Journal of Applied Research on Medicinal and Aromatic Plants

Comparative Estimation of Quality Control Parameters and Antioxidant Activity of

Unripe and Ripe Fruits of Morus alba L.
--Manuscript Draft--

Manuscript Number: JARMAP-D-20-00491

Article Type: Research Paper

Keywords: Mulberry; Physicochemistry; Phytochemistry; HPTLC; Flavonoids

Corresponding Author: Kamaruz Zaman, Ph.D.

Dibrugarh University
Dibrugarh, Assam INDIA

First Author: Arpita Paul

Order of Authors: Arpita Paul

Monami Rajiung

Anshul Shakya

Neelutpal Gogoi

Sushil Kumar Chaudhary, PhD.

Kamaruz Zaman, Ph.D.

Abstract: Morus alba L., fruits are used traditionally for its healing properties. The berries of the
plant contain numerous bioactive constituents such as flavonoids, tannins, phenolic
acids and ascorbic acid that escalated its significance in the global nutraceutical
market. Unfortunately, little is reported on the changes in the composition of these
phytoconstituents during the fruit maturation stages. The aim of our study is to
establish the variation in morphological, physicochemical, phytochemical parameters
and fluorescent analysis of ripe and unripe fruits of M. alba . In addition, a method for
HPTLC fingerprint analysis was developed for characterization and quantification of the
three bioactive marker compounds namely quercetin and rutin in ripe and unripe
mulberry fruits. The effect of maturation on antioxidant activity was also investigated.
The results of this study will provide information regarding the effect of maturation on
the accumulation of polyphenols and quantification of specific bioactive markers to
obtain the optimum time for harvesting so that the fruits contain the maximum amount
of nutrition for consumer use.

Suggested Reviewers: Liyaquat Ali, PhD.

Professor, Bangladesh University of Health Sciences
liaquat.phis@gmail.com

Shubhas Mandal, PhD.

Professor, Jadavpur University Department of Pharmaceutical Technology
scmandal1963@gmail.com

Shahid Ansari, PhD.

Professor, Jamia Hamdard Faculty of Pharmacy
shansari189@rediffmail.com

Roshan Sayeed, PhD.

Professor, Deccan School of Pharmacy
roshansalfi@yahoo.com

Opposed Reviewers:

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Cover Letter

COVER LETTER

To Date: 09/04/2020
The Editor,
Journal of Applied Research on Medicinal and Aromatic Plants
Sub: Submission of Manuscript entitled: “Comparative estimation of quality control
parameters and Antioxidant activity of unripe and ripe fruits of Morus alba L.”

Dear Editor,
We intend to publish an article entitled: “Comparative estimation of quality control
parameters and Antioxidant activity of unripe and ripe fruits of Morus alba L.” in your
prestigious journal. The manuscript is prepared based on the original research work carried
out by our team of researcher and is not under consideration for publication elsewhere. Our
study focuses on the changes in morphology, physical and chemical properties of M. alba
fruits during maturation. Comparative estimation of phenols, flavonoids and ascorbic acid
were carried out. Further, a HPTLC fingerprint method for routine quality control of the
radical scavengers namely, quercetin and rutin has been proposed. In addition, the effect of
maturation on antioxidant activity was also investigated. Our findings suggest valuable
information regarding the effect of maturation on fruit quality and advocates harvesting of M.
alba fruits at an appropriate stage to get the maximum nutritional benefit.
We declare no conflict of interest associated with this publication, and there has been no
significant financial support for this work. As corresponding author, I confirm that the
manuscript has been read and approved for submission by all the named authors.
We hope the article will find a suitable place in upcoming issue of your journal.

Yours faithfully,
Dr. Md. Kamaruz Zaman
Assistant Professor
Department of Pharmaceutical Sciences,
Dibrugarh University
Dibrugarh-786004, Assam, India
Tel: +916002441408
E-mail: kzaman91@dibru.ac.in
Manuscript File Click here to view linked References

Comparative Estimation of Quality Control Parameters and Antioxidant Activity of

Unripe and Ripe Fruits of Morus alba L.
Arpita Paul1, Monami Rajiung1, Anshul Shakya1, Neelutpal Gogoi1, Sushil Kumar
Chaudhary2, Kamaruz Zaman1*

1
Department of Pharmaceutical Sciences, Faculty of Science and Engineering, Dibrugarh
University, Dibrugarh - 786 004, Assam, India.
2
Faculty of Pharmacy, DIT University, Mussoorie-Diversion Road, Makkawala, Dehradun -
248 009, Uttarakhand, India.

