The present invention generally relates to Phytochemistry and method of extraction of secondary metabolite from plant part. More specifically it pertains to a novel process for extraction of high purity colchicoside from Gloriosa superba seed and extract thereof.

BACKGROUND OF THE INVENTION
Gloriosa superba and its importance:
Gloriosa superba also known as flame lily, climbing lily etc., is a flowering plant, belonging to Colchicaeceae family. Gloriosa superba plant is of great medicinal and economic importance as all the parts including roots, stem, leaves and seeds of this plant comprises of variety of secondary metabolites (phytochemicals) such as alkaloids, Glucosides, flavonoids, steroids, starch, terpinoids, tannins, saponins etc.
Gloriosa superba possesses antibacterial, anti-fungal, anti-pyretic, analgesic, anti-inflammatory, Neuro-protective, anticancer activity etc. It is widely used for the treatment of various health issues such as Gout, Arthritis, inflammation, ulcers, bleeding piles, skin diseases, cancer, leprosy, rheumatism, snake bite, etc. (https://irjponline.com/admin/php/uploads/3291_pdf.pdf)

Colchicine
Colchicine (C22H25NO6) is an alkaloid derived from plants of lily family, mainly from Colchicum autumnale or Gloriosa superba plant. The chemical formula of colchicine is [N-[(7S)-1,2,3,10-tetramethoxy-9-oxo-6,7-dihydro-5H-benzo[a]heptalen-7-yl]acetamide], it comprises of a 3 ring structure: A ring is trimethoxy-phenyl ring, B ring is a saturated seven membered ring and comprises of acetamide group and C ring is a tropolone ring (Fig 1). (https://www.ingentaconnect.com/content/ben/iddt/2018/00000018/00000002/art00005).
Colchicine is a medication approved by Food and Drug Administration (U.S. FDA) in 2009 for treatment of Familial Mediterranean fever (FML), acute gout flares and for prophylaxis of gout flares. Colchicine and its analogues are also used for treatment of cancer and inflammatory responses. But higher doses of colchicine are toxic and colchicine in-toxicity leads to certain gastrointestinal side effects, bone marrow depression, multiple organ failure, severe pancytopenia and even death of the individual. (https://accpjournals.onlinelibrary.wiley.com/doi/epdf/10.1002/j.1875-9114.1991.tb02629.x)

Colchicoside
Colchicoside having chemical formula [N-[(7S)-1,2,10-trimethoxy-9-oxo-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6,7-dihydro-5H-benzo[a]heptalen-7-yl] acetamide] (Fig 2) is a glycosylated alkaloid, a sugar derivative of colchicine, wherein the methoxy group at carbon-3 position in ring A is replaced with a sugar moiety, and termed as O-glucosyl colchicine. Colchicoside is present in the seeds of Colchicum autumnale and Gloriosa superba. The Alkaloid-specific glycosylation probably occurs in the tissues during maturation.(https://www.tandfonline.com/doi/abs/10.1080/00021369.1988.10868674)
Need for extraction of colchicoside:
- As reported in certain literatures, colchicine possesses severe toxicity and results in multiple organ failure and even death of the individual, which makes the use of colchicine limited. (https://pubmed.ncbi.nlm.nih.gov/20586571/). K. Yoshida et.al. (1987), reported that colchicoside, which is glycosylated derivative of colchicine, is less toxic and harmful to human health as compared to colchicine and possesses some anti-gout activity as that of colchicine. (K.Yoshida et.al;https://www.tandfonline.com/doi/abs/10.1080/00021369.1988.10868674). Hence there is a need for extraction of colchicine derivatives, which are less toxic as compared to colchicine.
- Colchicoside is commonly used as a starting material for production of its semi-synthetic derivative i.e., Thiocolchicoside comprising thio-analog at C-10 position. It has been used clinically for more than 35 years as an analgesic, skeletal muscle relaxant drug used in treatment of orthopedic, traumatic and rheumatologic disorders and helps in treatment of painful muscle contractions. (https://pubchem.ncbi.nlm.nih.gov/compound/Thiocolchicoside). Due to an increasing demand for Thiocolchicoside and limited use of Colchicine, there is a need for extraction of colchicoside (https://patents.google.com/patent/EP3086794B1/en).

