FIELD OF THE INVENTION:

The present invention relates to a process for extracting bio-active compound from Manilkara zapota (L.) Von Royen. More particularly the present invention relates to a process for extracting an anti-oxidative, anti-angiogenic, anti-cancer and anti-diabetic properties exhibiting compound from Manilkara zapota for treating angiogenic as well as Cancer and diabetic diseases.

BACKGROUND OF THE INVENTION AND PRIOR ART:

The family Sapotaceae consists of large evergreen trees and less commonly shrubs. Plants are distributed throughout the tropic of Asia, Africa and America. The family consists of about 40 genera. The important ones are Argania, Butyrospermum, Colacarpum, Chrysophyllum, Mimusops, Payenne, Sarcosperm and Sideroxylon. Mimusops is a genus of only trees and comprises of 30 species. Among these, Mimusops hexandera, Mimusops elengi and Mimusops manilkara (=Achras sapota) belongs to the same genus. All the three trees are of medium height of 12 to 15 meters and grow throughout India and Pakistan.

Within the genus Manilkara alone, there are numerous economically important taxa. M. bidentata, M. huberi, M. obovata and M. kauki are just a few of the many species which are used commercially for timber and are known for their heavy, durable, rot-resistant wood. The latex of Manilkara zapota (=Mimusops manilkara ; =Achras sapota) is tapped to form "chicle," (the original chewing gum) and M. bidentata latex is the source of "balata," formerly used in the manufacture of golf ball shells and machine belts. M. zapota fruit ("sapodilla") is also widely cultivated, while other species, although not in cultivation, are all edible. Additionally, a related species within the tribe Mimusopeae, Vitellaria paradoxa (syn =Butyrospermum parkii), is known in the cosmetics industry as the source of shea butter (a fat pressed from the seed), and is an important component of many skin creams (Pennington, 1991; Govaerts et a/., 2001; Mathews, 2009).

Manilkara zapota is found abundant in India and is a small to medium evergreen tree of slow growth. An infusion of the young fruits and the flowers is drunk to relieve pulmonary complaints. Sapodilla fruit regulates the metabolism and keeps the digestive tract clean. It also helps in regulating the secretion of gastric enzymes, thereby regulating metabolism. It is rich in minerals which help in the formation of essential enzymes and gastric juices.

Among its many benefits related to the digestive system, weight loss and obesity prevention can also be included in the benefits. This plant has anti-oxidative property and its fruit is preventive against biliousness and attacks of fever whereas seeds are diuretic. Because of the tannin content, young fruits are boiled and the decoction is taken to stop diarrhoea.

Fruits of Manilkara zapota, when quite ripe, are very delicious and sweet in taste. Jams, sherbets and syrups have also been prepared from the fruits industrially (Ginai, 1968). The parts of the plant species are reported to have more or less similar curative properties in folk medicine.

Activity-guided fractionation of a methanol extract from the fruit of Manilkara zapota cv. Tikal resulted in the isolation of two new antioxidants, methyl 4-O-galloylchlorogenate (1) and 4-O-galloylchlorogenic acid (2), along with eight known polyphenol^ antioxidants, namely, methyl chlorogenate (3), dihydromyricetin (4), quercitrin (5), myricitrin (6), (+)-catechin (7), (-)-epicatechin (8), (+)-gallocatechin (9), and gallic acid (10). Of the 10 polyphenols, 1 showed the maximum antioxidant activity (IC50 = 12.9 uM) in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free-radical assay and displayed cytotoxicity in the HCT-116 and SW-480 human colon cancer cell lines with IC50 values of 190 and 160 uM, respectively. Compound 2 showed high antioxidant activity (IC50 = 23.5 uM) in the DPPH free-radical assay and displayed cytotoxicity in the HCT-116 and SW-480 human colon cancer cell lines with IC50 values of 154 and 134 uM, respectively.

The prior art discussed above revealed that various bioactive components exhibiting pharmacological activity, are extracted from Manilkara zapota. So, there exists a need to extract useful bio-active medicated compounds from different parts of Manilkara zapota.