______ __________________________________________________________________
*Address for correspondence: Department of Pharmaceutical Sciences, Faculty of Science
and Engineering, Dibrugarh University, Dibrugarh - 786004, Assam, India.
Tel: +916002441408
E-mail: kzaman91@dibru.ac.in

Page 1 of 21
Abstract

Morus alba L., fruits are used traditionally for its healing properties. The berries of the plant
contain numerous bioactive constituents such as flavonoids, tannins, phenolic acids and
ascorbic acid that escalated its significance in the global nutraceutical market. Unfortunately,
little is reported on the changes in the composition of these phytoconstituents during the fruit
maturation stages. The aim of our study is to establish the variation in morphological,
physicochemical, phytochemical parameters and fluorescent analysis of ripe and unripe fruits
of M. alba. In addition, a method for HPTLC fingerprint analysis was developed for
characterization and quantification of the three bioactive marker compounds namely
quercetin and rutin in ripe and unripe mulberry fruits. The effect of maturation on antioxidant
activity was also investigated. The results of this study will provide information regarding the
effect of maturation on the accumulation of polyphenols and quantification of specific
bioactive markers to obtain the optimum time for harvesting so that the fruits contain the
maximum amount of nutrition for consumer use.

Key words: Mulberry; Physicochemistry; Phytochemistry; HPTLC; Flavonoids.

Abbreviations: UMFE: unripe mulberry fruit extract; RMFE: ripe mulberry fruit extract;
GAE: gallic acid equivalent weight; QE: quercetin equivalent weight; DPPH: 2, 2-diphenyl-
1-picrylhydrazyl; IC50: Inhibitory concentration 50 percentage.

Page 2 of 21
1. Introduction
White mulberry, Morus alba L., Moraceae, is a deciduous medium-sized tree cultivated in the
tropical countries for rearing silkworms and ruminants (Arabshahi-delouee and Urooj, 2007).
This plant holds a good position in the traditional system of medicine for its ability to prevent
and combat several diseases. The indigenous people use the fruits to treat anaemia, weakness,
fatigue and nourish the blood (Nadkarni, 1976). The fruits possess cleansing properties and
are used as a mouth wash and to prevent premature greying of hair (Arya, 1997). The fruits
for their great taste are eaten as such or made into fruit powder, jams, jellies, concentrated
juice or as a functional food. These berries are rich in polyphenols, flavonoids, vitamin C,
carotene, vitamin D, minerals and other health-promoting compounds (Butt et al., 2008).
Several reports have suggested that mulberry fruits have positive effects on metabolic,
proliferative and neuronal disorders (Jiao et al., 2017; Abbasi et al., 2009; Kim et al., 2010).
Further, in the European countries M. alba is welcomed as a ‘superfood’ due to the presence
of the high amount of bioactive constituents (Chen et al., 2018; Park et al., 2017), which are
beneficial to promote health and longevity (Natić et al., 2015).

Recently, there is an inclination towards plant-derived food, as epidemiological studies

suggest that diet rich in fruits and vegetables reduce the risk of several types of diseases such
as diabetes, cancer and cardiovascular diseases (Riboli and Norat, 2003; Bazzano and
Serdula, 2003; Faller and Fialho, 2009). Moreover, studies have shown that deep coloured
fruits like mulberry have higher content of polyphenols that play a prominent role in the
maintenance of human health (Bae and Suh, 2007; Dillard and German, 2000).

Fruit ripening is a complex process, it involves changes in the chemical composition, colour,
texture and flavour (Slimestad and Verheul, 2009). There is either a steady increase or a
decrease in polyphenol content as the fruits pass through the maturation phase (Serranoet al.,
2005; Wang and Zheng, 2001). This variation in phenolics is bought about not only by
cultivator, season or genetic differences but also by the degree of maturation (Aherne and
Brien, 2002; Zadernowski, 2005). However, there are no reports on changes in physical and
chemical properties of M. alba induced by ripening.

Therefore, our study focuses on the variation in organoleptic, physicochemical and

phytochemical parameters of mature ripe and unripe fruits of M. alba. Besides, a method for
HPTLC fingerprint analysis was developed for characterization and quantification of the two
bioactive marker compounds namely quercetin and rutin in ripe and unripe mulberry fruits.

