Marine organisms are rich sources of bioactive compounds with antioxidant properties. This study analyzed the antioxidant activity of compounds extracted from marine bioresources such as algae, fungi, bacteria, and invertebrates. Phenolic compounds, carotenoids, and anthraquinones with antioxidant effects were isolated from marine fungi using solvent extraction. The extracts were tested for antioxidant activity using methods like DPPH radical scavenging and reducing power assays. Compounds with potential antioxidant properties were identified that could serve as natural antioxidants or lead to new drugs.

1. PROJECT WORK
2020-2021
ANTIOXIDANT ACTIVITY OF MARINE BIOACTIVE
COMPOUNDS
SWAMI RAMANAND TEERTH MARATHWADA UNIVERSITY, NANDED
BY
Mr . SURYAWANSHI PRASHANT T.
Mr . GAIKWAD MAHESH B.
Mr . PAWAR AVINASH B.
Mr . MACHAPALE SUSHANT R.
B.PHARM FINAL
UNDER THE GUIDENCE OF
Prof. Salunke M.A
Asst. Professor,
Dept. of Pharmacognosy
Vilasrao Deshmukh Foundation, Group of Institutions,
VDF SCHOOL OF PHARMACY, LATUR

2. Marine organisms are rich sources of structurally diverse bioactive compounds with various
bio-Logical activities, and their importance as a source of novel bioactive substances is
growing rapidly. Marine natural products offer an abundant source of pharmacologically
active agents with great Chemical diversity and complexity and the potential to produce
valuable therapeutic entities.
The marine-originated compounds exhibit various biological activities. marine organisms Are
exposed to adverse environmental conditions such as light and high-oxygen concentrations
that Lead to the formation of free radicals.
Bioactives from marine bioresources such as macroalgae, microalgae, fungi, bacteria,
actinomycetes, inverteBrates, and vertebrates. marine algae are rich sources of bioactive
comPounds with various biological activities.
Moreover, marine-derived fungi have become an important Source of novel
pharmacologically active secondary metabolites and are considered as a rich source Of
bioactive compounds with medicinal and pharmaceutical benefits.
INTRODUCTION TO MARINE COMPOUNDS:-
Secondary Metabolites:-
They are highly hydrophilic components of molecular sizes ranging 126–650,000 Da.

3. PHARMACOLOGICAL ACTIVITIES OF MARINE-DERIVED PHARMACEUTICALS:-
Antioxidant Activity:-
Antioxidants may have a positive effect on human health as they can protect the human body
against damage by reactive oxygen species (ROSs), which attack macromolecules such as
membrane lipids,proteins, and DNA and lead to many health disorders such as cancer,
diabetes mellitus, and neuro-degenerative and inflammatory diseases with severe tissue
injuries. Marine-derived pharmaceuti-cally active compounds show promising antioxidative
effects.
Oxidant and Free Radicals:-
Free radicals and other reactive oxygen species which generated in living organism are
considered as the main cause for many pathological conditions for their oxidative properties
which actively act as oxygen donor to other compounds in living organism.
Some reactive oxygen species are free radicals. Most ofThe potentially harmful effects of
oxygen are believed to be due to the formation and activity of reactive oxygen species acting
as oxidants, There are, compounds with a tendency to donate oxygen to other substances.

4. Many reactive oxygen species are free radicals. A free radical is any chemical
species that has one or more unpaired electrons. Many free radicals are unstable
and highly reactive. Some of the free radicals and other important oxidants
found in living organisms.
Free Radicals Non radicals
Hydroxyl radical OH. Hydrogen peroxide H₂O₂
Suoeroxide radical o₂, nitric Singlet oxygen O₂
Oxide radical NO. Hypochlorus acid HOCL
Lipid peroxide radical LOO. Ozone (O₂)

