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Exploiting Plant Cell Culture Technology for Scalable Biosynthesis of Therapeutically

Relevant Compounds from Citrus maxima Peel: An Eco-Friendly Approach to Drug
Development and Nutraceutical Applications

Ranjit kumar 1, Dr.Saurabh Mishra2, Dr.Vijay Nema3

1,2Department of Biotechnology and Life sciences .Mangalayatan University, Aligarh

3ICMR-National Institute of Research in Tribal Health,Jabalpur

Abstract

The increasing demand for natural bioactive compounds with therapeutic and industrial
applications has driven the exploration of sustainable production methods. This study focuses
on the use of plant cell culture techniques to produce bioactive compounds from Citrus maxima
(pomelo) peel, an agricultural byproduct rich in flavonoids, phenolic acids, and essential oils
known for their antioxidant, anti-inflammatory, and antimicrobial properties. Traditional
extraction methods face challenges such as seasonal variability, environmental factors, and
depletion of natural resources, which plant cell culture systems can overcome by providing a
controlled and reproducible platform for metabolite production.
Callus cultures were successfully established using Murashige and Skoog (MS) medium
supplemented with 2,4-D and Kinetin. Optimization of growth regulators, sucrose concentrations,
and culture conditions enabled stable callus proliferation with enhanced biosynthetic potential.
Methanol-based extractions were most effective for isolating flavonoids such as Naringin and
Naringenin, whose presence and concentrations were confirmed through advanced analytical
techniques, including HPLC and LC-MS.
This research highlights the sustainable use of Citrus maxima peel, reducing environmental waste while
providing a valuable source of bioactive compounds. The findings demonstrate the potential of plant
cell culture techniques for scalable production, with applications in pharmaceuticals, nutraceuticals, and
functional foods. By establishing a reliable platform for bioactive compound biosynthesis, this
study lays the groundwork for further optimization and scale-up processes, offering an eco-
friendly solution to meet growing industrial and therapeutic demands.
Keywords
Plant cell culture, Citrus maxima, Pomelo peel, Phytochemical analysis, Flavonoids, Naringin,
Naringenin, Bioactive compounds, Sustainable production, Callus induction, Murashige and
Skoog medium, HPLC, LC-MS, Agricultural byproducts, Bioinformatics, Nutraceuticals,
Functional foods, Antioxidant properties, Anti-inflammatory, Pharmaceutical applications.
1. Introduction

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Consumption of fruits is essential for the prevention and treatment ofvarious diseases. Fruits
are affluent sources of secondary metabolites particularly, polyphenolic compounds like
flavonoids. Fruit flavonoids have been interesting as they have considerable bioactive
properties. The health benefits of flavonoids are attributed to their action as antioxidant,
antidiabetic and anticancer agents (Zhang et al., 2011). Modern research has shown that plant
flavonoids protect against cardiovascular diseases (Knekt et al., 2002). These low molecular
weight substances are phenylbenzo-pyrones with a group of structures based on a common
three-ring nucleus. Since ancient times, pomelo hasbeen used as appetizer, antitoxic, cardiac
stimulant, and stomach tonic (Arias and Ramon, 2005). The work of Xu et al., (2008), suggests
that the major flavanoids of pomelo are hesperidin, naringenin, and naringin (Xu et al., 2008).
Wide-ranging studies of pomelo have exposed its antioxidant properties using the ferric
reducing antioxidant power assay in vitro (Bailey, Oliveri and Levin, 2013). Hence it is clear
that Citrus maximais a good source of bioactive flavonoids.

The fruit rind consists of a leathery endocarp, having multiple oil glands. It possesses
antioxidant, anti-bacterial, anti-fungal, anti-platelets, hepatoprotective, anti-cancer, and anti-
diabetic properties. Abirami et al., (2013) reported the in vitro antibacterial activity of
methanolic extracts of C. maxima fruit peel extracts against S. aureus (Arumugam Abirami,
2013). In another similar study the antibacterial activity of the volatile constituents of C.
maxima fruit epicarp against Bacillus pumilus, was reported by Pandey et al., (2010) (Pandey,
Dubey and Saini, 2010). The Citrus maxima peel has more amount of antioxidants (phenols,
flavonoid, ascorbic acid) compared to the pulp. It was also reported that the white variety of
Citrus maxima had higher antioxidant content and capacity compared to the pink variety (Toh,
Khoo and Azrina, 2013). Aimee et al., (2014) reported the antioxidant activity of the
phytochemical constituents of the pericarp, mesocarp of ethanolic extracts of C. maxima fruit
peel (Barrion et al., 2014). In the present study also we find flavonones like Naringin and
Naringenin in C.maxima fruit peel.