But conventional methods used for production of colchicoside involves following problems:
Conventional methods used for colchicoside extraction are time consuming and also result in production of toxic and impure product.
The conventional colchicoside usually comprises of more than 3% impurities but not more than 5% total impurities. The major impurity of colchicoside is N-formyl analog at C-7, N-deacetyl-N-formyl colchicoside, which is tumorigenic in nature (https://patents.google.com/patent/US20090215904A1/en) and toxic to human health due to the presence of highly reactive aldehyde group (carbonyl compound) at carbon 7 (C-7) which readily reacts with enzymes/biomolecules and disrupts their normal functioning. Hence there is an immense requirement to reduce or completely remove the amount of N-formyl impurity from colchicoside.

Existing commercial approaches to overcome the problem of low purity of colchicoside and their limitations
There are three approaches used for purification which are stated as below:
1. Purification by Chromatography - This is achieved by subjecting the conventionally obtained colchicoside to cumbersome and complex chromatographic separation techniques such as Column chromatography, Neutral Alumina-column chromatography or performing column chromatography twice to remove N-formyl impurity from the desired product to produce pure form of colchicoside. But use of chromatography for purification is not desirable as these techniques are complex, cumbersome, time consuming, costly and require skilled and trained personnel’s for handling the chromatographic apparatuses and solvents.
2. Deformylation by acid catalysts – Another approach used for reducing N-formyl impurity from N-deacetyl-N-formyl colchicoside is selective deformylation by using certain acid catalyst such as Hydrochloric acids, sulphuric acid, phosphoric acid, perchloric acid or other organic acids such as acetic acid, citric acid, malic acid etc. but during deformylation step, two major problems are encountered, first is that the overall process becomes tedious and lengthy as it takes 20 to 32 hours for completion of deformylation and secondly the acid catalysts also attack and cleave the acid labile sugar moiety present at carbon 3 (C 3; ring A) leading to formation of 3-demethoxy colchicoside (one of the derivative of colchicoside), that appears as an impurity.
3. Synthetic glycosylation of colchicine - Various approaches for production of synthetic colchicoside by artificial/synthetic glycosylation of colchicine are existing. But the synthetic approaches used for glycosylation of colchicine are quite complex as these techniques require biotransformation reactions with help of certain microorganisms, which involve biotechnology based practices and are capital intensive moreover, highly trained and skilled persons are needed to carefully operate and monitor the biotransformation reactions.

Problem solved by present invention
Colchicoside extraction suffers from a major problem i.e., presence of high amount N-formyl impurity in the final product which is not pharmaceutically acceptable, tumorigenic and imposes negative impacts on human health. To overcome this problem and to obtain purified colchicoside, column chromatography or deformylation technique using acid catalysts but these techniques are not desirable as these are quite complex, cumbersome, and lengthy and results in cleavage of certain acid labile sugar moieties. The above problems have been solved in a very innovative manner in present invention as described below.

The present invention discloses a Non-column chromatographic technique to remove N-formyl impurity from N-deacetyl-N-formyl colchicoside in order to produce high purity colchicoside. It was observed that presence of water causes the hydrolysis of the sugars, hence in present invention, inventors came up with an approach of using acidic conditions (acid catalysts) with traces of water for refining and purification of colchicoside. The process of selective deformylation is being conducted by using selective acid catalyst in methanol along with traces of water. By using the technique of present invention, it was observed that the N-formyl group from N-deacetyl-N-formyl colchicoside was totally removed, leading to formation of pure colchicoside. Moreover the reagents used in present invention selectively attack the formyl group without attacking the N-acetyl. The process disclosed is economical, less time consuming and results in production of highly pure colchicoside.

