METHODOLOGY

METHODOLOGY

COLLECTION OF PLANT MATERIAL

The dried & authenticated roots of Hemidesmus indicus were obtained from Natural

Remedies Private Limited, Bangalore. A voucher specimen was deposited (PT/0707030).

EXTRACTION OF PLANT MATERIAL

The powdered plant material was extracted with methanol in a static extractor

successively each for 2 hrs. The process was first performed in small scale to find out the

yield and taken for pilot scale.

Lab scale extraction: The powdered roots of Hemidesmus indicus (100 gm) was

refluxed with Methanol (300 ml) for 2 hrs. The same process was repeated for three

times, then the filtration is carried out and after concentrating the solvent the % yield was

calculated which was found to be 10.97 %. On the basis of this the quantity of the raw

material to be taken for pilot scale extraction was decided.

Pilot scale extraction: The pilot scale extraction was carried out in static extractor. The

extraction vessel was made up of stainless steel, with steam jacket. It has a steel

perforated holder on which the plant material to be extracted was placed. The solvent

inlet is on top. Steam was supplied through a steam inlet from a steam boiler and

refluxing was done. The extract was collected from the outlet present at the bottom of the

vessel.

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METHODOLOGY

Procedure:

• Raw material of Hemidesmus indicus roots (20Kg) was refluxed with Methanol

(80Lit) in a static extractor for 2 hrs.

• After 2 hrs the methanolic extract was collected and the residue was again

charged for the second wash with 40 Lit methanol. The third was also done which

was same as the second wash and the extract was collected.

• All the methanolic extracts obtained were combined and filtered through muslin

cloth.

• The extract was concentrated under vaccum at 60ºC & dried in VTD.

Flow chart No. 1 - Extraction process of Hemidesmus indicus

Raw material (H.indicus, roots)

20 Kg

1 st wash 4 x lit, 2hrs

2 nd wash 2 x lit, 2hrs with 100 % MeOH

3 rd wash 2 x lit, 2hrs

Filtered each wash through fine cloth

Combined filtrate Spent

Concentrated under vacuum to thick paste

2.5 Kg of MeOH extract is obtained.

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METHODOLOGY

Flow chart No. 2 - Processing of MeOH extract

2 kg of MeOH extract of
Hemidesmus indicus

Refluxed with Acetone

1 st wash 4 lit Acetone

2 nd wash 2 lit Acetone each wash for 2 hrs.

3 rd wash 2 lit Acetone

Combined and filtered

Filtrate residues

Concentrated up to thick paste

Dried in VTD

234 gm

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METHODOLOGY

OPTIMIZATION OF TLC SYSTEM 70

Different solvent systems were tried to develop a TLC system for

identification of constituents in the extracts and the one showing maximum separation

was selected as mobile phase for the study. The following solvents were used for the

development of the TLC system:

Butanol : Ethanol : Acetic acid : water ( 4: 4 : 1 :1).

Acetone : water ( 9 : 1).

Ethyl acetate : Pet. ether (7:3).

Chloroform : Methanol (9 : 1, 8 : 2, 5 : 5).

Chloroform : Methanol (9.5 : 0.5).

Toluene : Chloroform : Ethanol (4 : 4 : 1).

Procedure:

The extract was dissolved in methanol and then spotted on the silica gel G 254

plates along with β-sitosterol, Lupeol, sitosterol-D-glucoside and ursolic acid with the

help of capillary tubes. TLC plates were developed and scanned at 254 and 366nm. The

ANS reagent was sprayed and the chromatogram was observed for separation.

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METHODOLOGY

ISOLATION OF PHYTOCONSTITUENTS 71

The acetone soluble part of the MeOH extract showed much more prominent

spots than that of the Acetone insoluble part in TLC (Chloroform : Methanol = 9:1).

Therefore this part was taken for the isolation by column chromatography.

COLUMN CHROMATOGRAPHY

The selected acetone soluble fraction was subjected to systematic column

chromatography using different solvent systems. The fractions collected were further

chromatographed using different stationary phases. Silica gel was used as the stationary

phase in the Column chromatography to isolate constituents.

Column chromatography was done using a glass column. The column was packed

with silica gel by wet packing method wherein a padding of cotton was placed at the

bottom of the column and then it is filled with eluting solvent of the lowest polarity

(pet ether). Then the required amount of stationary phase (silica) was poured into the

column to form a bed of silica. The solvent was eluted to the top of the bed. The extract

which was adsorbed in silica gel was then poured on to the bed of silica, a layer of cotton

covered it again and more amount of solvent was poured over it, the column was then run

by gradient elution technique.