Improvement of Citrus species by conventional breeding methods is hindered by a variety of

aspects like nucellar polyembryony, heterozygosity, sexual incompatibility etc (Koltunow
1993). In such conditions, in vitro culture procedures can be a solution to the problems. The
biotechnological production of precious secondary metabolites in plant cell cultures is an
attractive alternative to the extraction from whole plants (Ramachandra Rao and Ravishankar,
2002). Hence, plant tissue culture is a valid substitute for the production of pharmaceutically
useful phytonutrients. Apart from refining the existing cultivars, micropropagation is capable

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of mass scale generation in short time span. Thus, plant tissue culture is a influential tool for
propagation and enhancement of several useful plants.

This study intended to find the optimal growth regulator for effectual regeneration of Citrus
maxima. Also, the effect of sucrose concentration, media pH, and photoperiod on callogenesis
was studied. We developed an effective and reproducible method for callus induction and
regeneration from Citrus maxima peels. The effect of elicitors on production of bioactive from
callus was also determined. Further, phytochemical screening identified the compounds in the
callus and the promising range of properties that the callus may possess. Moreover, quantitative
estimations of bioactive compounds in the callus are done. Thus, the present work gives a
comparative analysis of the quantity of bioactive compounds in wild natural peel and that of
callus of C. maxima fruit peel.

2. Materials and methods

2.1 Culture Media

Plant cell culture medium contains inorganic components, organic compounds and growth
regulators. Modifying components of the medium like concentration and proportion is highly
useful for improving the effectiveness of plant cell cultures. For example, higher level auxin
stimulates cell growth, but frequently negatively impacts production of secondary metabolite.

The media used here was basal Murashige and Skoog (MS) Medium (1962).

2.2. Media prepararion

34 g of this PT010 MS media (Himedia) is weighed and dissolved in distilled water. To this,
0.8% agar was added. pH was adjusted to 5.75 ± 0.5 prior to autoclaving. Autoclaving is done
at 121° C and 15 psi of pressure for 20 minutes.

2.3. Surface sterilization

Fresh fruits from the C. maxima plants growing in the Shimoga and Chikkamagalur districts
were collected and soaked in water for 5 minutes. Then they were washed with antifungal
agent, Bavistin for 5 minutes, after which they were kept under running tap water for 30
minutes. Surface sterilization of peels was done with 0.1 % Mercuric chloride for 15 minutes
and rinsed with autoclaved double distilled water 3–4 times in Laminar air flow chamber.

2.4. Callus induction

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Peels were separated from surface sterilized fruits and excised with scalpel and inoculated on
MS media. Callus obtained was inoculated in culture bottles with 25 ml of MS media (0.8 %
Agar and 3 % sucrose). The sucrose concentration was varied to 5%, 6%, 8% and 10%)
accompanied with various concentrations and combinations of plant growth regulators like
Kinetin (KN), 2,4-Dicholorophenoxy acetic acid (2,4- D), Naphthalene Acetic Acid (NAA),
Indole-3-Acetic acid (IAA), Thidiazuron (TDZ) and Benzyl Amino Purine (BAP). Every
treatment had 10 culture bottles and the experiment was repeated three times. The cultures were
maintained at 23 ± 2°C with dark period for 1 weak and diurnal photoperiod later provided by
fluorescent tubes.

2.5. Subculture of callus

For subculture, healthy calli were cut into small pieces and cultured on MS semisolid media
supplemented with 0.8% agar and 8% sucrose along with growth regulators, 2µM 2,4-D and
5µM Kinetin concentrations. To understand the survival capacity of long-term callus cultures,
small pieces of calli of various age groups were transferred to optimized media (MS medium
with 0.8% agar and 8% sucrose and 2 µM 2,4-D and 5 µM Kinetin). Subcultures were
carriedout even by using 5µM IAA and 10 µM KN and 5 µM NAA and 10 µM KN. For each
treatment, 30 culture bottles were inoculated and the experiment was repeated 3 times. Cultures
were maintained at 23 ± 2 °C with dark period for 1 weak and diurnal photoperiod later
provided by fluorescent tubes. Amongthe above three combinations, only 2µM 2,4-D and 5
µM KN supported the better growth of callus.

2.6. Harvesting and Lyophilization of callus

Callus tissues were harvested and washed with distilled water 3 times to remove any residual
media. Then, the cells were lyophilized at – 40 °C till a constant weight was attained. The initial
weight of callus was 85g. The dry weight of callus after lyophilization was 20g.

2.7. Preparation of extracts

Aqueous extract

7g of the dried lyophilized callus was taken and was mixed with 100mldistilled water. The
mixture was boiled for 10 minutes in water bath and filtered (Whatman filter paper No.1). The
filtrate was cooled and extract was used for further tests. These extracts were stored in
refrigerator at 4°C.

