FIELD OF INVENTION:
The present invention relates in general to the improved method for isolation and
purification of berberine from the plant .More particularly the invention relates to the method for obtaining berberine from plant species Berberis aristata, as berberine hydrochloride, berberine sulphate and berberine tannatefrom the roots and stem bark.
DESCRIPTION OF THE RELATED ART:
Berberis aristata, a well known medicinal plant in IHR and its occurrence is reported from middle altitude areas (1800-3000 m) of the state of Uttarakhand and Himachal Pradesh (Samant et al.,1998; Chauhan, 1999). It is a spine scent shrub, 3-6 m in height with obovate to elliptic, toothed leaves, yellow flowers in corymbose racemes and oblong-ovoid, bright red berries. The extract from root-barks, roots and lower stem-wood, (known as Rasanjana or Rasaut or Rasavanti) is used as stomachic, laxative, hepatoprotective, antipyretic and in other ailments (Wang et al., 2004; Shahid et al., 2009; Semwal et al., 2009). It is usefiil in eye diseases particularly in conjunctivitis, indolent ulcers and in hemorrhoids (Rashmi et al., 2008). The plant is mostly collected from wild areas, and its agro-technique and cultivation is poorly known. Therefore, high demand for local usage as well as for pharmaceuticals creates a serious pressure on the natural resource which has already categorized the plant as endangered (Srivastava et al., 2006; Ali et al., 2008). As a remedial measure, exploration of new resource, conservation of the existing resources and establishment of cultivation are of prime importance for what systematic planning and management is essential and where disfribution modeling can play a key role.
It has been recognized that the possible influences of herbal medicines have not yet been thoroughly investigated compared to its potential use in health care. In many Asian countries the use of natural products from different plants and animals sources is likely to be in parallel with the use of conventional medicines (Zhu, 1999). These resources contain minerals, which could be additionally used as medicine therapy.
A number of alkaloids have been extracted from Berberis species that include berberine, baluchistanamine, chenabine, gilgitine, jehlumine, palmatine, punjabine and sindamine. Other alkaloids identified are umbellatine,

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oxyberberine, and berbamine. Three more alkaloids berbenine, berbericine hydrochloride and berbericinine hydro iodide have been isolated from its roots.
Berberine is a plant alkaloid with a long history of medicinal use concerning its significant antimicrobial activity against a variety of organisms including bacteria, viruses, fungi, protozoans, helminthes and Chlamydia (Huang et al., 2002). Berberine produced sedation in mice and conscious cats and potentiated phenobarbitone sleeping time.
Berberine was inhibited by 70 percent the secretary responses of the heat labile enterotoxins of Vibrio cholera and E. coli in experimental animals. It produced long lasting, dose related fall in blood pressure, when administered into an ear vein of anaesthetized rabbits. Berberine hydrochloride and sulphate help in the diagnosis of latent 11 malaria by releasing the parasites into the blood stream. It also inhibits HIV-1 reverse transcriptase (Gudima et al., 1994).
US Publication No. 20110158932 relates to methods and compositions containing a berberine compound or berberine related or derivative compound for treating and/or preventing hyperlipidemia as well as conditions or complications associated with hyperlipidemia in mammals. The berberine compound or berberine related or derivative compound of Formula I is berberine sulfate, berberine hydrochloride, berberine chloride, palmatine chloride, oxyberberine, dihydroberberine, 8-cyanodihydroberberine, tetrahydroberberine N-oxide, tetrahydroberberine, N-methyltetrahydroberberinium iodide, 6-protoberberine, 9-ethoxycarbonyl berberine, 9-N,N-dimethylcarbamoyl berberine and 12-bromo berberine, berberine azide, or berberine betaine. Berberine can be found in Hydrastis canadensis (goldenseal), Coptis chinensis (Coptis or goldenthread), Berberis aquifolium (Oregon grape), Berberis vulgaris (barberry), Berberis aristata (tree turmeric), Chinese Isatis, Mahonia swaseyi, Yerba mansa (Anemopsis californica) and Phellodendron amurense.
US Patent No. 7,862,838 relates to a composition comprising berberine extracted from Coptis Chinese, Coptis deltoidea, Coptis teeta, Hydrastis canadenses, ■ or Berberis vulgaris, berberis aquifolium, berberis aristata, etc with other constituents and regimen for the treatment of herpes simplex (herpes labialis and herpes genitalis) and herpes zoster, primarily by means of eliminating their