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METHODOLOGY

The general principle applied in column chromatography consists of following steps:

1. Pre-column preparation: The pre-column preparation included adsorption of the

selected extract/fraction, charging and saturation of the column.

a. Adsorption of the extract: The extract selected for further fractionation was

adsorbed on stationary phase in ratio 1:1 and dried at 60ºc under 600mm/Hg in

VTD.

b. Charging of column: A glass column was selected and rinsed with acetone

solvent. A cotton layer was placed at the bottom and the column was charged

with the solvent and stationary phase. The silica gel was used in the ratio 1:15 of

the extract to make the gel bed for complete separation. The solvent was eluted

up to the level of the column bed and the dried extract was charged in the

column. Another layer of cotton was placed over the charged matter to prevent

the disturbance of the extract bed while pouring the eluting solvent from the top.

c. Saturation of the column: The charge column was left for few hours for

complete saturation and removal of air bubbles to make the bed static.

2. Elution: The charged column was eluted with different mobile phases with gradual

increase in polarity. The fractions collected were dried in rotavapour (Buchi R-114).The

dried fractions were then weighed and recovered using methanol. All the fractions were

subjected to TLC for the identification of the desired bands.

COLUMN 1 (Fractionation and Isolation)

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METHODOLOGY

Requirements:

Stationary phase: Silica gel (60-200 mesh).

Mobile phase: Pet ether, Ethyl acetate, methanol.

Charged material: Acetone soluble fraction.

Volume of each fraction: 1000ml.

Procedure:

The column was first eluted with 100% pet ether. The polarity of mobile phase was

gradually increased with ethyl acetate and methanol. The fractions collected were dried in

rotavapour and weighed. The fractions collected from the column 1 are shown below.

Flow chart- 3 (Fractions collected from Column chromatography- 1).

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METHODOLOGY

CC-1
230 gm Acetone Ext. was
charged
PT/0612573

CC-1/F-1 CC-1/F-2 CC-1/F-3 CC-1/F-4 CC-1/F-5

100%PE+5 5% EA in 10% EA in 10% EA in 15% +20%
%EA in PE PE PE PE EA in PE
400 mg 500 mg 8.3 gm 38.7 gm 4.5 gm

CC-1/F-16 CC-1/F-17 CC-1/F-18 CC-1/F-19

20% MeOH 20%+50% 50% MeOH in 100% MeOH
in EA MeOH in EA EA 1.5 gm
5.1 gm 2.5 gm 18 gm
CC-1/F-6 CC-1/F-7 CC-1/F-8 CC-1/F-9 CC-1/F-10
30% EA in 30% EA in 50% EA in 50% EA in 75% EA in
PE PE PE PE PE
2.4 gm 3.2 gm 3.5 gm 2.3 gm 2.5 gm

CC-1/F-11 CC-1/F-12 CC-1/F-13 CC-1/F-14 CC-1/F-15

75% EA in 100% EA 5% MeOH 5% MeOH 10% MeOH
PE 5.8 gm in EA in EA in EA
2.9 gm 5.5 gm 2.7 gm 6.1 gm

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METHODOLOGY

TLC of all the fractions collected from CC-1 done with various solvent systems. It is

shown in the following table and the TLC images are shown in the Fig No. 5, 6 and 7.

Table No. 2 (TLC solvent systems for CC-1)

Solvent system Visualization

1. 5 % EA in Pet. Ether UV at 254 nm, 366 nm and ANS spray.

2. 15 % EA in Pet. Ether UV at 254 nm, 366 nm and ANS spray.

3. 30 % EA in Pet. Ether UV at 254 nm, 366 nm and ANS spray.

4. 10 % MeOH in Chloroform UV at 254 nm, 366 nm and ANS spray.

5. 30 % MeOH in Chloroform UV at 254 nm, 366 nm and ANS spray.

Depending upon the TLC pattern of all the fractions from column-1, two fractions (CC-

1/F-3 and CC-1/F-4) were taken for the next column chromatography to isolate the

targeted compound.

COLUMN 2 (Fractionation and Isolation)

Requirements:

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METHODOLOGY

Stationary phase: Silica gel (60-200 mesh).

Mobile phase: Pet ether, Ethyl acetate.

Charged material: CC-1/F-3 and CC-1/F-4 (40 gm).

Volume of each fraction: 500ml.

Procedure: The column was first eluted with 100% pet ether .The polarity of the solvent

was increased with ethyl acetate. The fractions collected were dried in rotavapour and

weighed. The fractions collected from the column 2 are shown below.

