chapter 5 isolation, separation and...

Chapter - 5

Isolation, Separation and Characterization of

Phytoconstituents

Section TITLE Pg.

No.

5.1. INSTRUMENTS AND EQUIPMENTS 76

5.2. CHEMICALS, REAGENTS AND MATERIALS 76

5.3. EXPERIMENTAL WORK 77-80

5.3.1. Chemical investigation for isolation and separation of

active phytoconstituents

77

5.3.2. Separation and Isolation phytoconstituents from

methanolic extract using chromatographic methods

77

5.3.3. Simple fractionation of prepared fractions 79

5.3.4. Identification and characterization of isolated

phytoconstituent using m.p., UV, FT-IR, Mass, NMR

spectroscopy

80

5.4. RESULTS AND DISCUSSION 81-94

5.4.1 Chemical investigation for isolation and separation of

active phytoconstituents

81

5.4.2 Column chromatographic method for separation of

phytoconstituent

82

5.4.3. TLC of compound obtained by Simple fractionation of

prepared extract

83

5.4.4. Identification and characterization of isolated

phytoconstituent using m.p., UV, FT-IR, Mass, NMR

spectroscopy (Designated as DI-I)

84

5.4.5. Introduction to betulinic acid 93

5.5. CONCLUSION 94

5.6. REFERENCES 94-95

Isolation, Separation and Characterization Chapter 5

75

5. ISOLATION, SEPARATION AND CHARACTERIZATION OF

PHYTOCONSTITUENT

Extraction is the crucial step for the analysis of medicinal plants, because it is

necessary to extract the desired chemical components from the plant materials for

further separation and characterization. Based upon literature survey, it was observed

that different prepared extracts of plant parts showed potent therapeutic effect.

Preliminary phytochemical screening which has been done for bark and leaves of

Dillenia indica and Dillenia pentagyna revealed presence of steroids, triterpenoids,

glycosides, phenolics etc. These phytoconstituents are required to be separated and

isolated. As the target compounds may be non-polar to polar and thermally labile, the

suitability of the methods of extraction must be considered. Various methods, such as

sonication, heating under reflux, soxhlet extraction and others are commonly used for

the plant samples extraction. In present chapter, bark of D. indica is used for

preparation of extract and for further separation. Prepared plant extract was separated

using column chromatography. In column chromatography, different fractions were

collected using increasing polarity of solvents which includes petroleum ether

(100%); graded mixtures of petroleum ether and toluene (10-90%v/v); toluene (100%)

graded mixtures of toluene and ethyl acetate (100%). Collected fractions were

checked using developed TLC method to obtain single phytoconstituent in pure form.

Another isolation method has been developed by using simple fractionation of

prepared extract and crystals obtained were analyzed using TLC. Pure crystals

obtained was characterized as betulinic acid using different spectroscopic techniques

like UV, IR, NMR, Mass spectroscopy.


76

5.1 INSTRUMENTS AND EQUIPMENTS

All the instruments were calibrated periodically as per in house SOP of Institute of

Pharmacy, Nirma University.

◘ Analytical balance CITIZEN Scale CX-220, manufactured by CITIZEN Private

Ltd.; India having weighing capacity of 10 mg to 220 mg.

◘ Soxhlet assembly

◘ Rotary Evaporator, Rota vapor R-200, Buchi Labortechnik, Switzerland was

used for the evaporation and concentration of eluents.

◘ Camag UV cabinet with dual wavelength UV lamp (254 nm and 366 nm).

◘ The Camag Twin-trough Chambers (20 cm×10 cm) with stainless steel lid

(Camag, Muttenz, Switzerland) were used throughout

◘ Hot air oven, EIE 108, EIE Instruments Pvt. Ltd., Ahmedabad India

◘ Melting point apparatus, T0603160; EIE Instruments Pvt Ltd., Ahmedabad

India

◘ Analytical balance, CX220, Citizen, USA.

◘ UV-visible spectrophotometer, Double beam, UV- 2450, Shimadzu, Japan

◘ Fourier Transform Infrared Spectroscope (FT-IR) with model no. 6100,

manufactured by JASCO Inc., JAPAN, with Spectra Manager software.

