Isolation and Identification of Boswellic Acids by Modern Analytical Techniques

Ms. Arshiya Zulfeen Mohd Fahim¹, Mr. Mohammed Shakir Ghouse², Mr. Syed Qumarul Islam¹,

Mr. Mohammed Abdul Mughni Danish¹,Mr. Shaikh Mehmood¹, Ms. Shaikh Saniya¹,

Ms. Quraishi Inshrah Fatima¹

¹Lecturer, Aayan Education and Welfare Trust’s, Aurangabad Pharmacy College, Mitmita, Aurangabad.

²Principal, Aayan Education and Welfare Trust’s, Aurangabad Pharmacy College, Mitmita, Aurangabad.

*Corresponding Author E-mail: arshiyasiddiqui01@gmail.com, mohammedshakir979@gmail.com

ABSTRACT:

Quality by Design (QbD) is a modern, scientific approach that formalizes product design, automates manual testing, and streamlines troubleshooting. It uses a systematic approach to ensure quality by developing a thorough understanding of the compatibility of a finished product to all of the components and processes involved in manufacturing that product. Olibanum also known as “Dhup”, Indian Frankincense is an oleo gum resin of Boswellia species. In India it is obtained from Boswellia serrata. Boswellia serrata (Burseraceae) The major use of Boswellia serrata in contemporary medicine is as an anti-arthritic and anti-inflammatory pharmacological agent. The anti-inflammatory properties of the gum resin are attributed to the presence of “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. Oleo gum resin was subjected for solubility studies in different solvents. In this 10 commonly available solvents were used for extraction including water. All of them showed same TLC pattern except water. In case of maceration no heat is employed, but the material has to be extracted multiple times with fresh solvent each time. In this method solvent requirement increases. Each time the extract was checked for presence of boswellic acids by TLC to ensure complete extraction. It took 4-5 times repeated extraction for complete extraction of Boswellic acids by maceration. 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.

KEYWORDS: Anti-arthritic, Anti-inflammatory.

INTRODUCTION:

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.

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. Vértesypublished 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. Bhattacharyaidentified 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.

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.

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.

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

S. No.	Solvent	Appearance of solution	Residue, comments
1.	Acetone	Milky (slight cloudy)	White
2.	Ethyl acetate	Milky (slight cloudy)	Yellowish
3.	Toluene	Transperant, colourless	Yellowish
4.	Methanol	Colourless	White
5.	Ether	Milky	White yellowish
6.	n Hexane	Slight cloudy	Brown
7.	Ethanol	Clear yellow transperant	Amorphus white
8.	Water	Yellowish turbid solution	Waxy lumps of white colour

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.

Photograph 4.2: 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

Mobile Phase: Toluene: Ethyl acetate: methanol (8:2:1)

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

Detection:

Visible (Vis.)-without any chemical treatment-

Anisaldehyde sulphuric acid (ASA)-Plate 2

UV 254 nm

Heat treatment is required after TLC plate is sprayed with ASA

Photograph 4.3: TLC of Boswellia extract (Toluene: Ethyl acetate: methanol)

Column Chromatography:

Stationary Phase: Silica 230-330 mesh size.

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

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 10ml each. Fraction 4, 5 shows single spot corresponds to Acetyl keto β boswellic acid Fraction 8-9 yields single spot corresponds to α and β boswellic acid

Isolation of acid fraction:

Following general procedure is employed for isolation of boswellic acid from oleogum resin

(a) Crushing the lumps of the gum resin of Boswelliaserrata and extracting the crushed lumps with ethanol.

(b) Removing insoluble material from above extract;

(d) Basifying the syrupy mass with an aqueous solution of an alkali to provide a solution having a pH in the range of 9 to 10.

(e) Extracting the solution with a solvents to provide an aqueous layer, and acidifying the aqueous layer with mineral acid to a pH in the range of 3-5 to provide a precipitate comprising boswellic acids;

(f) Washing the precipitate with water to provide said fraction being neutral to litmus;

(g) Separating individual boswellic acids from said fraction

Checking effect of alkali:

In the procedure mentioned above step (d) which indicates use alkali for basification. The aim of the present study is to check effect of different concentrations of alkali (NaOH and KOH) on separation of acid fraction.

