THE PATENTS ACT, 1970 COMPLETE SPECIFICATION
Section 10
"Method of Preparing Aloin"


Indian Council of Agricultural Research, an Indian organization of Krishi Bhavan, Dr. Rajendra Prasad Road, New Delhi-110001.

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is to be performed:


Field of invention
The present invention relates to a method of preparation of aloin with high purity and yield, more particularly to a method of extracting aloin from aloe leaf exudates / yellow sap, or of derived products. The aloin obtained from this method is of superior quality and higher yield and hence suitable for industrial and pharmaceutical purpose.
Background of the invention
Aloe refers to any species of the genus aloe, succulent perennials of the family Aloaceae, native to warm dry area of south and tropical Africa, but cultivated in many countries around the world. Among the numerous species of aloe, Aloe barbadensis (Syn. Aloe vera), Aloe ferox (Cape aloes) Aloe secundiflora and Aloe arborescens are commercially grown for gel and leaf yellow sap.
Aloe cultivation worldwide exceeds 23,600 ha (International Aloe Science Council). The main aloe producing countries are Mexico (10,700 ha), Dominican Republic (3,500 ha), Venezuela (3,400 ha), China (2000 ha), Thailand (1500 ha), United States of America (648 ha), Costa Rica (520 ha), India (300 ha), Malaysia (270 ha) and several other countries to a limited area. The areas under cultivation in many countries are increasing as the demand for aloe products are growing worldwide. Globally aloe products market is estimated to be in the range of $125 million. With a potential global market close to $1 billion at present, aloe is an important medicinal crop with great potential.
The juice of aloe leaves contains aloin, which is traditionally used as a purgative. The solidified juice originating in the pericycle and adjacent leaf parenchyma and flowing spontaneously from the cut of leaf, which is allowed to dry with or without the aid of heat. Aloin is the major anthraquinone compound present in the yellow sap of aloe having a chemical formula of 1,8 dihydroxy - 10 - ((3-D-Glucopyranosy) - 3 - (hydroxyl methyl) - 9(1 OH) anthracenone (Molecular formula C21H22O9). Dried aloe exudates of curracoa or barbados aloes aloe drived from Aloe barbadensis Mill contains not less than 28% of hydroxyanthracene derivatives, expressed as barbaloin. (British Pharmacopoeia) However cap aloe derived from A. forex Miller contains not less than 18% of anthracene derivatives. (Pharmacopiea franeaise, Paris, Adrapharma, 1996)
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Aloe paste or dried aloe juice with high aloin content is used in numerous product formulations by pharmaceutical industry. Aloin and its derivatives have important pharmaceutical properties and used in various medications. For example, rhein (9,10-dihydro-4,5-dihydroxy-9,10-dioxo'2-anthracenecarboxylic acid), diacerein its diacetylated derivative ( 4,5-bis (acetyloxy) 9,10-dihydro-4,5-dihydroxy-9,10-dioxo-2-anthracenecarboxylic acid) are described in numerous scientific publications as well as in patent FR-A-2,508,798 and several analogues thereof are important from a commercial point of view, are known for use in the treatment of abnormal degeneration of the connective tissue, e.g. inflammatory states of the joints and of the connective tissue, such as for example rheumatoid arthritis, osteoarthritis, osteoporosis, or of other diseases (GB 1,578,452), such as acute respiratory syndrome of adult (e.g: asthma) and pulmonary emphysema. A regular dose of 25 to 200mg of diacerhein daily administered as treatment. Patients treated have shown marked improvements in the clinical signs of osteoarthritis. There are no significant toxicities associated with rhein or diacerhein compounds, with the exception of a laxative effect found in a significant proportion of patients treated. Most research associated with rhein or diacerhein compounds has focused on attempting to alter administration or formulation to increase absorption and bioavailability, in an attempt to reduce this side effect.
These pharmaceutically important compounds can be derived from aloin. French patent (No. 2,508,798-Bl) and Belgian patent (No. 875,945) disclose the preparation of diacerein via dissolution of l,8-dihydroxyanthraquinone-3-carboxylic acid in excess acetic anhydride, in the presence of sulphuric acid as a catalyst. However, the diacerein obtained by means of the processes contain, as a by product considerable amounts of a aloe-emodin derivative, which has mutagenic activity even in amounts as low as 70 ppm. To produce diacerhein in high yields and especially free from said impurities was on a commercial scale is based on the use of aloin as raw material (European patent application No. 636,602 Al: US Pat. No. 3,089,879 & DE 80,407) was developed. Diacerein was synthesized via acetylation of aloin followed by oxidation and purification by means of crystallization. The said method requires high purity aloin of minimum 90%
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purity, as a sum of alpha and beta-barbaloin (or aloin), determined on the basis of the anhydrous product.
