Major project research - presentation Gives a brief understanding about our research work on isolating, characterizing and developing nanoparticles of a phytochemical called Hesperidin from Blood Orange (Citrus sinensis) peel extract which can be used as an effective inhibitor of binding of ACE 2 receptor with SARS CoV 2 and hence manage and prevent COVID-19 infection.

1. ISOLATION AND CHARACTERIZATION OF HESPERIDIN FROM Citrus
sinensis PEEL EXTRACT AGAINST CoV 2

2. CONTENTS
Introduction
Review of literature
Materials and methodology
Results and Discussion
Conclusion
References

3. INTRODUCTION
● A respiratory illness cluster has been seen within the Wuhan area of China since
2019. This global disease was spread by a lethal virus, and it was discovered that it
had been a modified variety of the severe acute respiratory syndrome (SARS), which
was declared eradicated in 2010.
History:
● A wholesale market for seafood, poultry, and wild animals has been connected to an
outbreak of viral pneumonia in China. The first 27 individuals were diagnosed with
an undetermined viral pneumonia and were admitted to the hospital. An
epidemiological study revealed a connection between them and Huanan, China.

4. Role of SARS-CoV-2 and ACE2 variations in COVID-19:
●Human Angiotensin-converting enzyme 2 (ACE2) serves as a receptor for cellular
entry for both SARS-CoV-1 and SARS-CoV-2.
●Human Angiotensin-converting enzyme 2 (ACE2) possesses cardiovascular
preventive properties, including the ability to prevent heart failure, thrombosis,
myocardial hypertrophy, fibrosis, arrhythmias, and atherogenesis. ACE 2 also defends
against lung damage and is down regulated after SARS-CoV-1 infection.
●Evidently, the virus has been mutating and adapting during this period. Significant
effort has been spent on identifying these variations and their impact on transmission,
virulence and pathogenicity of SARS-CoV-2.

5. • Binding of the SARS-CoV-2 spike protein to the angiotensin converting enzyme
2 (ACE2) promotes cellular entry. Some researchers investigated the potential of
“population-specific” ACE2 genetic variations to impact the SARS-CoV2
vulnerability and suggested no ethnicity enrichment for ACE2 polymorphisms
that could influence SARS-CoV2 S-protein binding. At the same time, some
studies use data mining to predict several ACE2 variants. On the other hand,
fewer studies revealed an association of ACE2 expression with COVID-19
outcome reporting higher expression levels of ACE2 in East Asians.
• The ACE2 gene variants and expression may modify the deleterious
consequences of SARS-CoV2 to the host cells. Many other environmental and/or
genetic factors could modify the disease outcome, as well as the presence or
absence of ACE2 in the body.

6. MECHANISM OF HESPERIDIN AGAINST SARS CoV 2
⮚ Hesperidin interferes with viral entry through ACE2 receptors, improves the host
cellular immunity, minimizes the release of inflammatory mediators and its mixture
with Diosmin protects against venous thromboembolism.
⮚ Hesperidin is one compound that could target the binding interface between SARS-
CoV-2 Spike and ACE2 human receptors. Based on virtual screening, Hesperidin may
disrupt the interaction of ACE2 with RBD of SARS-CoV-2 thus block its entry into
the lung cells.
⮚ Hesperidin with its high anti-inflammatory activity inhibited the secretion of pro-
inflammatory cytokines such as IFN-γ and IL-2. Besides, Hesperidin inhibited
IL-1β-stimulated inflammatory responses by inhibiting the activation of the NF-κB
signaling cascade.

8. Role of phytochemicals
● Herbal medicines and phytochemicals having antiviral activity against Corona
viruses. Aromatic herbs, herbal teas, culinary spices, and medicinal plants used in
ethno botanical treatments could all be beneficial in this regard.
● It was demonstrated how herbal therapy and phyto-medicine can be used to prevent
viral infections. As a result, some countries, including Algeria, are promoting the
use of herbal and medical products.
● Medicinal plants offer the most common treatment options since ancient times
against various viral diseases because of their safer, cheaper and less toxicity
profile. There are several phyto-constituents which have shown anti-viral activity
against different types of corona viruses. The National Health Commission of
China suggested traditional Chinese medicine as an alternative defense treatment
option.

9. AIM
• To isolate and characterize Hesperidin from Citrus sinensis fruit peel extract against
SARS CoV 2.
OBJECTIVES
• Isolation and characterization of Hesperidin from Citrus sinensis fruit peel extract
against SARS CoV 2.
• Phytochemical characterization of Hesperidin isolated from extract of Citrus
sinensis fruit peel.
• Design and evaluation of Hesperidin nanoparticles.

