UNIERSITY OFAGRICULTURE, FAISALABADCentreof Agricultural Biochemistry and Biotechnology (CABB)(Synopsisfor M.
Phil Biotechnology)TITLE: PolyphenolicContent And Wound Healing Activities of DaturaStramoniumName: Raheel AsgharRegd. No.: 2016-ag-626Supervisor: Dr. Sehar Nawaz ABSTRACTDatura (Datura stramonium) known as medicinalplant obtained sample from this plant dried at 20oC, and convertedinto powdered form by grinding. Ultra sonic bath used obtain extract from driedsample with 70% ethanol and methanol. Centrifuge the solution to obtain thesupernatant. To evaporate the solvent the vortex evaporator used and thecrystal will be dissolve in PBS for scratch assay. Human cell line use forscratch assay.
Cultured in a medium containing FBS (Fetal Bovine Serum). Then the cellswill be treated with glucose for 72 hours. Different concentrations of plantextract applied to the cell for 24 hours. In the cell monolayer a linear woundwill be generated. By using a sterile pipette tip a scratch will be created 1mmwide range wound. PBS (Phosphate Buffered Saline) used to wash the wells toremove the cellular debris caused due to occurrence of scratch.
The scratchedarea will be measured after regular intervals in order to study the cellproliferation, migration activities. Polyphenolic content of the plant assessedby the HPLC. UNIERSITY OF AGRICULTURE, FAISALABADCentreof Agricultural Biochemistry and Biotechnology (CABB)(Synopsisfor M. Phil Biotechnology) TITLE: PolyphenolicContent And Wound Healing Activities of DaturaStramonium a. Date of admission: 09-09-2016b. Date of initiation: 09-09-2016 c. Probable Duration: 2 YearsPERSONNELName: Raheel AsgharRegd.
No.: 2016-ag-626Supervisor: Dr. Sehar NawazSUPERVISORYCOMMITTEEDr. Sehar Nawaz (Chairman)Dr. Nisar Ahmed (Member)Dr. Muhammad Kashif (Member) Need of the project:Datura stramonium inventsin the America but is now originate around the whole world including the warmerareas of Northern, Central and South America, European, Asian, and Africancountries. Datura stramonium has long been known for it’s a drug thatcause hallucination & euphoric effects.
The weed was dried and smoked forhallucinations and total relaxation. Datura stramonium was used conventionallyto 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 connectivetissue. andsciatica as an ointment a smooth oily preparation that is rubbed on theskin for medicinal purposes or as a beautifying, and simplificationspasms of Parkinsons disease (Williams et al.
,2013). The juice of the greeneriesis in warm milk was used to eject intestinal larvae including cestodes, sporeswith palm greases used outwardly for pest bites and stings, in Nigeria (Rajbhandariet al.,2001).
The herb was used for the cure of ulcers, injuries, swelling, backache,wound on initiating discoloration of the skin membrane& inflammations, is ancircumstances affecting long-lasting,often irregular aching disturbingthe linkages tissue.,gout is characterized by unexpected, severe attacks of hurt,inflammation, painfulness in the joints, frequently the joint at the base ofthe large toe., asthma bronchitis (bronchitis & asthama aretwo provocative airway circumstances) and toothache ( getting something stuckin between the teeth) (Kirtikar et al.,1999).
D. stramonium L. is a perennial herbaceous herbthat plant live for more than two years.
Plants are modest, alternative, dark bottlegreen, approximately ovate, shallowly lobed & glabrous. Florae are huge, privateand trumpet shaped. In particular literature, this classes is named thorn apple& jimson weed (Singh et al,.2013).Lesions are inevitableactions in lifespan.
