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Title:
PROCESS FOR ISOLATION AND SEPERATION OF 3 BIOLOGICAL ACTIVE COMPOUNDS FROM ALOE VERA LEAF GEL FILET AND THE 3 COMPOUNDS
Document Type and Number:
WIPO Patent Application WO/2005/082919
Kind Code:
A1
Abstract:
Aloe vera leaf gel from Aloe barbadensis Miller is one of the most popular herbal material on the market. Most consumers worldwide are familiar with aloe’s use in skin-care products, but aloe can also be used as a beverage. Aloe products for internal use have been promoted for viral infections, wounds, ulcers, diabetes, cancer, headaches, arthritis, immune deficiencies, and many other conditions. The therapeutic effects of aloe gel has been scientifically well researched, and proven. But the major problem lay in the isolation und structure elucidation of the biological active compounds. Many postulate that there are over 100 compounds which work in a synergy to bring about these effects or an acetylated polymannose which has up to date not convinced. This invention has finally provided a method to isolate and elucidate three compounds from aloe gel with clear and independent therapeutic effects. Symbolic for this, is the Veracylglucan C (a oligomalyl hexasaccharide) which up to date has never been found in any other plant on this Planet.

Inventors:
ESUA MACNIELL FLORENTIN (DE)
Application Number:
PCT/IB2004/000095
Publication Date:
September 09, 2005
Filing Date:
January 29, 2004
Export Citation:
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Assignee:
ESUA MACNIELL FLORENTIN (DE)
International Classes:
A61K31/7024; A61P31/00; C07H1/08; C07H13/04; (IPC1-7): C07H13/04; A61K31/7024; A61P31/00; C07H1/08
Foreign References:
US5902796A1999-05-11
Other References:
F. TARRACH ET AL.: "Ueber die beim Erhitzen von Aepfelsäure oder Citronensäure mit Glucose entstehenden Reaktionsprodukte", Z. LEBENSM. UNTERS. FORSCH., 1987, pages 283 - 288, XP008036695
D.-W. PARK ET AL.: "Lipase-catalysed synthesis of beta-methylglucoside esters containing an alfa-hydroxy acid", BIOTECHNOLOGY LETTERS, vol. 23, 2001, pages 1947 - 1952, XP008036693
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Claims:
CLAIMS Aloe vera leaf gel filet has been used therapeutically, certainly since Roman times and some claim even long before. The literature research prior to this invention contained, on one hand many case reports and more or less anecdotal accounts of the healing properties of this plant material extending from complaints like skin diseases, sores and a series of other health <BR> <BR> problems (viral infections, stomach ulcers, diabetes etc. ) [T. Reynolds, A. C. Dweck, Journal of ETHNO-PHARMACOLOGY 68 (1999) 3-37, UK]. On the other hand many scientific works have been carried out both in labs and clinical settings to isolate, separate and investigate the biological active compounds of this very popular medicinal plant materia
1. l. The results obtained were in some cases the same and in many other cases very contradictory [USA <BR> <BR> 2004 {HYPERLINK"http : //www. uspto. gov/patft/index. html"}, T. Reynolds, A. C. Dweck, Journal of ETHNOPHARMACOLOGY, UK 1999]. Despite these facts many companies in the United States of America and some individuals there and around the world have filed (are still filing) and some received patents for their isolation methods which led to a mixture of compounds with no complete chemical structure elucidation, no uniformity in the production batches und contradictory results in the clinical examination (Acemannan and Carrisyn). Because of the above mentioned summary non of these researchers and patent owners have succeeded in obtaining an approval to market a drug for use on human beings suffering from the different diseases mentioned through out. The drug approving authorities have clear objective scientific criteria to evaluate a chemical compound with given biological activities; these are e. g a clear and concise chemical analysis with validated scientific pharmacopeias methods, which include proper and clear chemical synthesis schemes or. isolation and separating methods, mass spectrometry, optical spectrometry, a good use of nuclear magnetic resonance spectrometry (NMR) to unambiguously elucidate the chemical structure, batch and dosage uniformity and clear reproducible and non contradictory pharmacological results. From the above mentioned reasons, I as a pharmaceutical scientist believe that my invention has fulfilled all objective scientific requirements which all other researchers up till now did not succeed in doing. The compounds, Veracylglucan A, B, and C from Aloe leaf gel filet have been discovered and tested with clear und profound bioassay guided modern chemical analytical methods.
2. The following biological activities were observed; Veracylglucan A and B showed remarkable antiviral activities on Herpes virus.
3. (to mentioned is the fact the fraction contained more than 90% Veracylglucan B and more over Veracylglucan A is also very unstable, which will imply that Veracylglucan B is the actual antiviral component). Aloe vera leaf gel extracts have been reported to be effective on disease conditions caused by other DNA viruses (CytomaglyVirus etc.) and RNA viruses (HIV.