◘ Mass spectroscopy, The MS system consisted of API 2000 Q-Trap Mass

Spectrometer (Applied Bio systems, Perkin Elmer, Germany). The data was

collected and processed using ANALYST software.

◘ NMR spectroscopy, 1H and 13C NMR experiments were carried out at

processional frequencies of 300 MHz and 75 MHz, respectively in CDCl3 at 25 °C

temperature on BRUKER NMR (Bruker Ltd. Switzerland).

5.2 CHEMICALS, REAGENTS AND MATERIALS

◘ Analytically pure (≥98 %) Betulinic acid, Sigma-Aldrich (India).

◘ Methanol, HPLC grade and AR grade, Glacial acetic acid, formic acid, Sulfuric

acid, E Merck Ltd. (India).

◘ Anisaldehyde, Vanillin, AR grade, Central Drug House (P) Ltd., New Delhi

(India).


77

◘ Chloroform, Petroleum ether, Toluene, Ethanol, Acetone, Ethyl acetate, AR grade,

S.D. fine Chemicals, Mumbai (India).

◘ All the glasswares including round bottom flask, soxhlet assembly, column,

conical flask, volumetric flask, funnel, test tubes, pipettes, measuring cylinder,

beaker etc. were of Class A borosil glass.

◘ Silica gel, 60-120#, LOBA Chemie Pvt. Ltd for column chromatography.

◘ Silica Gel 60F254 for TLC, aluminium sheets (20x20) plates, Merck, Germany

◘ Calibrated micropipettes were used when required for accurate measurement and

transfer.

◘ Whatman no.1 filter paper, muslin cloth was used as filtering and straining media.

5.3 EXPERIMENTAL WORK

5.3.1 Chemical investigation for isolation and separation of phytoconstituents

5.3.1.1 Extraction procedure

500 g of powdered plant material (bark) of Dillenia indica was filled in a thimble and

extracted exhaustively by Soxhlet apparatus (8 h) using methanol at 60°C. The extract

obtained was collected and passed through Whatman no.1 filter paper to remove all

debris and unextractable matter, including cellular materials and other constitutions

that are insoluble in the extraction solvent. Filtered extract was concentrated using

rotary evaporator at 40° C to obtain dry extractives. Methanolic extract was dried for

further investigation and separation of phytoconstituents.

5.3.2 Separation and Isolation of phytoconstituents from methanolic extract

using Chromatographic Methods

Chromatographic methods were used to study and separate the components of the

methanol extract. Methods used include Thin-Layer-Chromatography (TLC) and

Column Chromatography (CC).

5.3.2.1 Thin-Layer Chromatographic Investigation (Development of mobile phase)

Phytoconstituents are investigated using thin layer chromatography. The extractive


78

was applied to a commercially prepared TLC plate with different solvent systems i.e.

the aims of this procedure were to identify the number of components in the extract,

distinguish the difference between extract, find out how close components of extract

are and to develop solvent systems which can further be used for column

chromatography. The gel plate used was Silica Gel 60F254.

Following general thin layer chromatographic method was applied to the prepared

methanol extract of bark using different solvent systems like CHCl3: Acetone: Formic

Acid (75:16.5:8.5), CHCl3: Ethyl acetate (60:40), Ethyl acetate: MeOH (90:10),

Toluene: Ethyl acetate (93:7), Toluene: CHCl3: EtOH (4:4:1), Ethyl acetate:

methanol: water (100:13.5:10) (Wagner H. C., Sabine Bladt, 1996).

Visualization was done under UV light or spraying with concentrated H2SO4,

anisaldehyde sulfuric acid (ANS) reagent, vanillin-sulfuric acid reagent etc. and

drying in the oven at 105°C. After the spots were visualized and labeled, their

retention factors (Rf value) were calculated and compared. The Rf values were

calculated according to the following formula:

5.3.2.2 Separation of phytoconstituents using Column chromatography

In order to isolate the bioactive compound from the crude extracts they were further

fractionated using column chromatography silica gel (40 g, 60-120 #, LOBA Chemie

Pvt. Ltd.) as stationary phase. A cleaned, dry column (60 X 3cm) was aligned in a

vertical position. A beaker was placed under the column outlet. The column was

partially filled with petroleum ether. A loose plug of cotton which had been washed

with petroleum ether was tamped down in to the bottom of the column. A small layer

of clean white sand was placed over the cotton wool by pouring sand in to the

column. The column was tapped gently to level the surface of the sand. The column

was then filled with petroleum ether and silica gel was added carefully from a beaker,

while solvent was allowed to flow slowly from the column. The column was tapped

as the silica gel was added till a desired height was attained.


