© 2016 Krishnendu Acharya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercial-
ShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/).
Journal of Applied Pharmaceutical Science Vol. 6 (11), pp. 185-190, November, 2016
Available online at http://www.japsonline.com
DOI: 10.7324/JAPS.2016.601129
ISSN 2231-3354
Pharmacognostic standardization of a well known edible mushroom,
Volvariella volvacea
Krishnendu Acharya
*
, Sandipta Ghosh, Ipshita Kundu
Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road,
Kolkata 700019, West Bengal, India.
ARTICLE INFO
ABSTRACT
Article history:
Received on: 06/09/2016
Revised on: 25/09/2016
Accepted on: 17/10/2016
Available online: 29/11/2016
From ancient times, Volvariella volvacea has been recognized as a dietary addition in many Asian and European
countries. Clinical evaluations have also revealed that the fruit body contains a great diversity of
pharmacologically active constituents that posses remarkable medicinal activities. However pharmacognostic
studies of the mushroom have not been carried out so far. So the present study was conducted with a view to
establish a pharmacognostic standard of the dried powder of V. volvacea. Study was executed on the basis of
microscopic, physical characters and phytochemical parameters using standard methods. Fluorescence analysis
of the dried powder was also conducted using different chemical reagents. On the other hand, a fresh methanol
soluble extract was prepared and prelimiminary screening of phytochemicals revealed the presence of phenols,
flavonoids, ascorbic acid, β-carotene and lycopene. In addition, HPLC profile was recorded and results showed
existence of 12 peaks as detected at 278 nm which might be of phenolic compounds. Furthermore, DPPH radical
scavenging activity (EC
50
-0.12 mg/ml) and total antioxidant capacity (23.75±1.25 μg ascorbic acid
equivalent/mg of extract) were conducted to evaluate antioxidant potentiality of the methanol extract. All these
above mentioned standards developed from this study will help in maintaining quality and purity as well as
ascertain the identity and authenticity of dried powder of V. volvacea.
Key words:
Antioxidant property, HPLC,
Medicinal edible mushroom,
Microscopic characters,
Pharmacognostic
standardization.
INTRODUCTION
In modern civilizations, natural products are becoming
an effective alternative to the toxic chemicals used in preparation
of synthetic medicine. Recently, many pharmaceutical companies
have started paying their attention towards the exploitation of
natural products in medicine formulation. The outputs are quite
successful as because the bioactive compounds from natural
resources are considered to be safe with no side-effects. But the
main hinderance in acceptance of alternative medicine is the lack
of standard documentation (Dahanurkar et al., 2000). Thus, there
is a need for quality control standardization of these
products which not only helps in proper identification but also in
* Corresponding Author
Krishnendu Acharya, Molecular and Applied Mycology and Plant
Pathology Laboratory, Department of Botany, University of Calcutta,
35, Ballygunge Circular Road, Kolkata 700019, West Bengal, India
E-mail: krish_paper @ yahoo.com
authentication of genuine material (Kushwaha et al., 2010).
However no single method can provide the absolute result. The
best solution is compilation of several different parameters of
pharmacognosy including qualitative as well as quantitative
measure to get a detailed and multidimensional view of the
product. Nowadays researchers have relied more on the therapeutic
values of mushrooms.
Different mushroom metabolites and active components
have shown strong medicinal prospects (Khatua et al., 2013; Mitra
et al., 2016; Chatterjee et al., 2014; Chatterjee et al., 2016; Mallick
et al., 2014; Mallick et al., 2015; Nandi et al., 2014; Nandi et al.,
2013). Volvariella volvacea is one such potential mushroom,
commonly known as paddy straw mushroom belongs to the family
Pluteaceae (Order: Agaricales).
It is a tropical cosmopolitan edible macrofungi and also
cultivated commercially throughout the Southeast Asia. This
mushroom is considered a delicacy in Asian cuisines because of its
delightful taste and flavor.
186
Acharya et al. / Journal of Applied Pharmaceutical Science 6 (11); 2016: 185-190
Besides this, the medicinal impact of this mushroom has
been scientifically evaluated by a number of scientists around the
globe establishing its antioxidative (Rai and Acharya, 2012, Sudha
et al., 2008, Ramkumar et al., 2012; Tripathy et al., 2016),
antimicrobial (Perera et al., 2001; Ayodele and Idoko, 2011),
hepatoprotective (Kalava et al., 2012a; Kalava et al., 2012b;
Acharya et al., 2012), anticancer (Wu et al., 2011; Maiti et al.,
2008) and immunomodulatory (Jeurink et al., 2008; Hao-Chi et
al., 1997) properties. Additionally, a variety of biologically active
compounds have been isolated from this mushroom (Hao-chi et
al., 1997).
