Biological activity and tissue specific accumulation of fluorescently labeled methyl jasmonate.

The C-7 position of jasmonate is practical for synthesis of a probe to use for chemical biological studies. To confirm the utility, we synthesized fluorescent-labeled methyl jasmonate. The synthesized compound exhibited Arabidopsis thaliana root growth inhibitory and meandering activity, and potent fluorescence was observed inside the root and root hairs.

Antitrypanosomal activities of acetylated bruceines A and C; a structure-activity relationship study.

The crude extract of Brucea javanica showed strong in vitro inhibitory activity against Trypanosoma evansi. Among the isolated quassinoids, bruceines A, C, and bruceantinol were found to be the most potent compounds against T. evansi. To gain a deeper understanding of the relationship between the free hydroxyl groups and the activity, several O-acetylated derivatives of bruceines A and C were synthesized and their in vitro antitrypanosomal activities against trypomastigotes of T. evansi were examined and compared with those of the original compounds. The following structure-activity relationships were observed: (1) the free hydroxyl groups at positions C-3, C-11, and C-12 are essential for antitrypanosomal activity; (2) the C-11 and C-12 hydroxyl groups are more important for the activity than the enolic hydroxyl group at C-3, and; (3) the free hydroxyl group at C-4' of bruceine C does not have any significant effect on the activity.

Bioconversion of proposed precursors into theobroxide and related compounds.

We have previously reported a tetraketide origin for theobroxide and its related compound. In the present study, bioconversion of natural and deuterium-labeled precursors of this proposed biosynthetic pathway by Lasiodipoldia theobromae was investigated. Theobroxide was quantified after bioconversion from each proposed precursor. The transformation of the isotopically labeled precursor to products was tracked by 2H NMR measurement.

Identification of a beta-glucosidase hydrolyzing tuberonic acid glucoside in rice (Oryza sativa L.).

Tuberonic acid (TA) and its glucoside (TAG) have been isolated from potato (Solanum tuberosum L.) leaflets and shown to exhibit tuber-inducing properties. These compounds were reported to be biosynthesized from jasmonic acid (JA) by hydroxylation and subsequent glycosylation, and to be contained in various plant species. Here we describe the in vivo hydrolytic activity of TAG in rice. In this study, the TA resulting from TAG was not converted into JA. Tuberonic acid glucoside (TAG)-hydrolyzing beta-glucosidase, designated OsTAGG1, was purified from rice by six purification steps with an approximately 4300-fold purification. The purified enzyme migrated as a single band on native PAGE, but as two bands with molecular masses of 42 and 26 kDa on SDS-PAGE. Results from N-terminal sequencing and peptide mass fingerprinting of both polypeptides suggested that both bands were derived from a single polypeptide, which is a member of the glycosyl hydrolase family 1. In the native enzyme, the K(m) and V(max) values of TAG were 31.7 microM and 0.25 microkatal/mg protein, OsTAGG1 preferentially hydrolyzed TAG and methyl TAG. Here we report that OsTAGG1 is a specific beta-glucosidase hydrolyzing TAG, which releases the physiologically active TA.

Potato tuber cell expansion-inducing activity of stereochemically restricted JA analogs.

Stereochemically restricted analogues of C-7 substituted 7-epi-jasmonate, together with 12-hydroxy jasmonic acid, 12-hydroxy jasmonic acid glucoside, and jasmonic acid conjugated with L-isoleucine (JA-Ile), were synthesized and then tested for potato tuber cell expansion-inducing activity. JA-Ile showed almost the same activity as JA, while the C-7 substituted 7-epi-jasmonates exhibited weaker activity than JA and showed an antagonist effect against JA.

Isolation of antibabesial compounds from Brucea javanica, Curcuma xanthorrhiza, and Excoecaria cochinchinensis.

One new curcuminoid, 3'-demethoxycyclocurcumin (1), was isolated from Curcuma xanthorrhiza as an antibabesial compound, together with p-hydroxybenzaldehyde (2) and cleomiscosin A (3) from Brucea javanica and (+)-epiloliolide (4) from Excoecaria cochinchinensis. The antibabesial activities were examined in vitro, and compounds 1-4, and diminazene aceturate were studied with IC(50) values of 16.6, 7.6, 15.6, 10.0, and 0.6 microg/ml, respectively.

Evaluation of efficacy of bruceine A, a natural quassinoid compound extracted from a medicinal plant, Brucea javanica, for canine babesiosis.

