materials and methods of pharmacognosy

8/13/2019 Materials and Methods of Pharmacognosy

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3.1. Collection of plant materials

Plant parts ofMadhuca longifolia were collected from 25-30 year old trees, from a temple-

owned grove, in a village, Rajendrum Arcot of Tanjavur district, Tamilnadu! Basella alba

and Cissus quadrangularis are collected from te plants maintained at "er#al $arden,

Tamil %niversity, Tanjavur!

&ollected plant specimens were identi'ed wit te use of local (loras vi)! $am#le *+./

and attew *+13/! dentity of plant specimens was con'rmed in consultation wit

otanical 4urvey of ndia *4/ &oim#atore! dentity of ariant of C. quadrangularis was

con'rmed #y te use of er#arium seets availa#le at 4, &oim#atore *4eets 6o7 "

6o! 35 and 8 6o! 119/!

3.2. Anatomical methods

&ollected plant parts were ':ed in (AA *5; 8tyl alcool 50 parts < $lacial acetic acid

5 parts < (ormalian +0 parts < =istilled water 35 parts/ in te 'eld itself! (ree-and

sections para>n em#edded sections and macerations were employed werever necessary!

=rawings were made wit te use of prism type camera ?ucida and micropotograps

were ta@en using ?eit)-rtoplan microscope! &alcium o:alates were potograped wit

polari)ed ligt in te same microscope! icro-measurements were made #y cali#rating a

stage micrometer wit ocular micrometer!

(i:ed plant parts were deydrated in 6-#utyl alcool *6A/ or tertiary #utyl alcool

*TA/ series!

6A deydration series was prepared as follows7

Sl. No. Ethyl Alcohol ml NBA ml Distilled water ml Time in hours

1 20 +0 .0 +

2 25 +5 0 +

3 30 25 95 +

30 90 30 +

! 25 55 20 2

" 20 .0 +0 2

# +5 15 -- 2

$ -- +00 -- 2

TA deydration series was prepared as follows7


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S1. NoDistilled %ater

&n mlTBA in ml

Ethyl Alcohol '!(

&n ml

Ethyl Alcohol

1))( mlTimes in hours

1 50 +0 90 -- 2

2 30 20 50 -- +23 +5 35 50 -- +

-- 55 95 -- +

! -- .5 -- 25 +

" -- +00 -- -- +

# -- +00 -- -- +2

$ -- +00 -- -- +

=eydrated materials were in'ltrated at 55-0B & and em#edded in para>n wa: of

melting point 5-51B&! Transverse and longitudinal sections were cut at 5-+5tic@nesses

using a rotary microtome! "auptCs adesive wit formalin *9;/ was used for a>:ing te

para>n sections on te slides!

3.!* +eneral Stainin,

Te staining scedules followed were,

+! Toluidine lue C0C

2! 4afranin CaC and (ast $reen C(&(C

3.!.1* Toluidine Blue -)-

=iDerential staining of sections was acieved witout removing te para>n #y using

0!05; toluidine #lue in water! After staining and wasing in distilled water, te slides

were dried, de-wa:ed in :ylene and mounted in =PE!

3.!.2* Safranin -)- and ast +reen -C-

2!25 gm of safranin was dissolved in 225 ml of 5; alcool! ne gm of fast green was

dissolved in a mi:ture of clove oil and a#solute alcool *in te ratio of .5725/! Te de-

wa:ed slides were stained in safranin for su>cient time and wased in alcool until te

e:cess stain was removed! Ten te slides were stained in fast green and diDerentiated in

clove oil and alcool! Te slides were #rougt down to :ylene and mounted in =PE!

3."* /istochemical stains3.".1* 0hloro,lucinol


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Tis stain was used to locali)e ligni'ed cells! 4ectioned plant parts were Fooded in + ;

solution of ploroglucinol in etanol for +-2 minutes! 8:cess stain was drained oD! To tis

few drops of "& was added! t was added and e:cess "& was drained oD! t was

mounted in wee@ glycerin and o#served!

3.".2* erric chloride

?ocali)ation of tannin was acieved #y use of ferric cloride! 4ectioned materials were

@ept in +0; formalin containing 2; ferric cloride! 4tained sections were wased and

mounted in water!

3.".3* &odine 0otassium &odide

t was used to stain starc grains in te cells! 2 gm of potassium iodide and 0!2 gm of

iodine was dissolved in +00 ml of water and used as reagent! 4ections of plant materials

were mounted in te a#ove reagent and o#served!

3.".* Sudan lac -B-

4udan #lac@ CC was used to identify oil content in te cells! 4ections of plant materials

were soa@ed in 50; etanol and ten in 4udan #lac@ CC dissolved in .0; etanol! Te

sections were diDerentiated in 50; etanol for one minute and o#served!

