Isolation of Vasicine, Vasicinone from Adhatoda zeylanica...

Isolation of Vasicine, Vasicinone from Adhatoda zeylanica.........

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Chapter 5

Isolation of Vasicine, Vasicinone from Adhatoda zeylanica leaf and Embelin from Embelia ribes

fruit 5.1 INTRODUCTION

Major pharmaceutical companies are not interested in the evaluation of plant extracts.

The reasons are quite simple. Firstly, when an extract shows activity in a bioassay, the

active principle must be isolated and characterized. This is expensive and may take a

long time, depending on the availability of appropriate amount of extract or plant

material, the time for bioassay, and the ease of unambiguously determining the

structure. By this time, the synthetic “hits” will have moved to the next stage of

decision making and the natural product is left behind. Moreover, concern over the

availability of enough quantity of a chemical entity required for development and

market needs of natural products, has been the one most limiting factor for the

pharmaceutical industry’s interest in natural products. Market demand can reach a

scale of hundreds to thousands of kilograms per annum. Total synthesis will not

economically provide the complex natural product to meet this market demand. As

compared to the minor compounds in the plants, the active compounds present in high

concentrations increase their probability to be used for clinical purpose. In contrast,

the present study provides an alternative, simple, rapid and inexpensive approach for

isolation, identification and activity of active compounds from plant extracts.

5.2 MATERIALS AND METHODS

5.2.1 Plant material collection and authentication

Adhatoda zeylanica leaf and Embelia ribes fruit were collected from Gujrat, India.

Their authenticity was confirmed by the taxonomist Dr. Sheetal Anandijiwala of our

department in year 2005-2006 and voucher specimen was deposited at the Department

of Pharmacognosy and Phytochemistry, B. V. Patel Pharmaceutical Education &

Research Development (PERD) Centre, Ahmedabad, India. The material was stored

in air tight containers at room temperature until use.


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5.2.2 Isolation of vasicine and vasicinone from methanolic extract of Adhatoda

zeylanica leaf and embelin from methanolic extract of Embelia ribes fruit

5.2.2.1 Preparation of methanolic extract

Dried leaves of A. zeylanica (500 g) were ground and extracted with methanol (1000

ml × 3) under reflux for 30 min at a maximum temperature of 50ºC. Dried fruits of E.

ribes (300 g) were ground and extracted with methanol (500 ml X 3) under reflux for

30 min at a maximum temperature of 50ºC. Both the extracts were filtered; the

filtrates were pooled and concentrated to dryness by removing the solvent under

reduced pressure at 50ºC. A mass of dark greenish sticky crude extract was obtained

from A. zeylanica leaf (93 g; 18.6 %) w/w and a mass of blackish sticky crude extract

of E. ribes (34 g; 11.3 % w/w). The extracts were stored in airtight glass bottles at 4ºC

in a refrigerator.

5.2.2.2 Fractionation of the methanolic extract of A. zeylanica and E. ribes

Methanolic extract of A. zeylanica (25 g) and E. ribes (8 g) were then suspended in

100 ml and 50 ml water respectively and partitioned successively with solvents in

increasing order of polarity viz, petroleum ether, chloroform, ethyl acetate, n-butanol

and the n-butanol insoluble fraction was also prepared. For A. zeylanica (100 ml X 3)

and for E. ribes 50 ml (X 3) solvent were used for liquid-liquid extraction. A.

zeylanica fractions were evaporated in vacuum to yield the residues of petroleum

ether (4 g; 16.0%), chloroform (0.8 g; 3.2 %), ethyl acetate (6.2 g; 25 %), n-butanol

(10 g; 40 %) fractions and n-butanol insoluble portion (0.9 g; 3.8 %) respectively. E.

ribes fractions were evaporated in vacuum to yield the residues of petroleum ether

(3.4 g; 42.5 %), chloroform (0.4 g; 5.3 %), ethyl acetate (1.6 g; 20 %), n-butanol (1 g;

12.4 %) fractions and n-butanol insoluble portion (0.7 g; 9.5 %) respectively. For

assay, fractions were dissolved in DMSO. Each fraction was bio-assayed for PfLDH.