KOH:

Acid separation by using 2% KOH:

10g of sample dissolved in ethanol. This solution was treated with 2% KOH solution(pH=10) and acidified with Conc. HCl.

Acid separation by using 5% KOH:

10g of sample dissolved in ethanol. This solution was treated with 5% KOH (pH 10.5) and acidified with conc. HCl. If no Precipitate is formed and extract remain as such without getting dissolved as viscous orange mass. This orange mass was separated and dissolved in ethanol and this solution was treated with Conc. HCl. Precipitate is formed which is separated and dissolved in ethyl acetate. It gave a soluble portion and insoluble precipitate. Insoluble precipitate was separated and dissolved in ethanol.

Acid separation by using 10% KOH:

10g of sample dissolved in ethanol. This solution was treated with 10% KOH (pH 10.5) and acidified with conc. HCl. If no Precipitate is formed and extract remain as such without getting dissolved as viscous orange mass. This orange mass was separated and dissolved in ethanol and this solution was treated with Conc. HCl. Precipitate is formed which is separated and dissolved in ethyl acetate. It gave a soluble portion and insoluble precipitate. Insoluble precipitate was separated and dissolved in ethanol.

Acid separation by using 20% KOH:

10g of sample dissolved in ethanol. This solution was treated with 10% KOH (pH 10.5) and acidified with conc. HCl. If no Precipitate is formed and extract remain as such without getting dissolved as viscous orange mass. This orange mass was very less which separated and dissolved in ethanol and this solution was treated with Conc. HCl. No precipitate was formed.

NaOH:

2% NaOH:

10g of sample dissolved in ethanol. This solution was treated with 2% NaOH (pH 10.5) and acidified with conc. HCl. If no Precipitate is formed and extract remain as such without getting dissolved as viscous orange mass. This orange mass was separated and dissolved in ethanol and this solution was treated with Conc. HCl. Precipitate is formed which is separated and dissolved in ethanol.

5% NaOH:

10g of sample dissolved in ethanol. This solution was treated with 5% NaOH and acidified with conc. HCl. The extract remain as such without getting dissolved as viscous orange mass. This orange mass was separated and dissolved in ethanol and this solution was treated with Conc. HCl. Precipitate is formed which is separated and dissolved in ethanol.

10% NaOH:

10g of sample dissolved in ethanol. This solution was treated with 10% NaOH and acidified with conc. HCl. The extract remain as such without getting dissolved as viscous orange mass. This orange mass was separated and dissolved in ethanol and this solution was treated with Conc. HCl. Precipitate is formed which is separated and dissolved in ethanol.

20% NaOH:

10g of sample dissolved in ethanol. This solution was treated with 10% NaOH and acidified with conc. HCl. The extract remained as such without getting dissolved as viscous orange mass. This orange mass was separated and dissolved in ethanol and this solution was treated with Conc. HCl. Precipitate is formed which is separated and dissolved in ethanol

Checking effect of acid:

In this parameter same above procedures were repeated for NaOH and KOH. Conc. H₂SO₄

was used instead of Conc. HCl.

Effect of strength of acid:

Here same procedures were repeated dilute acid was used instead of concentrated acid.

RESULT AND DISCUSSION:

Selection of suitable solvent:

As mentioned above in experimental work done, oleo gum resin was subjected for solubility studies in different solvents. In this 10 commonly available solvents were used for extraction including water. All of them showed same TLC pattern except water. This indicates that any solvent can be used for extraction of oleo gum resin except water (Use of any solvent here is recommended if the aim of extraction is isolation of Boswellic acid). Here one important point of cost can be considered with respect to solvent which will influence choice of solvent for extraction.

Details of TLC

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 254nm

(B)- Anisaldehyde sulphuric acid (ASA)

Heat treatment is required after TLC plate is sprayed with ASA

Photograph 5.1: TLC fingerprint for effect of solvents

Selection of extraction method of oleogum resin and completeness of extraction:

As mentioned in experimental conditions above different extraction methods were used for oleogumresin. Oleogum resin is mixture of polysaccharides gum, volatile oil and resin. Resin portion contains boswellic acids.

In case of soxhlet extraction and other methods of extraction employing heat like decoction problem of clogging and melting of material arises, which hampers process of extraction.