OBJECT OF THE INVENTION:

The main object of the present invention is to develop a novel process for extracting bio-active compound from Manilkara zapota.

Another object of the present invention is to develop a novel process for extracting anti-oxidative, anti-angiogenic, anti-cancer and anti-diabetic properties exhibiting compound from Manilkara zapota.
Yet another object of the present invention is to utilize Manilkara zapota leaves to extract anti-oxidative, anti-angiogenic and anti-diabetic properties exhibiting compound.

Further object of the present invention is to employ the extracted anti-oxidative, anti-angiogenic, anti-cancer and anti-diabetic properties exhibiting compound for treating angiogenic as well as Cancer and diabetic diseases.

BRIEF DESCRIPTION OF THE DRAWINGS:

FIG.1 illustrates the overall flowchart of the process for extracting Mazapotin from Manilkara zapota.

FIG.2 depicts the thin layer chromatography of Mazapotin (Formula 1) isolated from Manilkara zapota.

SUMMERY OF THE INVENTION:

The present invention disclose a process for extracting anti-oxidative, anti-angiogenic anti-cancer and anti-diabetic properties exhibiting compound, 6-(tetrahydro-3, 4, 5-trihydroxy-6-methyl-2H-pyran-2-yl)-3,5,7-trihydroxy-2-(4-hydroxy-3-(3-methylbut-2-enyl)phenyl)-4H-chromen-4-one ; Quercetin-2-enyl-6-rhamnoside (named as Mazapotin) of formula 1 from Manilkara zapota leaves. The process involves exhaustive extraction of dried powder of Manilkara zapota leaves with methanol to obtain methanol crude extract. The obtained methanol crude extract is washed with petroleum ether to remove petroleum ether soluble impurities to obtain purified methanol crude extract. To the purified methanol crude extract, butanol is added to extract the butanol fraction of the methanol crude extract. The obtained butanol fraction is mixed with silica gel and subjected to column chromatography using a series of solvents with increasing polarity. Mazapotin is eluted from the column using ethyl acetate and methanol in the ratio range of 35 - 40 : 15-10.

DETAILED DESCRIPTION OF THE INVENTION:
The present invention disclose a process for extracting anti-oxidative, anti-angiogenic anti-cancer and anti-diabetic properties exhibiting compound, Mazapotin [ 6-(tetrahydro-3, 4, 5-trihydroxy-6-methyl-2H-pyran-2-yl)-3,5,7-trihydroxy-2-(4-hydroxy-3-(3-methylbut-2-enyl)phenyl)-4H-chromen-4-one ; Quercetin-2-enyl-6-rhamnoside] of formula 1 from Manilkara zapota leaves.

The process of extractions (FIG. 1) is performed as follows. Leaves (1 kg) of Manilkara zapota (Sapotaceae) were taken and washed in running tap water and shade dried for 15 days. The dried powder of leaves (530 g) of Manikara zapota was exhaustively extracted with methanol (72 h) at room temperature. Methanol (2.5 L) was used, for the first time, for soaking the plant powder to get methanol supernatant liquid after 72 h. For repetition of the process for three times, 1 L of methanol was used each time for soaking. Totally, 5.5 L of methanol was used. The greenish extract was evaporated to dryness with the aid of a rotary evaporator at reduced pressure to yield a residue called methanol crude extract (62 g). The methanol crude extract was subjected to fractionation with increasing polarity of solvents such as petroleum ether, butanol and methanol and the three corresponding fractions such as petroleum ether (24.3 g), Butanol (15.2 g) and methanol (20.7 g) were obtained and evaporated to dryness. In this invention, the semisolid butanol fraction was subjected to column chromatography.

Chromatography is the term used to describe a separation technique in which a mobile phase carrying a mixture is caused to move in contact selectively with the adsorbent stationary phase In column chromatography, solvent was used as the mobile phase and silica gel was used as the stationary phase.

In column chromatography, the components are separated from one another by the column packing that involves various chemical and/or physical interactions between their molecules and the packing particles.

