diabetes

Phytochemical Screening

PRELIMINARY PHYTOCHEMICAL TESTS OF THE EXTRACTS FOR

VARIOUS CONSTITUENTS

The plant is a biosynthetic laboratory, not only for chemical

compounds such as carbohydrates, proteins and lipids that are utilized

as a food by man, but also for a multitude of compounds like

glycosides, alkaloids, volatile oils, tannins etc., that exert a

physiological and therapeutic effect .the compound that are

responsible for medicinal property of the drug are usually secondary

metabolites. A systematic study of a crude drug embraces, through

consideration of primary and secondary metabolites derived as a result

of plant metabolism. The plant material is subjected to preliminary

Phytochemical screening for the detection of various plant constituents

(Kokate et al., 2001).

The Phytochemical investigation of a plant may thus involve the

following: extraction of the plant material; separation and isolation of

the constituents of interest; characterization of isolated compounds;

investigation of the isolated compounds; investigation of the

biosynthetic pathways to particular compounds; and quantitative

evaluations (Trease and Evans, 1989).

In the Phytochemical investigation, the botanical identity of the

plants studied must be authenticated by an acknowledged authority at

some stage in the investigation so many mistake over plant identy

have occurred in the past that it is essential to authenticate the

material whenever reporting new substance from plants or even known

substances from new plant sources. For these reasons, it is now

common to deposit voucher specimen of a plant.

3.1 EXTRACTION PROFILE

Requirements for herbal medicines have been established within

the last few years, and the trend is to define the dosage form with

uniform amount of extract.Amoung extraction processes for large

20


amounts of dried extracts, percolation followed by lyophilization has

been the most common method for preparing herbal medicines.

Plant parts in fresh or dried form are used for extraction. Plant

may be dried before extraction the drying operation should be

performed under controlled conditions to avoid too many chemical

changes occurring. It should be dried as quickly as possible, without

using high temperatures, preferably in a good air draft.

Extraction of Withania coagulans fruit and acacia arabica bark is

done by using 50% alcohol i.e.hydroalcoholic, and water (boiling water

and cold water)using soxhlet apparatus and maceration. All the

extracts were dried in desiccators.

Extraction profile of Withania coagulans fruit and Acacia

arabica bark

3.1.1. Hydroalcoholic Extract

Withania Coagulans (Fruits)/Acacia Arabica (Bark)

Coarsely powdered (500gm)

Hot percolation(50% Methanol)

Dried in a water bath at temp 30-35°C (Stored in a cool dried place)

3.1.2. Hot Water Extract



Boiled for 2 hrs With Distilled water

Dried in a water bath at

21


Temp 30-35°C. Stored in a cool dried place.

3.1.3. Cold Water Extract



Macerated for 5 days in distilled water

Dried in a desiccator Stored in a cool dried place.

3.2 PARTICLE SIZE BASED EXTRACTION OF Acacia arabica

BARK

3.2.1.40# Particle size Bark

40# (Particle size) powder (20gm)

Macerated for 24 hours in distilled water

Dried in a desiccator Stored in a cool dried place

3.2.2 60# Particle sizeBark

60# Particle size powdered (20gm)


Dried in a desiccator

22


Stored in a cool dried place.

23


3.2.3.120# Particle size Bark

120# Particle size powdered (20gm)


Dried in a desiccator Stored in a cool dried place.

3.3 METHANOL EXTRACT OF Withania coagulans

3.3.1 Extraction using 75% Methanolic Extract

Fruits




3.3.2 Extraction using 100% Methanolic Extract

Fruits




24


3.4 PHYTOCHEMICAL SCREENING

Detection of Carbohydrates (Ronsenthaler, 1930; Wallis, 1985)

Five hundred mg of extract was dissolved in 5 ml of distilled water

and filtered. The filtrate was used to test the presence of

carbohydrates.

Molisch’s Test

Molisch’s reagent 10 gm of α-Napthol was dissolved in 100 ml of

95% alcohol to prepare Molisch’s reagent.

To one ml of filtrate, 2 drops of Molisch’s reagent was added in a

test tube and 2 ml of concentrated sulphuric acid added carefully along

the side of the test tube. Formation of violet ring at the junction

indicates the presence of carbohydrates.

Fehling’s Test

Fehling’s solution

(a) 34.66 gm of copper sulphate was dissolved in distilled water and

volume was made up to 500 ml.

(b) 173 gm of potassium sodium tartarate and 50 gm of sodium

hydroxide were dissolved in distilled water and volume was made up to

500 ml. (a) and (b) solutions were mixed in equal volume to give

Fehling’s solution.

To one ml of filtrate, 4 ml of Fehling’s reagent was added in a test

tube and heated for 10 minutes in a water bath. Formation of red

precipitate indicates the presence of reducing sugar.

Detection of Glycosides (Ronsenthaler 1930; Middeltone, 1956)

0.5 gm of extract was hydrolyzed with 20 ml of dilute

hydrochloric acid (0.1 N) and filtered. The filtrate was used to test the

presence of glycosides.

