Analytical Strategies for the Isolation and Identification of Bioactive Boswellic Acids

Medicinal plants continue to serve as a vital source of therapeutic agents, with triterpenoids representing a significant class of bioactive natural products. Among them, boswellic acids derived from Boswellia serrata, Boswellia carterii, and related species have gained considerable attention due to their potent inhibition of 5-lipoxygenase and other inflammatory mediators. Despite extensive pharmacological investigations, the therapeutic potential of boswellic acids largely depends on their purity, structural integrity, and accurate identification. Analytical challenges arise from the complex resin matrix, structural similarity among boswellic acid analogues, and their relatively low abundance. Consequently, robust analytical strategies integrating extraction, isolation, and advanced analytical techniques are essential. This paper aims to present a comprehensive overview of analytical methodologies applied for the isolation and identification of bioactive boswellic acids. Olibanum also known as “Dhup”, Indian Frankincense is an oleo gum resin ofBoswellia species. In India it is obtained from Boswellia serrata. Boswellia serrata (Burseraceae) is a large, much branched, deciduous tree that grows abundantly in the dry, hilly parts of India. It is or Indian Olibanum. Since ancient times, resins have been important in the preparation of incense, medicines, cosmetics and perfumes. The Egyptians, Hindus, Persians, Israelites, Greek, Romans and the Europeans of Queen Victoria’s times greatly valued these materials. Olibanum, the resin from the Boswellia species has been used as incense for centuries. However, its major use today is as a fixative in perfumes, soaps, creams lotions and detergents. In India, the gum resin exudates of Boswellia serrata, known in the vernacular as “Salai guggal”, has been used in the Ayurvedic system of medicine in the managements of several inflammatory conditions and as a topical anti-inflammatory agent. The major use of Boswellia serrata in contemporary medicine is as an anti-arthritic and anti-inflammatory pharmacological agent. Literature survey reveals that anti-inflammatory activities associated with this resin are completely restricted to presence of Boswellic acids. So focus in experimental work done is placed on isolation of acid fraction of oleo gum resin. As mentioned in the procedures above acid fraction was obtained as white precipitate. This white precipitate was separated, dried and weighed. Amount of acid fraction obtained was determined on weight basis. In the experimental work done isolation of acid fraction was carried out by procedures mentioned in section above. This is common procedure which uses treatment of resin with alkali to convert acid into its salt and then precipitating salt of acid by using mineral acid. Studies were carried out to check variations in amount of acid portion obtained when parameters were altered. Use of 2% KOH followed by dilute hydrochloric acid as mineral acid will be most suitable. The anti-inflammatory properties of the gum resin are attributed to the presence of “boswellic acids. It known as Gajabhakshya in Sanskrit, implying its ingestion by elephants has been used in the Ayurvedic medicine since antiquity. The interest in this herb was aroused by the fact that such a heavy animal carried its weight on its limbs for so long, yet lived longer than humans. This stimulated effort to find the ingredients in its diet, where Boswellia was found to be one. Boswellia has been mentioned in the ancient Indian Ayurvedic texts – the Sushruta Samhita and Charak Samhita. Boswellia is a tree of moderate height, which grows widely on dry hills of northwest India. In Ayurveda the oleo gum resin of BSE is known as ‘Salai Guggul’ or ‘Sallaki Guggul’. It has been used in the treatment of rheumatism, nervous diseases and as a topical anti-inflammatory agent. Preparations from the gum of Boswellia serrata Extract (BSE) have been used in traditional/folk medicine for treatment of inflammatory diseases. On stripping the bark, it yields gummy oleoresins, which contain oils, terpenoids and gums. Upto 16% of the resin is essential oil, the majority being α thujene and p-cymene. Four pentacyclic triterpene acids are also present, with β-boswellic acid being the major constituent. BSE showed anti-inflammatory and antibacterial activity while the non-phenolic fraction of gum resin exhibited sedative and analgesic effects when tested in rats. Animal and in vitro studies suggest its usefulness in many inflammatory and broncho-constrictive conditions. Animal studies performed in India show that ingestion of defatted alcoholic extract of BSE decreases polymorpho-nuclear leucocyte infiltration and migration, decreased antibody synthesis and caused almost total inhibition of the classical complement pathway. Recently, it has been shown to be the inhibitor of 5-lipoxygenase and also human leucocyte elastase and consequently it has been proposed in the treatment of various inflammatory conditions. In 1992, the active principles within the multi-component mixture of resin were identified, resulting inrecognition of Boswellic acids. The most important are Acetyl 11-Keto β-Boswellic Acid (AKBA) and 11- Keto β-Boswellic Acid (KBA). Boswellic acids were found to inhibit two pro-inflammatory enzymes, 5-lipoxygenase (which generates inflammatory leukotrienes) and Human Leukocyte Elastase (HLE). HLE is a serine protease that initiates injury to the tissue, which in turn triggers the inflammatory process. This dual inhibitory action on the inflammatory process is unique to boswellic acids.

