of Agricultural Biochemistry and Biotechnology (CABB)

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for M. Phil Biotechnology)

TITLE:       Polyphenolic
Content And Wound Healing Activities of Datura

Raheel Asghar

Regd. No.:                                                               2016-ag-626

Dr. Sehar Nawaz



Datura (Datura stramonium) known as medicinal
plant obtained sample from this plant dried at 20oC, and converted
into powdered form by grinding. Ultra sonic bath used obtain extract from dried
sample with 70% ethanol and methanol. Centrifuge the solution to obtain the
supernatant. To evaporate the solvent the vortex evaporator used and the
crystal will be dissolve in PBS for scratch assay. Human cell line use for
scratch assay. Cultured in a medium containing FBS (Fetal Bovine Serum). Then the cells
will be treated with glucose for 72 hours. Different concentrations of plant
extract applied to the cell for 24 hours. In the cell monolayer a linear wound
will be generated. By using a sterile pipette tip a scratch will be created 1mm
wide range wound. PBS (Phosphate Buffered Saline) used to wash the wells to
remove the cellular debris caused due to occurrence of scratch. The scratched
area will be measured after regular intervals in order to study the cell
proliferation, migration activities. Polyphenolic content of the plant assessed
by the HPLC.












of Agricultural Biochemistry and Biotechnology (CABB)

for M. Phil Biotechnology)


TITLE:       Polyphenolic
Content And Wound Healing Activities of Datura


a. Date of admission:                                                          09-09-2016

b. Date of initiation:                                                            09-09-2016    

c. Probable Duration:                                                          2 Years


            Raheel Asghar

Regd. No.:                                                              

Supervisor:                                                                          Dr. Sehar Nawaz


Dr. Sehar Nawaz                                                    

Dr. Nisar Ahmed                                                                 (Member)

Dr. Muhammad Kashif                                               (Member)


Need of the project:

Datura stramonium invents
in the America but is now originate around the whole world including the warmer
areas of Northern, Central and South America, European, Asian, and African
countries. Datura stramonium has long been known for it’s a drug that
cause hallucination & euphoric effects. The weed was dried and smoked for
hallucinations and total relaxation. Datura stramonium was used conventionally
to treat asthma, gastrointestinal complications, pains, abscesses, inflammation,
boils, headaches, hemorroids, rattlesnake bites, sprains, bulges, and cancers.
It was used for dismissing the pains of is an umbrella term for circumstances producing chronic,
often alternating discomfort affect the joins or connective
tissue. and
sciatica as an ointment a smooth oily preparation that is rubbed on the
skin for medicinal purposes or as a beautifying, and simplification
spasms of Parkinsons disease (Williams et al.,2013). The juice of the greeneries
is in warm milk was used to eject intestinal larvae including cestodes, spores
with palm greases used outwardly for pest bites and stings, in Nigeria (Rajbhandari
et al.,2001). The herb was used for the cure of ulcers, injuries, swelling, backache,
wound on initiating discoloration of the skin membrane
& inflammations,  is an
circumstances affecting long-lasting,
often irregular aching disturbing
the linkages 
gout is characterized by unexpected, severe attacks of hurt,
inflammation, painfulness in the joints, frequently the joint at the base of
the large toe., asthma bronchitis (bronchitis & asthama are
two provocative airway circumstances) and toothache ( getting something stuck
in between the teeth) (Kirtikar et al.,1999). D. stramonium L. is a perennial herbaceous herb
that plant live for more than two years. Plants are modest, alternative, dark bottle
green, approximately ovate, shallowly lobed & glabrous. Florae are huge, private
and trumpet shaped. In particular literature, this classes is named thorn apple
& jimson weed (Singh et al,.2013).

Lesions are inevitable
actions in lifespan. Lesions may rise due to physically, chemicaly or infectious
agents. Lesions curative is a procedure by which tissue redevelopment occurs.
It is complicated, dynamic process of restoring integrity and tissue layer. The
procedure of lesions reparation diverges slight from 1 type of tissue to alternative
& is commonly independent of the form of wound. Though the dissimilar stages
in the wound healing procedures happen in a continuous, integrated method, it
is appropriate to divide the overall procedure into three overlapping stages
and numerous natural components for imaginative purposes (Patil et al.,2009).