79

From dried extract obtained as per section 5.3.1.1., 1 g of extract was loaded on a

glass column (60 X 3 cm) packed with silica gel G (40 g, 60-120#, LOBA Chemie

Pvt. Ltd.) as a stationary phase. The extract was dissolved in a minimal quantity of

methanol and adsorbed on to silica gel G. When the sample had adsorbed to the silica

gel, small amount of sand was poured to cover sample. The mobile phase was poured

continuously to the top of the column by aid of a funnel. The bottom outlet of the

column was opened. The eluates (fractions) were collected in separate test tubes.

The column was eluted with different polarity of solvents; first eluted with petroleum

ether (100%); graded mixtures of petroleum ether and toluene (10-90%); toluene

(100%) graded mixtures of toluene and ethyl acetate (100%). The test tubes were

changed after 10 ml of each fraction and each collected fraction was analyzed by thin

layer chromatography technique. The components eluted were monitored using

Toluene: CHCl3: EtOH (4:4:1) mobile phase and visualized under UV light as well as

also derivatization with anisaldehyde sulfuric acid reagent followed by heating in

oven for 15 min. at 80 °C. Fractions were collected and pooled together on the basis

of similar results and the solvent was removed. They were then dried, weighed and

analyzed.

5.3.3 Simple fractionation of prepared fractions

Dried methanolic extract obtained as per section 5.3.1.1. was taken. The dried residue

was suspended in organic solvent like benzene (approx. 100 ml) added and fractioned.

Yellowish coloured benzene fraction was obtained. The procedure was repeated thrice

with same quantity of benzene and combined. The combined extracts were evaporated

to one third of its volume (approx. 50 ml) and semisolid mass obtained was kept aside

for 8 to 10 h which developed crude crystals. The crude crystals was re-crystallized

using methanol to obtain in pure form. Crystals obtained after recrystallization was

identified using IR, NMR and Mass spectroscopic technique. The compound was also

checked by performing TLC and Rf values were compared with compound obtained

using column chromatography.


80

5.3.4 Identification and characterization of isolated phytoconstituent using m.p.,

UV, FT-IR, Mass, NMR spectroscopy

Pure crystals obtained as per section 5.3.2.2 is designated as DI-I. The structure of a

purified compound can be determined using information from various spectroscopic

techniques. The isolated and purified crystals of DI-I was analysed by melting point

and different spectroscopic techniques like UV, FT-IR, Mass, 13C and 1H NMR

spectroscopy for characterization and structural elucidation.

5.3.4.1 Melting point

Crystals were checked for its melting point and checked with literature value.

5.3.4.2 UV Spectroscopy

The standard solution (200 ppm) of DI-I was prepared in methanol and used to do

analysis in UV-Vis. region from 200-800 nm to determine their ƛ max.

5.3.4.3 FT-IR spectroscopy

The FT-IR spectroscopic analysis was performed by diffused reflectance technique.

The FT-IR spectra of DI-I was recorded in the range of wave number 400 - 4000 cm-1.

FT-IR spectrum was recorded of compound DI-I, dried by mixing with KBr. A KBr

spectrum as a blank was taken. Then, mix the crystalline powder with standard

spectroscopic grade KBr powder in 1:100 ratio and take their spectra.

5.3.4.4 Mass spectroscopy

The analysis was performed in positive ionization mode with Electrospray interface.

The mass to charge (m/z) ratio was recorded in the range of 50-800 m/z. The

parameters for capillary and Rf voltage were 80 V, with nebulizer gas as air at a

pressure of 35 psi and curtain gas as nitrogen at a pressure of 10 psi.

5.3.4.5 NMR spectroscopy

10 mg of DI-I has been taken and dissolved in CDCl3. 1H NMR and 13C NMR

spectra of DI-I were recorded on instrument as described in section 5.1.


