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Objsective Glycyrrhizia uralensis, one of the most widely-used traditional Chinese medicines, is mainly cropped in China. However, many cultivars are less in glycyrrhizic acid than Chinese Pharmacopoeia requires. In this paper, we improved glycyrrhizic acid by regulating β-amyrin synthase gene (GuBAS). Methods Tobacco root-specific promoter TobRB7 and GuBAS cDNA were obtained and combined with linearized pCAMBIA1305.1 to construct root-specific plant expression vector which was later transformed into Agrobacterium rhizogenes ACCC10060 by electrotransformation. The cotyledons and hypocotyls of G. uralensis were infected by the recombinant A. rhizogenes ACCC10060 to induce hairy roots. The GA content was quantified by HPLC. Results The PCR and sequencing results both showed that three transgenic hairy root lines were obtained. The copy number of GuBAS in these transgenic hairy roots was intended by qRT-PCR to be 3, 7, and 4. GA was detected by HPLC, and the results showed that GA was present in the three transgenic hairy roots, while absent in wild hairy roots. Conclusion Over-expressing GuBAS root-specifically in hairy roots of G. uralensis enhanced GA accumulation.

Uvaia (Eugenia pyriformis Cambess.) leaves were subjected to extraction with supercritical fluid extraction (SFE) and ultrasound-assisted extraction (UAE) under different conditions and the results were compared with a conventional maceration extraction. The extracts obtained with SFE were chemically characterized by gas chromatography–mass spectrometry (GC–MS), which showed that β-amyrin (53.72%) and α-amyrin (24.63%) were their main compounds. The best results for SFE and UAE yieldings were 1.69% and 1.79%, respectively. The best results for amyrin content, by extract weight, were 81.19% for SFE and 77.03% for UAE. The highest amyrin content was 11.40 g of the isomers mixture per kg of dry leaf in SFE and 11.46 g kg−1 in UAE. Scanning electron microscopy analysis showed differences in leaf particles before and after the extraction process, caused by the removal of compounds by the extraction techniques studied.

Guiera senegalensis J.F. Gmel is a broad-spectrum African folk- medicinal plant, having activities against fowlpox and herpes viruses. Very recently, we have shown the anti-hepatitis B vius (HBV) potential of G. senegalensis leaves extract (GSLE). Here, we report the antioxidative and hepatoprotective efficacy of GSLE, including HPTLC quantification of four biomarkers of known antioxidative and antiviral activities. In cultured liver cells (HuH7) GSLE attenuated DCFH-induced oxidative stress and cytotoxicity. This was supported by in vitro DPPH radical-scavenging and β-carotene-linoleic acid bleaching assays that showed strong antioxidant activity of GSLE. Further, two simple and sensitive HPTLC methods (I and II) were developed and validated to quantify β-amyrin, β- sitosterol, lupeol, ursolic acid in GSLE. While HPTLC-I (hexane: ethylacetate; 75:25; v/v) enabled quantification of β-amyrin (Rf = 0.39; 20.64 μg/mg) and β-sitosterol (Rf = 0.25; 18.56 μg/mg), HPTLC-II (chloroform: methanol; 97:3; v/v) allowed estimation of lupeol (Rf = 0.47; 6.72 μg/mg) and ursolic acid (Rf = 0.23; 5.81 μg/mg) in GSLE. Taken together, the identified biomarkers strongly supported the antioxidant and anti-HBV potential of GSLE, suggesting its activity via abating the oxidative stress. To our knowledge, this is the first report on HPTLC analysis of these biomarkers in G. senegalensis that could be adopted for standardization and quality-control of herbal-formulations. Keywords: Guiera senegalensis, HPTLC, β-Amyrin, β-Sitosterol, Lupeol, Ursolic acid, Antioxidant, Antiviral, Anti-HBV

Anemone flaccida Fr. Shmidt (Ranunculaceae), known as 'Di Wu' in China, is a perennial herb which has long been used to treat arthritis. The rhizome of A. flaccida contains pharmacologically active components i.e. oleanane-type triterpenoid saponins. Oleanolic acid is natural triterpenoid in plants with diverse biological activities. The biosynthesis of oleanolic acid involves cyclization of 2,3-oxidosqualene to the oleanane-type triterpenoid skeleton, followed by a series of oxidation reactions catalyzed by cytochrome P450 monooxygenase (CYP450). Previously, we identified four possible cytochrome P450 genes belonging to CYP716A subfamily from the transcriptome of A. flaccida. In this study, we identified one of those genes "CYP716A254" encoding a cytochrome P450 monooxygenase from A. flaccida that catalyzes the conversion of the β-amyrin into oleanolic acid. The heterologous expression of CYP716A254 in yeast resulted in oxidation of β-amyrin at the C-18 position to oleanolic acid production. These results provide an important basis for further studies of oleanane-type triterpenoid saponins synthesis in A. flaccida.