Aloin and aloe emodin were reported to be lethal to insect pests and worms and used as biopesticide. Further, the use of rhein in the treatment of mycobacterial infections was also reported. Mycobacteria are aerobic organisms that include numerous species pathogenic for both humans and animals. Mycobacterium tuberculosis, the organism responsible for tuberculosis, is one species of mycobacteria that is presently of great concern to humans. It is resistant to many antibiotics currently available, and treatment often requires combinations of three or more drugs for periods exceeding one-year. Mycobacteria have a unique cell wall that has very high lipid content, creating a hydrophobic cell surface. This unique cell wall not only inhibits the permeability of nutrients into the cells, causing the mycobacteria to have a relatively slow growth rate, it also impairs the effectiveness of treatment with antibiotics. The continuing development of drug-resistant organisms, as well as the seriousness of mycobacterial infections in immunosuppressed individuals, confirms the need for additional pharmaceutical agents to treat these infections. Hence, the rhein and related anthroquinone compounds can be the target molecules for the treatment of drug resistant bacteria.
Recently, an aloe emodin derivative was reported in the treatment of cytotoxicity to tumor cells and effective against neoplasias. The said emodin derivative in position 3' exhibited improved solubility properties and at the same time showed in vitro cytotoxicity to tumor cells which were of neuroectodermal origin. Several reports are coming up with wide variety of uses for the anthroquinone derivatives from aloin in the recent times implying the increasing demand for this pharmaceutically important natural product.
Description of prior art
The conventional methods for extraction of aloin from aloe were mostly used the hardened dry sap as starting material. The yellow sap of aloe is heated in open air under atmospheric pressure and the concentrated residue is extracted and aloin is purified by
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recrystalization. An improved method over the traditional method for extraction of aloin and aloe emodin,from plants or plant extract was described in EP-A-374 890. The method used a hydrophilic solvent followed by filtration and evaporation to get thick syrup and subjecting it to several extractions with different solvents and recrystalization to obtain aloin. A further improvement for the extraction and purification was described in US patent 6,506,387. The invention essentially comprised of an extraction of an exudate of aloe in the presence of an aliphatic diol or triol of low molecular weight, followed by purification by crystallization. In the first step of the method an aliphatic diol or triol of low molecular weight is added to the yellow aloe juice, and a concentration was performed, then an extraction was realized in the second step. The extraction is followed by a recrystallization from an alcohol. The method claimed to produce aloin content of over 80%, generally between 85 and 90% under the conditions that are applicable when realizing the method on an industrial scale, the overall yield relative to the aloin present in the starting material (yellow aloe juice or derived product) is higher than 50%). The above said method had another advantage of allowing an important reduction in the amount of alcohol used in the purification step following extraction. Thus, in the case of a recrystallization in isobutanol, the amount of alcohol is between 10 and 20 volumes per volume of aloin present in the medium, as against 40 to 60 volumes in the usual methods.
Draw backs of the prior art
Conventional extraction methods of aloin from aloe produce inferior qualities of aloin and contain several impurities, which make them particularly unsuitable for industrial and pharmaceutical synthesis. As the impurities results in the production of undesirable intermediary compounds which are difficult to separate from the desired end products. These methods typically contain several extraction procedures with large quantities of solvents.
As an alternate route to natural aloin from plant sources, chemical synthesis of aloin was floated by various workers. Earlier attempts by various scientists to chemically synthesize the aloin and chrysophanic acid substrates initially met with little success. Particularly for
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aloin, all efforts to chemically synthesize the carbon-carbon linkage between the anthraquinone moiety and the glucose moiety were unsuccessful. One attempt of note was that published by Ayyanger et al., J. Sci. Ind. Res. (India) 20B:493-97 (1961). Unfortunately, poor yields and costly starting substances plague the chemical scheme utilized by Ayyanger et al., which starts with l-amino-5-chloro-anthraquinone for the synthesis of aloin.
Necessity to remove the drawbacks of the prior art
A more efficient and economical method is needed to fulfill the larger demand for aloin is urgently felt. Hence an improved method for preparation of aloin with higher purity and relatively higher yield was developed. The present invention described here related to a method of preparation of aloin with high purity of above 90% to 95% even upto 97% with yield of aloin over 90%) of starting material. Hence aloin obtained from this method is of superior quality and higher yield and will meet the demand for higher purity above 90% required for the production of diacerhein on a commercial scale.
