Title of the Invention

A method to isolate Isokaempferide from Acalypha alnifolia

Field of Invention

The invention relates to a column chromatographic method to isolate isokaempferide from new source comprising better yield. More specifically, the isokaempferide derived from a wild green leafy vegetable Acalypha alnifolia. In particular this invention relates to a greater quantity of isokaempferide isolated from acetone extract of the plant by chloroform:ethylacetate fraction of silica gel column chromatography.

Background of Invention

The information about the therapeutic properties and usage of medicinal plants arecommonly based on the empirical knowledge of ancient people, which was passed overseveral generations and originated the traditional medicine systems, utilized all over theworld (Traditional Chinese Medicine, Ayurvedic system, Western and African Herbalisms).The ethnobotanical documentation has been achieving in great extent and the traditional knowledge has also been proven side by side with the help of new technologies and methods. The potent of traditional plant has been revealed and the benefits haveto acquire in exact way by phytochemical isolation and evaluation. In present scenario, such medicinally important plants have focused by the researchers and the biological productshave been accepting by the people. The compounds derived from natural source is paving for better allopathic medicine too. A great dealof interest has been generated recently in the isolation, characterization and biological activity of thesephytochemicals. On the flip side, herbal drugs are discredited by most of the health related professionals owing to a lack of scientific research supporting its efficacy and safety (Kirley M. CanutoetaL, 2012).

Many medicinal plants have been evidenced for having antidiabetic activity with lipid lowering feature. Ethnobotanical information also indicates that more than 800 plants are used as traditional remedies for the treatment of diabetes throughout the world. The plant Acalypha alnifolia which is also known as A. capitata belongs to the family Euphorbiaceae, is traditionally used as a leafy vegetable by the inhabitants of Nilgiri District (Sasi and Rajendran, 2012). The leaf paste of the plant isan ingredient of the traditional formulation used to cureprickles by the Hoorali tribe of Sathyamangalam forest (Revathi et al.,2013)and leaf juice mixed with 150 ml boiled cow milk and given two times upto five months against diabetes (Balakrishnan et al.,2009). Evidently the use of A. capitata (A. alnifolia) in Southern Nigeria local communities as vegetable and it is used in the treatment of hypertension (Johnkennedy et al., 2011) are also the reason to focus on this plant for active compound isolation against diabetes.

The plant Acalypha alnifolia having significant antioxidant property which is coincidentally comparable with quantity of phenolics present in it, hence it should be with good pharmacological potential (Revathi et al., 2013) and In vivo antioxidant activity showed that A. alnifolia were effective in restoring activities of the oxidant stress to normal levels (Evanjelene and Natarajan, 2012). The previous reports proved their hypolipidemic ability on high cholesterol rats (Johnkennedy et al., 2011). The plant A. alnifolia has good nutritional values and acts as a better drug for proinflammatory mediator inhibition with its antinociceptive, anti-inflammaotry and antipyretic properties (Ponnusamy and Thangaraj, 2014). The acetone extract of A. alnifolia having ability to increase the insulin secretion as well as mitigating the diabetic complications which is supporting previous reports and confirms the therapeutic effects (Revathi and Parimelazhagan, 2015). As a result the research is needed to explore the active potential from this plant.

Bastos (1983) found as the most abundant component and described the isolation of compounds including isokaempferide. Canuto and Silveira (2006) carried out a phytochemical investigation of the ethanol extract from stem bark of Amburana cearensis. The ethanol extract was partitioned with water and ethyl acetate. The organic phase was dried and submitted to successive chromatographic columns on silica gel and dextran gels. These chromatographic separations led to the isolation of coumarin, two phenol acids and five flavonoids including isokaempferide. The stem bark of the plant yields 143 mg of isokaempferide from 3.4 kg which is calculated as 0.004%.

The seeds revealed Liquid-liquid partitioning from the ethanol extracts of Amburana cearensis followed by chromatography on Sephadex LH-20 and a reversed-phase HPLC chromatography of the ethyl acetate fraction, resulted the presence of isokaempferide (Canuto et al., 2010)

The cytotoxicity of isokaempferide from the A. cearensis, were evaluated on tumor cell lines where it inhibited thesea urchin egg development, as well as tumor cell lines (Costa-Lotufo et al., 2003).

Recently (Leal et al., 2006), it was demonstrated the relaxant action of the isokaempferide. The relaxation of the guinea-pig isolated trachea, induced by isokaempferide was a direct and an epithelium-independent phenomenon, resulting from several intracellular actions through a common pathway e.g., the opening of Ca and ATP-sensitive K channels.

