This invention relates to a method or a process for producing a fraction enriched upto 100% of 3-0-acetyl-ll-keto- -bosweHic acid from an extract containing a mixture of boswe!lic acids obtained from the gum resin of Boswel!ia species.

Related Art

Bosweilia trees are native to Ethiopia, Somalia, India and the Arabian Peninsula. There are a number of different Bosweilia species, and the Bosweilia sacra is the particular plant that is endemic to the Sultanate of Oman, which grows in the southern part of the country (Dhofar Province).

The gum resin of Bosweilia species is commonly known as Frankincense and has been used for many years as an anti-inflammatory agent, particularly in traditional Ayurvedic medicine.

In particular, studies that have been carried out have established the antiinflammatory activity of particular alcoholic extracts of the gum resin obtained from Bosweilia serrata in mice and rats.

A detailed study on the structure requirements for boswellic acids indicated that of all the six acids that are contained in the gum resin of Bosweilia species, 3-0- acetyl-ll-keto-p-boswellic acid (hereinafter referenced as "AKBA') shows most pronounced inhibitory activity (Sailer, E, ,, et al, British J. Pharmacology, HZ, 615-618, 1996).

AKBA was first isolated at the end of nineteenth century (1898), but the structure confirmation by NMR was only established at the beginning of the twenty first century in 2003. However, ever since AKBA was first isolated at the end of the nineteenth century, there has been growing interest in testing this compound as a potential drug candidate for a number of different diseases,

For example, AKBA has been indicated in apoptosis of cancer cells, in particular brain tumours and cells affected by leukaemia or colon cancer. AKBA has also been shown to exhibit anti-inflammatory behaviour and AKBA is also thought to decrease the symptoms of asthma.

However, despite the potential biological and pharmacological importance of AKBA, research and testing has been relatively limited because of difficulties in isolating AKBA via conventional methods. The relatively small amounts of AKBA that can be isolated using conventional methods has resulted in AKBA traditionally having a relatively high price, thereby limiting the use of this molecule for research purposes and particularly for use in commercial pharmaceutical treatments.

One of the most significant hurdles with conventional isolation techniques is that an organic solvent extract of the gum resin of Boswel ^' a species contains a total of six different boswellic acids. As such, concentration of AKBA is typically only found in a small amount, commonly in the range of between 1 to 10% by weight.

This low concentration of AKBA presents a particular issue with the large scale isolation of AKBA from the extracts of Boswellia gums resins, which has conventionally been a very difficult and time consuming task.

To date, almost all procedures followed the protocol developed by Winterstein el a/, and Jauch et al., which employ chemical conversions followed by chromatographic separation to enhance the percentage of the AKBA.

One prior art approach that has followed a different approach to the conventional protocol developed by Winterstein et al, and Jauch et al. is disclosed in International patent publication no. WO 2003/074063. This prior publication discloses a preparation of up to 100% 3-0-acety!-ll-keto-3- boswellsc acid from an extract containing a mixture of boswellic acids obtained from gum resin of a mixture of Bosweliia species. The preparation is obtained by oxidizing the mixture of boswel!ic acids, acetylating the fraction and separating cbromatographically to obtain a fraction enriched in AKBA in an amount between 10-100% by weight,

Prior to the oxidation, acetylation and separation steps, the process disclosed in WO 2003/074063 utilises rigorous acid/base treatments in order to obtain the boswellic acid cluster from the Bosweliia species gum resin.

Summary of the Invention

On the other hand, the present invention simply utilises a methanolic extract obtained from gum resin of Bosweliia species, which is not subjected to the same acid/base treatments disclosed in WO 2003/074063,

Further, the method of the present invention utilises recycled chromatographic separation and a unique solvent in the mobile phase, which allows for the purification of AKBA in a single step, using a single solvent. This is compared with conventional approaches in which there are many steps in the chromatographic separation process, which use multiple solvents in order to isolate and purify the AKBA,

It would be advantageous to provide alternative methods of enriching the concentration of AKBA in the boswellic acids fraction obtained from an extract containing a mixture of boswellic acids to a desired concentration up to 100%. This would overcome at least some of the disadvantages of previously known approaches in this field, or would provide a useful alternative.

