The Pentacyclic Triterpenes  ,  -amyrins: A Review of Sources and Biological Activities

Phytochemicals are biologically active compounds present in plants used for food and medicine. A great deal of interest has been generated recently in the isolation, characterization and biological activity of these phytochemicals. This book is in response to the need for more current and global scope of phytochemicals. It contains chapters written by internationally recognized authors. The topics covered in the book range from their occurrence, chemical and physical characteristics, analytical procedures, biological activity, safety and industrial applications. The book has been planned to meet the needs of the researchers, health professionals, government regulatory agencies and industries. This book will serve as a standard reference book in this important and fast growing area of phytochemicals, human nutrition and health.


Introduction
Pentacyclic triterpenes are ubiquitously distributed throughout the plant kingdom, in a free form as aglycones or in combined forms, and have long been known to have a number of biological effects. The compounds-amyrin and -amyrin are commonly found in medicinal plants and oleo-resin obtained by bark incision of several species of Bursera or Protium of the Burseraceae family. Both in vitro and in vivo studies have shown that amyrin also has important biological functions.
In light of the considerable interest recently generated in the chemistry and pharmacological properties of amyrins and their analogs, we have undertaken this review in an effort to summarize the available literature on these promising bioactive natural products. The review will detail the recent studies on the chemistry and bioactivity of , -amyrins, which is presented in the following sections: the isolation and distribution of -amyrin and -amyrin, giving a brief introduction to amyrins as natural products and the methods used in their isolation; the biological activities of amyrins, examining the biological properties associated with these compounds with a focus on their potential chemotherapeutic applications.

Detection
Amyrins are found in various plants and plant materials such as leaves, bark, wood, and resins. This material has to be pre-treated prior to isolation of the target compounds. First, the plant material is usually dried, then ground into a power and sieved. Second, extractions are carried out with dichloromethane, chloroformo, n-hexane, and methanol. The samples can be subjected to alkaline hydrolysis, derivatization and separation by thin layer chromatography, and the resulting material can be directly subjected to analysis. Gas chromatography (CG) and high performance thin layer chromatography (HPTLC) techniques are the most commonly employed methods to quantitate -, -amyrin in plants.
TLC provided an easy and rapid way to study plant extract profiles and partially identify compounds. The first step for the identification of -amyrin, -amyrin and 3-epi-lupeol was to compare R F values of reference standards with those of sample extracts. TLC on silica gel revealed that -amyrin on tracks 6 and 15, -amyrin on track 14 and the -, -amyrin mixture on track 16, as well as two standards, all had the same R F (Fig. 2). The -amyrin band was observed as brown, while the -amyrin band appeared violet, as did the band for the -, -amyrin mixture. TLC analysis revealed the presence of -amyrin, -amyrin and 3epi-lupeol by a comparison of the position and color of the triterpene spots with those of authentic compounds (Fig. 2). The bands of and -amyrin or their mixture were observed in all commercial resin tracks 1-5 and medicinal plant tracks 8-13, while 3-epi-lupeol track 7 was detected only in the commercial Mexican Copal resins tracks 1-4. Attempts were made to separate the -, -amyrin mixture, which had appeared homogenous on TLC, but without success. These results showed that TLC can be used as a simple method for a preliminary analysis of these triterpenes in extracts of commercial resins and plants, but cannot be employed for the analysis of the -, -amyrin mixture. (Hernández-Vázquez et al., 2010)  -Amyrin, -amyrin and other triterpenes were analysed by TLC and HPLC, the chromatographic techniques including silica gel and reversed-phase (C18RP) TLC and C18 RP-HPLC using UV and mass spectrometric (MS) detection with APCI (Martelanc et al., 2009). HPTLC combined with densitometry has been used to analyse the triterpenoids -, amyrin and the oleanolic acid content of acetone and ethyl acetate extracts of the leaves of Jovibara sobolifera (Sims) (Szewczyk et al., 2009). The detection and/or quantitation of -, amyrin either in plants or plant products using GC methods requires pre-derivatization of the samples, for example, acetylation or trimethylsilylation. Sometimes a sample clean-up employing silica gel columns or liquid-liquid partition is also necessary. 7=3-epi-lupeol; 8=Dandelion; 9=Olive; 10=Cancerina; 11=Nance wastes; 12=Bearberry; 13=Pot marigold; 14=-amyrin; 15=-amyrin and 16=mixture -, -amyrin.

