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Author(s): Srishti Verma1, Samay Tirkey2, Anushruti Satra3, Kamlesh Kumar Shukla*4

Email(s): 1srishtisipi@gmail.com, 2samay6789@gmail.com, 3anushrutisatra21@gmail.com, 4kshukla26@yahoo.co.in

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    1School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    2School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    3School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    4School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India
    *Corresponding Author Email- kshukla26@yahoo.co.in

Published In:   Volume - 4,      Issue - 1,     Year - 2022


Cite this article:
Srishti Verma, Samay Tirkey, Anushruti Satra, Kamlesh Kumar Shukla (2022) Secondary Metabolites Screening of Wild Mushrooms and Assessment of their In-Vitro Anti-Diabetic Property. NewBioWorld A Journal of Alumni Association of Biotechnology,4(1):28-34.

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 NewBioWorld A Journal of Alumni Association of Biotechnology (2022) 4(1):28-34            

RESEARCH ARTICLE

Secondary Metabolites Screening of Wild Mushrooms and Assessment of their In-Vitro Anti-Diabetic Property

Srishti Verma, Samay Tirkey, Anushruti Satra, Kamlesh Kumar Shukla*

 

School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur, Chhattisgarh, India

srishtisipi@gmail.com, samay6789@gmail.com, anushrutisatra21@gmail.com, kshukla26@yahoo.co.in

*Corresponding Author Email- kshukla26@yahoo.co.in

ARTICLE INFORMATION

 

ABSTRACT

Article history:

Received

07 May 2022

Received in revised form

17 June 2022

Accepted

25 June 2022

Keywords:

Secondary Metabolites;

Mushroom;

Anti-diabetic;

Blood Glucose

 

This study aimed to screen the secondary metabolites present in ten wild mushroom samples. All samples tested positive for phenols, flavonoids, and alkaloids, while six samples showed positive results for steroids and four for terpenoids. Phlobatannins were present in six samples, and glycosides were present in four. No anthraquinones were detected in any samples. In-vitro α-amylase inhibitory assay showed promising anti-diabetic potential, with inhibition ranging from 12.34 ± 0.50 to 93.86 ± 0.12% among the tested mushrooms. Samples 1M and 3M exhibited high inhibitory activity against α-amylase. These findings suggest that these wild mushrooms could be utilized to develop novel nutraceuticals with potential human health benefits.

 

 


Graphical Abstract:

Secondary Metabolites

Flavonoids

Phenols

Glycosides

Terpenoids

Anthraquinones

Steroids

Alkaloids

Antidiabetic


1.       Introduction

DOI: 10.52228/NBW-JAAB.2022-4-1-6

The world is facing numerous challenges to human health, including increasing populations, climate change, resource depletion, malnutrition, and pandemics. To address these issues proactively, identifying new sources of nutraceuticals is essential. Fungi, with an estimated number of over one million species, are an attractive option due to their diverse secondary metabolites that contribute to their survival in ecological niches (Boruta, 2018; Keller et al., 2005). These metabolites have demonstrated biological activity and show promise for drug development (Dias et al., 2012). Ascomycetes and basidiomycetes are the most prolific producers of secondary metabolites, with mushrooms offering the chance to investigate metabolites produced by both the sexual stage and vegetative mycelia. Mushrooms contain many bioactive components, including phenols, terpenoids, flavonoids, and more (Kumar et al., 2021; Öztürk et al., 2015; Nowak et al., 2014; Muszynska et al.; Ha et al., 2020). They are referred to as “biological response modifiers” for their ability to enhance and modulate the body’s reaction to infections. The health-enhancing properties of mushrooms make them a potential treatment option for COVID-19 and other diseases. With their diverse range of bioactive substances, mushrooms could be a part of everyday supplementation.

It's important to note that the consumption of mushrooms should not be considered as a replacement for medical treatment. While mushrooms contain bioactive compounds with potential health benefits, they should not be used as a substitute for prescribed medication. Now, let's take a closer look at some of the potential nutraceuticals found in mushrooms. One of the most well-known bioactive compounds found in mushrooms is beta-glucans, which are complex polysaccharides that have been shown to stimulate the immune system and potentially have anti-tumor properties. Beta-glucans have been found in many different mushroom species, including shiitake, reishi, and maitake (Misra et al., 2009; Qiang et al., 2009; Xiao et al., 2011).

In addition to beta-glucans, mushrooms also contain a variety of other bioactive compounds, including polysaccharides, terpenoids, phenolic compounds, ergosterol and enzymes (Verma at al. 2022; Ferreira et al., 2010; Barros et al., 2009). These compounds have been found to have a range of potential health benefits, including antioxidant, anti-inflammatory, and anti-cancer properties (Ferreira et al., 2010; Jeong et al., 2008). For example, ergosterol is a precursor to vitamin D2, which is important for bone health and immune system function. Some studies have also suggested that ergosterol may have anti-inflammatory and anti-tumor properties. Phenolic compounds found in mushrooms have also been studied for their potential health benefits. These compounds have been shown to have antioxidant properties and may also have anti-inflammatory and anti-cancer properties. Some of the phenolic compounds found in mushrooms include gallic acid, protocatechuic acid, and syringic acid.

