|Title of Invention||
"AN IMPROVED PROCESS FOR THE ISOLATION OF ANDROGRAPHOLIDES FROM ANDROGRAPHIS PANICULATA"
|Abstract||The present invention provides an improved process for the isolation of andrographolide from Andrographis paniculata plant. The steps are: extracting the powdered plant parts with polar solvent, partitioned with a non polar solvent, evaporating the non polar solvent, dissolving a residue in the solvent and crystallization, filtering the crystal of andrographolide and employing chromatography of filtrate after concentration to obtain remaining andrographolide and other compounds. The bulkness of non polar solvent extract reduced (35-40%) due to partitioning step, which improved the crystallization of andrographolide up 80%. This process improved the yield of andrographolide by 10-15% and decrease the cost of production due to reuse of solvent used in partition as well as extraction and reduced the quantity of silica gel needed for separation of remaining andrographolide through chromatography.|
|Full Text||The present invention relates to an improved process for the isolation of andrographolides from Andrographis paniculata.
Andrographis paniculata Nees (Acanthaceae) is a herb that has been commonly used in India. Thailand and China for the treatment of numerous ailments and diseases. Andrographispaniculata commonly known as 'Kalmegh' in Hindi is widely used in Indian system of medicine as a stomachic.tonic. antipyretic.anthelmintic. febrifuge and cholagoue for liver disorders general debility and colic pains. Over the past 20 years Andrographis paniculata has been evaluated for its analgesic anti-inflammatory, antibacterial and antiviral activities. (Habtemariam. S. Phytother. Res. 12. 37-40. 1998). A specific compound extracted from the plant Andrographis paniculata. Andrographolide, has been shown to have anti-HIV effect. Moreover.andrographolide has been shown to have anti-viral. Anti-neoplastic and hepatoprotective properties (wo 96/17605). Andrographolide is a diterpene containing a y lactone ring connected to a decalin ring system via an unsaturated C2 moiety. It found in different parts of the plant both in the free state and in glycoside.
Andrographolide can be isolated from the aerial part of the plant by extraction with alcohol or with alkaline solutions. Hydrolysis of andrographolide under cleavage of the lactone ring yields salts of andrographolic acid which can be reconverted into andrographolide by acidification.
Another method for the isolation of andrographolide from the powdered leaf of the plant involved extraction with petroleum ether, than with ethanol in soxhlet apparatus. On partial eyaporation and cooling, heavy crystals of andrographolide formed. The crystals were repeatedly washed with benzene and recrystallized in ethanol. (Ray. K.S and Datta, PC. Indian Drugs; 26(1). 7-91. 1987).
Another method reported for extraction of andrographolide involved extraction of leaf powder by cold maceration in (1:1) mixture of dichloromethane and methanol. The extract was filtered and solvent removed. The dark green crystalline mass obtained was washed with toluene several times to remove coloring material from the residue. The crystalline material was dissolved in methanol and crystal of andrographolide obtained. Rajani. M. et al. Pharmaceutical Biology. 38(3), 2004-2009, 2000).
However, the method of isolation of andrographolide through acid base treatment is not ideal because it is not possible to avoid some degradation of andrographolide or the loss of the compound. No any large scale process for the isolation of Andrographolide has been reported. The cold extraction involves the large amount of loss of solvent and recovery of andrographolide and its glycoside is less.
The main objective of the present invention is to provide an improved process for higher yield of andrographolide by selection of plant parts, use of most effecive solvent for extraction and aviod to chromatography.
The present invention provides an improved process for the isolation of andrographolide from the plant. Andrographis paniculata which comprises the extracting the powdered plant parts with polar solvent partitioned with a non polar solvent evaporating the non polar solvent dissolving a residue in the solvent and crystallization filter the crystal of andrographolide and employing chromatography of filtrate after concentration to obtained remaining andrographolide and other compounds.
The improved process consists of the following operation:
1. Shade drying and grinding of plant parts of Andrographis paniculata.
2. Extracting the powdered material with polar solvent.
3. Concentration of the total extract under vaccum to 25% of its original volume.
4. Addition of water in the polar solvent extract.
5. Partitioning of the aqueous polar solvent extract with non polar solvent.
6. Distillation of the non polar solvent to obtain the residue extract.
7. Crystallization of the andrographolide from the residual extract.
8. Filtration of crystals and concentration of filtrate.
9. Column chromatography of filtrate to recover remaining andrographolide and other compounds.
Accordingly, the present invention provides An improved process for the isolation of endrographolide from Andrographis paniculata which comprises extraction of air dried powdered aerial part of the plant Andrographis paniculata in a polar solvent as herein described in soxhlet,
concentrating the extracted solvent up to 25% of its original volume, adding water in the extract and partitioning the aqueous polar solvent extract with non polar solvent selected from n-hexane, toluene, benzene, dichlorimethane, chloroform , concentrating the non polar solvent fraction and dissolving the residue into methanol and crystals of andrographolide formed were filtered and mother liquor was concentrated to give residue, separating the andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide from the residue by chromatographing over silica gel. In an embodiment of the present invention the polar solvent used for the
extraction is selected from methanol acetonitile water and their combination etc. In an embodiment of the present invention the polar solvent extracts
diluted with water to make the extract 1.1 or 2.1 for the better transfer of
andrographolide to non polar solvent.
In another embodiment of the present invention, the non polar solvent
used for partitioning is to be selected from benzene, dichloromethane and
In another embodiment of the present invention the plant part for
extraction of andrographolide is selected from leave and stem.
In yet another embodiment of the present invention the separation of
endrographolide, neoandrographolide and 14-deoxy-11.12-
didehydroandrographolide over silica gel where as the ratio of crude extract to
silica gel is selected from 1:5 to 1:10.
In still another embodiment of the present invention, andrographolide isolated from different parts of Andrographis paniculate plant is in the rage of 2.8 -3.1% in leaves, 0..25-0.3° o from stem and root (trace).
In further embodiment of the present invention, the solvent used for the extraction is selected in different ratios of aqueous polar solvent from 0 to 10%.
By employing the aqueous polar solvent for extraction through soxhlet. improved the extraction of andrographolide with lesser amounts of coloring matter and total extract. The selection of plant part for extraction (leaves only), which contains higher yield of andrographolide rather using total aerial portion of the plant. Stem part contains 10 times less andrographolide where as stem part in the aerial portion present three to four times than leaves. The partitioning with non polar solvent selectively transfer andrographolide to non polar solvent and left other impurities. The partitioning step enriched the andrographolide in the extract.
The bulkness of non polar solvent extract reduced (35-40%) due to partitioning step, which improved the crystallization of andrographolide upto 80%. This process improved the yield of andrographolide by 10-15% and decrease the cost of production due to reuse of solvent, used in partition as well as extraction and reduced the quantity of silica gel needed for separation of remaining andrographolide through chromatography.
The details of the invention provided in the following examples are given by way of illustration only and should not be construed to limit the scope of the present invention:
Selection of the plant parts for the extraction:
A quantitative estimation of andrographolide in different parts (leaves, stem, root and seed) of the Androgrophis paniculata was done. Dried powdered sample of 0.1 gm of each pan was extracted with methanol. The extract was filtered and solvent was evaporated to dryness. The extract was dissolved in 5 ml HPLC methanol. The content of andrographoiide in each sample was estimated by HPLC - HP 1100 series equipped with multi-wave length UV detector unde the following operating conditions, mobile phase solvent used acetonitrie: water (70:30), flow rate 1 ml/min, detection at 230 nm,
column (C18). Quantification was performed using a calibration curve of standard andrographolide prepared as 1mg/ml solution in methanol. The content of andrographolide estimated in different plant parts are as follows leaves (2.8%), stem (0.3%). root (trace) and seed (0%).
Example - 2
Selection of the solvent:
Leaf powder of sample (l.Og) of Andrographis paniculata was extracted with 10 ml different solvents (acetonitrile:water (9:1), methanol, chloroform: methanol (1:1)} for cold percolation for 8 hr. three times. Each extract was filtered and evaporated under
vacuum. Each extract was dissolved in 20 ml HPLC solvent and estimated the andrographolide content by HPLC method described in example-1. Andrographolide content in leaf was recorded in different solventsis as follows: Acetonitrile:water (3.1%), Methanol (2.8%) and chloroform:methanol (2.7%).