Chloroform fraction of A. zeylanica and petroleum ether fraction of E. ribes

methanolic extract was found to have significant plasmodial lactate dehydrogenase

inhibitory activity as compared to other fractions of the respective plants. (Figure 5.1

& Figure 5.2)

5.2.2.3 Isolation of vasicine and vasicinone from methanolic extract of A. zeylanica leaf

TLC fingerprint profile


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TLC fingerprint profile of all the fractions of A. zeylanica were developed in solvent

system of ethyl acetate: methanol: ammonia (8: 0.5: 0.2) (at 25 ± 2°C temperature and

40 % relative humidity) and Co-TLC of all the fractions was carried out along with

reference standard vasicine and vasicinone (purchased from SPIC, Chennai India) on

precoated silica gel 60F254 TLC plates (E. Merck). Since, chloroform fraction of A.

zeylanica leaf exhibited good activity as compared to other fractions and vasicine and

vasicinone are the major constituents present in the chloroform fraction of A.

zeylanica leaf. Hence we decided to isolate vasicine and vasicinone from A. zeylanica

leaf.

Isolation of vasicine from methanolic extract A. zeylanica leaf

Vasicine was isolated from A. zeylanica leaf by the reported method of acid–base

extraction (Chaitali D et al., 2005). 10 gm of methanolic extract of A. zeylanica leaf

was extracted with 25 ml (X 3) 2N HCl (pH 2-2.5) under reflux for 30 min at a

maximum temperature of 50ºC, consequently this acidic fraction was basified with

liquor ammonia to pH 8-8.5. This basic solution was extracted with 150 ml (X 3)

chloroform, with subsequent TLC monitoring of each step for the presence of

vasicine. Chloroform extract was pooled and evaporated to dryness by removing the

solvent under reduced pressure at 50°C to get 3.1 gm of alkaloid fraction. Vasicine

was identified as the major compound in the alkaloid fraction by TLC in solent

system ethyl acetae: methanol: ammonia (8: 0.5: 0.2). The alkaloid fraction was

dissolved in a minimum quantity of ethanol and vasicine was crystallized out from the

alkaloid fraction. It was purified by repeated crystallization with ethanol. White

needles of vasicine (150 mg) was obtained having melting point of 210°C. For

isolation of vasicinone remaining fraction was subjected to column chromatography.

Isolation of vasicine from methanolic extract A. zeylanica leaf

Stationary phase

Silica gel (for flash chromatography) 230-400 mesh (Spectrochem, India), 100 g.

Sample load

2 g of fraction was dissolved in minimum volume of methanol. The solution was

mixed with 3 g of silica gel and the solvent was evaporated. The fraction adsorbed on

silica was loaded on silicagel column.


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Mobile phase

Remaining fraction was subjected to column chromatography over silica gel (Merck)

using mixtures of chloroform and methanol of increasing polarity. Vasicinone was

eluted from the column with chloroform: methanol (49:1) as a white solid and was

crystallised from chloroform: ethyl acetate.

Vasicine and vasicinone wee characterized by recording IR, MS and NMR spectra

and melting point and the identity of vasicine and vasicinone was confirmed by

comparison of their spectral data with those reported (Jain et al., 1980; Joshi et al.,

1994).

5.2.2.4 Isolation of embelin from methanolic extract of E. ribes fruit

TLC fingerprint profile

TLC profile of all the fractions of E. ribes was developed in solvent system of n-

propanol: n-butanol : ammonia (7: 1: 2) (at 25 ± 2°C temperature and 40 % relative

humidity) and Co-TLC of the fractions was carried out along with reference standard

embelin on precoated silica gel 60F254 TLC plates (E. Merck).

Since, petroleum ether fraction of E. ribes fruit exhibited good activity as compared to

other fractions and embelin was the major constituents present in the petroleum

fraction of E. ribes fruit. Hence we decided to isolate embelin from E. ribes fruit.