The individual components are retained by the stationary phase differently and separate from each other while they are running at different speeds through the column with the eluent. During the entire chromatography process, the eluent is collected in a series of fractions. The composition of the eluent flow can be monitored and each fraction is analyzed for dissolved compounds.

In the present invention, the column packing was done as follows. The silica gel was activated in hot air oven at 110°C for 1 h. The slurry of activated silica made in the hexane solvent at least 1.5 times the volume of solvent as silica, just measured out. The silica gel was thoroughly mixed by stirring vigorously to remove all the air from the silica. Before addition of the slurry, a small piece of the cotton was placed at the end of the column where the top of the knob of the column started. This was normally done by using a long stick or by a glass rod from the top of the column. Care was taken so that cotton should be compressed enough to support the column packing yet loose enough that the solvent flow will not be hindered. The cotton was placed in order to avoid slurry of the silica gel drain out while opening the knob
of the column and to allow the solvent alone to pass through it.

A funnel was placed on the top of the column and then, the slurry was poured into the column in small portions carefully and slowly, by keeping the knob open and with gentle taping after each addition of the slurry, in order to ensure the uniform packing. Frequently pouring of the slurry into the column was stopped and to swirl the slurry so that the silica gel was evenly mixed. A small quantity of solvent was allowed to remain at the top of the column in order to avoid the drying or cracking of the column. Tapping is necessary to avoid the air bubble formation in the column during packing otherwise can interfere in separation. The column was run fast for some time with the hexane solvent system (low polar solvent only) in order to remove any impurities.

For sample preparation, the semi-solid butanol fraction, 3 g was taken and ground with small amount of silica gel, which was used for the column chromatography. The butanol fraction and silica gel mixture was subjected to column chromatography (17 mm column diameter) using a series of solvents with increasing polarity.

The sample was then poured onto the silica gel (100-200 mesh) packed column (Stationary phase) in a minimum volume of 100% hexane by using a funnel and was allowed to settle. Then, it was eluted sequentially with solvent systems ( mobile phase) starting from non-polar to polar solvents such as Hexane, Ethyl acetate and Methanol in the following ratios.

1. 50 mL Hexane
2. 45 mL Hexane + 5 mL Ethyl acetate
3. 40 mL Hexane + 10 mL Ethyl acetate
4. 35 mL Hexane + 15 mL Ethyl acetate
5. 30 mL Hexane + 20 mL Ethyl acetate
6. 25 mL Hexane + 25 mL Ethyl acetate
7. 20 mL Hexane + 30 mL Ethyl acetate
8. 15 mL Hexane + 35 mL Ethyl acetate
9. 10 mL Hexane+ 40 mL Ethyl acetate
10. 5 mL Hexane + 45 mL Ethyl acetate
11. 50 mL Ethyl acetate
12. 5 mL Methanol + 45 mL Ethyl acetate
13. 10 mL Methanol + 40 mL Ethyl acetate
14. 15 mL Methanol + 35 mL Ethyl acetate
15. 20 mL Methanol + 30 mL Ethyl acetate
16. 25 mL Methanol + 25 mL Ethyl acetate
17. 30 mL Methanol + 20 mL Ethyl acetate
18. 35 mL Methanol + 15 mL Ethyl acetate
19. 40 mL Methanol + 10 mL Ethyl acetate
20. 45 mL Methanol + 5 mL Ethyl acetate
21. 50 mL Methanol
22. 5 mL Water + 45 ml_ Methanol
23. 10 mL Water + 40 mL Methanol
24. 15 mL Water + 35 mL Methanol
25. 20 mL Water + 30 mL Methanol
26. 25 mL Water + 25 mL Methanol
27. 30 mL Water + 20 mL Methanol
28. 35 mL Water + 15 mL Methanol
29. 40 mL Water + 10 mL Methanol
30. 45 mL Water + 5 mL Methanol

From the column, the eluted fractions of 15 mL each were collected. Each fraction was evaluated by DPPH (1.1-DIPHENYL-2-PICRYLHYDRAZYL) radical scavenging Dot-Plot assay (Bio-autography) to find out the active compounds in the fractions.