Since the method explained in the preferred embodiment is easy to perform and can be scaled up to industrial production and provide higher purity than the industrial requirement. This method is cheaper and less time consuming compared to earlier methods. In particular, the method according to the invention yields an improved selectivity of extraction and considerable decrease in the amounts of solvent used in the purification under the conditions under which the method is practiced on an industrial scale.
Summary of the Invention
The objective of the invention is to obtain pure aloin from aloe with minimum loss using easier method in a shorter time. The present invention is for an improved method for purification of aloin from aloe leaf exudates or the derived products thereof. The yellow sap of aloe or the derived product is dissolved in pyridine and homogenised thoroughly using a mechanical stirrer and centrifuged at room temperature (25-30°C) at 5000rpm for 10-20 minutes. The precipitate is discarded and supernatant is taken in a container. The
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supernatant is dissolved in excess of ethyl acetate and the resulting precipitate is removed and yellow solution is concentrated in reduced pressure (220-190 mbar) to get thick brown syrup. The syrup is added with excess of ethyl acetate and the contents are kept under dark for 15-20 minutes for the impurities to settle. The yellow solution is again filtered twice and concentrated under reduced pressure at 55° C. The resultant brown residue is either purified again with addition of excess ethyl acetate or dried further to get a yellow crystalline powder which is washed with diethyl ether and dried to get a free flowing yellow crystalline powder which contains above 90% pure aloin with aloin A to aloin B in ratio of 3:1 and suitable for industrial purposes. The powder so obtained can also be purified further by recrystallization in suitable alcohol to get a very high purity aloin product (95-97%).
Detailed Description of the invention
The present invention is for an improved method for purification of aloin from aloe leaf exudates or the derived products thereof. The yellow sap of aloe or the derived product is dissolved in pyridine and homogenised thoroughly using a mechanical stirrer and centrifuged at room temperature (25-30° C) at 5000rpm for 10-20 minutes. The precipitate is discarded and supernatant is taken in a container. The supernatant is dissolved in excess Of ethyl acetate and the resulting precipitate is removed and yellow solution is concentrated in reduced pressure (220-190 mbar) to get thick brown syrup. The syrup is added with excess of ethyl acetate and the contents are kept under dark for 15-20 minutes for the impurities to settle. The yellow solution is again filtered twice and concentrated under reduced pressure at 55° C. The resultant brown residue is either purified again with addition of excess ethyl acetate or dried further to get a yellow crystalline powder which is washed with diethyl ether and dried to get a free flowing yellow crystalline powder which contains above 90% pure aloin with aloin A to aloin B in ratio of 3:1 and suitable for industrial purposes. The powder so obtained can also be purified further by recrystallization in suitable alcohol to get a very high purity aloin product (95-97%).
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One of the solvent selected for the extraction of aloe exudates or derived product is pyridine which is an important industrial organic solvent, both as a fundamental building block or a starting material in the synthesis of compounds used as an intermediate in making pharmaceuticals, food flavorings, dyes, rubber chemicals, adhesives, paints, explosives, insecticides, herbicides and disinfectants and also as solvent in organic synthesis. Pyridine is widely used as a versatile solvent, since it is polar but aprotic. It is fully miscible with a very broad range of solvents including hexane and water. The experiments with selected organic solvents for extraction of aloin proved that pyridine was the most suitable solvent because of its solubilizing capacity of aloin and easy separation of impurities when suitable solvent is added during the extraction procedure. Since aloe exudates are highly soluble in pyridine, it acts both as a solvent and stabilizing agent for the starting material. In addition to the high solubility of this solvent, it is unique in separation of other phenolic compounds when ethyl acetate is added to the extraction medium.
The invention will be readily understood from the following examples.
Example 1:
In a beaker of IL, 100 ml of fresh exudates of aloe leaf is taken and 100ml of pyridine is added and homogenized thoroughly using a mechanical stirrer. The homogenized solution is then centrifuged at 5000rpm for 10 minutes and supernatant is taken for further extraction. To this mixture, 600 ml ethyl acetate is added and mixed. The contents are kept 20-30 minutes under room temperature (25-30° C) for the precipitates to settle. The precipitate is discarded by decanting or centrifugation or filtration and the supernatant is again filtered and concentrated in rotary evaporator under reduced pressure 240-190 mbar at 55° C until the concentrate became viscous (20-40 ml volume) brown syrup. The concentrated residue is re-extracted with 300 ml ethyl acetate. The mixture is kept in dark for 20-30 minutes in room temperature (25-30° C) for the impurities to settle as brown sticky precipitate which is filtered out and the yellow ethyl acetate fraction is again concentrated in the rotary evaporator until it becomes a yellow crystalline powder having 90% pure aloin with more than 90% recovery. Optionally one can dissolve the
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contents in hot isopropyl alcohol (60-70 C) and undissolved residue is discarded and the supernatant solution is dried again in evaporator under reduced pressure (80-60 mbar) at 50-60° C temperature preferably at 55° C. The dried yellow crystalline powder is washed twice with diethyl ether and dried until a free flowing crystalline yellow powder is obtained. The final product contains above 90% pure aloin and having up to 85-90% recovery.
