phytochemical analysis 2001, 12(6), 374-376

High-performance Thin Layer ChromatographicAnalysis of Anti-inflammatory Triterpenoidsfrom Boswellia serrata Roxb.

K. Krohn,1* M. S. Rao,1 N. V. Raman2 and M. Khalilullah2

1Universitat Paderborn, Fachbereich Chemie und Chemietechnik, 33098 Paderborn, Germany2Department of Chemistry, College of Engineering, JNT University, Kukatpally, Hyderabad, A.P, 500 072 India

A rapid and simple high-performance thin layer chromatographic (HPTLC) method was developed for thesimultaneous quantitative estimation of the biologically active triterpenoids �-boswellic acid, 3-O-acetyl-�-boswellic acid, 11-keto-�-boswellic acid and 3-O-acetyl-11-keto-�-boswellic acid from the gum resin ofBoswellia serrata. The assay combines the isolation and separation of boswellic acid derivatives on silica gel60F254-HPTLC plates with spot visualisation and scanning at 250 nm. Methanol was found to be the mostappropriate solvent for the exhaustive extraction of boswellic acid derivatives. Copyright � 2001 JohnWiley & Sons, Ltd.Keywords: High-performance thin layer chromatography; triterpene acids; boswellic acids; anti-inflammatory, anti-leukemia; Boswellia serrata.

INTRODUCTION

The gum resin of Boswellia serrata was used for thetreatment of inflammatory diseases in the traditionalAyurvedic medicine in India (Kiritikar and Basu, 1935;Chatterjee and Pal, 1984). A detailed pharmacologicalstudy by Singh and co-workers (Singh and Atal, 1986;Sharma et al., 1989) established that an alcoholic extractof the gum displayed marked anti-inflammatory activityin mice and rats and also inhibited the formation ofleucotrienes in rat peritoneal neutrophils in vitro (Macket al., 1990; Ammon et al., 1991). The constituents ofthe gum resin were reported to be monoterpenes,diterpenes and triterpenes (Pardhy and Bhattacharya,1978a–c).

In this paper the isolation of the pharmacologicallyimportant triterpene acids �-boswellic acid (1), 3-O-acetyl-�-boswellic acid (2), 11-keto-�-boswellic acid (3)and 3-O-acetyl-11-keto-�-boswellic acid (4) from B.serrata is described, and the development of an optimisedhigh-performance thin layer chromatographic (HPTLC)method for the simultaneous analysis of these fourimportant triterpenoids in gum resin is presented.

EXPERIMENTAL

Plant material. The gum resin of B. serrata wascollected from a tree in a southern region of India. Theplant material was identified by Dr V. S. Raju (Depart-ment of Botany, Kakatiya University, Warangal, India).Voucher specimen reference number: B.248.

Reagents and standards. Reagents were purchasedfrom Merck (Darmstadt, Germany). Compounds 1–4were isolated from a methanolic extract obtainedfrom 100 g of oleogum resin of B. serrata. A sample(45 g) of this extract was dissolved in 2% potassiumhydroxide solution (200 mL) and re-extracted with ethylacetate (5 � 150 mL). The ethyl acetate extract wasdiscarded and the aqueous solution was adjusted to pH 6with 2% hydrochloric acid and extracted again withethyl acetate (5 � 150 mL). The combined ethyl acetatesolution was washed with water, dried over anhydroussodium sulphate and evaporated to dryness to yield 18 gof residue which consisted of a mixture of boswellicacids. The residue was subjected to column chroma-tography over silica gel eluted with a gradient ofpetroleum ether:ethyl acetate to yield, with increasingpolarity, 3-O-acetyl-�-boswellic acid (2), acetyl-11-keto-

PHYTOCHEMICAL ANALYSISPhytochem. Anal. 12, 374–376 (2001)DOI: 10.1002/pca.606

Copyright � 2001 John Wiley & Sons, Ltd.

* Correspondence to: K. Krohn, Universitat Paderborn, Fachbereich Chemieund Chemietechnik, 33098 Paderborn, Germany.Email: [email protected]

Received 1 July 2000Revised 30 January 2001

Accepted 7 February 2001

�-boswellic acid (4), �-boswellic acid (1), and 11-keto-�-boswellic acid (3). The isolated boswellic acids wereidentified by comparison of their mass and IR spectraldata, [�]D values and melting points with those reportedin the literature (Pardhy and Bhattacharya, 1978a).

Extraction of oleogum resin for HPTLC analysis.Lumps or granules (1.0 g) of gum resin from B. serratawere extracted with solvents (3 � 25 mL each) of varyingpolarity (chloroform, ethyl acetate, methanol and petro-leum ether) in order to determine the most efficientextraction method for boswellic acid and its derivatives.Extracts were concentrated under vacuum, redissolved inmethanol, filtered, re-concentrated and evaporated to5 mL prior to HPTLC analysis.