10. REVIEW OF LITERATURE
● K. Chojnacka, et al (2020), in his review summarized current knowledge about
plant extracts containing polyphenols that inhibit Covid19. Natural products
(polyphenols) from many plants can provide a starting point for studies on the use
of plant extracts for the treatment and prevention of corona virus. Polyphenol
antiviral may inhibit corona virus enzymes, which are essential for viral replication
and infection.
● S. Pal, et al (2022), summarized that herbal medicinal plants received a noticeable
attention to treat COVID-19 in the Indian subcontinent. Their probable mechanism
and therapeutic approaches behind the activity of phytochemicals to stimulate
immune response as well as inhibition of viral multiplication discussed rationally.

11. ●Zhang et al (2020), studied the anti-coronavirus (anti-CoV) activity in the plant-
derived phytochemicals and medicinal plants gives a strong base to develop a novel
treatment employing these compounds for corona virus. Various phytochemicals and
medicinal plant extracts have been revised and are considered as potential anti-CoV
agents for effective control of the virus and future drug development.
●Agarwal et al (2020), abridged that an important source of dietary flavonoids,
including Hesperidin, Hesperetin, Taxifolin, Naringin, Naringenin, Diosmin,
Quercetin, Rutin, Nobiletin, Tangeretin, and others. Studies have demonstrated
antiviral activities of various flavonoids, including their use for respiratory diseases
and SARS-CoV. Among these, Hesperidin is the predominant flavonoid in citrus
fruits.
●Basu, A., et al(2020), described that natural products are safe and easily available to
treat coronavirus affected patients. Some flavonoids have been selected as small
molecules in molecular docking study of spike protein of SARS-CoV2 with its human
receptor ACE2 molecule. Their molecular binding sites on spike protein bound
structure with its receptor have been analysed. Hesperidin, Emodin and Chrysin are
selected as competent natural products from both Indian and Chinese medicinal
plants, effective to treat COVID-19. Among them, the phytochemical Hesperidin can
bind with ACE2 protein and bound structure of ACE2 protein and spike protein of
SARS-CoV 2 noncompetitively.

12. MATERIALS AND METHODOLOGY
Materials:
● Plant source: Fresh dry coarse and fine powder of Citrus sinensis fruit peel (250
gm.)
● Chemicals required : Petroleum ether ( 1000ml) ,Methanol ( 1000ml), Ferric
chloride, iodine, potassium iodine, acetone, distilled water , HPLC grade water ,
HPLC grade Acetonitrile, ammonia, PLGA-Chitosan polymer, PVA (polyvinyl
alcohol) , dichloromethane, silica gel-G.
● Equipment: Soxhlet apparatus , reflux condenser, HPLC heating mantle, magnetic
stirrer , volumetric flask , measuring cylinder , weighing balance , test tubes , conical
flask, electron microscope, hydrogen electrodes , microscope , seivers (No-22, 42,) ,
propeller system, freezer , butter paper ,filter paper , cotton plug , glass slide ,
Homogenizer.

13. A SLICED BLOOD
ORANGE
The blood orange is a variety of
orange (Citrus × sinensis) Citrus ×
sinensis also referred to as raspberry
orange with crimson, almost blood-
coloured flesh. The distinctive dark
flesh colour is due to the presence
of Anthocyanins, a family of
polyphenols pigments common to
many flowers and fruit, but
uncommon in citrus fruits
Chrysanthemin (cyanidin3-O-
glucoside) is the main compound
found in red oranges.

14. ● The flesh develops its characteristic maroon colour when the fruit develops with low
temperatures during the night.
● Sometimes, dark colouring is seen on the exterior of the rind, as well, depending on
the variety of blood orange.
● The skin can be tougher and harder to peel than that of other oranges.
● Blood oranges have a unique flavour compared to other oranges, being distinctly
raspberry-like in addition to the usual citrus notes.
● Hesperidin is a flavanone glycoside found in citrus fruits. Its aglycone form is called
as Hesperetin, derived from the word hesperidium for fruit produced by citrus trees.
● Its other name is Hesperetin 7- rutinoside.
● Hesperidin is a plant chemical that is classified as a bioflavonoid.
● Most commonly used for blood vessel conditions like hemorrhoids, varicose veins,
and poor circulation.