Isolation of embelin from methanolic extract E. ribes fruit

25 gm of E. ribes fruit was ground and extracted with 100 ml X 4 petroleum ether

under reflux for 30 min at a maximum temperature of 50ºC. The extract was filtered,

concentrated to 1/3 volume and kept overnight at room temperature. Embelin settled

at the bottom, and after decanting the supernatant, orange coloured mass was

obtained. It was purified by repeated crystallization with methanol, to obtain

glistening orange crystals of embelin (722 mg) having a melting point of 142-143°C.

It was characterized by recording UV, IR, MS and NMR spectra and melting point

and comparing with the reported data (Kumara Swamy HM et al., 2007; Haq et al.,

2007).


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5.2.3 Structure elucidation of vasicine, vasicinone and embelin

The melting point, Infra-red (IR), Mass, 1H-NMR and 13C-NMR spectra were

recorded for vasicine and embelin. The melting point was measured using a melting

point apparatus (Toshniwal, India). IR spectrum was recorded using KBr disk method

on IR spectrophotometer (Model 500, BUCK SCIENTIFIC). Mass spectra were

recorded in the positive ion mode on mass spectrophotometer (API 165, PERKIN-

ELMER). NMR spectra were recorded on FT-NMR spectrophotometer (DRX 300,

Bruker) at 300 MHz, using tetramethylsilane as internal standard.

5.2.4 In vitro testing of the antiplasmodial activity of vasicine, vasicinone,

embelin

The three isolated compounds were evaluated for antimalarial activity in schizont

maturation inhibition assay and Plasmodium falciparum lactate dehydrogenase

inhibition assay (PfLDH).

5.2.4.1 Schizont maturation inhibition assay

Experimental protocol

The test procedure for antimalarial screening described in section 2.2.5.1 was used.

Sample preparation

One mg of each compound was dissolved in 1 μl DMSO and 999 μl RPMI-1640 to

obtain a stock of 1 mg/ml (stock solution). A series of eight concentrations were

prepared from the stock solutions by 2-fold dilutions (0.0625–2 mg: ml) to give

concentrations around the range of 50% inhibition (IC50).

5.2.4.2 Plasmodium falciparum lactate dehydrogenase inhibition assay

Experimental protocol

The test procedure for antimalarial screening described in section 2.2.5.2 was used.

Sample preparation

The compounds were dissolved in DMSO separately to produce a stock solution of 1

mg/ml each. These stock solutions were subsequently diluted with culture medium

containing 10% human serum before being transferred in triplicate of 10 µl each at

concentrations of 1,10,25,50 and 100 μg/ml into two 96-well microtiter plates.

5.2.5 In vivo testing of the antiplasmodial activity of vasicine, vasicinone, embelin


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The three isolated compounds were evaluated for antimalarial activity in Plasmodium

berghei model.

5.2.5.1 Introduction

Plasmodium berghei belongs to a group of four Plasmodium species that infect

murine rodents. These species are P. vinckei, P. chabaudi, P. yoelii and P. berghei.

After the original discovery of P. berghei was made by Vincke and Lips in 1948,

since then a number of isolates (strains) have been collected from the wild. P. berghei

infects laboratory hamsters, rats and mice. A susceptible mosquito vector for P.

berghei, which is widely used in the laboratory, is Anopheles stephensi (Sinden,

1996).

These rodent parasites exhibited similarity to human malaria parasites with respect to

both proteomic and genomic level. For example, a number of (surface) proteins

demonstrate conservation both in structure and function between rodent and human

malaria parasites (such as CS, TRAP, P45/48, CTRP, P25, P28, AMA-1, MSP-1)

(Sinden, 1978; Aikawa and Seed, 1980; Lin et al., 2000). Also studies demonstrated a

high level of conservation of genome organisation between rodent and human

parasites. The genome of both P. falciparum and the four rodent parasites are

organised into 14 linear chromosomes, ranging in size from 0.5-3.8 Mb. They possess

identical telomeric repeats [CCCT (A/G) AA], organised into tandem arrays.

Comparative mapping of genes located in the central regions of the chromosomes has

shown that both linkage and gene order appear to be well conserved between human

and rodent parasites, resulting in significant level of synteny between these species

(Carlton et al., 1998; Lin et al., 2001; Rich and Ayala; 2003). Moreover, conservation

of specific gene domains, regulatory control elements (Janse et al., 1994),

organisation of complex genomic loci and the presence of conserved multi-gene

families in rodent and human parasites (Homewood and Neame, 1980) all emphasize

the high similarity of genome organisation, gene content and gene-regulation.

