FIELD OF THE INVENTION
The present inventions relates to the microbial conversion for the preparation of
Glycosylated form of Colchicine, Thiocolchicine , N- formyl N-deacetyl thiocolchicine
and other related compounds possessing colchicine skeleton.
BACKGROUND OF THE INVENTION
Colchicine is a secondary plant metabolite extracted from the plant of Colchicaceae
family. Gloriosa superba and Iphigenia stelata are two important sources of colchicine.
Colchicine as such is a toxic compound and finds its application in number of disease
prevention particularly for gut. The colchicine and its related compound particularly 3-O
glycosyl thiocolchicine is a very interesting molecule in pharmaceuticals mainly for the
therapy of diseases of muscle-skeletal system. The 3-O-glycosyl thiocolchicine is also an
valuable starting molecule for preparation of novel antidepressant, anti psoriasis,
immunosuppressive and anti inflammatory medicines.
The method of synthetic conversion of glycosylated form of colchicine commonly
known as colchicoside to 3-O glycosylated thiocolchicine is well established art
throughout the globe including India. India Glycols is one of the manufacturer of
thiocolchicoside prepared from Gloriosa superba active constituent Colchicoside. At
present the demand for thiocolchicoside is increasing continuously though the raw
material availability is limited. Besides, the increasing demand for thiocolchicoside, the
colchicine which is one constituent of Gloriosa superba and has limited applications
thereby limiting its global demand. To cope with the above scenario alternative use of
colchicine by its conversion to more potent molecule is to be worked out. Therefore,
2
with the aim of sustainable process to
reduce the extra burden on the scarce resource of Gloriosa superba seed and to utilize the
colchicine extracted from Gloriosa superba in a efficient way alternative application of
colchicine is needed. In this context an efficient method for the conversion of colchicine
to its glycosylated form which find its application for various diseases will reduce the
extra burden on this precious resource. To achieve this target many efforts have been
made by different inventors by the application of bacteria, fungi or yeast.
3
The work already attempted by different researchers/organization is mentioned
below.
US patent 6372458, Bombardelli and Ponzone :Discloses biotransformation of
colchicinoid compounds with Bacillus megaterium. Their study report conversion of
colchicinoid compound into its 3-O-glycosyl form using Bacillus megaterium.
WO/15642 : Discloses a process of production of colchicinoid glycosyl derivative in
high conversion yield ( up to 90%). Starting from colchicinoid compound such as
colchicine, thiocolchicine and derivative thereof, with an initial concentration of 1g/l.
The process is based on a preliminary regioselective demethylation at C-3 position.
EP 1745140B1: Ponzone : Discloses biotransformation of colchicine or its derivative
effected by selected microbial strain for preparation of 3-O –glycosyl derivative of
colchicinoid compounds.
WO/2012/038982 A2; Invention discloses method for the biotransformation of
thiocolchicine a colchicinoid comprising a selective microorganism Providentia
vermicola. Use of bacterial strain ,Providentia vermicola for microbial biotransformation
of thiocolchicine and other related compounds to their corresponding glycosylated
derivative is investigated.
US/2009/011479 A1:Ponzone :Discloses biotransformation process , effected by means
of selected microbial strain ,und into its glycosylated form
WO2015097567 A1 : Prabhakaran Krishnamutri et al, :Discloses bioprocess for the
preparation of glycosylated colchicine its analog and derivative the invention more
particularly relates to the transformation of colchicine with the microbial strain of
Bacillus aryabhattai.
4
US patent 5777136: Discloses chemical synthesis of colchicoside and thiocolchicoside
by glycosylation. The process involves the use of hazardous reagents such as sodium
fluoride, boron trifluoride and pyridine for the glycosylation reaction.
3292/KOLNP/2006 (2006), Ponzone: Disclosed biotransformation process, effected
by means of selected microbial strains, for the preparation of 3-0-glycosyl
derivatives of colchicinoid compounds.
CN43/200480042995: Discloses biotransformation of colchicine, thiocolchicine or
derivative with Bacillus megaterium. The product achieved have high purity as well the
productivity is high.