25


Modified Borntrager’s Test

To one ml of filtrate, 2 ml of 1% ferric chloride solution was

added in a test tube and heated for 10 minutes in boiling water bath.

The mixture was cooled and shaken with equal volume of benzene.

The benzene layer was separated and treated with half of its volume of

ammonia solution. Formation of rose pink or cherry color in the

ammoniacal layer indicates the presence of anthranol glycoside.

Legal’s Test

To one ml of filtrate, 3 ml of sodium nitropruside in pyridine and

Methanolic alkali (KOH) was added in a test tube. Formation of pink to

blood red color indicates the presence of cardiac glycoside.

Keller-Killiani Test

Small portion from the respective extracts was shaken with 1 ml

glacial acetic acid containing a trace of ferric chloride. 1 ml of conc.

H2SO4 was added carefully by the sides of the test tube. A blue colour

in the acetic acid layer and red colour at the junction of the two liquids

indicate the presence of glycosides.

Detection of Alkaloids (Ronsenthaler, 1930; Peach and Tracey,

1955)

0.5 gm of extract was dissolved in 10 ml of dilute hydrochloric

acid (0.1 N) and filtered. The filtrate was used to test the presence of

alkaloids.

Mayer’s Test

Mayer’s reagent

(a) Dissolve 1.36 gm of mercuric chloride in 60 ml of distilled water

(b) Dissolve 5 gm of potassium iodide in 20 ml distilled water, mix

(a) and (b) and adjust the volume to 100 ml with distilled water.

Filtrates were treated with Mayer’s reagent; formation of yellow cream

colored precipitate indicates the presence of alkaloids.

26


Dragendorff’s Test

Dragendorff’s reagent

(a) Dissolve 8 gm of bismuth nitrate in 20 ml of nitric acid

(b) Dissolve 27.2 gm of potassium iodide in 50 ml distilled water,

mix (a) and (b) and adjust the volume to 100 ml with distilled

water.

Filtrate were treated with Dragendorff’s reagent, formation of red


Hager’s Test

Hager’s reagent: Saturated solution of picric acid in distilled water

Filtrates were treated with Hager’s reagent; formation of yellow


.Detection of Phytosterols and Triterpenoids

(Paech and Tracey, 1955; Finar, 1959)

0.5 gm of extract was treated with 10 ml chloroform and filtered.

The filtrate was used to test the presence of Phytosterols and

triterpenoids.

Libermann’s Test

To 2 ml filtrate in hot alcohol, few drops of acetic anhydride was

added. Formation of brown precipitate indicates presence of sterols.

Libermann’s Burchard Test

100 mg of extract was treated with 2 ml of chloroform and

filtered. To the filtrate few drops of acetic anhydride was added, boiled

and cooled. Concentrated sulphuric acid was added through the sides

of the test tube. Formation of brown ring at the junction indicates the

presence of steroidal saponins.

27


Salkowaski Test

To the test extract solution added few drops of Conc.H2SO4

shaken and allowed to stand, lower layer turns red indicating the

presence of sterols.

Detection of Protein and Amino Acids

(Ronsenthaler, 1930; Finar, 1959; Hawk et al., 1954)

100 mg of each extract was taken in 10 ml of water and filtered.

The filtrate was used to test the presence of protein and amino acids.

Millon’s Test

Millon’s reagent: Dissolve 1 gm mercury in 9 ml of fuming nitric acid,

keeping the mixture well cooled during the reaction. When the action is

complete, add equal volume of distilled water.

2 ml of filtrate was treated with 2 ml of Millon’s reagent in a test

tube and heated in a water bath for 5 minutes, cooled and added few

drops of NaNO2 solution. Formation of white precipitate, which turns to

red upon heating, indicates the presence of proteins and amino acids.

Ninhydrin Test

Ninhydrin reagent: 0.25% solution of n-butanol.

To 2 ml of filtrate, 0.25% Ninhydrin reagent was added in a test

tube and boiled for 2 minutes. Formation of blue color indicates the

presence of amino acids.

Biuret test

2 ml of filtrate was treated with 2 ml of 10% sodium hydroxide

solution in a test tube and heated for 10 minutes. A drop of 7% copper

sulphate solution was added in the above mixture. Formation of

purplish violet color indicates the presence of proteins.

Detection of Fixed Oils and Fats (Ronsenthaler, 1930)

28


Oily Spot Test

One drop of each extract was placed on filter paper and solvent

was allowed to evaporate. An oily stain on filter paper indicates the

presence of fixed oil.

Detection of Phenolics and Tannins

(Kokate, 2001; Handa and Kapoor, 2000)

100 mg of each extract was boiled with 1 ml of distilled water

and filtered. The filtrate was used for following tests.

Ferric Chloride Test

To 2 ml of filtrate, 2 ml of 1% ferric chloride solution was added

in a test tube. Formation of bluish black color indicates the presence of

phenolic nucleus.

Lead Acetate Test

To 2 ml of filtrate, few drops lead acetate solution was added in a

test tube. Formation of yellow precipitate indicates the presence of

tannins.