Page 3 of 21
The effect of maturation on antioxidant activity was also investigated. The results of this
study will provide information regarding the effect of maturation on the accumulation of
polyphenols and quantification of specific bioactive markers to obtain the optimum time for
harvesting so that the fruits contain the maximum amount of nutrition for consumer use.

2. Materials and methods

2.1. Collection and identification of the plant material

The fruits were harvested at an interval of one week in March 2018 from Dibrugarh
University campus (27°47ʺ latitude and 94°91ʺ longitude), Assam, India. The plant was
identified and authenticated by the Botanical Survey of India, Eastern Regional Centre,
Shillong (No: BSI/ERC/2018/Tech/468, dated 22.10.2018).

2.2. Preparation of the extracts

After picking the ripe and unripe fruits were separated. One part of the sample was used for
determination of organoleptic properties. While the other part of the sample was dried
separately under shade. The dried materials were subjected to pulverization and then passed
through sieve no. 30 to get a coarse powder of the crude drug. The dried powder was
extracted with 70% methanol by cold maceration at room temperature for 48 hrs. The extracts
were filtered through Whatmann no.1 filter paper. This was repeated for multiple times and
similar extracts were pooled together, concentrated and vacuum evaporated using a rotary
evaporator at a temperature of 45°C and the solvent traces were removed by lyophilisation.
The freeze-dried extracts thus obtained were stored at low temperature in airtight containers.

2.3. Organoleptic evaluation

Visual inspection provides the simplest and quickest means to establish identity, purity and
quality of the crude drug. The sensory parameters like colour, odour, taste, size, shape and
texture should be evaluated. For the morphological studies, fresh fruits of M. alba L. were
investigated for different organoleptic features by repeated observations. The weight of the
fruits was also measured. The experiments were replicated three times and the results were
expressed in mean ± standard deviation.

2.4. Physicochemical analysis

Page 4 of 21
The physicochemical parameters such as ash value (total ash, water-soluble ash, acid
insoluble ash), extractive value and moisture content of the ripe and unripe fruit powders
were carried out as per WHO guidelines on quality control methods for medicinal plants
(WHO, 2007). Each experiment was carried out in triplicates and the results were expressed
as mean ± standard deviation.

2.5. Preliminary phytochemical screening

The unripe mulberry fruit extract (UMFE) and ripe mulberry fruit extract (RMFE)were
evaluated for the presence of plant secondary metabolites such as carbohydrates, proteins,
alkaloids, flavonoids, tannins, steroids and glycosides as described by Harbone, Evans and
Trease, and Software (Harborne, 1973; Evans, 1989; Sofowora, 1996).

2.6. Fluorescence analysis

Fluorescence spectroscopy is a rapid and non-destructive technique allowing the screening of

a large number of plant samples (Karoui and Blecker, 2011). Thus, fluorescence spectroscopy
has been utilized for the authentication of different products. For the study a small quantity of
the powdered drug was placed on a non-fluorescent glass slide, a few drops of freshly
prepared reagent was added and mixed properly with a glass rod. Then the mixture was
allowed to stand for few minutes, observed under visible light, short (254 nm) and long (366
nm) UV radiations in a UV chamber and were recorded.

2.7. Quantitative estimation

An analytical method was developed and validated to determine the content of total phenols,
total flavonoids and ascorbic acid in UMFE and RMFE, by the UV/Vis spectrophotometric
method.

2.7.1. Estimation of total phenolic content

The Folin-Ciocalteu method is used to measure the total concentration of phenolic content
with slight modifications (Singleton and Rossi, 1965). The sample solution (1mg/ml) was
mixed with 100 μl of FolinCiocalteu’s reagent and 4 ml of distilled water in a test tube. After
5 minutes, 1.5 ml 0f 20% of sodium carbonate was added and the reaction mixtures were
allowed to incubate for 30 minutes at room temperature. Later, the absorbance was measured
at 760 nm in UV-Vis spectrophotometer. Thereafter, the calibration curve was plotted using
gallic acid (10-100 μg/ml). The total phenolic content of the extract was determined using Eq

Page 5 of 21
1. The results were expressed as mg of gallic acid equivalent weight (GAE)/ g of the extract.
The analysis was done in triplicate and recorded as mean ± standard deviation.
𝐶×𝜂
𝑇= 𝑤
…………. Eq 1
Where,

T represents total phenolic content expressed as mg of gallic acid equivalent weight (GAE), c
represents calculated concentration from the standard curve of gallic acid (μg/ml), η
represents the volume of the extract (ml), and w represents the weight of extract (g).

