Extract from a plant of the genus <i>Boswellia </i>and related products and uses

Abstract

The invention relates to a novel extract from the resin of a plant of the genus Boswellia (Burseraceae). The invention further relates to a composition comprising the novel extract, in particular a pharmaceutical composition or a cosmetic composition, or a food supplement, a food or a beverage comprising the extract. Further, the invention concerns the use of the extract and the respective compositions, in particular the use of the extract or a composition comprising the extract in the cosmetic or therapeutic field as well as in food supplements. The invention also relates to the novel extract and the respective compositions for use in the treatment and/or prevention of a disease. A method of treatment and/or prevention of a neurodegenerative disease is further provided. A process is also provided, by which the novel extract is obtained.

Claims

1. An extract from a resin of a plant of genus Boswellia, wherein the limonene content, the linalool content, and the thujone content are each less than 20 ppm in the extract and the estragol content is less than 100 ppm in the extract, wherein the limonene content, the linalool content, the thujone content, and the estragol content are each calculated based on the dry weight of the extract.

2. The extract according to claim 1, wherein the limonene content and the thujone content are less than 10 ppm, wherein the limonene content and the thujone content are calculated based on the dry weight of the extract.

3. The extract according to claim 1, wherein the plant is selected from the group consisting of: Boswellia papyrifera, Boswellia serrata, Boswellia sacra, and Boswellia carterii.

4. The extract according to claim 1, wherein the plant is not Boswellia papyrifera.

5. The extract according to claim 1, wherein the essential oil content of the extract is reduced in a relative amount with respect to the resin.

6. The extract according to claim 1, wherein at least one protein interacting polyphenol is reduced in amount in comparison to a conventional extract of the resin.

7. The extract according to claim 6, wherein the protein interacting polyphenol content in the extract is less than 0.10% (w/w), and wherein the protein interacting polyphenol content is calculated based on the dry weight of the extract.

8. The extract according to claim 6, wherein the at least one protein interacting polyphenol is a tannin.

9. The extract according to claim 1, wherein the extract comprises a triterpenic acid or a derivative thereof.

10. The extract according to claim 1, wherein the extract comprises a cyclic diterpenoid or a derivative thereof.

11. The extract according to claim 1, wherein the extract comprises a monocyclic diterpenoid or a derivative thereof.

12. The extract according to claim 1, comprising at least one cyclic diterpenoid or derivative thereof in an amount of at least 4% (w/w) and at least one triterpenic acid or derivative is present in an amount of at least 15% (w/w), calculated based on the dry weight of the extract.

13. The extract according to claim 1, comprising at least one cyclic diterpenoid or derivative thereof in an amount of 10 to 25% (w/w) and at least one triterpenic acid or derivative is present in an amount of 20 to 40% (w/w), calculated based on the dry weight of the extract.

14. A composition comprising the extract of claim 1.

15. The composition according to claim 14, wherein the composition is a pharmaceutical composition and comprises at least one additional pharmaceutically acceptable excipient.

16. The composition according to claim 14, further comprising a second active ingredient.

17. The composition according to claim 14, wherein the composition is selected from the group consisting of a food, a beverage and a food supplement.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The Figures shown in the following are merely illustrative and shall describe the present invention in a further way. These Figures shall not be construed to limit the present invention thereto.

(2) FIG. 1: Effect of Boswellia extracts on LPS-stimulated IL6 release in THP-1 cells

(3) FIG. 2: Effect of Boswellia extracts on LPS-stimulated TNF-α release in THP-1 cells

(4) FIG. 3: Effect of Boswellia extracts on LPS-stimulated IL1-β release in THP-1 cells

(5) FIG. 4: ROS induction in HepG2 cells HepG2 cells were exposed to Boswellia extracts and controls for two hours and ROS levels were determined. A. Positive control Menadione. B. Negative control ethanol. C. Boswellia serrata extract according to Example 12. D. Boswellia serrata extract according to Example 15.

(6) FIG. 5: ROS induction in HepG2 cells HepG2 cells were exposed to Boswellia extracts and controls for four hours and ROS levels were determined. A. Positive control Menadione. B. Negative control ethanol. C. Boswellia serrata extract according to Example 12. D. Boswellia serrata extract according to Example 15.

EXAMPLES

(7) The following Examples are intended to illustrate the invention further. They are not intended to limit the subject matter of the invention thereto.

(8) The examples and results provided herein are based on data obtained using resin extracts prepared with ethanol/water as extraction solvent as defined in the ESCOP Monograph for Olibanum indicum (Olibanum indicum). Resins from India (Boswellia serrata Roxb.ex Colebr., examples 3 and 10), Somalia (Boswellia carteri syn. sacra Birdwood, Example 6), Oman (Boswellia sacra syn. carteri Birdwood, Example 1), served as starting materials. The obtained standard ethanolic liquid extracts (Examples 1, 3, 6, and 10) were analyzed on their content of undesired compounds as well as on their content of boswellic acids and cembrenes. The extract from Example 10 was also processed using state of the art procedures to obtain a standard solvent free dry powder extract (Example 14) or a standard solvent free oily extract (Example 15). Further this extract and the extracts of Examples 1, 3 and 6 were also purified according to the inventive process to obtain the new extract composition.