1.1.     Diabetes mellitus (DM)

Diabetes mellitus is a serious disorder of carbohydrate metabolism characterized by hyperglycemia, high glycated hemoglobin, and an elevated risk of morbidity and mortality (Papoutsis et al. (2021)). There are mainly two types of diabetes, type 1 or insulin-dependent diabetes mellitus (IDDM), resulting from selective destruction of insulin-producing β cells in the pancreas, and type 2 or non-insulin-dependent diabetes mellitus (NIDDM), resulting from insulin resistance and impaired insulin secretion (Deveci et al., 2021). The majority of cases are type 2, and symptoms include hunger, increased thirst, frequent urination, fatigue, and blurred vision. If left untreated, diabetes can ultimately lead to several acute complications, including ketoacidosis, stroke, heart disorders, kidney failure, eye damage, foot ulcers, impotence, and even death.

Diabetes was first documented by the Egyptians and is characterized by weight loss and polyuria. The Greek physician Aretaeus introduced the term diabetes mellitus, with diabetes meaning "to pass through" and mellitus referring to sweetness. Medieval Greco-Arab physicians recognized diabetes by its main symptoms of increased thirst, frequent urination, and tiredness. Today, the prevalence of diabetes is rising among adults over 18 years of age, with an estimated 8.5% affected in 2014, and the percentage increasing every year. Diabetes at least doubles a person’s risk of early death, with approximately 1.5–5.0 million deaths each year resulting from diabetes from 2012 to 2015 (WHO). The global economic cost of diabetes in 2014 was estimated to be $612 billion USD, with DM accounting for 2.2% of deaths worldwide and being one of the major causes of death among humans. It is estimated that by the year 2025, over 300 million people will be affected by diabetes in the world, and the estimated number of diabetic patients in 2030 will be more than double that in 2005. In the United States, diabetes is the seventh leading cause of death, affecting 25.8 million (8.3%) of the population (Papoutsis et al., 2021).

Furthermore, diabetes imposes an increasing economic burden on national healthcare systems worldwide. According to the International Diabetes Federation, the global health expenditure on diabetes is expected to be a total of at least $490 billion USD in 2030. However, currently available modern drugs used for diabetes treatment are often associated with limitations such as high cost, inadequate efficacy, and toxicity, making the need for the development of novel strategies with fewer side effects, such as ethnobotanical medications, more pressing (Sokovic, 2019). Diabetes causes gradual degradation of the kidneys, with classic symptoms such as excessive urination. Eventually, the degree of degradation is enough to cause complete kidney failure, a progression called Diabetic Nephropathy (Misra et al., 2009; Qiang et al., 2009; Xiao et al., 2011).

The use of mushrooms for the treatment and prevention of diabetes is becoming increasingly important, as research has shown that they contain active components such as polysaccharides, dietary fibers, and other compounds with anti-hyperglycemic activity (Lo and Wasser, 2011). Polysaccharides, particularly β-glucans, found in mushrooms can help restore the functions of pancreatic tissues, increase insulin output, and improve the sensitivity of peripheral cells to circulating insulin, leading to a decrease in blood glucose levels. Various mushrooms, including Pleurotus ostreatus, Ganoderma lucidum, Grifola frondosa, and Lentinus edodes, contain compounds that act as anti-diabetic agents by reducing cholesterol synthesis, absorption, and feeding (De Silva et al., 2012). Other mushrooms, such as Poria cocos and Cordyceps, have been used in traditional Chinese medicine to cure diabetes and other related disorders due to the presence of triterpenoids and polysaccharides, respectively (Lindequist et al. 2005; Sato et al., 2002). The secondary metabolites found in mushrooms, such as phenol, lectins, flavonoids, terpenoids, alkaloids, tannins, and metal-chelating agents, are responsible for their anti-diabetic properties and have potential for use as therapeutic agents. Further research is needed to develop anti-diabetic mushroom nutraceuticals.

2.       Materials and Methods

2.1.              Mushroom sample- A total of ten mushroom samples were selected for screening purposes.

2.2.              Sample preparation- To prepare the samples, the mushrooms were sun-dried and ground into a fine powder using a blender. The resulting powder was then extracted with various organic solvents at a ratio of 1:100 w/v. specifically, 2 grams of mushroom powder were incubated with 200 ml of organic solvent for 24 hours in a shaking incubator. After filtration, the obtained extract was concentrated using a rotary evaporator under pressure to produce a concentrated extract. This extract was then used for further analysis, including secondary metabolites screening, quantification of secondary metabolites, and in-vitro α-amylase inhibitory assay.

2.3.              Secondary metabolites screening-

2.3.1.         Test for phenols and Tanin- Ferric chloride test was done according to Evans (2009) and Suman et al. (2013), with some modifications, Mushroom samples (10mg) were dissolved in 1 mL of solvent, then filtered. The filtration (2 mL) is added with 1 mL FeCl3 3%. The presence of green to slightly blackish deposits indicate the presence of tannins and polyphenols

2.3.2.         Test for flavonoid- Sodium hydroxide test was done according to Joseph et al. (2013), with some modifications, 2ml of mushroom extracts were treated with few drops of 20% sodium hydroxide solution. An intense yellow colour was formed. It becomes colourless on addition of dilute hydrochloric acid, indicating the presence of flavonoids.