2.7.2. Estimation of total flavonoid content

The aluminium chloride colourimetric assay method was used to measure the total flavonoid
content (Makris et al., 2007). The sample solution (1mg/ml) was mixed with 4 ml of distilled
water and 0.3 ml of 5% sodium nitrite solution in a test tube. After 5 minutes, 0.3 ml of 10%
of aluminium chloride was added. Then 2 ml of 1 M sodium hydroxide was added and the
volume was made up to 10 ml with distilled water. Finally, the absorbance was measured at
510 nm against a blank prepared by replacing the sample solution with distilled water.
Thereafter, the calibration curve was plotted using quercetin (10-100 μg/ml). The total
phenolic content of the extract was determined using Eq 1. The total flavonoid content of the
extract was expressed as mg of quercetin equivalent weight (QE)/ g of the extract. The
analysis was done in triplicate and recorded as mean ± standard deviation.
2.7.3. Estimation of ascorbic acid content
The ascorbic acid content in M. alba fruit extracts was determined by 2, 6-
dichloroindophenol method with slight modifications (Yen and Chen, 1996). The sample
solution (1 mg/ml) was mixed with 9 ml of 50 μM of 2, 6 –dichloroindophenol dye was
added and incubated at room temperature 3 min. the absorbance was measured at 515 nm
against a blank prepared by replacing the sample solution with distilled water. Later, the
concentration of the ascorbic acid in the fruit extracts were determined by comparison with
the absorbance of standard ascorbic acid at different concentrations. The analysis was done in
triplicate and recorded as mean ± standard deviation.
2.8. HPTLC analysis
In this study, the quantification of two flavonols were carried out by HPTLC.
Chromatographic analysis was performed on TLC aluminium plates (20 × 10 cm) pre-coated
with silica gel 60 F254 (Merck) (0.2mm thickness) equipped with Linomat V sample
applicator (CAMAG, Muttenz, Switzerland) with a 100 μL (Hamilton, USA)syringe. 100 mg

Page 6 of 21
of UMFE and RMFE were sonicated separately in 1 mL methanol for 20 min followed by
centrifugation (RM-03 Plus, REMI, India) for 10 min. The supernatant was used for the
study. Quercetin (1mg/ml) and rutin (1mg/ml) were used as standards. Applications were
made to the plates as bands of 8 mm width and 11.5 mm apart and 10.0 mm from the bottom
edge of the same chromatographic plate. The chromatograms were developed in ascending
method to a distance of 70 mm, in a CAMAG glass twin-trough chamber previously saturated
with mobile phase vapour for a period of 30min, at a temperature of 25±2° C with a relative
humidity of 33%. Here, the mobile phases Toluene: Ethyl acetate: Formic acid (10:8:0.4);
Hexane: Ethyl acetate: Glacial acetic acid (8:12:0.2) were used for identification and
quantification of quercetin and rutin respectively. After development, the plates were dried
and were observed under CAMAG UV cabinet (254 and 366 nm). Quercetin (detection
wavelength = 262 nm) and rutin (detection wavelength = 262 nm) were identified by
superimposing the UV spectra of the standards and the samples within the same Rf range and
quantified utilizing calibration plot.
2.9. Antioxidant assay
The antioxidant characteristics of M. alba extracts were assessed by determining DPPH (2, 2-
diphenyl-1-picrylhydrazyl) radical scavenging activity and nitric oxide scavenging activity.
2.9.1. Determination of DPPH radical scavenging activity

The DPPH radical scavenging activity of the extracts was measured according to the method
described by Brakuet al. (2013) with slight modifications (Barku et al, 2013). At first sample
solutions (100 μg/ml) was prepared by dissolving 1 mg of each extract in 10 ml of methanol.
Then 0.5 ml of the sample solution was mixed with 0.5 ml of DPPH radical (0.1 mM solution
of DPPH prepared in methanol) in a 96-well plate, and incubated in dark for 30 minutes at
room temperature. Later the absorbance was measured at 517 nm against a blank prepared by
replacing the sample solution with methanol. Quercetin was used as the standard anti-oxidant.
The IC50(Inhibitory concentration 50 percentage) values were calculated. The assay was
carried out in triplicate and values were expressed as mean ± standard deviation. The
percentage of inhibition was calculated using the following expression:

𝐴 (𝑏𝑙𝑎𝑛𝑘) − 𝐴 (𝑠𝑎𝑚𝑝𝑙𝑒)
% 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = × 100 ..……… Eq 2
𝐴 (𝑏𝑙𝑎𝑛𝑘)

Where A (blank) and A (sample) stands for the absorption of the blank sample and absorption
of the tested extract sample respectively.