(9) Briefly, standard ethanolic liquid extracts were prepared (Examples 1, 3, 6 and 10), which underwent an evaporation step followed by adjusting the Ethanol content of the resulting concentrate to an ethanol concentration between 20 and 50% (w/w) ethanol (this process step results in first reduction of the undesired volatile oil compounds). The resulting suspension was purified from protein interacting polyphenols by addition of an aqueous solution of gelatin (tannin-gelatin crosslinking reaction, also called hardening) and subsequently cellulose powder was added, which binds the protein-polyphenol-complexes to form a fine dispersion (the suspended cellulose particles hydrate and are form little granules with the gelatin and the polyphenol crosslinked gelatin resulting in a fine disperse hydrophilic adsorbate). In the following, ethanol was added until a concentration of 80-90% (w/w) was reached (the addition of ethanol leads to dehydration and precipitation of the granules on the one side and dissolving of the lipophilic extractives on the other side). A precipitate and a clear supernatant were obtained, the latter representing the purified extract solution. The supernatant was separated from the precipitate by filtration in order to obtain a clear purified ethanolic liquid extract (Examples 2, 4, 7 and 11) as a process intermediate. Typically, the filtration was done in two steps: a first filtration for obtaining a clear liquid and a second filtration after mixing the extract solution with diatomaceous earth (Celite®), which binds potentially remained traces of non-precipitated gelatin. Said extract solution was subjected to an initial evaporation step followed, after water addition, by a water steam distillation at 90-100° C. In some examples, this process was carried out in the presence of an additionally added lipid phase, which protects cyclic diterpenoids, namely monocyclic diterpenoids, such as cembranoids, but not the undesired mono and sesquiterpenes, from evaporation during steam distillation. During said process step, residues of undesired steam volatile compounds (mono- and sesquiterpens) evaporated together with the water steam and were so eliminated from the extract solution. Finally, the remaining solution was evaporated at 40-70° C. until the it was solvent free. Subsequently the purified solvent free oily liquid extract (Example 5, 8 and 12) or the purified solvent free dry powder extract (Example 9 and 13) was obtained.

(10) Material and Methods

(11) The following examples illustrate the present invention. Beside the detailed description of the extraction and purification procedures and their yields the resulting intermediate extracts and finished extracts were analyzed on their content of limonene, linalool, thujone and estragol, their content of serratol, incensol and incensol-acetate as well as boswellic acids (see Table 1). The intermediate products were analyzed additionally on their content of polyphenolic compounds. The analysis of mono- and sesquiterpenes was performed by Labor Veritas, Engimattstrasse, Zürich, Switzerland using gas chromatography coupled to MS readout (Labor Veritas GC-Reports No 214-0721), whereas the analysis of pentacyclic triterpenic boswellic acids (11-keto-ß-boswellic-acid (KBA), acetyl-11-keto-ß-boswellic-acid (AKBA), ß-boswellic-acid, acetyl-ß-boswellic-acid, α-boswellic-acid and acetyl-α-boswellic-acid have been measured individually and been additively mentioned as “triterpenic acids” in the following examples) and cembrenes (serratol, incensole and incensole-acetate have been measured individually and been additively mentioned as “cembrenes” in the following examples) was performed in house by high pressure liquid chromatography (Method/Top Alpinia No. TOP-215: reports no. SR-2029) and the analysis of polyphenol was performed in house by UV/VIS spectroscopy according to the method in the European Pharmacopoe 7.0/2.08.14.00, in house reports no.: AR-1131. The following resins were used for the experiments: Example 1 and 2 OBM001 (Boswellia sacra, Oman); Examples 3, 4 and 5 OBN001 (Boswellia serrata, India), Examples 6, 7, 8, 9 BAX019 (Boswellia carteri, Somalia) and Examples 10, 11, 12, 13, 14 and 15 BSR037 (Boswellia serrata, India), documented in in-house release documents AR-1065.01, 1037.01, 1097.01, 1096.01, 1128.01, and 1091.01. Unless otherwise specified, the percentages indicated in the Examples refer to weight percent (% w/w).

Example 1 (Preparation of a Standard Ethanolic Liquid Extract OBM001: LN00226)

(12) 20.0 g milled resin (particle size <2 mm) were extracted with 200.01 g Ethanol 80% w/w in an 500 ml Erlenmeyer under stirring at 250 rpm, 50° C. for 60 min. The preparation was then filtrated twice over a deep layer cellulose filter (AF15 Filtrox®).