Botanical Aspects of Boswellia Species:

The botanical origin of Boswellia species has been characterized as:

Division: Spermatophyta

Subdivision: Angiospermae

Tribe: Rosopsida

Subtribe: Rosidae s. lat.

Overclass: Rutanae

Class: Anacardiales

Family: Burseraceae

Genus: Boswellia

The family of Burseraceae is represented in the plant kingdom with 17 genera and 600 species, widespread in all tropical regions. The species are often a predominant component of the vegetation in dry, lowland areas. Some species of the two most important genera of this family, Commiphora and Boswellia, produce resins that are of considerable commercial value as raw materials of balm, myrrh and frankincense.

 The Chemical History of Olibanum:

Although the oil of olibanum had occupied the shelves of the 16th century pharmacies as “oleum thuris”, the first investigation on its chemical composition was performed in 1788 by Johann Ernst Baerat the University of Erlangen. Following his work, the first elementary analysis was carried out by F.W. Johnston in 1839. The constituents of the essential oil were first investigated by J. Stenhouse in 1840, and he identified depending on the origin of the resin fourteen monoterpenoic constituents including pinene, dipentene, phellandrene and cadinene. In 1898, A. Tschirch and O. Halbey published for the first time that olibanum had an acidic constituent, boswellic acid, with a molecular formula of C₃₂H₅₂O₄ but they could not suggest a structure at that time. At the beginning of the 1930’s, the olibanum resin was investigated in more detail. The study of A. Winterstein and G. Stein in 1932 drew the attention to the resin acids, the pentacyclic triterpenoic α- and β-amyrin like skeletons with different functional groups, which were attempted to be isolated and identified with the analytical methods possible for that time. Nevertheless, by the 1960´s several of these acids such as α- and β-boswellic acids, 11α- hydroxy-β-boswellic acid and 3-O-acetyl-11-hydroxy-β-boswellic acid were identified by various derivatisation methods. In 1967, G. Snatzke and L. Vértesy published the structures of acetyl-11-keto-β-boswellic acid as well as epi-α- and epi-β-amyrin and their acetates, α- and β-amyrenone and viridiflorol from the neutral fraction of olibanum, adding that it is composed of 5-9% essential oil, 15-16% resin acids, 25-30% of material insoluble in ether containing the polysaccharides and 45-55% ether soluble compounds. In 1978 R.S. Pardhy and S.C. Bhattacharya identified tirucallic acids as well as β-boswellic acid, acetyl-β-boswellic acid, 11-keto-β-boswellic acid, acetyl-11-keto-β-boswellic acid from B. serrata Roxb.and a diterpenoic cembrene derived alcohol, “serratol”. Studies on the isolation and identification of the boswellic acids with modern analytical techniques and on their pharmacological effects are still going on. Therefore, these topics will be further discussed in the following parts of this work. The first important and comparative study on the essential oil of olibanum of different origins was performed by H. Obermann from Dragoco (Holzminden, Germany) in 1977. He investigated two different commercial brands of olibanum, “Eritrea” and “Aden” by GC-MS, which corresponded to B. carterii and B. serrata resins, respectively. As a result of this investigation it was reported that not only the fragrance of these two qualities but also the composition of the constituents in the oils were different. Boswellic acids are pentacyclic triterpenic acids primarily belonging to the ursane and oleanane skeletons. Major boswellic acids include β-boswellic acid, acetyl-β-boswellic acid, 11-keto-β-boswellic acid (KBA), and 3-O-acetyl-11-keto-β-boswellic acid (AKBA). Among these, AKBA is considered the most pharmacologically active constituent. The presence of closely related structural analogues necessitates the use of high-resolution analytical tools to ensure precise identification and differentiation. The “Eritrea” oil was reported to have octylacetate as the major constituent (52%) as well as α-pinene, camphene, p-methoxytoluol, hexyl acetate, limonene, 1,8-cineole, octanol, linalool, bornyl acetate, cembrene A, incensole, incensyl acetate and an unknown diterpenoic constituent. In contrast, “Aden” oil was found to contain α-pinene as the major constituent (43%), camphene, β-pinene, sabinene, o-cymol, limonene, 1,8-cineole, p-cymol, campholenaldehyde, verbenone, octyl acetate and cembrenol, a diterpene alcohol with cembrene skeleton which was identified later by the same group, which was expected not be different than “serratol” described before. In 1985 a detailed review was published by P. Maupetit on the “Aden” brand of olibanum. He reported 47 new constituents identified in the resinoid and in the oil of olibanum in addition to 169 formerly identified substances including the pyrolysis products. Recent studies by Verghese on B. serrata oil and by A.M. Humprey et al. comparing B. carterii oil with cumin, ginger, rosemary oil, were reinvestigations of known facts. These studies pointed to the difficulties in the identification of the origin of olibanum resin as well as in the determination of standard olibanum oil. The gum resin of Boswellia serrata is known to contain:

Monoterpenes (α thujene)

Diterpenes (macrocyclic diterpenoids such as incensole, incensole oxide, inoincencole oxide, a diterpene alcohol (serrtol))

Triterpenes (such as α- and β-amyrins)

Pentacyclic triterpenic acids (boswellic acids)

Tetracyclic triterpenic acids (tirucall-8, 24-dien-21-oic acids)

 Boswellia and Boswellic acids:

The four major pentacyclic triterpenic acids present in the acidic extract of Boswellia serrata gum resin.

β-Boswellic Acid

Acetyl-β-Boswellic Acid

11-keto-β-Boswellic Acid

Acetyl-11-keto-β-Boswellic Acid

Apart from this oleogum resin of boswellia also contains monoterpenes, Diterpenes and tetracyclic triterpenes (described above). These compounds are responsible for anti-inflammatory activities of resin¹.

Description:

a) Macroscopic:

Drug occurs in globular, transparent, tears forming agglomerates of various shapes and sizes, brownish-yellow, upto 5cm long, 2cm thick, fragrant, fracture brittle; fractured surface waxy and translucent; burns readily and emanates an agreeable characteristic, balsamic resinous odor; taste, aromatic and agreeable.

Chemical Constituents:

Essential oil 8-12 %, Polysaccharides (45-60%), higher terpenoids (25-35%).

Objective:

Checking the effect of alkali on acid extraction.

Chemical Profile of Boswellic Acids

Boswellic acids are a class of pentacyclic triterpenoid acids predominantly found in the oleo-gum-resin of Boswellia species such as Boswellia serrata, B. sacra, and B. carterii. They are biosynthesized via the cyclization of squalene and are chemically classified into two major structural types: ursane and oleanane triterpenoids, distinguished by their core ring system and functional substituents. Recent reviews emphasize the structural diversity and therapeutic relevance of these compounds within the resin matrix.

Major Boswellic Acid Constituents

The principal boswellic acids of pharmacological interest include:

• β-Boswellic acid (β-BA): The basic oleanane triterpenoid with a carboxylic acid group at C-24 and a hydroxyl at C-3.
• Acetyl-β-boswellic acid (Aβ-BA): An acetylated derivative, contributing to altered polarity and bioactivity.
• 11-Keto-β-boswellic acid (KBA): A key keto analogue with enhanced activity in inflammatory models.
• 3-O-acetyl-11-keto-β-boswellic acid (AKBA): Widely reported as the most pharmacologically active boswellic acid, particularly in anti-inflammatory pathways.

