RESULTS

RESULTS

COLLECTION OF PLANT MATERIAL

Authenticated dried roots of Hemidesmus indicus (PT/0707030) were collected

from Natural Remedies Pvt. Ltd. and subjected to methanolic extraction.

EXTRACTION OF PLANT MATERIAL

The dried roots of Hemidesmus indicus were coarsely powdered and 20 kg was

subjected for extraction with methanol as shown in Flow chart-1. The yield was found to

be 12.5%.

FRACTIONATION OF THE EXTRACT

The 2 kg of methanolic root extract of Hemidesmus indicus was refluxed with

Acetone as shown in Flow chart- 2. The acetone layer was selected for further

fractionation.

OPTIMIZATION OF TLC METHOD

Optimization of TLC method was done using different mobile phases. The

solvent system (chloroform: methanol, 9:1) and ANS as visualizing agent showed best

resolution. β-sitosterol, Lupeol and sitosterol-D-glucoside were found in the extracts (lab

scale and pilot scale) corresponding to the Rf values as follows:

Sitosterol-D-glucoside = 0.236.

β-sitosterol = 0.789.

Lupeol = 0.868.

TLC images are shown in the Fig No. 2, 3 and 4.

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RESULTS

TLC OF LAB SCALE, PILOT SCALE EXTRACTS WITH SOME STANDARDS

Solvent system = Chloroform : MeOH (9 : 1).

L = Lab scale extract, P = Pilot scale extract, B = β-sitosterol, Lu = Lupeol,

Sd = Sitosterol-D-glucoside, U = Ursolic acid.

Fig No. 2 (Visualization UV at 254 nm.).

Fig No. 3 (Visualization UV at 366 nm.).

Fig No. 4 (Visualization ANS Spray).

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RESULTS

ISOLATION OF PHYTOCONSTITUENTS

The present study aimed at phytochemical analysis of Hemidesmus indicus to

isolate the marker constituents. The acetone fraction of MeOH extract was first subjected

for isolation of phytoconstituents using column chromatography. Systematic column

chromatography of the acetone fraction was performed using 4 different columns. The

isolation scheme is shown in Flow chart 3 to 7.

Column chromatography

Acetone fraction was subjected to column chromatography using different

columns (1-4) and different mobile phases with increasing the polarity from Pet. Ether to

Ethyl acetate / Methanol.

Isolation /Fractionation using column-1(CC-1)

19 fractions were collected from column-1 using acetone fraction (Flow chart

3). These fractions were concentrated under vacuum. The weights of the residue of the

fractions are shown in Flow chart 3. The TLC of the fractions collected from column-1

are shown in the Fig No. 5, 6 and 7. Two major spots corresponding to the Rf values

0.235 and 0.558 were found in the fractions CC-1/F-3 and CC-1/F-4. So these were

selected from the column-1 for further purification by using column-2.

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RESULTS

TLC OF CC-1 (ALL FRACTIONS TOGETHER)

Solvent system: 5% Ethyl acetate in Pet. Ether.

Fig No. 5 (Visualization UV at 254 nm.).

Fig No. 6 (Visualization UV at 366 nm.).

Fig No. 7 (Visualization ANS Spray).

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RESULTS

Isolation /Fractionation using column-2(CC-2)

8 fractions were collected from column-2 using fractions CC-1/F3- and CC-1/F4

as starting material (Flow chart 4). These fractions were concentrated under vacuum.

The weights of the residue of the fractions are shown in Flow chart 4. Fig No. 8 and 9

shows the TLC of the fractions collected from column-2. By processing the ppt obtained

in CC-2/F-5 and CC-2/F-6, a pure compound was isolated and coded as HI-004. The

fraction CC-2/F-2 was selected from the column-2 for further purification by using

column-3.

Isolation /Fractionation using column-3(CC-3)

14 fractions were collected from column-3 using fraction CC-2/F-2 as starting

material (Flow chart 5). These fractions were concentrated under vacuum. The weights

of the residue of the fractions are shown in Flow chart 5. The fraction CC-3/F-10 yields

2.3 gm of amorphous powder with a single spot on TLC having the R f value 0.162. TLC

of the fractions collected from column-3 is shown in the Fig No. 10.