Chromatographic conditions. Chromatography wasperformed on pre-activated (110°C) silica gel 60F254HPTLC plates (10 � 10 cm; 0.25 mm layer thickness;Merck). Samples and standard compounds were appliedto the layers as 8 mm wide bands, positioned 10 mm fromthe bottom of the plate, using a Camag (Mutten,Switzerland) Linomat IV automated TLC applicator withnitrogen flow providing delivery from the syringe at aspeed of 10 �L/s. These parameters proved critical andwere maintained for all analyses performed.

Detection and quantification of compounds 1–4. TLCplate development was performed using a Camag twin-trough glass tank which had been pre-saturated withmobile phase for ca. 2 h. Solvent was allowed to run upthe plate to a height of 8 cm. TLC analyses were madeunder laboratory conditions of 20 � 5°C and 50%

relative humidity. The composition of the mobile phasewas optimised by testing different solvent compositionsof varying polarities. A mixture of hexane:acetone (7:3,v/v) was employed in the optimised method.

After development, the layers were dried and the com-ponents were visualised by UV irradiation at 250 and365 nm. Compounds 1–4 were quantified on the plateusing a Camag model-3 TLC scanner equipped withCamag CATS 4 software. The slit width was set to8 � 0.4 mm and the scan wavelength was 250 nm. In orderto calibrate the method, stock solutions (1.0 mg/mL) ofcompounds 1–4 were prepared in methanol and variousamounts of these solutions (containing 1–25 �g ofstandard) were analysed by HPTLC exactly as describedabove and calibration curves constructed.

The precision of the instrumentation was checked byrepeated scanning of the same spots of boswellic acids(10 �g/spot) seven times each, and the coefficient ofvariation (CV) calculated. The repeatability of themethod was tested by replicate scanning (n = 3) ofstandard solutions of boswellic acids (10 �g/spot) afterapplication to a TLC plate. The variability of the methodwas studied by analysing aliquots of different concentra-tions of standard solutions of boswellic acids (1, 5, 10, 20and 25 �g) on the same day (intra-day precision) and ondifferent days (inter-day precision) and the relativestandard deviation (RSD) values were calculated.

RESULTS AND DISCUSSION

Different compositions of the mobile phase for HPTLCwere tested and the desired resolution of compounds 1–4(see Fig. 1), together with symmetrical and reproduciblepeaks, was achieved using hexane:acetone (7:3, v/v) asthe mobile phase. The calibration curves for compounds1–4 were linear over the range 1–25 �g and are presentedin Table 1, whilst the inter- and intra-day precisions ofthe determination are given in Table 2. The recoveryrates, determined by adding known amounts of stocksolutions of each of the compounds to an extract of B.

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Table 1. Linear regression equations and Rf values for boswellic acids 1–4 analysed by HPLTC

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Table 2. Inter- and intra-day precisions of the determina-tion of �-boswellic acid by HPTLC

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DETERMINATION OF BOSWELLIC ACID DERIVATIVES BY HPTLC 375

Copyright � 2001 John Wiley & Sons, Ltd. Phytochem. Anal. 12: 374–376 (2001)

serrata followed by replicate (n = 3) quantitative ana-lyses, were determined to be 97, 98, 96 and 98%,respectively, for compounds 1–4. The purities of thepeaks corresponding to each of the four compounds wereconfirmed by the demonstration that the UV absorptionspectra recorded on the CAMAG TLC scanner at thestart, middle and end position of each band werecompletely superimposable.

Different solvents of varying polarities were used forthe extraction of the boswellic acids from the gum resinof B. serrata, and the average of three replicates ispresented in Table 3. Methanol was found to be the mostappropriate solvent for the optimal extraction ofboswellic acids.

In summary, the HPTLC method for the simultaneousanalysis of compounds 1–4 from B. serrata, reported herefor the first time, is simple, rapid and accurate. Differentsolvents of varying polarity were used for the extractionof the gum, but methanol was found to be most suitablefor the extraction of the boswellic acids.

Acknowledgements

The authors are grateful to Ram Reddy (Principal) and M. S. N. Raju(Head, Department of Chemistry) at the JNTU College of Engineering,Kukatpally, Hyderabad for their kind cooperation.

REFERENCES

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Table 3. Effect of the extracting solvent on the determination of boswellic acids 1–4 in Boswellia serrata

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376 K. KROHN ET AL.

Copyright � 2001 John Wiley & Sons, Ltd. Phytochem. Anal. 12: 374–376 (2001)

phytochemical analysis 2001, 12(6), 374-376

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