These and other advantages are met with the present invention, a broad form of which is set out herein, which additionally discloses optional and preferred aspects of the invention. These embodiments are not necessarily limiting on the invention, which is described fully in this entire document. FIG 1 shows the chemical structure of 3-0-acetyl-ll-keto-3-boswellic acid (AKBA),

FIG 2 shows the six boswellic acids (Bl to B6) present in an extract obtained from the gum resin,

FIG 3 is a flow chart showing the chemical modification and purification steps to enhance the yield of AKBA.

FIG 4 is a bar graph showing the yield enhancement of AKBA following the isolation steps of the current invention.

Detailed De cri tion

The present invention relates to large scale isolation of the most active boswellic acid, 3-0-acetyl-l l-keto-3-boswellic acid (AKBA) from extracts obtained from the gum resin of Boswe!lia species. The chemical structure of AKBA is shown in FIG 1.

Furthermore, the present invention is aimed at enriching the concentration of AKBA in the boswellic acids fraction to a desired concentration up to 100% by weight. The invention also relates to the process of removing inactive or less potent boswellic acids from the fraction by converting them to AKBA. This is achieved through a combination of chemical reactions and physical separations by chromatographic methods. This method of enriching the concentration of AKBA in the boswellic acids fraction is shown in FIG 3,

In particular, the present invention utilises vacuum liquid chromatography (VLC) to directly obtain the boswellic acids (BAs) cluster from the gum resin of the Bosweliia species without any need for further acid/base treatments. This approach reduces the number of steps in the purification/isolation process and reduces the loss of the yield of AKBA in the final sample.

The chemical modification of the crude extract enhances the yield of AKBA via a two-step approach, firstly by oxidation followed by temperature-controlled NEOS microwave-assisted acetylation. IMEQS microwave is used to assist acetylation of secondary alcohols.

As shown in FIG 2, the extract obtained from the gum resin of Boswellia species contains a ^W BA cluster" (which includes BA, ABA, KBA, AKBA) along with some other metabolites. As shown in FIG 3, in the first step of oxidation, the BA cluster is converted to a "KBA cluster" (KBA and AKBA), with double enhancement in the overall percentage of AKBA, This "KBA cluster" is then subjected to temperature-controlled NEOS microwave assisted (MWA) acetylation, which converts the KBA cluster into the AKBA.

The method or process of the present invention isolates and purifies AKBA in an extract obtained from the gum resin of Boswellia species, particularly Boswellia sacra using recycled HPLC. This method very effectively removes the other boswellic acids closely associated with AKBA, providing quantitative isolation as well as high purity AKBA in the final sample. Recycled chromatographic separation allows the recycling of the sample, in part or full, and increases the separation efficiency of the process while keeping the peak dispersion to a minimum. Specifically, the present invention utilises a novel solvent of 1% ethanol blended chloroform in the mobile phase during the separation stage, which provides a very efficient isolation of AKBA.

As discussed above, the organic solvent extract obtained from the gum resin of Boswellia species contains six boswellic acids. These acids are shown in FIG 2 as Bl, B2, B3, B4, B5 and B6.

As shown in FIGS 3 and 4, the concentration of AKBA (B2) in the natural boswellic acids fraction ranges from 1 to 10%. However, the present invention demonstrates a significant increase in the percentage of AKBA from the original fraction to the final product. The enhancement procedure as well as the purification steps were followed-up by analytical HPLC. The crude resin was found to contain around 4% of AKBA, while the methanolic extract contains around 8% AKBA. After the VLC separation, chemical enhancement and recycling preparative HPLC purification 100% purity of the sample was achieved.

As discussed above, the present invention relates to a method for isolating and purifying 3~0~acetyl~ll-keto~3~ boswellic acid (AKBA) from an extract obtained from the gum resin of Boswellia species containing a mixture of boswellic acids. The extract is a methanolic extract obtained by vacuum-liquid chromatography from gum resin of Boswellia species. The extract is preferably a Frankincense resin of certified botanical origin obtained from the Dhfoar region in the Sultanate of Oman.