Detection of Flavonoids (Shellard, 1957)

Shinoda Test

To 100 mg of extract, few fragments of magnesium metal were

added in a test tube, followed by drop wise addition of concentrated

hydrochloric acid. Formation of magenta color indicates the presence

of flavonoids.

Alkaline Reagent Test

To 100 mg of extract, few drops of sodium hydroxide solution

were added in a test tube. Formation of intense yellow color that

becomes colorless on addition of few drops of dilute acid (HCl),

indicates the presence of Flavonoids.

Detection of Saponins (Kokate, 2001)

29


Foam Test

Dilute 1 ml of respective separately with distilled water to 20 ml

and shake in a graduated cylinder for 15 minutes. A one cm layer of

foam indicates the presence of Saponins.

30


Table 3.1 Qualitative chemical Test for Withania coagulans fruits

extract.

S.N. Test WCHAE WEHWE WCCWE

1. Test for carbohydrates

Molisch’s test Molisch’s test ++ ++ ++

Fehling test Fehling test ++ ++ ++

2. Test for Glycoside

ModifiedModifiedBorntrager Borntrager

++ ++ ++

Kellar-killiani test Kellar-killiani test ++ ++ ++

Legal’s test Legal’s test ++ ++ ++

3. Test for Saponins

Foam test Foam test ++ ++ ++

4. Test for alkaloidsTest for alkaloids

Mayer’s test Mayer’s test ++ ++ --

Hager’s reagentHager’s reagent ++ ++ --

Dragondroff’s test Dragondroff’s test + + -

5. Test for Flavonoids

Shinoda test Shinoda test ++ -- --

Alkaline reagent testAlkaline reagent test ++ -- --

6. Test for Phenolic compounds and Tannins

Lead acetate solutionLead acetate solution -- -- --

Ferric chloride solution Ferric chloride solution testtest

-- -- --

7. Test for Phytosterols and triterpenoids

Libermann’s testLibermann’s test ++ ++ ++

Salkowski’s test Salkowski’s test ++ ++ ++

Libermann Burchard’s testLibermann Burchard’s test ++ ++ ++

8. Test for Fixed oil and fats

Saponification test Saponification test -- -- --

*Denotes absence; + = denotes presence.

WCHAE:-Withania coagulan hydroalcoholic extract.

WCHWE:- Withania coagulan hot water extract

WCCWE:- Withania coagulan Cold water extract

31


Table 3.2 Qualitative chemical Test for the different extracts of

Acacia arabica Bark

S.N. Test AAHAE AAHWE AACWE

1. Test for carbohydrates

Molisch’s test Molisch’s test ++ ++ ++

Fehling test Fehling test ++ ++ ++

2. Test for Glycoside

ModifiedModifiedBorntrager Borntrager

-- -- --

Kellar-killiani test Kellar-killiani test -- -- --

Legal’s test Legal’s test -- -- --

3. Test for Saponins

Foam test Foam test ++ ++ ++

4. Test for alkaloidsTest for alkaloids

Mayer’s test Mayer’s test -- -- --

Hager’s reagentHager’s reagent -- -- --

Dragondroff’s test Dragondroff’s test -- -- --

5. Test for Flavonoids

Shinoda test Shinoda test -- -- --

Alkaline reagent testAlkaline reagent test -- -- --

6. Test for Phenolic compounds and Tannins

Lead acetate solutionLead acetate solution ++ ++ ++

Ferric chloride solution Ferric chloride solution testtest

++ ++ ++

7. Test for Phytosterols and triterpenoids

Libermann’s testLibermann’s test -- -- --

Salkowski’s test Salkowski’s test -- -- --

Libermann Burchard’s testLibermann Burchard’s test -- -- --

8. Test for Fixed oil and fats

Saponification test Saponification test -- -- --

* Denotes absence; + = denotes presence.

AAHAE: - Acacia arabica hydroalcoholic extract

AAHAE:- Acacia arabica hot water extract

AAHAE: - Acacia arabica cold water extract

32


3.5 CHROMATOGRAPHY

Chromatography may be regarded as an analytical technique

employed for the purification and separation of organic and inorganic

substances. It is also found useful for the fractionation of complex

mixture, separation of closely related compounds, such as isomers and

in the isolation of unstable substances (Sharma, 2001).

Chromatography is essentially a group of techniques for the

separation of the compounds of mixtures by their continuous

distribution (differential affinities of the solutes) between two phases,

one of which is moving past the other. The fixed phase is called the

stationary phase, and the moving one is termed as mobile phase. In

chromatography, the underlying mechanism is the partitioning of the

moving compounds between two liquid phases and are being

reversible bound on the surface of the adsorbent. Among various

chromatographic techniques, the thin layer chromatography (TLC) is

best suited for the analysis of drugs in a pharmaceutical laboratory.

This method requires consumes little time for completion of analysis

(15-60 min) and requires minimal amounts of sample (0.1 g). (Stahl,

1965)

The system associated with this definition is: (Beckett, 2001)

a) A solid stationary phase and a liquid or gaseous mobile phase

(adsorption chromatography).

b) A liquid stationary phase and a liquid or gaseous mobile phase

(partition chromatography).

c) A solid polymeric stationary phase containing replaceable ions

and an ionic liquid mobile phase (ion exchange chromatography).

d) An inert gel which act as a molecular sieve, and a liquid mobile

phase (Gel chromatography).