Page 7 of 21
2.9.2. Determination of Nitric oxide scavenging activity

The capacity of extracts to scavenge nitric oxide was assessed by the method of Habu and
Ibeh (2015). Sample solutions (100 μg/ml) were prepared by dissolving 1 mg of each extract
in 10 ml of methanol. Then 0.5 ml of sodium nitroprusside (10 mM in Phosphate buffer
saline of pH 7.4) was mixed with 1 ml of different concentration of extract solutions including
the blank and incubated at 25° C for 120 minutes. After incubation, 1.2 ml of Griess reagent
was added to all the reaction mixtures and the absorbance was measured at 546 nm(Habu and
Ibeh, 2015). Gallic acid was used as the standard anti-oxidant. The IC50 values were
calculated. The assay was carried out in triplicate and values were expressed as mean ±
standard deviation. Percentage inhibition was calculated using Equation 2.

3. Result and Discussion

3.1. Organoleptic evaluation

M. alba is multiple fruits classified as a drupe. As the fruits undergo the maturation phase,
change in the size, shape, colour, texture and taste takes place. The unripe mature fruits are
white, slowly turn to pink and finally to red/purple when they are completely ripe. The ripe
fruits are succulent and are sweet. The reduction in astringency during the fruit ripening
process is due to the reduction of tannin content, as tannins are responsible to impart
astringent taste to unripe fruits (Jelled et al., 2017).There was a significant increase in the
weight of ripe fruits and our results were comparable with Jiang and Nie (2015). The
macroscopic features which were observed are listed in Table 1.
3.2. Physico-chemical analysis

Physico-chemical evaluation of crude drug serves as a tool for their identification. The ash
content helps in the identification of contaminants or adulterants present in the crude drug.
The total ash is the residue left after incineration of the crude drug. The total ash, acid
insoluble and water-soluble analysed in this study are summarized in Table 2.

Extractive value represents the weight of crude extract obtained after extracting with different
solvents (Baidoo et al., 2019). The results of the study reveal that the polar solvents like
methanol, ethanol and water have high extractive power than the non-polar solvents such as
petroleum ether, chloroform and ethyl acetate (Table 2). This indicates that the mulberry
plant contains more of polar phytoconstituents in their fruits. Moreover, the extractive value
of the ripe fruits was found to be higher than unripe fruits suggesting that when the ripe fruits

Page 8 of 21
of mulberry pass through their maturation phase more amount of polar secondary metabolites
get accumulated in them.

The moisture content should be determined, as high moisture present in the crude drug can
lead to deterioration of the crude material during its processing and storage. The moisture
content was found to be high in the ripe fruits than the unripe fruits (Table 2) indicating that
they are highly susceptible to microbial contamination. Thus, preventive measures should be
taken during the processing and storage of the ripe mulberry fruits.

3.3. Preliminary phytochemical screening

Mulberry fruits are reported to contain alkaloids and structures of most of these alkaloids
have been identified (Kim et al., 2014).The preliminary phytochemical studies prove the
presence of the major classes of phytoconstituents such as alkaloids, carbohydrates, proteins,
flavonoids, tannins and glycosides in both ripe and unripe fruits of M. alba(Table 3).
Polyphenols are present in ample amounts in these fruits (Yang et al., 2010). As in our study
polyphenols are detected in both ripe and unripe fruits so further quantitative and HPTLC
analysis was carried out to detect their amounts which will ultimately determine the right
harvesting time.

3.4. Fluorescence analysis

Fluorescence analysis of powdered ripe and unripe fruits of M. alba was performed using
various chemical reagents. The fluorescent characteristic of the chemical treated crude drug
was then observed in visible, short (254 nm) and long (366 nm) UV light (Table 4).

3.5. Quantitative estimation

Phenolic compounds present in fruits play an important role in the maintenance of health and
their content helps in determining the fruit quality (Voca et al., 2008). Mulberry fruits are rich
in phenolic compounds which are associated with antioxidant, anti-diabetic, antiviral,
nephroprotective activities(Yang et al., 2010; Lee et al., 2018; Abbasi et al., 2009). In the
present study, the total phenolic content of the unripe and ripe mulberry fruits was evaluated
using Folin-Ciocaletu’s reagent. The polyphenol content gradually increased during the
maturation of the mulberry fruits (Gerasopoulos and Stavroulakis, 1997) and the results of the
study confirm it (Table 5). According to Lou et al. (2012) the total phenolic content increase
when the fruits are still in an immature stage, then there is a decrease in the phenolic content
as they pass through the immature stage to mature stage and finally, there is a steady increase

Page 9 of 21
when the matured fruits start to ripen(Lou et al., 2012). Even in our study, an increase in
phenolic content was examined when the mature fruits change to the edible ripe state.