In case of maceration no heat is employed, but the material has to be extracted multiple times with fresh solvent each time. In this method solvent requirement increases. Each time the extract was checked for presence of boswellic acids by TLC to ensure complete extraction. It took 4-5 times repeated extraction for complete extraction of Boswellic acids by maceration.

After extraction concentration of material was carried out. During concentration much of the volatile oil evaporates.

Photograph 5.2: TLC fingerprint for completeness of extraction

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

Isolation of total acids (boswellic acid):

Literature survey reveals that anti-inflammatory activities associated with this resin is 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 these parameters were altered.

Effect of KOH:

Following are observations when KOH is used as basifying agent

Table 5.1: Effect of percentage of KOH as basifying agent on acid fraction

	Percentage of KOH	Amount of acid
1.	2% KOH	1.4g
2.	5% KOH	1.0g
3.	10% KOH	0.6g
4.	20% KOH	0.1g

Graph 5.1: Effect of different percentage of alkali on acid fraction isolation

Photograph 5.3: TLC fingerprint for effect of KOH

1. 2% KOH

2. 5 % KOH

3. 10% KOH

4. 20 % KOH

5. Whole extract

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

Effect of NaOH:

Following are observations when NaOH is used as basifying agent

Table 5.2: Effect of percentage of NaOH as basifying agent on acid fraction

	Percentage of NaOH	Amount of acid
1.	2% NaOH	1.0g
2.	5% NaOH	0.9g
3.	10% NaOH	0.5g
4.	20% NaOH	0.1g

Graph 5.2: Effect of different percentages of NaOH on acid fraction isolation

Photograph 5.3: TLC fingerprint for effect of NaOH

1. 2% NaOH

2. 5 % NaOH

3. 10% NaOH

4. 20 % NaOH

5. Whole extract

Thin Layer Chromatography:

Thin layer chromatography is carried out under following conditions

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

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

3. Detection:

(A)-UV 254 nm

(B)- Anisaldehyde sulphuric acid (ASA)

Heat treatment is required after TLC plate is sprayed with ASA

Effect of Change in mineral acid (H₂SO₄):

Table 5.3: Effect ofChange in mineral acid on acid fraction

S. No.	Percentage of Alkali	Conc. HCl	Conc. H₂SO₄
1.	2% KOH	1.4g	1.2g
2.	5% KOH	1.0g	0.8g
3.	10% KOH	0.6g	0.5g
4.	20% KOH	0.1g	0.1g
5.	2% NaOH	1.0g	1.0g
6.	5% NaOH	0.9g	0.7
7.	10% NaOH	0.5g	0.4
8.	20% NaOH	0.1g	0.1

Graph 5.3: Effect of Change in Mineral acid on isolation of acid fraction (KOH)

Graph 5.4: Effect of Change in Mineral acid on isolation of acid fraction (NaOH)

Photograph 5.4: TLC fingerprint for effect of Change in mineral acid on acid fraction (KOH)

1. 2% KOH

2. 5 % KOH

3. 10% KOH

4. 20 % KOH

5. Whole extract

Photograph 5.5: TLC fingerprint for effect of Change in mineral acid on acid fraction (NaOH)

1. 2% NaOH

2. 5 % NaOH

3. 10% NaOH

4. 20 % NaOH

5. Whole extract

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

Effect of Change in strength of mineral acid

Table 5.4: Effect of strength of mineral acid on acid fraction

Sr. No.	Percentage of Alkali	Dil HCl	Conc. HCl	Dil H₂SO₄	Conc. H₂SO₄
1.	2% KOH	1.5	1.4g	1.0	1.2
2.	5% KOH	1.0	1.0g	0.8	0.8
3.	10% KOH	0.4	0.6g	0.3	0.5
4.	20% KOH	0.05	0.1g	0.1	0.1
5.	2% NaOH	0.8	1.0g	1.0	1.0
6.	5% NaOH	0.6	0.9g	0.6	0.7
7.	10% NaOH	0.5	0.5g	0.3	0.4
8.	20% NaOH	0.1	0.1g	0.05	0.1

Graph 5.5: Effect of strength of mineral acid on isolation of acid fraction (HCl + KOH)