3.5.1. Thin Layer Chromatography

Thin layer chromatography (TLC) is a method of analysis in which

the stationary phase, a finely divided solid, is spread as a thin layer on

33


a rigid supporting plate and the mobile phase, a liquid, is allowed to

migrate across the surface of the plate (Dailey, 1995).

Thin layer chromatography is a technique in which a solute

undergoes distribution between two phases, stationary phase acting

through adsorption and a mobile phase in the form of a liquid. The

adsorbent is a relatively thin, uniform layer of dry finely powdered

material applied to a glass, plastic or metal sheet or plate. Glass plates

are most commonly used. Separation may also be achieved on the

basis of partition or a combination of partition and adsorption,

depending on the particular type of support, its preparation and its use

with different solvent.

Identification can be effected by observation of spots of identical

Rf value and about equal magnitude obtained, respectively, with an

unknown and a reference sample chromatographed on the same plate.

A visual comparison of the size and intensity of the spots usually

serves for semi quantitative estimation.

Advantages of TLC (Sharma, 2001, Wagner and Bladt, 1995)

It is simple and can be performed on an analytical as well as on a

preparative scale.

TLC can readily detect compounds, which are encountered in

trace amounts, due to its high sensitivity.

It may be applied to almost the entire spectrum of chemical

compounds.

TLC provides a chromatographic drug fingerprint. It is therefore

suitable for monitoring the identity and purity of drugs and for

detecting adulterations and substitutions.

With the aid of analyze drug combinations and Phytochemical

preparations.

The time required for the demonstration of most of the

characteristic constituents of a drug by TLC is very short.

34


In addition to qualitative detection, TLC also provides semi

quantitative information on the major active constituents of a drug

or drug preparation, thus enabling an assessment of drug quality.

Experimental (Stahl, 1969)

(i) Preparation and activation of plates

Silica gel G with distilled water (1:3) was triturated in a glass

pestle mortar and spread over the glass plates (10 cm x 20 cm.) by

Pouring till a uniform layer was obtained and allowed to air-dried. The

plates were activated for one hour at 110-1200 C before application of

samples.

(ii) Preparation of sample solution

The sample solution was prepared by dissolving small quantity of

extract (0.1%) in respective solvent. Suspended impurities if any were

filtered off.

(iii) Saturation of chamber

The solvent system was prepared and poured into the TLC

chamber. A filter paper sheet was placed into it to provide rapid

saturation and to prevent edging effect. Chamber was sealed by

placing a glass plate at the mouth of chamber with paraffin wax.

(iv) Application of spots

The spots of sample solution were applied with the help of thin

capillaries on the activated plates, at a distance of about 1.5 cm. from

the bottom and were allowed to dry in air. The distance between two

spots was kept at least 10 mm. Detecting Reagents

a. Anisaldehyde–sulphuric acid reagent: 0.5 ml Anisaldehyde

was mixed with 10 ml of glacial acetic acid followed by 85 ml

methanol and 5 ml conc. Sulphuric acid drop wise.

b. 5% Ferric chloride solution in methanol

c. 10% Sulphuric acid in methanol

35


(v) Developing of Chromatograms

After saturation, the plates were placed in the chamber and the

solvent was allowed to run until attaining a solvent height approx. 15

cm from the point of spotting. Then it was allowed to dry in air and

sprayed with detecting reagent and kept at 1000C for 5 minutes. Rf

values for each spot was calculated as follows:

Distance traveled by solute Rf value = Distance traveled by solvent frontTable 3.3: Solvent system for Withania coagulans

Solvent SystemNo. of Spots

ResolutionRf

valueColour

Benzene:Methanol: Toluene (8:1:1)

7 Very Good0.90

Pink

Benzene: Methanol (2:1) 5 Good 0.87

Acetonitrile:Water (8:2) 5 Good 0.71

Butanol:Glacial acetic acid : Water (4:1:5)

4 Poor0.58


4 Poor041


4 Poor0.24

Benzene : Acetone (36:13)

3 Poor018

Table 3.4: Solvent system for Acacia arabica

Solvent SystemNumber of

SpotsResolution

Rf

valueColour

Acetonitrile:Water (8:2) 4 Good 0.14

Black


4 Poor 0.28


4 Poor 0.40


4 Very Good 0.88

36


Withania Coagulans: TLC Profile of Hydro-alcoholic

Extract

Acacia arabica: TLC Profile of cold water Extract

Solvent System: Benzene: Methanol: Toluene (8:1:1)

(No. Of Spots: 7)

Spraying Reagent: Anisaldehyde in sulfuric acid and heating at 110°C for 5

min

Solvent System: Butanol: Glacial acetic acid: Water

(4:1:5)(No. Of Spots: 4)

Spraying Reagent: Anisaldehyde in sulfuric acid and heating at 110°C for 5

min.