During an initial oxidation step, the boswellic acids in the extract are oxidised, thereby forming a fraction containing keto-boswellic acids. In a subsequent acetylatlon step, these keto-boswellic acids in the fraction are converted into 3-0-acetyl-ll-keto- -boswellic acid (AKBA) by microwave assisted temperature-controlled acetylatlon.

Finally, the fraction is purified by recycled HPLC chromatographic separation, which utilises 1% ethanol blended chloroform in the mobile phase to enrich the concentration of 3~0-acetyl~ll~keta~ ~boswellic acid (AKBA) in the fraction. In this final sample, the AKBA in the fraction may be enriched up to 100% by weight.

Boswellic acids exist in frankincense as a cluster of triterpenic acids (BAs cluster) and other trsterpenoids with similar Rf values. The difficulty in isolating individual BAs using normal HPLC is due to their close polarity in which the peaks appeared merged. Utilising recycled HPLC, the separation of peaks appears from the first cycle. However, good separation that allows isolation is achieved after four cycles.

The first step in the process or method of the present invention involves the oxidation of the boswellic acid mixture to keto-boswellic acids. An oxidant conventionally used for allylic oxidation is preferably used for this step. The preferred oxidant used for this step is AZ-Bromosuccinsmide (NBS) and dioxane, but other oxidants with similar characteristics can also be used within the scope of the present invention, For example, oxidizing agents such as selenium dioxide in a suitable solvent, sodium dichromate AcQH-AczO or t-butylchromate in CC1 - AcGH-Ac ₂ G, CrOs- Pyridine can also be utilised to conduct oxidation step. Dry material obtained after oxidation showed 10-12% by weight AKBA by HPLC analysis.

The second step of the process or method of the present invention involves conversion of the ll-keto- -boswellic acids obtained from the oxidation step into 3-0-acetyl-ll-ketO" -boswellic acid (AKBA) by microwave assisted acetylation. The microwave reactor allowed the selection of energy, solvent and time. Temperature was monitored by an infrared sensor. The oxidised keto-boswellic acids, pyridine, 4~(dimetbylammo)-pyridine and acetic anhydride in dichloromethane were heated for 1 min. The solvent was set for dichloromethane and the power was fixed for 500W as optimum microwave power density.

The NEOS temperature-controlled (MWATC-NEOS) acetylation is used in this invention for the first time to assist the standard acetylation. This method allowed the selection of the solvent and temperature. This modification allowed the reduction of time from 4 hours (conventional acetylation) to 1 min and enhancement of yield. Dry material obtained after acetylation showed 30-40% by weight AKBA by HPLC analysis.

Alternately, the first step in the process involved acetylation of the boswel!ic acids mixture to acetylated bosweilic acids. This step could be executed by any typical acetylating agent like acetic anhydride/pyridine.

The third step of the process or method of the present invention involves chromatographic separation, specifically by recycling HPLC utilising novel solvent 1% ethanol blended chloroform in the mobile phase. In this way, higher grade AKBA is obtained from the acetylation mixture compared with conventional chromatographic methodology. Solid supports such as one or more of silica gel, reversed phase silica, alumina, sephadex and TOYOPEA L ^® can be used in the process.

The present invention utilises recycling HPLC, which allows the removal of the other boswel!ic acids closely associated with AKBA that appeared as shoulders via 4 purification cycles. The use of 1% ethanol blended chloroform in the mobile phase, afforded more than 99% pure AKBA via a single purification step using one solvent.

Whilst HPLC is the preferred separation method, other chromatographic techniques selected from gravity column, flash chromatography, reversed phase chromatography, preparative high pressure liquid chromatography and the combinations thereof can also be utilised by this invention.

While the invention has been described with reference to preferred embodiments above, it will be appreciated by those skilled in the art that it is not limited to those embodiments, but may be embodied in many other forms.

In this specification, unless the context clearly indicates otherwise, the word "comprising" is not intended to have the exclusive meaning of the word such as "consisting only of", but rather has the non-exclusive meaning, in the sense of "including at least". The same applies, with corresponding grammatical changes, to other forms of the word such as "comprise", etc.