Advantages offered by present invention
i. Overcomes the problem of Impurity - By using method of present invention, it was observed that N-formyl impurity was completely removed from the desired product, hence present invention helps in obtaining highly pure colchicoside free from N-formyl impurity.
ii. Overcomes the problems of existing techniques - Unlike already existing techniques used for manufacturing and purification of colchicoside, the process used in present invention is simple, less time consuming and more economical and results in production of high purity end product.

PRIOR ART
US Patent No. 2,734,014 discloses “Glucoside Colchicoside and a method of making same”
The above cited patent discloses a method of extraction of colchicoside and preparation of other acyl derivatives of the colchicoside. The colchicoside in above cited patent is extracted from seeds or bulb-tubers of Colchicum autumnale. The starting material such as seeds are first dried and defatted using petrol ether or other hydrocarbons. Further the plant material is extracted using first solvent that might be halogenated aliphatic hydrocarbon including chloroform, tetrachloro ethane, trichlotomethylene etc. in order to remove colchicine but not colchicoside. The remaining material after colchicine extraction is again extracted using 2nd solvent that may be water, lower aliphatic alcohol, and mixture of solvents, preferably mixture of alcohol and chloroform in ratio (1:3), the extract is then stirred with water, so that colchicoside moves to aqueous solution and is recovered in crude state by evaporating water/solvent. Further for purification, the obtained extract is recrystallized multiple times or purification is achieved by using activated charcoal.

In present invention, a method for removal of N-formyl from N-formyl-N-deacetyl colchicoside is disclosed, whereas above cited invention discloses a method of preparation of colchicoside, and further purification is conducted using multiple recrystallization or using activated charcoal. In present invention, the extracted colchicoside is purified using acidic conditions with traces of water, preferably using hydroxylamine hydrochloride in methanol with 1% water.

Indian Patent Application No.: 1450/DEL/2011 discloses “A novel non-column chromatographic process to reduce N-Formyl-N-Deacetyl Thiocolchicoside and obtain high purity pharmacopoeial grade 3-O-(ß-D-Glucopyranosyl)-3-O-Demethyl Thiocolchicine from colchicoside”.

The above cited patent discloses a non-chromatography method for preparation of high purity Thiocolchicoside from natural colchicoside. The invention involves a process of thiomethylation to convert colchicoside to Thiocolchicoside followed by selective deformylation reaction using acid catalyst to reduce the content of N-formyl-N-deacetyl Thiocolchicoside impurity. In above invention unrefined that is only 50% pure colchicoside is used as a starting material. The acid catalyst is selected from perchloric acid, hydrochloric acid, citric acid, acetic acid, formic acid, Sulphuric acid, phosphoric acid or mixture of these acids. The acid catalyst only attacks the formyl group without unwanted point chemical distortions to reduce the high level impurity N-formyl-N-deacetyl thiocolchicoside below 1,5% ; selective crystallization for purification of thiocolchicoside to remove the impurities mainly N-formyl-N-deacetyl thiocolchicoside and its polar form; refining of the (3-O-ß-D-glucopyranosyl)-3-O-demethyl thiocolchicine crystals in ethanol or methanol to reduce level of impurity N-formyl-N-deacetyl thiocolchicoside below 0.35%; and obtaining Pharmacopoeial grade 3-O-(ß-D-glucopyranosyl)-3-O-demethyl thiocolchicine having composition of 99% purity containing colchicoside less than 0.2% and N-Formyl-N-deacetyl thiocolchicoside below 0.35% besides all other unknown impurities less than 0.1.

The present invention deals with purification of colchicoside itself without using complex column chromatography techniques, whereas above cited invention involves refining of Thiocolchicoside which is obtained after chemical processing of colchicoside. The present invention involves addition of acid catalyst along with traces of water by employing hydroxylamine hydrochloride in methanol with 1% water

US Patent Application No.: 8,415,395 B1 discloses “Colchicine compositions and methods”

The above cited patent discloses a method of preparation of ultrapure colchicine comprising not more than 2.0% impurities and colchicine compositions. Ultrapure colchicine disclosed in above cited invention comprises not more than 0.42% N-deacetyl-N-formyl colchicine. In above cited patent, conventional colchicine is extracted using chloroform, the extract is then washed with NaOH, NaCl and acetic acid, filtered the extract, washed twice using ethyl acetate and the resulting distillate was crystallized. Further the extracted colchicine is subjected to purification by using column chromatography or Neutral alumina column chromatography technique followed by distillation and crystallization of the ultrapure colchicine.