37


3.6. HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY

(HPTLC)

High performance thin layer chromatography (HPTLC) is a

modern, powerful analytical technique with separation power,

performance and reproducibility superior to classic TLC. Based on the

use of high performance TLC plates with small particles sizes (3-5 µm)

and precise instruments for each step of the chromatographic process

(Sample application, chromatogram development, chromatogram

evaluation). HPTLC provides the means not only for flexible screening

procedures and qualitative analyses but also for demanding

quantitative determinations. Instruments can easily be validated and

are fully compliant with GMP. HPTLC features highly sensitive scanning

densitometry and video technology for rapid chromatogram evaluation

and documentation. Today most HPTLC instruments are computer

controlled and can therefore, offer dramatically improved

reproducibility of the analytical result.

It is the only chromatographic method offering the option of

presenting the result as an image. Furthermore, TLC is the sole

technique in which all the components of the sample are included in

the chromatogram. In contrast, HPLC and GC are selective and not all

of the compounds in the sample are included in the display. The real

breakthrough in TLC came through the work of Stahl, who introduced

the use of calcium sulphate as binder, and standardized layer

thickness and chromatographic development (Stahl, 1958). Not only

does the technique give visual results but it excels in its simplicity and

is low in cost. Parallel analysis of samples is possible, sample capacity

is high and results are obtained rapidly.

TLC is flexible and multiple detection is possible. It is an ideal

screening method in biological and chemical analysis, providing

identification and qualitative results, determination of adulteration,

together with quantitative and semi-quantitative determination. In

conjunction with microorganisms and other biological agents, TLC

38


bioautography can be used to screen for bioactivities (Hostettmann

et al., 1997). The disadvantages of TLC are a lack of automation, the

problems of reproducibility which sometimes occur and the lack of

accuracy in quantization. Nevertheless, TLC will remain a fast and

simple micro technique of chromatography. In HPTLC, the plates are

precoated with stationary phase with a typical mean particle size of 5

lm. The plates give better separations and reproducibility than normal

precoated TLC plates (mean particle size 12 lm) and they also allow

more sensitive detection. Shorter developing distances are required.

The number of theoretical plates is in the 5000 range (Reich and

Schibli, 2007), while for HPLC the range is 6–10,000.

The separation power of HPTLC is still lower than that of HPLC

and the latter is preferred for quantitative determination. Merck also

offers HPTLC plates with spherical particles, which gives faster

chromatography and better separation power. A water-resistant layer

is available from Merck and for RP-18 W supports, 100% water can be

used with these plates. For herbal extracts, regulatory agencies often

recommend fingerprint chromatography for proper identification

purposes. HPTLC is ideal in this instance and excellent examples can

be found in the literature (Wagner and Bladt, 1995; Reich and

Schibli, 2007).

Preparation of standard sample

Working standard solutions were prepared by serial dilution of a

10% Methanolic stock solution(100μg/ml).Different aliquots of 10, 20,

30, 40, 50μg was then prepared and 10μl of these sample were applied

to the plate.

Preparation of test sample

Test sample of the extract is prepared by dissolving different

extracts 10mg in 10ml of 10% methanol and 10μl of the test sample is

applied in the plate.

39


HPTLC analysis

Solvent system: upper phase butanol: acetic acid: water (4:1:5).

Plate: prepared aluminum F254 plate.

(1) Preparation of standard curve

The different aliquots is applied on the prepared plate and run

manually in the TLC chamber upto the height 90 mm.

After complete running the plate is scanned in HPTLC scanner

and standard curve is plotted between concentration and Area

under curve.

(2) Preparation of test curve.

Different extracts of Acacia arabica were applied on the prepared

plate and runed manually in the TLC chamber upto the height 90

mm after complete running the plate is scanned in HPTLC

scanner.

Table 3.5: HPTLC profile of ℓ-epicatechin at 254 nm

Trace

Start position (Rf)

Start heigh

t (AU)

Max position (Rf)

Max heigh

t (AU)

Max (%)

End position (Rf)

End heigh

t (AU)

Area (AU)

Area (%)

1 0.79 33.6 0.88 184.4

100 0.90 0.1 6923 100

2 0.79 33.6 0.88 118.2

100 0.90 0.1 11234

100

3 0.79 33.6 0.88 190.2

100 0.90 0.12 17564

100

4 0.79 33.6 0.88 196.2

100 0.90 0.1 23487

100

5 0.79 33.6 0.88 225. 100 0.90 0.1 2985 100

40


2 4

(Note: On the basis of Sastry et al. (1963) work on the Acacia arabica bark

we have selected ℓ-epicatechin as a standard).