Flavonoids possess strong antioxidant, antiproliferative and antibacterial activities, which

tend to increase with an increase in stress levels in plants (Harborne and Williams, 2000).
The total flavonoid content of M. alba fruits is shown in Table 5. The flavonoid content was
found to be higher in the ripe fruits and our results were comparable with (Mahmood et al.,
2012). In contrast, the flavonoid content was found to decrease in a study conducted by
(Jelled et al., 2017). This variation in the flavonoid content may be influenced by changes in
the gene, climate, temperature and time of harvest (Lakenbrink et al., 2000).

Ascorbic acid is the most potent antioxidant detected in the fruits of M. alba(Abbasi et al.,
2009). The ascorbic the acid content in the unripe and ripe fruits of M. alba are given in
Table 5. The ascorbic acid content detected in unripe fruits was higher than the ripe fruits, as
found in sour orange, cashew, mango, pineapple, orange and guava but it was not correlated
with water melon, where there is a reduction in ascorbic acid as the progress of the fruit
towards the end of maturation (Muhammad et al., 2014). The reason for the reduction in
ascorbic acid content during ripening might be due to enzymatic changes, stabilization of
ascorbic acid by higher levels of citric acid ad malic acid, breakdown of ascorbic acid by
fructose, increase in time and the temperature of exposure (Igwe, 2014).

3.6. HPTLC analysis

Berries are rich in polyphenolic compounds. Quercetin and rutin are present in mulberries
(Park et al., 2017) but as per the author’s knowledge, their variation during the fruit
maturation phase is not yet reported by HPTLC method. So, an HPTLC fingerprint method
for quantification of these flavonols in the extracts is carried out with the markers quercetin
and rutin. The HPTLC fingerprints of quercetin were characterized by light brown colour
bands under UV at 254 nm (Figure 1). The plate was scanned at 262 nm and the calibration
plot indicated a linear function of concentration in the range 100 to 1200 ng of quercetin. The
linear regression equation that was obtained, y = 5.647×10-9x+ 2.232×10-4 and the regression
coefficient (R) was found to be R2 = 0.998015. The identification of quercetin was confirmed
by superimposing the UV spectra of the standards and the samples within the same Rf͠ 0.334.
Figure 2. The content of quercetin detected in unripe fruits (209.4µg/ml) was higher than the
ripe fruits (79 µg/ml). Our work is in close agreement with that of (Mahmood et al., 2012).

Page 10 of 21
Rutin, a glycosidic form of quercetin present in M. alba is reported to have antioxidant,
antidiabetic, nephroprotective and neuroprotective effects (Wang et al., 2013)(Lee et al.,
2018)(Seo et al., 2015). In our study, the bands of standard rutin were found to match with
that of the extracts (Figure 3). The calibration curve for rutin indicated a linear function of
concentration in the range 100 to 1000 ng of rutin. The linear regression equation obtained
was, y = 6.607×10-9x+ 9.98×10-4 and the regression coefficient (R) was found to be R2 =
0.996172. The identification of rutin was confirmed by superimposing the UV spectra of the
standards and the samples within the same Rf 0.235, Figure 4. The content of rutin was found
to increase in the ripe fruits as in tomatoes (Bhandari and Lee, 2016). The content of
quercetin and rutin quantified by HPTLC method are summarized in Table 6.The time of
harvest is seen to have a strong impact on the content of the two flavonols, quercetin and
rutin.
3.7. Antioxidant assay
Antioxidant capacity of a food product should be evaluated to examine the process of
oxidation and thus to check its health benefits. Several antioxidants such as phenolic
compounds, flavonoids, ascorbic acid are present in M. alba (Iqbal et al., 2012). DPPH is
widely used to evaluate the antioxidant activity of natural products. The effect of antioxidants
on DPPH is due to their hydrogen donating capacity (Bauman et al., 1980). In our study,
UMFE and RMFE were evaluated for their free radical scavenging activity against quercetin
as a standard compound. Several reports suggest that lower the IC50 value stronger is the
antioxidant activity (Alara et al., 2018). The IC50 value for UMFE, RMFE and quercetin were
found to be 71.94 µg/ml, 54.18 µg/ml and 45.60 µg/ml respectively. Although IC50 value of
quercetin is lower than UMFE and RMFE, the extracts showed antioxidant activity. The
IC50value of RMFE was found to be lower than UMFE, this indicates that RMFE shows
better antioxidant activity than UMFE.
A group of researchers conducted antioxidant study of M. alba fruits at different stages of
maturation. They observed that the ripe fruits of M. alba gave the best antioxidant activity
when compared to ripe M. nigra and M. rubra fruits (Jelled et al., 2017). Another report
suggests that there is a steady increase in antioxidant capacity of M. alba in later stages of
fruit development (Lou et al., 2012) and the results of our study were comparable with the
works cited above.
In the nitric oxide scavenging assay, the nitric oxide generated from sodium nitroprusside
reacts with oxygen to form nitrite. These nitrite ions then diazotize with sulphanilamide and
naphthyl ethylenediamine, a pink colour formed, which is measured at 546 nm (Habu and

