Field of the invention
The present invention relates to the method of separation and isolation of indirubin from
Strobilanthes flaccidifolius with high yield.
Background of the invention
Indirubin is a compound of the indigoid family. It has been isolated from many indigo plants at
different places. It has been proved to be effective in cancer, skin lesions and Alzheimer's
disease. It inhibits the enzyme CDK (Cyclin dependent kinase) which in turn inhibits tumoral
growth in case of human lung, mammary carcinoma cell and chronic myelocytic leukemias. It
also collectively inhibits CDK and GSK (Glycogen Synthase Kinase) and inhibits the abnormal
hyperphosphorylation of the microtubule-binding protein to be observed in psoriatic lesions of
the skin.
US6849096 teaches a new hair dye using natural indigo plant which does not tains skins when
split thereon and is for example capable of preventing white hair from turning yellowish and of
dyeing hair with natural tone while hiding white hair and which provides long-lasting, durable
colours. According to '096 fresh leaves were heat treated to deactivate enzyme and dried.
Moreover, US'096 also consist of use of metals which is not green method. Further the heat
treated leaves results in low yield.
Thus there is a need to provide a method for separation and isolation of indirubin from S.
flaccidifolius with high yield.
Objects of the invention
It is an object of the present invention to overcome the drawbacks of the prior arts.
It is another object of the present invention to provide a method of separation and isolation of
indirubin having high yield from S. flaccidifolius.

It is another object of the present invention to provide a method of separation and isolation of
indirubin having less number of method steps.
It is a further object of the present invention to provide a method of separation and isolation of
indirubin using less number of substrates.
Summary of the invention
It is an aspect of the present invention to provide a method for the separation and isolation of
indirubin, resulting in high yield from Strobilanthes flaccidifolius comprising the steps of:
(i) taking fresh leaves of Strobilanthes flaccidifolius in a closed stoppered flask and
adding chloroform such that the reaction of indoxyl with β-glucosidase occurs in
the presence of chloroform;
(ii) collecting the product intermittently after 3 days for 3 times;
(iii) concentrating the obtained liquid product of step (ii) with the help of a freeze
drier;
(iv) loading the said product in an open column chromatograph;
(v) eluting indirubin first with petroleum ether and then with 50% chloroform in
methanol.
Brief description of the accompanying figures
Figure 1 illustrates a mass spectra showing that the molecular ion peak was found at 263 as MH
ion so the molecular weight was 262 peak at 219 corresponds to the fragment left after the
cleavage of (-CO-NH2).
Figure 2 illustrates UV-Visible spectra of indirubin with peak around 207 nm and 536nm.

Figure 3 illustrates infra red spectra wherein N-H stretching, carbonyl stretchings and C-H
stretching were observed.
Figure 4 illustrates nuclear magnetic resonance spectra wherein C-H and N-H protons were seen
Figure 5 illustrates chromatogram of the chloroform extract of kum leaves, which indicates
presence of brown indigo and indirubin at wavelength of UV detector at 598 nm.
Figure 6 illustrates Powder XRD pattern of indirubin
Detailed description of the invention
The present invention relates to a new method of separation and isolation of indirubin resulting
in high yield from S. flaccidifolius. The method relates to reaction with chloroform on fresh
leaves of S. flaccidifolius wherein the fresh leaves are added with chloroform and the reddish
chloroform and a reddish chloroform extract is prepared. The liquid extract is then concentrated
with the help of freeze drier.
According to the present invention, indoxyl obtained by the cleavage of indicant and other indigo
precursors reacts with β-glucosidase enzyme present in the fresh leaves in the presence of
chloroform forms indirubin. The combination of reaction with chloroform which is favourable
for indirubin and from fresh leaves where enzymes are not deactivated results in higher yield.
Separation and extraction method of indirubin from Strobilanthes Jlaccidifolius
Method of Extraction
The fresh leaves of Strobilanthes flaccidifolius are taken and extracted with the help of
chloroform. The extract is collected intermittently after three days for three times and
concentrated with the help of a freeze drier as it is found to be sensitive to heat when
concentrated on a normal water bath.
Method of Separation