81

5.4 RESULTS AND DISCUSSION

Isolation and separation of phytoconstituent from bark of D. indica was done using

column chromatographic method. Results obtained by performing preliminary

phytochemical screening (section 4.4.7.2) and extractive values (section 4.4.4),

methanol was taken as a solvent for separation and further extraction. Prepared extract

has been investigated by TLC and results are reported below. (Table 5.1)

5.4.1 Chemical investigation for isolation and separation of phytoconstituents

5.4.1.1 TLC investigation

Separation of compounds has been achieved by using different solvent systems

mentioned in Table 5.1.

Many solvent has been tried to achieve separation of phytoconstituents mentioned in

table 5.1, Toluene: CHCl3: EtOH (4:4:1) after derivatization with anisaldehyde

sulfuric acid (ANS) reagent showed remarkable separation of compounds present in

methanolic extract of the bark of D. indica. (Figure. 5.1)

TABLE 5.1 Spots obtained from extracts by thin layer chromatography

Sr.

No. Solvent System

No. of

Spots Rf Values

254 nm 366 nm After

Derivatization

1

CHCl3: Acetone:

Formic Acid

(75:16.5:8.5)

2 -- 0.5 0.55

2 CHCl3: Ethyl acetate

(60:40) 3 -- 0.5 0.6, 0.7

3 Ethyl acetate: MeOH

(90:10) 2 -- -- 0.3, 0,45

4 Toluene: Ethyl

acetate (90:10)

4

0.2 0.8 0.6, 0.65

5 Toluene: CHCl3:

EtOH (4:4:1) 5 0.15, 0.4 0.8

0.35, 0.5, 0.7,

0.84, 0.9


82

FIGURE 5.1 TLC plates of methanolic extract of bark showing spots at different Rf

a) 366 nm, b & c) after Derivatization with ANS reagent

5.4.2 Column chromatographic method for separation of phytoconstituents

Total about 50 fractions were collected in test tubes using increasing polarity of

solvents mentioned as per section 5.3.2.2. Each test tube was checked for presence of

phytoconstituents using TLC and results are reported in Table 5.2.

TABLE 5.2 TLC investigation of fractions eluted using column chromatography

Test

tube no. Description No. of Spots Figure

1 16, 17 Light green band was

eluting No Spot

2 20, 21 Light orange colored

band was eluting

One fluorescent spot at

366 nm at Rf 0.8 A

3

27, 28,

29, 30,

31, 32,

33

Colourless band

One magenta color spot

after Derivatization with

ANS reagent at 0.65 Rf

B

5.4.2.1 Thin Layer Chromatography of fractions collected by column

chromatography

Test tube numbers 27-33 were showing single spot after derivatizing with ANS

reagent. In TLC, using mobile phase- Toluene:CHCl3:EtOH (4:4:1) isolated

compound resolved a single spot at Rf 0.60. All fractions were combined and


83

concentrated. Upon keeping aside for few hours crude crystals were obtained. This

was further recrystallized using methanol to get pure compound.

FIGURE 5.2 TLC plate of fraction eluted using column chromatography

(a) Rf 0.8 at 366 nm (b) Rf 0.6 after Derivatizing with ANS reagent

5.4.3 TLC of compound obtained by Simple fractionation of prepared extract

Crystals obtained using simple fractionation was analyzed using TLC showed single

spot. Rf value of obtained crystals correlated with compound obtained using column

chromatography (Figure 5.3). Around 1.72 g of pure crystals obtained using simple

fractionation method which was found to be 7.37 % w/w in MeOH extract and 0.43

%w/w in bark of D. indica. Column chromatography and simple fractionation of

methanolic extract of Dillenia indica bark yielded one compound designated as DI-I.

FIGURE 5.3 (A) TLC of crystals [column chromatography at Rf 0.6];

(B) TLC of crystals [simple fractionation at Rf 0.6]


84

5.4.4 Identification and characterization of isolated compound DI-I using m.p.,

UV, FT-IR, Mass, NMR spectroscopy

5.4.4.1 Melting point determination of DI-I

Determination of melting point of DI-I was carried out using melting point apparatus

using open capillary method. [Cichewicz, R. H. and Kouzi, S. A., 2004]

TABLE 5.3 Melting point of compound DI- I

Compound Observed Melting Point

DI-I 316-318 0 C

5.4.4.2 Results of UV spectroscopy

UV spectrum of DI-I (200 ppm) in methanol was taken and scanned in the range of

200-400 nm on UV spectrophotometer. (Figure 5.4) UV-Vis spectra of compound

indicates that absorptivity value is very less at all wavelength (Table 5.4).