Flow chart- 4

CC-2
40 gm of CC-1/F-3 + CC-1/F-4
was charged

CC-2/F-1 CC-2/F-2 CC-2/F-3 CC-2/F-4

100% PE + 5% EA in 5% EA in 5% EA in
1%-4% EA PE PE PE
in PE 24.6 gm 3.6 gm 800 mg
100 mg

CC-2/F-5 CC-2/F-6 CC-2/F-7 CC-2/F-8

10% EA in 10% EA in 10% EA in 100% EA
PE PE PE in PE
6.7 gm 1.2 gm 100mg 500 mg

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METHODOLOGY

TLC of all the fractions of Column-2 is shown in the Fig No. 8 and 9.

Processing of CC-2/F-5 and CC-2/F-6

CC-2/F-5 and CC-2/F-6 showed precipitates at the bottom of the test tubes. The ppt was

separated and dissolved in Pet. Ether and kept over night for recrystallization. Pet. Ether

wash given further to purify it. TLC of the crystals showed a sharp single spot which was

sensitive at UV 254 nm and also after spraying with ANS reagent. It was a pure

compound and the code given to this was HI-004.

COLUMN 3 (Fractionation and Isolation)

Depending upon the TLC pattern of all the fractions from column-2, CC-2/F-2 was taken

for the next column chromatography to isolate the targeted the compound.

Requirements:

Stationary phase: Silica gel (60-200 mesh).

Mobile phase: Pet ether, Ethyl acetate.

Charged material: CC-2/F-2 (24 gm).

Volume of each fraction: 250ml.

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METHODOLOGY

Flow chart-5

CC-3
24 gm of CC-2/F-2 was charged

CC-3/F-1 CC-3/F-2 CC-3/F-3 CC-3/F-4

1-8 (100% 9-14 (100% 15-16 17
PE) PE) (100% PE) (100% PE)
wt. none wt. none wt. 50 mg wt. 50 mg

CC-3/F-5 CC-3/F-6 CC-3/F-7 CC-3/F-8

18-26 27 28-32 33-68
(100% PE) (100% PE) (100% PE) (0.5% EA)
wt. 50 mg wt. 50 mg wt. 50 mg 18.6 gm

CC-3/F-9 CC-3/F-10 CC-3/F-11 CC-3/F-12

69-70 71-73 74-76 77-81
(0.5% EA) (0.5% EA) (0.5% EA) (2% EA)
900 mg 2.3 gm 500 mg wt 50 mg

CC-3/F-13 CC-3/F-14
82-84 85-90 (5%
(5% EA) EA)
wt none wt none

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METHODOLOGY

The TLC of all the fractions of CC-3 is shown in Fig No. 10.

Processing of CC-3/F-10

TLC of CC-3/F-10 showed a sharp single spot which was not UV sensitive but could be

visualized after spraying with ANS reagent. It was a pure compound and recrystalized.

Recrystallization was tried with various solvents such as Pet. Ether, Chloroform and

Acetone. Finally it was recrystalized with acetone. The code given to this was HI-01.

COLUMN 4 (Fractionation and Isolation)

Depending upon the TLC pattern of all the fractions from column-1, CC-1/F-18 was

taken for the next column chromatography to isolate the targeted two compounds which

given prominent spots in TLC.

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METHODOLOGY

Requirements:

Stationary phase: Silica gel (60-200 mesh).

Mobile phase: Pet ether, Ethyl acetate, Methanol.

Charged material: CC-1/F-18 (18 gm).

Volume of each fraction: 500ml.

Flow chart-6

CC-4
18 gm of CC-1/F-18 was charged

CC-4/F-9 CC-4/F-10 CC-4/F-11 CC-4/F-12

CC-4/F-13
15% of Pharmacognosy
Department 20% - PESCP 30% 50% 51
100%
MeOH in MeOH in MeOH in MeOH in
MeOH
EA EA EA EA
400 mg
4.9 gm 2.9 gm 1 gm 600 mg
METHODOLOGY

CC-4/F-2 CC-4/F-3 CC-4/F-4

CC-4/F-1 20% EA in 40-100% 5% MeOH
100% PE PE EA in PE in EA
none 100 mg 400 mg 1 gm

CC-4/F-5 CC-4/F-6 CC-4/F-7 CC-4/F-8

10% 10% 10% 15%
MeOH in MeOH in MeOH in MeOH in
EA EA EA EA
1.7 gm 1.8 gm 400 mg 3.8 gm

The TLC of all the fractions from this column is shown in Fig No. 11, 12 and 13.

Processing of CC-4/F-10

The CC-4/F-10 showed 2 major targeted spots in TLC. This fraction was further

processed as below to get pure compounds.