The Journal of Phytochemistry 1993, 33(4) 817-820 discloses the glycosylation of
thiocolchicine by a cell suspension culture of Centella asiatica.
Ponzone et.al. (2014) disclosed an efficient, direct biotransformation of thiocolchicine
into thiocolchicoside, performed by a specific strain of Bacillus megaterium. The same
process, with minor modifications, can be used to convert the by-product 3-O-demethylthiocolchicine
into thiocolchicoside. In addition, they describe the B. megaterium strain
selection process and the best conditions for this effective double biotransformation. The
final product is claimed to be of pharmaceutical quality.
Kashyap Kumar Dubey (2014) disclosed a recombinant strain of Escherrichia coli with
CYP102A1 gene for the demethylation of colchicine.
Bouhouche (1998) disclosed that exogenously supplied 3-demethylthiocolchicine
converted into 3-O-glucosylthiocolchicine (thiocolchicoside) by a cell suspension culture
5
of Centella asiatica. Around 30% of 3-demethylthiocolchicine (136 μM) was
glucosylated after an 11-day incubation period. In vitro glucosylation by cell-free
extracts
V. Lukic (1992) reported metabolic transformation of N-deacetyl-N-Formylcolchicine
is the oxidative cytochrome P450 dependent O-demethylation of substituents in the
aromatic ring A and tropolone ring C. It was found that O-demethylation (both, in vitro
and in vivo) takes place predominantly in the aromatic ring, especially at position 2.
OTHER REFERENCES:
Ponzone C., Berlanda D., Donzelli F, Biotransformation of colchicinoid compounds
to their 3-O-glucosyl derivative by selective strains of Bacillus megaterium,
Molecular Biotechnology,(2014) 56 (7) 653-659.
Bouhouche N., JM Solet, A. Simon-Ramiasa, J. Bonaly ,J. Bonaly ,Conversion of 3-
dimethylthiocolchicine into thiocolchicoside by Centella asiatica suspension
cultures. Phytochemistry; (1998); 47(5):743-747.
Kashyap Kumar Dubey, Shafiul Haque, Bhanu P. Singh, B.K. Behera Construction
of recombinant Escherichia coli for enhanced bioconversion of
colchicine into 3-demethylated colchicine, Process Biochemistry (2010) 45, 1036-1042.
Helmi Yousif Alfarra and Muhammad Nor Omar Alfarra Greener Journal of
Biological Sciences ISSN: 2276-7762 Vol. 3 (10), pp. 357-364,
V. Lukic, A. Husek,, O. GaŠić,, D. Walter ova The in vitro and in vivo
biotransformation of N-deacetyl-N-formylcolchicine, European (1992) 17 (4) 243-246.
6
Patrick J Davis, Antimicrobial Agents and Chemotherapy, (1981) 19 (3), 465-469.
Jawed, KK Dube, M Wahid, M Y Areeshi, S. Haque, Efficient solvent system for
maximizing 3-demethylated colchicine recovery using response surface methodology,
Process Biochemistry, (2015)
The above mentioned methods for conversion of thiocolchicine, colchicine and other
related compounds having colchicine skeleton require of high quantity of raw materials.
The purification of the glycosylated product mentioned under above is also a point of
concern. In addition, the process time reported for bioconversion are very large. To
overcome all these issues in an efficient way, in the present invention, the raw material
quantities like carbon and nitrogen source were optimized and reduced substantially
without impacting the conversion. The total process time which is reported 30-40 hours
was reduced to 26-36 hours with high level of conversion. In the process of present
invention the microbiologically converted thiocolchicoside and colchicoside is isolated
and purified employing environmentally safe green chemistry techniques. Ion exchange
resin which have long life and could be used process after process was used for isolation
and purification. The Resin have high affinity for glycosylated thiocolchicine and are
absorbed in the same. This overcome the vast consumption of alumina and silica
which are traditionally used for the isolation and purification. The liquid-liquid extraction
for isolation of glycosylated compounds used traditionally have the disadvantage of
application chlorinated solvents. The Liquid-liquid exchange also need large quantities
of solvent for complete isolation of active compound. However, the process of present
invention uses only green solvent water and ethanol for entire isolation and
purification process thereby completely replacing the use of chlorinated solvents which
are currently used for isolation of non-polar compounds of the reaction mass. The other
advantage of present process of invention is, it recovers the entire active molecule
7
absorbed in the adsorbent -01 in comparison to the traditional methods which result in
the loss of substantial quantity of active due to the retention in the adsorbent. Therefore,
the present invention lead to the increased efficiency of isolation thus higher quantity of
recovery of the glycosylated compounds.