As the demand of this mushroom is increasing day by
day for its immense medicinal importance, a strong
pharmacognostic profiling is of utmost needed. Therefore, the
main objective of the present work is to establish the
pharmacognostic parameters of this mushroom using standard
methods, which could be helpful in preparation of a monograph
for the proper identification of the mushroom.
MATERIALS AND METHODS
Mushroom materials
Basidiocarps of V. volvacea were collected during the
rainy season from coastal villages of West Bengal.
Identification and authentication of the basidiocarp was done using
standard literature (Shaffer 1997, Dutta et al. 2011) and the
voucher specimen present in CUH herbarium. A
representative specimen (CUH AM036) was deposited in the same
herbarium for future reference as per the method by
Pradhan et al., 2015. For powder analysis basidiocarps were first
dried by field drier at 40°C for overnight, then pulverized to make
them crispy using electric blender and this coarse powder was
sieved into a fine powder by using a160 mesh. Then the
finely sieved powder was stored in an airtight container for future
use.
Microscopic and organoleptic characterization of powdered
basidiocarps
Microscopic features were obtained from dried powder
of the basidiocarps in 5% KOH, Melzer’s reagent, and Congo red.
The slide was then examined under Carl Zeiss AX10 Imager A1
phase contrast microscope and images were captured with a digital
camera at desired magnifications. Several other features like
colour, odour, taste, and nature of dried powder were also
evaluated.
Fluorescence analysis
Fluorescence study of dried sieved powder was
performed as per standard procedure (2007). A small quantity of
sieved powder was placed on a grease free slide and treated with
16 different chemical agents and mixed properly by gentle tilting
the slide. Then the slide was placed inside the UV viewer chamber
and exposed to visible, short (254 nm) and long (365 nm)
ultraviolet light to study their fluorescence behavior.
Preparation of methanolic extract
5 g of pulverized dried powder was soaked in 100 ml
methanol for 72 h before extraction. Thereafter, it was filtered
through Whatman No. 1 filter paper. The filtrate was concentrated
under reduced pressure at 80°C using rotary evaporator and
preserved in refrigerator at 4°C in a dark bottle for further use.
Extractive value was also recorded.
Quantitative estimation of mycochemicals
Methanolic extract was subjected for quantitative
estimation of mycochemicals such as phenol, flavonoids, ascorbic
acid, β carotene and lycopene. The content of total phenolic
compounds in extract was estimated using Folin-Ciocalteu reagent
and gallic acid as standard. The results were expressed as μg of
gallic acid equivalents per mg of dry extract. Total flavonoid
content was determined using aluminium nitrate and potassium
acetate. Quercetin (5–20 μg/ml) was used to calculate the standard
curve.
The results were expressed as μg of quercetin equivalents
per mg of dry extract. β- carotene and lycopene were estimated by
measuring absorbance at 453, 505 and 663 nm. Ascorbic acid was
determined by titration against 2, 6-dichlorophenol indophenol dye
(Khatua et al., 2015).
High performance liquid chromatographic (HPLC) profile of
methanolic extract
The extract was filtered through 0.2 μm filter paper and
20 μl filtrate was loaded on the HPLC system (Agilent, USA).
Separation was achieved on an Agilent Eclipse Plus C18 column
(100 mm × 4.6 mm, 3.5 μm) using a flow rate of 0.8 ml/min at
25°C using 11 standards. The mobile phase consisted of eluent A
(acetonitrile) and eluent B (aqueous phosphoric acid solution,
0.1% v/v). A gradient program was used for elution: 0-2 min, 5%
A; 2-5 min, 15% A; 5-10 min, 40% A; 10-15 min, 60% A; 15-18
min, 90% A. The absorbance of sample solution was measured at
280 nm (Khatua et al., 2015).
DPPH radical scavenging activity
A solution of DPPH was freshly prepared by dissolving
DPPH dye in required volume of methanol (about 0.1 mM). 2 ml
reaction mixture consisted of freshly prepared methanol solution
of DPPH (0.1 mM) and various concentrations of extract. After 30
minutes of incubation in dark at room temperature absorbance was
measured at 517 nm against blank (Shimada et al., 1992). The
percentage inhibition of radicals was calculated using the
following formula:
% inhibition = (A
control
– A
sample
)×100/ A
control
Where A
control
is the absorbance of DPPH solution without extract
and A
sample
is the absorbance of sample with DPPH solution. The
EC
50
value was reported as the effective concentration at which
DPPH radicals were scavenged by 50%. Ascorbic acid was used
for comparison. All tests were performed at least in triplicate, and
graphs were plotted using the average of three determinations.