Example 2:
The method according to the present invention follows the principle of extraction described in example No.l, and instead of fresh yellow sap of aloe, the sun or oven dried exudates is made into powder and used for extraction.
One proceeds as indicated in example No. 1, but uses 20g of dried powder of aloe leaf exudates. This powder is added with 75-100ml of pyridine and homogenized thoroughly using a mechanical stirrer. The homogenized solution is then centrifuged at 5000rpm for 10 minutes and supernatant is taken for further extraction. To this mixture 600 ml ethyl acetate is added and mixed. The contents are kept 20-30 minutes under room temperature (25-30° C) for the precipitates to settle. The precipitate is discarded by decanting or centrifugation or filtration and the supernatant is again filtered and concentrated in rotary evaporator under reduced pressure 240-210mm Hg at 55°C until the concentrate became viscous (30-50ml volume) brown syrup. The concentrated residue is re-extracted with 300 ml ethyl acetate. The mixture is kept in dark for 20-30 minutes at room temperature (25-30° C) for the impurities to settle as brown sticky precipitate which is filtered out and the yellow ethyl acetate fraction is again concentrated in the rotary evaporator until it becomes a yellow crystalline powder having 90% pure aloin with more than 90%
recovery. Optionally one can dissolve the contents in hot isopropyl alcohol (60-70 C) and undissolved residue is discarded and the supernatant solution is dried again in evaporator under reduced pressure (80-60 mbar) at 50-60 C temperature preferably at 55 C. The dried yellow crystalline powder is washed twice with diethyl ether and dried
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until a free flowing crystalline yellow powder is obtained. The final product contains above 90% pure aloin and having up to 85-90% recovery.
Example 3:
One proceeds as indicated in example No. 2, but uses 20 g of freeze dried powder of aloe leaf exudates. This powder is added with 100 ml of pyridine and homogenized thoroughly using a mechanical stirrer. The homogenized solution is then centrifuged at 5000rpm for 10 minutes and supernatant is taken for further extraction. To this mixture 600 ml ethyl acetate added and mixed. The contents are kept 20-30 minutes under room temperature (25-30° C) for the precipitates to settle. The precipitate is discarded by decanting or centrifugation or filtration and the supernatant is again filtered and concentrated in rotary evaporator under reduced pressure 240-210 mm Hg at 55° C until the concentrate became viscous (30-50 ml volume) brown syrup. The concentrated residue is re-extracted with 300 ml ethyl acetate. The mixture is kept in dark for 20-30 minutes at room temperature (25-30° C) for the impurities to settle as brown sticky precipitate which is filtered out and the yellow ethyl acetate fraction is again concentrated in the rotary evaporator until it becomes a yellow crystalline powder having 90% pure aloin with more than 90% recovery. Optionally one can dissolve the contents in hot
isopropyl alcohol (60-70 C) and undissolved residue is discarded and the supernatant solution is dried again in evaporator under reduced pressure (80-60 mbar) at 50-60 C temperature preferably at 55 C. The dried yellow crystalline powder is washed twice with diethyl ether and dried until a free flowing crystalline yellow powder is obtained. The final product contains above 90% pure aloin and having up to 85-90% recovery.
Example 4:
One proceeds as indicated in Example 1 and obtains the yellow crystalline powder. The crystalline powder so obtained is dissolved in hot alcohol (60-70° C) like ethanol / iso-propanol / iso-butanol and preferably iso-butanol up to saturation. The alcohol solution is cooled to 5° C and kept for Vi to 1 hour. The aloin crystals are collected by decanting the
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supernatant and washed with diethyl ether. The dried final product is so obtained contains 95% or more pure aloin with 80-90% recovery.