These facts suggest that murine malaria models are particularly valuable in studies of

the erythrocytic stage of malaria infection, because the morphology and parasite

development stages are similar to those in human malaria infections. An important

advantage with murine malaria models is that all parasite stages can be observed in

the peripheral circulation (Gibbons et al., 2007; Mackenstedt et al., 1989). Also, it is


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easy and safe to handle and manipulate any stage of the life cycle of murine malaria

parasites in the laboratory, and such models often represent the only practical means

towards in vivo experimentation (Janse and Waters, 1995). Thus, species of malaria

parasites that infect rodents provide models for the study of the biology of malaria

parasites that infect humans (Sinden, 1996).

5.2.5.2 Experimental

Parasite density determination and inoculum preparation

A standard inoculum was prepared from a donor mouse with P. berghei parasitized

erythrocytes. Infected blood from the donor mouse was obtained by cardiac puncture

after anesthesia with chloroform Parasitaemia was established by microscopic

examination of a thin blood film under oil immersion at x 100 magnification and

measured as a percentage of infected erythrocytes in fields of 500 erythrocytes. Each

mouse was infected with a standard inoculum of the 106 parasitized erythrocyte

suspension in phosphate buffered saline (0.2 ml) from a donor mouse that was

prepared based on percentage parasitaemia and number of erythrocytes counted per

microlitre of blood using an improved Neubear Haematocytometer.

Treatment

A 4-day suppressive test according to Peter et al (1975) was used. The animals were

divided into 5 groups of 6 mice each. Animals in group 1 were given the vehicle only

(i.e. 0.2 ml PBS) and served as the uninfected control. Animals in group 2 were

administered 20 mg/kg b wt. suppressive dose of chloroquine and those in groups 3,4

and 5 were administered vasicine (40 and 100 mg/kg b wt.), vasicinone (500 mg/kg b

wt.) and embelin (500 mg/kg b wt.) respectively. Treatments commenced

immediately after inoculation (Day 0) and continued on days 1, 2 and 3 via an oral

route using a canula. Tail blood films of the infected animals were prepared, fixed in

methanol, stained with 4% Giemsa stain for 20 min and examined microscopically

under oil immersion on day 4 post inoculations. Parasite density and percentage

reduction in parasitaemia relative to chloroquine control on group by-group basis

were calculated.

5.3 RESULTS AND DISCUSSION


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5.3.1 Isolation of vasicine and vasicinone from methanolic extract of Adhatoda

zeylanica leaf and embelin from methanolic extract of Embelia ribes fruit

Methanolic extract of A. zeylanica leaf (IC50 5.80 µg/ml) and E. ribes fruit (IC50 13.10

µg/ml) showed good antiplasmodial activity in inhibiting PfLDH

5.3.1.1 Fractionation of the methanolic extract

Methanolic extracts of A. zeylanica and E. ribes were fractionated in different polarity

solvents viz, hexane, chloroform, ethyl acetate, n-butanol fractions and n-butanol

insoluble fraction. IC50 values for A. zeylanica fractions were as follows (Figure 5.1):

petroleum ether fraction 243.70 µg/ml, chloroform fraction 6.76 µg/ml, ethyl acetate

fraction 971.16 µg/ml, n-butanol fraction 99.84 µg/ml and n-butanol insoluble

fraction 1283.73 μg/ml. IC50 values for E. ribes fractions were as follows: petroleum

ether fraction 5.26 µg/ml, chloroform fraction 100.33 µg/ml, ethyl acetate fraction

130.00 µg/ml, n-butanol fraction 3883.87 µg/ml and n-butanol insoluble fraction

6431.99 μg/ml (Figure 5.2).