2.3.3.         Test for glycosides- Keller-Killiani test was performed according to Tiwari et al. (2011), in brief, 5ml of each mushroom extract was added with 2 ml of glacial acetic acid. It was followed by the addition of few drops 5% ferric chloride solution. 1ml of concentrated Sulfuric acid. Formation of brown ring at interface confirms the presence of glycosides.

2.3.4.         Test for terpenoid and steroid- Liebermann-Burchard Test was done followed to Suman et al. (2013), 5mg of the sample was extracted with chloroform and then filtered. 2 ml of filtrate formed which were then added with 1-2 ml of acetic anhydride and 2 drops of concentrated H2SO4 from the side of the tube. The formed colour is red, then it confirms the presence of terpenoid and green colour shows the presence of sterols.

2.3.5.         Test for alkaloids- Wagner’s test was performed according to Suman et al. (2013), 1mL of extract was taken and placed into a test tube. Then 1mL of potassium mercuric iodide solution (Wagner’s test reagent) was added and shaken. Emergence of whitish or cream precipitate implies the presence of alkaloids.

2.3.6.         Test for phlobatannins- HCl test was done, to each extract, 1% aqueous hydrochloric acid was added and each sample was then boiled with the help of Hot plate stirrer. Formation of red coloured precipitate confirmed a positive result (Tiwari et al., 2011).

2.3.7.         Test for anthraquinone- Borntrager test was used, 1gm of mushroom sample then add 5-10 ml of dilute HCl. After that it was boiled on water bath for 10 min and filtered   benzene was added along with equal amount of ammonia solution, then it was properly shaken. Appearance of pink to red colour indicate presence of anthraquinone moiety (Sheel et al., 2014).

2.4.              In-vitro α-amylase inhibitory assay- The inhibition assay was conducted using the chromogenic DNSA method described by Sokovic, M. (2019). This method measures enzyme activity by determining the amount of reducing sugars produced. Stock solutions of all extracts (1000 ppm) were prepared by dissolving 1 g of each extract in 1 L of phosphate buffer saline (PBS), and various concentrations of the extract were prepared.

Next, 100 μl of α-amylase solution (0.001 g of α-amylase was dissolved in 100 ml of 0.02 M sodium phosphate buffer pH 6.9 with 6.7 mM sodium chloride) were added to each test tube. Then, 100 μl of different concentrations of the test samples (100 μg, 200 μg, 300 μg, and 400 μg) were added to each test tube separately. All tubes were incubated at 25°C for 10 minutes.

After the incubation period, 100 μl of 1% starch solution was added to each tube. The tubes were then incubated at 37°C for 10 minutes. Next, 200 μl of 3,5-dinitrosalicylic acid (DNSA) reagent was added to each tube, and the tubes were incubated in a hot water bath (85°C). After 5 minutes, the mixture changed color to orange-red, and the tubes were removed from the water and diluted with 2 ml of distilled water bath. The samples were then cooled to room temperature

The absorbance was measured at 540 nm. A control was prepared using the same procedure, but replacing the extract with distilled water. A blank was performed by replacing the enzyme with buffer. The α-amylase inhibitory activity was calculated as the percentage of inhibition using the following formula:

% Inhibition = (Abs control – Abs extracts / Abs control) × 100

3.       Result and Discussion

3.1.              Mushroom sample - There are ten wild mushroom samples, which can be classified into two orders: Polyporales and Agaricales. Samples 1M, 2M, 3M, 4M, and 6M belong to the order Polyporales, while samples 5M, 7M, 8M, 13M, and 14M belong to the order Agaricales (Figure 1).

3.2.              Secondary metabolites screening- These mushroom samples were dried and ground to a fine powder. This powder was extracted using polar and non-polar organic solvents. These extracts were concentrated under pressure using a rotary evaporator and used for the various studies.

The secondary metabolites were conducted to detect phenols, flavonoids, alkaloids, steroids, terpenoids, anthraquinones and glycosides. All the ten samples reported positive for phenols, flavonoids and alkaloids (Figure 2). Samples 4M, 6M, 7M, 8M, 13M and 14M reported a high concentration of phenols. Flavonoid was present in  large quantities in samples 5M and 8M. Alkaloids showed a strong result in samples 6M and 13M and in moderate amounts in the rest of the samples. Samples 1M, 4M, 5M, 6M, 7M and 8M showed a positive result for steroids, while terpenoids were detected in 2M, 8M and 13M samples. The positive test of phlobatannins was given by samples 1M, 2M, 3M, 7M, 13M and 14M. All the samples gave a negative result for anthraquinones while, glycosides were present in samples 1M, 3M, 6M and13M. The results for these tests are shown in table1. The esults align with the studies conducted by Sheel et al. (2014), Tiwari et al. (2011), Suman et al. (2013), and Joseph et al. (2013).