(13) Results: 184.54 g of a clear amber liquid extract with a solid content (dry weight of the extract after evaporation) of 6.54% were obtained. A sample of the extract was analyzed and the following content (calculated based on the dry weight of the standard ethanolic liquid extract obtained in Example 1) was obtained: estragol: 138.2 ppm; linalool: 98.1 ppm; limonene: 12.6 ppm; thujone: 34.8 ppm (gas chromatography); triterpenic acids: 21.49% (HPLC); cembrenes: 4.18% (HPLC) and polyphenols (Ph.Eur.): 0.19% (spectrophotometry).

Example 2 (Preparation of a Purified Ethanolic Liquid Extract OBM001:LN00227)

(14) 50.1 g of the liquid extract from Example 1 (OBM001:LN00226) was transferred to a 250 ml glass flask and 75-85% of the solvent was evaporated at 150 mbar, 100 rpm and 60° C. until an residual suspension of 15-25%, calculated to the starting amount, was reached. The flask was transferred to a balance and ethanol 40% w/w was added until a total filling weight of 25 g was reached. A clear amber liquid was obtained. Subsequently 5.2 g cellulose powder (Sanacel®) and 6.7 g of a gelatin solution (gelatin Ph.Eur.: 10% w/w aqueous solution) were added and rotated at 60° C. and 100 rpm for 45 min. After said time 110 g of ethanolum absolutum (PhEur.) were added and rotated at 100 rpm, 20-30° C. for 15 min. The mixture was filtered twice over a deep layer cellulose filter (AF15 Filtrox®) and a clear yellowish liquid extract was obtained (117.5 g). Said extract was transferred in a 250 ml glass flask and 2.0 g of a diatomaceous earth (Celite®) were added. This suspension was rotated at 20-30° C. for 10 min and was then filtered twice over a deep layer cellulose filter (AF50 Filtrox®).

(15) Results: 106.8 g of a clear yellowish liquid extract with a solid content (dry weight of the extract after evaporation) of 2.49% was obtained. A sample of the extract was analyzed and the following content (calculated based on the dry weight of the extract obtained in Example 2) was obtained: estragol: 103.8 ppm; linalool: 179.6 ppm; limonene: <10.0 ppm; thujone: 33.3 ppm (gas chromatography); triterpenic acids: 23.90% (HPLC); cembrenes: 4.63% (HPLC) and Polyphenols (Ph.Eur.): 0.00% (spectrophotometry). Compared to the standard extract, 111% of triterpenic acids and 111% of cembrenes were resulting.

Example 3 (Preparation of a Standard Ethanolic Liquid Extract OBN001: LN00228)

(16) 20.03 g milled resin (particle size <2 mm) were extracted with 200.01 g Ethanol 80% w/w in an 500 ml Erlenmeyer under stirring at 250 rpm, 50° C. for 60 min. The preparation was then filtrated twice over a deep layer cellulose filter (AF15 Filtrox®).

(17) Results: 191.41 g of a clear amber liquid extract with a solid content (dry weight of the extract after evaporation) of 6.31% were obtained. A sample of the extract was analyzed and the following content (calculated based on the dry weight of standard ethanolic liquid extract obtained in Example 3) was obtained: estragol: 7388.6 ppm; Linalool: 481.3 ppm; Limonene: <5.0 ppm; thujone: 14.5 ppm (gas chromatography); triterpenic acids: 27.46% (HPLC); cembrenes: 8.62% (HPLC).

Example 4 (Preparation of a Purified Ethanolic Liquid Extract OBN001:LN00228.1)

(18) 50.1 g of the liquid extract from Example 3 (OBN001:LN00228) were transferred to a 250 ml glass flask and 75-85% of the solvent were evaporated at 150 mbar, 100 rpm and 60° C. until an residual suspension of 15-25%, calculated to the starting amount, was reached. The flask was transferred to a balance and ethanol 40% w/w was added until a total filling weight of 25 g was reached. A clear amber liquid was obtained. Subsequently 5.2 g cellulose powder (Sanacel®—to form granules with precipitating gelatin) was suspended in the liquid and then 6.7 g of a gelatin solution (gelatin Ph.Eur.: 10% w/w aqueous solution) were dissolved and rotated at 60° C. and 100 rpm for 45 min. After said time, 110 g of ethanolum absolutum (PhEur.) were added and the mixture was rotated at 100 rpm, 20-30° C. for 15 min, resulting in a fine disperse precipitate and a clear supernatant. The mixture was filtered twice over a deep layer cellulose filter (AF15 Filtrox®) and a clear yellowish liquid extract was obtained (117.5 g). Said extract was transferred in a 250 ml glass flask and 2.0 g of a diatomaceous earth (Celite®—to bind residual traces of gelatin) were added. This suspension was rotated at 20-30° C. for 10 min and then filtered twice over a deep layer cellulose filter (AF50 Filtrox®).