Injury therapeutic
is the interface of a multifaceted cascade of cellular and biochemical activities
leading to the reestablishment of structural and purposeful integrity with recover
of strength of injured tissues. It encompasses continuous cell-cell cooperation
and cell matrix borders that allow the process to development in dissimilar overlying
phases and processes including inflammation, injury shriveling,
reepithelialization, tissue remodeling, and development of granulation tissue
with angiogenesis. The stages of injury healing usually development in

a predictable, appropriate
method, and if they do not, curative may development improperly to either a long-lasting
injury such as a venous ulcer or pathological scarring for example a keloid wound.

mixtures have concerned the courtesy of researcher as ordinary anti-oxidants.
They have shown enormous potential as probable protecting features in
contradiction of numerous terrible infections comprising tumor, heart illnesses,
infectious infections etc. (Aslam et al., 2012; Baskar et al., 2012). The
antiradical probable of remedial florae is regularly related with their
phenolic acids, flavonoids is class of plant & fungus secondary
metabolites, tannins, and vitamins E & C contents. Herbal polyphenols mainly
flavonoids have ability to act as excellent free radical scavengers. Flavonoids
stop lipid peroxidation. It is also suggested that flavonoids have anti-inflammatory
used to decrease inflammation, anti-allergic used to decrease allergic agent,
anti-viral and anti-cancer used to decrease cancer properties (Sana et al.,
2014). There is opposite connection b/w the intake of polyphenols and existence
of heart infections. Solid suggestions about the welfares of polyphenols has advised
the search for natural antioxidants that element inhibits oxidants leading to
the identification of natural properties and abstraction, separation of
antioxidant particles (Jahan et al., 2011).

Many in vitro
and in vivo examines have been planned, and are now obtainable for estimation of
the wound healing process in mammalian skin. Among these, the scratch assay is
a valuable and low-priced process to learning and measure procedures that
belong to the 2nd stage of injury curative, specifically the proliferation and
migration of cells. (Van Horssen et al., 2006).

Examines of
polyphenolic substances in the Datura stramonium
removes were performed using the HPLC diode array recognition.( Zubair et

The chief
objective of this work to evaluate the polyphenolic content as well as the
injury curative action of Datura



Review of literature:

Datura is
a wild weed belongs to family Solanaceae, its term derived from Sanskrit term
“Dhutra” (divine inebriation) is useing for its curative properties. Numerous class
of Datura are identified & broadly employed for their therapeutic
and poisonous properties that are based upon more than thirty alkaloids any of
a class of nitrogenous organic mixtures of plant source that have marked
physical activities on human being. (Maheshwari et
al.,2013). D. stramonium seeds are used for acne diseased
sebaceous glands in the skin membrane and bronchitis in
Sakarya region of North-west Turkey and locally named “Tatala”, although the fuel
ether extraction is also create to possess anti-microbial actions in
contradiction of Esherichia coli ( Uzun et al., 2004) Datura herbal portions are used to
treat numerous syndromes including asthma chronic infection of lungs airways,
skin syndromes, jaundice yellow tinge in skin, piles another form of
hemorrhoids, and diabetes problem with harmone insulin. Datura seeds
have been used as a prophylactic amount to treat animal bites, specifically
Canis familiaris bites. Somebody bitten by dog and that dog is full of madness
dog is managed with juice of Datura along with butter milk & jiggery.
Also, the site of bite is messy with a paste of the fruit. (Maheshwari et

The antimicrobial that kill
micro-organism its action of the aqueous and ethanolic extract of the stem-bark
of Datura stramonium was examination in contradiction of Staphylococcus
aureus, Salmonella typhi, Shigella spp, Eschericia coli, Klebsiella pneumonia &
Neisseria gonorrhea. Ethanolic extraction presented more antiseptic action
than the aqueous extract. It presented antiseptic action in contradiction of
all the verified microbes excluding Neisseria gonorrhea. (Shagal et al., 2012).