FIGURE 5.4 UV spectra of compound DI-I (200-400 nm)

TABLE 5.4 λmax of compound DI I by spectrophotometer

Compound Observed λmax

DI –I 210 nm


85

5.4.4.3 Results of FT-IR Spectroscopy

FT-IR spectroscopy was carried out to ascertain functional groups. FT-IR spectrum of

DI-I (Figure 5.5) was recorded in diffused reflectance mode. The FT-IR spectrum of

DI-I presents dominant IR absorption bands in the high wave region at 3456 cm-1, and

2939 cm-1; attributed to -OH, -CH3 and -CH2 asymmetric and symmetric stretching

vibrations respectively.

In finger print region, the FT-IR spectrum presets dominant bands at 1679, 1445,

1368, 1035, 885, 792 cm-1 and many other bands of medium to weak intensity. The

observed band at 1679 cm-1 can be assigned to C=O stretching of –COOH functional

group. Other bands in the spectral range are assigned to bending vibrations of –OH, -

CH2 and CH3 groups as well as to skeletal bending bonds. The band at 1368 cm-1 is

due to C-O stretching among others. The intense band as 885 cm-1 in IR spectrum is

due to vibration of the CH2 in alkene group. Theoretical wave numbers responsible

for functional groups are compared with observed wave numbers and presented in

Table 5.5 [Falamaş A., 2011; Elvira E. Kovac-Besovic et al., 2009].

FIGURE 5.5 Recorded FT-IR spectra of compound DI-I from D. indica bark.


86

TABLE 5.5 Important frequencies of DI-I obtained in FT-IR spectra

Compound

Parameter

Theoretical

frequency (cm-1) Functional group

IR (cm-1)

3459.67 O-H stretching

2939.95 C-H stretching of aliphatic compounds

1679 C=O stretch of -COOH group

1455 OH bending

1139 C-O stretching of -COOH group

5.4.4.4 Results of Mass spectroscopy

Mass spectroscopy (MS) was carried out to determine the molecular weight of

isolated phytoconstituent. The MS and MS/MS spectra of DI-I are shown in Figure

5.6. The molecular ion (M+1) peak was obtained at 455.2 m/z which confirms

molecular weight of DI-I at 455.2 [Cichewicz RH and Kouzi SA, 2004].

5.4.4.5 Results of NMR spectroscopy

The 1H and 13C NMR experiments of DI-I were carried out results obtained

mentioned in figure 5.7 to figure 5.12. The 1H NMR spectrum showed fine tertiary

methyl singlets at δ 0.63.0.65, 0.68, 0.74, 0.90, 0.97 and one more secondary

hydroxyl group showed broad triplet at δ 3.34 and two olefinic protons at δ 4.55 and

4.68 representing the exocyclic double bond (Figure 5.6). The appearance of carbonyl

group at δ 177.3 in 13C NMR spectrum suggested the presence of acid group in its

structure [Md. Muhit A. et al., 2010; Parvin N. et al., 2009; Cichewicz, R. H. and

Kouzi, S. A., 2004].

TABLE 5.6 1H and 13C NMR spectral assignments for DI-I

Compound Observed NMR spectral assignments

1H NMR(δ) CDCl3; δ 4.65 (1H, s), δ 4.513 (1H, s); δ 3.033 (1H, d);

δ 0.989 (3H, s, Me-26)

13C NMR(δ) CDCl3; δ 178.3, δ 150.67, δ 109.17, δ 78.24, δ 77.66,

δ 76.81, δ 55.87, δ 48.98, δ 42.27


87

FIGURE 5.6 Mass Spectra of compound DI-I


88

FIGURE 5.7 1H NMR Spectra of compound DI-I



89


FIGURE 5.10 13C NMR Spectra of compound DI-I


90

FIGURE 5.11 13C NMR Spectra of compound DI-I (zoomed upto 9 m/z)

FIGURE 5.12 13C NMR Spectra of compound DI-I


91

5.4.4.6 Summarized Results of Spectral analysis

From results of m.p., IR, NMR and Mass spectra of the isolated phytoconstituent, It

was proposed that the structure may be of Betulinic acid (Figure. 5.4, 5.5, 5.6, 5.7,

5.8, 5.9, 5.10, 5.11 and 5.12) (Table 5.7)

Structure of DI-I has been elucidated from obtained results which were compared

with reported values of betulinic acid and confirmed. Figure 5.13 shows the structure

of betulinic acid.