The present invention does not involve the use of high cumbersome, costly, and time consuming technique such as column chromatography for reduction/removal of N-formyl impurity, whereas the present invention implies a simple and less time consuming method using acidic conditions along with traces of water for the purification of the colchicoside, which is glycosidic derivative of colchicine.

Indian Patent Application No.: 2966/MUM/2012 discloses “Colchicum seed treatment, extraction, isolation of Colchicoside from Gloriosa superba seed with high yield and purity”

The above cited patent discloses a process of obtaining colchicine and colchicoside from seed of Gloriosa plant without using column chromatography. The process first involves removal of colchicine by immersing seeds in chlorinated solvent such as dichloromethane for 48-50 hours, filtration and distillation of solvent under reduced pressure and further crystallization using methanol. For extraction of colchicosides, the treated seeds are sun dried, grinded, de-hulled as the hull comprises of the colchicosides. The hull is further pulverized, charged into the reactor and extracted using polar solvent i.e., Aqueous methanol comprising water and methanol (90:10) so that colchicoside is leached out, the reactor is repeatedly charged with fresh methanol until whole colchicoside is extracted, the extraction is monitored using TLC, after extraction, the solvent in distilled under reduced pressure, the obtained aqueous mass is washed repeatedly using chloroform and methanol (90:10). The obtained chloroform layer is concentrated under reduced pressure to obtain brown syrupy mass which is further crystallized using methanol. On HPLC, the chromatogram in above cited invention showed that the purity of colchicine and colchicoside is about 97%.

Present invention involves utilization of acid catalyst along with traces of water for reducing the N-Formyl impurity from colchicoside which is not disclosed in the above cited invention hence present invention differs from the above cited invention.

Journals, Books and Publications

Nautiyal, O.P. Isolation of 3-demethylcolchicine from Gloriosa superba sludge and coupling with a-acetobromoglucose to yield colchicoside and Thiocolchicoside; Journal of Natural Products, Vol. 4(2011): 87-93

The cited paper discloses a method for synthetic production of colchicoside using a-acetobromoglucose. In cited paper, The starting material Gloriosa superba sludge was added to silica gel in methanol and concentrated under vacuum to get dry mixture, the mixture is then loaded on column chromatography of silica gel conditioned with chloroform, the obtained fractions are analyzed using TLC, concentrated using rotary evaporator and crystallized to obtain 3-demethyl colchicine of 95% purity which is used for synthesize of colchicoside. 3-demethyl colchicine was further dissolved in water and dioxane mixture. The mixture is then exposed to basic medium of TEA in nitrogen atmosphere. Further acetobromoglucose was added to the reaction mixture and same was agitated for about 24-48 h after completion of reaction, the obtained product is washed, dried and concentrated using rotary evaporator. The obtained brown syrupy liquid is dissolved in methanol and de-protected using 1% NaOH and finally the product is recovered using methanolic chloroform. The recovered product is dried to obtain off white colchicoside which upon crystallization yielded 1.5 g of pure colchicoside of 99% assay.

The present invention deals with purification by removing N-formyl impurity from colchicoside using acidic conditions with traces of water, whereas the above cited paper discloses preparation of colchicoside using acetobromoglucose. Hence present invention differs from the above cited invention.

Keiichiro Yoshida, Takahisa Hayashi & Konosuke Sano; Colchicoside in Colchicum autumnale Bulbs; Agricultural and Biological Chemistry 52 (2), 593 ~ 594, 1988

The above cited paper discloses a method used for extraction of colchicoside from Colchicum autumnale and structure elucidation of the same. Colchicine alkaloids were extracted with ethanol at 80°C for 30 min from Colchicum autumnale bulbs and were directly chromatographed on reverse phase Hiber Lichrosorb RP-18 with CH3CN-CH30H-50 mM phosphate buffer (pH 6.0), 35: 5 :60. The colchicoside fraction was further purified by HPLC using a 5 ~ 35% CH3CN-H20 linear gradient containing 5% CH30H.

