Claims:We claim:
1. A process for isolating seselin from Aegle marmelos comprising the steps of:
a) Extracting comminuted material of Aegle marmelos with a low polar solvent to
obtain crude extract;
b) reconstituting the crude extract with a low polar solvent;
c) filtering the resulting solution;
d) allowing seselin to crystallize;
e) separating the seselin crystals; and
f) washing and drying the crystals to get pure seselin.
2. The process for isolating seselin from Aegle marmelos as claimed in claim 1, wherein the
comminuted material is chosen from dry or fresh fruits, leaves, bark or roots of Aegle
marmelos, preferably leaves.
3. The process for isolating seselin from Aegle marmelos as claimed in claim 1, wherein the
low polar solvent is chosen from hexane and petroleum ether, preferably hexane.
4. The process for isolating seselin from Aegle marmelos as claimed in claim 1, wherein the
crystallization is at room temperature (27±1°C).
5. The process for isolating seselin from Aegle marmelos as claimed in claim 1, wherein
yield of seselin isolated from the method is 1.5% to 12%. , Description:A PROCESS FOR ISOLATION OF SESELIN WITH ANTIVIRAL ACTIVITY
FROMAegle marmelos CORREA
Field of invention
The present invention discloses an inexpensive process for efficient isolation of seselin from
parts of plant, particularly the process elucidates isolation of seselin from leaves other than bark
and root ofAegle marmelos Correa.
Background
Aegle marmelos Correa belonging to the family Rutaceae, is a moderately sized, slender,
aromatic tree, 6.0-7.5 m in height and 90-120 cm in girth, native to the Indian subcontinent. The
leaves of Aegle marmelos Correa, contains ?-sitosterol, aegelin, lupeol, rutin, marmesinin,
marmeline, ß-sitosterol, flavone, glycoside, isopentenyl halfordiol, marmeline and phenylethyl
cinnamamides, a-Phellandrene, Limonene and p-cymene.
Seselin, an angular pyranocoumarin is found in Rutaceae, Thymelaeaceae, Apiaceae, Fabaceae
and Moraceae families and is commonly found in plants like Plumbago zeylanica, Sigmatanthus
trifoliatus, Zingiber officinale, Skimmia repens, Rauvolfia verticillataetc.
Seselin is reported to have interesting biological activities such as antiviral, peripheral
anti-inflammatory, antinociceptive, DNA damaging agent, antitumor, anti-inflammatory,
antifungal, larvicidal activities. Seselin has been previously identified with inhibitory activity in
both indole acetic oxidase and peroxidase enzyme systems, ovicidal against red spider mite,
Tetranynchus urticae, anti-fungal, anti-tumor and anti-HIV, weak to moderate cytotoxicity,
antinociceptive and vasodilatory, anti-fungal, peripheral anti-inflammatory and inhibits
phyto-hemagglutinin stimulated cell proliferation in human blood mononuclear cells,
anti-feedant and mosquito larvicidal activity and interact with DNA groove binding through
non-interaction mode.
Seselin (IUPAC name 8,8-Dimethyl-2H, 8H-pyrano[3,2-f]chromen-2-one) an angular
pyranocoumarin is represented by the molecular formula C14H12O3, (Figure 1a) and has
molecular weight 228.24 m/z. The molecular ion peaks at (m/z): 29 [M+H]+, 228 (M+), 213 [M CH3]+ (100) 185, 171, 157, 141, 128, 115, 102, 92, 77 and 63 with melting point of 120ºC,
having peaks at ? 265, 289, 328 nm in UV/visible absorption spectrum; a strong absorption at
? 2977, 1721, 1633, 1595, 1481, 1438, 1371, 1340, 1260, 1217, 1154, 1113, 1071, 907, 855,
832, 733 cm-1 in IR spectrum.; 1H NMR (CDCl3, 500 Hz) 1.47 (6H S, 2 × Me), 6.22 (1H, d, J=9.0
Hz, H-3), 7.60 (1H, d, J = 9.0 Hz, H-4), 7.21 (1H, d, J = 8.5 Hz, H-5), 6.72 (1H, d, J= 8.5Hz,
H-6), 6.87 (1H, d, J = 10.0 Hz, H-9) and 5.73 (1H, d, J=10.0 Hz, H-10);13C NMR spectra
(CDCl3, 125 Hz) 28.12 (2 × Me), 109.28 (C-8), 112.59 (C-3), 143.93 (C-4), 112.59 (C-4a),
130.76 (C-5), 113.53 (C-6), 156.31 (C-7), 150.10 (C-8a), 114.99 (C-9), 127.78 (C-10), 77.32
(C-11), 161.03 (C-2).