Lesions may rise due to physically, chemicaly or infectiousagents. Lesions curative is a procedure by which tissue redevelopment occurs.It is complicated, dynamic process of restoring integrity and tissue layer. Theprocedure of lesions reparation diverges slight from 1 type of tissue to alternative& is commonly independent of the form of wound. Though the dissimilar stagesin the wound healing procedures happen in a continuous, integrated method, itis appropriate to divide the overall procedure into three overlapping stagesand numerous natural components for imaginative purposes (Patil et al.,2009).Injury therapeuticis the interface of a multifaceted cascade of cellular and biochemical activitiesleading to the reestablishment of structural and purposeful integrity with recoverof strength of injured tissues. It encompasses continuous cell-cell cooperationand cell matrix borders that allow the process to development in dissimilar overlyingphases and processes including inflammation, injury shriveling,reepithelialization, tissue remodeling, and development of granulation tissuewith angiogenesis.
The stages of injury healing usually development ina predictable, appropriatemethod, and if they do not, curative may development improperly to either a long-lastinginjury such as a venous ulcer or pathological scarring for example a keloid wound.Polyphenolicmixtures have concerned the courtesy of researcher as ordinary anti-oxidants.They have shown enormous potential as probable protecting features incontradiction of numerous terrible infections comprising tumor, heart illnesses,infectious infections etc. (Aslam et al., 2012; Baskar et al.
, 2012). Theantiradical probable of remedial florae is regularly related with theirphenolic acids, flavonoids is class of plant & fungus secondarymetabolites, tannins, and vitamins E & C contents. Herbal polyphenols mainlyflavonoids have ability to act as excellent free radical scavengers. Flavonoidsstop lipid peroxidation. It is also suggested that flavonoids have anti-inflammatoryused 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 existenceof heart infections. Solid suggestions about the welfares of polyphenols has advisedthe search for natural antioxidants that element inhibits oxidants leading tothe identification of natural properties and abstraction, separation ofantioxidant particles (Jahan et al.
, 2011).Many in vitroand in vivo examines have been planned, and are now obtainable for estimation ofthe wound healing process in mammalian skin. Among these, the scratch assay isa valuable and low-priced process to learning and measure procedures thatbelong to the 2nd stage of injury curative, specifically the proliferation andmigration of cells. (Van Horssen et al., 2006).Examines ofpolyphenolic substances in the Datura stramoniumremoves were performed using the HPLC diode array recognition.( Zubair etal.,2012).
The chiefobjective of this work to evaluate the polyphenolic content as well as theinjury curative action of Daturastramonium. Review of literature:Datura isa wild weed belongs to family Solanaceae, its term derived from Sanskrit term”Dhutra” (divine inebriation) is useing for its curative properties. Numerous classof Datura are identified & broadly employed for their therapeuticand poisonous properties that are based upon more than thirty alkaloids any ofa class of nitrogenous organic mixtures of plant source that have markedphysical activities on human being.
(Maheshwari etal.,2013). D. stramonium seeds are used for acne diseasedsebaceous glands in the skin membrane and bronchitis inSakarya region of North-west Turkey and locally named “Tatala”, although the fuelether extraction is also create to possess anti-microbial actions incontradiction of Esherichia coli ( Uzun et al., 2004) Datura herbal portions are used totreat numerous syndromes including asthma chronic infection of lungs airways,skin syndromes, jaundice yellow tinge in skin, piles another form ofhemorrhoids, and diabetes problem with harmone insulin.
Datura seedshave been used as a prophylactic amount to treat animal bites, specificallyCanis familiaris bites. Somebody bitten by dog and that dog is full of madnessdog 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 etal.,2013)The antimicrobial that killmicro-organism its action of the aqueous and ethanolic extract of the stem-barkof Datura stramonium was examination in contradiction of Staphylococcusaureus, Salmonella typhi, Shigella spp, Eschericia coli, Klebsiella pneumonia gonorrhea. Ethanolic extraction presented more antiseptic actionthan the aqueous extract. It presented antiseptic action in contradiction ofall 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 nationstate after heart infections and tumor. Upon wound in mammalian skin membrane amultifaceted process of injury curative begins. The procedure contains numerousevents overlying with each other. The main actions of injury curative procedureare; tenderness, cell proliferation is rise the amount of cells as a result ofcell development & migration and change the appearance its remolding. Anumber of cytokines and development factors are linked with these actions(Dinda et al., 2015). Wound curative is a complicated process and cellproliferation is an significant stage during the curative procedure of wound.