6. Based on the number of substituents, they can be found as simple phenols or
polyphenols.
These well-known phytochemicals can be classified into different structural groups
ranging from simple phenols to complex molecules such as phenolic acids,
coumarins, tannins, lignins, lignan, stilbenes, flavonoids.
Polyphenois are biosynthesized mainly through two basic pathways that in chide the
shikimic acid and acetate-malonate pathways.” Simple phenolic compounds and
phenolic acids are considered as metabolites of the shikimic acid pathway.
However, the biosynthesis of comples polyphenol compounds such as flavonoids
requires both the shikimic acid malonate pathways and acetate.
A) Antioxidant from marine algae
PHENOLIC COMPOUNDS:-

7. Experimental studies have sevealed that the an tioxidant radical scavenging activity of
these compounds mainly proceeds through hydrogen atom transder or through
electron transfer mechanisms.
The hydrogen atom transfer mechanism indicates the ability of the phenolic
derivatives (ArOH) to transfer an H atom from the phenolic -OH group. The stability
of the resulting phenolic radicals (Ar) govems the radical scavenging ability of these
compounds.
RADICAL SCAVENGING ANTIOXIDANT ACTIVITY OF PHENOLIC COMPOUNDS:-
TYPES OF POLYPHENOLIC COMPOUNDS:-
Phenolic acids:-
Phenolic acids consist of a phenolic ring with carboxylic acid functional groups. The
presence of phenolic acids. including gallic, protocatechuic, gentisic,
P-hydroxybenzoic, chlorogenic, vanillic, syringic, caffeic, salicylic, coumaric,and
ferulic acids. has been identified from 16 dif-ferent algae species that possess
profound antioxidant activityas revealed by antioxidant assays,

8. Including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity,
Fe2+chelating assay, reducing power assay and have been found to be inhibitors of
lipid peroxidation in iron-induced liposomes.
Measurement of antioxidant activity of these fractions has revealed that the
antioxidant capacity of these compounds relates with their structural properties.
A higher antioxidant activity could be observed for phenolicacids with an attached -
CH = CH-COOH moiety that increases the H atom donating ability and extends the
radical Halogenated phenolic compounds have been found in brown algae and in red
algae species.
Most of them have indicated pronounced radical scavenging activities. Brominated
and fully substituted mono- and bis-phenols from the red alga
Symphyocladialatiuscula have shown potential DPPH radical scavenging activities.

9. Flavonoids:-
Flavonoids are polyphenolic compounds that are characterized by the C-15 skeleton,
arranged into three rings With C6–C3–C6 units composed of two phenyl rings on
Either side with one heterocyclic ring (benzoic-c-pirone) in the Middle that are
generally abbreviated as A, B, and C, respectively.
Flavonoids can be classified into several Groups based on the presence/absence of the
carbon–carbon Double bond between C2 and C3 positions, a hydroxy group On C3
position, and a carbonyl carbon at the C4 position.
These compounds represent a remarkably diverse group of Secondary metabolites
with a diverse array of biological Activities mainly related to their antioxidant
activity. Flavonoids are found to be present mainly in land plants, but Recent
investigations indicate the presence of flavonoids in Marine flora.
Flavonol glycosides Are compounds where a sugar molecule is bound with the
Flavonoid group through aglycosidic bond. In addition, Isoflavones have also been
identified from several marine And fresh water algae and cyanobacteria. the phenolic
OH group in the flavonoid moiety can donate HH Radicals indicating radical
scavenging ef-Fects.

10. Flavonoids are well known for their superoxide and hydroxyl radical scavenging
activities both in hydrophilic and Lipophilic systems.
These compounds can donate hydrogen radicals to peroxyl, hydroxyl, and
peroxynitrite radicals, stabilizing them and producing a stable semiquinone Radical
that undergoes resonance stabilization as indicated in Termination of further
reactions proceeds Through the flavonoid radical that reacts with available free
Radicals.
Flavonoids have the ability toact as antioxidants by chelating transition metal ions,
there by suppressing the Fenton type reactions that are believed to be some of the
most active pathways of ROS generation.