Previous reports on methods of isolation of seselin from different plants and their parts primarily
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involve chromatography and multiple steps. It has been observed that several other compounds
frequently are found together with seselin, making their isolation difficult. In general, isolation of
seselin depends initially upon successive extraction of dried plant with commonly used solvents
of increasing polarities (petroleum ether, benzene, ether, methanol and ethanol etc.). It has been
observed that non-polar solvents for extraction of the plant materials were employed, resulting in
less recovery. Still, polar solvents (methanol and ethanol) used for the extraction resulted in a
higher amount of total extract having more colour and fatty material. The separation of fatty and
colouring matter is a difficult task. This renders the process of isolation very expensive, tedious
and poor yielding.
Isolation of seselin crystals from the roots of Sigmatanthus trifoliatus has been reported by
extracting with methanol and partitioning using hexane. Nonetheless the yield reported is only
1.9% considering 800g of raw material.
Seselin was isolated from the roots of Plumbago zeylanica (2.2 kg), with 95% EtOH (40 lx3).
The solvent was concentrated in vacuo and the residue was successively partitioned between
H
2O (1 l) and n-hexane (1 lx3), followed by ethylacetate (1 lx3) and n-BuOH (1 lx3). The
ethylacetate extract (21 g) was subjected to silica gel column chromatography with a gradient of
ethylacetate in n-hexane, and 10 fractions were collected. Fraction 1 was applied to a silica gel
column, eluting with n-hexane - ethylacetate (9:1) to yield a solid, which on crystallization from
an n-hexane – ethylacetate mixture gave plumbagin (4, 1.54 g) and fraction 3 was further
purified by Sephadex LH-20 column (acetone) and preparative HPLC to give seselin (9, 25.8
mg), 5-methoxyseselin (10, 3.6 mg), suberosin (11, 26.4 mg), xanthyletin (12, 76.2 mg) and
xanthoxyletin (13, 16.5 mg).
Root bark of Pleiospermium alatum extracted with hot hexane and further elution over silica gel
with gradient hexane and ethylacetate gives 9.24% yield of seselin. Likewise, Decatropis bicolor
extracted with hexane and further elution over silica gel chromatography with gradient hexane
and ethylacetate gave seselin in the 4th and 5th fractions. Similarly, leaf extracts Clausena anisata
hexane or petroleum ether extract when eluted with hexane and ethylacetate over silica gel
column the 12th fraction is seselin.
Thus, the present invention overcomes the existing difficulties by using a simple solvent
extraction, yielding a comparatively higher amount of seselin, in crystalline form, making the
isolation of seselin directly from leaves of Aegle marmelos efficient and cost effective. The
invention thus paves way for direct processing of Aegle marmelos on a large scale for the
isolation of pure seselin.
Brief description of figures
Figure 1a:Structure of seselin.
Figure 1b:Seselin crystals under Microscope (40X)
Figure 2a:GC Chromatograms of seselin.
Figure 2b: TLC profile of seselin.
Figure 3a:UV spectrum of seselin.
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Figure3b: FT-IR spectrum of seselin.
Figure 4a and 4b:NMR spectrum ofseselin1H and 13C.
OBJECTS OF THE PRESENT INVENTION
The main objective of the present invention is to provide a simple, cost-effective and efficient
process for the isolation of seselin from fresh/dry and mature/immature Aegle marmelos leaves
apart from bark and roots.
Another objective of the present invention is to obtain pure seselin in a yield economically
considerable.
Another objective of the present invention is to utilize the seselin isolated from Aegle marmelos
against NPV infection in the larvae of silkwormBombyx mori.