Analysis
Gas chromatography (CG) is applied to determine the concentration of -, -amyrin and -, -amyrin mixtures. The chemical composition of the essential oil of Lemon Catnip (Nepeta cataria L. var. citriodora Balbis) was determined by CG-MS, and -amyrin was detected in the hydrodistilled volatile of Lemon Catnip (Wesolowska et al., 2011). -amyrin has also been determined in the kernel fats of the shea tree (Vitellaria paradoxa; Sapotaceae) of sub-Saharan countries (Akihisa et al., 2010). CG of the apolar extract from Clusia Minor L. leaves led to the identification of 25 compounds, lupeol and -amyrin being the most abundant triterpenoids (Mangas-Marin et al., 2008). CG-MS fingerprints for cerumen from the stingless bee Tetragonula carbonaria in South East Queensland, Australia, showed trace quantities of TMS ethers of -amyrins (Massaro et al., 2010). Studies on the constituents of yellow Cuban propolis by CG-MS revealed the presence of large amounts of triterpenic alcohols including -amyrin, (Márquez-Hernández et al., 2010). Solid-phase extraction and GC-MS were developed to separate and enrich only sterols from unsaponifiables of vegetable, hazelnut and olive oils, detecting sterols, lupeol and -, -amyrin (Azadmard- Damirchi et al., 2010). Epicuticular and intracuticular waxes from both adaxial and abaxial surfaces of Kalanchoe daigremontiana leaves (Hamet et Perr. De la bathie) were analyzed by CG. All wax mixtures were found to contain triterpenoids and fatty acids, the triterpenoid fraction containing small amounts of -amyrin (van Maarseveen & Jetter., 2009). Fatty acid, phytosterol, and polyamine conjugate profiles of corn edible oils were analyzed by GC-MS and HPLC, and a few minor sterols and -amyrin were identified and quantified using GC-FID (Moreau et al., 2009). Fatty acids, phytosterols and tocopherols of Milk thistle (Silybum marianum) seeds were determined in four varieties grown in Ardebil-Iran. In this study using TLC-GC, dimethylsterols were predominant followed by cycloartenol and -amyrin, (Fathi-Achachlouei, Azadmard-Damirchi., 2009). CG proved an effective method for quantitative measurement of the -sitosterol content of white mulberry (Morus alba) leaves and bark without derivatization. Sterols, lupeol and , -amyrin were identified in leaves and bark by GC-FID analysis (Böszörményi et al., 2009). GC and CG-MS were used to analyse hexane extracts from seven oleoresins of Protium species. High concentrations of and -amyrin were identified in P. strumosum (64%) and P. tenuifolium (66.7%) ). Finally, the analytical performances of three atmospheric-pressure sources, electrospray (ESI), atmospheric-pressure chemical ionization (APCI) and atmosphericpressure photoionization (APPI), were evaluated for the analysis of pentacyclic triterpenes in liquid chromatography-mass spectrometry (LC-MS) (Zarrouk et al., 2010). The developed LC-MS method was used to characterize pentacyclic triterpenes in tree plant extracts. The main component of birch bark was betulin and the extracts of Okume resin exhibited high amounts of and -amyrin (Rhourri- Frih et al., 2008). Other technique used to quantitate and determine amyrins is Reversed-Phase High Performance Liquid Chromatography (RP-HPLC). HPLC was used for analysis of some isomeric plant triterpenoids (-amyrin and amyrin -amyrin, lupeol, lupenon, lupeol acetate, cycloartenol acetate, ursolic acid oleanolic acid and two sterols) (Martelanc et al., 2009), other studies was for analysis of medicinal plants and Mexican Copal resins (Hernández-Vázquez et al., 2010) and resin obtained from species of the genus Protium (Burseraceae) (Dias et al., 2011).
An-and -amyrin mixture has been detected in the following plants: n-hexane and chloroform extracts of the epicuticular wax layer of Mandevilla guanabarica and Mandevilla moricandiana, (Cordeiro et al., 2011), an ethanolic extract of roots of Salacia amplifolia, , and chloroform extracts of Blue Honeysuckle (Lonicera caerulea L.) (Palíkova et al., 2008).
A multitude of extraction and isolation schemes have been used for the procurement of amyrin, -amyrin and an / amyrin mixture. Typically, dry material (resins, leafs and stem barks) is extracted with hexane or another non-polar solvent, (Fig. 3), and the resulting extract is directly subjected to column or thin layer chromatography. An alternative procedure is sequential fractionation by silica gel columns using various solvents. The