Isokaempferide from A. cearensis seems to occur by an inhibitory action on the release of inflammatory mediators, and/or alternatively by interfering with a certain phase of the neutrophil migration into the inflammatory focus (Leal et al., 2003; Leal, 2006).Other data (Leal et al., 2008) corroborated this hypothesis showing that both the isokaempferide exert their anti-inflammatory activities mainly by inhibiting the lipopolysaccharide-induced release of TNF-a, although the involvement of other inflammatory mediators cannot be excluded.

In another prior art (patent no. US 8,299,119 B2; Leach et al., 2012) biologically active compounds includes isokaempferide derived from a plant Centipeda cunninghamii filled particularly for antioxidant and anti-inflammatory activity.

The study of phytochemicals quantity variation in different field has done with Dracocephalum kotschyi where the isokaempferide contents ranged from 124 to 766 |ig/g DW (Fattahi et al., 2013).

The prior art of the flavone compound isokaempferide separated from liquoriceinhibits the Protein Tyrosine Phosphatase IB for preventing and treating diabetes and obesity (Publicaiton No. CN 101982465 A).

The prior art regarding this compound emphasize the pharmacological properties and also supports the importance of present finding. Hence the present amendment is new and feasible to get theisokeampferide specifically. This invention relates to find a new source among existing would bring out isokaempferide in greater quantity bythemethod of column chromatography.

Objects of invention

The compound isokaempferide has already been proposed with one or more of the problems of the prior art can be overcome by the various embodiment of the present invention.

The principal object of this invention is a method to isolate the compound isokaempferide comprising extraction methods and column chromatographic method particularly the mobile phase is being the key to get the compound.

Another object of this invention is new source for isokaempferide which is from the plant Acalypha alnifolia.

A further object of this invention is identifying high yielding source of the compound where the yield is calculated as 0.14%.The plant leaf acetone extract 15 g yields 21mg of isokaempferide.

Statement of invention

Pharmaceutical^ effective active compound isokaempferide isolated from a wild leafy vegetable Acalypha alnifolia acetone extract in greater quantity by a method of column chromatography comprising the usage of solvents to extract and gradual changes in mobile phase is paving to get the compound without difficulty and noteworthyyield.

Summary of Invention

• The isolation of isokaempferide from Acalypha alnifolia is the first report.

• The present invention is isolation of isokaempferide by a new method from anew source-a pharmaceutically potent flavone compound.

• The isokaempferide extracted from Acalypha alnifolia acetone extract by column chromatographic technique

• Successive solvent extraction followed by gradual changes in mobile phase of chromatography from low polarity solvent, the method is novel invention to get isokaempferide.

• Gradual enhancement of polarity of the solvent sensation on acetone extract is the key to derive the compound in 45% ethylacetate elution.

• Methods:Among successive solvent extraction of A. alnifolia, acetone extract having antioxidant, antidiabetic property against Streptozotocin induced diabetic rats. Hence, acetone extract has selected for active compound separation; the extract on silica column chromatography, the Chloroform: ethylacetate (55:45) mobile phase elution yields isokaempferide crystal which was confirmed with FTIR, NMR spectrum and structural formula has shown below.

• Chemically the name of the structure is 537-dihydroxy-2-(4-hydroxyphenyl)-3-methoxy-4H-chromen-4-one

• Molecular formula: C^HuOe

• By this isolation method the compound isokaempferide derived at maximum is also the advantage to get remarkable quantity.

• According to the method described above acetone extract yields 0.14% of isokaempferide; hence the plant must be the good alternative source for the particular compound.

• The prior art of the flavone compound isokaempferide separated from liquorice having antidiabetic property due to the Protein Tyrosine Phosphatase IB inhibition ability (Patent Publication No. CN 101982465 A). This concludes the isokamepferide might be the reason for the antidiabetic property of acetone extract.

Detailed description of invention

Preparation of acetone extract

A. alnifolia was collected from the Bharathiar University campus. The voucher specimen was deposited and then identified and authenticated by the Botanical Survey of India,Southern Circle, Coimbatore, India (authentication numberBSI/SRC/5/23/2011-12/Tech.-1137). The leaves were separatedfrom the plant, washed in running tap water, and shade-dried.Then it was powdered and packed in small thimbles separately and extracted successively with different solvents such as petroleum ether, chloroform, acetone, and methanol in the increasing order of polarity using the Soxhlet apparatus each for 72 hours at 30°C. The extract obtained was dried and used for further assessments.