Page 11 of 21
Ibeh, 2015). The antioxidants present in the mulberry fruit extract donate its proton to nitrite
radical leading to a reduction in absorbance. The reduction in the absorbance is a measure of
the extent of nitrite radical scavenging activity. In our study, gallic acid was used as a
standard compound. The IC50 value of RMFE was found to be lower than UMFE (Table 7).
The underlying reason behind the superiority in antioxidant activity of RMFEmay be due to
the presence of higher amounts of antioxidant compounds’ or may be due to the synergistic
effect of the phytoconstituents present in the extract that are more potent in quenching NO
radical than RMFE. To author’s knowledge nitric oxide radical scavenging test was not used
for mulberry fruits.

4. Conclusion
The pattern of accumulation of phenolic compounds varied during the maturation of M. alba
fruits. The variation in phenolic, flavonoid and antioxidant capacity can be correlated with
fruit colour, taste, texture, moisture content, total phenolic, total flavonoid and ascorbic acid
content. Ascorbic acid and quercetin contents decreased as the matured unripe fruits entered
the ripening stage. In contrast, rutin increased from unripe to ripe stage. The phenolic,
flavonoid content was higher in the ripe fruits. These results suggest that the ripening stage
play a significant role in the levels of phenolics, flavonoids, ascorbic acid, quercetin and rutin
contents. The mature ripe stage is proper picking period as they have a higher content of
phenols, flavonoids and ascorbic acid with better taste and antioxidant capacity. But the
matured unripe ones can also serve as natural antioxidants when processed.

Page 12 of 21
Conflict of interest
The authors declare that they have ‘‘no conflict of interest’’ with the article.

Funding
This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.

Acknowledgement

The authors are also grateful to University Grants Commission (UGC) for providing the
HPTLC facility under UGC-SAP (DRS-1) Programme.

Page 13 of 21
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Tables
Table 1: Evaluation of Organoleptic features of M. alba fruits.

Organoleptic features Unripe fruits Ripe fruits

Colour White Pink/red

Odour Characteristic Characteristic
Taste Sour/ astringent Sweet
Size 1-5 cm 2-5 cm
Shape Cylindrical/ ellipsoidal Cylindrical/ ellipsoidal
Texture/ appearance Unpalatable/ unjuicy Succulent/ juicy
Weight 1.59 ± 0.32 4.18 ± 0.47

Table 2: Evaluation of physicochemical parameters of M. alba fruits.

Physicochemical Unripe fruits Ripe fruits

parameter(s) Mean ± SD (%w/w) Mean ±SD (%w/w)
Ash value
Total ash 10.91 ± 0.07 9.97 ± 0.46
Acid-insoluble ash 5.31 ± 0.05 6.39 ± 0.56
Water-soluble ash 3.14 ± 0.03 2.79 ± 0.47
Extractive value
Petroleum ether 1.79 ± 0.82 2.30 ± 0.53
Chloroform 6.30 ± 0.76 5.10 ± 0.98
Ethyl acetate 8.39 ± 0.83 9.40 ± 0.75
Ethanol 11.37 ± 0.08 13.16 ± 0.07
Methanol 14.02 ± 0.03 14.76 ± 0.41
70% methanol 15.59 ± 0.74 17.17 ± 0.77
Water 13.63 ± 0.50 15.80 ± 0.09
Moisture content 5.25±0.47 8.41 ± 0.18
SD represents standard deviation and each experiment was performed in triplicates.