15. SPECIES Citrus sinensis
CULTIVATION Blood orange cultivators
COLLECTION Dried fruit peel can be cut into
two grades by propeller system.
ORIGIN Southern Mediterranean ,18th
century
CULTIVATION MEMBERS Farmers of Tarocco (Italy)
Farmers of Moro (Spain)
Farmers of sanguinello (Sicily)
SUPPLIER INDIAMART
AMAZON
FLIPKART

16. Isolation of Hesperidin from Citrus sinensis fruit peel
Modern Method: -
1. 800 ml Petroleum Ether (40 – 60°C) is filled in a 250 ml round bottom flask with
magnetic stir bar.
2. 250g dried and powdered orange peels are placed in the extraction sleeve of a
Soxhlet extractor and covered with a little glass wool.
3. A reflux condenser is put on the Soxhlet extraction unit, and then the reaction
mixture is stirred and heated for 4 hours under strong reflux.
4. The petroleum ether extract is discarded. In order to remove the adherent
petroleum ether, the content of the extraction sleeve is laid out in an extensive
crystallization dish.
5. Afterwards the substance is placed again in an extraction sleeve and, like before
methanol is used, allowed to percolate until the solvent changed to colourless.

17. Identification and characterization of Hesperidin from Citrus
sinensis fruit peel extract
● Ferric chloride test
The isolated Hesperidin extract was treated with FeCl3 (ferric chloride
Reagent) hence due to the Hesperidin in the extract , reaction between
Hesperidin and Fecl3 to form "wine red colour" .
● Shinoda test
2ml of Shinoda reagent is treated with 10 ml of Hesperidin extract which leads
to formation of "Bright pinkish violet colour”.
● Thin layer chromatography
The mobile phase composition is optimized to establish a suitable and accurate
densitometry
TLC method for analysis of Hesperidin.

18. Design of Hesperidin nanoparticles:
PREPARATION:
▪ HPD-NPs were synthesized by emulsification and evaporation methods.
▪ 20 mg HPD and 80 mg PLGA-CHITOSAN were dissolved in 5 ml of
dichloromethane and homogenized for 40 seconds to form the oil phase
emulsification.
▪ The oil phase emulsification was combined with 20 ml PVA (1% w/w) containing
0.2% of Chitosan and homogenized for another 40 seconds to form the second
water phase emulsification.
▪ This water phase emulsification was added to 100 ml water and stirred for 6
minutes to achieve organic reagent evaporation and nanoparticle hardening.
▪ Finally, HPD/NPs were harvested by centrifugation at 12,000 rpm for 20 min and
washed 3 times with ultrapure water then stored for 48 hrs for storage at 4‘C.

19. Characterization of Nanoparticles
Scanning electron microscope
(SEM) Zeta potential Size distribution
For the purpose of
studying the particle
morphology and imaging
of the synthesized Hsp-
NPs, the scanning
electron microscopy
(SEM) was performed.
Characterizing the Hsp-
NPs, zeta potential and
particle size were
measured using the Zeta-
sizer machine.
Nanoparticle size plays an
important role in the
formulation and drug
distribution.
Size distribution of
nanoparticles analyzed
based on SEM, is
determined.

20. RESULTS AND DISCUSSION
Isolation of Hesperidin from Citrus sinensis fruit peel extract:
⮚ Crude extraction is collected in a round bottom flask which is mild yellow in
colour. This crude extraction is stored in 1°c in the freezer overnight. Crude
Hesperidin was isolated and is given in figure below:

21. Identification of Hesperidin phytochemical:
TEST OBSERVATION INFERENCE
FERRIC CHLORIDE
TEST
WINE RED COLOR Isolation of peel
extract contains
flavonoid
(Hesperidin)
SHINODA TEST BRIGHT PINKISH
VIOLET COLOUR
Hesperidin
bioflavonoid -
present
THIN LAYER
CHROMATOGRAPHY
Butanol:acetic
acid:water(3:1:1)
TWO SPOTS
OBSERVED
Hesperidin Rf value
was found to be
0.69

22. FERRIC CHLORIDE TEST THIN LAYER CHROMATOGRAPHY

24. Estimation of Hesperidin by HPLC
The qualitative and quantitative analysis of Hesperidin was performed on a HPLC
instrument equipped with Chemstation HPLC software provided by Chemstation
solutions company, was used with Eurospher 100 and a thermostatted column oven was
used with a reverse-phase C18 column (4.6 mm I.D. x 250 mm, 5 mm) and the
temperature of the column was controlled at 30 °C.
Compounds were monitored at 256 nm using a UV/Visible detector or PDA
(photodiode array).
An isocratic mobile phase condition consisting of methanol (Solvent A) and water
containing (0.1% O-phosphoric acid) (Solvent B) were used as 50% A and 50% B, the
total run time was 10 min at flow rate of the mobile phase 1 ml/min and injection
volume was 20 µ.

