Introduction
In 1991, Sabinsa Corporation pioneered Boswellin^®, a standardized extract containing boswellic acids, for the U.S. market. The prevalently used nomenclature for the anti-inflammatory constituents in Boswellia serrata extracts is "boswellic acids". Standardized extracts currently marketed are labeled as containing about 50% to70% boswellic acids. A titrimetric analytical procedure is used to arrive at this value. This procedure measures the total content of organic acids and therefore projects a cumulative organic acids value. Isolation of the individual components and detailed analytical methodology, developed by Sabinsa Corporation, have now provided the necessary data to redefine this extremely useful natural product.

Boswellia serrata (N.O. Burseraceae) is a large, branching, deciduous tree which grows abundantly in the dry, hilly parts of India. It is known as "Dhup", Indian Frankincense or Indian Olibanum. The gum resin exudate of Boswellia serrata, known in the vernacular as "Salai guggal", has been used in the Ayurvedic system of medicine for the manage-ment of rheumatism, respiratory diseases, and liver disorders. The major use of Boswellia serrata in contemporary medicine is as an anti-arthritic and anti-inflammatory pharmacological agent.

Discussion
The anti-inflammatory properties of the gum resin is attributed to the presence of "boswellic acids". 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 which initiates injury to the tissues, which in turn triggers the inflammatory process. This dual inhibitory action on the inflammatory process is unique to boswellic acids.

Detailed Chemistry of Boswellin®

The gum resin of Boswellia serrata is known to contain:

• Monoterpenes (a-thujene)
• Diterpenes (macrocyclic diterpenoids such as incensole, incensole oxide, iso-incensole oxide, a diterpene alcohol (serratol))
• Triterpenes (such as a-and b-amyrins)
• Pentacyclic triterpenic acids (boswellic acids)
• Tetracyclic triterpenic acids (tirucall-8,24-dien-21-oic acids)

The four major pentacyclic triterpenic acids present in the acidic extract of Boswellia serrata gum resin (Boswellin® ) are:

• b-Boswellic Acid (I)
• Acetyl-b-Boswellic Acid (II)
• 11-keto-b-Boswellic Acid (III)
• Acetyl-11-keto-b-Boswellic Acid (IV)

(1)	(2)
(3)	(4)

Two other pentacyclic triterpenic acids have also been isolated:

a-Boswellic Acid (V)

g-Boswellic Acid (VI)

(5)

(6)

In addition to the above six pentacyclic triterpenic acids, four tetracyclic triterpenic acids have also been identified. These are:

3 a-tirucall-8,24-dien-21-oic acid (VII)

3-ketotirucall-8,24-dien-21-oic acid (VIII)

3 a-hydroxytirucall-8,24-dien-21-oic acid (IX)

3 b-hydroxy tirucall-8,24-dien-21-oic acid (X)

(7)	(8)
(9)	(10)

In the manufacture of Boswellin® , Boswellia serrata gum resin is separated into two fractions by solvent extraction and alkali treatment:

• An acid fraction
• A neutral fraction

The total acid fraction (I-X) is pharmacologically significant and all available clinical documentation is based on this fraction. However, additional research done in the 90’s indicates that the boswellic acids from the total acid extract are the principal pharmacologically active components to give the therapeutic benefits.

Analysis of Boswellin® (ref 6)

The following observations from the structural profiles of the pentacyclic triterpenic acids are analytically significant:

• All these compounds have a hydroxyl functional group either in free form (OH) or as acetate (OAc) in the "3" position and a carboxyl functional group (COOH) in the "4" position.
• Two of these acids have a keto (C=O) functional group in the "11" position, adjacent to the vinylic double bond.
• One boswellic acid can be transformed to another by simple chemical reactions.

The standard assay procedure for the acid fraction of Boswellia serrata extract involves:

• Titration - for total organic acids (I-X)
• A recently developed HPLC procedure for quantifying the four biologically active b-boswellic acids (I-IV)

Pioneering efforts by Sabinsa Corporation have resulted in improved methods for isolation and HPLC assay of the individual b-boswellic acids. Working HPLC standards for individual boswellic acids have also been meticulously prepared.

This involves the following steps:

• Physical separation of the mixture into individual components
• Elution of the separated components from the chromatographic column at different rates.
• Using UV detector, only the eluted components (one particular compound at a time) is quantitated. The peaks for b-Boswellic Acid and Acetyl- b-Boswellic Acid are recorded at 210 nm, while those for 11-keto- b-Boswellic Acid and Acetyl-11-keto- b-Boswellic Acid are recorded at 254 nm. Detection at two different UV absorptions are necessitated because of the spectral properties of the four compounds and their interactions.

This procedure ensures that each component is quantitated separately. The working standards used for comparison are characterized on the basis of measured physical and chemical properties.