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Table 3: Preliminary phytochemical screening of extracts of unripe and ripe fruits of M. alba.
Phytoconstituent(s) UMFE RMFE
Alkaloids + +
Carbohydrates + +
Amino acids / Proteins + +
Flavonoids + +
Steroids + -
Tannins and Phenolic + +
compounds
Glycosides + +
Key: + Detected; - Not detected
Table 4: Fluorescence analysis of powdered ripe and unripe fruits of M. alba.
Powder+ Reagent/Solvent Visible light UV-254 nm UV-366 nm
M. alba ripe fruits
No reagent Brownish NF NF
yellow
Acetic acid Yellow Grey White
Distilled water Orange Violet Grey
Methanol Pale yellow Grey Grey
Ethanol NF NF White
Dil. Hcl Reddish Dark blue Grey
brown
Dil. H2SO4 Brick red Dark blue Grey
Dil. Ammonia Brown Violet Grey
Dil. HNO3 Reddish Dark blue Grey
brown
1N NaOH Reddish Violet Grey
brown
Hexane NF NF NF
Chloroform Pale yellow NF Grey
Toluene Yellow Violet White
Carbon tetrachloride NF Violet NF
Potassium dichromate Yellow Violet Black
5% Fecl3 Greenish Blue NF
yellow
5% Picric acid Yellow Violet Black

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5% KOH Brown Violet Grey
5% Iodine Reddish Blue Green
brown
M. alba raw fruits
No reagent Brown NF NF
Acetic acid Yellow White White
Distilled water Yellow Violet Grey
Methanol Yellow White Grey
Ethanol Green Grey Grey
Dil. Hcl Peach Blue Violet
Dil. H2SO4 Peach White Grey
Dil. Ammonia Brown Grey Green
Dil. HNO3 Orange Blue Blue
1N NaOH Red Green Blue
Hexane Olive green Grey Grey
Chloroform NF NF NF
Toluene Green Green Violet
Carbon tetrachloride NF NF NF
Potassium dichromate Yellow Black Blue
5% Fecl3 Yellow Blue Blue
5% Picric acid Yellow Black Green
5% KOH Yellow Blue Blue
5% Iodine Red Violet Violet
NF – No fluorescence
Table 5: Total phenolic, flavonoid and ascorbic acid content of unripe and ripe fruits of M.
alba.
Parameter(s) UMFE RMFE
Total Phenolic Content 26.54 ± 0.23 31.73 ± 0.56
(mg/g equivalent of gallic acid)
Total Flavonoid Content 14.55 ± 0.31 22.41 ± 0.60
(mg/g equivalent of quercetin)
Ascorbic acid content (mg/g) 26.56 ± 0.47 18.14 ± 0.63

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Table 6: Amount of bioactive marker compounds present in M. alba fruit extracts quantified
by HPTLC method.
Bioactive marker UMFE RMFE
(μg/100 mg of extract) (μg/100 mg of extract)
Quercetin 209.40 79.00
Rutin 31.67 91.57

Table 7: Antioxidant activity of extract from unripe and ripe fruits of M. alba.
IC50 of DPPH (µg/ml) IC50 of Nitric oxide (µg/ml)
Quercetin 45.60 --
Gallic acid -- 41.89
UMFE 71.94 73.21
RMFE 54.18 56.39

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Figure

Figures

Figure1: Visualization of TLC plate under 254 & 366 nm. S1-S7: 100-1200 ng/band of
quercetin; T1 -T3: 5µL of unripe mulberry fruit extract (UMFE); T4-T6: 5µL of ripe mulberry
fruit extract (RMFE).

Figure 2: Fingerprint chromatogram of quercetin, unripe mulberry fruit extract (UMFE) and
ripe mulberry fruit extract (RMFE) at 262 nm.

Page 1 of 2
Figure 3: Visualization of TLC plate under 254 & 366 nm. S1-S7: 100-1600 ng/band of rutin;
T1 –T2: 5µL of unripe mulberry fruit extract (UMFE); T3-T4: 5µL of ripe mulberry fruit
extract (RMFE).

Figure 4: Fingerprint chromatogram of rutin, unripe mulberry fruit extract (UMFE) and unripe
mulberry fruit extract (UMFE) at 262 nm.

Page 2 of 2
Conflict of Interest

Conflict of interest
The authors declare that they have ‘‘no conflict of interest’’ with the article.