Graph 5.6: Effect of strength of mineral acid on isolation of acid fraction (H₂SO₄ + KOH)

Graph 5.7: Effect of strength of mineral acid on isolation of acid fraction

(HCl + NaOH)

Graph 5.8: Effect of strength of mineral acid on isolation of acid fraction

(H₂SO₄ + NaOH)

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 5.6: TLC fingerprint for effect of strength of mineral acid on acid fraction (KOH+ Dil HCl)

1. 2% KOH

2. 5 % KOH

3. 10% KOH

4. 20 % KOH

5. Whole extract

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 5.7: TLC fingerprint for effect of strength of mineral acid on acid fraction

(KOH+Dil H₂SO₄)

1. 2% KOH

2. 5 % KOH

3. 10% KOH

4. 20 % KOH

5. Whole extract

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 5.8: TLC fingerprint for effect of strength of mineral acid on acid fraction

(NaOH+Dil HCl)

1. 2% NaOH

2. 5 % NaOH

3. 10% NaOH

4. 20 % NaOH

5. Whole extract

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 5.9: TLC fingerprint for effect of strength of mineral acid on acid fraction

NaOH+Dil H₂SO₄

1. 2% NaOH

2. 5 % NaOH

3. 10% NaOH

4. 20 % NaOH

5. Whole extract

Isolation of individual boswellic acids:

As per literature survey acid portion of resin contains number of boswellic acid derivatives but the acetyl keto β boswellic acid (AKBA) is reported to have potent anti-inflammatory activity. So hers focus is made on isolation of AKBA and α and β boswellic acid mixture.

Literature survey gives an idea about separation of these two individual boswellic acids by TLC. Those mobile phases were utilized in column chromatography for separation of these two boswellic acids.

Column Chromatography1:

The mobile phase Toluene: Ethyl acetate: n heptane: Formic acid (8:2:1:0.3) was used for separation of individual acids, details of experimental conditions were mentioned in section above in Column Chromatography 1. Here formic acid was not added in mobile phase because it is immiscible with remaining solvents.

Pure AKBA and α and β boswellic acid can be isolated from column chromatography. As mentioned in experimental conditions these two compounds were isolated from fraction 4 and 5, 6-8 respectively. These compounds were isolated and confirmed by R_f values as well as by spectroscopic studies. This procedure was repeated twice and same fraction yields these two compounds.

Pure AKBA and α and β boswellic acid can be isolated from column chromatography. As mentioned in experimental conditions these two compounds were isolated from fraction 4 and 5, 8 and 9 respectively. These compounds were isolated and confirmed by R_f values as well as by spectroscopic studies. This procedure was repeated twice and same fraction yields these two compounds.

Photograph 5.10: TLC fingerprintof fractions of column 1

w- Whole Extract, Numbers corresponds to fraction numbers

Thin Layer Chromatography:

Thin layer chromatography is carried out under following conditions

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

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

3. Detection: UV 254 nm

Heat treatment is required after TLC plate is sprayed with ASA

Column Chromatography 2:

Isolation of pure AKBA and α and β boswellic acid can also be done by different solvent systems in column chromatography. Here Toluene: Ethyl acetate: Methanol (8:2:1) was used as mobile phase. Procedure was repeated twice and nearly same fractions yield pure compounds. AKBA was isolated from fractions 6-9 and mixture of α and β boswellic acid was obtained from fractions 10-13. Identity of these two was confirmed by TLC running them with standard.

Spectroscopic studies:

β-Boswellic acid:

Chemical Name: 3a-hydroxy-urs-12-en-23-oic acid

Molecular Formula: C₃₀H₄₈O₃

Melting Point: 226-228⁰C

FTIR (in KBr) cm^-1:3500, 1699

UV (methanol): maxima at 208nm

GC MS: 394, 218, 203, 189, 175, 161

HNMR (CDCl₃): 11, 5.29, 3.58, 2-1, 1-0.9

In above spectroscopic data FTIR values 3500 stands for OH group and 1699 stands for COOH.