In above cited paper, cumbersome and time consuming chromatographic methods are disclosed for purification of colchicoside, whereas in present invention, acid catalyst along with trace of water is being used for removal of N-formyl impurity from extracted colchicoside.

OBJECT OF THE PRESENT INVENTION

The main object of the present invention is to disclose a process for extraction of high purity colchicoside from Gloriosa superba with no N-formyl analog as impurity.
Another object of present invention is to disclose an extraction process of colchicoside from Gloriosa superba with no N-formyl impurity.
Another object of present invention is to disclose a process of extracting high purity colchicoside from Gloriosa superba which is simple and less time consuming.

SUMMARY OF THE INVENTION
The present invention discloses a non-chromatography process for extraction of non-toxic, high purity colchicoside from Gloriosa superba and extract thereof. The already existing methods used for purification and refining of colchicoside are capital intensive, time consuming and cumbersome. The present invention discloses a simple, less time consuming and easy method for extraction and purification of the colchicoside without chromatography. In present invention, inventors came up with an approach of using acid catalysts with traces of water optimally employing hydroxylamine hydrochloride in methanol with 1% of water and it was observed that the N-formyl group from N-deacetyl-N-formyl colchicoside was completely removed, leading to the formation of pure colchicoside. Moreover, the reagent used only selectively cleaves the N-formyl group without attacking the N-acetyl or other moieties present within colchicoside.
The process disclosed in present invention involve extraction of G. superba seeds using methanol, isolation of colchicine using non-polar solvent chloroform, isolation of colchicoside using n-butanol, removal of N-formyl impurity from colchicoside by using acid catalyst optimally hydroxylamine hydrochloride in methanol with traces of water optimally 1% water and further NMR spectroscopic analysis to detect the presence or absence of N-formyl impurity within the extracted pure product. By employing the method of present invention colchicoside with no N-formyl impurity is obtained.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Structure of colchicine
Figure 2: Structure of colchicoside
Figure 3: NMR Spectrum of purified colchicoside
Figure 4: NMR Spectrum impure colchicoside

DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses a non-chromatographic novel process of extraction of high purity colchicoside from Gloriosa superba and extract thereof. The process disclosed is simple, less time consuming and cost effective as compared to other lengthy and cumbersome already existing processes.
In present invention, alkaloid colchicine is first isolated from the de-fatted Gloriosa superba seeds by methanolic extraction, concentrating to a syrupy liquid, dilution with water in the ratio 1:4 and thereafter, extraction with the help of non-polar solvent “Chloroform”. Further colchicoside is extracted using n-butanol from remaining aqueous extract left after colchicine isolation. The inherent advantage of using n-butanol is that highly polar primary metabolites materials are left behind in the aqueous part and n-butanol selectively extract colchicoside with a little impurity, which makes the overall purification easier. After isolation and crystallization of colchicoside, the N-formyl group of its natural analog, N-formyl N-deacetyl colchicoside, is cleaved by selective de-formylation reaction (leading to the formation of N-deacetyl colchicoside) using acid catalyst i.e., hydroxylamine hydrochloride in methanol along with traces of water i.e. about 1% of water which is simple, less time consuming and is not disclosed in any of the prior art studies. The residual N-deacetyl colchicoside, so formed, is easily converted to colchicoside by acetylation.
The inventors carried out reactions with hydrochloric acid, anhydrous zinc chloride (Lewis acid) but on analysis of the product with HPLC, it was found that substantial amount of glycoside moiety is also cleaved. So, hydroxylamine hydrochloride salts, which is known to contain hydroxylamine as free base has been used, which is responsible for the cleavage of N-formyl group.