For performing Bio-autography, aliquots of fractions (1 uL) were spotted carefully on TLC plates and dried for 3 min. After drying, 0.4 mM DPPH solution (2 uL) was spotted onto the dry spots of the fractions and the layers were dried for 2 min. The stained silica gel layers revealed purple backgrounds with yellow spots showed presence of active compound.

From the column chromatography of butanol fraction, 30 fractions were obtained
DPPH (1.1-DIPHENYL-2-PICRYLHYDRAZYL) radical scavenging Dot-Plot assay (Bio-autography) of all the 30 fractions revealed that only 2 fractions, 18th fraction (35 mL Methanol + 15 mL Ethyl acetate) and 19th fraction (40 mL Methanol + 10 mL Ethyl acetate) contained the active novel flavonoid, Mazapotin (Formula 1) of Rf value 0.69 (FIG. 2).

The physical characteristics of the compound were determined and found to be yellow powder in nature with melting point of 285±2°C . The compound is soluble in methanol and water. The yield of the compound by the above process is 30 mg. The Rf value is 0.69 with the solvent system Ethyl acetate : Methanol (1.8 : 0.2).

The compound Mazapotin is characterized by UV-Vis, IR, NMR and Mass spectra to ascertain the chemical structure of the compound.

Based on the comparison of the physical characteristics and spectral data, the isolated compound Mazapotin (Formula 1) was conformed to be a flavonoid. It was identified as 6-(tetrahydro-3, 4, 5-trihydroxy-6-methyl-2H-pyran-2-yl)-3,5,7-trihydroxy-2-(4-hydroxy-3-(3-methylbut-2-enyl)phenyl)-4H-chromen-4-one ; Quercetin-2-enyl-6-rhamnoside (Formula 1) .

6-(tetrahydro-3, 4, 5-trihydroxy-6-methyl-2H-pyran-2-yl)-3,5,7-trihydroxy-2-(4-hydroxy-3-(3-methylbut-2-enyl)phenyl)-4H-chromen-4-one ; Quercetin-2-enyl-6-rhamnoside.

Biological activities of the compound Mazapotin were studied to determine its anti-oxidative, anti-angiogenic, anti-cancer and anti-diabetic properties. The antioxidant activity assay was determined by DPPH radical scavenging assay. CAM assay (chorioallantoic membrane) was used to evaluate anti-angiogenic activity. MTT assay was used to evaluate the anti-cancer activity. Alpha-amylase enzyme inhibition assay was used to evaluate anti-diabetic activity. The results are as follows.

The DPPH free radical scavenging activity of Mazapotin at 7 µg/mL concentration was found to be 98.34±0.37% and for the standard Quercetin, it was 67.17±1.01% at the same concentration.

At 0.5 mg/pellet concentration of Mazapotin, the f angiogenesis inhibition of the compound was found to be 57.25±1%.

At 25 µg/ mL concentration of Mazapotin, the MCF7 cell death was 85.42%.

The inhibition of a-amylase activity of Mazapotin at 35 µg/mL concentration was found to be 66.66±2.63% and for the standard Acarbose, it was 98.16±0.28% at the same concentration.

In one of the preferred embodiment, the present invention shall disclose a process for extracting anti-oxidative, anti-angiogenic, anti-cancer and anti-diabetic properties exhibiting compound, Mazapotin [6-(tetrahydro-3, 4, 5-trihydroxy-6-methyl-2H-pyran-2-yl)-3,5,7-trihydroxy-2-(4-hydroxy-3-(3-methylbut-2-enyl)phenyl)-4H-chromen-4-one ; Quercetin-2-enyl-6-rhamnoside] of formula 1 from Manilkara zapota leaves. The process involves exhaustively extraction of dried powder of Manilkara zapota leaves with methanol to obtain methanol crude extract. The obtained methanol crude extract is washed with petroleum ether to remove petroleum ether soluble impurities to obtain purified methanol crude extract.