Molecular ion peak of 394 is due to loss of CO₂(-44) andH₂O(-18). 218 is base peak. Proton NMR δ values of 11 indicates OH of COOH. 5.29 indicates presence of C=C i.e. vinyl proton. 3.58 indicates CH-OH. Other values from 2 to 1stands for methylenes and methines 23 protons and from 1-0.9 stands for methyls i.e 21 protons. Other NMR values are mentioned in structure.

Acetyl-11-keto-β-boswellic acid:

Chemical Name: 3a-Acetoxy-urs-12-en-11-keto-23-oic acid

Molecular Formula: C₃₂H₄₈O₅

Melting Point: 271-274⁰C

FTIR (in KBr) cm^-1: 1740, 1701, 1647

UV (methanol): maxima at 250nm

GC MS: 394, 218, 203, 189, 175, 161

HNMR (CDCl₃): 5.5, 3.58, 2.5, 2-1, 1-0.9

In the above spectroscopic data IR values of 1740 indicates presence of acetyl group, 1701 is for COOH and 1647 for α, β unsaturated carbonyl. Molecular ion peak of 408 indicates loss of CO₂ (-44) and H₂O (-18). NMR δ values of 11 indicates OH of COOH. 5.29 indicates presence of C=C i.e. vinyl proton. 3.58 indicates CH-OH. Other values from 2 to 1stands for methylenes and methane’s 21 protons and from 1-0.9 stands for methyl’s i.e 21 protons. Other NMR values are mentioned in structure.

CONCLUSION:

In the present study optimization of process parameters in isolation of boswellic acid/acid fraction was carried out. A general process for isolation of boswellic acid from Boswellia serrata oleo gum resin involves use of alkali and acid one after the other same parameters were optimized during the course of the study.

Isolation of boswellic acids/Acid fraction involve/s:

Extraction of oleo gum resin with a suitable solvent: Here selection of a suitable solvent becomes important parameter to be optimized. Selection of a suitable solvent was based on different parameters like potency of the solvent i.e. ability to extract desired component.

Apart from potency of solvent its economic point of view should also be considered during extraction process. Selection of a suitable solvent was carried out by extracting oleo gum resin with different commonly available solvent in laboratory. Section 5.1 describes extraction of resin with different solvents effect of which is seen in Photograph 5.1. From TLC it can be concluded that extraction can be done with any commonly available lab level solvent like acetone, ethyl acetate, toluene, ethanol, methanol except water. If we observe TLC of water extract it do not shows presence of desired boswellic acid in TLC finger print so water cannot be here a suitable solvent for extraction. In case of other solvent when economic point of view is considered apart from potency, Ethanol becomes most suitable solvents for extraction.

Selection of suitable extraction process:

In the previous section for selection of suitable solvent maceration was used as a method of extraction because maceration is least hazardous and mild process when extraction is considered so if a solvent is able to extract desired component by this process it will be able to extract the same component by other methods of extraction also. During selection of suitable method of extraction different commonly available extraction method like maceration, Soxhlet extraction, distillation were used for extraction of oleo gum resin with ethanol. When these methods were tried maceration seems to be more suitable method for extraction because in other methods of extraction which involves use of heat it creates difficulty in extraction due to clogging of crude Oleogumresin contains waxes, gum which melts during extraction same problem happens when steam distillation is used. Steam distillation not a method of extraction but it is a method to fractionate the extract as this Oleogumresin also contains volatile oil. Some methods of extraction reported in literature also mentions use of steam distillation for removal of volatile oil prior to extraction but this volatile oil can also be removed during concentration of extract. In case of selection of suitable extraction process completion of extraction is also one of the most important parameter to be optimized. Completion of extraction process can be checked by carrying out TLC after extraction [here maceration]. During maceration oleo gum resin was kept in contact with ethanol for 24 hrs with shaking in this case maceration was not carried out once but successively till it shows absence of any further desired chemical component in the residue here it seems that it takes 6 successive maceration of same type to extract completely the desired components which can be explained on the basis of TLC.

Isolation of acid fraction:

Literature survey reveals that the pharmacological activity of oleo gum resin resides mainly in acid fraction of the extract so in the present study focus is made on extraction of acid fraction of extract. Out of the various methods reported in literature most suitable and simplest method of extraction is followed, which is mentioned in Section 4.7. Method described as,

(h) Crushing the lumps of the gum resin of Boswellia serrata and extracting the crushed lumps with ethanol.