4. etc. ), and I believe Veracylglucan A and B would be more or less a new remedy for ill health due to these viruses. Veracylglucan C showed a clear activation of human fibroblast, which are important cells for the production of wound healing compound in humans. This was observed alongside with the anti inflammatory effect of the mixture of the three compounds. These observations were clear and I can conclude that in disease conditions were tissue damage plays a central role, Veracylglucan C is a new remedy. All three compounds showed clear antibacterial activities (exact investigations are still in progress) Veracylglucan C has shown immune stimulating properties in the first screening phase (exact investigations are still in progress) It was finally observed that after the full chemical analysis, the Veracyglucan A and B had in a 10% watery solution a PH value of 3.78 at 21 degrees Celsius und Veracylglucan C a PH value of 5.5 at the same temperature. The Compounds were also studied in water (6%) at room temperature over three days the results showed that Veracylglucan C is the most stable of all the compounds followed by Veracylglucan B and the most unstable Veracylglucan C. The ester structure of the compounds are simply hydrolyzed to give malic acid, glucose, maltose etc. (these mixture is comparable to apple juice!). The three compound are very hygroscopic and become by room temperature in a space of hour amorphous as a result of relatively high temperature and moisture in the air. Due to these observations, that is why this method is the only way one can isolate and characterize these compounds and why many researchers working at normal ambient conditions did not succeed over the years to get proper hold of these compounds. Conclusively it was also noted that the compounds made just 0,8% of the total content of a full Aloe vera leaf gel filet, whereby Veracylglucan A 2%, Veracylglucan B 38% and Veracylglucan C 60% is available in this 0,8% special extract. Since we know concentration is very important parameter to qualify a biological effect, this is another reason why direct application of aloe vera gel filet extracts do not always yield the expected results. To my conviction these results of this invention give an answer to the thousands of questions surrounding aloe vera leaf gel filet and its therapeutic properties. These compounds have a very great market value, that's why they have to be protected from uncontrolled chemical syntheses.
Description:
Description of the Invention The leaf gel filet was care fully obtained using a kitchen knife from a whole leaf an adult aloe vera barbadensis Mill. , which was commercially obtained. This Gel was immediately homogenized with a common kitchen mixer, it was then centrifuged at 4 degrees Celsius by 10000 * g (gravitational constant), the supernatant was then immediately introduced in a simple commercially available cellulose ester dialysis membrane tubes with a molecular cut of weight (MCOW) between 6000-7000 Da. (the size of the tubes used were adapted to the quantity of matrial used) Dialyses was then carried out for 48 hours at 8 degrees Celsius against"aqua distillata" (distillated water). The external water phase and not the water phase in the tube! was then collected an immediately frozen with liquid nitrogen or in a freezer at minus 30 degrees Celsius. This ice mass containing Veracylglucan A, B and C was properly freeze dried (lyophilized) this resulted to a very hygroscopic and low density mixture of the three compounds. These compounds were then immediately separated with absolutely water free methanol 30 minutes under stirring and another 30 minutes was used for sedimentation in the system at 8 degrees Celsius, Veracylglucan A and B are very soluble in methanol while Veracylglucan C is totally insoluble in methanol under these conditions the methanol phase (supernatant) was then separated from the insoluble counterpart. The methanol solution was then immidiately treated to obtain the compounds by use of a rotary evaporator at 27 degrees Celsius and pressure 5 m Bar. The methanol insoluble Veracylglucan C was kept in the main time in the refrigerator at 8 degrees Celsius. After completely evaporating methanol, the three compounds were then dissolved separately (A and B making on fraction) in distillated water (prior to biological testing the three compounds were exhaustively-3 times 9 hours-dialyzed against distilled water at 8 degrees Celsius using tube membranes with MCOW= 100 Da for A and B and MCOW=1000 Da. in other to get rid of rests of methanol, A and B respectively) and immediately freeze dried. The dialysis was carried out all along in a ratio of 10 probe solution units in the Tubes to 90 external distillated water phase units.

The lyophilize compounds were then ready for Chemical analysis, a fraction was also lyophilized containing all three compounds. The mixture of compound were first analyzed using a gradient RP-18-HPLC-PDA Method with following parameters; acetonitrile /water (distilled) 100-70% (starting with acetonitril at 100 %), flow rate 0,5 ml/Min. at room temperature. These analysis gave the first indications that the mixture contained thre compounds. After one and two dimensional (for Veracylglucan C partial hydrolysis was carried out to solve structure elucidation problems) NMR analysis (experiments used; 1H, 13C, APT, 1H-1H-COSY, HMBC and HMQC) and mass spectrometry with ESI-MS for all three compounds and MALDI-TOF-MS only for Veracylglucan C. With these data clear chemical structures of the compounds could be produced. This the led to the application of capillary electrophoresis which became quite useful to complete this work. The analysis was carried out with a borate buffer at PH 10.5 for seven minutes this gave rise to three good peaks which could be determined with the PDA detector and distinct UV-spectra of the three compounds were then obtained during this process.

For the investigation of the biological activities the works were carried out with concentrations of 10,6, 4, and 2% relative to rest of the test media. The compounds were only removed from their storage at minus 30 degrees Celsius just a couple of minutes before thier introduction to the respective test systems.