To attain all these targets, the present inventors has done screening of number of
microorganism and found that Bacillus subtilis has the property to convert colchicine and
thiocolchicine to their glycosylated form in specific and highly efficient manner.
SUMMARY OF THE INVENTION:
The present invention relates to the microbial conversion for the preparation of
Glycosylated form of Colchicine, Thiocolchicine , N- formyl N-deacetyl thiocolchicine
and other related compounds possessing colchicine skeleton.
The colchicine and or thiocolchicine intended to be converted to their 3-O-Glycosylated
form are dissolved in water containing minimum amount of polyethylene glycol or
polypropylene glycol and aliphatic alcohol C1-C4, preferably C1-C2. The same is
sterilized and all nutrient are added after properly sterilizing the same. The strain of
Bacillus incubated and the process continued. The progress of microbial conversion was
regularly monitored by applying TLC of sample undergoing conversion. Finally, it is
monitored quantitatively by HPLC.
The fermentation culture broth is stopped once the TLC shows more than 92%
conversion.
8
The fermented broth is diluted by adding of reverse osmosis water and collected in a
sterilized tank. This harvested culture broth is subjected for filteration by incorporating
Cartridge filtration, micro-filteration and ultrafiltration. The volume reduction of filtered
mass is carried out by evaporation and or by nano- filteration to concentrate the mass.
The filtrate which is almost clear no sediments visible to the eye is loaded to a adsorbent
column having cation exchange resin. The active molecule in glycosylated form of
thiocolchicine is separated by gradient elution of the mass. The eluted mass is further
concentrated and stripping of water done by stripping with ethanol and the dried mass
crystallized in the ethanol. The filteration and finally drying of filtered crystals resulted
in the active thicolchicoside.
DETAILED DESCRIPTION OF THE INVENTION:
The conversion of thiocolchicine and colchicine to their glycosylated compounds by
chemical route was attempted initially to prepare the glycosylated compound. The
reaction resulted in demethylation at 2- and 3- position in addition to the number of
other compounds formed. This non- selectivity of 3-O-demethylation by chemical
reaction opened the route and necessity for the conversion of thiocolchicine or
colchicine by micro-organism. In this field already many researchers have successfully
done the conversion of colchicine and thiocolchicine employing various microbial
strains. In our quest to screen a new micro-organism after conducting innumerable
trials we found that bacteria of genus Bacillus, – Bacillus subtilis efficacy for
conversion of thiocolchicine, colchicine to their 3-O- demethyl form and further to
glycosylated form. We found that Bacillus subtilis led in selective glycosylation at C-3
position of the aromatic ring of the colchicine and thiocolchicine.
9
Fig 1: Thiocolchicine
Fig 2: Colchicine
The glycosylation at C-3 position of aromatic ring with the Bacillus sp. of present
invention provide an economically viable technology which incorporate environment
friendly and sustainable green process for isolation and purification of desired
glycosylated compound thicolchicoside, colchicoside and other variants respectively. The
main emphasis of the invention is environment friendly processing without using
chlorinated solvents.
The present invention further provide a process of isolation of 3-O-glycosyl derivative
mentioned above from the fermentation broth once the thiocolchicine is converted to
thicolchicoside implying the microbial species of Bacillus subtilis.
10
Fig 3:Thiocolchicoside
In another embodiement, the thicolchicoside prepared from thiocolchicine by the process
of present invention finds its use as muscle relaxant. The bacteria Bacillus subtilis
converts thiocolchicine, colchicine and their related compounds bearing colchicine
skeleton to their 3-O-glycosylated compound.