Advantages of the present disclosure:
The present invention reports a commercially feasible process for isolation of seselin directly
fromAegle marmelos leaves.
The extraction from leaves directly is a single step process rendering it more economical yielding
considerably high purity seselin as compared to the use of other solvents like methanol or
ethanol.
Crystallization of seselin directly from the crude extract results in good crystals with high yield
and purity.
Other advantages being:
1. The yield is very high.
2. The source is leaf, which is renewable and available in abundance.
3. Utilization of low polar solvents or a solvent mixture.
4. Crystals can be obtained at room temperature.
5. Provide a cost-effective method for selective extraction of seselin.
6. An economically feasible process for the isolation of pure seselin for therapeutic use.
Summary
The present invention discloses an inexpensive process for efficient isolation of seselin from
leaves other than bark and root of Aegle marmelos Correa for utilization specifically against
NPV infection in the larvae of silkworm Bombyx mori or for any other therapeutic purpose. The
present invention provides a novel and cost-effective process for the isolation of seselin from
Aegle marmelos to overcome the drawbacks of hitherto known process from other sources. The
process involves extracting comminuted plant material of Aegle marmelos with a low polar
solvent hexane or petroleum ether, reconstitution of the crude extract with a low polar solvent or
solvent mixture like hexane or petroleum ether, filtering the resulting solution, allowing the
crystallization of seselin, separating the crystallized seselin, washing the crystals to get pure
seselin.
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DETAILED DESCRIPTION
The present invention provides a novel process for the isolation of compound seselin, which can
be used as potential antiviral enriched fraction or an active molecule from Aegle marmelos
against NPV infection in the larvae of silkworm Bombyx mori from leaves apart from root and
bark ofAegle marmelos, the said process comprising the steps:
1. extracting comminuted plant material of Aegle marmelos with a low polar solvent like
hexane or petroleum ether to obtain crude extract;
2. reconstitution of the crude extract with a low polar solvent or a solvent mixture like
hexane or petroleum ether;
3. filtering the resulting solution;
4. crystallisation of seselin;
5. separation of the seselin crystals;
6. washing and drying the crystals to get pure seselin.
The present invention provides a novel and cost-effective process for the isolation of seselin
from Aegle marmelos to overcome the drawbacks of hitherto known process from other sources.
The invention more particularly provides a process, which gives a cheaper and higher yield of
potent candidate for antiviral enriched fraction or an active molecule from Aegle marmelos
against NPV infection in the larvae of silkwormBombyx mori, seselin from the natural source.
The main embodiment of the present invention provides a novel process for the isolation and
crystallization of seselin from Aegle marmelos, which can be used as potential antiviral
compound against NPV infection in the larvae of silkwormBombyx mori,
One embodiment of the present invention is to obtain crude extract using hexane or petroleum
ether from comminuted dry and mature/immature Aegle marmelos leaves apart from bark and
roots.
Another embodiment of the present invention is direct crystallisation of seselin from the crude
hexane or petroleum ether extract with a solvent or a solvent mixture of hexane or petroleum
ether.
In one embodiment of the invention the solvent used viz., hexane or petroleum ether, is 100%.
In another embodiment the crude extract is dried at room temperature.
Another embodiment of the present invention is direct crystallisation of seselin from the crude
hexane or petroleum ether extract with a solvent of hexane or petroleum ether.
In one embodiment the dried crude extract obtained is resuspended in hexane and concentrated
through centrifugation.
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In another embodiment the dried crude extract obtained is resuspended in hexane and filtered
through a double layered muslin cloth.
Another embodiment is utilization of low polar solvent mixture to obtain seselin with high
purity.
Another embodiment of the present invention is to obtain high purity crystals of seselin at room
temperature. The yield of seselin isolated by the disclosed method is 9%.
Another embodiment of the present invention is to provide a method for obtaining potent
antiviral enriched fraction or an active molecule from Aegle marmelos against NPV infection in
the larvae of silkwormBombyx mori.