TABLE 5.7 Summary Data of M.P and Spectral analysis of DI-I

Parameter Betulinic acid Literature value

(Reported value) Reference

M.P. 3200 C 316-318 0 C

(Cichewicz, R.

H. and Kouzi, S.

A., 2004)

UV (λ max)

Does not show any

absorbance so,

chromophore group is

absent but can obtain

spectra at 210 nm

210 nm

(Cichewicz, R.

H. and Kouzi, S.

A., 2004; Simona

Cinta-Pinzaru,

2012 )

IR (cm-1)

3469 cm-1 (OH str), 1679

(C=O), 1452 (OH ben),

1375 (CH2=CH-CH3),

1139 (C=O)

1136 cm-1, 1373

cm-1, 1450 cm-1,

1670 cm-1, 3465

cm-1

(Falamaş A.,

2011; Elvira E.

Kovac-Besovic

et al., 2009)

1H NMR(δ)

CDCl3; δ 4.65 (1H, s), δ

4.513 (1H, s); δ 3.033 (1H,

d); δ 0.989 (3H, s, Me-26)

δ 4.75, δ 4.62, δ

3.20, δ 3.00, δ 0.99 (Md. Muhit A. et

al., 2010; Parvin

N. et al., 2009;

Cichewicz, R. H.

and Kouzi, S. A.,

2004)

13C

NMR(δ)

CDCl3; δ 178.3, δ

150.67, δ 109.17, δ

78.24, δ 77.66, δ 76.81, δ

55.87, δ 48.98, δ 42.27

δ 178.8, δ 151.3, δ

109.9, δ 78.4, δ

77.0, δ 76.81, δ

56.01, δ 49.8, δ

42.9

MASS 455.2 (M-1), 439, 413, 206 455 m/z

(Cichewicz, R.

H. and Kouzi, S.

A., 2004)


92

FIGURE 5.13 Structure of DI- I identified as Betulinic Acid

5.4.4.7 Comparison of FT-IR spectra of isolated BA and Standard BA

Obtained crystals of BA has been taken for reconfirmation using FT-IR spectroscopy.

FT-IR spectra of isolated BA by different extraction methods as well as BA procured

from market is shown in Figure 5.14.

FIGURE 5.14 Overlay of FT-IR spectra of Isolated BA and BA (Sigma)


93

5.4.5 Introduction to betulinic acid

Betulinic acid (3β, hydroxy-lup-20(29)-en-28-oic acid) is a pentacyclic triterpenoid of

plant origin.

TABLE 5.8 Physicochemical properties of betulinic acid

Sr. No. Physicochemical properties Result

1 Molecular Formula C30H48O3

2 IUPAC Name (3β)-3-Hydroxylup-20(29)-en-28-

oic acid

3 Molecular weight 455.7 g/mol

4 Category Pentacyclic triterpenoid

5 Appearance White or off white fine powder

6 Solubility

i. Pyridine and acetic acid

ii. Water

iii. Organic solvents like alcohols, ethers

Highly soluble

0.02 µg/ml

Soluble upon warming

7 Optical rotation +7˚ - +9˚ (c= 0.9 in pyridine)

8 Nature Acidic

Category: [Cichewicz RH et al., 2004]

Analgesic, Non-Narcotic, Anti-HIV agent, Anti-infective agent, Anti-inflammatory

agent, Anti-retroviral agent, Anti-inflammatory agent, Antimalarial, Antineoplastic

agent, Antiparasitic agent, Antiprotozoal agent, Antirheumatic agent, Antiviral agent,

Hormone substitute, and Hormone antagonist, Prostaglandin antagonist

Official Status: [Merck Index, 2006]

Official in Quality Standards of Indian Medicinal Plant; Vol. 7; ICMR 2008; p.g.no.:

78-85 and in The Merck Index, An encyclopaedia of chemicals, drugs and biological;

Merck research laboratories; USA; 14th edition; 1192.