11. Compensive list of phenolic compounds isolated from marine algae
Seaweed Identified compounds
Phenolic Halimeda monile Salicylic,gallic, & caffeic acids
Laminaria digiata Gallic , protocatechuic, gentisic,p-hydroxybenzoic,
vallinic
Dictyota dichotoma Gallic, protocatechuic, gentisic, p-hydroxybenzoic,
chlorogenic, vallic, caffeic
Fucus vesiculosus Gallic, protocatechuic, gentisic, chlorogenic, vallic,
caffeic
Flavonoids Acanthophora soicfera Acanthophorin A, Acanthophorin B,triliroside,
catechin,quercetin
S. muticum, hypnea
spinella, porophyra sp.,
C. crispus
Daidzin, genistin ,ononin,
sissotrin,genistein,formononetin,bochanin

12. Phlorotannins:-
Phlorotannins are a class of marine algal polyphenolic Compounds mainly confined to the
brown algae.
These Compounds are oligomers of phloroglucinol (1,3,5-Trihydroxybenzene) monomers that
get biosynthesized Through the acetate–malonate pathway.
Phlorotannins can be categorized into several classes, in-Cluding fuhalols (phloroglucinol units
joined with ether Bridges at ortho and para positions with an extra OH-group in Each third
ring), phlorethols (phlorotannins, which containan ether linkage), fucols (that contain a phenyl
linkage), fu-Cophloroethols (that contain an ether and phenyl linkage), and Eckols (that contain
a dibenzodioxin linkage).
Phlorotannins are highly hydrophilic components due to the pres-Ence of -OH groups that can
form H bonds with water. The Molecular sizes of these compounds range between 126 and 650
kDa.
The brown alga Ecklonia cava is one of the most abundantSources of polyphenolic compounds,
including phlorotanninsSuch as phloroglucinol, eckol, fucodiphloroethol G,
phlorofucofuroeckol A, dieckol, 7-phloroeckol, and 6,6 bieckol Phlorotannins, including
phloroglucinol, eckol, Phlorofucofuroeckol.

13. Halogenated phenolic compounds:-
Halogenated phenolic compounds have been Found in brown algae and in red
algae species.
Most of Them have indicated pronounced radical scavenging activities. Brominated
and fully substituted mono- and bis-Phenols from the red-alga Symphyocladia
Latiuscula have shown potential DPPH radical scavenging Activities.
The red alga Polysiphonia urceolata was alsoFound to contain bromophenols (BPs)
with potent antioxi-Dant activity to DPPH radical scavenging activity.Li et al.
Report the isolation of five nitrogen-containing BPs from Rhodomela confervoides
with DPPH and 2,20-azino-bis(3-Ethylbenzothiazoline-6-sulfonate) (ABTS) radical
scavenging activity

14. B) Antioxidant from Marine Fungi:-
It has been observed That most marine sponges harbor often fungi with
representatives of Acremonium, Aspergillus, Fusarium, Penicillium, Phoma, and
Trichoderma.
Moreover, thanks to the accumulation of fungi in sponges. These macro-
organisms have been also used to find fungi belonging to the less common taxa,
such as Beauveria, Botryosphaeria, Epicoccum, Tritirachium,
Paraphaeosphaeria, Bartalinia and Volutella.

15. Extraction Techniques:-

16. Phenolic Compounds:-
Phenolic compounds are known for their innate antioxidant activity, and several recent studis
Report a direct proportionality between phenolic compounds content in fruits and their
antioxidant Properties.
They are naturally produced by many plant species and are in antioxidant Complex signalling
web between plants and the surrounding environment chaetopyramin, a metabolite with
scavenging properties obtained from the marine fungus C. globosum isolated from the red
algae Polysiphoniaurceolata. Chaetopyramin was obtained together with known derivatives
isotetrahydroauroglaucin and2-(203-epoxy-10,30-heptadienyl)-6-hydroxy-5-(3-methyl-2-
butenyl)benzaldehyde,showing IC50 valuesat the DPPH assay of 35, 26 and 88 µg/mL,
respectively.
Carotenoids:-
Carotenoids are cluster of fat-soluble pigments primarily present in photosynthetic
organisms And found produced also by many fungal species (especially pigmented yeasts).
Carotenoids are Biochemically terpenoids pigments synthesized from a 5-carbon precursor,
isopentenyl pyrophosphate (IPP) from the parent compound mevalonate by isoprenoid
pathway. Among the known carotenoids derived from marine fungi, Astaxanthin, a
xanthophyll carotenoidis the strongest anti-oxidants due to its structure and better
scavenging activity.