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Soxhlet extracts

7g of the dried lyophilized callus was used for solvent extraction using 80% Hexane, 80%
Methanol, and 100% Ethyl acetate separately in a standard Soxhlet apparatus set-up. The
extraction was done for 2-3 hours at a temperature not exceeding the boiling point of the
solvent. Each extract was transferred to sterile glassvials and kept at 4°C before use.

2.8. Phytochemical analysis of callus extracts of Citrus maxima fruit peel.

Tests for phytochemical screening of different extracts of callus of Citrus maxima fruit peel,
using different solvents were achieved by the same method used earlier for phytochemical
screening of wild fruit peel as explained in material & methods chapter of present study.

Quantitative phytochemical analysis of callus of C.maxima fruit peel with different solvents
involved, Estimation of Total flavonoid content (TFC) and DPPH radical scavenging assay.
These two assays were done by following the same procedure as explained in the material &
methods chapter of present study.

This quantitative analysis was further continued with the Thin Layer Chromatographic
technique, which was employed by comparing callus extract with the standards using the
technique as mentioned in material & methods chapter of present study.

3. RESULTS

3.1 Callus induction and subculture

The peel explants of Citrus maxima showed the following response withrespect to the different
concentrations of auxins and cytokinins. The carbohydrate concentrations were also varied to
develop a proper protocol for the maximum production of callus. Table 3.1. represents the
callus growth response of Citrus maxima peel explants on MS medium with Auxins. The
growth is expressed involume (cc) and percentage callus induction.

Table 3.1. Callus induction from C.maxima peel explants on MS mediacomplemented

with different concentrations of Auxins.

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Concentration Growth response of callus in cc Callus

induction
2,4-D percentage
1st set of 2nd set of 3rd set of Mean

bottles bottles bottles

0.5 µM 2.18 2 2.1 2.07 30%

1 µM 2.4 2.3 2.3 2.33 35%

2 µM 2.9 3 2.9 2.9 85%

5 µM 2.5 2.55 2.4 2.48 45%

10 µM 2.5 2.4 2.3 2.4 50%

20 µM 2 2.1 2.2 2.1 20%

IAA

0.5 µM 0.9 0.9 1 0.93 20%

1 µM 1.1 1.2 1 1.1 35%

2 µM 1.9 1.8 1.9 1.8 40%

5 µM 2.3 2.1 2.2 2.2 50%

10 µM 2 2.3 2.4 2.23 65%

20 µM 2.3 1.9 1.8 2 45%

NAA

0.5 µM 0.6 0.5 0.6 0.55 10%

1 µM 0.7 0.6 0.7 0.6 15%

2 µM 1 0.9 0.9 0.93 20%

5 µM 0.6 0.7 0.7 0.63 30%

10 µM 0.9 1 1.2 1.03 35%

20 µM 0.8 0.5 0.6 0.63 10%

From the above results it is clear that, among the above mentioned auxins, only 2,4-D
supported well for the induction and growth of C.maxima peel explants. This was followed
by IAA and NAA did not support well for the induction and growth of callus. Growth
response of C.maxima to different Cytokinins is shown in Table. 3.2.

Table 3.2: Callus induction from C.maxima peel explants cultured on MS mediumwith
different Cytokinins.

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Concentration Growth response of callus in cc Callus

induction
KN 1st set of 2nd set of 3rd set of Mean percentage
bottles bottles bottles

0.5 µM 0.5 0.6 0.5 0.53 10%

1 µM 1 0.9 1.1 1 30%

2 µM 1.3 1.4 1.3 1.33 40%

5 µM 2.8 2.6 2.7 2.7 80%

10 µM 2.3 2.2 2.1 2.1 50%

20 µM 2 1.9 1.7 1.8 10%

TDZ

0.5 µM 0.5 0.6 0.5 0.53 5%

1 µM 0.6 0.7 0.6 0.63 10%

2 µM 0.5 0.8 0.6 0.63 20%

5 µM 0.9 0.8 0.7 0.7 15%

10 µM 0.9 1 0.9 0.93 10%

20 µM 0.6 0.7 0.63 10%

BAP 0.6

0.5 µM 0.5 0.5 0.5 0.5 5%

1 µM 0.6 0.5 0.5 0.53 10%

2 µM 0.5 0.7 0.6 0.6 20%

5 µM 0.5 0.7 0.5 0.56 15%

10 µM 0.6 0.7 0.5 0.6 10%

20 µM 0.5 0.5 0.7 0.56 10%

Among cytokinins, KN strongly supported the induction and growth of C.maxima peel. The
growth response of peels of Citrus maxima to different concentrations of combinations of plant
growth regulators is represented in Table.3.3 Highest percentage of response was observed in
combination of 2 µM 2,4-D and 5 µM Kinetin