Adhatoda zeylanica

Leaf 480 g

Petroleum ether 4.0 g, 16 %

IC50 243.70 µg/ml

Chloroform 0.8 g, 3.2 %

IC50 6.76 µg/ml

Ethyl acetae 6.25 g, 25 % IC50 971.16

µg/ml

n-Butanol 10.0 g, 40 %

IC50 99.84 µg/ml

Subjected to fractionation 25 g

Extract with methanol

n-Butanol insoluble portion

0.9 g, 3.8 % IC50 1283.73 µg/ml

Crude methanolic extaract 89 g, 18.6 %

IC50 5.80 µg/ml

Figure 5.1: Schematic diagram of fractionation of methanolic extract of the leaf of Adhatoda zeylanica.

Yield of each fraction and % w/w – in parenthesis; IC50 of the fractions screened in PfLDH inhibition assay


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5.3.1.2 Isolation of vasicine and vasicinone

Since chloroform fraction of A. zeylanica showd better activity than the other

fractions, we checked for the type of chemical constituents present therein. From Co-

TLC of the chloroform fraction with vasicine and vasicinone, it was found that

vasicine and vasicinone were the major compounds found in the chloroform fraction

of A. zeylanica leaf methanolic extract. After repeated recrystallization of alkaloid

fraction with ethanol, 150 mg (0.26 % w/w) white needles of vasicine was obtained.

Remaining fraction was subjected to column chrmatography using silicagel and eluted

with chloroform with increasing proportion of methanol. Vasicinone was eluted from

the column with chloroform: methanol (49: 1) as a white solid and was crystallised

from chloroform: ethyl acetate (3:1).

5.3.1.3 Isolation of embelin

Repeated crystallization and purification of petroleum ether extract of Embelia ribes

fruits with methanol resulted in isolation 722 mg embelin (yield 2.88 % w/w).

Chloroform 0.4 g, 5.3 % IC50 100.33

µg/ml

Embelia ribes Fruit 285 g

Petroleum ether

3.4 g, 42.5 % IC50 5.26 µg/ml

Ethyl acetae 1.6 g, 20 % IC50 130.00

µg/ml

n-Butanol 1 g, 12.4 %

IC50 3883.87 µg/ml

Subjected to fractionation 8 gm

Extract with methanol

n-Butanol insoluble portion

0.7 g, 9.5 % IC50 6431.99 µg/ml

Crude methanolic extaract 32 g, 11.2 %

IC50 13.10 µg/ml

Figure 5.2: Schematic diagram of fractionation of methanolic extract of the fruits of Embelia ribes.

Yield of each fraction and % w/w – in parenthesis; IC50 of the fractions screened in PfLDH inhibition assay


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5.3.1.4 Spectral details of vasicine

The mass spectrum (atmospheric pressure ionization) of vasicine (Figure 5.3) showed

molecular ion peak at m/z 189.5 [M - H].

The proton nuclear magnetic resonance (H1 NMR) spectra of vasicine (Figure 5.4) in

CDCl3 exhibited four singlet between δ 6.95-7.25 resonances at 6.95 (1H, m, Ar H-8),

7.02 (1H, m, Ar H-7), 7.15 (1H, m, Ar H-6), 7.25 (1H, m, Ar H-9) which were

attributed to the four aromatic protons. One proton triplet at 4.9 due to (1H, t, H-1).

Two multiplet at δ 2.15 and 2.45 due to methylene (1H, m, H- 3) and (1H, m, H-2)

respectively and two multiplet at δ 3.28 and δ 3.45 due to (1H, m, H- 5) and (1H, m,

H-4) respectively. One at δ 4.63 due to methylene (2H, brs, H-10). One at δ 5.5 due to

hydroxyl (1H). The δ values of the 13C NMR of vasicine were compared with those of

vasicine given in earlier reports (Figure 5.5) (Joshi et al., 1994). The δ values for

vasicine are: δ 29.1 (C-1), 47 (C-2), 48.5 (C-3), 77.8 (C-4), 122 (C-5), 126 (C-6),

127.4 (C-7), 129.4 (C-8), 131 (C-9), 150 (C-10), and 164 (C-11). Note: TLC densitometrc scan and overlay absorption spectra of vasicine and vasicinone of isolated compound with standard compound are incorporated at the end of the chapter (Page no. C5-A and C5-B).