The following example illustrates one of the best methods of carrying out the process according to this invention.

The samples of various grades of frankincense were collected from different locations in Dhofar. The freshly collected air-dried material (500 g) was crushed and extracted by maceration in 80% methanol for 10 days (3x5 L). The combined methanol extract was evaporated and the concentrated viscous extract (200 g) was fractionated by VLC over silica gel (1.4 kg), and eluted with hexane and gradients of chloroform up to 100% and methanol up to 20%. Various sub-fractions and the semi-pure compounds were collected by elutlng the various polarity organic solvents in the order of increasing polarity.

The semi-pure compounds were subjected to further purification by using recycling preparative HPLC utilizing 1% ethanol blended chloroform. The removal of the other boswellic acids closely associated with AKBA that appeared as shoulders was achieved after 4 purification cycles. The idea of recycling is to separate the peaks in the chromatogram to allow better separation. The optimum separation was achieved after 4 cycles and the pure compound, AKBA was obtained.

The pure AKBA showed pink colour on the TLC when sprayed with cerric sulphate reagent followed by heating. This pink colour was changed to dark black on further heating. This observation was indicative of the presence of a terpenoid skeleton of the molecule.

The El-MS of AKBA showed a molecular son peak at m/z 512. The molecular mass was further confirmed through FAB-MS and thus the molecular formula C32H48O5 was deduced by the FAB-MS studies (m/z 511 [M - H] ) combined with ¹³ C NMR spectral data of AKBA.

The Retro Diels Alder (RDA) fragmentation was identified by the presence of base peak at m/z 232. This RDA fragment is characteristic of Δ ¹² ursene type triterpenes with COOH group at C-24, The IR spectrum showed characteristic absorption bands for hydroxyl group (3410 cm ^-1 ), carbonyl groups (1735 and 1688 cm ^-1 ) and the tri-substituted double bond (1628 and 812 cm ^"1 ),

Five tertiary methyls were resonated as singlets in the ¹ H NMR spectrum of AKBA.The secondary methyl (Chh-29) was observed as doublet at δ 0,78, whereas the other secondary methyl (CH3-3O) was observed as overlap signal with the tertiary methyls. All these observations were indicative of the ursane- type skeleton in the molecule. The olefinic proton (H-12) was observed at δ 5.53 as a broad singlet, whereas the proton geminal to the hydroxyl group was observed at δ 5.28 (1H, m). Table 1: IMMR Data and Key HHBC Correlations for AKBA

The ¹³ C NMR spectrum (BB, and DEPT) of AKBA showed thirty two signals, including eight methyl, nine methylene, seven methine and eight quaternary carbons (Table 1). The seven methyl signals (C-23 and C-25 to C-30), and one acetyl methyl in the range of δ 13 ppm and 29 ppm, one downfield quaternary carbon (C-24) at δ 181,2 along with one methine carbon (C-3) at δ 73.0, proposed that the compoundis likely to be a boswellic acid derivative. The oiefinic moiety in the ring-C was confirmed by the presence of signals at δ 165,0 in the ¹³ C NMR spectrum for quaternary carbons (C-13), and the methine carbon (C-12) at δ 130.5. The downfield shift of C-13 and a signal at δ 199.3 in ¹³ C NMR spectrum indicated the presence of the α-β unsaturated system with a!kene and ketone moieties. Furthermore, the quaternary carbon at δ 170.2, coupled with the indication of acetyl group in ¹ H NMR spectrum also confirmed the presence of acetyl substitution in the molecule.

The ¹ H- ¹³ C connectivity was determined through HSQC spectrum and the long- range ¹ H- ¹³ C correlations (HMBC) were helpful for assigning the positions of different sub-structures in the molecule to confirm the final structure. H-3 proton showed long range heteronuclear correlations with C-l (δ 34.6), C-2 (δ 23.5), C- 4 (δ 46.4), CH ₃ -23 (δ 23.8) and C=0 (δ 170.2); H-12 proton showed interactions with C-ll (δ 199,3), C-13 (δ 165,0), and C-14 (δ 43.7), thus indicating the relative positions of these groups in the molecule,