Table 3.3: Callus induction response of C.maxima peel explants cultured on MSmedia
accompanied with different mixtures of Auxins and KN

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Concentration Growth response of callus in cc Callus

induction
2,4-D + KN 1st set of 2nd set of 3rd set of Mean percentage
bottles bottles bottles

1µM +1 µM 1 1.3 1.7 1.33 30%

1µM +2 µM 1.5 1.3 1.6 1.46 35%

2µM +4 µM 2.5 2.2 2.6 2.43 80%

2µM +5 µM 3.2 3.5 3.6 3.4 90%

5µM +10 µM 2.4 2 2.1 2.1 60%

10µM +5 µM 1 0.9 0.8 0.9 20%

IAA+KN

1µM +1 µM 0.6 0.7 0.6 0.63 10%

1µM +2 µM 0.7 0.8 0.6 0.7 20%

2µM +4 µM 0.9 1 0.9 0.93 10%

2µM +5 µM 1.8 1.9 2.3 2 20%

5µM +10 µM 2.3 2.4 2.5 2.4 30%

10µM +5 µM 0.7 0.6 0.6 0.63 10%

NAA+KN

1µM +1 µM 0.3 0.2 0.4 0.3 10%

1µM +2 µM 0.1 0.3 0.2 0.2 20%

2µM +4 µM 0.4 0.3 0.5 0.4 10%

2µM +5 µM 0.8 1.0 0.7 0.83 20%

5µM +10 µM 0.6 0.4 0.5 0.5 30%

10µM +5 µM 0.6 0.4 0.3 0.43 10%

From the above results, it is evident that among the three combinationsof growth regulators,
2,4-D and KN supported the induction and growth of callus of C. maxima peel. This was
followed by IAA and KN combination and NAA and KN did not support much callus
induction and growth. The graphical representation of percentage induction of callus growth
with combinations of growth regulators isshown in Fig.1.

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Fig. 1. Percentage yield of callus with combinations of growth regulators

The concentration of carbohydrate in the media is essential in the induction and growth of
callus. The effect of different concentrations of sucroseon the growth and percentage yield of
callus of C.maxima peel is given in Table 3.4.

Table 3.4: Callus growth response of C.maxima peel explants with respect to
Carbohydrate concentration

Sucrose Growth response response of callus in cc Callus

induction
concentration 1st set of 2nd set of 3rd set of Mea
4th percentag
in g/L bottles bottles bottles n e
bottle

30g/L 1.2 1.3 1.4 1.3 1.3 30%

50g/L 2 2.3 2.2 2.3 2.2 50%

60g/L 2 2.4 2.5 2.3 2.3 60%

80g/L 2.9 3.9 3.3 3.6 3.42 90%

100g/L 1 0.9 1.4 1.6 1.2 10%

By observing the results of effect of carbohydrate concentration to the induction and growth of
callus, it is clear that MS medium with 8% of sugar strongly supports the callus growth of
C.maxima peel explants. The graphical representations of effect of different concentrations of
sugar on growth of callus is expressed in Fig.2 and that of percentage growth response is shown
in Fig. 3.

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Fig.2. Effect of different concentrations of sucrose on callus growth

Fig.3. Percentage yield of callus with different concentrations of sucrose.

Elicitors were used to increase the yield of bioactive compounds from the callus explants. The
two different elicitors used were Chitosan and Jasmonic acid. The growth response and
percentage yield of callus of C.maxima with respect to two different elicitors are shown in the
Table.3.5.

Table.3.5: Effect of elicitors on induction of callus from C.maxima peelex plants

Concentration % of Yield Growth ofcallus in

cc
0.5 µM 30% 2
1 µM 40% 2.33
Chitosan 2 µM 40% 2.4
5 µM 85% 2.9
10 µM 50% 2.5

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20 µM 20% 2.1
Concentration % of Yield Growth ofcallus in
cc

0.5 µM 10% 0.5

1 µM 15% 0.7

Jasmonic acid 2 µM 20% 1

5 µM 10% 0.8

10 µM 5% 0.5

20 µM 3% 0.5

The growth response of callus with different elicitors is expressed graphicallyin the Fig.4.

Fig.4. Effect of elicitors on percentage yield of callus.

From the above results it is clear that Chitosan supports the growth of callus but Jasmonic acid
did not support the growth of callus of Citrus maxima peel.

3.2. Statistical Analysis

Data is represented in the means ± SE values of a minimum of 3 replicates. One-way analysis

of variance (ANOVA) was performed using SPSS program V16. The significance between
control and treatment was examined by Duncan’s Multiple Range Tests (DMRT) at 5% level
of significance. Results are expressed with 95 % confidential level. The groups with mean
values having different letter in bracket are significantly different at the 5 % level according to
DMRT.