Additional α-boswellic acids (ursane type) and acetylated derivatives also contribute to the overall chemical complexity.

Physicochemical and Bioavailability Considerations

Boswellic acids are generally crystalline, non-volatile compounds with limited water solubility, resulting in poor oral bioavailability. This has significant implications for both therapeutic efficacy and analytical measurements. Extensive phase I and II metabolism of AKBA and KBA has been characterized in vitro, revealing metabolites with distinct profiles that impact bioactivity and systemic exposure.

Structure–Activity Relationships (SAR)

The presence of the 11-keto group and acetyl moiety at C-3 is strongly correlated with anti-inflammatory and immunomodulatory activity. In vitro studies demonstrate that AKBA and KBA can modulate immune cell activation and regulatory T-cell markers (FoxP3) at micromolar concentrations, underscoring the importance of specific functional groups in receptor interactions and biological response.

Analytical Implications

Because structural analogues of boswellic acids often co-occur in complex plant matrices, advanced chromatographic separation combined with spectroscopic identification is essential to resolve and accurately identify each compound. Recent analytical work has highlighted streamlined HPLC methods with improved sensitivity for routine quantification of AKBA in biological and formulation samples.

MATERIALS AND METHODS:

Procurement of Material:

Oleogumresin of Boswellia serrata (Commonly known as Loban) was purchased from Medical Shop of crude drugs at Gulmandi, Aurangabad. 500g of material was purchased. It was available as yellowish brown to blackish masses, opaque and transparent.

Collection and Authentication: Oleo-gum-resin samples of Boswellia species are collected and authenticated using macroscopic and microscopic evaluation, followed by taxonomic confirmation.

Extraction Techniques

Extraction plays a crucial role in the recovery of boswellic acids. Commonly employed methods include:

Solvent extraction: Using methanol, ethanol, or hydroalcoholic solvents

Soxhlet extraction: For exhaustive extraction

Ultrasound-assisted extraction (UAE): Enhances yield and reduces extraction time

Supercritical fluid extraction (SFE): Offers selectivity and reduced solvent usage

Optimization of extraction parameters significantly influences the yield and purity of boswellic acids.

Identification test:

General Test: (90%) a tear of' Kunduru is not altered much in form but becomes almost opaque and white; when a drop of con. H₂SO₄ is added on a freshly fractured surface, it becomes cherry red which, when washed with water changes to a white emulsion, then turn to a buff color.

Fluorescence Test:

Brownish-yellow color in day light; aqueous extract under U.V. light (366nm) light green and in (254nm) shows dark blue color; alcoholic extract under U.V. light (366nm) is colorless and in (254nm) shows light green color.

Positive identification tests are given by oleo gum resin

Solubility studies of Oleogumresin:

For solubility study gum was crushed by mortar and pestle and its solubility was tested in different solvents. Following are the solvents used with observation. These solutions were subjected to TLC studies.

Table 4.1: Solubility studies of Oleogumresin

Thin Layer Chromatography (TLC):

Sample Preparation:

In case of dried extract dissolve a small quantity of the sample in the least polar solvent in which it is soluble. Alternatively, sample can also be prepared by extracting the small amount of crude drug material (say 1g) with solvent.

Sample Application:

Sample thus prepared is applied in very small amount (in microlitres) as spot or as lane on TLC plate just 1cm-2cm above the base of it. This is called as solute front.

Development of TLC Plate:

After the TLC plate is spotted it has to be transferred to the development tank. This tank holds the mobile phase. The solute front is not allowed to sink in to the solvent system otherwise the compound will diffuse in to the solvent system. Large variety of development tanks are commercially available, the cheapest way is to use a beaker with a watch glass as lid. To saturate the inside atmosphere of the tank, the tank can be lined with filter paper.