Introduction Amyrins are triterpenes that have attractive pharmacological potential; however, their low water solubility and erratic stomach absorption hinders their use as a drug. The aim of this paper was to develop a novel α-amyrin-loaded nanocapsule for intestinal delivery and evaluate, preliminarily, its cytotoxic ability against leukemic cells. Material and methods Five nanocapsule formulations were designed by the solvent displacement-evaporation method. Poly-e-caprolactone, Eudragit® E100, and Kollicoat® Mae 100 P were used as film-former materials. Particle size, polydispersity index (PdI), zeta potential, and the pH of all formulations were measured. The cytotoxic potential of the nanocapsules was evaluated in vitro using different leukemic lineages Results Nanocapsules coated with Kollicoat® Mae 100 P presented the smallest particle size (130 nm), the lowest zeta-potential (−38 mV), and the narrowest size distribution (PdI = 0.100). The entrapment efficiency was 65.47%, while the loading capacity was 2.40%. Nanocapsules release 100% of α-amyrin in 40 min (pH 7.4), by using a possible mechanism of swelling-diffusion. The formulation showed excellent on-shelf physicochemical stability during one year. Additionally, nanocapsules produced a selective cytotoxic effect on a human leukemia lineage Kasumi-1, an acute myeloid leukemia cell line, and produced cell death by apoptosis Conclusion α-amyrin-loaded nanocapsules appear to be a promising nanoformulation that could be used against leukemia.

Pentacyclic triterpenes have a diverse biological activity and they are promising targets for the medicinal applications in numerous disorders. The high amount of triterpenes is found in Lamiaceae and Ericaceae family; however, the new sources of these compounds are continuously being looked for. Different species of Carlina genus have been commonly used in traditional medicine in many countries and their pharmacological properties may be related to pentacyclic triterpenes; thus, the objective of this work was the analysis of various species of Carlina genus regarding the presence of triterpenic compounds and their distribution in different parts of plant. So far, the chemical study of these plants refers only to the determination of polyphenols and the essential oils composition. HPTLC was used as a screening method for qualitative investigations. The quantification of triterpenes was done using RP-HPLC-PDA on spherical and monolithic stationary phases. The identity of compounds was established by HR-APCI-MS and 1H NMR techniques. Our investigation reveals the occurrence of α-amyrin, β-amyrin, β-amyrin acetate, lupeol, lupeol acetate, betulinic, oleanolic and ursolic acid mostly in aboveground parts of the studied species. Carlina genus turned out to be a rich source of biologically active triterpenes.

The present study explores the effect of varying concentrations of soil cadmium (50−200 mg/kg soil) and lead (200−800 mg/kg soil) on glasshouse grown plantlets of Centella asiatica. The plantlets were fairly tolerant to soil cadmium and lower concentrations of lead in terms of biomass accumulation. Application of 200 mg/kg soil cadmium concentration, led to enhanced biomass/two-fold increase in shoot length and a significant accumulation of cadmium in shoots and roots. Specific activities of anti-oxidant enzyme systems such as superoxide dismutase, ascorbate peroxidase, and guaicol peroxidase were also found to be elevated under treated conditions. Interestingly, the ratio of cadmium concentration in roots: soil (bioaccumulation coefficient factor) was more than 1, Centella asiatica could be a potential bioaccumulator of cadmium. Also since translocation of cadmium from roots to shoots was less than 40 %, the leaves, accumulating more than twenty five fold more centellosides (in 200 mg/kg soil cadmium v/s control conditions) could also be potentially exploited for phytochemicals with minimum post-harvest processing. Real time PCR analysis also revealed higher population of centelloside biosynthetic gene transcripts such as squalene synthase, farnesyl pyrophosphate synthase, β amyrin synthase, as compared to control conditions. Plant growth, although tolerant to lower concentrations of soil lead could not effectively accumulate lead in roots, and centelloside contents was also found to be only marginally higher than control. This study reports the possibility of exploiting C. asiatica as potential plant systems that may be cultivated on cadmium contaminated soils to reap phyto-pharmaceutical benefits.