41

http://0-web5s.silverplatter.com.wagtail.uovs.ac.za/webspirs/doLS.ws?ss=SASTRY-G-P+in+AU


Fig. 3.1: Standard curve of ℓ -epicatechin at 254 nm

Fig. 3.2: HPTLC profile of ℓ-epicatechin of conc. 10 μg/ml at 254 nm

y = 581.15x + 377.9

R2

= 0.9959

0

5000

10000

15000

20000

25000

30000

35000

0 20 40 60CONC (µg/ml)

AUC

42




43


Fig. 3.5: HPTLC profile of ℓ-epicatechin of conc. 40 μg/ml at 254

nm

44



Table 3.6: HPTLC profile of Acacia arabica (40≠size) extract at 254 nm

Peak

Start Position (Rf)

Start Height(AU)

Max positio

n(Rf)

Max Heigh

t(AU)

Max (%)

End Positio

n(Rf)

End Heigh

t(AU)

Area(AU)

Area(%)

1 0.07 5.5 0.10 31.4 6.89 0.09 27.9 603.7 3.31

2 0.11 33.9 0.14 56.7 12.4

3

0.20 1.8 2599.9 13.5

0

3 0.25 4.5 0.28 18.3 4.01 0.30 7.1 618.5 3.21

4 0.63 15.5 0.66 25.5 5.60 0.67 18.1 700.3 3.64

5 0.67 18.1 0.70 34.1 7.48 0.71 30.8 863.7 4.48

6 0.76 49.4 0.88 289.9 63.6

0

0.91 8.6 13876.

6

72.0

4

45


Fig. 3.7: HPTLC profile of Acacia arabica (40≠size) extract at 254nm

46


Table 3.7: HPTLC profile of Acacia arabica (60≠size) extract at 254 nm

Peak

Start Position (Rf)

Start Height(AU)

Max positio

n(Rf)

Max Heigh

t(AU)

Max (%)

End Positio

n(Rf)

End Heigh

t(AU)

Area(AU)

Area(%)

1 0.05 0.2 0.10 39.6 7.59 0.11 35.6 862.3 4.68

2 0.11 35.6 0.14 62.3 11.9

4

0.20 5.6 2829.0 15.3

5

3 0.25 6.0 0.28 30.6 5.86 0.29 25.6 785.8 4.26

4 0.29 25.6 0.66 30.5 5.84 0.33 6.9 563.4 3.06

5 0.44 7.3 0.70 17.8 3.41 0.48 14.7 437.0 2.37

6 0.48 14.7 0.86 25.1 4.81 0.52 13.1 639.5 3.47

7 0.57 13.3 0.66 32.7 6.26 0.61 26.3 856.6 4.65

8 0.69 27.0 0.71 39.9 7.65 0.72 37.9 811.0 4.40

9 0.77 58.6 0.88 243.4 46.6

4

0.91 8.9 10647.

8

57.7

7

47



48


Table 3.8: HPTLC profile of Acacia arabica (120≠size) extract at 254nm

Peak

Start Position (Rf)

Start Height(AU)

Max positio

n(Rf)

Max Height(AU)

Max (%)

End Positio

n(Rf)

End Height(AU)

Area(AU)

Area(%)

1 0.05 0.2 0.12 58.2 14.0

6

0.11 49.1 1533.

7

10.7

0

2 0.13 35.6 0.14 61.2 14.7

8

0.20 10.6 2220.

0

15.4

9

3 0.24 6.0 0.28 31.8 7.68 0.29 14.6 1295.

4

9.04

4 0.69 27.0 0.71 39.0 9.42 0.72 33.9 1117.

4

7.79

5 0.81 58.6 0.88 223.9 54.0

7

0.91 7.3 8169.

3

56.9

8

49



50


Table 3.9: HPTLC profile of Acacia arabica (crude) extract at 254nm

Peak

Start Position (Rf)

Start Height(AU)

Max positio

n(Rf)

Max Height(AU)

Max (%)

End Positio

n(Rf)

End Height(AU)

Area(AU)

Area(%)

1 0.07 14.4 0.14 59.7 18.4

0

0.19 4.4 3815.

4

26.3

5

2 0.23 1.0 0.28 35.8 11.0

3

0.33 5.6 1389.

2

9.50

3 0.38 2.1 0.40 17.4 5.37 0.42 5.9 329.0 2.25

4 0.79 61.3 0.88 211.5 65.2

1

0.93 6.0 9048.

8

61.9

0

51


Fig 3.10: HPTLC profile of Acacia arabica (crude) extract at 254 nm

52


4.1 RESULTS AND DISCUSSIONAfter the extraction of both the plant the yield and nature of

extracts was found to be:

Withania Coagulans

Properties

HAE HWE CWE 75% HAE 100% AE

Yield 25.54% 9.92% 14.02% 30.23% 20.93%

NatureSemi-solid

Semi-solid

Semi-solid

Semi-solid

Semi-solid

Colour Brown Brown Brown Brown Brown

Acacia Arabica

Properties

HAE HWE CWE40#CW

E60#CWE

120#CWE

Yield 22.62% 5.35% 2.1% 2.1 2.6 1.8

Nature Solid Solid Solid Solid Solid Solid

ColourReddish brown

Reddish

brown

Reddish

brown

Reddish

brown

Reddish brown

Reddish brown

Solvent system for Withania coagulans

Solvent SystemNo. of Spots

Resolution Colour

Benzene:Methanol: Toluene (8:1:1)

7 Very Good

PinkRfvalue 0.90, 0.87, 0.71, 0.58, 041, 0.24, 018.