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Table 3.6: ANOVA analysis of callus induction response of C.maxima peel explantscultured
on MS medium with different growth regulators

Sl. Growth Mean ±SE

No regulator
. s
Different concentrations of different growth regulators

O.5µM 1.0µM 2.0µM 5.0µM 10µM 20µM

1 2,4-D 2.09±0.05 2.33±.0.03 2.93±0.03e 2.40±0.04c 2.40±0.05 2.10±0.05

c c a
b
2 IAA 0.93±0.03 1.10±0.05b 1.86±.0.03 2.48±004c 2.23±0.12 2.00±0.15
b d a
B

3 NAA 0.56±0.03 0.66±0.03a 0.93±.0.03 0.66±0.03a 1.03±0.08 0.63±0.08a

a b b
b

4 KN 0.53±0.03 1.00±0.05b 1.33±0..03 2.70±0.05d 2.20±0.05 1.86±0.08

a c a
B

5 TDZ 0.53±0.03 0.63±0.03a 0.63±.0.08 0.80±0.05b 0.93±0.03 0.63±0.03a

a a
b

6 BAP 0.50±0.00 0.53±0.03a 0.60±0.05a 0.56±0.06a 0.60±0.05 0.56±0.06a

a c

F Value 327.950 241.840 309.90 398.317 113.760 72.279

Table 3.7: ANOVA analysis of callus growth response of C. maxima peel explants with respect
to Carbohydrate concentration

Sl.No. Concentrations Sucrose (Mean ±SE )

1 30g/L 1.40±0.05a

2 50g/L 2.16±0.14b

3 60g/L 2.26±0.08b

4 80g/L 3.50±0.26c

5 100g/L 1.03±0.08a

F Value 41.096

Table 3.8: ANOVA analysis of callus induction response of C. maxima peel explantswith
respect to different combinations of Auxins and KN
Sl. Growth Growth with different combinations of Auxins and KN

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No regulators Mean ±SE

1+1 µM 1+2 µM 2+4 µM 2+5 µM 5+10 µM 10+5 µM

1 2,4D+KN 1.33±0.20b 1.46±.08a 2.43±0.12a 3.43±0.12a 2.16±0.12a 0.90±0.05b

2 IAA+KN 0.63±0.03a 0.70±0.05b 0.93±0.03b 2.00±0.15b 2.40±0.05a 0.63±0.03a

3 NAA+KN 0.30±0.05a 0.20±0.05c 0.40±0.05c 0.83±0.08c 0.50±0.05b 0.43±0.08a

F Value 18.31 84.53 176.52 111.6 152.57 13.45

Fig. 5 (a). Explant of peels of C.maxima on first day of inoculation

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Fig. 5 (b). Induction of callus from explants after 12 days

Fig. 5 (c).Proliferation of callus after 22 days.

Fig. 5 (d) Subculture of callus

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Fig. 5 (e)Proliferation of callus after elicitation

Fig. 5 (f) Callus taken for lyophilization

3.3. Qualitative Phytochemical analysis of different extracts of callus of Citrus maxima

fruit peel.

Phytochemical screening of different extracts of callus of C. maxima fruit peel, with diverse
solvents yielded different results. There were alterations in intrinsic phytochemicals in the
extracts of different solvents. The yield is measured based on the appearance of dark or light
colour in the tests. The phytochemicals of C. maxima fruit peel callus extracts of solvents are
listed in Table 3.9.

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Table 3.9: Phytochemical constituents of callus of Citrus maxima fruit peel.

Solvents
Sl Phytochemica Tests performed Ethyl
. ls acetate
Aqueou Methan Hexane
N s ol
o.

1 Tannins Ferric chloride Test - - - +

2 Flavonoids Sodium hydroxide + ++ ++ +

Test

Lead acetate Test + ++ + +

3 Triterpenoids Chloroform Test - + - +

4 Coumarins Ferric chloride Test + - + -

5 Alkaloids Hager’s Test + - - -

6 Carotenoids Chloroform Test - - - -

Ninhydrin Test + ++ + -

7 Proteins and Lead sulphide Test + + + +

amino acids

Biuret Test - - - +

8 Carbohydrates Benedict’s Test ++ + - +

9 Saponins Foam Test + + ++ -

Key:-++ = Highly detected: based on dark color, + =Less detected: based on light color,

- = Not detected All tests were carried out in triplicates.

The above tests exposed the presence of flavonoids more in Methanolic extract of callus,
followed by Ethyl acetate when compared to the other solvent extracts. Whereas the other
bioactive compounds were found lesser or absent in callusextracts of different solvents.