(19) Results: 110.2 g of a clear yellowish liquid extract with a solid content (dry weight of the extract after evaporation) of 2.44% was obtained. A sample of the extract was analyzed and the following content (calculated based on the dry weight of the extract obtained in Example 4) was obtained: estragol: 3218.3 ppm; linalool: 391.3 ppm; limonene: <10.0 ppm; thujone: <10.0 ppm (gas chromatography); triterpenic acids: 27.77% (HPLC); cembrenes: 8.76 (HPLC). Compared to the standard extract, 101% of triterpenic acids and 102% of cembrenes were resulting.

Example 5 (Preparation of a Purified Solvent Free Oily Liquid Extract OBN001:LN002211.2)

(20) 75.0 g of the purified ethanolic liquid extract from Example 4 (OBN001:LN0228) were transferred to a 250 ml glass flask and 1.68 g middle-chain triglycerides (Labrafac®- to dissolve the extractives and prevent evaporation of diterpenoids) plus 0.545 g middle chain mono and diglycerides (Peceol®- to dissolve the extractives and prevent evaporation of diterpenoids) were added. The mixture was evaporated under reduced pressure at 200 mbar, 250 rpm and 55° C. for 20 min. Then 60.0 g of distilled water (steam distillation of mono and sesquiterpenes) were added and distillation was started at 600 mbar, 90° C. for 30 min. Subsequently, the vacuum was reduced to 200 mbar and evaporation was performed for further 30 min. Then the bath temperature was reduced to 50° C. and the residual solvent was evaporated at 150-5 mbar during an additional time of 60 min.

(21) Results: 4.05 g of a viscous clear amber liquid extract were obtained. A sample of the extract was analyzed and the following content (calculated based on the dry weight of the purified ethanolic liquid extract (i.e. 75.0 g×0.0244=1.83 g) obtained in Example 4) was obtained: estragol: 76.7 ppm; linalool: 15.0 ppm; limonene: <5.0 ppm; thujone: <5.0 ppm (gas chromatography); triterpenic acids: 27.13% (HPLC); cembrenes: 10.62% (HPLC). Compared to the standard ethanolic liquid extract of Example 3, 99% of triterpenic acids and 123% of cembrenes were resulting.

Example 6 (Preparation of a Standard Ethanolic Liquid Extract BAX019: LN0017127)

(22) 100.20 g milled resin (particle size <1 mm) were extracted with 1001.14 g ethanol 80% w/w in an 2000 ml Erlenmeyer under stirring at 250 rpm, 50° C. for 90 min. The preparation was then filtered twice over a deep layer cellulose filter (Beco® Filter CPKS) after addition of 8.0 g crospovidone (Ph.Eur.) and 17.14 g Sanacel®.

(23) Results: 974.31 g of a clear amber liquid extract with a solid content (dry weight of the extract after evaporation) of 5.57% were obtained. A sample of the extract was analyzed and the following content (calculated based on the dry weight of the standard ethanolic liquid extract obtained in Example 6) was obtained: estragol: 36.0 ppm; linalool: 40.2 ppm; limonene: <12.0 ppm; thujone: 60.0 ppm (gas chromatography); triterpenic acids: 21.06% (HPLC); cembrenes: 21.14% (HPLC) and polyphenols (Ph.Eur.): 0.41% (spectrophotometry).

Example 7 (Preparation of a Purified Ethanolic Liquid Extract BAX019:LN0017128)

(24) 500.24 g of the liquid extract from Example 6 (BAX019:LN0017127) were transferred to a 2000 ml glass flask and 75-85% of the solvent were evaporated at 150 mbar, 100 rpm and 60° C. until an residual suspension of 15-25%, calculated to the starting amount, was reached. The flask was transferred to a balance and ethanol 40% (w/w) was added until a total filling weight of 250 g was reached. A clear amber liquid was obtained. Subsequently, 50.31 g cellulose powder (Sanacel®) and 67.10 g of a gelatin solution (gelatin Ph.Eur.: 20% w/w aqueous solution) were added and rotated at 60° C. and 100 rpm for 30 min. After said time, 1108.38 g of ethanolum absolutum (PhEur.) were added and rotated at 100 rpm, 20-30° C. for 15 min. The mixture was filtered twice over a deep layer cellulose filter (AF15 Filtrox®) and a clear yellowish liquid extract was obtained. Said extract was transferred in a 2000 ml glass flask and 20.6 g of a diatomaceous earth (Celite®) were added. This suspension was rotated at 20-30° C. for 10 min and then filtered over a deep layer cellulose filter (AF50 Filtrox®).

(25) Results: 1090.97 g of a clear yellowish liquid extract with a solid content (dry weight of the extract after evaporation) of 1.91% were obtained. A sample of the extract was analyzed and the following content (calculated based on the dry weight of the purified ethanolic liquid extract obtained in Example 7) was obtained: estragol: 36.0 ppm; linalool: 52.8 ppm; limonene: <12.0 ppm; thujone: <12.0 ppm (gas chromatography); triterpenic acids: 22.57% (HPLC); cembrenes: 22.75% (HPLC) and polyphenols (Ph.Eur.): 0.08 (spectrophotometry). Compared to the standard ethanolic liquid extract of Example 6, 107% of triterpenic acids and 108% of cembrenes were resulting.