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pathogenetic conditions, taking into account both pathogenesis and etiology thereof.
US Patent No. 6,395,772 relates to a method for blocking attachment of leukocytes to endothelial cells/lining of the blood vessels comprising administering an amount of berberine to a mammal or its pharmaceutically acceptable salts effective to block attachment of leukocytes to endothelial cells. Berberine is isolated from a medicinal plant, Berberine aristata and is also commercially available. Berberine and its pharmaceutically acceptable salts can also be produced synthetically Berberine is isolated from a medicinal plant, Berberine aristata and is also commercially available. Berberine and its pharmaceutically acceptable salts can also be produced synthetically.
US Patent No. 6,291,483 relates to a method of treating TNF mediated or induced septic shock comprising suppressing TNF induced effects or inhibiting TNF-mediated effects by administering a therapeutically effective amount of berberine or a pharmaceutically acceptable salt thereof to a patient in need of such treatment. Berberine and its pharmaceutically acceptable salts are commercially available. Berberine may also be extracted and isolated from Berberis aristata, a plant belonging to family Berberidaceae. One method of extracting and isolating berberine from Berberis aristata comprises coarsely grinding the air dried stems/roots of Berberis aristata and subjecting this material to thorough defatting with non-polar solvents like hexane or petroleum ether at 60-80.degree. C. followed by extraction with 95% ethanol or 95% methanol in water. The alcoholic extract is then concentrated in a water bath and the basic constituents are extracted from the residue preferentially with a 7% aqueous solution of cifric acid or acetic acid or dilute HCl/H.sub.2 SO.sub.4.
US Publication No. 20090136469 relates to a formulation for oral administration to exert a beneficial effect on the cardiovascular system, owing to a combination of activities such as eulipidaemic, cholesterol-lowering and triglyceride-lowering, antioxidant and protective of the vasal endothelium comprising berberine in combination with a policosanol and/or red yeast and preferably containing an antioxidant, such as astaxanthin and/or folic acid. Berberine is obtained from the

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bark of Berberis aristata by aqueous-alcoholic extraction and then crystallization processes which leave the chemical structure of the molecule unchanged
Publication No. RU2423992 relates to a method of preparing Pakistan amine and berberine chloride by extraction of Siberian barberry (Berberis sibirica Pall.) roots with organic solvents (ethanol, methanol, acetone, and also their mixtures with water) followed by separation of a precipitate and Pakistan amine sedimentation by a concentrated ammonia solution and Pakistan amine purification by aluminium oxide column chromatography and further berberine chloride sedimentation from the remained filtrate by concentrated hydrochloric acid, with recrystalliszation purification of the settle down salt.
Publication No. JP62012777 relates to method for depositing and separating berberine alkaloid as an ionic salt, by treating a tissue piece or cell of a plant containing the berberine alkaloid with sulfuric acid and adding a salt exchange agent containing chlorine ion, sulfuric acid ion, etc., to the resultant extract solution.
The article entitle "Alkaloids of berberis arista-isolation of aromoine and oxyberberine" talks about five alkalids isolated from extract of the root bark of Berberis aristata which have been characterized as aromoline, oxyberberine, oxyacanthine, berbine and berbine chloride.( Atta-ur-Rahman and Akbar Ali Ansari, HEJ Research Institute of Chemisty, Univerity of Karachi-32, Pakistan, 1983).
As diversity is. present in natural products due to their distinct physiochemical properties, for example solubility, there is a need for an efficient extraction system to release the compounds from biomass. Several approaches can be used to extract the plant material. Although water is used as an extractant in many protocols but different organic extractions are also developed for the various solubilites of plant constituents.
Hence the present invention provides the improved method for isolation and purification of berberine of high yield and purity from the plant.

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OBJECTS OF THE INVENTION:
The principal object of the present invention is to provide an improved method for the isolation and purification of berberine from plant Berberis aristata
Another object of the present invention is to provide an improved method for isolation and purification of berberine from plant species Berberis aristata also comprising salts of berberine hydrochloride, berberine sulphate and berberine tannate, from the roots and stem bark.
Still another object of the present invention is to provide a method for extracting berberine from the roots of Berberis aristata which is of high yield and with high purity.
Another object of the present invention is to provide the purified berberine extract used for therapeutic and high commercial value compound used in the treatment of cancer, regulating glucose and lipid metabolism in cancer cells as well as nano medicine.
Still another object of the present invention is to provide berberine compound with 99.68% pure isolated from the plant Berberis aristata.
SUMMARY OF THE INVENTION
Accordingly the present invention provides an improved method for isolation and purification of berberine from the plant species Berberis aristata berberine as berberine hydrochloride, berberine sulphate and berberine tannate from the roots and stem bark. The method is based on selective pH extraction by water at a temperature in the range of 20-85*'C. Berberine is a compound that has been identified as a source of therapeutic and high commercial value compound, used in the treatment of cancer, regulating glucose and lipid metabolism in cancer cells, and currently the predominant clinical uses of berberine include bacterial diarrhea, intestinal parasite infections, and ocular trachoma infections, as well as nano medicine.