EXAMPLE 1: Isolation of seselin through direct method
800gm of dried leaf powder of Aegle marmelos were taken and it was extracted with 500 mL of
100% hexane to obtain crude extract. It was allowed to dry at room temperature. The dried
crude extract was collected and it was centrifuged at 1500 rpm for 10 min and supernatant was
removed. The pellet was washed with 100 ml of 100% hexane. Alternatively, the dried crude
extract was suspended with 100% hexane. It was filtered with double layered of muslin cloth
attached with the filtration chamber. The pellet was air dried and stored. It was reconstituted in
ethyl acetate and observed under the 40X objective for the presence of crystals (Figure 1b) and
confirmed by GC-MS analysis.
EXAMPLE 2: Isolation of seselin using column chromatography
400g of Aegle marmelos was extracted with hexane or petroleum ether. The hexane or petroleum
ether extracts (10g) were fractionated using a silica gel column (230-400 mesh, 50 x 2 cm) and
successively eluted with solvents of hexane or petroleum ether, hexane or petroleum ether – ethyl
acetate gradient elution (90:10 to 0:100, 100 ml collected) to yield 40 fractions. These fractions
subjected to TLC were then pooled to 14 fractions having similar Rf profiles. The pooled fraction
4, stored at room temperature to obtain seselin crystals. Table 1 provides details of various
fractions.
Table 1. Column chromatography fractionation of crude hexane or petroleum ether extract of
leaves ofAegle marmelos using sequential partition with solvents.
Ratio of
solvents (%)
Initial
fractions
Final
fractions
TLC profile with Rf value
100% Hexane or
petroleum ether 1-4 1 0.707, 0.171
90 : 10 7-9 2 0.712, 0.163
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90 : 10 8-11 3 0.40, 0.475, 0.550, 0.625, 0.80
90 : 10 12 4 0.40 (seselin)
90 : 10 13 5 0.102, 0.205, 0.307, 0.345, 0.487
90 : 10 14-18 6 0.075, 0.150, 0.355, 0.403, 0.500, 0.550
80 : 20 19-20 7 0.095, 0.142, 0.333, 0.381, 0.452
80 : 20 21-22 8 0.523, 0.666, 0.733
70 : 30 23-24 9 0.227, 0.295
70 : 30 25 10 0.133, 0.333, 0.400, 0.444, 0.622
60 : 40 26-27 11 0.324, 0.594, 0.891, 0.943
50 : 50 28-30 12 0.184, 0.342, 0.500, 0.658, 0.763, 0.895
40 : 60 31 13 0.230, 0.769, 0.948
30 : 70 32-34 - -
20 : 80 35-37 - -
10 : 90 38-39 - -
100%
Ethyl acetate 40 14 0.722, 0.944
EXAMPLE 3: Isolation of seselin
10gm of dried leaf powder of Agele marmelos was directly extracted with hexane or petroleum
ether and ethylacetate (90:10) ratio. The solution was filtered and allowed to rest at room
temperature. No crystals were observed.
EXAMPLE 4: Calculation of % yield of compound
% of yield calculation by considering the quantity of raw material used:
Raw material = 800 gm
Crude extract obtained = 13.280gm
Compound obtained = 1201 mg
% of yield = Compound obtained / Raw material used X 100
= 1201 / 80,000 X 100
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% of yield = 1.501
% of yield calculation by considering the quantity of crude extract used:
Raw material = 800 gm
Crude extract obtained = 13.280gm
Compound obtained = 1201 mg
% of yield = Compound obtained / crude extract used X 100
= 1201 / 13,280 X 100
% of yield = 9.043
Example 5: TLC of seselin
About 1 mg of hexane or petroleum ether extract of leaves of Aegle marmelos was dissolved in
10 µl of hexane or petroleum ether. The sample was spotted onto the TLC plate and the plates
eluted with solvent system of hexane and ethyl acetate in the ratio 9:1, and the eluted plates were
exposed to iodine vapour and also visualized under UV light at wavelength of 254 nm and 366
nm. Figure 2a shows the TLC chromatogram of seselin.