Acharya et al. / Journal of Applied Pharmaceutical Science 6 (11); 2016: 185-190 187
Total antioxidant assay for methanolic extract
The reaction mixture consisted of 0.3 ml sample solution
and 3 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium
sulphate and 4 mM ammonium molybdate), according to the
method by Prieto et al., 1999. Tubes were capped and incubated at
95ºC for 90 min. Samples were cooled at room temperature and
absorbance was measured at 695 nm against blank. Concentrations
of ascorbic acid (1-30 μg/ml) were used to obtain a standard curve.
Total antioxidant activity was expressed as the number of
equivalents of ascorbic acid.
Statistical analysis
All assays were carried out in triplicate. The results are
expressed as mean values and standard deviation (SD). Results
were compared by means of a Student’s t test to determine the
significant difference among samples. The analysis was carried out
using Microsoft
®
Office Excel (Microsoft
®
, USA), where values of
p ≤ 0.05 were considered as statistically significant.
RESULTS AND DISCUSSION
Authentication of natural material is a crucial
prerequisite step before using in research field or for medicinal
purpose. Therefore, in this study we have performed the different
parameters of pharmacognostic investigation to standardize
this macrofungus to some extent. Organoleptic characterization of
dried powder is considered as an important part of
pharmacognostic standardization. The crude sieved powder was
light brown in colour, salty taste without any significant odour and
powdery in texture. The dried sieved powder of V. volvacea under
microscopic investigation with 5% KOH and Congo red showed
following characters (Figure 1): Several fragmented thin-walled
hyphae lacking clamp connection, spores ovoid to ellipsoid (5.13
µm x 2.38 µm) ochraceous salmon, smooth with thickened wall
and guttulate contents, basidia (14.08 µm - 4.95 µm x 2.61 µm -
1.24 µm) clavate, tetrasterigmatic. Melzer’s reaction showed
negative result which indicates that the spores are non-amyloid in
nature.
Fluorescence analysis is also an important tool for
qualitative assessment of crude drug and it provides an idea about
their chemical nature (Sonibare and Olatubosun, 2015,
Bhattacharya and Zaman, 2009). If substance themselves do not
give fluorescence; they may often be converted into fluorescent
derivatives or decomposition products by applying different
chemical reagents. This distinguishing character is useful in
identification of authentic samples and recognizing adulterates.
The fluorescence characteristics of dried powder of V. volvacea
are summarized in Table 1.
Freshly prepared methanolic extract was subjected for
organoleptic characterization and quantitative estimation of
phytochemicals. The fraction was brownish in colour, sticky in
nature with extractive value of 15 ± 2.3%.
Fig. 1: A) Sieved powder of Volvariella volvacea. Microscopic characterization: B) Hyphae C) Fragmented hyphae, basidia and spores D) Spores.
Table 1: Fluorescence profile of dried powder of Volvariella volvaceae.
Serial No.
Reagents
Visible
UV
Long
(365 nm)
Short
(254 nm)
1
Powder as such
Brownish-yellow
Blackish brown
Deep brown
2
Hager’s
Brown
Brownish black
Dark brown
3
Mayer’s
Rust brown
Greyish violet
Rust brown
4
Dragendroff’s
Brownish yellow
Greyish magenta
Greenish brown
5
Iodine solution
Brownish orange
Purplish black
Greenish brown
6
1(N) HNO
3
Reddish brown
Purplish black
Brown
7
50% HNO
3
Golden brown
Brownish black
Greenish brown
8
Phloroglucinol
Chocolate brown
Greyish brown
Greenish brown
9
Barfoed
Brownish green
Purplish black
Greyish brown
10
Sodium nitroprusside
Light brown
Reddish brown
Greenish brown
11
FeCl
3
Muddy brown
Purplish Brown
Brownish green
12
1(N) NaOH
Rust brown
Purplish black
Brownish green
13
Acetic acid
Brown
Greenish black
Yellowish-brown
14
1(N) HCl
Brown
Blackish brown
Blackish brown
15
Methanol
Rust brown
Purplish black
Blackish brown
16
1(N) NaOH in Methanol
Brown
Blackish brown
Brownish green
188
Acharya et al. / Journal of Applied Pharmaceutical Science 6 (11); 2016: 185-190
Quantification of different signature phytochemicals
present in the methanolic extract of V. volvacea showed three
major antioxidant compounds that are present in the following
order: ascorbic acid > phenol > flavonoid. β carotene and lycopene
were found in negligible amount (Table 2).