Black cumin (Nigella sativa) is a medicinal herb belongs to the Ranunculacea which raised attention due to its pharmaceutical properties. Medical significance of N. sativa mainly attributed to its oxygenated monoterpenes and triterpene saponins which are biosynthesized via the methyl erythritol phosphate (MEP) and MVA (mevalonic acid) pathways, respectively. In order to improve our understanding of monoterpene and triterpene metabolism the partial sequence of a hypothetical monoterpene synthase (MTS) was isolated from N. sativa for the first time and subsequently the relative gene expression of two genes of monoterpene metabolism including geranyl diphosphate synthase (GDS), the isolated monoterpene synthase (MTS) and two genes of triterpene saponins biosynthesis including squalene epoxidase (SQE) and β- amyrin synthase (βAS) were monitored in response to two universal abiotic elicitors including methyl jasmonate (MJ) and salicylic acid (SA), also in different tissues of N. sativa including leaves, stems, roots, capsules and immature seeds. Results of semi quantitative reverse transcription PCR revealed differential regulation of these genes in N. sativa tissues and also in response to abiotic elicitors. Each gene followed a different expression profile and expression level in target tissues. The expression of all studied genes were up-regulated by exogenous application of MJ and SA on N. sativa leaves (at 24 h after treatments), except of GDS which its transcript was not induced by SA. The higher expression of these genes in response to abiotic elicitors might reflect an elevation in the corresponding metabolites; hence elicitation is an applicable tool for enhancement of pharmaceutically active mono/triterpenes in N. sativa.

Pentacyclic triterpenes (PT), ursolic acid (Urs), and α-amyrin (AMalf) are natural products exhibiting broad spectrum of antibacterial activity. These compounds are membrane-active and can disorder bacterial membranes when incorporated; however, the exact mechanism of their membrane activity is unknown. In our studies, we applied Langmuir monolayer technique supported by Brewster angle microscopy to model the interactions of the selected PT with the lipid matrix of E. coli inner membrane. As the model membrane, we applied mixtures (75/25 mole/.mole %) of the representative Escherichia coli phosphatidylethanolamine (POPE), with the cardiolipin (ECCL) or phosphatidylglycerol (ECPG) extracted from the E. coli inner membrane. On the basis of the recorded isotherms, we performed thermodynamic analysis and calculated free energy of mixing ΔGexc. It turned out that the phospholipids forming the inner membrane of E. coli are ideally miscible, whereas in binary systems composed of PT and POPE, negative deviations from ideality indicating attractive interactions between the investigated PT and POPE molecules were observed. On the other hand, in ternary systems composed of PT, POPE and one of the E. coli anionic phospholipids large positive changes in ΔGexc were observed. Thus, both PT exhibit disorganizing effect on the model E. coli membrane. It was also proved that at low terpene proportion, AMalf can be more active than Urs. However, at higher proportion Urs incorporation can lead to the disintegration of cardiolipin-rich domains present in bacterial membrane.

β-Amyrin isolated from Symplocos cochinchinensis Moore. leaves was evaluated for the effects on free radicals, antioxidant enzymes and liver protection on CCl 4 induced oxidative stress in Wistar rats. β-Amyrin showed very good scavenging effects on 2,2-diphenyl-picrylhydrazyl (DPPH) (IC 50 89.63 ± 1.31 μg/ml, AAI = 1.11), hydroxyl (IC 50 76.41 ± 1.65 μg/ml, AAI = 1.30), nitric oxide (IC 50 87.03 ± 0.85 μg/ml, AAI = 1.14) and superoxide (IC 50 81.28 ± 1.79 μg/ml, AAI = 1.23) radicals as well as high reducing power and strong suppressive effect on lipid peroxidation. In in vivo study, pre-treatment of rats with β-amyrin at 50 mg/kg for 7 days showed significant reduction in the levels of SGOT, SGPT and LDH compared to CCl 4 treated rats. SOD, CAT, GSH and GPx levels were increased significantly after treatment with β-amyrin. These results clearly demonstrated that β-amyrin possessed marked antioxidant activity.

