INTRODUCTION:
The genus Fulvifomes Murrill belongs
to Hymenochaetaceae family, Basidiomycotina phylum are showing medicinal values1.
According to recent phylogenetic studies, the genus Fulvifomes is very
much closely related to Aurificaria D.A. Reid and Phylloporia
Murrill. Earlier this was not treated as an independent genus but a subgenus of
Phellinus Quél2.
The circumscription of Fulvifomes as
a genus shown morphological characters like the combination of annual to
perennial and effused-reflexed, sessile to substipitate sporophore with
solitary to imbricate pilei and an homogeneous or duplex context, a dimitic
hyphal system, lack of hymenial setae, and subglobose to ellipsoid, yellowish
to brown, slightly thick to thick-walled basidiospores. Recently, several
species of Fulvifomes were recorded3,4,5,6. The present study
aims at phenotypical identification, mycochemical bioactive compounds,
proximate composition, and antifungal activity was tested.
MATERIALS AND METHODS:
Collection and Phenotypical identification:
The sporophore was collected from Andhra
Pradesh, India, during the rainy season (July–September) of the years 2015 to
2019. Field characters like habit, host, name of locality and other
macro-morphological characteristics were recorded for sample specimens. For
Phenotypical identification of sporophore, different Macroscopic features like
abhymenial, hymenial surfaces, context, and pore tubes of species were
examined. Microscopic features like hyphae, basidiospores and pilear crust were
observed by preparing crush mounts and free-hand sections in water, 5% KOH
solution, and staining was done with cotton blue (1%, in lactophenol), Congo
red (1%, in distilled water), phloxine (1%, in distilled water), and Melzer’s
reagent7,8,9,10,11. Voucher specimen of F. fastuosus (ALC 35)
has been deposited at the herbarium of the Museum of Botany Department, Andhra
Loyola College, Vijayawada, Andhra Pradesh, India (ALC).
Extraction of bioactive compounds:
The sporophore of F. fastuosus was
initially rinsed thrice in distilled water and dried on paper toweling and
samples was cut into fine pieces and powdered. For preparing the extracts
methanol, ethanol and water was used as solvents. For every 1gram of powder,
50ml of solvent was used and was subjected to extraction using maceration.
After the completion of extraction, the supernatant was filtered through
Whatman No. 1 filter paper and the filtrates stored at 4°C for further use to
perform various assays for determination of bioactive mycochemicals and
antifungal activity.
Mycochemical tests:
The screening of bioactive mycochemicals in
fresh sporophores of F. fastuosus is tested by using standard methods of
Indian Pharmacopoeia followed by Evans and Trease12, Gokhale13,
Trease, and Evans14, Kokate et al.,15, Harborne16,
Khandelwal17 and Shanmugam et al.,18. Nagadesi et al.,19
Test for Carbohydrates:
Molisch’s test:
To a small amount of the extract few drops
of Molisch’s reagent was added followed by the addition of conc. H2SO4
along the sides of the test tube. The mixture was then allowed to stand for 2
min and then diluted with 5ml of distilled water. Formation of red or dull
violet colour at the inter phase of two layers indicates the presence of
carbohydrates. First yellow then brick red precipitate was observed.
Fehling’s test:
The extract was treated with 5ml of
Fehling’s solution (A and B) and kept in boiling water bath for 5-10 min. The
formation of yellow or red colour precipitate indicates the presence of
reducing sugar.
Test for Proteins:
Biuret test:
Test sample (3ml) was mixed with 4% NaOH
and few drops of 1% CuSO4 solution were added. Violet or pink color
not appeared. To 3ml of the extract few drops of 10% sodium chloride and 1%
copper sulfate was added for the formation of violet or purple colour. On
addition of alkali, it becomes dark violet.
Tests for Amino Acids:
Ninhydrin test:
Test sample (3ml) and 3 drops of 5%
ninhydrin solution were heated in boiling water for 10 min. Purple color
appeared.
Test for
lipids:
Brown Bag test:
Certain kinds
of paper such as a piece of brown paper bag can readily absorb lipids and can
be used to test for the presence of lipids.