Throughoutthe procedure keratinocytes not only migrate and proliferate to protect thewound but also rapid cytokines & growth factors (Gurtner et al., 2008)Thescratch assay includes: (1) formation of a synthetic gap termed scratch, on aconfluent cell monolayer, (2) Cell movement on the ends of the anew producedgap towards the open to close the scratch (3) finally the restoration of a cellto cell interaction. The simple stages in this assay are: formation of ascratch on the cell mono layer, taking the pictures at the start and afterregular break to study cell migration, and lastly linking the pictures in orderto regulate the level of cell migration. The cell proliferation abilities of humanoidcell line were evaluated using a scratch wound curative assay a little adaptedfrom (Lianget al., 2007).Phenolic mixturesare generally present in numerous florae and exhibition many therapeutically abilities(Kahkonen et al., 1999).
Many therapeutic plants are abundant foundationsof phyto chemicals for example phenolic & poly phenolic mixtures, severalof which have solid antioxidant activities which are regularly used in nutritiongoods and in many healing cures (Wong et al., 2006; Liu et al.,2008).
Theseeds, shrubberies, shoot and blossom extracts of D. stramonium exhibiteda wide-ranging of Total polyphenolic contents & inhibit the oxidationpotential. All shares of plant under learning process displayed considerable quantityof whole polyphenols. Among dissimilar portions, blossom extracts organized byreflux 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 sixpercent), Nitric oxide radical (eighty six percent) & Linoleic acidperoxidation (56.
45 percent). Methanolic extract of florae also displayed extremefalling potential. These outcomes designate that florae of D. stramonium arepromising cause of regular anti-oxidants (Sana et al.,2014)Polyphenols& flavonoids are used for the inhibition & treatment of many infectionswhich is mostly related with free radicals (Havesteen, 1983).
Examines of polyphenolic insides in the Plant ago leafextracts were achieved using the similar apparatus, circumstances and proceduresas in our earlier studies (Zubair et al.,2011, 2012). Therefore, examines were achievedusing an HPLC diode array discovery technique. When phenolic compounds isseprated, a Synergi hydro-RP 80A (250 mm ×4.60 mm, 4! m) column wasused at a temperature of 24 ?C. The mobile stage contained ofsolution 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 0–10min; 79% A, 21% B for 10–13 min; 20% A, 80% B for 13–14 min; 82% A, 18% B for14–16.
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 mainleaf extracts had before been did by Zubair etal. (2012). MATERIAL AND METHODS The research will be conducted in LAB 1, in the Centreof Agricultural Biochemistry and Biotechnology (CABB), University ofAgriculture, Faisalabad.PlantMaterial:Leafs of Datura Stramoniumcollected from the Botanical garden of University of Agriculture,Faisalabad. PolyphenolicStudy:The polyphenoic content of the Datura stramonium examined by the HPLC diodearray recognition technique.ScratchAssay:The wound healing action of Datura stramonium can be measured by scratch assay.Literature Cited:Williams DG.
Larvicidal potential ofthe leaf extract of Datura stramonium and Occimum gratissimum against Culex quinquefasciatus mosquitospecies. MSc thesis, Faculty of Science, Amadu Bello University- Zaria 2013 Rajbhandari KR Ethnobotany of Nepal.Kathmandu: Kishor Offset Press Private Limited, 2001: 142-143Kirtikar KR and Basu BD. Indianmedicinal plants. 2nd ed. Volume III. Dehradun: International Book Distributors1999: 1783-1787.
Singh, L.R. ; Singh, O.M. Datura stramonium:An overview of its phytochemistry and pharmacognosy. Research J. Pharmacognosyand Phytochemistry, 5.3.
, 143-148, 2013.Patil, S. B.