Figure 5.3: Atmospheric pressure ionization mass spectrum (API-MS) of Vasicine.


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Table 5.1 1H NMR chemical shift assignment for vasicine (CDCl3) Position Experimental

values (δ - ppm) Data from the literature (δ - ppm)

H-1 4.9 4.83 H-2 2.45 2.43 H-3 2.15 2.12 H-4 3.28 3.33 H-5 3.45 3.43 H-6 to 9 6.95-7.25 6.84 – 1.14 H-10 4.63 4.53 Table 5.2 13C NMR chemical shift assignment for vasicine (CDCl3) Position Experimental


C-1 29.1 28.8 C-2 47 47 C-3 48.5 48.1 C-4 77.8 70.2 C-5 122 119 C-6 126 125.7 C-7 127.4 124 C-8 129.4 128.3 C-9 131 123.6 C-10 150 142.3 C-11 164 163.9

H8

H6

Figure 5.4: Proton nuclear magnetic resonance (1H NMR) spectrum of vasicine.

H1

H10

H4 H5 H2 H3

H7

H9


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Thus, identity of vasicine was confirmed by recording IR, MS and NMR spectra and

by comparison of the spectral data with those reported (Jain et al., 1980; Joshi et al.,

1994). 5.3.1.5 Spectral details of vasicinone

The IR (KBr) spectrum of vasicinone (Figure 5.6) showed the characteristic

absorption at 1470 cm-1 (due to CN bond), 1660, 1684 cm-1 (due to stretching

vibration of C = O), 2871, 3163 cm-1 (due to stretching vibration of OH group). The

mass spectrum (atmospheric pressure ionization) of vasicinone [Figure 5.7] showed

molecular ion peak at m/z 203.5 [M - H]-.

The proton nuclear magnetic resonance (H1 NMR) spectra of vasicinone (Figure 5.8)

in CDCl3 exhibited four singlet at between δ 7.29-8.35 resonances at δ 7.29 (1H, m,

Ar H-6), δ 7.8 (1H, m, Ar H-7), δ 7.53 (1H, m, Ar H-8), δ 8.35 (1H, m, Ar H-9)

which were attributed to the four aromatic protons. One proton triplet at 5.3 due to

methane (CH) (1H, t, H1). Two multiplet at δ 2.32 and 2.68 due to methylene (1H, m,

H- 3) and (1H, m, H-2) respectively and two multiplet at δ 4.0 and 4.8 due to

C1 C3 C4 C2

C6 C7

C9

C10 C11

CDCl3

C8

C5

Figure. 5.5: Corbon nuclear magnetic resonance (13C NMR) spectrum of vasicine.


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methylene (1H, m, H- 4 and (1H, m, H-5) respectively. One multiplet at δ 6.0 due to

hydroxyl (1H, m, H- 3α).

The δ values of the 13C NMR of vasicinone (Figure 5.9) were compared with those of

vasicine given in earlier reports (Joshi et al., 1994). The δ values for vasicinone are: δ

29.2 (C-1), 43.86 (C-2), 71.76 (C-3), 118 (C-4), 125.94 (C-5), 126.73 (C-6), 127.2 (C-

7), 134.69 (C-8), 147.28 (C-9), 156 (C-10), 160.76 (C-11),

Figure 5.6 Infrared spectrum of vasicinone.

Figure 5.7: Atmospheric pressure ionization mass spectrum (API-MS) of Vasicinone.


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Table 5.3 1H NMR chemical shift assignment for vasicinone (CDCl3) Position Experimental


H-1 5.3 5.25 H-2 2.68 2.62 H-3 2.32 2.31 H-4 4.0 4.02 H-5 4.8 4.38 H-6 to 9 7.29-8.35 7.48 – 8.28 Table 5.4 13C NMR chemical shift assignment for vasicinone (CDCl3) Position Experimental


C-1 29.2 29.3 C-2 43.86 43.4 C-3 71.76 71.6 C-4 118 120.8 C-5 125.94 126.5 C-6 126.73 126.9 C-7 127.2 134.4 C-8 134.69 126.4 C-9 147.28 148.3 C-10 156 160.7 C-11 160.76 160.5

Figure. 5.8: Proton nuclear magnetic resonance (1H NMR) spectrum of vasicine.