The yield percentage was measured by the weight obtained at each solvent extraction. Methanol (16%)was yielded higher than acetone (9.7%). Petroleumether (3.3%) and chloroform (2%) yield was very leastwhere both were low polar solvents whereas, hot water yields 7.5%. Besides, acetone and methanol extractshave potent antioxidant capacity with good quantity ofsecondary metabolites (Revathi et al., 2013).The acetone extract potent good analgesic, anti-inflammatory, antipyretic properties (Ponnusamy and Thangaraj 2014) and against diabetes on normal and STZ induced diabetic rats (Revathi and Parimelazhagan, 2015). Hence this extract has taken for active compound isolation.

A complete compound isolation method

Column chromatographic isokaempferide isolation

The acetone extract of A. alnifolia about 15 g blended with 30 g of silica gel for compound separation with column chromatography S The column size is about 50cm x 30 mm, A wet column was prepared by using 60-120 mesh silica gel in 1:5 ratio with extract (30 g). The 150 g of silica gel was mixed with petroleum ether. S A perfect packing has done with the silica gel by pouring the above said admixture on top of the column and continuous drawing of petroleum ether for 2 days on perfect settlement.

The petroleum ether has detained over the column 10 cm from silica gel in order to avoid column dryness and the stopper has applied and checked often. Acetone extract has dissolved in acetone and blended with silica gel (1:2) and dried. The blended acetone extract has carefully loaded over the packed column after silica gel settlement. A piece of cotton has put over the silica pack, inside the detained solvent.

A perfect packing has done with petroleum ether by pouring the solvent on top of the column and continuous drawing of petroleum ether for 3 days on perfect settlement of silica gel along with extract.

The next mobile phase is chloroform which is gradually replaced where each step has increased 5 %. The low polar compounds have cleaned off by these two solvents completely.

The column has run with each ratio of the mobile phase about 200 mL Every ten mL of the elution has taken in separate test tubes.

The ethylacetate has introduced next with chloroform like previous. The flow rate is a drop per 2 seconds.

The elutions which are having same Rf value in Thin layer silica gel (G 254) chromatographic plate has poured together and concentrated

The chloroform: ethylacetate (55:45) yields colorless elution (20 Test tubes) where poured together and solvent was evaporated in room temperature.

The fraction finds a crystal with little impurities which was removed by dissolved in ethylacetate and poured in a watch glass. The peripheral crystal has scrapped out and stored. The centralcreamy green impure residue was dissolved again with ethylacetate and does the same.

The crystal has taken for structure elucidation with the help of FTIR and 13C and 'H- NMR data.

The scrapped crystal is green shaded white fine substance and the characters are matched with isokaempferide.

The 15 g of acetone extract which was taken for compound isolation yields 21 mg of isokaempferide (0.14%).

S FTIR-Figure!

i. 3389.89 cm"1- -OH-

ii. 2924.52 cm-1 --CH-iii. 1692.23 cm-1 --C=0-H-NMR - Figure 2 i. 3.397 (3H, s, for-OCH3) ii. 9.16 (lH,s, for C8-H) iii. 731(lH,s,forC6-H) iv. 6.625-6.920-4-H(m) (C2\ C3', C5' and C6' protons) v.l2.19(BSfor-OH) proton

S. 13C-NMR - Figure 3 -OO- 167.906 Carboxyl -0-CH3 63.262 Methoxy Carbon S 5,7-dihydrdxy-2-(4-hydroxyphenyl)-3-methoxy-4H-chromen-4-one(Figure4)

• The isolation of isokaempferide from Acalypha alnifoliais the first report. ■S Successive extraction followed by gradual changes in mobile phase from low polarity solvent is the novelty of the present invention to get isokaempferide.

Gradual enhancement of polarity of the solvent sensation on acetone extract is the key to derive the compound in 45% ethylacetate elution. S By this isolation method the compound isokaempferide derived at maximum is also the advantage to get remarkable quantity.

Claims

We claim,

1. The compound isokaempferide was isolated from the column chromatographic elution of Acalypha alnifolia leaf acetone extract.

2. Column chromatographic mobile phase is gradually changed from low polar to polar solvent by 5% in each step. The isokaempferide has obtained in chloroform:ethylacetate (55:45) fraction.

3. According to claim 2, preparation of column

a. A wet column has prepared with silica gel and petroleum ether solvent.

b. A finely packed column has loaded with acetone extract of A. alnifolia where acetone extract has dissolved in acetone and then blended with two parts of silica gel continuously until complete evaporation of acetone.

4. According to claim 3, The column chromatographic mobile phase running method is as follows,

a. The column has run out with petroleum ether which is gradually changed to chloroform by 5 % each step followed by ethyl acetate.

b. In each step 200 mL of solvent has run and each 10 mL have collected in separate vial.

c. A complete 20 fractions of 45% ethylacetate has isokaempferide substances which have to pour in watch glass for dry.