3.#* 4aceration

GeDreyCs maceration metod was adopted! 4mall pieces of specimens not more tan one

nun in tic@ness were @ept in a solution of eHual parts of +0; cromic acid and +0;

nitric acid for overnigt or until te materials #ecameC musyC in te:ture! After

maceration, te tissues were wased in water and stained wit safranin! Te macerated

and stained tissues were spread on slides and mounted in glycerin for o#servation!

3.$* 5uantitati6e microscopy

Peelings of upper and lower leaf epidermal layers were used for calculating te stomatal

inde:! ?eaf segments of 2 E +0 mm in si)e were soa@ed in sodium ypoclorite solution,

until tey got #leaced! Tese #leaced materials were used for calculating te palisade

ratio, vein islet num#er and vein termination num#er! &alcium o:alate crystals were also

o#served in tese materials and measured!

3.'* Analytical 4ethods

Te procedures recommended in ndian Parmacopoeia *+/ were followed for

calculating total as, acid-insolu#le as, loss on drying at ++0B& and alcool and water-


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3.11*5ualitati6e phytochemical studies

Kualitative pytocemical analyses were done using te procedures of Jo@ate *+9/!

Al@aloids, car#oydrates, tannins and penols, ':ed oils and fats, saponins and gums and

mucilageLs were Hualitatively analy)ed! 4inodaCs test was followed to analy)e Favonoids!

3.11.1* Alaloids

Te e:tracts were dissolved in diluted sulpuric acid and 'ltered! Te 'ltrate was treated

wit ayerCs, =ragendorfrs, "agerCs and IagnerCs reagents separately! Appearance of

cream, orange #rown, yellow and reddis #rown precipitates in response to te a#ove

reagents respectively indicate te presence of al@aloids!

3.11.2* Carohydrates

300 gm of aHueous and alcoolic e:tracts were dissolved in water and 'ltered! Te 'ltrate

was treated wit concentrated sulpuric acid and ten wit oliscCs reagent!

Appearance of pin@ to violet color indicates te presence of car#oydrates!

Te 'ltrate was #oiled wit (elingCs and wit enedictCs solutions! (onnation of #ric@ red

precipitate in (elingCs and enedictCs solutions is te positive result for reducing sugars

and non-reducing sugars respectively!

3.11.3* Tannins and phenols

4mall Huantity of alcoolic and aHueous e:tracts was dissolved in water and to tat ferric

cloride solution *5;/ or gelatin solution *+;/ or lead acetate solution *+0;/ was added!

Appearance of #lue color wit ferric cloride or precipitation wit oter reagents

indicates te presence of tannins and penols!

3.11.* la6onoids

Te e:tract wit few ml of alcool was eated wit magnesium and ten concentrated

ydrocloric acid was added under cooling! Appearance of pin@ color indicates te

presence of Favonoids!

3.11.!* +ums and 4ucila,e9s

A#out +0 ml of e:tract was slowly added to 25 ml of a#solute alcool wit constant

stirring! Precipitation indicates te presence of gums and mucilageLs!

3.11."* i:ed oils and fats


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A drop of concentrated e:tract was pressed in-#etween two 'lter papers and @ept

undistur#ed! il stains on te paper indicate te presence of oils and fats!

3.11.#* Saponins

A#out one ml of te alcoolic and aHueous e:tracts were dissolved separately in 20 ml ofwater and sac@ed in a graduated cylinder for +5 minutes! (ormation of one cm layer

oDoam indicates te presence of saponins!

3.12*5uantitati6e phytochemical tests

3.12.1* Estimation of Ascoric acid or 7itamin -C-

Ascor#ic acid or vitamin C&C is estimated following te procedure in AA& *Anonymous,

+10/!

;ea,ents

+!


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Estimation in sample

An aliHuot of te sample e:tract containing not more tan 0!+ mg of tannic acid was used

and te percentage of tannin was determined!

Calculation

Tannin

as

annic

acid ;

N

mg of tannic acid E dilution E +00

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

ml of sample ta@en for color

developmentE Ieigt of sample ta@en E +000

3.12.3* Estimation of 0henol

Penol was estimated using te metod of ray and Torpe, +59!

;ea,ents

+! olinCiocalteau rea,ent*4tandard Kualigens reagent was #ougt from scienti'c

supplier!

2! Sodium caronate solution*20; solution in water!

3! Standard solution*+00 mg of catecol in +00 m of water! t was diluted to +0

times for wor@ing standard!