Storage Condition: Store in cool, well-ventilated area. Keep away from direct

sunlight. Keep container tightly sealed until ready for use. Recommended storage

temperature is at +4°C.


94

Mechanism of Action: BA selectively induces apoptosis in tumor cells by directly

activating the mitochondrial pathway of apoptosis through p53 and CD95 independent

mechanism. [Nick, A et al., 1995; Bringmann G et al., 1997; Peng C et al., 1998;

Cinta PS et al., 2002]

5.5 CONCLUSION

In present chapter, two methods has been developed for separation of phytoconstituent

using bark of D. indica. Thin layer chromatographic method developed showed

remarkable separation of phytoconstituents which can be used for method development.

One potent anticancer compound, betulinic acid, was obtained using two different

methods like column chromatography and simple fractionation which has been

characterized using m.p., IR, NMR and Mass spectroscopy. Column chromatography

yielded good amount of betulinic acid and another simple fractionation method proved

to simple and reproducible method for isolation of betulinic acid. Developed TLC

method for separation of betulinic acid can be further applied for quantification in

different parts of plant species.

5.6 REFRENCES

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"Betulinic Acid: Isolation from Triphyophyllum peltatum and Ancistrocladus

heyneanus, Antimalarial Activity, and Crystal Structure of the Benzyl Ester*."

Planta medica 63.03 (1997): 255-257.

Cichewicz RH, Kouzi SA. “Chemistry, biological activity, and chemotherapeutic

potential of betulinic acid for the prevention and treatment of cancer and HIV

infection”. Med. Res. Rev., 24 (2004): 90–114. DOI: 10.1002/med.10053.

Cı̂ntă Pı̂nzaru, S., N. Leopold, and W. Kiefer. "Vibrational spectroscopy of betulinic

acid HIV inhibitor and of its birch bark natural source." Talanta 57.4 (2002): 625-

631.

Elvira E. Kovac-Besovic, Duric K, Kalodera Z, Sofic E. “Identification And Isolation

Of Pharmacologically Active Triterpenes In Betuale Cortex, Betula Pendula

Roth., Betulaceae” Bosnian Journal of Basic Medical Sciences 2009; 9 (1): 32-38.


95

Falamaş, A., Pinzaru, S. C., Dehelean, C. A., Peev, C. I. and Soica, C. (2011), Betulin

and its natural resource as potential anticancer drug candidate seen by FT-Raman

and FT-IR spectroscopy. Journal of Raman Spectroscopy, 42: 97–107.

doi: 10.1002/jrs.2658

Kumar, D.; Mallick, S.; Vedasiromoni, R. J.; Pal, C. B. Anti-leukemic activity of

Dillenia indica L. fruit extract and quantification of betulinic acid by HPLC.

Phytomedicine 2010, 17, 431-435.

Md. Muhit A, Tareq SM, Apu AS, Basak D, Islam MS. Isolation and Identification of

compounds from the leaf extract of Dillenia indica Linn. Bangladesh Pharm J.

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Nick, Andre, Wright, A. D., Rali, T., & Sticher, O."Antibacterial triterpenoids from

Dillenia papuana and their structure-activity relationships." Phytochemistry

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Parvin N, Md. Rahman S, Md. Islam S, Md. Rashid A. Chemical and biological

investigations of Dillenia indica Linn. Bangladesh J Pharmacol 2009; 4:122-5

Peng, C.; Bodenhausen, G.; Qiu, S.; Fong, H. H. S.; Farnsworth, N. R.; Yuan, S.

"Computer‐assisted structure elucidation: application of CISOC–SES to the

resonance assignment and structure generation of betulinic acid." Magnetic

Resonance in chemistry 36.4 (1998): 267-278.

Simona Cinta-Pinzaru, Cristina A Dehelean, Codruta Soica, Monica Culea and Florin

Borcan, Evaluation and differentiation of the Betulaceae birch bark species and

their bioactive triterpene content using analytical FT-vibrational spectroscopy and

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