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The Pentacyclic Triterpenes , -amyrins: A Review of Sources and Biological Activities 493 amyrins are not readily visible on TLC plates UV ( = 254 and 365 nm) but are easily detected following exposure to iodine vapors, anisaldehyde-H 2 SO 4 or vanillin-H 2 SO 4 spray reagents.

Anti-microbial and anti-fungal
The antimicrobial properties of n-hexane and methanol extracts of Bombax malabaricum flowers were examined against different bacterial, fungal and yeast strains. The methanol extract was highly active against Staphylococcus aureus, Bacillus subtilis, Stretoccocus faecalis, Neisseria gonorrehea, Pseudomonas aeruginosa and Candida albicans, whereas the n-hexane extract displayed moderate-to-weak activities against the same test microorganisms. An n-hexane extract afforded sterols including -amyrin (El-Hagrassi et al., 2011). A bioassay-guided fractionation of n-hexane extracts of Bursera simaruba (L) Sarg. leaves resulted in the isolation and identification of five sterols and -amyrin. Additionally, n-hexane extracts have displayed anti-inflammatory activity on adjuvant-carrageenan-induced inflammation in rats (Carretero et al., 2008). -Amyrin and other compounds have been proposed as possible biomarkers for the fungal resistance of grape-vine leaves (Vitis vinifera) (Batovska et al., 2008).
In a recent study on the leaves of Siraitia grosvenorii, -amyrin and other bioactive compounds were obtained, and their activities against the growth of oral bacterial species Streptococcus mutans, Actinobacillus actinomycetemcomitans, and Fusobacterium nucleatum and the yeast C. albicans were evaluated in vitro. -amyrin only exhibited a slight inhibition of Streptococcus mutans and Fusobacterium nucleatum (Zheng et al., 2011). Bioassay-guided fractionation of the methanol extract of the stem bark of Klainedoxa gabonensis Pierre ex Engl.
(Irvingiaceae) afforded 12 compounds: four flavonoids and eight (including -amyrin) triterpenes. Antimicrobial activities in the triterpenoids ranged from low to non-existent (Wansi et al., 2010). In this study, the in vitro antibacterial activity of the methanolic extract and isolated compounds from the bark of Byrsonima Crassifolia against twelve bacteria and the yeast C. albicans was investigated. Eight known compounds, -amyrin, betulin, betulinic and oleanolic acid, quercetin, epicatechin, gallic acid and-sitosterol, were isolated and evaluated for their antimicrobial activity. Bacterial growth was inhibited by -amyrin, olenolic and gallic acid at concentrations ranging from 64 to 1088 g.mL -1 (Rivero-Cruz et al., 2008).