0reparation of standard cur6e

=iDerent aliHuots of wor@ing standard were ta@en in test tu#es and teir volume was

made up to 3 m wit water! 0!5 m of (olin-&iocalteau reagent was added to eac tu#e

and @ept for 3 minutes! To eac tu#e, 2 ml of 20; sodium car#onate solution was added

and mi:ed torougly! Te test tu#es were @ept in #oiling water e:actly for one minute

and cooled down! Te a#sor#ance was measured at 50 nm against #lan@ and a standard

grape was plotted!

0reparation of sample

ne gm of sample was ground in a pestle and mortar wit 10; alcool! Alcoolic e:tract

was centrifuged at +0000 RP for 20 minutes! Te supernatant was saved separately and

te residue was re-e:tracted wit 10; alcool and centrifuged for more tan 've times!

Te supernatants collected was evaporated to dryness and dissolved in @nown volume of

water!


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Estimation in sample

AliHuots of sample were ta@en in test tu#es diluted wit water and a#sor#ance read as

a#ove and using te standard grape concentration of penol *as mgM+00 gm sample/ was

calculated #y using te standard grap!

3.12.* Estimation of Sucrose as &n6ert Su,ar

8stimation of sucrose as invert sugar was carried out #y using modi'ed (elingCs solution

*4o:let/ *Anonymous, +10/!

;ea,ents

+! So:hlet modi=cation of ehlin,-s solution* Prepared fresly #y mi:ing eHual

Huantity of copper sulpate solution and al@aline tartrate solution *so:let reagent/!

2! Copper sulphate solution*39!3 gm of copper sulpate was dissolved in water

and diluted to 500 ml!

3! Alaline tartrate solution*+. 3 gm of potassium sodium tartrate and 50 gm of

sodium ydro:ide were dissolved in water and diluted to 500 mi! ?et it stand for two days

and te clear solution was decanted and used!

9! Standard solution*!5 gm of pure sucrose wit 5 m of "& was dissolved in

water and made up to +00 mi! t was stored for tree days at 20-25& and ten diluted to

+000 ml! 6ecessary Huantity can #e neutralised wit 6 6a" #efore use!

Standardi>ation of standard solution

+0 or 25 ml of mi:ed 4o:let solution was pipette out in a conical Fas@! To tis was

added neutrali)ed and diluted standard solution as to reduce te copper content of

reagent! t was #oiled on a wire-gauge at moderate #oiling for 2 minutes! ne ml of

*0!02;/ metylene #lue was added and ended te titration #y adding few drops of sugar

solution to get faintest #lue precipitate!

0reparation of sample and estimation

Te powder was #oiled in alcool, 'ltered and made up to +00 ml! Te volume *ml/ of

4o:let reagent reHuired to reduce all te copper content as per te procedure for

standardi)ation was calculated!

Calculations

mg of

sugar N

Total reducing sugar reHuired E +00

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO


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in

+00

ml of

sampl

e

Titer value

3.12.!* Estimation of Total Saponins

4aponins were Huantitatively estimated #y te procedure followed #y amuji et al! *+15/

wit some modi'cations!

Estimation

Jnown Huantity of te sample powder was e:tracted wit alcool in a 4o:let e:tractor!

Te e:tract was concentrated in vacuum and re-e:tracted wit petroleum eter, eter and

etyl acetate seHuentially! Te defatted residue was dissolved in least amount of etanol

and poured in drops in e:cess acetone wit constant stirring! Te precipitate was 'ltered

and re-e:tracted for many times to get te puri'ed saponins in acetone! Te 'nal

precipitate was 'ltered in a pre-weiged 'lter paper and te percentage was calculated

wit te weigt of te sample ta@en!

3.13*T8C studies

Alcoolic e:tracts were spotted on 4ilica $elC $C coated plates! 6-#utanol, glacial acetic

acid and water in te ratio of 97 +72 are used as mo#ile pase for identi'cation of

antocyanins *Iagner et al!, +19/!

3.1* Standardi>ation of oil from seed ernels

est solvent for e:tracting more percentage of oil from @ernels was determined, #y

e:tracting te coarsely powdered seed @ernels in a 4o:let e:tractor wit diDerent

solvents li@e e:ane, petroleum eter, and cloroform!

Refractive inde: was found out wit an A##eCs type refractometer and speci'c gravity was

calculated wit a standard pycnometer! 4aponi'caion value and iodine value are

calculated #y te standard procedures *Anonymous, +10/!

3.1.1* Saponi=caion 6alue

ne gm of oil was dissolved in +0 ml of eterMetanol *27+ vMv/ mi:ture! To tis, 25 ml of

0!5 6 alcoolic potassium iodide was added and #oiled in water #at for 30 minutes, wit

an air condenser, wic was used to avoid loss #y evaporation! After #oiling, it was


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materials and methods of pharmacognosy

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