While evaluating the toxicity of the tuberous root extracts of Hemidesmus indicus, a traditional medicinal plant, the glucose lowering property of the root was observed by the investigators. Therefore, it was thought of interest to isolate the anti-hyperglycemic principle from the root and determine its utility to develop an anti-diabetes mellitus medicine. The active principle was isolated from H. indicus root extract by anti-hyperglycemic activity guided chromatographic techniques. Glucose tolerance test in rats was used to evaluate the anti-hyperglycenic property. Anti-diabetes mellitus property was evaluated in alloxan-induced diabetic rats as well as streptozotocin-induced (type-2 model) diabetic rats. The active principle was isolated and identified with spectral data as β-amyrin palmitate. Although it is a known compound, its presence in H. indicus is not known previously. It was observed for the first time that β-amyrin palmitate has remarkable anti-hyperglycemic activity in orally glucose loaded rats. Further, interestingly, it exhibited excellent anti-diabetes mellitus activity in both alloxan-diabetic and streptozotocin-diabetic rats at a very low concentration (50µg/kg body weight). One of the mechanisms of action of β-amyrin palmitate appears to be blocking the entry of glucose from the intestine. β-Amyrin palmitate is very promising to develop a medicine for diabetes for combination therapy and/or mono-therapy.

Pressurized liquid extraction (PLE) of Arctium lappa leaves using aqueous ethanol is examined and presented for the first time in this work. In addition, global extraction yields, chemical composition, phenolic compounds and antioxidant activity of the PLE extracts were compared with those obtained by supercritical fluid extraction (SFE). PLE extractions were evaluated at different conditions of pressure (15, 20 and 25) MPa and temperature (313.15, 333.15 and 353.15) K. The maximum global yield (37.40 wt %) was obtained by PLE at 15 MPa and 353.15 K. Furthermore, the PLE extracts contained higher concentrations of chlorogenic acid (1.84%) and rutin (1.46%) and exhibited considerably high DPPH free radical scavenging activity (160.54 μmol Trolox g−1). PLE optimum extract exhibited considerable concentrations of phytol, lupeol and amyrin, making this a promising alternative for obtaining biologically active extracts from natural sources such as Arctium lappa leaves.

Glycyrrhiza uralensis is considered to be one of the most important herbs in traditional Chinese medicine due to its numerous pharmacological effects particularly its ability to relieve cough and act as a mucolytic. Based on previous research, these effects are mediated by a number of active ingredients, especially glycyrrhizic acid (GA). In the present study, a gene encoding β -amyrin synthase ( β -AS) involved in GA biosynthesis in G. uralensis has been cloned and expressed in Saccharomyces cerevisiae . The cloned enzyme showed similar activity to native enzymes isolated from other Glycyrrhiza species to catalyze the conversion of 2,3-oxidosqualene into β -amyrin. In fact the β -AS gene is particularly important in the GA biosynthetic pathway in G. uralensis . The complete sequence of the enzyme was determined and a phylogenetic tree based on the β -AS gene of G. uralensis and 20 other species was created. This showed that Glycyrrhiza glabra had the closest kinship with G. uralensis . The results of this work will be useful in determining how to improve the efficacy of G. uralensis by improving its GA content and in exploring the biosynthesis of GA in vitro .

A pot experiment was conducted to investigate into effects of molybdenum (Mo) on the secondary metabolic process of glycyrrhizic acid (GA). One-year-old seedlings were grown in pots with washed vermiculite and sand. Hoagland nutrition solution was irrigated with four concentrations: 0, 0.52, 5.2 and 10.4 mg L −1 . The accumulations of GA and its biosynthetic precursors ( β -amyrin and squalene) and then expression of the key synthase ( β -amyrin synthase, β -AS) were studied on 35, 70 and 105 d. In the early stage, that was on the 35 and 70 d, the contents of squalene and GA, and the expression of β - AS gene under 0.52 and 5.2 mg L −1 Mo treatments were significantly higher than that under 0 and 10.4 mg L −1 Mo. There was a contrary result of β -amyrin. However, the content of squalene under 0 mg L −1 Mo was the highest on 105 d. Thus, it suggested an appropriate concentration of Mo could promote the accumulation of GA, by affecting the biosynthetic process of GA at a certain time. Practically, the time and amount of application of Mo on Glycyrrhiza uralensis should be the noted.