The gene sequencing of Bacillus subtilis MTCC is
11
12
The compound with colchicine skeleton described in the present investigation is
colchicine, thiocolchicine, 3-O- dimethyl colchicine and 3-O- demethyl thiocolchicine.
As observed in the present invention the strains of Bacillus subtilis could grow in very
high concentration of thiocolchicine. Further it is observed that species Bacillus of
present invention have very high and specific conversion efficacy for converting
thiocolchicine and related compounds to their glycosylated form. The conversion of
thiocolchicine to thicolchicoside or colchicine to colchicoside, more specifically
conversion of thiocolchicine was more than 92% as observed in the present invention.
Another advantage of the present invention is the use of gram positive strain (Bacillus
subtilis.) which could be handled safely.
13
The process of invention for conversion of colchicine and thiocolchicine to their
glycosylated form is carried out by suspending/ dissolving the above compound in water
containing a small quantity of C2-C4 aliphatic alcohol containing polyethylene glycol
and/ or polypropylene glycol.
In another embodiement, we found that the conversion process takes place in one cycle
thereby converting 3 -O- demethyl intermediate to 3-O-glycosyl compound of
thiocolchicine and related compounds having colchicine skeleton.
The present invention discloses that the Bacillus subtilis can be selected on different
agar media containing a suitable carbon source. In the present invention glucose,
fructose, dextrose and starch were attempted as carbon source. The dextrose among the
different carbon sources gave better results. Among the nitrogen source yeast extract,
peptone, meat extract were attempted. The bio-conversion process was studied at a
temperature 18-45 degree C and at a pH 3.7-8.5, preferably at a pH 5.2-7.0.
The culture media used for storage and conservation of culture are peptone, meat
extract and yeast extract. Carbon source mentioned above is used for conservation and
storage.
Screening and Isolation of Bacteria:
Soil samples from different localities around Uttarakhand State ,India, particularly from
Dehradun, Haridwar and from Ponta Sahib(Himachal Pradesh) were collected from
river soil and agricultural field in the following manner:
14
1. Wet soil samples about 10 - 20 gm. i.e. 2 - 6 inches from the surface were collected in
sealable plastic bags from different location and subjected for screening.
The soil samples thus collected were brought to the lab and stored in refrigerated
conditions for further screening and bacterial isolation work.
The soil samples were screened for their physiological activity to convert
Colchicine / Thiocolchicine to their respective glycosylated forms viz. Colchicoside /
Thiocolchicoside. Throughout the screening studies we have used 1 - 2 g of the collected
soil samples; soil sample was transferred to 50 ml test tube/Erlenmeyer flask having 10
ml of sterile saline solution; the soil suspension was mixed carefully to get uniformity of
suspension. The soil suspension was allowed to stand for about 10 min and 1 ml of the
supernatant from the soil suspension was transferred to 50 ml sterile LB broth (10 g
peptone, 5 g yeast extract, 5 g sodium chloride in 1000 ml water+10 g/L Dextrose) in
500ml conical flask with 100mg of the substrate thiocolchicine and the flask was
incubated on a rotary shaker at 25-37°C and 180 rpm for 24-72 hr. A good mixed
bacterial growth was observed after 72 hrs of incubation; the broth was then aseptically
serial diluted up to 10 -8 with saline and plated on to LB agar medium having
thiocolchicine in the concentrations ranging from 1.0 g to 4.0 g per lit. The LB plates
were incubated at 25-37°C for 72 hr. The individual bacterial colonies were isolated
separately on LB agar slants with Thiocolchicine. Likewise, many individual bacterial
colonies were selected from various soil samples and re-inoculated in to LB broth with
thiocolchicine. The broth samples diluted with methanol(1:5 ratio) were centrifuged and
the supernatant was tested for the presence of Thiocolchicoside by TLC (Thin Layer
Chromatography) as standard screening procedure for identifying potentially active
bacteria with trans-glycosylation activity on thiocolchicine. By using the above
mentioned screening procedure, we found one bacterium which showed strong trans-
15
glycosylation activity and converted Colchicine to Colchicoside and Thiocolchicine
to Thiocolchicoside. The isolated bacterium was further purified by Λ Single colony'
isolation technique and found to have the following morphological characters.