Example 6: GC-MS of seselin
Gas chromatography and mass spectrum (GC-MS) analysis of seselin from a fraction of hexane
or petroleum ether crude extract of Aegle marmelos using HP 5-MS capillary standard non-polar
column, dimension: 30 Mts, ID: 0.25 mm, film thickness: 0.25µm. Flow rate of mobile phase
(carrier gas: He) was set at 1.0 ml/min. In the gas chromatography, temperature program (oven
temperature) was set at 50°C and raised to 250°C at the rate of 10°C/min. 10 mg of sample was
dissolved in 500 µl of methanol and the injection volume was 1 µl. The sample was run fully at a
range of 50-650 m/z and the results were compared using NIST 14 Mass Spectral Library search.
Figure 2b shows the GC-MS spectrum of seselin.
Example 7: UV spectroscopy analysis of seselin
A diluted sample of 10 mg of the purified compound in 1 ml of methanol was scanned from 200
nm to 600 nm using a spectrophotometer containing double beam in identical compartments,
each for reference and test solution fitted in 1 cm path length quartz cuvettes. Figure 3a shows
the UV spectrum of seselin.
Example 8: Fourier Transmission Infrared (FT-IR) spectroscopic analysisof seselin
The purified compound (5 mg) was ground with IR grade potassium bromide (KBr) (1:10 v/w)
pressed into discs under vacuum using a Pelletizer. The spectrometer was continuously purged
with dry nitrogen to eliminate atmospheric water vapour and carbon dioxide. The IR spectrum
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was recorded in the region between 4000 - 600 cm-1 and the experiment was performed at room
temperature. Figure 3b shows the IR spectrum of seselin.
Example 9: NMR of seselin
Seselin (0.2 g) was dissolved in 400 µl of deuterated chloroform and shaken in a vortex mixer.
The resulting mixture was placed into a 5 mm diameter ultra-precision NMR sample tubes. The
temperature of the sample in the probe (broadband gradient probe head 5 mm; ‘BBO’, with VT
& auto tune and quadrupole inverse probe) with gradient was 30°C. The chemical shifts (d) were
reported in ppm using solvent proton signal as standard. All figures of the 1H-NMR spectra and
of the expanded 1H-NMR spectrum regions were plotted at a fixed value of absolute intensity to
validate comparative analysis. Figure 4a and b shows the1H and13C NMR spectrum respectively.
Example 10: Insect Feeding Bioassay on seselin
For the bioactivity assay – leaf disc no choice method was used. Freshly moulted fifth instar
larvae were starved for 3 h and were fed with 1×105 POBs/10µl (50% lethal dose). After three
hours of viral inoculation through feeding, the larvae were allowed to feed the fresh mulberry
leaf discs (4 cm in diameter) sprayed with 100 µg of Seselin (fraction-4). The larvae fed with
leaves alone and leaves with 0.4% DMSO were served as positive controls. The larvae fed with
POBs alone were served as negative control. 12 larvae with triplicates were maintained for each
treatment. After complete consumption of leaf area by the larvae both in viral inoculation and
Seselin (fraction-4) treated (experiment), they were allowed to feed the mulberry leaves
normally. The larval mortality was recorded. In addition, cocoon weight (g), shell weight (g),
shell ratio (%) and adult emergence (%) were recorded (Table 2).
Table 2:
Control Positive control Negative
control
Seselin
Cocoon weight (g) 1.742 ± 0.06b 1.740 ± 0.01b -- 1.810 ± 0.03a
Shell weight (g) 0.311 ± 0.02b 0.312 ± 0.01b -- 0.344 ± 0.0a
Shell ratio (%) 17.85 ± 0.10b 17.93 ± 0.20b -- 19.04 ± 0.43a
Adult emergence (%) 100 100 -- 100
Numbers followed by the same letter in each column do not differ significantly from each other
by the Tukey’s test (P <0.05). Each value is the mean ± SD of three replications.
Control - Normal feeding only with mulberry leaves.
Positive control (mulberry leaves treated with 0.4% DMSO, medium used to dissolve solvent
extracts).
Negative control (mulberry leaves treated with OBs at its LD50 value i.e., 1 × 105 OBs of NPV)
-- 100% larval mortality resulted in no economic characters.
POBs/OBs – polyhedral occlusion bodies/occlusion bodies