An extraction process for Emblica Officinalis Gaertn
Technical Field:
The present invention relates to novel process for extraction of active principles selected from tannins, gallic acid, ellagic acid, emblicol, phyllantidine of Emblica Officinalis Gaertn. The present invention also relates to a process of extraction, which is resistant to microbial load development.
Background & prior art:
Emblica Officinalis Gaertn is found both in wild and cultivated state in the mixed deciduous forest in India, ascending to 1300 meters on the hills .It is a hepatoprotective herb. The herb is a major constituent of phytochemicals and secondary metabolic bearing pharmacological activities. Although literature is available on the physiological effects of the active constituents present in the plant but little is reported on the mass production of extract from Emblica Officinalis Gaertn and presence of pathogens into the extract. Pathogens such as Streptococern, S. Robrinus, Steptococei Prevotella oris are human's carcinogens agents. Listeria monocytogene is also a gram positive rod -shaped bacterium that causes listeriosis, a potentially life threaten illness Microbes are ubiquitous and some of them cause serious disease in humans, the members of enterobacteriacea, organism that cause urinary tract infections and dental caries, The common species of S aureus are pathogenic and cause endcarditis, urinary tract infection pyelonephritis (Inflammation of Kidney). The end product of S. aurus are also pathogenic, they cause clot formation, lyse WBC.
Since the mid 1960's, medicinal herbs have been the subject of phytochemicals research to determine the active constitutes and its pharmacological activities. It is

well known in prior art that Emblica Officinalis Gaertn has analgesic properties. Hypoglycemic effect of methanol extract of phyllanthis amours has been reported by Kuttan et al (2002). However not much is known in order to prepare a safe herbal preparations and avoid presence of microbial contamination.
The aim of the present study is to develop a novel method for extracting the active ingredients in Emblica Officinalis Gaertn using suitable pH and a solvent system, which yields extract that exhibits resistance against microbial loads and residual toxin for long time. The selection of solvent for specific herb plays a key role in extraction. In the present study several solvents have been compared in terms of their efficiency in extraction wherein the extract is found to be microbial load free.
Extraction without any elevation in temperature only provides the actual and pure active principle in their natural form. The use of elevated temperature could change or could degrade the primary active components, which may undergo change in metabolism and result in altered property of molecule and their pharmacology in the preparations. However prior art teaches the extraction at elevated temperatures.
The Ninhyderine test demonstrated that the boiling of vegetable material could denature the basic property of material.
1. 1 mg methanolic solution of Ninhyderine when apply to the raw material of amla
it exhibits violet color.
2. 1 mg methanolic solution of Ninhyderine when apply to the boiled raw material of
amla it did not exhibits any color.
Objectives:
1. An objective of the present invention is to provide an efficient extraction system to provide a microbe free extract.

2. Another objective of the present invention is to provide a scheme for tracking
the sources of contamination.
3. A further objective of the present invention is to provide a routine monitoring
of microbial flora in raw and formulated herbs.
Detailed Description:
The present invention relates to a novel process for extraction of active principles selected from Tannin, Gallic acid, Ellagic acid, Emblicol, Phyllantidine of Emblica Officinalis Gaertn, which is resistant to microbial load development.
The present invention relates to an efficient solvent system for extraction of the active principle, wherein although eight solvent were studied, only one solvent extract exhibited complete resistant against harmful pathogens. Extract of two solvents have broad activity as compared to of six solvent extracts. The alcoholic extract has greatest activity followed by aqueous extracts. Use of proper preparation techniques could provide the safe and pharmacologically active herbal preparations.
The solvent for extraction was selected from ethanol, petroleum ether, benzene, acetone, water, acetonitirle, ethyl acetate and chloroform.
The extraction by ethanol at pH 7.2 exhibited the resistance against microbial load and residual toxins, aqueous extract at pH 8.2 also exhibits less resistance against microbial load and residual toxins as compared to ethanol extracts. The pH of rest of the solvents was also kept from about 6 to about 8.7.
On the other hand, acetone and ether exhibited significant development of e-coli at room temperature in the intervals of five days, fifteen days and after one month.

Microbial determination is done by taking sample of the extract in Soybean casein digest medium and incubated in test tube in a BOD Incubator. (NSW-151, Universal) at 35°C - 37°C for about 24 hrs. The enriched sample is poured in following specific media for different bacterial pathogens.
1) TAMC (Total Aerobic Microbial Count)-SCDA (Soybean Casein Digest Agar
(Pour plating)
2) TYMC (Total Yeast Mold Count)-SDA (Sabouraud Dextrose Agar)
3) E. coli-MacConkey Agar
4) S. aureus- Mannitol salt agar
5) P. aeruginosa- Cetrimide agar
6) Salmonela- Brilliant Green Agar
PI writ the full form of TAMC & TYMC-SDA
Table 1 shows the comparative statement of Detection of bacteria and fungi in the extract
(Table Removed)