A. b-boswellic acids BY HPLC*

B. Total Organic Acids BY TITRATION*

Total organic acids : 70.9%

*Results averaged over 7 random samples drawn from batches prepared between 1995 and 1999

The clinical studies reported in literature used the acid fraction of the extract of the gum resin from Boswellia serrata labeled "boswellic acids". The above discussion however reveals the complex chemistry of the acid fraction. In light of these facts, the clinical effectiveness of Boswellia serrata acidic fraction could be viewed in two ways:

• The total acid constituents are clinically significant (constituted by the titrimetric assay constituents (about 70%)). However, this is not supported by recent scientific evidence.
• The comparatively low levels of pure b-boswellic acids present (about 25%) are the anti-inflammatory constituents, contributing entirely to the clinical effectiveness.

The anti-inflammatory properties of the gum resin are attributed to the presence of total organic acids, erroneously referred to as boswellic acids throughout the initial literature¹.

Although all clinical documentation was based on total organic acids (erroneously termed boswellic acids) from the methanolic extract, recent studies on isolated Boswellic Acid components show that only the four b-boswellic acids are effective anti-inflammatory components.

In a preliminary study by H.P.T. Ammon and others, it was shown that among the boswellic acids, Acetyl-11-keto-b-Boswellic Acid was observed to have the most pronounced inhibition of 5-lipoxygenase product formation.² The comparative inhibitory effects of various b-boswellic acids on 5-lipoxygenase product formation are indicated in Figure 1³.

 
Figure 1 : Biological activity of various b-Boswellic acids in inhibiting the enzyme 5-Lipoxygenase

In another pioneering study⁴ using isolated boswellic acids, Y. Shao and others also showed that the pharmacological properties reside only in the four b-boswellic acids. This study focused on the anti-cancer activity of the isolated boswellic acids. Studies by H. Safayhi et al⁵ showed that Acetyl-11-keto-b-boswellic acid decreased the activity of human leukocyte elastase (HLE) in vitro with an IC₅₀ value of about 15 mM.

In conclusion, the total acid value of the extract is useful as a standardization tool, while sophisticated analytical methodology should be used to determine the actual amount of therapeutically useful pentacyclic triterpenic acids, the b- boswellic acids.

Composition of Commercial Samples : Comparative Evaluation:

Commercial samples of Boswellia serrata extracts from various sources were tested for b-Boswellic acids content and composition using HPLC methods. There are many companies offering Boswellia serrata extracts, ranging in potency from 50% to 100% boswellic acids. The analytical results for a few samples are indicated in Figure 2 and Figure 3, in terms of content and composition, respectively. In many commercial samples, the most active b-Boswellic acids are available in negligible quantities only.

Figure 2 : b-Boswellic acids Content in Commercial Samples of Boswellia serrata extract as determined by HPLC

Figure 3 : b-Boswellic acids Composition in Commercial Samples of Boswellia serrata extract as determined by HPLC

The total organic acids content in these samples as determined by titration is indicated in Figure 4.

Figure 4 : Total Organic Acids in Commercial Samples of Boswellia serrata extract as determined by titration

Thus it is evident that the active components in Boswellia serrata extract cannot be accurately predicted based on titrimetric method analysis. It is equally interesting to note that while the titrimetric method gives more than 50% organic acids, several of the commercially available products contain only negligible amounts of the two key boswellic acids, namely 11- keto- beta- and acetyl- 11- keto- beta- boswellic acids (Figure 3). These compounds are pharmacologically the more potent anti-inflammatory compounds.

Nomenclature labeling and dose recommendations:

For scientific accuracy, the following nomenclature is recommended for labeling purposes:

"Boswellia serrata extract containing 65-70% organic acids"

Based on the evaluation of all data and documentation so far, the dose of Boswellic acids for anti-inflammatory purposes is as follows:

As total organic acids: 200 mg x 3 times daily

equivalent to total boswellic acids: 50 mg x 3 times daily

That is, Boswellia serrata powdered extract equivalent to 286 mg of the 70% total organic acids extract (286x0.7=200.2 mg).

However, to determine the supply of spurious extracts (such as those labeled as containing Boswellic acid 100%), companies are encouraged to perform a HPLC assay of Boswellia serrata extracts.