H1

H2 H3

H4H5

H6

H7 H8

H9


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Identity of vasicinone was confirmed by recording IR, MS and NMR spectra and by

comparison of the spectral data with those reported (Jain et al., 1980; Joshi et al.,

1994). 5.3.1.5 Spectral details of embelin

The IR (KBr) spectrum of embelin (Figure 5.10) showed the characteristic absorption

at 1193 cm-1 (due to C-O bond), 1339, 1617 cm-1 (due to stretching vibration of C =

O), 2857, 2930 (due to stretching vibration of methyl C-H) 3307 cm-1 (due to

stretching vibration of OH group). 1H NMR spectrum showed signal at δ 5.28 for proton (e and f), signal at δ 7.78 is due

to proton d. There is a triplet at δ 2.4 ppm corresponds proton (c), remaining all

aliphatic hydrogen atoms appeared between δ 0.9-1.9 integrated for protons a and b

(Figure 5.11). The δ values of the 13C NMR of embelin (Figure 5.12) were compared

with those of embelin given in earlier reports. The δ values for embelin are: δ 30 (C-

a), 104 (C-b), 117.8 (C-c), 130.6 (C-d), 154.4 (C-e), 181 (C-f), 181 (C-g).

C6

C10

Figure. 5.9: Expanded carbon nuclear magnetic resonance (13C NMR) spectrum of vasicinone.

C1 C2 C3

C4

C5 C7

C8

C9 C11

CDCl3 N

N

O

OH

C1

C2

C3

C4C5

C6

C7

C8C9 C10

C11

C6

C10


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Figure 5.16: Carbon nuclear magnetic resonance (13C NMR) spectrum of embelin.

Figure 5.10: Infrared spectrum of embelin.

a, b

Figure. 5.11: Proton nuclear magnetic resonance (1H NMR) spectrum of embelin.

c e, f

d

Table 5.5 1H NMR chemical shift assignment for embelin (CDCl3) Position Experimental

values (δ - ppm) Data from the literature

H-a & b 0.9-1.9 1.2 – 2.1 H-c 2.4 2.4 H-d 7.78 7.6 H- e & f 5.28 5.35

a, b


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Thus, identity of embelin was confirmed by recording IR, MS and NMR spectra and

by comparison of the spectral data with those reported (Swamy et al., 2007; Haq et

al., 2005).

5.3.2 In vitro testing of the antiplasmodial activity of vasicine, vasicinone,

embelin

Further, the isolated compounds were evaluated for the antimalarial activity on

schizont maturation and PfLDH inhibition assay. Vasicine showed IC50 value of 8.75

µg/ml in schizont maturation inhibition assay. At 15.6 µg/ml vasicine completely

inhibited schizont maturation while no live parasites were observed above 31.2 µg/ml.

Vasicinone showed IC50 value of 63.20 µg/ml in schizont maturation inhibition assay.

Embelin showed IC50 value of 9.97 µg/ml in schizont maturation inhibition assay. At

31.2 µg/ml schizont maturation was compleately inhibited while no live parasites

were observed above 62.5 µg/ml. In Plasmodium falciparum lactate dehydrogenase

inhibition assay vasicine showed 86.53 % inhibition at 50 µg/ml and there was

complete inhibition at 100 µg/ml. Vasicinone and embelin showed 63.09 and 93.91 %

inhibition respectively at 100 µg/ml. In PfLDH inhibition assay vasicine, vasicinone Note: TLC densitometrc scan and overlay absorption spectra of embelin of isolated compound with standard compound are incorporated at the end of the chapter (Page no. C5-C).

f

Figure 5.12: Carbon nuclear magnetic resonance (13C NMR) spectrum of embelin.

O

O

OH

HO

b

(CH10) CH3

a

c

d

ef

g

a

b c d e g


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and embelin showed an IC50 of 2.05, 57.20 and 3.33 µg/ml respectively. The results

indicate that vasicine and embelin showed dose dependent schizonticidal activity. At

higher concentrations embelin was found to kill all the parasites. In vasicine treated

samples a few dead parasites were seen.