Bacterial Morphological characters:
• Round, wavy, Entire, Rough, translucent, creamish colour
The isolated colony was stored in 20 % glycerol and stored at -20°C and the bacterial LB
slants are stored at 4°C - 8°C. The slant culture was used for experimental purpose for
media optimization. The isolated organism was sent to Microbial Type Culture
Collection (MTCC), Indian Institute of Microbial Technology, Chandigarh, India for the
purpose of identification. The organism was identified as Bacillus subtilis. To our
knowledge, this organism Bacillus subtilis has not been reported in the literature
searched to have trans-glycosylation activity through which Thiocolchicine was
converted to Thiocolchicoside and Colchicine to Colchicoside. The organism has been
deposited with MTCC as per Budapest treaty on the International recognition of the
deposit of microorganisms for the purposes of patent procedure and the MTCC accession
number assigned is MTCC 25074.
The isolated organism was maintained on LB agar medium and used for further studies.
In a similar fashion many colonies are selected from various soil samples in addition to
the soil from two of the above areas. The efficacy of isolated micro-organism was
studied for its conversion of thiocolchicine to thiocolchicoside analyzed for the by Thin
layer chromatography. The mobile phase was 5% methanol on chloroform. Employing
the above screening method we observed that above strain Bacillus subtilis possesses
the capability to convert thiocolchicine to thiocolchicoside.
The conversion process of invention can also be performed under similar condition
with the strain by carrying out mutation by physical means (UV).
16
The process of conversion can also be performed under the similar conditions as
conducted by other inventors for bioconversion of colchicinoid compounds with
bacterial species of Bacillus megaterium and Providentia vermicola as disclosed in US
6150140 and WO 2015097567A1 respectively.
The strain used under the present invention is capable of proper growth once grown in
the substrate containing nitrogen and carbon source.
It was found that the carbon source (glucose, glycerin, maltose and Dextrose ) are useful.
As a nitrogen source yeast extract, peptone, beef extract) could be implied. The process
of conversion could be attempted at a pH ranging fro 3.5 to 8.5 preferably, 5.2 to 7.2 and
more preferably at a pH 6.4 to 7.2. The temperature for the conversion is in the range of
12-50 degree Celsius, preferably 22 to 38 degree Celsius.
The micro-oraganism pertaining to present invention is not reported in the literature for
its capability and efficacy for conversion of colchicinoid compounds. However, this
organism was first reported by Nakamura (1989).
Further the present invention describes the microbial conversion of colchicinoid
compounds to their 3-O-glycosylated compound by contacting the colchicinoid
compound with the strain of Bacillus subtilis at a temperature of 12-50 degree Celsius
and at a pH of 3-8.5 for a time duration of 12-30 hours.
EXAMPLES:
Example 1:
Soil samples were collected from different location of Uttarakhand state particularly
from Dehradun and Haridwar district. Soil samples were also collected from Ponta
Sahib Himachal Pradesh. For comparison soil from Tamilnadu state was also arranged.
The
17
collected soil samples were screened for isolation of micro-organism. For isolation 5-10g
soil sample is suspended in water containing 0.1% Polyethylene glycol and from the
supernatant different dilution were prepared. This was plated on nutrient agar and
dextrose containing 0.5-12g thiocolchicine The plated were placed for incubation at 22-
36 degree Celsius,
Preferably at 26-32 degree C for 48-70 hours. The growth of micro-organism was
observed intermittently. Once achieving required growth, the colonies were transferred
to another plate/slant and their efficacy for converting colchicine or thiocolchicine was
noted. Thus, the isolated pure culture of bacteria were inoculated in a media containing
monosaccharide 5-30g/L, Soya peptone 3-7g/l and yeast extract 5-8g/l. thiocolchicine or
colchicine 0.5g is added to this and growth observed. Growth of micro organism is
continued from 48-70 hours and now transferred to fresh media containing 50g glucose,
peptone 12g and yeast extract 10-12g in which was having 0.8 to 1.0g thiocolchicine and
growth continued for 70-140 hours at 18-32 degree C in a rotary shaker at 150-225RPM.