The ethanolic extract and fractions of Wrightia t omentosa Roem. & Schult (Apocynaceae) leaves were tested in vivo for their antidyslipidemic activity in high fat diet (HFD) induced dyslipidemic hamsters. Activity guided isolation resulted in identification of antidyslipidemic compounds β-AA and β-AP. Compounds β-AA and β-AP decrease the levels of LDL by 36% and 44%, and increase the HDL-C/TC ratio by 49% and 28%, respectively, at a dose of 10 mg/kg. In addition, the isolated compounds β-AA and β-AP showed significant HMG-CoA-reductase inhibition, which was further established by docking studies.

Previous studies from our group investigated the antinociceptive property of amyrin octanoate, a synthetic compound derivative from natural precursor alpha, beta-amyrin, against nociceptive response induced by acetic acid and formalin. Here, we investigated some of the mechanisms of action underlying the antinociceptive effects of amyrin octanoate. Amyrin octanoate given intraperitoneally (0.001-1 mg /kg) or intrathecally (10-1000 ng /site) caused dose-dependent and long-lasting inhibition of acetic acid-induced visceral nociception, with mean ID(50) values of 0.003 (0.001-0.005) mg/kg and 122.4 (60.8-246.6) ng/site, respectively. In the capsaicin- and glutamate-induced paw licking, amyrin octanoate caused significant and dose-dependent inhibition of both nociceptive responses, with ID(50) values of 1.36 and 0.04 mg/kg, respectively. Furthermore, amyrin octanoate also reduced significantly the nociception caused by intrathecal injection of glutamate, substance P and capsaicin, with inhibitions of 36+/-11%, 67+/-10% and 78+/-5%, respectively. The antinociception caused by amyrin octanoate in the acetic acid test was significantly attenuated by neonatal pretreatment of mice with capsaicin, but seems to involve mechanisms independent of G(i/o) protein, opioidergic, serotonergic, noradrenergic and cholinergic system, since it was not affected by pertussis toxin, naloxone, yohimbine, mecamylamine or atropine. In addition, amyrin octanoate reduced thermal and mechanical hyperalgesia induced by bradykinin and phorbol myristate acetate in rats, without affecting similar responses caused by prostaglandin E(2). Taken together, the present results shown that octanoate amyrin produces antinociceptive and antihyperalgesic effects, through an interaction with capsaicin-sensitive fibers and the inhibition of the PKC signaling pathway.

The effects of alpha,beta-amyrin, a pentacyclic triterpene isolated from Protium heptaphylum was investigated on rat model of orofacial pain induced by formalin or capsaicin. Rats were pretreated with alpha,beta-amyrin (10, 30, and 100mg/kg, i.p.), morphine (5mg/kg, s.c.) or vehicle (3% Tween 80), before formalin (20 microl, 1.5%) or capsaicin (20 microl, 1.5 microg) injection into the right vibrissa. In vehicle-treated controls, formalin induced a biphasic nociceptive face-rubbing behavioral response with an early first phase (0-5 min) and a late second phase (10-20 min) appearance, whereas capsaicin produced an immediate face-rubbing (grooming) behavior that was maximal at 10-20 min. Treatment with alpha,beta-amyrin or morphine significantly inhibited the face-rubbing response in both test models. While morphine produced significant antinociception in both phases of formalin test, alpha,beta-amyrin inhibited only the second phase response, more prominently at 30 mg/kg, in a naloxone-sensitive manner. In contrast, alpha,beta-amyrin produced much greater antinociceptive effect at 100mg/kg in the capsaicin test, which was also naloxone-sensitive. These results provide first time evidence to show that alpha,beta-amyrin attenuates orofacial pain at least, in part, through a peripheral opioid mechanism but warrants further detailed study for its utility in painful orofacial pathologies.

Fifteen different derivatives of an alpha- and beta-amyrin mixture were synthesized by acylation with appropriate anhydride or acid chlorides and oxidation in the presence of tert-butyl chromate or PCC. The molecular structures of the obtained compounds were confirmed by means of IR and (1)H NMR spectra. The compounds were screened for antinociceptive activity using the acetic acid pain model. The 3-O-acyl derivatives alpha- and beta-amyrin propionate 4, alpha- and beta-amyrin hexanoate 6, and alpha- and beta-amyrin octanoate 7 were found to be the most active compounds of the series. In addition, we also have found that alpha- and beta-amyrin octanoate 7 was able to reduce acetic acid-induced abdominal constriction when administered by oral route. Furthermore, this compound reduced the nociceptive response induced by intraplantar injection of formalin.