3.4. Quantitative Phytochemical analysis of different extracts of callusof Citrus maxima

fruit peel

3.4.1. Total Flavonoid Content (TFC)

Total flavonoid contents of callus of C. maxima fruit peels using solvents Methanol and Ethyl

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acetate were examined by Aluminum chloride colorimetric method. The results were
articulated as quercetin as reference to standard curve. Quercetin standard curve for the solvent
methanol is shown in Fig.6 and that of Ethyl acetate in Fig.7. Table.3.10 represents flavonoid
content of Methanol and Ethyl acetate extracts of Citrus maxima fruit peel callus.

Fig.6. Calibration curve of Quercetin in methanol.

Concentratio
n
Fig.7. Calibration curve of Quercetin in Ethyl acetate.

Table 3.10. Total flavonoid content of callus of C. maxima fruit peels in twodifferent solvents

Solvents OD Total flavonoidcontent (mg/g)

Value

Methanol 0.45 5.4

Ethyl acetate 0.16 1.9

It is proven that the methanolic extract of callus showed higher flavonoid content than the Ethyl
acetate extract.

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3.4.2. DPPH radical scavenging assay

The free radical scavenging effect of methanolic extract of callus of Citrus maxima peels was
determined using the DPPH radical scavenging method. The scavenging effect of different
concentrations of callus extracts on DPPH free radical was quantified with gallic acid standard.
The results were expressed as % inhibition. Fig.8. shows the DPPH radical scavenging activity
of callus extracts of Methanol in comparison with the standard gallic acid curve. The
percentage inhibition of DPPH free radical by callus extracts of Citrus maxima peel is shown
in Table 3.11.

Table 3.11: DPPH Radical Scavenging activity of Callus and standard Gallic acid

Percentage Inhibition
Concentration Gallic acid Callus

0.2µM 9.4 7.1

0.4µM 11.3 9.5

0.6µM 13.2 11.9

0.8µM 15.0 14.2

1µM 20.7 16.6

Fig. 8: DPPH radical scavenging activity of Citrus maxima peel callus

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DPPH Radical Scavenging assay showed very good antioxidant activity incallus of C.maxima
peel in comparison with the standard Gallic acid.

3.4.3. Thin layer chromatography for flavonoids of callus of C.maxima fruitpeel.

TLC was employed with the use of standard silica gel G layers with binder gypsum. On these
layers, Methanolic extract of callus was applied together with standards, Naringin and
Naringenin. The plates were developed at room temperature in a vertical separating chamber,
in the selected solvent system of Methanol: Chloroform: Hexane, in the ratio 7:2:1 by
ascending technique. The plates were dried post development and the spots were detected by
holding it against a bright light. Standards and callus extract applied on TLC plates and related
for their Rf values. Comparison with Rf values, and colour flavonoid compounds in callus were
identified.

TLC of methanolic extracts of callus of Citrus maxima peels exposed the following results.
Flavonoid compounds found in the callus were Naringin and Naringenin. This was confirmed
by comparing the spots obtained in the elution of standards and their Rf values (Table 3.11).
Fig.9 shows the TLC of methanolicextract of callus of Citrus maxima peel in comparison with
the standards Naringin andNaringenin.

Standard Naringin band

Callus elution bands

Standard Naringenin ba

Fig. 9.TLC of callus of Citrus maxima peels in comparison with the standards

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The Rf values of the two spots of callus were found to be 0.32 and 0.63. TheRf values of
standards were found to be 0.31, that of Naringenin and 0.62, that of Naringin. By
comparing these values it is clear that the flavonoids present in callusare Naringin and
Naringenin.

3.4.4. LC-MS analysis of Callus of Citrus maxima fruit peels.

Liquid Chromatography Mass Spectrophotometric Analysis (LCMS) allowed the

determination of the major flavonones in callus of C.maxima peel extract in methanol on the
basis of their molecular weight. The results yielded by LC-MS analysis is given in the Fig. 10.

Fig 10. LCMS analysis of callus of C.maxima peel extract.

The LCMS results revealed the presence of flavonones, Naringin and Naringenin. The
molecular weight of Naringin is 580 and that of Naringenin is272.The callus extract showed
the presence of molecules with the above molecular weights, indicating their presence.

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Fig 11: LCMS chromatogram ofStandard Naringin and Naringenin

Fig 12: LCMS chromatogram of callus

LC-MS analysis was also carried by comparing with standards. The results showedthe
chromatogram of standards and that of callus. By comparing the peaks of standard
chromatogram of Naringin and Naringenin with that of callus peaks, it was clear that both
Naringin and Naringenin are found in callus.

3.4.5. HPLC analysis of Callus of C.maxima fruit peels

The LCMS analysis revealed the presence of flavonones Naringin and Naringenin in
Methanolic extract of callus of C. maxima peels. Further quantification of these two flavonones
was done by HPLC analysis. HPLC chromatogram of standard Naringin and Naringenin is
shown in Fig. 13. HPLC analysis of methanolic extract of Callus of Citrus maxima peels with
and without elicitor are shown in Fig. 14 and Fig. 15.