(i) Removing insoluble material from above extract;

(j) Concentrating the extract till a reddish brown syrupy mass is obtained;

(k) Basifying the syrupy mass with an aqueous solution of an alkali to provide a solution having a pH in the range of 9 to 10.

(l) Extracting the solution with a solvents to provide an aqueous layer, and acidifying the aqueous layer with mineral acid to a pH in the range of 3-5 to provide a precipitate comprising boswellic acids;

(m) Washing the precipitate with water to provide said fraction being neutral to litmus;

(n) Separating individual boswellic acids from said fraction.

The method involves treatment of extract with alkali first and then acid. The method was optimized by selection of these 2 parameters. Studies when carried out to check change in type of the alkali that is used in first step of separation, here KOH and NaOH was used as alkalies.

Effect of NaOH and KOH was checked at different concentrations. In case of KOH from observation it seems that amount of acid fraction obtained at 2 percent KOH is more as compare to higher percentages (see table 5.1). So conclusion can be made that isolation of acid fraction is dependent on percentage of KOH but not directly proportional on it. When alkali is changed to NaOH seems that from table No. 5.2. isolation of acid fraction is also dependent on specific concentration of alkali but not directly proportional to concentration of alkali the conclusion can also be made that isolation of acid fraction is pH dependent in case of both NaOH and KOH but when amount of acid fraction is compared in both cases KOH seems to be more suitable than NaOH.

Effect of mineral acid:

The second step of isolation after treatment with alkali involves use of mineral acid for precipitation of acid fraction.ere parameters which was optimized are selection of suitable mineral acid. Commonly used mineral acid in laboratory are HCl and H₂SO₄. These where selected for the studies. When HCl is used as mineral acid at different concentrations (see table 5.3 on page 42) of KOH effect will be same but when H₂SO₄ is replaced with HCl decrease in amount of acid fraction is observed (see table 5.3 on page 42 and corresponding TLCs) so conclusion can be made of HCl will be more suitable as compared to H₂SO₄.

Effect of strength of acid:

In this study strength of mineral acid is used as parameters for optimization. Dilute and concentrated acid where used (see Table 5.4) from table it can be concluded that dilute acid seems to be more suitable as compared to concentrated acid but from table this applies only in case of HCl. In case of H₂SO₄ amount of acid fraction obtained by use of Conc.H₂SO₄ is more than that of dilute H₂SO₄ but if we observe all 4 values of acids use of dil.H₂SO₄ yields more acid fraction as compared to remaining 3. This applies to KOH when it is used in low concentration i.e. 2 percent and effect of which already mentioned. When NaOH is used as alkali and dilute HCl is used as mineral acid. Acid fraction obtained was a little bit lesser a compared to H₂SO₄ [Dilute and concentrated] and also concentrated HCl. When 2 percent NaOH is used in initial stage of separation followed by use of H₂SO₄ amount of acid fraction obtained seems to be more as compared to dilute HCl but still it is lesser than the amount of acid fraction obtained from 2 percent KOH as an alkali followed by dilute HCl as a mineral acid.

Isolation of individual Boswellic acid:

In this case column chromatography is used as method of isolation. Isolation of Acetyl Keto β boswellic acid (AKBA) and α and β boswellic acid was performed. Because α and β boswellic acid is major boswellic acid and Acetyl Keto β boswellic acid (AKBA) is most potent anti-inflammatory agent. Separation pattern in column chromatography can be predicted from TLC. If TLC is optimized and column chromatography is performed any optimized solvent system can be used. in case where Toluene: Ethyl acetate: n heptanes: Formic acid (8:2:1:0.3) was used in TLC formic acid was not added in column chromatography. In present study two optimized solvent systems were used (see section 4.6.1 and 5.4). Individual boswellic acid isolated was confirmed by spectroscopic studies.

ACKNOWLEDGEMENTS:

I would like to express my gratitude to the management, Principal, staff, and lab technicians of Aurangabad Pharmacy College, who helped me a lot to publish this article.

Financial Disclosure statement:

The author received no specific funding for this work.

Conflict of Interest:

The authors declare that there is no conflict of interest regarding the publication of this article.

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