QUANTITATIVE AND QUALITATIVE DETERMINATION OF 3-O-ACETYL-11-KETO-Β-BOSWELLIC ACID (AKBA) AND OTHER BOSWELLIC ACIDS IN BOSWELLIA SACRA FLUECK (SYN. B. CARTERI BIRDW) AND BOSWELLIA SERRATA ROXB.
The main constituents of B. serrata and B. sacra are volatile oils, composed of monoterpenes and sesquiterpenes, diterpenes including incensole, incensole acetate and cembrenol (serratol), lipophilic pentacyclic triterpene acids of the oleanane- (α-boswellic acids), ursane- (β-boswellic acids) and lupane-type (lupeolic acids), as well as an ether-insoluble fraction containing polysaccharides (arabinose, galactose, xylose).
Among triterpenoids, bioactive boswellic acids are of particular interest, with particular reference to 3-O-Acetyl-11-Keto-β-Boswellic Acid (AKBA), 11-Keto-beta-Boswellic Acid (KBA), and the various β-boswellic acids (βBAs), and α-boswellic acids (αBAs) and their esters. (Figure 1 and Table 1).
Figure 1: Chemical structure of the boswellic acids identified in Boswellia serrata and Boswellia sacra. Numbers correspond to compounds listed in Table 1.
Table 1: Molecular mass and fragmentation pattern of compounds identified in the methanolic extracts of B. sacra and B. serrata.
|#||Compounds||RT (min)||[M-H]-(m/z)||ions (m/z)||Occurence in|
|01||11-Keto-Ursolic acid||24||469||451, 407, 391||x||x|
|02||11-Keto-β-boswellic acid||25.8||469||451, 407, 391||x||x|
|03||3-O-Acetyl-11-keto-β-Boswellic Acid||28.8||511||451, 361||x||x|
|04||3-α-Hydroxy-7,24-dien-tirucallic acid||34.5||455||453, 437, 373||x|
|05||3-α-Hydroxy-8,24-dien-tirucallic acid||36.5||455||453, 437, 373||x||x|
|06||3-β-Hydroxy-7,24-dien-tirucallic acid||39.4||455||453, 437, 373||x|
|07||3-O-Acetyl-oleanolic acid||41||497||479, 437||x||x|
|08||Lupeolic acid||41.6||455||437, 409||x|
|09||3-O-Acetyl-ursolic acid||43.3||497||479, 437||x||x|
|10||α-Boswellic acid||44.5||455||437, 409, 377||x||x|
|11||β-Boswellic acid||45||455||437, 409, 377||x||x|
|12||3-O-Acetyl-α-boswellic acid||52||497||459, 437||x||x|
|13||3-O-Acetyl-β-boswellic acid||52.9||497||459, 437, 395||x||x|
The precise identification and quantification of boswellic acids is usually obtained with liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) by using selected ion monitoring (SIM) detection (Figure 2).
Figure 2. Analytical equipment used by Biosfered for the qualitative and quantitative determination of boswellic acids: HPLC coupled to electrospray ionization and tandem mass spectrometry.
Table 2 shows for both species the quantitative determination of the main Boswellic acids. The total amount of the main Boswellic acids was statistically (P < 0.05) higher in B. sacra than in B. serrata. In B. sacra, the total amount of the main Boswellic acids in the methanolic extract was lower than 50% and this value was reduced to about 29% when the amount was considered on the total gum resin dry weight (Table 2). In B. serrata the total content of the main Boswellic acids in the methanolic extract was lower than 30% and the value dropped to 16% when Boswellic acids were calculated on the gum resin dry weight (Table 2). In both species, the major Boswellic acids were represented by αBA and βBA, in agreement with literature data . A direct comparison between the two species shows that the contents of B. sacra AKBA (about 10 fold), αBA (1.5 fold) and βBA (1.6 fold) were statistically (P < 0.05) higher than in B. serrata.
Table 2. Quantitative determination of boswellic acids in Boswellia sacra and Boswellia serrata by HPLC-DAD-ESI-MS/MS, by using calibration curves from pure standards. Data are expressed as g kg-1 gum resin dry weight. (Standard deviation), in the same row, different letters indicate significant (P < 0.05) differences.
|Compound||Boswellia sacra||Boswellia serrata|
|Extract content||Gum-resin content||Extract content||Gum-resin content|
|KBA||35.50 (1.26)a||21.26 (0.76)b||34.62 (2.57)a||19.03 (1.41)c|
|AKBA||70.81 (4.66)a||42.41 (2.79)b||7.35 (0.89)c||4.04 (0.49)d|
|αBA||184.34 (11.27)a||110.40 (6.75)b||126.00 (7.77)b||69.27 (4.27)c|
|βBA||186.19 (4.98)a||111.50 (2.98)b||113.21 (7.37)c||62.24 (4.06)c|
|A-αBA||5.40 (0.35)a||3.23 (0.21)b||2.92 (0.19)b||1.60 (0.11)c|
|A-βBA||9.95 (0.23)a||5.96 (0.14)b||11.33 (0.28)a||6.23 (0.15)b|
|Totale||491.20 (14.75)a||294.77 (8.83)b||295.25 (23.00)b||162.29 (12.64)c|
Boswellia sacra and Boswellia serrata extracts are widely used in pharmaceutical and nutraceutical preparations.