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Fig.13. Chromatogram of standard Naringin and Naringenin

Fig.14: Chromatogram of Naringin and Naringenin in callus without elicitor.

Fig.15: Chromatogram of Naringin and Naringenin in callus with elicitor

4. Discussion

Different strategies including composition of media,type of growth hormones, carbohydrate

concentration, choice of elicitors, temperature, pH etc are used to increase production of
secondary metbolites. Growth regulator concentration is generally a decisive factor in growth

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and secondary Metabolite production. Auxins and cytokinins show the most outstanding effects
on productivity and growth of plant metabolites. Among different concentrations of 2,4-D
tested, extreme callus induction response (85%) and highest growth response (2.9cc) was
observed with 2,4-D at 2µM concentration. Similarly among different concentrations of IAA
tested, maximum callus induction response (65%) and highest growth response (2.23cc) was
observed with IAA at 10µM concentration. Likewise among different concentrations of NAA
tested, maximum callus induction response (35%) and highest growth response (1.03cc) was
observed with NAA at 10µM concentration. All the above results shows that among auxins,
2,4-D supported very well for the induction and growth of C.maxima peel explants.

Among cytokinins, between different concentrations of Kinetin tested, maximum callus

induction response (80%) and highest growth response (2.7cc) was observed with Kinetin at
5µM. Likewise among concentrations of TDZ tested, maximum callus induction response
(20%) was observed with TDZ at 2µM and highest growth response (0.93cc) was observed
10µM. Similarly among concentrations of BAP tested, maximum callus induction response
(20%) and highest growth response (0.6cc) was observed with BAP at 2µM concentration.

The above response were increased to 90% of callus induction and highest growth response of
3.4cc, when 2,4-D (2 µM) was used in combination with Kinetinat 5µM. But the response
was reduced to 20% and growth response to 2cc, when IAA(2µM) was used in combination
with Kinetin at 5µM. The NAA and KN did not support the callus induction as well as its
growth. The above concentration of these two combinations of growth regulators, i.e 2 µM
NAA and 5µM KN supported for only 20% callus induction response. The callus induced from
Citrus maxima fruit peels were green and fragile, and suitable for regeneration.

In overall, among the all the growth regulators, maximum 90% of callusinduction was observed
in 2,4-D and Kinetin (2µM + 5µM) combination and lowest percentage of callus induction
(5%) was obtained from BAP (0.5µM) and TDZ(0.5µM). Sativaet al (2010) observed that 2µM
concentrations of Kinetin had a maximum callus induction (86.33%) on C. jambhiri in MS
medium. Work of Ali and Mirza (2006), reveal that highest callus induction was observed on
MS media, supplemented with 1.5mg/L 2,4-D (92%).All these observations are in line with
the results of the present study, where highest percentage of callus induction (90%) was
supported by 2,4-D and Kinetin (2 µM + 5µM) combinations.

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The level of sucrose affected the growth and productivity of secondary metabolites in cultures
(Mantell et al., 1983). Sucrose concentrations of 2.5% and 7.5% in Coleus blumei media
supported the growth of callus (Misawa, 1985). 8% sucrose was found to be optimal for cell
culture of Catharanthus roseus (Knobloch and Berlin, 1980). These studies were supported by
the results of the present study, that showed the highest percentage of callus induction of 90%,
MS media, accompanied with 8% of sucrose concentration. Lower concentrations of sucrose
supports the callus induction and growth, but very high concentrations were found to be
detrimental for callus inductionas seen in 10% sucrose. The optimum callus induction and
growth was seen only with 8% of sugar.

Elicitation is a process which activates synthesis of secondary metabolites in plant cells to

guarantee their survival, persistence and competitiveness (Namdeo, 2007). Chitosan is a
polysaccharide, used as elicitor in tissue cultures to induce the accumulation of bioactive
secondary metabolites. The work of Chakraborty et al. (2009) reported that chitosan mimics
the effects of some pathogenic microorganisms to stimulate plants to manufacture defense-
related secondary metabolites. Here we studied, Chitosan and Jasmonic acid with the basal MS
media along with the phytohormone combinations of 2,4-D and KN (2µM + 5µM) with 8%
sucrose. Among different concentrations, maximum callus induction response (85%) and
highest growth response (2.9cc) was observed with Chitosan at 5µM concentration. Significant
increase of callus dry weight (0.102 g) was observed at sucrose and Chitosan 100 mM
containing medium when compared with the control (Sayed et al., 2016). Similarly highest
accumulation of flavonones, Naringin and Naringen were also found in the same concentration.
The present study is in line with these references as Chitosan in higher concentration reduced
the callus induction to 20% and lesser flavonone accumulation. On the other hand Jasmonic
acid did not supported callus induction. The highest callus induction was only 20% with 2µM
concentration and lowest was only 3% with 20µM concentration. This result is also in
accordance with the results of Collinge et al., (1987) and Mukandan et al., (1990).

