8/16/2019 Investigating Antimicrobial Activity of Plant Extracts (Pre-post)

1/5

Pre - Report

Investigating Antimicrobial Activity of Plant Extracts

2016 / 04 / 04ection 1 / 201!10110" / Ra#man $ony %&r

8/16/2019 Investigating Antimicrobial Activity of Plant Extracts (Pre-post)

2/5

1. TitleInvestigating Antimicrobial Activity of Plant Extracts'

2. PurposeInvestigation of antimicrobial activity of plant extracts on t#e Escherichia coli.

3. Theorya' (&lt&re met#o)* prea)ing

+ne ,ay to isolate p&re colonies is t#e sprea) plate tec#ni &e' tarting from a li &i) c&lt&re of bacteria. a series of tenfol) )il&tions is ma)e. an)a small amo&nt of eac# )il&tion is place) )irectlyon t#e s&rface of separate agar plates' $#esample is sprea) over t#e s&rface of t#e plate ,it#a #eat-sterili e). bent glass ro)' $#e early)il&tions. t#ose containing t#e most bacteria. ,ill

pro)&ce confluent gro,t# t#at covers t#e entireagar s&rface' ater )il&tions. containing fe,er an) fe,er organisms. yiel) in)ivi)&al colonies'As ,e s#all see later. sprea) plates not only enable &s to isolate p&re c&lt&res b&t also can be&se) to en&merate t#e n&mber of viable bacteria in t#e original gro,t# t&be' A viable organismis one t#at s&ccessf&lly replicates to form a colony' Thus, each colony on agar plate representsone viable organism present in the original liquid culture.

b' train* E. coli 213 Protease eficient 5 lon ompT 3* E' coli strains are 7nat&rally7 )eficient in t#e lon protease,#ic# in 8-12 strains serves to )egra)e misfol)e) proteins an) to prevent some cell cycle-specific proteins from acc&m&lating' $#e +mp$ protease resi)es at t#e s&rface of ,il) type E.coli in bot# 8-12 an) strains. pres&mably #elping t#e cells to )erive amino aci)s from t#eir external environment' (ells )eficient in bot# t#ese proteases are m&c# more amenable to t#e

pro)&ction of proteins from clone) genes' $1 P#age Resistant fhuA23* $1. an extremely vir&lent p#age re &ires t#e E. coli ferric#y)roxamate &pta9e receptor for infectivity' eletion of t#is gene confers resistance to t#istype of p#age. b&t )oes not significantly affect t#e transformation or gro,t# c#aracteristics of

t#e cell'

c. (lear one :one of In#ibition3

8/16/2019 Investigating Antimicrobial Activity of Plant Extracts (Pre-post)

3/5

+n t#is agar plate. a bacterial isolate is teste) for resistance to eac# of t,elve )ifferent antibiotics' $#eclear ones aro&n) eac# )isc are t#e ones of in#ibitiont#at in)icate t#e extent of t#e test organism;s inability tos&rvive in t#e presence of t#e test antibiotic' A3$#e )is9 s#o,s a large one of in#ibition< ,#ereas 3 s#o,s noone of in#ibition. in)icating resistance of t#e isolate tot#e test antibiotic' Presence of one of in#ibition is nota&tomatically interprete) as s&sceptibility to t#e

antibiotic< t#e one ,i)t# #as to be meas&re) an) compare) against a reference stan)ar) ,#ic#contains meas&rement ranges an) t#eir e &ivalent &alitative categories of s&sceptible.interme)iately s&sceptible or resistant'=or example. t#is E'coli isolate on t#e rig#t #as a one of in#ibition of 10'1mm aro&n) ampicillin A>3< since t#eone )iameter interpretation c#art is as follo,s*

Resistant* 1?mm or lessInterme)iate* 14-16 mm&sceptible* 1@ mm or more$#is partic&lar E'coli isolate is rea) as resistant to ampicillin'

d. (#aracteristics an) mec#anism of t#e anti-bacterial activity of S. baicalensis an)C. chinensis.

Scutellaria baicalensis: 1' 2g )issolve) in 4mlB 3 - Coptis chinensis: 1'!6g )issolve)in 4ml B 3'

$#e (#inese name for Coptis sp' =ranc#. from t#eRan&nc&leae family. is C&ang lian' $#ere are 16species ,orl),i)e. 21 six of t#ese gro, in mainlan)(#ina. an) only Coptis quinquefolia in $ai,an' +f t#ese. C. chinensis . C. deltoidea an) C. tetoides aretypically fo&n) in #erbal mar9ets in (#ina an)

$ai,an' $#e r#i ome #as been fo&n) to be a ric# so&rce of t#e al9aloi) berberine' In reference boo9s. C&ang ian is specifie) most often as C. chinensis ' It is mainly &se) tra)itionally as a#eat p&rgative. anti-inflammatory. )e-toxicant an) to clear )amp' $#e (#inese name for S.

baicalensis Deorgi. from t#e amiaceae family is C&ang in an) #as been fo&n) to contain#ig# levels of t#e flavones ,ogonosi)e an) baicalin' ot# t#ese #erbs are tra)itionally &se) totreat con)itions bro&g#t abo&t from excess #eat an) )amp in t#e bo)y. incl&)ing bacterialgastroenteritis'

e. >et#o) of extracting of t#e antimicrobials in plants'i' olvent extraction

$#e plant parts are )rie) imme)iately eit#er in an artificial environment at lo,temperat&re !0-60F(3 or )rie) preferably in s#a)e so as to bring )o,n t#e initial largemoist&re content to enable its prolonge) storage life an) ' $#e )rie) berries are p&lverise)

by mec#anical grin)ers an) t#e oil is remove) by solvent extraction' $#e )efatte) materialis t#en extracte) in a sox#let apparat&s or by soa9ing in ,ater or alco#ol "!G v/v3' $#e