5.3.3 In vivo testing of the antiplasmodial activity of vasicine, vasicinone, embelin

The results of the previous in vitro experiments provided the rationale for determining

whether vasicine, vasicinone and embelin could prevent or suppress in vivo growth of

the rodent malarial parasite P. berghei. This In vivo evaluation of antimalarial

compounds typically begins with the use of rodent malaria parasites. Of these, P.

berghei, P. yoelii, P. chabaudi and P. vinckei have been used extensively in drug

discovery and early development (Childs et al., 1984). These rodent models have been

validated through the identification of several antimalarials-for example, mefloquine,

halofantrine and artemisinin derivatives (Peters et al. 1977; Peters et al., 1987;

Vennerstrom et al., 2000; Posner et al., 2003). In view of their proven use in the

prediction of treatment outcomes for human infections, these models remain a

standard part of the drug discovery and development pathway.

Of these above mentioned models, Plasmodium berghei model is the most extensively

used model. Swiss mice are tolerant of P. berghei infection at a low to moderate level

of parasitaemia (<5%), with organ failure and death occurring only at high

parasitaemia (Cox, 1988; Landau and Gautret, 1998; Peters and Robinson, 1999).

Hence, the features of P. berghei infection model are beneficial for pharmacodynamic

studies because tolerance of the infection allows the mice to reach a parasitaemia of

up to 5% before drug treatment strategies are commenced and the parasitaemia can be

monitored down to a level of approximately 0.005%.

Five groups of mice were infected with 106 parasitized erythrocytes from the blood of

a single mouse parasitized with P. berghei. After treatment with compounds and drug,

the suppression of parasitemia (chemo-suppression) and mean survival rate (time)

were used as measures of efficacy. The effect of the drug was considered ineffective

if: before and after treatment no significant difference in degrees of parasitaemia or a

continuous increase of parasitaemia is noted. In the present experiment animals

treated with compounds showed decrease in parasitaemia. Samples were categorized

as highly active when chemo-suppression was above 60% or moderately active


87

between 30 and 60%, but lowly active below 30%. Mean survival time of the

experimental groups of test animals correlated to treatment.

(Table 5.1) shows a summary of parasitaemia suppression (%) for mice on day 5 p.i.

and their corresponding survival on day 9 p.i., when 100% mouse-mortality of the

untreated control occurred.

Table 5.6 Effect of vasicine, vasicinone and embelin on parasitaemia suppression post infection with Plasmodium berghei

DRUG (mg/kg b.wt.) 5th DAY 9th DAY Survival

rate

Vasicine (100) 20.44 ± 0.00 19.27 ± 0.00 12 Vasicine (40) - 10.38 ± 0.00 13 Vasicinone (500) 12.1 ± 1.88 45.4 ± 1.88 10 Embelin (500) 9.2 ± 1.30 49 ± 1.30 20 Chloroquine (20) 29.99 ± 0.00 64.83 ± 0.00 -

The survival period of mice that received compounds shown chemo-suppression lived

longer compared to those with no treatment. As compared to 6 days average survival

period of untreated group, average survival period for embelin is 10 days and for

vasicinone it is 20 days. Even though, parasitaemia suppression (%) is more with

embelin is more as compared to vasicinone at the same dose, its survival period is

less.

5.4 CONCLUSION

The present investigation demonstrates that vasicine, vasicinone and embelin are

potential candidates for the discovery of effective anti-malarial drugs or lead

compounds. To our knowledge, this is the first report of the anti-plasmodial activity

of vasicine, vasicinone and embelin. Structurally new scaffolds acting as

antiplasmodial agents viz., vasicine, pyrrolquinazoline alkaloid and embelin, a

benzoquinone were isolated. Vasicine and embelin exhibited consistent

antiplasmodial activity in scizont maturation inhibiton assay and lactate

dehydrogenase inhibition assay, which are based on morphological criteria and

biochemical reaction respectively. In view to determine the mechanism of action by

which these causes antiplasmodial activity are given in the further section.


88

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Isolation of Vasicine, Vasicinone from Adhatoda zeylanica...

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