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METHODOLOGY

Flow chart-7

CC-4/F-10

Dissolved in Ethyl Acetate

Precipitate found at the bottom of mother liquor (MLR)

Filtered and PPT washed 3-4 times with EA

MLR concentrated and again dissolved in EA to get PPT

Above process followed again for two times

All the PPT combined and All MLR combined

PPT dried (HI-02) MLR concentrated and dried (HI-03)

TLC ANALYSIS OF ISOLATED COMPOUNDS

TLC was done for all the isolated compounds with various suitable solvent systems.

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METHODOLOGY

TLC of compound HI-01 and HI-004 (Fig No. 14 - 17)

Stationary phase: Precoated silica gel G plates.

Mobile phase: 5% Ethyl acetate in Pet. Ether.

Visualization: UV at 254 nm, 366 nm and after spraying with ANS reagent.

TLC of compound HI-02 and HI-03 (Fig No. 18)

Stationary phase: Precoated silica gel G plates.

Mobile phase: Butanol : Ethanol : Acetic acid : Water (4:4:1:1).

Visualization: UV at 254 nm, 366 nm and after spraying with ANS reagent.

DETERMINATION OF MELTING POINT OF ISOLATED COMPOUNDS

The isolated compounds were subjected to melting point analysis using capillary tube,

closed at one end, and small amount of the sample was placed into it. The capillary tube

was then dipped at the closed end into liquid paraffin along with a thermometer. The

paraffin was then heated and the temperature at which the sample melted was noted

down.

HPLC ANALYSIS OF THE ISOLATED COMPOUNDS

The isolated compounds HI-01, HI-02, HI-03 and HI-004 were taken for the HPLC

analysis for the identification and purity assessment.

Analytical HPLC for HI-01

Requirements

Column: Kromasil C-18.

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METHODOLOGY

Mobile Phase: Methanol : Buffer (9:1).

(Buffer = 380 ml water + 20 ml Acetonitrile

+ 100 ml Orthophosphoric acid).

Elution: Isocratic.

Flow rate: 1.2 ml/min.

Injection vol.: 20 µl.

Detector: SPD-M10Avp photodiode array detector at 210 nm.

Sample preparation: 1.88 mg of HI-01 was weighed and dissolved in 2 ml of HPLC

grade methanol with the help of sonicator.

Procedure: The HPLC of the isolated compounds were performed using above protocol

and the chromatogram was recorded.

Analytical HPLC for HI-02, HI-03 and HI-004

Requirements

Column: Kromasil C-18.

Mobile Phase: Pump A (0.136 gm Potassium dihydrogen

Orthophosphate + 0.5 ml Orthophosphoric Acid +

Water q.s to 1000 ml.)

Pump B (Acetonitrile).

Elution: Gradient.

Time (min.) B-Conc. (%) A-Conc. (%)

0.01 5 95
15 30 70
25 80 20
35 30 70
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52 Stop Stop
METHODOLOGY

Flow rate: 1.2 ml/min.

Injection vol.: 20 µl.

Detector: SPD-M10Avp photodiode array detector at 270 nm.

Sample preparation: 2.16 mg of HI-02, 2.24 mg of HI-03 and 1.48 mg of HI-004 were

separately weighed and dissolved separately in 2 ml of HPLC grade methanol with the

help of sonicator.

Procedure: The HPLC of the isolated compounds were performed using above protocol

and the chromatogram was recorded.

SPECTROSCOPIC ANALYSIS OF THE ISOLATED COMPOUNDS

As the purity of the HI-01 and HI-004 was up to the mark, they were taken for further

characterization by NMR (13C and 1H), Mass spectrometry, C.H.N analysis etc using

CDCl3 as the solvent. These were done at Sophisticated Instrument Facility, QUEST

Research and Training Institute, Bangalore and IISc, Bangalore.

ESTIMATION OF HI-01 AND HI-004 IN ACETONE FRACTION BY HPLC

METHOD

Requirements

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METHODOLOGY

All the requirements and parameters were same as that of described in the HPLC analysis

of the HI-01 and HI-004.

Sample preparation (extract): 11.34 mg and 11.05 mg of the acetone soluble fraction of

the methanolic extract of the roots of the plant was weighed and dissolved separately in 2

ml of HPLC grade methanol with the help of sonicator for the estimation of the acetone

fraction with respect to HI-01 and HI-004 respectively.

Standard preparation: HI-01 (1.88 mg) and HI-004 (1.48 mg) were used as standards

for the estimation of the acetone fraction. The procedure was same as described in sample

preparation of HI-01 and HI-004 and chromatogram was recorded using the same

protocol respectively.

The estimated amount was calculated by using the formula =

Peak area of sample X standard dilution X purity of compound

Peak area of standard sample dilution

(Standard = HI-01 and HI-004, sample=Acetone extract).

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