Open column chromatography method is used for separating indirubin. Further it is carried out in
a number of smaller columns than in a larger volume column for faster separation. The
concentrated extract is made into slurry with the help of silica gel 60-120 mesh (Merck) for
column chromatography. It is then packed on a column. The elution is started with eluent of 100
percent petroleum ether, then polarity is increased with chloroform by making it 10% v/v
chloroform, then to cent per cent chloroform, then the polarity is further increased by adding
50% methanol where indirubin was eluted. Then the elution was completed with cent percent
methanol. The different fractions are monitored with Thin Layer Chromatography with silica gel
G(Merck) and with the help of Ultraviolet lamp to detect pure indirubin.
Identification
Indirubin is confirmed with help of infra red, ultra violet, NMR, Mass and Powder XRD. The
separated indirubin is collected from the fractions collected.
According to the present invention, the most effective way to get good yield of indigo paste was
done by maceration of small cut pieces of fresh leaves and young shoots of plant material with
water. Maceration of fresh S. flaccidifolius for 72 hours gave highest amount of indigo paste with
bluer color (Table 1)
Table 1. The characteristic of crude indigo paste in different freshness of plant materials and
different periods of maceration in water


The yields of the indigo dye from fresh plant material were highest. The semi-dried and dried
plant material gave significantly low yields (Table 2). Moreover, the quantity of indigo
precursors is also dependent on the cultivars and period of harvest. The high yield of fresh leaves
is due to the increased activity of the enzyme β-glucosidase. However, the lower yield could
result from a number of other factors such as that the dye molecules aggregated and did not come
out in the solution or the dye pigments were trapped within the dried plant tissues. Further the
addition of bacteria to the fermentation process did not affect the indigo yield and addition of
some acids decreased the indigo. It could be concluded that the natural enzyme and bacteria of
the plant material were effective enough for fermentation but the addition of acid might destroy
the enzyme and bacteria instead.
Table 2. Indigo dye yield from 72 hrs maceration of 100 g S. flaccidifolius Nees. and I. tinctoria
Linn.

Both blue and red pigments were highly soluble in methanol. The red pigment could be separated
from the blue pigment by dissolving the crude indigo powder in chloroform and evaporating to
dryness. The residual powder containing most blue pigment was finally dried. The separation of
blue and red pigments from the powder of leaves with water, the enzyme might have been
deactivated in the drying process as there was no indigo obtained after solvent extraction with
chloroform.
Examples
The present invention is further described by way of non-limiting illustrating examples.

Process for the separation of indirubin from S. flaccidifolius
25 gm of fresh leaves of S. flaccidifolius was weighed and taken in a closed stoppered flask.
Approximately 150 ml of chloroform was added and extracted. The reddish chloroform extract
was collected intermittently after three days for three times and the extract was collected in a
beaker. The liquid extract was concentrated with the help of a freeze drier as indirubin was found
sensitive to heat when concentrated on a normal water bath into a paste.
Open column chromatography method was used for separating indirubin. The separation was
performed with optimized conditions lowering the band broadening factors in the Van Deemters
equation. Further it was carried out in more number of smaller columns than in a larger volume
column for faster separation. The concentrated extract was made into a slurry with the help of
silica gel for column chromatography 60-120 mesh for open column chromatography. The slurry
was loaded on a open glass column. The elution of the loaded column was started with petroleum
ether followed by the following in the pattern

The different fractions were monitored with Thin Layer Chromatography on TLC plates and
with the help of Ultraviolet lamp to detect pure indirubin. Indirubin structure was confirmed with
help of IR, UV, NMR, Mass Spectra and Powder XRD pattern. The separated indirubin was
collected from the fractions collected. The fractions were then concentrated with the help of a
freeze drier.
Calculation

The separated indirubin was collected from the fractions collected. For approximate quantitative
yield 25 g of fresh leaves were taken in a closed flask and extracted with chloroform. The yield
was found to be around 0.095 g from 25 g of fresh leaves. The percentage yield was found to be
0.095/25 x 100 = 0.38%. The moisture content was found to be 84.74%.

WE CLAIM:
1. A method for the separation and isolation of indirubin, resulting in high yield from
Strobilanthes flaccidifolius comprising the steps of:
(i) taking fresh leaves of Strobilanthes flaccidifolius in a closed stoppered flask and
adding chloroform such that the reaction of indoxyl with glucosidase occurs in
the presence of chloroform;
(ii) collecting the product intermittently after 3 days for 3 times;
(iii) concentrating the obtained liquid product of step (ii) with the help of a freeze
drier;
(iv) loading the said product in an open column chromatograph;
(v) eluting indirubin first with petroleum ether and then with 50% chloroform in
methanol.
2. The method as claimed in claim 1, the chloroform in step (i) is used in an amount of 150
mL for three reactions.
3. The method as claimed in claim 1 wherein said method, the activity of β-glucosidase is
retained.