Emulsion test:
Suspended the sample in ethanol which
allows lipids to dissolve in it, and then decanted the liquid into water. Since
lipids do not dissolve in water, it falls out of the solution as cloudy white
emulsion.
Test for Glycosides:
Free content of the sugar extract was
determined. The sample was hydrolyzed with mineral acid (dilute hydrochloric or
dilute sulfuric acid). Again the total sugar content of the hydrolyzed extract
was determined. Increase in the sugar content indicated the presence of
glycoside in the extract.
Glycoside +H2O Aglycon
(genin) + Glycon (sugar)
Liebermann’s Test:
We added 2.0ml of acetic acid and 2ml of chloroform
with whole aqueous plant crude extract. The mixture was then cooled and we
added H2SO4 concentrated. Green color showed the entity
of aglycone, steroidal part of glycosides.
Legal’s test:
Aqueous or alcoholic sample extract was
mixed with 1 ml of pyridine sodium nitroprusside was added. Pink to red color
appeared.
Test for cardiac glycosides:
Keller-Kiliani Test:
A solution of glacial acetic acid (4.0ml)
with 1 drop of 2.0% FeCl3 mixture was mixed with the 10ml aqueous
plant extract and 1ml H2SO4 concentrated. A brown ring
formed between the layers which showed the entity of cardiac glycosides.
Test for Steroids:
Salkowski’s test:
Sample (2ml) was mixed with 2ml of
concentrated Sulfuric acid, it was well shaken then chloroform layer appeared red
and acid layer shown greenish yellow fluorescence.
Lieberman-Buchard reaction:
Sample (2ml) was mixed with chloroform.
1-2ml of acetic anhydride was added and 2 drops concentration sulfuric acid was
added from the sides of the tube. First red then blue and finally green colour
appeared.
Tests for Sterols:
The sample was treated with 5% potassium
hydroxide solution appearance of pink colour indicated the presence of sterols.
Test for Saponin:
Foam test:
To 1ml of the extracts 5ml distilled water
was added and shaken vigorously. Formation of foam indicated presence of
saponins.
Phosphate test:
Ammonium
molybdate test:
A small amount
of the sample is acidified with concentrated nitric acid, to which a little ammonium
molybdate is added. The presence of phosphate ions is indicated by the formation
of a bright yellow precipitate layer of ammonium phosphomolybdate. The
appearance of the precipitate can be facilitated by gentle heating
Silver nitrate test:
To a small amount of sample add silver
nitrate solution. Silver phosphate is formed as a yellow precipitate by the
reaction between a soluble silver compounds, such as silver nitrate with a
soluble orthophosphate.
Proximate composition:
To establish standards for their identity,
quality, purity of hymenophore powder the proximate composition was carried out
for F. fastuosus collected from ALC, Vijayawada, Andhra Pradesh, India.
The pulverized sporophore of F. fastuosus was used for the
standardization of physicochemical parameters in triplicate. Foreign matter, moisture
content, extractive values, ash values20,21 dry matter22,
absorption properties, foaming properties23, emulsion values24,
dispersibility25, flow characteristics, swelling index26
were determined.
Antifungal test:
To test the antifungal activity of F.
fastuosus, the method described by Nagadesi and Arya27 was used.