In the search of hepatoprotective agents from natural sources, alpha- and beta-amyrin, a triterpene mixture isolated from the trunk wood resin of folk medicinal plant, Protium heptaphyllum was tested against acetaminophen-induced liver injury in mice. Liver injury was analysed by quantifying the serum enzyme activities and by histopathological observations. In mice, acetaminophen (500 mg/kg, p.o.) caused fulminant liver damage characterized by centrilobular necrosis with inflammatory cell infiltration, an increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, a decrease in hepatic glutathione (GSH) and 50% mortality. Pretreatment with alpha- and beta-amyrin (50 and 100 mg/kg, i.p. at 48, 24, and 2 h before acetaminophen) attenuated the acetaminophen-induced acute increase in serum ALT and AST activities, replenished the depleted hepatic GSH, and considerably reduced the histopathological alterations in a manner similar to N-acetylcysteine, a sulfhydryls donor. Also, the acetaminophen-associated mortality was completely suppressed by terpenoid pretreatment. Further, alpha- and beta-amyrin could potentiate the pentobarbital (50 mg/kg, i.p.) sleeping time, suggesting the possible suppression of liver cytochrome-P450. These findings indicate the hepatoprotective potential of alpha- and beta-amyrin against toxic liver injury and suggest that the diminution in oxidative stress and toxic metabolite formation as likely mechanisms involved in its hepatoprotection. In conclusion, this study supports the traditional use of Protium heptaphyllum resin as a medicinal agent and suggests the feasibility of developing herbal drugs for treatment of liver disorders.

The sterol composition of spores from 16 species of arbuscular mycorrhizal fungi belonging to the order Glomales were examined by GC–MS. The major compound was found to be 24-ethylcholesterol (up to 85%) followed by cholesterol (up to 15%). Several other sterols such as 24-methylcholesterol, Δ5-avenasterol and 24-ethylcholesta-5,22-dien-3 β -ol were also detected. Significant amounts of α -amyrin, a common vascular plant triterpene, were present in the spores of all the fungal species analyzed. The absence of ergosterol, a classical fungal sterol, is discussed in relation to fungal evolution.

A 2274 bp Arabidopsis thaliana cDNA was isolated that encodes a protein 57% identical to cycloartenol synthase from the same organism. The expressed recombinant protein encodes lupeol synthase, which converts oxidosqualene to the triterpene lupeol as the major product. Lupeol synthase is a multifunctional enzyme that forms other triterpene alcohols, including β -amyrin, as minor products. Sequence analysis suggests that lupeol synthase diverged from cycloartenol synthase after plants diverged from fungi and animals. This evolutionary order is the reason that fungi and animals do not make lupeol.

A convergent synthesis provided a C 25 and a C 35 oxidopolyprene in which a farnesyl C 15 unit is connected in a head-to-head fashion to a geranyl C 10 or a geranylgeranyl C 20 unit. When incubated with recombinant β-amyrin synthase from Pisum sativum the C 25 oxidopolyprene was enzymatically converted to an unnatural novel tetracyclic sesterterpene, while the C 35 analogue did not afford any cyclization product.

A series of Δ2-triterpenoids (oleana-2,12-diene, oleana-2,13(18)-diene, oleana-2,18-diene, ursa-2, 12-diene, and taraxera-2,14-diene) has been identified in Holocene buried mangrove sediments (Florida, USA) by comparison of the mass spectra of and by co-injections with authentic standards. In the Oligocene Brandon lignite (Vermont, USA) oleana-2,12-diene and oleana-2,13(18)-diene were also identified, along with two A-ring contracted amyrin derivatives. By comparison with the sedimentary occurrence of related analogues of oxyallobetulin and betulin and the demonstrated diagenetic formation of lup-2-ene from lupan-3-one, these novel terrigenous biological markers are considered diagenetic products formed by dehydration of precursor molecules, such as α- and β-amyrin, and taraxerol. The identification of these early intermediates brings us one step closer to the full understanding of the diagenetic fate of terrigenous triterpenoids.