The bioactivity of these Boswellia extracts is based on the content of Boswellic acids. Clearly, the dose of bioavailable BAs is central to the issue of Boswellia efficacy.
Claims of 70% Boswellic acids or even 30-40% AKBA are currently found, but this work confirms that the Boswellic acids content never exceeds 50% of the methanolic extract, whereas lower percentages are obtained when Boswellic acids are expressed on the gum resin weight.
Moreover, the highest percentage of AKBA found in B. sacra was below 8%.
ONLY ANALYTICAL METHODS BASED ON HPLC FOLLOWED BY MASS SPECTROMETRY ALLOW THE PRECISE QUANTIFICATION AND IDENTIFICATION OF BAS IN BOSWELLIA SACRA AND BOSWELLIA SERRATA EXTRACTS.
THEREFORE, ANY CLAIM OF BOSWELLIC ACIDS CONTENT OF EITHER BOSWELLIA SACRA AND BOSWELLIA SERRATA GUM RESINS EQUAL OR HIGHER THAN 70% OR AKBA CONTENTS OF 30% ARE SIMPLY UNREALISTIC OR BASED ON A WRONG QUANTITATIVE DETERMINATION.
For further reading on the quantitative analysis of boswellic acids refer to the publication:
MANNINO, G., OCCHIPINTI, A., MAFFEI, M.E. (2016)
Quantitative determination of 3-O-Acetyl-11-Keto-β-Boswellic Acid (AKBA) and other boswellic acids in Boswellia sacra Flueck (syn. B. carteri Birdw) and Boswellia serrata Roxb.
Molecules. 21:1329 – (download pdf).
MECHANISM OF ACTION AND EFFICACY OF BOSLIQ
The boswellic acids have long been studied for their healing properties. The AKBA for example is known for its anti-inflammatory, neuroprotective, chemopreventive, antioxidant, inhibitor of 5-lipoxygenase, and antibiotic properties for the treatment of retinopathies and wounds (over 65 citations in ISI Web of Science to November 2016).
Recently, Appendino and colleagues (Pollastro F, Golin S, Chianese G, et al. 2016. Neuractive and anti-inflammatory Frankincense cembranes: A structure-activity study. J Nat Prod 79:1762-1768.) have shown that serratol (or cembrenol) present in B. serrata is a potent agonist of the transient receptor potential vanilloid 3 (TRPV3), a heat-sensitive ion channel expressed in keratinocytes and in the central nervous system (Figure 3).
Figura 3. Activators, inhibitors and modulators of TRPV3. Quaternary structure of TRPV3 with compounds and signalling pathways known to activate, inhibit or modulate the receptor. From Broad et al., Pharmaceuticals 2016, 9(3), 55; doi:10.3390/ph9030055.
The TRPV3 receptor has important physiological implications: from genetic diseases with inflammatory processes to weight management, to depression up to brain functions. In addition serratol acts at submicromolar concentrations. The incensole acetate (typical of B. sacra) is a selective agonist of TRPV3 and can stimulate calcium influx mediated by TRPV3, while the incensole is a potent inhibitor of STAT3 induced by IL-6 and a potent activator of NF-KB.
THESE RESULTS INDICATE THAT BESIDES THE PROVEN EFFECT OF BOSWELLIC ACIDS, EXTRACTS CONTAINING BOSWELLIA DITERPENES AS INCENSOLE, INCENSOLE ACETATE (AS IN BOSLIQ®-AKBA) AND SERRATOL (OR CEMBRENOL, AS IN BOSLIQ®-BA) PROVIDE GREATER CONTRIBUTION AS SUPPORT TO REDUCE INFLAMMATORY PROCESSES.
Pre-clinical and clinical studies on the effectiveness of BosLiq®-AKBA and BosLiq®-BA are under way. Recently, in a pilot study, the use of BosLiq®-AKBA (in combination with chondroitin, collagen type II and bromelain) for 24 weeks resulted in a significant reduction of pain and stiffness on 40 subjects with mild or moderate chronic lateral epicondylitis and 18 patients suffering from tendonitis in the Achilles tendon (Guelfi M. 2016. Efficacy and safety of DAIGO® Tendiplus a dietary supplement agent in patients with lateral epicondylitis elbow or Achilles tendinopathy. a pilot study. Int. J. Exper. Clin . Res. 5: 9-12).