,Naikwade, N. S., Kondawar, M. S., Magdum, C. S., & Awale, V.
B. (2009).Traditional uses of plants for wound healing in the Sangli district,Maharashtra. International Journal of PharmTech Research, 1(3),876-878.
Aslam, S., N. Jahan, S. Ali and K.
U. Rehman. 2012.
An innovative microwave-assisted extraction and antioxidant potential ofpolyphenols from different parts of Ocimum basilicum. J. Med.
Plants Res.6:2150-2159 Baskar, A.A., K.S.A. Numair, M.
A. Alsaif, S.Ignacimuthu. 2012.
In vitro antioxidant and antiproliferative potential ofmedicinal plants used in traditional Indian medicine to treat cancer. Red. Rep.17:145-156. Sana, F., Nazish, J.,Nosheen, S.
, & Saba, A. (2014). Comparative antioxidant potential and totalpolyphenolic contents of different parts of Datura stramonium. PakistanJournal of Agricultural Sciences, 51(3), 719-724.P.
Martin, “Wound healing—aiming for perfect skin regeneration,”Science, vol. 276, no. 5309, pp. 75–81, 1997.
Jahan, N., K. Rehman, S.
Ali and I.A. Bhatti. 2011.Antioxidant activity of gemmo therapeutically treated indiginous medicinalplants. Asian J.
Chem. 23:3461- 3470. VanHorssen, R., Galjart, N., Rens, J.A.P., Eggermont, A.
M.M., Ten Hagen, T.L.M.,2006.
Differential effects of matrix andgrowth factors on endothelial and fibroblast motility: application of amodified cell migration assay. Journal of Cellular Biochemistry 99, 1536–1552. Zubair, M., Ekholm, A., Nybom, H., Renvert, S.,Widen, C., & Rumpunen, K.
(2012). Effects of Plantago major L. leafextracts on oral epithelial cells in a scratch assay. Journal ofEthnopharmacology, 141(3), 825-830.
Maheshwari, N. O., Khan, A., & Chopade, B.A. (2013). Rediscovering the medicinal properties of Datura sp.
: Areview. Journal of Medicinal Plants Research, 7(39),2885-2897.Uzun E, Sariyara G,Adsersenb A, Karakocc B, Ötük G, Oktayoglua E, Pirildara S(2004). Traditional medicine in Sakarya province (Turkey) and antimicrobialactivities of selected species.
J. Ethnopharmacol. 95:287. Shagal MH, Modibbo UU and Liman AB. Pharmacologicaljustification for the ethnomedical use of Datura stramonium stem-bark extractin treatment of diseases caused by some pathogenic bacteria. Int Res PharmPharmacol 2012; 2(1): 16-19. Barbosa-pereira L, Pocheville A, Anugulo I,Peseiro-Losada P, Cruz JM.
2013.Fractionation and Purification of BioactiveCompounds Obtained from a Brewery Waste Stream. BioMed Research International2013;2013:408491DindaM, Dasgupta U, Singh N, Bhattacharyya D, Karmakar P. 2015.
PI3K-mediatedproliferation of fibroblasts by Calendula officinalis tincture: implication inwound healing. Phytother Res 29(4): 607–616. GurtnerGC, Werner S, Barrandon Y, Longaker MT. 2008. Wound repair and regeneration.Nature 453, 314–321.
Liang, C.C., Park, A.
Y., Guan,J.L., 2007. In vitro scratch assay: a convenient andinexpensive method for analysisof cell migration in vitro. Nature Protocols 2,329–333.HavesteenB (1983). Flavonoids, a class of natural products of high pharmacologicalpotency.
Biochem. Pharmacol. 30:1141–1148.Wong, P.S., L.P. Leong and J.
H.W. Koh.2006. Antioxidant activities of aqueous extracts of selected plants. Food Chem.99:775-783.Liu, H.
, N. Qiu, H. Ding and R. Yao. 2008.Polyphenols contents and antioxidant capacity of 68 Chinese herbals suitablefor medical or food uses. Food Res.