The regulation of triterpenoid saponin biosynthesis in Gypsophila paniculata and Saponaria officinalis suspension cells has been investigated by studying the effects of adding gypsogenin 3,O-glucuronide, a ubiquitous saponin precursor in plants of the genus Gypsophila which contain gypsogenin saponins, to the cultures. Treatment of suspended G. paniculata cells with gypsogenin 3,O-glucuronide 24 hr before administration of [ 14 C]acetate resulted in a marked reduction in the incorporation of radioactivity into saponins and their precursors but not into sterols and steryl glucosides. 2,3-Oxidosqualene-cycloartenol and -amyrin cyclase activities were measured in microsomal fractions obtained from suspension cultures of the two species. Pretreatment of cultures with gypsogenin 3,O-glucuronide was shown to lead to inhibition (in G. paniculata cells) or stimulation (in S. officinalis cells) of the 2,3-oxidosqualene-amyrin cyclase activity and was without any marked effect on 2,3-oxidosqualene-cycloartenol cyclase activity. These results strongly suggest that in these species 2,3-oxidosqualene-cyclases are regulating steps in the isoprenoid pathway orienting the biosynthetic flux towards either tetracyclic or pentacyclic triterpenes.

With a view to obtain new compounds bearing potential applications in the biogenetic type synthesis of natural products, acetylmethyl ursolic acid ( 1 ) was subjected to prolonged treatment with H 2 O 2 /AcOH on boiling water bath. Several such products ( 2 , 3 , 5 & 7 ) have thus been obtained. A new one-pot partial synthesis of 15α-hydroxy Δ 12 -analogues of pentacyclic triterpenes of amyrin series has been achieved. 13 C-NMR spectral data of the major product are also described.

Drugs were traditionally derived from natural molecules. Over half of the new drugs approved in the past 20 years can trace their structural origins to sources as disparate as poisonous sea snails, soil fungus, and tree bark. In recent years, pharmaceutical companies turned their backs on natural products because of the difficulty of rapidly isolating and synthesizing molecules from natural sources on a large scale. However, a number of drugs and drug intermediates including artemisinin, capsaicin, arachidonate, and amyrin can be made using genetic techniques coupled to chemistry.

Helichrysum kraussii is used to treat several diseases including tuberculosis. The biologically active compounds and their possible role as phytoalexins were investigated. Jasmonic acid was used as an elicitor to activate the resistance system of the plant, and to initiate the synthesis of defense compounds. To eliminate the risk of pathogen attack and activation of plants in the field, tissue cultures were initiated with the aim of producing cell suspension cultures. Different time intervals and concentrations of jasmonic acid were tested to determine the strongest activation. Two compounds were present in the activated cell cultures that were not present in the controls. After isolation the compounds were identified as the terpenoids α-amyrin and β-amyrin. These terpenoids were tested against the bacterium Bacillus cereus to determine their antibacterial activity. This is the first report of their presence in a Helichrysum species.

As part of ongoing studies of the medicinal aspects of Maritime flora, particularly the herbal remedies of the Micmac and Malecite Indians, the sterols and triterpenes of Achillea millefolium L. (Com-positae), a widely used herbal remedy known commonly as yarrow, were determined. Using modern techniques, including nuclear magnetic resonance spectroscopy and combined GC-mass spectrometry, β -sitosterol was identified as the major sterol and α -amyrin as the major triterpene of this plant. The sterols stigmasterol, campesterol, and cholesterol and the triterpenes β -amyrin, taraxasterol, and pseudotaraxasterol were also identified. Successful therapeutic application of yarrow may be partly due to the presence of one or more of these compounds since many sterols and triterpenes exhibit a wide range of pharmacological activities. This is the first reported occurrence of cholesterol, campesterol, and the four triterpenes in yarrow.

The distribution of deuterium in squalene and β-amyrin, biosynthesized from mevalonic acid-6,6,6-d3 in Pisum sativum, has been examined b

Hydrogen peroxide-selenium dioxide in t -butanol has been found to be a good reagent for the preparation of 11α,12α, -oxidotriterpenoids of oleanane and ursane skeletons; whereas α -amyrin and β-amyrin acetates furnished 11α,12α -epoxyurs-14-en-3β-yl acetate ( 4c ) and 11α, 12α -epoxytaraxer-14-en-3β-yl acetate ( 4a ) respectively, uvaol and ursolic acid/methyl ester gave 11α, 12α -epoxyurs-28→13-olide-3β-ol ( 5d ); erythrodiol ( 3c ) and oleanolic acid ( 3b )/methyl ester ( 3d ) afforded 11α, 12α-epoxy-oleanam-28→13-olide-3β-ol ( 5 ) ; oleam-12-en-2α, 3β, 28-yl triacetate ( 3j ) on similar reaction furnished 11α, 12α -epoxytaraxer-14-en-3β-ol-2α, 28-yl diacetate ( 4d ) and 11α, 12α-epoxytaraxer-14-en-2α,3β-diol-28-yl acetate ( 4e ).