This procedure was followed for screening of all soil samples collected from different
locations.
Example 2:
Production of thiocolchicoside in Shake Flask from 20% glycerol stock
Thiocolchicoside in shake flask by using Bacillus subtilis.
One vial of 1ml inoculum from 20% glycerol stock ( working cell bank) to 50ml media
in 1000ml which is sterilized for 20 minutes at 121.5 degree C.
1. Glucose: Peptone Yeast Extract pH 6.8
18
Table 1:
Seed Media:
Sr. No. Ingredients Concentration,g/L
1. Tryptone 10.0
2. Yeast Extract 5.0
3. NaCl 10.0
4. Glucose 10.0
pH :6.9
Table2:
Conversion Media
Sr. No. Ingredients Concentration, g/L
1 Soya peptone 7.0
2 Yeast Extract 3.5
3. NaCl 5.0
4. MgSO4 0.5
4. Glucose 20.0
5. Thiocolchicine 1.5
pH :6.7-7.2
Inoculated flask was incubated at 28-32 degree C and kept in rotary shaker for 30 hours
at 180-220RPM. The optical density is measured at regular interval and once optical
density of 25 achieved the seed were transferred at 1-4%inoculum to conversion
19
medium. The conversion media was dispensed at 100ml media in 1000ml
Erlenmeyer flask.
For conversion the conversion media is incubated at a temperature 28-32 degree C at 220
RPM for 30 hours. Samples were taken every two hours for TLC observation for
conversion of thiocolchicine. TLC was performed by spotting samples on silica gel plates
and putting in mobile phase containing 80:20 chloroform: methanol. For analyzing the
conversion of thiocolchicine HPLC on normal phase in silica column. The content of the
product was estimated by reverse phase HPLC analysis in isocratic condition. To analyze
the sample 15ml sample is methanol to was centrifuge for 10 minutes and the supernatant
decanted and used for analysis.
Example 3:
Production of thiocolchicoside in 5 L fermenter:
One vial of 1ml inoculum from glycerol stock is transferred to 1000ml Erlenmeyer flask
containing 100ml of seed medium.
Medium was sterilized at 121 degree Celsius for 30 minutes. After inoculation the
Erlenmeyer flask is inoculated at 30 degree Celsius and kept in a rotary shaker at
230RPM for 30 hours. Optical density of 20-25 shows good growth. The seed on
achieving optical density of 20 was transferred at 1-5% inoculum to conversion
media.
The conversion media was prepared in 5L fermenter with 3.5L of media and sterilized at
121 degree C for 30 minutes. The fermenter was run at 28 degree Celsius for 32-36 hours
at 250-700RPM. The optical density was measured regularly and maintained above 20.
Aeration of the fermenter was maintained between 0.7 to 1.5vvm. The back pressure of
fermenter was maintained below 1bar. RPM and aeration pressure were
20
increased step by step to maintain the dissolved oxygen level within the fermenter
to obtain optimal growth.
The samples from the fermenter were taken every two hour and TLC observation was
done for conversion of thiocolchicine. TLC was performed by spotting samples on silica
gel plates and putting in mobile phase containing 80:20 chloroform: methanol. For
analyzing the conversion of thiocolchicine HPLC on normal phase in silica column,
mobile phase 2% acetic acid in N-heptane, chloroform: methanol at different gradient is
run. The content of the product was estimated by reverse phase HPLC analysis in
isocratic condition. (Water: Acetonitrile) 90:10, C-8 column. To analyze the sample
15ml sample is methanol was centrifuged for 10 minutes and the supernatant decanted
and used for analysis.
Example 4:
Production of Thiocolchicoside in 50L fermenter:
500ml of inoculum from 20% glycerol stock (WCB) is transferred aseptically to 50ml of
seed media (Table-1).