The Aspergillus niger was causing soft rot of carrot, A. oryzae
was causing mold in paddy seeds, Mucor racemosus causing soft rot if
bitter guard, Rhizopus stolonifer was causing soft rot in lady’s finger
and R. artocarpi causing fruit rot in Jack fruit was isolated and used
for antifungal test. The fungal extracts were mixed with appropriate volume of
medium (PDA) to obtain concentrations ranging from 5 to 25% in the final volume
of 100ml of medium. This 100ml medium was dispensed into 100mm Petri plates
with triplicates. Plant pathogenic fungi were placed in the centre of each
plate. Control sets were also prepared without fungal extract. The plates were
incubated at 25±20C and growth of colony was measured after 7 days
of inoculation. The radial growth of mycelium was measured at two points along
the diameter of the plate and mean of these two readings was taken as the
diameter of the colony. The growth of colony in control sets was compared with
that of various treatments and difference was converted into percent inhibition
by following formula
Diameter of control set - diameter of treated set
Percent inhibition =
-------------------------------------------- ×100
Diameter of
control set
RESULTS:
Phenotypical identification:
The sporophore was found on the living tree trunk of Peltophorum
roxburghii (G. Don) Degener causing white rot. The sporophore is identified
as Fulvifomes fastuosus (Lév.) Bondartseva and S. Herrera, belonging to
Hymenochaetaceae family. The description of the sporophore was given below
Fulvifomes fastuosus (Lév.) Bondartseva and S. Herrera:
The sporophore is
perennial, pileate, broadly attached, in group forming single fruiting body,
woody, and hard. The Pilei are applanate, projecting up to 17.3 x 14.5 x 15.5cm
in size and 2cm thickness at base. Pileal surface is dark brown when dry,
concentrically sulcate with wide zones and margin is curved, obtuse and thick
brown (Plate I Fig, B). The pore surface is dark brown; pores
circular, 8 – 9per mm, context golden yellow, concolorous with tubes and up to
1–2mm thick. Tubes are paler than pores, up to 1.5cm long, tube layers distinct
(Plate I Fig, C).,
Hyphal system is dimitic; In the context, the generative hyphae are yellowish, thin walled, simple
septate, negative in Melzer’s reagent, acyanophilous, 2.5 – 3.65μm in
diameter; skeletal hyphae dominant, brown, thick walled with wide lumen,
unbranched, negative in Melzer’s reagent, acyanophilous, 3.85–5.65μm in
diameter Setae absent.
Basidia and Basidioles not seen. Basidiospores subglobose, thick walled, golden
brown, smooth, negative in Melzer’s reagent, acyanophilous, 5.29 x 4.65 μm
in size.
Proximate composition:
The hymenophore powder of F. fastuosus shows the proximate composition in Table 2. Foreign
matter present in sporophore powder of F. fastuosus is 1.5% whereas moisture content in sporophore powder
of F. fastuosus is 5.5%. The higher the dry matter in
sporophore of F. fastuosus indicate presence of less moisture. The
extractive value of F.
fastuosus powder shows
higher ethanol soluble value when compared to water soluble content. The ash
content of F. fastuosus sporophore powder is 7.89%, showing higher
water soluble ash when compared to acid insoluble ash. The absorption capacity
of F. fastuosus sporophore powder shown higher water
absorption when compared to oil absorption. The emulsion formation capacity of F. fastuosus sporophore powder shown high dispersibility when
compared to emulsion stability. The flow properties of F. fastuosus sporophore powder of F. fastuosus shown tapped density is more when compared to bulks
density. The foam formation capacity of F. fastuosus sporophore powder shown higher swelling capacity when
compared to foaming capacity.
Antifungal activity:
The antifungal activity of F. fastuosus on
plant pathogenic fungi was shown in Table 3. The methanolic extract of F.
fastuosus shown best antifungal activity when compared to water extract.
The 15%, 20%, 25% concentration of methanolic extract of F. fastuosus shown
100% inhibition of plant pathogenic fungi tested. The 20%, 25% concentration of
water extract of F. fastuosus has shown 100% inhibition of plant
pathogenic fungi. Except 5% concentration all methanolic extracts of F.
fastuosus shown 100% inhibition of A. niger and M. racemosus
(Plate I Fig, A).
DISCUSSION:
The distinctive morphological characters of Fulvifomes
species are the dimitic hyphal system and coloured basidiospores2.
In the present study also the basidiocarp shown the morphological characters of
Fulvifomes genus, so the sample collected from Andhra Pradesh was
identified as F. fastuosus. The
first report on the occurrence of species F. fastuosus from south India
and found in the host trees of Albizzia sp28. The F.
fastuosus has been reported in the trees of Xylocarpus granatum29.