Example 5:
In a 50 litres container 10 kg of aloe exudates is taken and 3-5 litres of pyridine is added and mixed thoroughly for 15 minutes to get a homogenous solution. After leaving the contents for 20-30 minutes in room temperature (25-30° C), the supernatant is collected in a 200 liters vessel after decanting / ceritrifugation. To the supernatant, 150-200 litres of ethyl acetate are added slowly with constant mixing. The mixture is kept in dark for aside for 30-45 minutes at room temperatures (25-30° C) for the precipitation of impurities. The solution is filtered thrice to remove the impurities and concentrated in a vacuum
evaporator at 220-190 mbar pressure in the temperature range of 50 -70 C until thick
brown syrup is obtained. To this brown viscous material, 50-75 litres of ethyl acetate is added and kept in the dark for 20-30 minutes at room temperature (25-30°C) again filtered to remove any impurities. The yellow transparent solution is dried like the earlier
steps and the resultant thick brown syrup is added with 15-20 litres of hot (60-70 C)
isopropyl alcohol and the contents are dissolved. The undissolved residue is discarded and the supernatant solution is dried again in evaporator under reduced pressure (60-80 mbar) at 50-60 C temperature preferably at 55 ° C. The dried yellow crystalline powder is washed twice with diethyl ether and dried until a free flowing crystalline yellow powder is obtained. The final product contains above 90% pure aloin and having up to 90% recovery. Ethyl acetate is recovered during concentration is distilled and can be reused for extraction.
Description of final product obtained:
The final product is a free flowing yellow crystalline powder with aloin purity over 90%. The final product contains aloin A and aloin B in the ratio of 3:1. The product is suitable for most of the industrial uses and for pharmaceutical purposes. The final product after recrystallization in alcohol is of superior quality with 95-97% purity.
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Table: Extraction purity and yield of aloin from different sources of aloe leaf exudates

Material used for extraction Aloin
content
(%) Purity of aloin obtained following extraction procedure (%) Yield of aloin when compared with starting material
Fresh aloe leaf exudates 45-55 90-95 -up to 90%
Dried aloe leaf exudates 25-30 Up to 90 -85-90%
Freeze dried exudates 40-50 90-95 -up to 90%
Yellow powder after first purification -90 95-97% 80-90%
Having thus described a few particular embodiments of the invention, various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements, as are made obvious by this disclosure, are intended to be part of this description though not expressly stated herein, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description is by way of example only, and not limiting. The invention is limited only as to the scope of the following claims.
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We Claim:
1. A method of preparing aloin comprising the steps of:
(a) adding azotrophic organic solvent to aloe exudates or derived products thereof,
(b) mixing homogenously the contents,
(c) centrifuging the mixed content and obtaining supernatant 1,
(d) adding ethyl acetate to said supernatant 1 and mixing,,
(e) decanting/centrifuging/filtering the contents to obtain supernatant 2,

(f) concentrating supernatant 2 in rotary evaporator under reduced pressure at temperature 50-60° C,
(g) mixing concentrated residue with organic solvent,
(h) decanting/centrifuging/filtering the contents to obtain supernatant 3,
(i) concentrating said supernatant 3 in rotary evaporator to obtain aloin as dry
crystalline powder, (j) optionally washing the powder with diethyl ether to obtain purified aloin.
2. The method as claimed in claim 1 wherein said aloin is more than 90% pure.
3. The method as claimed in claim 1 wherein said aloe exudates or derived products is selected from fresh exudates of aloe leaf, oven or sun dried aloe exudates or freeze dried powder of aloe leaf exudates.
4. The method as claimed in claim 1 wherein said azotrophic organic solvent is pyridine.
5. The method as claimed in claim 1 wherein in step f the pressure is progressively reduced from 240mbar to 160mbar at 55° C temperature.
6. The method as claimed in claim 1 wherein the concentrated residue obtained in step (f) is viscous brown syrup.
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7. The method as claimed in claim 1 wherein the washing in step (j) is preferably two times.
8. The method as claimed in claim 1 wherein the aloin obtained in step (j) is further purified using the steps of:

(a) dissolving aloin crystalline powder in alcohol,
(b) cooling solution to about 5° C,
(c) allowing the cooled solution to stand for about 30 min,
(d) decanting the supernatant,
(e) washing the aloin crystals with diethyl ether.

9. The method as claimed in claim 8 wherein said aloin is more than 95% pure.
10. The method as claimed in claim 1 wherein the extraction is followed by re-extraction by an organic solvent and precipitation in alcohol.
11. The method as claimed in claim 1 wherein the organic solvent is selected from ethyl acetate, acetone, methanol, ethanol n-propanol, iso-propanol, n-butanol, tertiary butanol or iso-butanol.
12. The method as claimed in claim 1 wherein the aloe used in step (a) is Aloe barbadensis , Aloe capernsis, Aloeferox or Aloe Africana.
13. Method for preparing aloin substantially as herein described with reference to
accompanied drawings and examples.
Dated this 12 day of2008 rh jjp-
(Neeti Wilson)
Of Anand And Anand Advocates
Agent for the Applicant
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