Medium was sterilized at 121 degree celsius for 20 minutes. After inoculation the
Erlenmeyer flask is incubated at25-38 degree celsius and kept in a incubator shaker at
180-250 RPM for 30 hours. Optical density of 20-25 shows good growth. The seed on
achieving optical density of 20 was transferred at 1-5% inoculum to conversion media.
The conversion media was prepared in 50L fermenter with 35-40L of media. The
fermenter was run at 28-37 Degree Celsius for 28-36 hours at 250-700RPM The optical
density at 600nm is measured spectrophotometrically at regular intervals to check the
growth. Aeration of the fermenter was maintained between 0.7 to 1.5vvm. The back
21
pressure of fermenter was maintained below 1bar. With the progress of cycle aeration,
RPM and back pressure were increased step by step to maintain the dissolved oxygen
level within the fermenter for the optimal growth.
The samples from the fermenter were taken every two hour and TLC observation was
done for conversion of thiocolchicine. TLC was performed by spotting samples on silica
gel plates and putting in mobile phase containing 80:20 chloroform: methanol. For
analyzing the conversion of thiocolchicine HPLC on normal phase in silica column ,
mobile phase 2% acetic acid in N-heptane, chloroform: methanol at different gradient is
run. The content of the product was estimated by reverse phase HPLC analysis in
isocratic condition (water: acetonitrile) 90:10, C-8 column. To analyze the sample 15ml
sample in methanol was centrifuged for 10 minutes and the supernatant decanted and
used for analysis.
The conversion in 50 L after 34 hours was 92% as analyzed by HPLC in a silica
column.
Isolation of Thiocolchicoside:
After achieving a conversion of 92-95% fermentation process was terminated. The
fermentation broth is diluted with reverse osmosis water. The biomass from the
fermented broth is subjected to micro-filteration, process followed by ultra-filteration to
attain clear permeate liquid. The filtrate is further concentrated by evaporation under
vacuum in distillation unit/20L Buchi rotavapour to reduce the volume to 1/10 o to 1/20
of the original volume. From this reduced volume further separation of solid mass is
done by adding 3-4 volume methanol to settle the solid and oily mass. This was
filtered/centrifuged. The filtered mass is taken in distillation unit for solvent recovery.
The solvent is further removed by stripping with water. The active molecule is now in
22
water. For isolation and purification of thiocolchicoside from aqueous mass
environment friendly process was used. For the same, the aqueous mass containing
thiocolchicoside is absorbed in ion exchange resin having hydrophobic nature. The ion
exchange resin is coded as adsorbent-01 and the aqueous mass is absorbed in resin and
washed of all inorganic impurities with water washing. The resinous impurities and oil
were removed by washing with aqueous caustic soda solution pH 9.5-10.0. This is
further washed to remove the alkali and finally gradient ethanol: water run to remove the
impurities. TLC observation of the eluted mass was done regularly. Finally pure
thiocolchicoside is eluted with ethanol: water (60:40 to 20:80) preferably 35:65. The
eluted solvent is concentrated for solvent recovery and finally crystallized with ethanol
get pure crystals of thiocolchicoside Filteration and drying of crystals carried out with
nutche filter/ centrifuge.
From 50 L fermenter having 60-80g thiocolchicine for fermentation at 1.5 to 2.0g/l, 55-
75 g thiocolchicoside is obtained at a recovery of 92-94% respectively. Further trials to
improve the thiocolchicoside recovery are continuously run.
The thiocolchicoside prepared by microbial conversion was subjected to purity and
content analysis. The purity and assay was determined by HPLC analysis.
Spectral data of thiocolchicoside:
Mass: 564.12
Proton-NMR (DMSO) 1.80 (3H-17,s); 2.40 (3H-18,m) 2.53 (4H-5a\5b,6a\6b,m)’314-
3.63(sugar protons,2’/\3’,4’,3H,m), 3.83(3H, 13-OMe,s) 4.32 (H-7,m), 4..35(2H,6’m),
4., 494 (glu-1H,d), 5.09 (5H,d), 5.37 (OH, d) 6.85 (H-4,s) 7.01 (H-8,s) 7.14 (H-12,d)
7.25 (H-8,s) 862 (NH, d)
C-13NMR : (DMSO<6) 181.66, 169.22, 157.89, 151.59,151.54, 150.84, 141.64, 137.89,
134.63,134.54, 134.5, 128.25, 127.10, 126.92, 111.54, 77.79, 77.63, 73.79, 70.33, 61.43,
23
61.2, 56.52, 51.83, 36.00 ,29.64, 22.87, 14.80
The product shows 99.2% purity and 99.0% thiocolchicoside by HPLC analysis.