Example 8 (Preparation of a Purified Solvent Free Oily Liquid Extract BAX019:LN0017132)

(26) 200.03 g of the purified ethanolic liquid extract from Example 7 (BAX019:LN0017128) were transferred to a 500 ml glass flask and 0.97 g middle-chain triglycerides (Labrafac®) plus 0.97 g middle chain mono and diglycerides (Peceol®) were added. The mixture was evaporated under reduced pressure at 200 mbar, 250 rpm and 60° C. for 30 min. Then 45.0 g of distilled water were added and distillation was started at 900 mbar for 30 min, then 600 mbar, 90° C. for 30 min. Subsequently, the vacuum was reduced to 200 mbar and evaporation was performed for further 20 min. Then the bath temperature was reduced to 60° C. and the residual solvent was evaporated at 150-5 mbar during an additional time of 60 min.

(27) Results: 5.89 g of a viscous clear amber liquid extract were obtained. A sample of the extract was analyzed and the following content (calculated based on the dry weight of the purified ethanolic liquid extract (i.e. 200.03 g×0.0191=3.82 g) obtained in Example 7) was obtained: estragol: 11.6 ppm; linalool: <5.0 ppm; limonene: <5.0 ppm; thujone: <5.0 ppm (gas chromatography); triterpenic acids: 20.33% (HPLC); cembrenes: 19.44% (HPLC). Compared to the standard ethanolic liquid extract of Example 6, 97% of triterpenic acids and 92% of cembrenes were resulting. The polyphenol content of the extract of Example 8 is 0.08%, as the extract is prepared from the extract obtained in Example 7.

Example 9 (Preparation of a Purified Solvent Free Dry Powder Extract BAX019:LN0017130)

(28) 200.09 g of the purified ethanolic liquid extract from Example 7 (BAX019:LN0017128) were transferred to a 250 ml glass flask. The extract solution was evaporated under reduced pressure at 200 mbar, 250 rpm and 60° C. for 60 min. Then 45.0 g of distilled water were added and distillation was started at 900 mbar for 30 min, then 600 mbar, 90° C. for 30 min. Subsequently, the vacuum was reduced to 200 mbar and evaporation was performed for further 20 min. Then the bath temperature was reduced to 60° C. and the residual solvent was evaporated at 150-5 mbar during an additional time of 60 min.

(29) Results: 4.31 g of a yellow powder were obtained. A sample of the extract powder was analyzed and the following content (calculated based on the dry weight of the purified ethanolic liquid extract (i.e. 200.09 g×0.0191=3.82 g) obtained in Example 7) was obtained: estragol: 17.8 ppm; linalool: <11.5 ppm; limonene: <5.0 ppm; thujone: 17.1 ppm (gas chromatography); triterpenic acids: 18.92% (HPLC); cembrenes: 18.45% (HPLC). Compared to the standard ethanolic liquid extract of Example 6, 90% of triterpenic acids and 87% of cembrenes were resulting.

Example 10 (Preparation of a Standard Ethanolic Liquid Extract BSR037: LN0017121)

(30) 100.87 g milled resin (particle size <1 mm) were extracted with 1000.14 g ethanol 80% w/w in a 2000 ml Erlenmeyer under stirring at 250 rpm, 50° C. for 90 min. The preparation was then filtered twice over a deep layer cellulose filter (Beco® Filter CPKS) after addition of 8.02 g crospovidone (Ph.Eur.) and 17.02 g Sanacel®.

(31) Results: 962.09 g of a clear amber liquid extract with a solid content (dry weight of the extract after evaporation) of 5.98% were obtained. A sample of the extract was analyzed and the following content (calculated based on the dry weight of the standard ethanolic liquid extract obtained in Example 10) was obtained: estragol: 3987.3 ppm; linalool: 367.0 ppm; limonene: 11.9 ppm; thujone: 86.7 ppm (gas chromatography); triterpenic acids: 24.02% (HPLC); cembrenes: 9.16% (HPLC) and polyphenols (Ph.Eur.): 0.05 (spectrophotometry).

Example 11 (Preparation of a Purified Ethanolic Liquid Extract BSR037:LN0017122)

(32) 500.10 g of the liquid extract from example 10 (BSR037:LN0017121) were transferred to a 2000 ml glass flask and 75-85% of the solvent was evaporated at 150 mbar, 100 rpm and 60° C. until an residual suspension of 15-25%, calculated to the starting amount, was reached. The flask was transferred to a balance and ethanol 40% w/w was added until a total filling weight of 250 g was reached. A clear amber liquid was obtained. Subsequently, 50.3 g cellulose powder (Sanacel®) and 67.2 g of a gelatin solution (gelatin Ph.Eur.: 10 w/w aqueous solution) were added and rotated at 60° C. and 100 rpm for 30 min. After said time, 1100.8 g of ethanolum absolutum (PhEur.) were added and rotated at 100 rpm, 30° C. for 30 min. The mixture was filtered twice over a deep layer cellulose filter (AF15 Filtrox®) and a clear yellowish liquid extract was obtained. Said extract was transferred to a 2000 ml glass flask and 20.0 g of a diatomaceous earth (Celite®) were added. This suspension was rotated at 20-30° C. for 10 min and then filtered over a deep layer cellulose filter (AF50 Filtrox®).

