrose agar (PDA), malt extract agar (MEA) and yeast extract agar (YEA).

6. The process as claimed in claim 4, wherein the first liquid medium comprises potato dextrose broth (24 gm/L), peptone (5 gm/L), yeast extract (3 gm/L), KH2PO4 (1 gm/L), MgSO4 (0.5 gm/L) and Vitamin B1 (50 mg/L).

7. The process as claimed in claim 4, wherein the substrate is selected from brown rice, wheat and corn.


8. The process as claimed in claim 4, wherein the second liquid medium comprises glucose (30 gm/L), peptone (5 gm/L), yeast extract (3 gm /L), KH2PO4 (1 gm/L), tri ammonium citrate (1 gm/L), MgSO4 (0.5 gm/L), vitamin B1 (15 mg/L) and vitamin B12 (10 mg/L).

9. The process as claimed in claim 4, wherein the bio-larvicide is used for controlling the vector of dengue and malaria.

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