Anti-inflammatory activity
Hexane extracts of Bursera simaruba (L.) Sarg. leaves display an anti-inflammatory effect on adjuvant-carrageenan-induced inflammation in rats. In order to isolate and identify the active compounds of the hexane extract, we performed a preliminary phytochemical study and a bioassay-directed fractionation using the carrageenan-induced paw oedema test in mice. From the nine fractions (A-I) obtained, A and E showed the strongest antiinflammatory activity, comparable to that of the reference drug phenylbutazone. Sterols and-amyrin have been isolated and characterized from these fractions, the evidence suggesting that these bioactive compounds may play a key role in the anti-inflammatory effects of B. Simaruba extracts (Carretero et al., 2008).
Ligustrum (privet) plants are used by Chinese physicians to prevent and cure hepatitis and chronic bronchitis. Three common Ligustrum plant spp., namely Ligustrum lucidum Ait. (LL), L. pricei Hayata (LP) and L. sinensis Lour. (LS) were collected to assess their analgesic/antiinflammatory effects on chemical-induced nociception and carrageenan-induced inflammation in rodents. The methanol extracts from Ligustrum plant leaves effectively inhibited nociceptive responses induced by 1% acetic acid and 1% formalin. LP and LL reduced the edema induced by 1% carrageenan. The most potent Ligustrum plant was LP, which also reduced abdominal Evans blue extravasations caused by lipopolysaccharide, lipoteichoic acid, autocrines and sodium nitroprusside. The triterpenoid content of the three Ligustrum spp. was measured by HPLC, the highest content of -amyrin, betulinic acid and lupeol being found in LP. This work suggested that these three triterpenoids are responsible for the anti-inflammatory potency of LP .
A recent report describes that the roots of Calotropis gigantea (Linn.) R.Br, traditionally used in India to treat asthma, possess anti-lipoxygenase activity, it was found that intraperitoneal administration of indomethacin did not block edema formation, but edema was inhibited by montelukast and methanolic extracts of C. gigantea roots. This result indicates that the extract from C. gigantea was responsible for the inhibition of the lipoxygenase pathway in the arachidonate metabolism. Therefore, it can be concluded that C. gigantea may have a similar mechanism of action as dexamethasone as well as antioxidant and anti-lipoxygenase effects, possibly due to the presence of -amyrin and -amyrin (Bulani et al., 2011).
Aqueous and organic extracts of Acacia visco Lor. Ap Griseb (Fabaceae) were tested for antiinflammatory activity in experimental rat models. The extracts revealed an antiinflammatory effect against carrageenan-induced oedema, phospholipaseA-induced oedema, and cotton pellet-induced granuloma without any acute toxic effects. Among the class of compounds characterized from A. visco leaves, the triterpenoids lupeol, -amyrin and -amyrin may be mainly responsible for these anti-anflammatory properties (Padernera et al., 2010). -, -Amyrin ameliorates L-arginine-induced acute pancreatitis in rats. It has been demonstrated that the crude resin of Protium heptaphyllum (March.) has an and amyrin ratio of 63:37. The mixture of both compounds and methylprednisolone treatments significantly (P < 0.05) attenuated the L-arginine-induced increases in pancreatic wet weight/body weight ratio, and decreased the serum levels of amylase and lipase, and TNFand IL-6, in comparison with the vehicle control. Also, pancreatic levels of MPO activity, TBARS, and nitrate/nitrite were significantly lower. The conclusion of this study is that , amyrin has the potential to combat acute pancreatitis by acting as an anti-inflammatory and antioxidant agent (Melo et al., 2010).
Another study has shown the systemic preventive or therapeutic anti-inflammatory action of the triterpenes and -amyrin in TNBS-induced colitis in mice. It was found that , amyrin is as efficacious as dexamethasone in reversing the macroscopic and microscopic outcomes of TNBS-induced colitis, including the restoration of cytokine balance. Furthermore, the results also indicate that inhibition of NF- and CREB activation is certainly the main mechanism through which these triterpenes exert their anti-inflammatory action (Vitor et al., 2009). Another report demonstrated for the first time that , -amyrin isolated from Protium heptaphyllum modulates acute periodontal inflammation in rats by reducing neutrophil infiltration, oxidative stress and the production of proinflammatory cytokine TNF-a, and suggests that these triterpenes might be useful as a therapeutic agent for the treatment of gingivitis and to retard the progression of periodontitis (Holanda-Pinto et al., 2008).