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In a preferred embodiment of the present invention the method for the extraction of berberine is provided. The plant tissue Berberis aristata is chopped into pieces, and extracted with acidic water ( acetic acid and sulfUric acid) having at 70-95°C, with three times volume, at thrice, each for three hours with stirring and filtered. The filtrate is charcoalized at high temperature, again and filtered using celite as filter-aid under vacuum.
In still another embodiment of the invention, the filtrate obtained is treated with ammonia solution and then filtered. The basic filtrate is adjusted to pH-2 with hydrochloric acid, kept to 10°C for 8 hours and filtered.
In an embodiment of the present invention, the precipitate is provided a bed-wash with 50% water and acetone under vacuum and the final precipitate is dried at 80°C for 8 hours followed by grinding and sieving and finally assayed for berberine compound with HPLC and is found to be > 99.68% purity.
In still another embodiment of the present invention the purified berberine compound isolated from the plant has possible therapeutic application in the treatment of cancer, regulating glucose and lipid metabolism in cancer cells, and also as antibacterial and anti diarrheal as well as for nano medicinal use.
BRIEF DESCRIPTION OF THE DRAWINGS:
It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered for limiting of its scope, for the invention may admit to other equally effective embodiments. Figure 1 illustrates the HPLC analysis of the standard and with Berberis aristata with acidic water extract.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
In the present invention, an improved method for isolation and purification of berberine from plant species is provided containing berberine with specific reference oiBerberis aristata, as berberine hydrochloride, berberine sulphate and

-8-berberine tannate, from the roots and stem bark. The method is based on selective pH extraction by water at a temperature in the range of 20-85° C. Berberine is a compound that has been identified as a source of therapeutic and high commercial value compound, used in the treatment of cancer, regulating glucose and lipid metabolism in cancer cells. Currently the predominant clinical uses of berberine include bacterial diarrhea, intestinal parasite infections, and ocular trachoma infections, as well as nano medicine.
The invention is described in detail with reference to the example given below. The example is provided just to illustrate the invention and therefore, should not be construed to limit the scope of the invention. Example 1.
Collection of plant material
The plant Berberis aristatawas collected from the Uttarakhand state district
Pithoragarh at barbey village.
Extraction Method:
The plant tissue was chopped into pieces, and extracted with acidic water (acetic
acid and sullliric acid) at 70-95°C. The process v^/as repeated 3 times for 3 hours
each using 3 times volume with stirring. Filtered filtrate was charcoalized at high
temperature and again filtered using celite as filter-aid under vacuum.
The filtrate was treated with ammonia solution and filtered. The basic filtrate was
adjusted to pH=2 with hydrochloric acid, kept to 10°C for 8 hours and filtered.
The ppt. was provided a bed-wash with 50% water and acetone under vacuum.
Finally ppt. was dried at 80°C for 8 hours, ground and sieved. It was assayed for
berberine with HPLC . It had a purity of > 99.68%
Example 2.
1 .OKg chopped underground tissue of Berberis aristata, previously cleaned with
water, was extracted with 3.0 liter DM water having 3.0 ml glacial acetic acid and
6 ml sulfuric acid, at 95 °C for three hours with stirring. Filtered at high
temperature , method was repeated twice with similar operating parameters.
The filtrate was mixed, 3 gm of activated charcoal was added to it and it was
filtered using celite, at high temperature. The filtrate was basified with ammonia
solution and filtered. Filtrate was mixed with hydrochloric acid LR, grade till
pH=2.