(33) Results: 931.55 g of a clear yellowish liquid extract with a solid content (dry weight of the extract after evaporation) of 1.84% were obtained. A sample of the extract was analyzed and the following content (calculated based on the dry weight of the extract obtained in Example 11) was obtained: estragol: 2482.2 ppm; linalool: 281.1 ppm; limonene: <13.0 ppm; thujone: 27.0 ppm (gas chromatography); triterpenic acids: 26.37% (HPLC); cembrenes: 9.05% (HPLC) and polyphenols (Ph.Eur.): 0.00% (spectrophotometry). Compared to the standard ethanolic liquid extract of Example 10, 110% of triterpenic acids and 99% of cembrenes were resulting.

Example 12 (Preparation of a Purified Solvent Free Oily Liquid Extract BSR037:LN0017125)

(34) 200.02 g of the purified ethanolic liquid extract from Example 11 (BSR037:LN0017122) were transferred to a 500 ml glass flask and 0.96 g middle-chain triglycerides (Labrafac®) plus 0.96 g middle chain mono and diglycerides (Peceol®) were added. The mixture was evaporated under reduced pressure at 200 mbar, 250 rpm and 60° C. for 30 min. Then 45.0 g of distilled water were added and distillation was started at 900 mbar, 90° C. for 30 min, then 600 m bar, 90° C. for 30 min. Subsequently, the vacuum was reduced to 200 mbar and evaporation was performed for further 20 min. Then the bath temperature was reduced to 60° C. and the residual solvent was evaporated at 150-5 mbar during an additional time of 60 min.

(35) Results: 6.10 g of a viscous clear amber liquid extract were obtained. A sample of the extract was analyzed and the following content (calculated based on the dry weight of the purified ethanolic liquid extract (i.e. 200.02 g×0.0184=3.68 g) obtained in Example 11) was obtained: estragol: 65.1 ppm; linalool: 12.6 ppm; limonene: <5.0 ppm; thujone: 9.5 ppm (gas chromatography); triterpenic acids: 24.10% (HPLC); cembrenes: 9.02% (HPLC). Compared to the standard ethanolic liquid extract of Example 10, 100% of triterpenic acids and 98% of cembrenes were resulting. The polyphenol content of the extract of Example 12 is 0%, as the extract is prepared from the extract obtained in Example 11.

Example 13 (Preparation of a Purified Solvent Free Dry Powder Extract BSR037:LN0017124)

(36) 200.4 g of the purified ethanolic liquid extract from Example 11 (BSR037:LN0017122) were transferred to a 500 ml glass flask. The extract solution was evaporated under reduced pressure at 200 mbar, 250 rpm and 60° C. for 60 min. Then 45.0 g of distilled water were added and distillation was started at 900 mbar at 90° C. for 30 min, then 600 mbar, 90° C. for 30 min. Subsequently, the vacuum was reduced to 200 mbar and evaporation was performed for further 20 min. Then the bath temperature was reduced to 60° C. and the residual solvent was evaporated at 150-5 mbar during an additional time of 60 min.

(37) Results: 3.95 g of a yellow dry powder were obtained. A sample of the extract powder was analyzed and the following content (calculated based on the dry weight of the purified ethanolic liquid extract (i.e. 200.04 g×0.0184 g=3.68 g) obtained in Example 11) was obtained: estragol: 281.1 ppm; linalool: 96.2 ppm; limonene: <10.0 ppm; thujone: 17.1 ppm (gas chromatography); triterpenic acids: 22.20% (HPLC); cembrenes: 8.41% (HPLC).). Compared to the standard ethanolic liquid extract of Example 10, 92% of triterpenic acids and 92% of cembrenes were resulting.

Example 14 (Preparation of a Standard Solvent Free Dry Powder Extract BSR037:LN0017123)

(38) 109.10 g of the standard ethanolic liquid extract from Example 10 (BSR037: LN0017121) were transferred to a 250 ml glass flask. The extract solution was evaporated under reduced pressure at 200 mbar, 250 rpm and 60° C. for 60 min. Subsequently, the vacuum was reduced to 150 mbar and evaporation was performed for an additional time of 60 min.

(39) Results: 6.05 g of a yellow powder were obtained. A sample of the extract powder was analyzed and the following content (calculated based on the dry weight of the standard ethanolic liquid extract (i.e. 109.10 g×0.0598=6.52 g) obtained in Example 10) was obtained: estragol: 2777.2 ppm; linalool: 325.1 ppm; limonene: 18.0 ppm; thujone: 27.0 ppm (gas chromatography); triterpenic acids: 22.29% (HPLC); cembrenes: 8.40% (HPLC). Compared to the standard ethanolic liquid extract of Example 10, 93% of triterpenic acids and 92% of cembrenes were resulting.