24

WE CLAIM:
1. The process of conversion of colchicine skeleton compound to their glycosylated form
by the microorganism of Bacillus genus--- wherein, the compound with colchicine
skeleton are supplemented with a strain of Bacillus sp. at a temperature of 100C -45 0C
and pH between 3.7-8.5.
2. The compound with colchicine skeleton with a strain of Bacillus sp. as claimed in
Claim 1 is kept between 150C -400Cand more preferably between 250C to 38 0C .
3. The Microbial process as claimed in Claim1, is conducted with microbial strain
Bacillus subtilis having MTCC accession No. 25074 deposited at Institute of Microbial
Technology, Chandigarh.
4. The Process as claimed in claim 1, the concentration of colchicine or thiocolchicine
and related compound with colchicine skeleton varies from 0.2 to 4.0g/liter.
5. The concentration of colchicine or thiocolchicine and related compound with
colchicine skeleton as claimed in Claim 4, is preferably1.0 to 2.5g/L in shake flask.
6. The process as claimed in claim 1, wherein microbial conversion lead to
glycosylation (addition of hexose) at C-3 position resulting in the formation of
thicolchicoside.
7. The Process as claimed in claim 1,wherein, trans glycosylation process is carried out
using Bacillus subtilis.
8. The process as claimed in claim 1, the compound with colchicine skeleton is selected
from a group consisting thiocolchicine, colchicine and other similar compounds.
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9. The process as claimed in claim 1, wherein, conversion of compound with colchicine
skeleton, to thicolchicoside applying Bacillus subtilis is conducted in aqueous medium
using protein, malt extract, yeast extract, sugar etc.
10. The process as claimed in claim 1, wherein the glycosylation at position C-3of
aromatic ring, takes place solubilizing/suspending, thiocolchicine and or colchicine and
other compound in water, aliphatic alcohol having 1-4 carbon atoms, with catalytic
amount of polyethylene and polypropylene glycol (0.2-1.0%). Adding the media in
presence of Sugar preferably, glucose, fructose and monosaccharaides to get the active
Molecule with in 15-40 hrs.
11. The process as claimed in claim 10, wherein, the conversion of thiocolchicine and
colchicine to their glycosylated form respectively, is done at a concentration of 0.5-
2.75g/L in Shake flask and 5.0L fermenter and more specifically at 1.0-2.5g/L.
12. The process as claimed in claim 11,wherein, the conversion of compound bearing
colchicine skeleton is carried out in the stock solution 0.5-2.5g/L capacity to pilot
fermenters 50L.
13. The process as claimed in claim 1, wherein the process of microbial conversion of
compound bearing colchicine skeleton is carried out in pilot fermenter of 50L capacity
within a time span of 15-30hours.
14. The process as claimed in claim 1, wherein the conversion of compound with
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colchicine skeleton carried out in aqueous medium, with different medium, formulations
containing one or
more organic nitrogen source (casein hydroxyl ate, meat extract, peptone, soya peptone)
either alone or in combination and one or more carbon source (glucose, glycerol, starch
etc.,) inorganic phosphorous and inorganic salt of various ions.
15. The process as claimed in claim 1, wherein conversion of thiocolchicine suspended in
water is done by selective trans glycosylation by adding monosaccharaides in the media
to get 3-O- glycosylated product within 12-30 hours , preferably 26 hours.
16. The process as claimed in claim 1, wherein, the conversion of thiocolchicine to
thicolchicoside with bacterial strain of Bacillus subtilis in water based medium consisting
of different carbon, nitrogen sources and inorganic salt of various ions (K+ Na + and
Mg++).