Injury living tissue caused by a cut,
blow or other effect is the 3rd main purpose for medicinal visits in developed nation
state after heart infections and tumor. Upon wound in mammalian skin membrane a
multifaceted process of injury curative begins. The procedure contains numerous
events overlying with each other. The main actions of injury curative procedure
are; tenderness, cell proliferation is rise the amount of cells as a result of
cell development & migration and change the appearance its remolding. A
number of cytokines and development factors are linked with these actions
(Dinda et al., 2015). Wound curative is a complicated process and cell
proliferation is an significant stage during the curative procedure of wound. Throughout
the procedure keratinocytes not only migrate and proliferate to protect the
wound but also rapid cytokines & growth factors (Gurtner et al., 2008)

scratch assay includes: (1) formation of a synthetic gap termed scratch, on a
confluent cell monolayer, (2) Cell movement on the ends of the anew produced
gap towards the open to close the scratch (3) finally the restoration of a cell
to cell interaction. The simple stages in this assay are: formation of a
scratch on the cell mono layer, taking the pictures at the start and after
regular break to study cell migration, and lastly linking the pictures in order
to regulate the level of cell migration. The cell proliferation abilities of humanoid
cell line were evaluated using a scratch wound curative assay a little adapted
from  (Liang
et al., 2007).

Phenolic mixtures
are generally present in numerous florae and exhibition many therapeutically abilities
(Kahkonen et al., 1999). Many therapeutic plants are abundant foundations
of phyto chemicals for example phenolic & poly phenolic mixtures, several
of which have solid antioxidant activities which are regularly used in nutrition
goods and in many healing cures (Wong et al., 2006; Liu et al.,

seeds, shrubberies, shoot and blossom extracts of D. stramonium exhibited
a wide-ranging of Total polyphenolic contents & inhibit the oxidation
potential. All shares of plant under learning process displayed considerable quantity
of whole polyphenols. Among dissimilar portions, blossom extracts organized by
reflux used methanol as solvent, displayed the uppermost polyphenolic substances
(880 mg GAE/g of extract) & showed maximum antioxidant potential.
Methanolic extracts of florae at 100?g/mL concentration repressed the extreme 2,2-diphenyl-1-picrylhydrazyl redical (ninety nine percent), Super oxide radical (ninety six
percent), Nitric oxide radical (eighty six percent) & Linoleic acid
peroxidation (56.45 percent). Methanolic extract of florae also displayed extreme
falling potential. These outcomes designate that florae of D. stramonium are
promising cause of regular anti-oxidants (Sana et al.,2014)

& flavonoids are used for the inhibition & treatment of many infections
which is mostly related with free radicals (Havesteen, 1983). Examines of polyphenolic insides in the Plant ago leaf
extracts were achieved using the similar apparatus, circumstances and procedures
as in our earlier studies (Zubair et al.,
2011, 2012). Therefore, examines were achieved
using an HPLC diode array discovery technique. When phenolic compounds is
seprated, a Synergi hydro-RP 80A (250 mm ×4.60 mm, 4! m) column was
used at a temperature of 24 ?C. The mobile stage contained of
solution A: acetic acid (one percent) & acetonitrile (five percent) in H2O & solution B: methanol (five percent) and acetonitrile
(ninety five percent). The gradient used was as follows: 82% A, 18% B for 0–10
min; 79% A, 21% B for 10–13 min; 20% A, 80% B for 13–14 min; 82% A, 18% B for
14–16.1 min and then 82% A, 18% B to the end of the run (18 min).
Identification of main original polyphenolic mixtures in the plant ago main
leaf extracts had before been did by Zubair et
al. (2012).



The research will be conducted in LAB 1, in the Centre
of Agricultural Biochemistry and Biotechnology (CABB), University of
Agriculture, Faisalabad.


Leafs of Datura Stramonium
collected from the Botanical garden of University of Agriculture,




The polyphenoic content of the Datura stramonium examined by the HPLC diode
array recognition technique.


The wound healing action of Datura stramonium can be measured by scratch assay.

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