Isolation /Fractionation using column 4(CC-4)

14 Fractions were collected from column-4 using fraction CC-1/F18 as starting

material (Flow chart 6). These fractions were concentrated under vacuum. The weights

of the residue of the fractions are shown in Flow chart 6. The fraction CC-4/F-10 was

taken for the further purification by precipitation method and two purified compounds

(amorphous powder) were isolated which showed the single spots in the TLC having the

Rf values 0.465 and 0.558. The TLC of the fractions collected from column 4 is shown in

the Fig No. 11, 12 and 13.

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RESULTS

TLC OF CC-2 (ALL FRACTIONS TOGETHER)

Solvent system: 10% Ethyl acetate in Pet. Ether.

Fig No. 8 (Visualization UV at 254 nm.).

Fig No. 9 (Visualization ANS Spray).

TLC OF CC-3 (ALL FRACTIONS TOGETHER)

Solvent system: 2% Ethyl acetate in Pet. Ether.

Fig No. 10 (Visualization ANS Spray).

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RESULTS

TLC OF CC-4 (ALL FRACTIONS TOGETHER)

Solvent system: Butanol : Ethanol : Acetic acid : Water = 4 : 4 : 1 : 1

Fig No. 11 (Visualization UV at 254 nm.).

Fig No. 12 (Visualization UV at 366 nm.).

Fig No. 13 (Visualization ANS Spray).

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RESULTS

ISOLATED COMPOUNDS

Altogether four compounds were isolated from acetone fraction namely HI-01,

HI-02, HI-03 and HI-004. The TLC of HI–01 and HI–004 was done along with MeOH

ext.(M) and acetone fraction(A) and also along with water extract(W) of the roots of the

plant Hemidesmus indicus.

The compounds HI–01 and HI–004 were found in the MeOH ext.(M) and

Acetone fraction(A) corresponding to the Rf values as follows:

HI-01 = 0.564. (Visualized after spraying with ANS reagent).

HI-004 = 0.230. (Visualized at UV 254 nm and after spraying with ANS reagent).

The TLC is shown in the Fig No. 14 and 15.

These compounds were absent in the water ext.(W). Fig No. 16 and 17.

TLC of HI-02 and HI-03 was done and Rf values were found to be as follows:

HI-02 = 0.462.

HI-03 = 0.554.

The TLC is shown in the Fig No. 18.

The yields of isolated compounds are shown in Table No. 3 and the physical

properties of these compounds are shown in the Table No. 4.

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RESULTS

TLC OF HI-01 AND HI-004

Solvent system: 5% Ethyl acetate in Pet. Ether.

Fig No. 14 (UV 254 nm.) Fig No. 15 (ANS)

Fig No. 16 (UV 254 nm.) Fig No. 17 (ANS)

TLC OF HI-02 AND HI-03

Solvent system: Butanol : Ethanol : Acetic acid : Water = 4 : 4 : 1
Fig No. 18 (ANS)

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RESULTS

Table No. 3. List of isolated compounds and their yields.

No. Code of isolated compounds Yield

1 HI-01 2.3 gm

2 HI-02 150 mg

3 HI-03 800 mg

4 HI-004 1.2 gm

Table No. 4. Physical properties of the isolated compounds

Code Solubility State Odour Melting point

Pet. Ether, Mild 208-210 0C
HI-01 Hexane, White crystals Characteristic
Chloroform.
Acetone, Brown amorphous
HI-02 Ethanol, powder None _
Methanol.
Ethyl acetate, Brown amorphous
HI-03 Ethanol, powder None _
Methanol.
Chloroform, Colourless crystals Aromatic, 39-40 0C
HI-004 Ethyl acetate. Characteristic

ANALYTICAL HPLC FOR HI-01

Table No. 5 Analytical HPLC results of HI-01

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RESULTS

Compound code Time (min) Concentration (%) Area

1.228 0.5814 65360

HI-01
1.690 99.4186 11176075

ANALYTICAL HPLC FOR HI-02, HI-03 AND HI-004

The process followed was same for all HI-02, HI-03 and HI-004. The results

found are as follows:

Table No. 6.