Example 15 (Preparation of a Standard Solvent Free Oily Extract BSR037:LN0017126)

(40) 100.00 g of the standard ethanolic liquid extract from Example 10 (BSR037: LN0017121) were transferred to a 250 ml glass flask and 1.49 g middle-chain triglycerides (Labrafac®) plus 1.49 g middle chain mono and diglycerides (Peceol®) were added. The mixture was evaporated under reduced pressure at 600 mbar, 250 rpm and 60° C. and then the vacuum was reduced to 10 mbar in the course of 50 min and the final vacuum was maintained at 60° C. and 250 rpm for 15 min.

(41) Results: 9.27 g of a viscous clear amber liquid extract were obtained. A sample of the extract powder was analyzed and the following content (calculated based on the dry weight of the standard ethanolic liquid extract (i.e. 100.00 g×0.0598=5.98 g) obtained in Example 10 (100.00 g×0.0598=5.98 g)) was obtained: estragol: 1156.6 ppm; linalool: 176.7 ppm; limonene: <10.0 ppm; thujone: 15.7 ppm (gas chromatography); triterpenic acids: 22.83% (HPLC); cembrenes: 8.69% (HPLC). Compared to the standard ethanolic liquid extract of Example 10, 95% of triterpenic acids and 95% of cembrenes were resulting.

(42) TABLE-US-00001 TABLE 1 Summary of the analysis results desired compounds undesired compounds Triterpenic Extract Estragol Linalool Limonene Thujone Polyphenols acids Cembrenes Ex. derived type resin type (ppm) (ppm) (ppm) (ppm) (% w/w) (% w/w) (% w/w) 1 SEL Arabian sacra 138.2 98.1 12.6 34.8 0.19 21.49 4.18 2 (from 1) PEL Arabian sacra 103.8 179.6 <10 33.3 0 23.9 4.63 3 SEL Indian serrata 7388.6 481.3 <5 14.5 n.d. 27.46 8.62 4 (from 3) PEL Indian serrata 3218.3 391.3 <10 <10 n.d. 27.77 8.76 5 (from 4) POL Indian serrata 76.7 15 <5 <5 0 27.13 10.62 6 SEL Somalia carteri 36 40.2 <12 60 0.41 21.06 21.14 7 (from 6) PEL Somalia carteri 36 52.8 <12 <12 0.08 22.57 22.75 8 (from 7) POL Somalia carteri 11.6 <5 <5 <5 <0.08 20.33 19.44 9 (from 7) PDP Somalia carteri 17.8 11.5 <5 17.1 n.d. 18.92 18.45 10 SEL Indian serrata 3987.3 367 11.9 86.7 0.05 24.02 9.16 11 (from 10) PEL Indian serrata 2482.2 281.1 <13 27 0 26.37 9.05 12 (from 11) POL Indian serrata 65.1 12.6 <5 9.5 0 24.1 9.02 13 (from 11) PDP Indian serrata 281.1 96.2 <10 17.1 n.d. 22.2 8.41 14 (from 10) SDP Indian serrata 2777.2 325.1 18 27 n.d. 22.29 8.4 15 (from 10) SOL Indian serrata 1156.6 176.7 <10 15.7 n.d. 22.83 8.69 (SEL: standard ethanolic liquid extract; PEL: purified ethanolic liquid extract; POL: purified solvent free oily liquid extract; PDP: purified solvent free dry powder extract; SDP: standard solvent free dry powder extract)

CONCLUSIONS

(43) As shown in the technological Examples 2, 4, 7, and 11 Boswellia extracts processed with the new purification process methodology, already the purified liquid extracts (intermediate step of the process) show reductions of limonene, linalool and thujone as well as estragol and also show very low levels or even absence of protein interacting polyphenols when normalized to the differing initial load of polyphenols of the standard extracts of Examples 1, 3, 6, and 10. Furthermore, the final extracts prepared according to the inventive process (oily extract Examples 5, 8 and 12) contain less than 20 ppm of each of limonene, linalool and thujone and less than 100 ppm of estragol. Also the purified dry powder extracts (Examples 9 and 13) contain considerably lower amounts of the undesired compounds than the standard extracts. The purified extracts also show very low levels of protein interacting polyphenols and, importantly, with small deviations between the different examples, independently of the initial load of polyphenols in the standard extracts. On the other hand, the inventive process retains the desired secondary plant metabolites of Olibanum, namely the triterpenic acids as well as the cyclic diterpenoids such as cembrenes (>90% w/w of the standard extract content) in the extract up to the final oily extracts (Examples 5, 8 and 12), and in the purified dry powder extracts (Example 9 and 13 with at least 87% recovery of triterpenic acids). These results reflect the robustness and validity of the process of the present invention with respect to the production of novel Boswellia extracts free or nearly free of undesired compounds. The process according to the invention thus provides extracts from the resins of Boswellia species, wherein undesired volatile mono- and sesquiterpenoids as well as polyphenolic compounds are reduced or eliminated, while it retains desired secondary plant metabolites of Olibanum, namely triterpenic acids as well as cyclic diterpenoids, such as the group of cembrenes.