The incorporation of mevalonic acid-2- 14 C into cut flower stalks of Taraxacum officinale L., whose principal sterol and triterpene respectively in the dried form is β-sitosterol and β-amyrin, was studied at intervals of 0·5 hr to 2 weeks. A trace of product possibly identical with lanosterol was indicated by gas chromatography. However, it was only weakly radioactive at all intervals studied, and it was concluded that lanosterol was not an obligatory intermediate in the biosynthesis of β-sitosterol under the conditions of these particular experiments. Several compounds which may be transient 4α-methyl sterols and therefore possible precursors of β-sitosterol were indicated by gas chromatography, but none could be identified with the methyl sterols or triterpenes available as reference compounds. The 14 C-content of β-sitosterol slowly increased to a relatively constant value after 8 hr, thereafter remaining stable. The 14 C-content of β-amyrin, on the other hand, rose slowly to a maximum in about 20 hr and then declined. The data suggest a moderate rate of turnover of β-amyrin.

Label from geraniol- 14 C was incorporated into squalene, β-amyrin and sterol, principally β-sitosterol, to the extent of 0·41, 1·41 and 0·09 per cent respectively in germinating seeds of Pisum sativum . Modes of utilization of this substrate are discussed.

The biosynthesis of α-spinasterol and β-amyrin has been investigated in Camellia sinensis . Administration of doubly labeled 4R,4- 3 H-2- 14 C-mevalonic acid led to the formation of radioactive α-spinasterol and β-amyrin. In the case of α-spinasterol, the 3 H/ 14 C ratio was found to be 2·44:5·00 rather than 3:5. The 3 H/ 14 C ratio of the biosynthesized β-amyrin was found to be 1·0:1·0, which is consistent with an earlier study in Pisum sativum .

The distribution of triterpene methyl ethers in several generations of interspecific hybrids of Cortaderia indicates dominant gene control of their synthesis. The hybrid C. richardii × C. toetoe is an exception because synthesis of α- and β-amyrin methyl ethers is suppressed in F 1 and F 2 , but is restored in the backcross F 1 × C. toetoe ; this backcross generation was heterozygous for genes for the amyrin methyl ethers, and on selfing segregated in a simple Mendelian ratio.

Label appeared in several cell fractions isolated from the cotyledons of pea seeds germinated for 48 hr with mevalonate-[2-14C]. The major radioactive metabolite in each fraction was amyrin. In a similar experiment, a fraction sedimenting between 1000 and 25 000 g and a microsomal pellet were labeled with 3H from mevalonate-[2-3H]. Each of these tritiated fractions on incubation with UDP-glucose-[U-14C] yielded CHCl3-MeOH-soluble material bearing 14C and 3H. TLC of the extracts gave a compound chromatographically identical with a glucoside and bearing the two isotopes. Acid hydrolysis of this compound gave an ether-soluble material carrying 3H alone. On TLC it co-chromatographed with amyrin. Of the two tritiated cotyledon fractions, the microsomal pellet had the lower glucosyltransferase activity. The labeled amyrin residing in this fraction served as an acceptor for glucose from UDP-glucose in the presence of a glucosyltransferase from pea seedling axis tissue. In such a mixed preparation, the axis tissue transferase suffers a marked inhibition by the cotyledon preparation.

Squalene 2,3-epoxide incubated with microsomes from bramble cell suspension cultures is shown to be converted into α-amyrin.

Experiments have shown that the variation in the ratios of α- to β-amyrin which are found in plants is not controlled by S -adenosylmethionine. The radioactive compound produced in peas after feeding with radioactive methionine is 24-methylene-lanosterol.

Air-​dried whole plants of S. acmella were extd. and chromatographed to yield α- and β-​amyrin esters (with acetic, lauric, myristic, palmitic, linoleic, and linolenic acids)​, myricyl alc., α- and β-​amyrins, stigmasterol, and sitosterol-​O-​β-​D-​glucoside. The absence of any sesquiterpene was considered significant.

It was shown that the aglycone of the main saponin of Primula elatior (L.) Hill is not primulagenin A but protoprimulagenin A with an oxide ring between C-13 β and C-28 in the β amyrin ring system.