Solvent system for Acacia arabica

Solvent SystemNumber of

SpotsResolution Colour


4 GoodBlack

Rf value0.14, 0.28, 0.40, 0.88.

HAE – Hydro alcoholic extract AA – Acacia arabica HWE – Hot water extract WC – Withania coagulansCWE – Cold water extract

53


The maximum yield was shown by hydro alcoholic extracts of

both the drug. The nature of the extracts in case of Withania coagulans

was semisolid and brown in colour. But in case of acacia arabica nature

of extract was dry powder and reddish Brown in colour.

Thin layer chromatography:

After Phytochemical analysis thin layer chromatography was

performed and suitable solvent system was formed which in the case

of WCHAE is Benzene: Methanol: Toluene (8:1:1) and seven spots are

observed and resolution is Very Good. And in case of AACWE Butanol:

Glacial acetic acid: Water (5:1:4) and four spots are observed and

resolution is very good.

After preliminary TLC detection and development of suitable

mobile phases, HPTLC analysis was carried out. The samples were

prepared in a 10mg/10ml concentration of cold water extract of Acacia

arabica bark powder of 40 mesh size. The HPTLC profile of cold water

extract of Acacia arabica bark powder of 40 mesh size revealed the

presence of six spots, the maximum concentration was found to be of

component 6 at Rf value 0.88 and other values shown in table (Fig.

6.7).

The HPTLC profile of cold water extract of Acacia arabica bark

powder of 60 mesh size revealed the presence of nine spots, the

maximum concentration was found to be of component 9 at Rf value

0.88 and other values shown in table (Fig. 6.8).


powder of 120 mesh size revealed the presence of five spots, the

maximum concentration was found to be of component 5 at Rf value

0.88 and other values shown in table (Fig. 6.9).


crude powder revealed the presence of four spots, the maximum

54


concentration was found to be of component 4 at R f value 0.88 and

other values shown in table (Fig. 6.10).

BIBLIOGRAPHY 48

1. Goodman. L.S., Gilman., 1995. The pharmacological basis of therapeutics, 9th edition Macgraw hill publication, .1487-1513.

2. Lebovitz, H.E., 2004.Therapy for Diabetes Mellitus and Related Disorders. 4th edition. Alexandria:American Diabetes Association.

3. Goodman. L.S., Gilman., 1995. The pharmacological basis of therapeutics, 9th edition Macgraw hill publication, .1487-1513.

4. Sachdewa, A., Raina, D., Srivastava, A.K and. Khemani, L.D., 2001. Effect of Aegle marmelos and Hibiscus rosa sinensis leaf extract on glucose tolerance in glucose induced hyperglycemic rats (Charles foster), Journal of Environmental Biology 22, 53–57.

5. Kamalakkanan, N., Rajadurai, M and Prince, P.S., 2003. Effect of Aegle marmelos fruits on normal and streptozotocin-diabetic Wistar rats, Journal of Medicinal Food 6, 93–98.

6. Augusti, K.T., 1973. Studies on the effects of a hypoglycemic principle from Allium Cepa Linn, Indian Journal of Medical Research 61, 1066–1071.

7. Rabinkov. A., Miron.T., Konstantinovski, L., Wilchek, M., Mirelman, D and Weiner L., 1998. The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins, Biochimica and Biophysica Acta. 1379, 233–244.

8. Rajasekaran, S., Sivagnanam, K., Ravi, K and Subramanian, S., 2004. Hypoglycemic effect of Aloe vera gel on streptozotocin-induced diabetes in experimental rats, Journal of Medicinal Food. 7, 61–66.

9. Endres, H and H ilal, M., 1963.the tannins of acacia arabica. Part ii. The isolation of several polyhydroxyphenols from the fruit pods phytochemistry 2, 151-6.

55

http://0-web5s.silverplatter.com.wagtail.uovs.ac.za/webspirs/doLS.ws?ss=Phytochemistry-+in+SO

http://0-web5s.silverplatter.com.wagtail.uovs.ac.za/webspirs/doLS.ws?ss=HILAL-M+in+AU

http://0-web5s.silverplatter.com.wagtail.uovs.ac.za/webspirs/doLS.ws?ss=ENDRES-H+in+AU


BIBLIOGRAPHY 49

10. Choudhary, M. I., Dur-e-Shahwar., Zeba Parveen., Abdul, Jabbar., Irshad Ali., Atta-ur-Rahman.,1995.Antifungal steroidal lactones from Withania coagulanc e . Phytochemistry, 40(4),1243-1246.

11. Mustafa, N. K., Tanira, M. O. M., 1999. Antimicrobial activity of Acacia nilotica subspp. nilotica fruit extracts. Pharmacy and Pharmacology Communications. Sept. 5, 583-586.

12. Bhargava, A., Srivastava, A., 1998. Antifungal activity of polyphenolic complex of Acacia nilotica bark. Indian Forester 124(5), 292-298.