The purified methanol crude extract was extracted with butanol to obtain butanol fraction of the methanol crude extract. Then a mixture comprising of the obtained butanol fraction and silica gel was loaded on to a column chromatography packed with silica gel and more than one appropriate organic solvent was added into the column to remove the fraction containing Mazapotin from the column. Finally, the eluate containing Mazapotin was collected from the column and purified to obtain purified Mazapotin.

As per the invention, the organic solvents are selected from the group comprising of hexane, ethyl acetate, methanol or combinations thereof.

In accordance with the invention, the organic solvents are ethyl acetate and methanol in the ratio of 35 - 40 :15 -10.

According to the invention, the purified Mazapotin has a spot around Rf value 0.69 in thin layer chromatogram with the solvent system of ethyl acetate : methanol in the ratio of 1.8 : 0.2, and spraying reagent of DPPH (1,1-Diphenyl-2-picrylhydrazyl).

In another preferred embodiment the present invention shall disclose an anti-oxidative, anti-angiogenic, anti-cancer and anti-diabetic properties exhibiting compound, Mazapotin [ 6-(tetrahydro-3, 4, 5-trihydroxy-6-methyl-2H-pyran-2-yl)-3,5,7-trihydroxy-2-(4-hydroxy-3-(3-methylbut-2-enyl)phenyl)-4H-chromen-4-one ; Quercetin-2-enyl-6-rhamnoside] of formula 1, prepared from the process as discussed above.

While a preferred embodiment of the invention has been shown and described, modifications thereof can be made by one skilled in the art without departing from the spirit of the invention.

We Claim:

1. A process for extracting anti-oxidative, anti-angiogenic, anti-cancer and anti-diabetic properties exhibiting compound, Mazapotin [ 6-(tetrahydro-3, 4, 5-trihydroxy-6-methyl-2H-pyran-2-yl)-3,5,7-trihydroxy-2-(4-hydroxy-3-(3-methylbut-2-enyl)phenyl)-4H-chromen-4-one ; Quercetin-2-enyl-6-rhamnoside] of formula 1

Formula 1 from Manilkara zapota leaves comprises of following steps:

a. exhaustively extracting dried powder of Manilkara zapota leaves with methanol to obtain methanol crude extract,

b. washing the obtained methanol crude extract with petroleum ether to remove petroleum ether soluble impurities to obtain purified methanol crude extract,

c. extracting the purified methanol crude extract with butanol to obtain butanol fraction of the methanol crude extract,

d. loading a mixture comprising of butanol fraction of step (c) and silica gel to a column chromatography packed with silica gel,

e. adding at least one appropriate organic solvent into the column to remove the fraction containing Mazapotin from the column,

f. collecting the eluate containing Mazapotin from the column,

g. purifying the eluate of step (f) to obtain purified Mazapotin.

2. The process as claimed in claim 1 wherein the said organic solvents are selected from the group comprising of hexane, ethyl acetate, methanol or combinations thereof.

3. The process as claimed in claim 1 wherein the said organic solvents are ethyl acetate and methanol in the ratio of 35 - 40 :15 - 10.

4. The process as claimed in claim 1 , wherein the said purified Mazapotin has a spot around Rf value 0.69 in thin layer chromatogram with the solvent system of ethyl acetate : methanol in the ratio of 1.8 : 0.2, and spraying reagent of DPPH (1,1 -Diphenyl-2-picrylhydrazyl).

5. An anti-oxidative, anti-angiogenic, anti-cancer and anti-diabetic properties exhibiting compound, Mazapotin [ 6-(tetrahydro-3, 4, 5-trihydroxy-6-methyl-2H-pyran-2-yl)-3,5,7-trihydroxy-2-(4-hydroxy-3-(3-methylbut-2-enyl)phenyl)-4H-chromen-4-one ; Quercetin-2-enyl-6-rhamnoside] of formula 1, prepared from the process as claimed in claim 1.