1.
Introduction
DOI: 10.52228/NBW-JAAB.2022-4-1-6
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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).