The method used for the extraction of high purity colchicoside is illustrated by way of example as below:

Example 1
1. Procurement of Raw Material- Gloriosa superba seeds were procured from Sudha Herbal Traders, (2/206A, Vellipalyam, Alangkombu, Mettupalayam, Coimbatore, Tamil Nadu, Pin Code 641302

2. Extraction of G. superba seeds- Gloriosa superba seeds were collected, dried and powdered. Further the powder is extracted using 2 liters of 95% alcoholic solvent “Methanol” for 6 times to obtain an extract with Colchicine 0.80% w/w and Colchicoside 0.75% w/w

3. Defatting- The methanolic extract was further defatted using 2 liters of non-polar solvent i.e., “Hexane” for 5 times. The solvent methanol is further reduced off by the process of distillation in order to obtain a syrupy fluid. The syrupy fluid is further diluted with water (1:4 v/v) prior to extraction of colchicine and colchicosides.
We can use Ethanol or Ethyl Acetate or Chloroform or Toluene etc. instead of Methanol to produce same results. This is well established and reported in literature / patents (Refs.. US patent 2734014; Natural Product Extractions: Principle and Applications; Edited by Mauricio A. Rostagno and Juliana M. Prado; The Royal Society of Chemistry 2013: Chapter 2 and Bioactive Natural Products: Detection, Isolation, and Structural Determination; Edited by Steven M. Colegate Russell J. Molyneux; 2nd edition, CRC, Taylor & Francis Group, Boca Raton, CRC Press, 2008, London New York.

4. Isolation of Colchicine- Colchicine was isolated by using conventional method i.e., it was extracted using chloroform. The chloroform-colchicine extract was monitored repeatedly using thin layer chromatography until the analysis showed no more presence of colchicine in chloroform. After complete isolation of colchicine, it was crystallized using alcohol.

5. Isolation of Colchicoside - The leftover part after colchicine extraction was re-extracted using n-butanol. Rationale being that the highly polar material after colchicine extraction remains in the aqueous part and n-butanol selectively extracts the colchicoside with a little impurity, which makes the further process of purification easy. Colchicoside is extracted multiple times and repeatedly monitored using thin layer chromatography (TLC) until TLC show the absence of colchicoside in n-butanol extract. The extracted colchicoside was further crystallized using alcohol.

6. Removal of N-formyl group - For removal of N-formyl impurity, Hydroxylamine hydrochloride in methanol (25% by weight) with 1% water is used. 2.0 g of extracted colchicoside with 3.86% N-formyl impurity (analyzed by HPLC) was dissolved in 35 ml of methanol as minimum volume and further 500 mg of Hydroxylamine hydrochloride was added to the solution mixture. In a ratio of 4:1 w/w.
The mixture was further refluxed for six hours. The mixture was then cooled down and excess of solvent methanol was reduced off by distillation. Further 100 ml of cold water was added to the residual mass and pH was adjusted to 7.00 by adding 0.1 (N) NaOH solution. Again 100 mL methanol was added to make the volume ratio 1:1 and colchicoside was extracted using chloroform.

7. Distillation and Crystallization - The organic layer after colchicoside extraction was removed by distillation and the desired pure product purified from a bed of silica gel column (mobile phase methanol: chloroform = 1:9) and then crystallized using ethanol. The product was dried under vacuum at 45-50 degree. Choice of ethanol is a well thought of choice as Ethyl acetate or methanol could also have been used but needed in huge quantity to dissolve the material, while methanol resulted substantial quantity of impurities along with, leading to color deposition on the product. Ethanol did not have these problems.

8. NMR Analysis – The extracted and dried product was analyzed using Nuclear Magnetic Resonance Spectroscopy. And it was observed that 1H NMR Spectrum showed the absence of any singlet peak at 8.5 ppm, which is characteristic chemical shift value of N-Formyl proton (-N-CHO), while the corresponding NMR Spectrum of colchicoside before subjecting to this reaction shows a singlet at 8.5 ppm, confirming the present of N-formyl group.
The total time required in the above complete process is 3-4 days.

Present invention offers a significant advantage as the process is quite simple, less time consuming and economical over the existing methods for removing impurity from colchicoside. Moreover, the product is highly pure hence is of great economical and pharmaceutical importance.