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The acidic phase was kept at 10°C for 8 liours, filtered and ppt. was provided bed-wasii of 30 ml of 50% acetone with water, using vacuum.
The ppt. was dried at 75°C for 10 hours under vacuum, ground, sieved through 60 mesh. 3.5 gm yellow free flowing powder of assay, HPLC, 99.8% was obtained.
Example 3.
I .OKg chopped under grounded tissue of Berberis aristata, previously cleaned
with method water, was extracted with 3.0 litre DM water having 3.0 ml glacial
acetic acid and 6 ml sulfuric acid, at 98 °C for three hours with stiiring. Filtered at
high temperature , the method was repeated twice, with similar operating
parameters.
The filtrate was mixed gm of activated charcoal was added to it and it was filtered
using celite, at high temperature .The filtrate was basified with ammonia solution
and filtered. Filterate was mixed with hydrochloric acid LR, grade, pH=2.
The acidic phase was kept at 5°C for 10 hours, filtered and ppt. was provided
bed-wash of 30 ml of 50% acetone with water, using vacuum.
The ppt. was dried at 80°C for 10 hours under vacuum, grounded and sieved
through 60 mesh. 3.54 gm yellow free flowing powder of assay, HPLC, 99.83%
was obtained.
Example 4.
1 .OKg chopped aerial and under ground tissue of Berberis aristata, previously
cleaned with method water, was extracted with 3.0 liter DM water having 3.0 ml
glacial acetic acid and 6 ml sulfuric acid, at 80°C for three hours with stirring.
Filtered at high temperature, the method was repeated twice, with similar
operating parameters. The filtrate was mixed, 3 gm of activated charcoal was
added to it and it was filtered using celite, at high temperature
Filtrate was mixed with hydrochloric acid LR, grade, pH=2.
The acidic phase was kept at 10°C for 10 hours, filtered and ppt. was provided
bed-wash of 30 ml of 50% acetone with water, using vacuum.
The ppt. was dried at 80°C for 10 hours under vacuum, ground and, sieved
through 60 mesh. 2.98 gm yellow free flowing powder of assay,, 99.86% was
obtained.

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Example 5.
] .OKg chopped aerial and under ground tissue of Berheris aristata, previously cleaned with method water, was extracted with 3.0 liter DM water having 3.0 ml glacial acetic acid and 6 ml sulfuric acid, at 85°C for three hours with stirring. Filtered at high temperature , the method was repeated twice, with similar operating parameters.The filtrate was mixed, 3 gm of activated charcoal was added to it and it was filtered using celite. at high temperature Filtrate was mixed with hydrochloric acid LR, grade, pll-2. The acidic phase was kept at 10°C for 10 hours, filtered and ppt. was provided bed-wash of 30 ml of 50% acetone with water, using vacuum. The ppt. was dried at 80°C for 10 hours under vacuum, grounded, sieved through 60 mesh, 2.16 gm yellow free flowing powder of assay, HPLC, 99.91% received.
Numerous modifications and adaptations of the system of the present invention will be apparent to those skilled in the art and thus it is intended by the appended claims to cover all such modifications and adaptations which fall within the true spirit and scope of this invention.

WE CLAIM:
1. An improved method for isolation and purification of berberine compound
from the plant Berberis aristata comprising the steps of:
a. chopping the ground tissue of plant into pieces, and extracting the extract with
acidic water having temp ranging 60-100°C, with three times volume, thrice ,
each for three hours with continuous stirring;
b. filtering out the filtrate in high temperature and the same step is repeated
twice, with similar operating parameters as in step a,
c. obtaining filtrate of step b which is further mixed and added with activated
charcoal and filtered using filter aid and then mixing the treated filtrate with
ammonia solution;
d. drying the precipitate to get the final product.
2. The improved method for isolation and purification of berberine compound from the plant as claimed in claim 1, wherein the ground tissue Berberis aristata in the range of 0.5 to 2.0Kg is chopped and extracted with 3.0 liter DM water having 3.0 ml glacial acetic acid and 6 ml sulfuric acid, at 95 °C for three hours, thrice t and with three time volume with continuous stirring.
3. The improved method for isolation and purification of berberine compound from the plant as claimed in claim 1, wherein the filtrate obtained in step c is mixed and added with 3 gm activated charcoal to absorb the impurities and pigments and also filtered out with filter aid using celite, at hot condition.
4. The improved method for isolation and purification of berberine compound from the plant as claimed in claim 1, wherein the filtered obtained in step c is also further treated with hydrochloric acid of LR grade at pH= 2.
5. The improved method for isolation and purification of berberine compound from the plant as claimed in claim 1, wherein the bed wash is provided to the precipitate with a solvent mixture in the range of 20 to 50ml of 50% acetone with water by using vacuum.
6. The improved method for isolation and purification of berberine compound from the plant as claimed in claim 1, wherein the precipitate is dried at a temperature in the range of 60 to 90.degree.C under vacuum for a duration ranging from 6 to 12hrs and grounding to a size of 60mesh for obtaining berberine in the range of 1 to 5gm with at least 99% purity.
7. The improved method for isolation and purification of berberine compound
from the plant, as claimed in claim 1, wherein the purified berberine having
possible application in pharmaceutical formulation with antitumor activity and
antibacterial and also used in nano medicine.
8. An improved method for isolation and purification of berberine compound
from the plant Berberis aristata substantially as herein described with reference to
the examples and figure accompanying this specification.