a. Key +++=marked,++=moderate,+=slight, —absent
b. A to J represent the extract code number.
Examples:
Example 1:
Powdered Amla (100 g) is sieved in cloth and the dried fine material is gradually ground and weighed (50g) and aqueous slurry is prepared before loading for solvent extraction. Petroleum ether (250 ml) is added at a temperature of about 25°C to 35°C and both the layers are collected separately in Petri dish. Then these Petri dishes are kept in open at room temperature for five, fifteen & thirty days before the testing of microbial load.
Example 2:
Powdered Amla (100 g) is sieved in cloth and the dried fine material is gradually ground and weighed (50g) and aqueous slurry is prepared before loading for solvent extraction. Benzene (250 ml) is added at a temperature of about 25°C to 35°C and both the layers are collected separately in Petri dish. Then these Petri dishes are kept in open at room temperature for five, fifteen & thirty days before the testing of microbial load.
Example 3:
Powdered Amla (100 g) is sieved in cloth and the dried fine material is gradually ground and weighed (50g) and aqueous slurry is prepared before loading for solvent extraction. Chloroform (250 ml) is added at a temperature of about 25°C to 35°C and both the layers are collected separately in Petri dish. Then these Petri dishes are kept in open at room temperature for five, fifteen & thirty days before the testing of microbial load.
Example 4:
Powdered Amla (100 g) is sieved in cloth and the dried fine material is gradually ground and weighed (50g) and aqueous slurry is prepared before loading for solvent
extraction. Ethyl acetate (250 ml) is added at a temperature of about 25°C to 35°C and both the layers are collected separately in Petri dish. Then these Petri dishes are kept in open at room temperature for five, fifteen & thirty days before testing of microbial load.
Example 5:
Powdered Amla (100 g) is sieved in cloth and the dried fine material is gradually ground and weighed (50g) and aqueous slurry is prepared before loading for solvent extraction. Benzene (250 ml) is added at a temperature of about 25°C to 35°C and both the layers are collected separately in Petri dish. Then these Petri dishes are kept in open at room temperature for five, fifteen & thirty days before testing of microbial load.
Example 6:
Powdered Amla (100 g) is sieved in cloth and the dried fine material is gradually ground and weighed (50g) and aqueous slurry is prepared before loading for solvent extraction. Acetone (250 ml) is added at a temperature of about 25°C to 35°C and both the layers are collected separately in Petri dish. Then these Petri dishes are kept in open at room temperature for five, fifteen & thirty days before the testing of microbial load.
Example 7:
Powdered Amla (100 g) is sieved in cloth and the dried fine material is gradually ground and weighed (50g) and aqueous slurry is prepared before loading for solvent extraction. Ethanol (250 ml) is added at a temperature of about 25°C to 35°C and both the layers are collected separately in Petri dish. Then these Petri dishes are kept in open at room temperature for five, fifteen & thirty days before testing of microbial load.
Example 8:
Powdered Amla (100 g) is sieved in cloth and the dried fine material is gradually ground and weighed (50g) and aqueous slurry is prepared before loading for solvent extraction. Acetonitrile (250 ml) is added at a temperature of about 25°C to 35°C and both the layers are collected separately in Petri dish. Then these Petri dishes are kept in open at room temperature for five, fifteen & thirty days before testing of microbial load.
Example 9:
Protocol for Microbial study
1gm of sample is incubated in 9 ml Soybean casein digest medium and
incubated in test tube in a BOD Incubator.(NSW-151,Universal) at 35°C - 37°C for
24hrs.
The enriched sample is poured in following specific media for different bacterial
pathogens.
1) TAMC-SCDA (Pour plating)
2) TYMC-SDA
3) E. coli-MacConkey Agar
4) S. aureus- Mannitol salt agar
5) P. aeruginosa- Cetrimide agar
6) Salmonela- Brilliant Green Agar
Example 10:
Determination bacterial activity on extract
A well of 6 mm. diameter is prepared in nutrient agar with cork barer in steroid condition and sealed with 2% agar. 100 ml of log face broth culture of respective bacterial strains is spread on the plate 0.50mg extract is poured with absolute alcohol (AR grade), which serves as control. The plate is incubated at 37°C for 24-48 hours.
The microbial growth inhibition is determined as the diameter of zone of inhibition around the well in mm.

We Claim:
1. A process for extraction of the active principle of Emblica Officinalis Gaertn comprising:
a. Grinding Powdered Emblica Officinalis Gaertn;
b. Preparation Aqueous slurry;
c. Loading the slurry onto an organic solvent;
d. Extracting the organic layer and
e. Drying the organic layer.
2. The process as claimed in Claim 1, wherein the organic solvent is selected from
Ethanol, acetone and acetonitrile.
3. The process as claimed in Claim 1, wherein the drying is carried out at a
temperature between about 25°C and about 35°C.