13. Chandel,.B.S ., shah,N.M and T ripathi,R.M ., 1993.in vitro antibacterial activity of acacia arabica bark. Indian journal of indigenous medicines 9(1/2), 77-79.

14. Shah, B. H., Safdar,B., 1997. The antiplatelet aggregatory activity of Acacia nilotica is due to blockade of calcium influx through membrane calcium channels. General Pharmacology 29,251-255.

Glotter, E., Natural Products Reports, 8415, 1991

15. Kirtikar, K.R and Basu, B.D., 1993. Indian Medicinal Plants vols. 1–4, Periodical Experts, Delhi.

16. Dymock, W., Warden, C.J.H. and Hooper, D., pharmacographia indica’, reprinted by institute of health and tibbi research, Karachi 306, 10972.

17. Atta-Ur-Rahman, Choudhary,M.Iqbal ., Yousaf, Muhammad., Gui,Waseem and Qureshi,Samina., 1998. New withanolides from Withania coagulans. Chemical and Pharmaceutical Bulletin (Tokyo) 46(12), 1853-1856.

56

http://web5.silverplatter.com.hmlproxy.lib.csufresno.edu/webspirs/doLS.ws?ss=Chemical-and-Pharmaceutical-Bulletin-+Tokyo++in+SO

http://web5.silverplatter.com.hmlproxy.lib.csufresno.edu/webspirs/doLS.ws?ss=Qureshi-Samina+in+AU

http://web5.silverplatter.com.hmlproxy.lib.csufresno.edu/webspirs/doLS.ws?ss=Gui-Waseem+in+AU

http://web5.silverplatter.com.hmlproxy.lib.csufresno.edu/webspirs/doLS.ws?ss=Yousaf-Muhammad+in+AU

http://web5.silverplatter.com.hmlproxy.lib.csufresno.edu/webspirs/doLS.ws?ss=Atta-Ur-Rahman-M-Iqbal-Choudhary+in+AU

http://0-web5s.silverplatter.com.wagtail.uovs.ac.za/webspirs/doLS.ws?ss=Indian-Journal-of-Indigenous-Medicines+in+SO

http://0-web5s.silverplatter.com.wagtail.uovs.ac.za/webspirs/doLS.ws?ss=Indian-Journal-of-Indigenous-Medicines+in+SO

http://0-web5s.silverplatter.com.wagtail.uovs.ac.za/webspirs/doLS.ws?ss=Tripathi-R-M+in+AU

http://0-web5s.silverplatter.com.wagtail.uovs.ac.za/webspirs/doLS.ws?ss=Shah-N-M+in+AU

http://0-web5s.silverplatter.com.wagtail.uovs.ac.za/webspirs/doLS.ws?ss=Chandel-B-S+in+AU


BIBLIOGRAPHY 50

18. Nur-e-Alam, M., Muhummed Yousaf, Samina, Qureshi., Irfan,Baig., Shama, Nasim., Atta-ur-Rahman., Choudhary,M.I., 2003.A novel dimeric podophyllotoxin-type lignan and a new withanolide from Withania coagulans . Helvetica Chimica Acta 86(3), 607-614.

19. Choudhary, M. I., Dur-e-Shahwar., Zeba Parveen., Abdul, Jabbar., Irshad Ali., Atta-ur-Rahman.,1995.Antifungal steroidal lactones from Withania coagulanc e . Phytochemistry, 40(4),1243-1246.

20. Kokate, C.K., Purohit, A.P and Gokhale, S.B., 2001. Text Book of Pharmacognosy. Carbohydrate and derived products, drugs containing glycosides, drugs containing tannins, lipids, and proteins alkaloids. 7th Ed., Nirali Prakashan, India, pp. 133-166, 167-254, 255-269, 272-310, 428-523.

21. Ronsenthaler, L., 1930. Chemical investigations of plants. G. Bell and Sons, London., pp. 23-29, 119-132.

22. Middeltone, H., 1956. Systematic Qualitative Analysis. Edward Arnold Publishers Ltd., London, pp. 91-94.

23. Peach, T and Tracey, M.V., 1955. Modern Methods in Plant Analysis. Springer Verlog, Berlin, pp. 387

24. Finar, I.L, 1959. Organic Chemistry. 2nd Ed., the English Language Book Society, London, pp. 280-431.

25. Shellard, E.J., 1957. Practical Plant Chemistry for Pharmacy Students. Pitman Medical Publishing Co. Ltd, London., pp. 34-80.

26. Sharma, B.K., 2001.Instrumental Methods of Chemical Analysis, 20th edition. Goyal publishing house., pp 96-102.

27. Stahl, Ergon., 1969. Thin Layer Chromatography, 2nd edition, Springer Verlag Belin Heidelberg, New York., pp 63-65, 311-333.

57


28. Beckett, A.H., Stenlake, J.B., 2001.Practical Pharmaceutical Chemistry, Ist Edition, vol.-II, CBS publisher, 85.

58

diabetes

Documents

distilled water

dried form

water bath

amounts of dried extracts

phytochemical investigation

particle size bark

large phytochemical

gm hot percolation