Visualization of TLC Plate/ Detection of Spots:

It is easy to visualize colored compounds but all compounds are not colored. There are numerous reagents available to visualize TLC plates. Even when the material being analyzed is colored it is necessary to treat the TLC plate to visualize any no-colored spots that may be present in the sample. The two most useful means of analysis are ultraviolet-light and iodine vapour. The TLC plate can be dipped into a stock solution of the reagent or the plate can be sprayed with a diffuser. Commonly used reagents are anisaldehyde sulphuric acid reagent, vanillin sulphuric acid reagent, antimony trichloride reagent. After the plate has been sprayed, it is heated for 10 minutes at 110^oC. The compounds produce spots visible under ultraviolet light 360nm and 254nm.

Stationary phase:

Precoated silica gel plate with fluorescent indicator (F₂₅₄) from Merck PSGF₂₅₄

Mobile Phase: Toluene: Ethyl Acetate: Methanol (8:2:1)

Detection:

1. UV 254nm

2. Anisaldehyde sulphuric acid (ASA) (Heat treatment is required after TLC plate is sprayed with ASA).

Extraction Studies:

Crude drug material is subjected for extraction studies by employing different methods of extraction commonly available in laboratory.

Following methods were tested:

1. Maceration

2. Hot continuous extraction (Soxhlet extraction)

3. Steam distillation and reflux

4. Direct heating (Decoction)

Maceration:

In this method material 250g of Oleogumresin is subjected for maceration for 20 hrs in ethanol (500ml) with continuous shaking at 100rpm on shaker. This process is repeated successively for three times.

Hot continuous Extraction:

This is also called as hot continuous percolation or Soxhlet extraction. Crude drug material is placed in body of extractor. Heating is applied.

Steam distillation or reflux:

In this method Oleogumresin is placed in RBF fitted with condenser and heat is applied to RBF.

Direct Heating:

In this method oleo gum resin is placed in a glass container and direct heat is applied to container.

Isolation of Boswellic Acid by Column Chromatography:

For running of column optimization of solvent system is carried out by Thin Layer Chromatography.

Thin Layer Chromatography:

Thin layer chromatography is carried out under following conditions

Mobile Phase: Toluene: Ethyl acetate: n heptane: Formic acid (8:2:1:0.3)

Stationary phase: Precoated silica gel plate with fluorescent indicator (F254) from Merck PSGF₂₅₄

Detection:

A.   UV 254 nm

B.    Anisaldehyde sulphuric acid (ASA)

Heat treatment is required after TLC plate is sprayed with ASA

Photograph 4.1: TLC of Boswellia extract

(Toluene: Ethyl acetate: n heptane: Formic acid)

Column Chromatography

Procedure:

In column chromatography, the mobile phase is again a solvent, and the stationary phase is a finely divided solid, such as silica gel or alumina. Chromatography columns vary in sizes. There is an element of trial and error involved in selecting a suitable solvent and adsorbent for the separation of the constituents of a particular mixture. A small volume of the sample whose constituents are to be separated is placed on top of the column. The choice of the eluting solvent should ensure that the sample is soluble. However, if the sample was too soluble the mobile phase (solvent) would move the solutes too quickly, resulting in the non-separation of the different constituents. Usually, one should start with a less polar solvent to remove the less polar compounds, and then slowly increase the polarity of the solvent to remove the more polar compounds.

Steps in Column Chromatography

Column Chromatography 1

Stationary Phase:

Silica 230-330 mesh size.

Mobile phase: Toluene: Ethyl acetate: n heptane (8:2:1)

Column: Glass column

Column diameter: (internal diameter of column):1.5cm

Height of column: 10.5cm

Height of extract loaded: 1.5cm (Height of column refers to height at which silica, the stationary phase is filled)

Number of fractions collected: 32 fractions of 20ml each. Fraction 4, 5 shows single spot corresponds to Acetyl keto β boswellic acid Fraction 8-9 yields single spot corresponds toα and β boswellic acid

Column Chromatography 2:

This column was run on Toluene: Ethyl acetate: Methanol (8:2:1). This mobile phase was optimized with TLC. 

Thin Layer Chromatography:

Thin layer chromatography is carried out under following conditions