25. HESPERIDIN HPLC CHROMATOGRAPH

26. Formulation of Hesperidin nanoparticles (HPD-NP)
HPD-NPs were synthesized by emulsification and evaporation methods.
To improve the water solubility and bioavailability of HPD, biodegradable polymer
PLGA was employed to encapsulate HPD to form soluble NP carriers, and Chitosan
was employed to modulate the surface zeta potential.
The steps performed during formulation of Hesperidin nanoparticles are given in
figures below:

27. Characterization of Hesperidin nanoparticles
SCANNING ELECTRON MICROSCOPE:
For the purpose of studying the particle morphology and imaging of the synthesized
Hsp-NPs, the scanning electron microscopy (SEM) measurement was performed.
The image of SEM exhibited relatively a spherical shape of the nanoparticle that
were formed with a diameter in the range of 194 to 355nm, while Hsp demonstrated
uninformed shape with aggregation and irregular distribution of the particles in
comparison with Hsp-NPs that showed a spherical, smooth, and almost
homogenous structure.
SEM analysis results are given in figures below:

28. SEM ANALYSIS OF HESPERIDIN
NANOPARTICLES

29. Determination of Zeta potential
Zeta potential and particle size were measured using the Zetasizer machine. The zeta
potential was found to be −24.40 mV, indicating that the loaded nanoparticles were stable
in solution, as described by the guideline. Zeta potential is an essential physicochemical
indicator of the stability of Nano suspensions. Zeta potential measurement results are
given in figure below:

30. Determination of Hesperidin nanoparticles size
distribution
Nanoparticle size plays important role in the formulation and drug distribution,
size distribution of nanoparticles analysed based on the scanning electron
microscope (SEM) based on SEM, size distribution ranges from 194 nm to
355nm and average size distributed in formulation is determined as 274.5nm .
Size distribution is determined on the bases of scanning electron microscope
reports, analysed results are given in figure below:

31. CONCLUSION
●The outbreak of Covid-19 disease, caused by SARS-CoV-19 has motivated
researchers to look for new antiviral formulations due to a lack of targeted medicines.
The SARS-CoV-2 spike protein enhances cellular entrance by binding to the
angiotensin converting enzyme 2 (ACE2). As a result, human ACE2 variants might
influence virus susceptibility or resistance.
●Hesperidin and Hesperetin are flavonoids found in abundance in citrus fruits. These
phytochemicals may bind to various receptors of the Severe Acute Respiratory
Syndrome coronavirus 2 (SARS-CoV-2) such as spike protein, angiotensin-
converting enzyme 2, and proteases. Hence, the present study was designed to isolate,
characterize and further design and evaluate nanoparticles of Hesperidin from fruit
peel extracts of Citrus sinensis.

32. ●In the present study Hesperidin was isolated from Citrus sinensis fruit peel extract by
using modern isolation method protocol.
●Further phytochemical characterization and identification of Hesperidin in the extract
was done by various qualitative and quantitative tests such as Ferric chloride test,
Shinoda test and thin layer chromatography (TLC) from this TLC Rf value calculated
as 0.69 and HPLC.
●Zeta potential was found to be −24.40 mV, indicating that the loaded nanoparticles
were stable in solution, as described by the guideline.
●Overall findings show that Hesperidin based Nanoparticles could be a promising
strategy to treat SARS- CoV 2.

33. REFERENCES
1. Wu, Yi-Chia; Chen, Ching-Sunga; Chan, Yu-Jiuna,b,c,* The outbreak of
COVID-19: An overview, Journal of the Chinese Medical Association.
2. Antony, Priya, and Ranjit Vijayan. "Role of SARS-CoV-2 and ACE2
variations in COVID-19." biomedical journal 44.3 (2021): 235-244.
3. Boukhatem, M.N.; Setzer, W.N. Aromatic Herbs, Medicinal Plant-Derived
Essential Oils, and Phytochemical Extracts as Potential Therapies for Corona
viruses: Future Perspectives. Plants 2020, 9,800.
4. Boukhatem, Mohamed Nadjib, and William N Setzer. “Aromatic Herbs,
Medicinal Plant-Derived Essential Oils, and Phytochemical Extracts as
Potential Therapies for Corona viruses: Future Perspectives.”
5.Basu, A., Sarkar, A. & Maulik, U. Molecular docking study of potential
phytochemicals and their effects on the complex of SARS-CoV 2 spike protein
and human ACE2.