The process disclosed in present invention for preparation of colchicoside with high purity is novel, as evident from non-disclosure in the prior art.
The inventors carried out reactions with hydrochloric acid, anhydrous zinc chloride (Lewis acid) but on analysis of the product with HPLC, it was found that substantial amount of glycoside moiety is also cleaved. So, hydroxylamine hydrochloride salts, which is known to contain hydroxylamine as free base, and it is responsible the cleavage of N-formyl group, was chosen.
Therefore the inventive step by way of technological advancement lies in utilization of acidic conditions with traces of water by employing hydroxylamine hydrochloride in methanol with traces of water, which helps in removal of N-formyl impurity without attacking the N-deacetyl group and other important moieties of colchicoside. Hydroxylamine hydrochloride contains free hydroxyl amine, which is responsible this transformation. The reaction condition is not conducive for hydrolysis of the sugar moiety part.
Moreover, the process has significant economic advantages being simple, cost-effective and less time consuming as it does not involve utilization of cumbersome and complex chromatography techniques.
Industrial application is duly clear as purified colchicoside is of great pharmaceutical importance it is less toxic and can be used as an anti-gout agent for treatment of gouts moreover it is used as a starting material for the production of thiocolchicoside, which is used as a muscle relaxant and anti-inflammatory drug in many parts of world.

I CLAIM:

1. A process for extraction of non-toxic high purity colchicoside from Gloriosa superba seed consisting of following steps:
i) Gloriosa superba seeds are dried and powdered;
ii) powdered seeds are extracted 95% alcoholic solvent for 6 times;
iii) methanolic extract is de-fatted using non-polar solvent for 5 times;
iv) alcoholic solvent is distilled off by the process of distillation in order to obtain a syrupy liquid;
v) syrupy fluid is diluted with water (1:4 v/v) prior to extraction of colchicine and colchicosides;
vi) Colchicine is isolated completely by extracting diluted liquid obtained in step v with chloroform-methanol (4:1);
vii) crystallization of colchicine obtained in step vi is carried out using alcohol;
viii) the leftover part after colchicine extraction after step vii is re-extracted using n-butanol multiple times, until thin layer chromatography (TLC) show the absence of colchicoside in n-butanol extract;
ix) crystallization of extracted colchicoside obtained in step viii is carried out using alcohol;
x) extracted colchicoside with its N-formyl analog, as impurity, are dissolved in methanol;
xi) Hydroxylamine hydrochloride is added to the solution mixture in a ratio of 4:1 w/w;
xii) the mixture obtained in step xi is refluxed for six hours, cooled down and excess of solvent methanol was distilled off by rotary evaporator.
xiii) cold water is added to the residual mass and pH is adjusted to 7.00 by adding 0.1 (N) NaOH solution;
xiv) methanol is added to make the volume ratio 1:1 and colchicoside is extracted using chloroform;
xv) organic layer after colchicoside extraction is removed by distillation and the desired pure product purified from a bed of silica gel column (mobile phase methanol: chloroform = 1:9) and then crystallized using ethanol.
xvi) the crystallized product is dried under vacuum at 45-50 degree to obtain colchicoside of purity >90% and free from N-formyl impurity.

2. The process for extraction as claimed in claim 1 WHEREIN the alcoholic solvent used in step 1(ii) and step 1(vii) is selected from group of Methanol, Ethanol, Ethyl Acetate, Chloroform, and Toluene
3. The process for extraction as claimed in claim 1 WHEREIN the non polar solvent used in step 1(iii) is selected from group of Hexane, Heptane, Petroleum Ether (boiling range 60-80 degree) and aromatic hydrocarbons like Toluene, Xylenes.
4. The process for extraction as claimed in claim 1 WHEREIN the alcohol used for crystallization of colchicine used in step vii and step ix is carried out is selected from a group of Methanol, Ethanol, Isopropanol, Ethyl Acetate, Dimethyl Sulfoixide.
5. The extract colchicoside extracted with process claimed in claim 1 WHEREIN the same has purity >90% and totally free from N-formyl impurity.