17. Anthraquinones and Xanthones:-
Anthraquinones represent a large group of quinoid pigments with about 700
molecules describedThat differ in the nature and position of substituent groups,
which are produced by plants, lichens,Insect, bacteria and fungi.
Anthraquinones are widespread in fungi, as they were found in several genus
including AspergillusSpp., Eurotiumspp., Fusarium spp., Dreschlera spp.,
Penicillium spp., Emericellapurpurea, Culvularialunata,Mycosphaerella rubella,
Microsporum sp. As in the case of other polyketide compounds,In fungi they
are synthetized through the acetate-malonate pathway.

18. Application Of Marine Fungal Antioxidants:-
Source strain Antioxidant molecules Extraction
methodologies
Phenolic compound
Callyspongia
siphonella(sponge)
Penicillium
brevicompactum
Syringic
acid,acetosyringone,
sinapic acids
Eb.extraction:ErOAc
Hypospongia
communis(sponge)
Gymnacella dankaliensis,
nigrospora
oryzae,chaetomium
globosum,engyodontium
album
Crude extracts Mycelium
extraction:EtOAC
Lomentaria catenate(red
algae)
Microsporum sp. Flavoglaucin&
isodihydroaurpglaocin
Eb. Extraction:EtOAc
Focus vesuculosus(brown
algae)
Epicoccum sp. 4,5,6-trihydroxy-7-
methylpthalide,
E.b. & mycelium
homogenized extraction
:EtOAc

19. C) Antioxidant from Marine Bacteria:-
It is estimated that more Than 50% sponge tissue are symbiont microorganisms.
Bioactive compounds mostly found in sponge symbiont microorganisms are generally
derived from alkaloids, terpenes, terpenoids and peptides.
Numerous types of infectious diseases caused by pathogenic bacteria or yeast are one of
basic problems in biomedical field that need to be seriously controlled. These bacteria are
widely reported to be highly potential as the producer of antimicrobial bioactive compound.
Materials and Methods:-
Sponge Sample Collection:-
The marine sponge Sylotella sp specimens used in This study were collected from
Bira island of Kepulauan Seribu, Indonesia. The sponges were Manually obtained from
1.5 to 3.0 m depth.

20. Hemolytic Test:-
The bacterial isolates that produced antimicrobial Compound were tested to know the
possibility of the Bacteria include in pathogene bacteria using hemolytic Test. The
bacterial isolates were streaked onto blood Agar medium and incubated overnight (24 h)
at room Temperature.
Extraction of Bioactive Compounds from Bacteria:-
The bacterial isolates were cultured in 1 L SWC Broth medium. The cultures were
incubated and shaked At 100 rpm for 72 h at 27°C. After incubation, bacteria Cultures
were mixed with ethyl acetate solvent 1: 0.75 (v/v) ratio in two replications.
Mixed solvent and bacteria Cultures were stirred at 250 rpm for 12 h at room
Temperature.

21. Antimicrobial Activity Test:-
Antimicrobial activities of crude extracts were tested Using agar diffusion method on
paper disc (6 mm in Diameter).
Positive control used was 5 mg mL−1 ampicillin while the negative control was ethyl
acetate solvent soaked-peper disc. Each palte was then incubated at 37°C for 24 h.