Standard HPLC chromatograms for the four b-Boswellic acids are shown in Figures 5 and 6:

Figure 5: HPLC CHROMATOGRAM OF b-BOSWELLIC ACIDS WORKING STANDARD AT 210 nm

Figure 6: HPLC CHROMATOGRAM OF b-BOSWELLIC ACIDS WORKING STANDARD AT 254 nm

b-Boswellic Acid

1. Chemical structure:    (I) 2. Chemical name 3a-Hydroxy- urs-12-en-23-oic acid 3. Molecular formula C30H48O3     Molecular weight 456.7 4. Melting point 226 - 2280C (Lit. mp 228 - 230OC) 5. Specific rotation + 106.80 (Lit. + 108O) 6. FTIR (in KBr) 3500 cm-1, (OH) 1699.5 cm-1, (COOH) 7. UV (methanol) Maxima at 208 nm (Lit. 208 nm) 8. NMR (in CDCl3) ¶ 5.15 (CH=C, vinylic proton) 4.08 (CH-OH) 2.3-1.1 (Methylenes and methines 23 protons) 1.1-0.7 (Methyls, 21 protons) 9. GC-MS 394 (M-68[44 due to-CO2 & 18 due to –H2O]) 218 (base peak, due to retro-Diels-Alder fragmentation). Other characteristic fragments: 203, 189, 175, 161.

Acetyl-b-Boswellic Acid

1. Chemical structure (II) 2. Chemical name 3a-Acetoxy-urs-12-en-23-oic acid 3. Molecular formula C32H50O4 Molecular weight 498.74 4. Melting point 252 - 2550 C (Lit. mp 2530C) 5. Specific rotation + 1380 (Lit.  + 141.30) 6. FTIR (in KBr) 1732 cm-1 (OAc), 1701 cm-1 (COOH) 7. UV (methanol) Maxima at 208 nm (Lit. 208 nm) 8. NMR (in CDCl3) ¶ 5.31 (CH=C, vinylic proton) 5.2 (CH-OAc) 2.1 (COCH3) 1.9-1.25 (Methylenes and methines, 23 protons) 1.2-0.7 (Methyls, 21 protons) 9. GC-MS 394 (M-104[44 due to-CO2 & 60 due to –HOAc]) 218 (base peak, due to retro-Diels-Alder fragmentation).

11-Keto-b-Boswellic Acid

1. Chemical Structure   (III) 2. Chemical name 3a-Hydroxy-urs-12-en-11-keto-23-oic acid 3. Molecular formula C30H46O4 Molecular weight  470.69 4. Melting point 195 - 1970C (Lit. mp 195°C) 5. Specific rotation +78.50 (Lit. + 79.5o) 6. FTIR (in KBr) 3460 cm-1 (OH), 1693 cm-1 (COOH) 1647 cm-1 (a,b unsaturated carbonyl) 7. UV (in Methanol) Maxima at 250 nm (Lit. 250 nm) 8. NMR (in CDCl3) ¶ 5.55 (CH=C, vinylic proton) 4.08 (CH-OH) 2.6-1.4 (Methylenes and methines 21 protons) 1.25-0.75 (Methyls, 21 protons) 9. GC-MS 408 (M-68[44 due to-CO2 & 18 due to –H2O]) 232 (base peak, due to retro-Diels-Alder fragmentation). Other characteristic fragments  217, 175, 161, 135.

Acetyl-11-Keto-b-Boswellic Acid

1. Chemical structure  (IV) 2. Chemical name 3a-Acetoxy-urs-12-en-11-keto-23-oic acid 3. Molecular formula C32H48O5 Molecular weight  512.73 4. Melting point 271 - 2740C (Lit. mp 2710C) 5. Specific rotation + 88.50 (Lit.  +87.00) 6. FTIR (in KBr) 1740 cm-1 (Ac), 1701 cm-1 (COOH) 1647 cm-1 (a,b unsaturated carbonyl) 7. UV (in Methanol) Maxima at 250 nm (Lit. 250 nm) 8. NMR (in CDCl3) ¶ 5.55 (CH=C, vinylic proton) 5.2 (CH-OAc) 2.6-1.4 (Methylenes and methines 21 protons) 1.25-0.75 (Methyls, 21 protons) 9. GC-MS 408 (M-68[44 due to-CO2 & 18 due to –HOAc]) 232 (base peak, due to retro-Diels-Alder fragmentation). Other characteristic fragments  217, 175, 161, 135.

1. Singh, G.B. et al. Boswellic acids - A new class of anti-inflammatory agents with a novel mode of action. Paper presented at the International Seminar : Traditional Medicine, Calcutta, India, 7-9 November, 1992, pp 81-82.

2. Safayhi, H. et al. (1992) Boswellic acids: novel, specific, non-redox inhibitors of 5-lipoxygenase. J. Pharmacol. Exp. Ther. 261:1143-6.

3. Patent No. 0 552 657 A1 (1993), European Patent Office

4. Shao, Y., et al. (1998). Inhibitory activity of boswellic acids from Boswellia serrata against human leukemia HL-60 cells in culture. Planta Medica, 64(1):328-331

5. Safayhi, H. et al. (1997) Inhibition by boswellic acids of human leukocyte elastase. J. Pharmacol. Exp. Ther. 281:460-463.

6. SAMI Labs Ltd., India – Research Reports and Analytical method development studies (1999).