Other pharmacological activities
and -Amyrin have been tested for a variety of other biological activities. An anti-ulcer effect of Cytocarpa procera and Amphipterygium adstringens was assayed on experimental gastric injury in rats and phytochemical analysis allowed the identification of -amyrin and -sitosterol in A. adstringens (Rosas-Acevedo et al., 2011). The triterpenoids -amyrin, cohulupone and garcinielliptone were isolated from the pericarp, heartwood and seed of Garcinia subelliptica, respectively, and the three compounds showed an inhibitory effect on xanthine oxidase. Treatment of NTUB1, a human bladder cancer cell, with -amyrin or amyrin in cotreatment with cisplatin for 24 h resulted in a reduced viability of cells. This work suggested that -amyrin exhibited weak cytotoxic activities against NTUB1 cells . The antiproliferative effects of n-hexane, chloroform and aqueous methanol extracts prepared from the whole plant of Centaurea arenaria M.B. ex Willd. were investigated against cervix adenocarcinoma (HeLa), breast adenocarcinoma (MCF7) and skin epidermoid carcinoma (A431) cells, using the MTT assay. Only the flavonoids and lignans showed moderate activity against these cell lines and -amyrin was inactive (Csapi et al., 2010). From the ethyl acetate fraction of the stem bark of Camellia japonica, three new triterpenoids, 3--O-acetyl-16b-hydroxy-12-oxoolean, 3-O-acetyl-16-hydroxy-11-oxoolean-12-ene, and 3--O-acetyl-16-hydroxyolean-12-ene, along with seven known compounds, 3--hydroxy-1-oxofriedelan, friedelin, 3--friedelanol, canophyllol, 3-oxofriedelan-1(2)-ene, amyrin, camellenodiol, and camelledionol, were isolated. Their structures were established on the basis of spectroscopic analysis and chemical evidence. The isolated compounds were tested in vitro for their cytotoxic activities against the A549, LLC, HL-60 and MCF-7 cancer cell lines. Among them, -amyrin exhibited weak cytotoxicity against A549 and HL-60 cancer cell lines with IC (50) values of 46.2 and 38.6 M, respectively (Thao et al., 2010). Another report showed that the methanol extract obtained from soxhlet extraction of leaves of Ardisia elliptica Thunberg (Myrsinaceae) contained and -amyrin, determined by GS-MS. The leaf extract inhibited platelet aggregation with an IC 50 value of 167 g/mL, using bioassay guided fractionation. -Amyrin was isolated and purified showing an IC 50 value of 4.5 g/mL, while that of aspirin was found to be 11 g/mL, indicating that -amyrin is more potent that aspirin in inhibiting collagen-induced platelet aggregation (Ching et al., 2010). Two triterpenes, -amyrin and 12-oleanene 3, 21-diol, were isolated as a mixture from the chloroform soluble fraction of an ethanolic extract of Duranta repens (Verbanaceae) stem. The mixture was highly effective against the larvae of Culex quinquefasciatus Say (Diptera: Culicidae) as a mosquitocide. C. quinquefasciatus is a potential vector of Wuchereria bancrofti (Filarioidae), the causative agent of human lymphatic filariasis (Nikkon et al., 2010). One study has examined the potential trypanocidal activity of different plant species growing in the Brazilian Cerrado, after in vitro screening of 20 extracts obtained from 10 plants. The phytochemical analysis of the most active extracts (hexane extracts) allowed the identification of -amyrin, -amyrin, lupeol and other triterpenes and sterols. The results showed that pure amyrins are inactive whereas the n-hexane leaf extract of Tibouchina stenocarpa cogn. Melastomataceae was active. The trypanocidal activity of the extract may be due to the presence of other compounds (Cunha et al., 2009).

Conclusion
and -Amyrin are bioactive compounds commonly found in leaves, barks and resins.
Such plant material is an interesting source of these triterpenoids, as it allows for easy extraction. Extensive research over the last four years has identified and -amyrin in several plants and the pure compounds have shown anti-microbial, anti-inflammatory and other interesting biological activities. Amyrins are also involved in the biosynthetic pathways of other biologically active compounds such as avenacine, centellosides, glycyrrhizin or ginsenosides. The development of biotransformation systems to convert amyrins into these or other compounds would open new ways for using and -amyrins as a source of bioactive plant secondary metabolites more scarcely distributed in the plant kingdom. In this context, the bioconversion of -amyrin into centellosides in Centella asiatica cell cultures has been recently reported (Hernandez-Vazquez et al., 2010).

Acknowledgement
We thank the PAPIIT-UNAM, IN223611 and CONACYT, CB2009 IN 129061, for financial support. Phytochemicals are biologically active compounds present in plants used for food and medicine. A great deal of interest has been generated recently in the isolation, characterization and biological activity of these phytochemicals. This book is in response to the need for more current and global scope of phytochemicals. It contains chapters written by internationally recognized authors. The topics covered in the book range from their occurrence, chemical and physical characteristics, analytical procedures, biological activity, safety and industrial applications. The book has been planned to meet the needs of the researchers, health professionals, government regulatory agencies and industries. This book will serve as a standard reference book in this important and fast growing area of phytochemicals, human nutrition and health.