22. METHODS IF ANTIOXIDANTS ACTIVITY DETERMINATION:-
Antioxidant capacity
assay
Principle of the method End-product determination
Spectrometry
DPPH Antioxidant reaction with an organic redical Colometry
ABTS Antioxidant reaction with an organic cation radical Colometry
FRAP Antioxidant reaction with a Fe(|||) complex Colometry
CUPRAC Cu (|||) reduction to cu (|) by antioxidants Colometry
ORAC Antioxidant reaction with peroxy radicals, induced
by AAPH
Loss of fluorescence of
fluorescence of
Fluorimetry Emission of light by a substance that has absorbed
light or other electromagnetic radiation of a
different wavelength
Recording of fluorescence
excitation/emission spectra

23. Spectrometric Techniques:-
The DPPH method:-
DPPH(2,2-diphenyl-1-picrylhydrazyl) is a stable free radical, due to the delocalization of the
spare electron on the whole molecule. Thus, DPPHdoes not dimerize, as happens with most
free radicals. The delocalisation on the DPPH• molecule determines the occurence of a purple
colour, with an absorbtion band with a maximum around 520nm.
When DPPH reacts with a hydrogen donor, the reduced (molecular) form (DPPH) is
generated, accompanied by the disappearance of the violet colour. Therefore, the absorbance
diminution depends linearly on the antioxidant concentration. Trolox is used as standard
antioxidant
The ABTS method:-
The ABTS cation radical (ABTS•+) which Absorbs at 743 nm (giving a bluish-green colour)
is formed by the Loss of an electron by the nitrogen atom of ABTS (2,2’-azino-bis(3-
Ethylbenzthiazoline-6-sulphonic acid)).
In the presence of Trolox (or of Another hydrogen donating antioxidant), the nitrogen atom
quenches The hydrogen atom, yielding the solution decolorization.

24. The FRAP (ferric reducing antioxidant power) method:-
The FRAP (ferric reducing antioxidant power) method relies on the reduction by The
antioxidants, of the complex ferric ion-TPTZ (2,4,6-tri(2-pyridyl)-1,3,5-triazine). The
binding of Fe2+ to the ligand creates a very intense Navy blue color. The absorbance can
be measured to test the amount Of iron reduced and can be correlated with the amount
of antioxidants . Trolox or ascorbic acid were used.
The ORAC(oxygen radical absorption capacity)assay:-
The peroxyl radical, induced by 2,2’-azobis-(2-amidino-propane) Dihydrochloride
(AAPH). Fluorescein was used as the Fluorescent probe. The loss of fluorescence was an
indicator of the Extent of the decomposition, from its reaction with the peroxyl radical.
The lipid peroxidation inhibition assay:-
The lipid peroxidation Inhibition assay method uses a Fenton-like system (Co(II) +
H2O2), to Induce lipid (e.g. fatty acid) peroxidation Α-linolenic acid was chosed as a
model substrate. It was mixed with The analysed sample, as well as with the Fenton-like
mixture, to induce Lipid peroxidation. After the end of the incubation, the
concentration thiobarbituric acid-reactive substances (TBARS) was measured, as the
index of lipid peroxidation.

25. The CUPRAC (cupric reducing antioxidant power) assay:-
The standard antioxidants or extracts are mixed with CuSO4 and Neocuproine. After 30min,
the absorbance was measured at 450nm. In The assay, Cu(II) is reduced to Cu(I) through the
action of electron-Donating antioxidants. Results are expressed in milligrams of Trolox Per
liter of extract.
Fluorimetry:-
Fluorescence is the emission of light by a substance That has absorbed light or other
electromagnetic radiation of a different Wavelength. In most cases, emitted light has a
longer wavelength, and Therefore lower energy, than the absorbed radiation.
Fluorescence Emission occurs when an orbital electron of a molecule relaxes to its
Ground state, by emitting a photon of light after being excited to a Higher quantum state
by some type of energy. Fluorescence assay has Been used to antioxidant content
determination.