Compound code Time (min) Concentration (%) Area

12.883 32.8840 63074
14.746 20.1505 38650
HI-02
25.845 19.2725 36966
27.069 20.0882 38531
14.155 46.8234 25821
16.466 17.9841 9917
HI-03
19.401 35.1925 19407
26.031 99.8465 23110515
27.275 00.1176 27214
HI-004
33.946 00.0360 8322

SPECTROSCOPIC ANALYSIS OF THE ISOLATED COMPOUNDS 72, 73

All the spectroscopic data obtained for the compound HI-01 suggested that the

structure of the compound may be the following:

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RESULTS

8
CH2
H3C
a
l
k m n b
7 3
i CH3 CH3 CH3
h c 1
j
d
CH3
e 2
H3COOC g
f
6 H3C CH3
4
5

The supporting integral values (1H NMR) for this structure were as follows

δ = 3.22 – m – 11H (at a, b, 1, c, d).

δ = 1.40 – s – 4H (at e and f).

δ = 0.30 – s – 1H (at g).

δ = 1.18 – d – 4H (at h and i).

δ = 0.35 – s – 1H (at j).

δ = 1.51 – s – 4H (at k and l).

δ = 0.31 – s – 1H (at m).

δ = 0.32 – s – 1H (at n).

δ = 2.63 – m – 6H (at 2 and 3).

δ = 2.92 - m – 12H (at 4, 5, 7 and 8).

δ = 1.00 – m – 3H (at 6).

δ = 4.58 –s – 2H (at =CH2).

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RESULTS

The supporting IR values were as follows

2940.61 = Aliphatic C-H of -CH3, =CH2.

1736.97 = C=O.

875.72 = Aliphatic C-C.

1366.62 = Aliphatic C-H.

The mass peaks obtained at 409 m/z and 424 m/z. The 13C NMR data were also

supportive to the above structure.

The CHN analysis report showed that Carbon was 90.532% and Hydrogen was

9.377%. Nitrogen was absent in the compound.

So all these data clearly suggested that the above structure was may be the right

one for the compound HI-01. The chemical name of this may be 20(30)-Ursen-3-acetate.

All the spectroscopic data obtained for the compound HI-004 suggested that the

structure of the compound may be the following:

O
1
OH
6
2

5 3
4

O
H3C

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RESULTS

The supporting integral values (1H NMR) for this structure were as follows

δ = 3.86 – s – 3H (Aromatic O- CH3 at 4).

δ = 6.43 – s – 1H (Aromatic H at 3).

δ = 6.52 – d – 2H (Aromatic H at 5).

δ = 7.40 – d – 2H (Aromatic H at 6).

δ = 9.41 – s – 1H (Aromatic CHO at 1).

δ = 11.48 – s – 1H (Aromatic OH at 2).

The supporting IR values were as follows

3050 = Aromatic –OH.

1633 = Aldehydic C=O.

1326.12 = Aromatic C=C.

2850 = Aldehydic C-H.

1505.51 = Aromatic C-O.

2902 = C-H of O- CH3 at 4.

The mass peaks obtained at 153 m/z, 291 m/z and 300 m/z. The 13C NMR data

were also supportive to the above structure.

So all these data clearly suggested that the above structure was may be the right

one for the compound HI-004. The chemical name of this structure may be 2-Hydroxy-4-

Methoxy benzaldehyde.

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RESULTS

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

METHOD

Peaks were observed in the similar retention time corresponding to the values as

observed in the HI-01 (1.690 min in compound and 1.694 min in Acetone fraction) and

HI-004 (26.031 min in compound and 26.021 min in Acetone fraction). It confirms the

presence of the compounds in the acetone fraction of MeOH extract of the plant part.

Hence the process can be said as standardized.

In the HPLC chromatogram of the Acetone fraction, the area of HI-01 was

found to be 11081622. So the HI-01 present in the Acetone fraction was found to be

14.34 %.(By calculating according to the formula given in methodology).

Again in the HPLC chromatogram of the Acetone fraction, the area of HI-004 was found

to be 22364910. So the HI-004 present in the Acetone fraction was found to be 12.94 %.

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