Table 2: Mycochemical estimation of methanolic extract of Volvariella
volvaceae.
Mycochemical constituents
Amount
Total phenols (μg GAE/mg of extract)
8.75 ± 0.44
Total flavonoids (μg QE/mg of extract)
1.56 ± 0.22
β carotene (μg/mg of extract)
0.286 ±0.022
Lycopene (μg/mg of extract)
0.231 ± 0.021
Ascorbic acid (μg/mg of extract)
9.7125 ± 0.528
In comparison with already published reports of
mushrooms such as Griffola frondosa (0.37 μg/mg of extract)
(Acharya et al., 2015a), Ganoderma lucidum (2.77 μg/mg of
extract) (Acharya et al., 2015b), Macrocybe crassa (1.81 μg/mg of
extract) (Acharya et al., 2015c), Lentinula edodes (3.96 μg/mg of
extract) (Acharya et al., 2015d), Pleurotus ostreatus (11.1 μg/mg
of extract) (Acharya et al., 2016a), and Laetiporus sulphureus (2
μg/mg of extract) (Acharya et al., 2016b), ascorbic acid content of
this mushroom was found to be the highest one. According to
earlier reports, besides fruits and vegetables, mushrooms are also
considered as a huge resource antioxidant compounds. As a part of
this category, ascorbic acid is one of the strong water soluble
antioxidant.
Chromatographic study also can be categorized as a part
of chemical fingerprint. The HPLC fingerprint showed a
characteristic fingerprint profile that serves as markers for quality
control standardization of the crude drug. Qualitative analysis of
the chromatogram revealed existence of 12 peaks with two mobile
phase peaks as presented in Figure 2. R
f
value of each
corresponding peak helps to identify the chemical compounds
present in the drug. R
f
value along with area of each
corresponding peak are given in the Table 3. In order to
investigate the antioxidant activity, the methanolic fraction was
subjected for two different in vitro assays. DPPH radical
scavenging assay constitutes the most simple and widely used
method to evaluate antioxidant activities in comparatively short
time than others. Stable DPPH radical shows absorption maxima at
517 nm. Antioxidant compounds present in the extract decreases
its absorbance due to their hydrogen donating ability. It can be
visualized by discoloration of purple colored solution to yellow.
As shown in Figure 3, scavenging effect of methanolic extract of
V. volvacea increased in a concentration dependent manner. EC
50
value of the methanol extract was 0.12 mg/ml. Our result also
coincides with the earlier works which proves that the methanolic
extract of different mushrooms have strong antioxidant capacity
(Khatua et al., 2013).
Another assay was also carried out to test antioxidant
potentiality of the methanolic extract termed as total antioxidant
capacity assay using phosphomolybdenum method. The theory is
based on the reduction of Mo (IV) to Mo (V) and formation of
green phosphate / Mo (V) complex with characteristic absorption
at 695 nm. The total antioxidant capacity was determined using
linear regression equation expressed as the number of equivalent
of ascorbic acid which was found to be 23.75±1.25 ascorbic acid
equivalent (AAE) per mg of extract. Therefore high content of
ascorbic acid can justify the good antioxidant potentiality of the
extract.
Table 3: HPLC profile of methanol extract of Volvariella volvacea at 278 nm.
Peak no.
Retention time (min)
Area (AU)
1
4.949
724.45807
2
5.207
2581.14868
3
6.165
155.25165
4
8.688
376.34503
5
10.037
314.59995
6
10.354
128.91489
7
10.504
259.02283
8
10.834
392.47763
9
11.282
582.46271
10
12.355
18.17178
11
12.597
10.77141
12
15.806
216.32178
Fig. 2: Enlarged HPLC chromatogram of methanol extract from Volvariella volvacea at 278 nm.
Acharya et al. / Journal of Applied Pharmaceutical Science 6 (11); 2016: 185-190 189
Fig. 3: DPPH radical scavenging activity of methanol extract from Volvariella
volvacea.
CONCLUSION
The present study provides pharmacognostic standards of
V. volvacea powder with the help of modern sophisticated
techniques and relevant parameters. Different pharmacological
parameters including microscopy, organoleptic, mycochemical
quantification as well as chromatographic profiling were reported
for the first time for this mushroom. All these aggregated data will
help to authenticate the crude powder before processing for drug
formulations and also to identify the genuine species.
ACKNOWLEDGEMENT
Financial support and sponsorship: Nil.
Conflict of Interests: There are no conflicts of interest.
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