Example 16 (Cytokine Analysis in Lipopolysaccharide-Stimulated THP-1 Cells)

(44) THP-1 cells were stimulated with lipopolysaccharide (LPS) and cytokine levels were measured after incubation with Boswellia extract BSR037: LN00125 (Example 12) or Boswellia extract BSR037: LN00126 (Example 15).

(45) THP-1 cells (ATCC® TIB202™), a human monocyte cell line (from acute monocytic leukemia), were cultured in RPMI-1640 Medium (Lonza) supplemented with 10% Fetal Bovine Serum (Life Technologies), 0.05 mM 2-mercaptoethanol (Life Technologies), 4.5 g/L glucose (Sigma), 10 mM HEPES (Sigma), and 1 mM sodium pyruvate (Sigma).

(46) In 96 well round-bottom plates (cell culture treated), 7×105 THP-1 cells per well were plated in 180 μl/well THP-1 medium. Cells were incubated for 1-2 hours at 37° C. and 5% CO.sub.2 until extract dilutions were ready. Dilutions of extracts and ethanol (as solvent control, Fluka) were prepared in Phosphate Buffered Saline (without Ca++ and Mg++, BioConcept).

(47) 20 μl/well of extract dilutions and controls, respectively, were added in duplicate wells. Ethanol was added to a final concentration of 0.2% as the solvent control. Cells were incubated for one hour at 37° C. in a 5% CO.sub.2 incubator.

(48) Lipopolysaccharide stock was prepared (Sigma, LPS from E. coli 055:65, 10 mg/ml in ultrapure water). Shortly before use, the stock solution was sonicated for 2 minutes. LPS was added to 10 g/ml final concentration, and incubated at 37° C. and 5% CO.sub.2 for 24 hours.

(49) LPS was added to all sample wells except two wells. These wells were the unstimulated control.

(50) After 24 hours, THP-1 cell culture supernatants were collected for cytokine analysis. The samples in the wells were mixed. The plate was centrifuged to pellet the cells. Cell-free supernatants (140 μl per well) were removed and transferred to a new round-bottom 96 well plate. Plates were sealed, and the supernatants were stored at −80° C. until measurement.

(51) Cytokines were measured in the THP-1 supernatants by DuoSet ELISA from R&D Systems. The levels of IL6 (FIG. 1), TNF-α (FIG. 2) and IL1-β (FIG. 3) were determined.

(52) As a result, Boswellia extracts BSR037: LN00125 (Example 12) and BSR037: LN00126 (Example 15) showed anti-inflammatory cytokine patterns as indicated by a dose-dependent reduction of IL6, IL1-β and TNF-α. In comparison to BSR037: LN00126 (Example 15), the extract BSR037: LN00125 (Example 12) showed a more pronounced anti-inflammatory pattern, especially in regard of the reduction of IL1-β and TNF-α cytokine levels.

Example 17 (Determination of Reactive Oxygen Species (ROS) in HepG2 Indicator Cells)

(53) The potential of Boswellia extract BSR037: LN00125 (Example 12) and Boswellia extract BSR037: LN00126 (Example 15) of inducing reactive oxygen species (ROS) was tested. The effects were compared with ROS generation upon exposure to positive control Menadione as well as two solvent ethanol. To this end, HepG2 cells were incubated in presence of different concentrations of the respective Boswellia extract for two hours. ROS levels were determined by using the ROS-Glo system (Promega).

(54) HepG2 cells were seeded in 296-well flat-bottom black microtiter plates at a density of 10000 cells per well, 100 μl per well, and allowed to attach to the plate for 24 hours.

(55) Test item dilutions were prepared in separate plates. The maximum concentration was a 1:100 dilution in media (final solvent concentration: 1% ethanol) followed by a 1:3 serial dilution in media containing 1% ethanol. Positive control compound Menadione was used at a maximum concentration of 500 μM followed by a 1:2 serial dilution. Cells exposed to media containing 1% ethanol served as untreated control.

(56) After overnight adherence, media was completely removed from cells by vacuum aspiration and 80 μl of compound dilution, positive and negative controls were added to the cells. Test items were tested in two replicates per concentration (A and B). In independent experiments, ROS levels were measured after two hours of incubation (see FIG. 4A-D) or after four hours of incubation (see FIG. 5A-D), respectively).

(57) As a result, ROS induction was detected for the positive control Menadione as well as for Boswellia extracts BSR037: LN00125 (Example 12) and BSR037: LN00126 (Example 15), wherein BSR037: LS00125 (Example 12) showed a higher potency of ROS induction.