Extraction and isolation of andrographolide:
Andrographis paniculata leaves were groundee into powder of mesh size (3-4mm). The powdered material (1.0kg) was extracted with 10% aqueous acetonitrile in soxhlet. The total extract was concentrated under vacuum upto (700 ml) 25% of its original volume. Water was added (600 ml) to make the solution (1:1). The aqueous acetonitrile extract was partitioned with n-hexane (3 times x 300 ml) followed by benzene (300 ml x 5 times). The total benzene extract was evaporated upto 20% and left over night for crystallization it room temperature. The process of crystallization repeated three times to get 80% of andrographolide present in the extract. The crystalline compound was filtered and dried to get pure andrographolide. The remaining andrographolide in filtrate (3.0g) was isolated by column chromatography over silica gel in the ratio of 1:10 and andrographolide(6.0g) was eluted with 4-6% methanol in chloroform(8-10%), solvent system eluted neoandrographolide(2.8g) and 2-4% solvent eluted 14-deoxy-l 1,12-
didehydroandrographolide. Thereby 88% of pure andrographolide(27g) was isolated from leaf powder containg 3.05% andrographolide. The andrographolide was identified by mmo. IR. 'H & 13CNMR and mass spectral d
Andrographis paniculata leaf powder (lOOg) was extracted with 10% aqueous acetonitrile in soxhlet. The total extract was concentrated upto 25% of its original volume (125 ml). Water (45 ml) wasadded to make the solution (2:1). The aqueous acetonitrile extract was partitioned with n-hexane (50ml x 3times) followed with chloroform (75 ml x 3). The total chloroform soluble extract was dried over anhydrous sodium sulphate and solvent was evaporated. The residue (5.0g) obtained was dissolved in methanol and left for crystallization. The crystalline compound (2.0g, 67%) was filtered and dried to get pure andrographolide. The remaining andrographolide was obtained through column chromatographv over silica gel in the ratio of 1:10 and andrographolide was eluted with 4% methanol in chloroform. Thereby 85 % of pure andrographolide was isolated.
Andrographis paniculata leaf powder (lOOg) was extracted with methanol. The total extract obtained was converted into (1:1) solution aqueous methanol. The aqueous methanol was partition with n-hexane (75 ml x 3) followed by dichloromethane (100 ml x 3). The total dichloromethane extract was concentrated after drying over sodium sulphate. The extract was dissolved in methanol, left for over night and the crystal formed was filtered. The process of crystallization was repeated twice to obtain 80% of andrographolide. The remaining compound was isolated from the mother liquor
chromatography as described in example 3. The total yield 82% of the andrgrapholide was obtained from its content present in the material.
The improved process for isolation of andrographolide, the subject matter of this patent, afforded a number of advantages.
1. Andrographolide is obtained more than 80% without chromatography.
2. Acid base treatment is avoided in this process, which decompose the andrographolide,
a sensitive γ lactone.
3. Selective transfer of andrographolide into non polar solvent phase leaving polar
constituents in aqueous polar solvent phase, which results into better crystallization of
4. The process allows the reuse of the solvent.
5. Simple, economic and efficient method for the isolation of the andrographolides.
We claim :
1. An improved process for the isolation of endrographolide from Andrographis paniculata which comprises extraction of air dried powdered aerial part of the plant Andrographis paniculata in a polar solvent as herein described in soxhlet, concentrating the extracted solvent up to 25% of its original volume, adding water in the extract and partitioning the aqueous polar solvent extract with non polar solvent selected from n-hexane, toluene, benzene, dichlorimethane, chloroform , concentrating the non polar solvent fraction and dissolving the residue into methanol and crystals of andrographolide formed were filtered and mother liquor was concentrated to give residue, separating the andrographolide, neoandrographolide and 14-deoxy-11,12-didehydroandrographolide from the residue by chromatographing over silica gel.
2. A process claimed in claim 1, wherein the andrographolide neoandrographolide and 14-deoxy-H, 12-didehydroandrographolide were extracted from aerial parts of the Andrographis paniculata, selected from leaves and stem.
3. A process claimed in claims 1 and 2, wherein the polar solvent used for extraction was selected from methanol, acetonitrile, water and their combination.
4. A process claimed in claims 1 to 3, wherein the addition of water in the polar extract to make it 1:1 to 2:1.
5. A process claimed in claims 1 to 4, wherein the recovery of andrographolide from the filtrate obtained through chromatography over silica gel is in the ratio of 1:10.
6. A process as claimed in claims 1 to 5, wherein 82 % to 88% yield of andrographolides may be obtained.
7. A process claimed in claim 1, wherein the andrographolide, eluted from column from different solvent ratjo CHCI3:MeOH (96:2).
8. An improved process for the isolation of andrographolides from Andrographis paniculata substantially as here is described with references to the examples accompanying the specification.
543-del-2003-complete specification (granted).pdf
|Indian Patent Application Number||543/DEL/2003|
|PG Journal Number||20/2009|
|Date of Filing||31-Mar-2003|
|Name of Patentee||COUNCIL OF SCIENTIFIC & INDUSTRIAL RESEARCH|
|Applicant Address||RAFI MARG NEW DELHI, INDIA|
|PCT International Classification Number||A61K 35/78|
|PCT International Application Number||N/A|
|PCT International Filing date|