The callus was characterized for the presence of various phytonutrients present in them. The
phytochemical analysis of callus of C. maxima peel revealed the presence of good amounts of
flavonoids than other bioactive compounds in Methanolic and Ethyl acetate extracts. This is in
line with the study of Adhikari et al., (2017), which explores the presence of flavonoids in
callus of C. junos. Further, the quantitative estimations of the callus involving estimation of

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Total Flavonoid Content with these two solvents, exposed highest yield of flavonoids in
Methanolic extract. This result was supported by the results of Meratan et al., (2009) who found
better amounts of TFC in in vitro plantlets of C. reticulata. Hence further investigations of
flavonoids were carried out by taking only Methanolic extract of callus. There is a linear
correlation between flavonoid concentration and radical-scavenging activity (Kumaret al.,
2015). Citrus junos callus extract exhibited strong antioxidant activity in DPPH radical
scavenging assay (Adhikari et al., 2017). Likewise the present study revealed an excellent
antioxidant activity in the Methanolic extract of callus of C.maxima by DPPH radical
scavenging assay.

TLC profiles of callus extracts of A. oleraceae showed the presence of flavonoids that exhibited
a prominent, white color spot with Rf value of 0.49, which was characteristic for callus extract
of A. oleraceae (Abeysinghe, Wijerathne and Dharmadasa, 2014). In accordance with this the
present study exposed the presence of flavonones, Naringin and Naringenin when the callus
sample was run with standards in the silica gel. The presence of these two flavonones was
confirmed by LC-MS analysis using the standards. Furthermore quantifications of Naringin
and Naringenin in callus without elicitors and callus with elicitors were conducted using
standards by HPLC analysis. The result revealed more amounts of Naringin and Naringenin in
callus with elicitor, Chitosan.

HPLC analysis that was applied to trace flavonoids in both control and elicitors treated calli,
showed that flavonoids were detected only in callus elicited with fructose and Chitosan (Sayed
et al., 2017). On the other hand, the present study revealed higher percentage of flavonoids in
callus of C. maxima peel, i.e. 9.17% when compared to the callus of S. khuzistanica. In overall,
from the above results it could be concluded that the callus of Citrus maxima peel is a
significant source of flavonones, especially Naringenin and Naringin. The elicitor Chitosan has
played a significant role in accumulation of these two flavonoids in the callus. Thus the in vitro
culture of C. maxima peel is a good development in the field of biotechnology, as it is a better
alternative for the wild peel extract.

5. Conclusion

The plant cell culture provides an effective alternative to demand of the bioactive secondary

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metabolites. They have an scale-up potential, thus commercialization of plant cells embodies
an alternative to secondary metabolites. This also aids to save the genetic diversity of the wild
population of medicinal plants. Here, an efficient method has been developed for callus
induction and production of Naringin and Naringenin in callus culture of C. maxima fruit peel
for the first time. In-vitro callus induction was attained from peel explants cultured on MS
media complemented with different concentrations of growth regulators, Auxins and
Cytokinins. MS media supplemented with 8% sucrose, fortified with 2 µM 2,4-D and 5µM
Kinetin was the most promising media for callus formation.

The phytochemical analysis of callus of C. maxima peel revealed the presence of good amounts
of flavonoids than other bioactive compounds in Methanolic and Ethyl acetate extracts.
Further, the quantitative estimations of the callus involving estimation of total flavonoid
content with these two solvents, exposed highest yield of flavonoid in Methanolic extract.
Hence, further investigations of flavonoids were carried out by taking only Methanolic extract
of callus. DPPH radical scavenging assay showed that the Methanolic extract of callus had
flavonoids with excellent antioxidant activity. The flavonoids present in the callus of C.maxima
peel was investigated by TLC method. It revealed the presence of flavonones, Naringin and
Naringenin when the sample was run with standards in the silica gel. The presence of these two
flavonones was confirmed by LC-MS analysis using the standards. LC-MS analysis confirmed
the presence of Naringin and Naringenin based on their molecular weight by showing same
peaks as that of the standards. Furthermore quantificationsof Naringin and Naringenin in
callus without elicitors and callus with elicitors were conducted using standards. The result
revealed more amounts of Naringinand Naringenin in callus with elicitor, Chitosan.Thus, the
present study results revealed that the in-vitro callus culture of C.maxima peel was an effective
alternative in producing flavonones, Naringin and Naringenin than their mother plant fruit peel.

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