In the present study the F. fastuosus was found on the living tree of P.
rouxbergii causing white rot from Andhra Pradesh, South India. The fruiting
bodies of F. fastuosus were reported for the first time from Andhra
Pradesh, India. It is also a new host record reported from India
Members of the Hymenochaetaceae family are known to
possess strong bioactive properties including antioxidant and anticancer
activity which is strongly attributed to polyphenolic substances contained in
the mushrooms30. In the present study the sporophore of F.
fastuosus is also the member of the Hymenochaetaceae family; so it shown
the Phenols and Polyphenolic compounds in all extracts. The screening of
mycochemical composition of each tested Phellinus species extract
revealed the presence of various biochemicals and secondary metabolites such as
carbohydrates, reducing sugars, proteins, amino acids, steroids, terpenoids,
phenolic compounds, flavonoids, tannins, anthraquinone glycosides, cardiac
glycosides and alkaloids31. In the present study the fruiting bodies
of the Fulvifomes is sister group to Phellinus so it shown
different secondary metabolites like Carbohydrates, Proteins, Amino acids,
Lipids, Alkaloids, Cardiac glycerides, Flavonoids, Phenols, Terpinoids,
Steroids, Sterols, saponins, Tannins, and Phosphate. Since the polyphenolic
secondary metabolites produced by F. fastuosus mushroom exhibit largely
diverse structural differences, the isolation and separation of metabolites can
be lengthy and tedious32. In the present study also the fruiting
bodies collected from Andhra Pradesh, South India show excellent concentration
of polyphenolic compounds.
The findings regarding the physicochemical properties
showed low values of foreign matter and moisture content indicating good
quality of these mushrooms. High foreign matter and moisture content impairs
the purity of the sample making it undesirable for use. Young mushroom samples
showed high moisture content and low dry weight as compared to mature fruiting
bodies33,34. In the present study sporophore is matured one, the
foreign matter is 1.5% and moisture is 5.5% so the Fruiting bodies of F.
fastuosus powder are good quality and purity also good. As per literature
reports, the moisture content of wild mushrooms ranged from 26.2–90.26%35
and dry matter from 91.25–95.54%36. The moisture content of F.
fastuosus is 5.5% and dry matter is 90.9%. The value of bulk density is a
measure of the heaviness of powdered sample. The higher is the bulk density of
a powder, the greater is its dispersibility. Both the tested mushrooms showed
good values of bulk density and dispersibility31 which are
comparable to the values reported for Ganoderma species37,38.
in the present study also the sister group of F. fastuosus showed higher
bulk density and dispersibility values. Extractive values indicate the type of
biochemical constituents present in the sample. Phellinus fastuosus
contained more alcohol soluble while P. sanfordii consisted of more
water soluble polar biochemical components as indicated by the extractive
values of each species31. In the present study the F. fastuosus shown high alcohol
soluble mycochemicals. The absorption and emulsion properties form a criterion
for the suitability of powder to be used in pharmaceutical and nutraceutical
formulations. Each tested mushroom was observed showing comparable absorption
and emulsion capabilities31. In the present study the F.
fastuosus showed good absorption and emulsion properties so the powder have
good quality for usefulness in pharmaceutical formulations. The values of ash
content of a powder give an estimation of inorganic or earthy material present
in it. The value of ash content in each mushroom was found in the range
recorded31 for sixteen wild mushrooms collected from Anatolia 3.37 –
15.46%39. In the present study also the ash content is found in the
range recorded results of earlier work. The formation of foam and swelling of
powder was not observed in any of the tested mushroom 31. In the
present study the F. fastuosus shown formation of foam and swelling of
powder values.
In case of human fungal pathogen Candida albicans
sesquiterpenoid extract of Phellinus species was found to be better than
with potent activity followed by diterpenoid extract of Phellinus species.
The triterpenoid extract of Phellinus shown low to moderate activity
against human fungal pathogen40. In the present study the extracts
of F. fastuosus was showing high inhibition of plant pathogens like A.
niger and M. racemosus which is causing soft rot in fruits and vegetables.
ACKNOWLEDGEMENTS:
The authors are thankful to the Principal, Vice
principal (P.G), Andhra Loyola college, Vijayawada - 8, for laboratory
facilities in Botany Department. Prof. Arun arya, Head, Environmental Science,
Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, for
identification conformation of fungi.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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