8/16/2019 Investigating Antimicrobial Activity of Plant Extracts (Pre-post)

4/5

res&lting alco#olic extract is filtere). concentrate) in vac&o or by evaporation. treate) ,it#C(l 12%3 an) refl&xe) for at least six #o&rs' $#is can t#en be concentrate) an) &se) to)etermine t#e presence of p#ytoconstit&ents' Denerally. t#e saponins )o #ave #ig#molec&lar ,eig#t an) #ence t#eir isolation in t#e p&rest form poses some practical)iffic&lties' $#e plant parts t&bers. roots. stems. leave etc3 are ,as#e) slice) an) extracte),it# #ot ,ater or et#anol "!G v/v3 for several #o&rs' $#e res&lting extract is filtere).concentrate) in vac&o an) t#e )esire) constit&ent is precipitate) ,it# et#er'

ii' &percritical fl&i) extraction =E3 &per (ritical =l&i) Extraction =E3 involves &se of gases. &s&ally (+2. an)compressing t#em into a )ense li &i)' $#is li &i) is t#en p&mpe) t#ro&g# a cylin)er containing t#e material to be extracte)' =rom t#ere. t#e extract-la)en li &i) is p&mpe) intoa separation c#amber ,#ere t#e extract is separate) from t#e gas an) t#e gas is recovere)for re-&se' olvent properties of (+2 can be manip&late) an) a)H&ste) by varying t#e

press&re an) temperat&re t#at one ,or9s at' $#e a)vantages of =E are. t#e versatility itoffers in pinpointing t#e constit&ents yo& ,ant to extract from a given material an) t#e fact

t#at yo&r en) pro)&ct #as virt&ally no solvent resi)&es left in it (+2 evaporatescompletely3'

iii' >icro,ave-Assiste) extraction >AP applications incl&)e t#e extraction of #ig#-val&e compo&n)s from nat&ral so&rcesincl&)ing p#yton&trients. n&trace&tical an) f&nctional foo) ingre)ients an) p#armace&ticalactives from biomass' >AP tec#nology offers some combination of t#e follo,inga)vantages* 1' Improve) pro)&cts. increase) p&rity of cr&)e extracts. improve) stability of mar9er compo&n)s. possibility to &se less toxic solvents< 2' Re)&ce) processing costs.increase) recovery an) p&rity of mar9er compo&n)s. very fast extraction rates. re)&ce)energy an) solvent &sage' Examples incl&)e antioxi)ants from )rie) #erbs. carotenoi)s

from single cells an) plant so&rces. taxanes from tax&s biomass. p#ytosterols fromme)icinal plants. polyp#enols from green tea. an) many more'

4. Experimental Method (Reagent & pparatus!a. Reagent Apparat&s

Alco#ol lamp. plate. loop. et#anol. sprea)er. micropipette. !mm paper )iscs. t,ee er.)eioni e) ,ater. agar plate. pre-c&lt&re) E. coli brot#. plant extract sol&tions . (3. )il&te)extract sol&tions 1/10 . 1/10 (3. inc&bator'

". Experimental >et#o)s1' abel at t#e bottom of t#e plate 1/10 . 1/10 (. . (. B3'2' Po&r "!G et#anol to a bea9er an) immerse t#e sprea)er' terili e t#e sprea)er &sing flame

an) c#ill it to ambient temperat&re' Jo& s#o&l) #an)le t#e sprea)er caref&lly ,#en &sing et#anol ,it# flame' terili e t#e sprea)er an) cool it )o,n in t#e air for 1! secon)s'

?' rop 200 of t#e pre-c&lt&re) E. coli brot# on agar plate &sing micropipette an)micropipette tips'

4' prea) t#e brot# on agar plate'!' P&t ! paper )iscs ! mm3 on t#e agar plate &sing flame-sterili e) t,ee er'6' rop 10 of )eioni e) ,ater control3. plant extract sol&tions . (3. )il&te) extract

sol&tions 1/10 . 1/10 (3 on eac# paper )isc' rop t#e sol&tion slo,ly'

@' Inc&bate t#e agar plate for overnig#t at ?@ an) observe t#e formation of t#e clear one'meas&re t#e si e3

8/16/2019 Investigating Antimicrobial Activity of Plant Extracts (Pre-post)

5/5

o not let t#e plate be &psi)e-)o,n

#. Re$erence1' Koan ' lonc e,s9i. Ko#n B' =oster< >icrobiology* An Evolving cience 2n) e)' < B' B'

%orton (ompany< 2011< p126-122' =rancesca ' eac#< Anti-microbial properties of c&tellaria baicalensis an) (optis c#inensis.

t,o tra)itional (#inese me)icines< #ttp*//bio#ori ons'oxfor)Ho&rnals'org/content/4/2/11"?' #ttp*//amrls'cvm'ms&'e)&/microbiology/)etecting-antimicrobial-resistance/test-

met#o)s/examples-of-antibiotic-sensitivity-tesing-met#o)s4. #ttps*//,,,'neb'com/pro)&cts/c2!?0-bl21-competent-e-coli

https://www.neb.com/products/c2530-bl21-competent-e-colihttps://www.neb.com/products/c2530-bl21-competent-e-coli