Bruceine A, a natural quassinoid compound extracted from the dried fruits of Brucea javanica (L.) Merr., was evaluated for its antibabesial activity in vitro and in vivo. Bruceine A inhibited the in vitro growth of Babesia gibsoni in canine erythrocytes at lower concentration compared with the standard antibabesial drug diminazene aceturate and killed the parasites within 24 hr at a concentration of 25 nM. Oral administration of bruceine A at a dosage of 6.4 mg/kg/day for 5 days resulted in no clinical findings in a dog with normal ranges of hematological and biochemical values in the blood. Three dogs were infected with B. gibsoni and two of them were treated with bruceine A at a dosage of 6.4 mg/kg/day for 6 days from day 5 post-infection. An untreated dog developed typical acute babesiosis symptoms including severe anemia, high fever, and complete loss of appetite and movement. However, the two bruceine A-treated dogs maintained their healthy conditions throughout the experimental period of 4 weeks although complete elimination of parasites from the peripheral blood was not achieved and decreases in the packed cell volume and the erythrocyte and platelet counts were observed. Since natural quassinoid compounds have been used as traditional medicines for the treatment of various ailments including cancer and malaria, the present results suggest that bruceine A or other related compounds are potential candidates for the treatment of canine babesiosis.

Purification and cDNA cloning of a wound inducible glucosyltransferase active toward 12-hydroxy jasmonic acid.

Tuberonic acid (12-hydroxy epi-jasmonic acid, TA) and its glucoside (TAG) were isolated from potato leaflets (Solanumtuberosum L.) and shown to have tuber-inducing properties. The metabolism of jasmonic acid (JA) to TAG in plant leaflets, and translocation of the resulting TAG to the distal parts, was demonstrated in a previous study. It is thought that TAG generated from JA transmits a signal from the damaged parts to the undamaged parts by this mechanism. In this report, the metabolism of TA in higher plants was demonstrated using [12-(3)H]TA, and a glucosyltransferase active toward TA was purified from the rice cell cultures. The purified protein was shown to be a putative salicylic acid (SA) glucosyltransferase (OsSGT) by MALDI-TOF-MS analysis. Recombinant OsSGT obtained by overexpression in Escherichia coli was active not only toward TA but also toward SA. The OsSGT characterized in this research was not specific, but this is the first report of a glucosyltransferase active toward TA. mRNA expressional analysis of OsSGT and quantification of TA, TAG, SA and SAG after mechanical wounding indicated that OsSGT is involved in the wounding response. These results demonstrated a crucial role for TAG not only in potato tuber formation, but also in the stress response in plants and that the SA glucosyltransferase can work for TA glucosylation.

In vitro antitrypanosomal activities of quassinoid compounds from the fruits of a medicinal plant, Brucea javanica.

The medicinal plant Brucea javanica (L.) Merr. (Simaroubaceae) is widely distributed throughout Asia where its bitter fruits have been used in traditional medicine for various ailments. Fifteen C-20 quassinoids were isolated from the fruits of B. javanica and examined for their in vitro antitrypanosomal activities against trypomastigotes of Trypanosoma evansi. Bruceine A, bruceantinol, bruceine C, brusatol, and bruceine B showed strong antitrypanosomal activities with IC(50) values in the range of 2.9-17.8nM, which compared well with the standard trypanocidal drugs diminazene aceturate (IC(50)=8.8nM) and suramin (IC(50)=43.2nM). However, dehydrobruceine A, dehydrobruceine B, and dehydrobrusatol were about 2100, 900, and 1200 times less active, respectively, than bruceine A, bruceine B, and brusatol. The relationship of the structure and antitrypanosomal activity of these quassinoid compounds suggested that the presence of a diosphenol moiety in ring A and the nature of the C-15 side chain are important for their activities against T. evansi. This is the first report on the antitrypanosomal activity of isolated quassinoids.

New potato micro-tuber-inducing cyclohexene compounds related to theobroxide from Lasiodiplodia theobromae.

Two new cyclohexene compounds related to theobroxide (3) were isolated from the mycelia of Lasiodiplodia theobromae OCS71. The structures of these compounds were determined to be (4S,5S)-4,5-dihydroxy-2-methyl-cyclohex-2-enone (1) and (3aS,4R,5S,7aR)-4,5-dihydroxy-7-methyl-3a,4,5,7a-tetrahydrobenzo[1,3]dioxol-2-one (2) by means of spectroscopic analyses and chemical correlation to 3. Compound 2 was shown to take up the carbonate ion to form a carbonic acid ester non-enzymatically. The compounds also showed potato micro-tuber-inducing activities at a concentration of 10(-3) M, using a culture of single-node segments of potato stems in vitro.

Temperature regulates tuber-inducing lipoxygenase-derived metabolites in potato (Solanum tuberosum).

Temperature is one of the major environmental factors affecting potato tuberization. It has been suggested that lipoxygenase (LOX) mediates between temperature and tuber induction. In this study, the contents of the LOX-derived metabolites hydroperoxylinolenic acid (HPOT), jasmonic acid (JA), tuberonic acid (TA) and tuberonic acid glucoside (TAG) were analyzed in leaves of potatoes growing at different temperatures. At low, tuber-inducing temperature, endogenous levels of JA, TA and TAG rise, indicating their crucial role in tuber induction. The concentration of 13(S)-HPOT seems not to be directly affected by temperature. Instead, the molecule has only a short half-life in leaves and is readily metabolized.

Screening of Indonesian medicinal plant extracts for antibabesial activity and isolation of new quassinoids from Brucea javanica.

Boiled extracts derived from 28 Indonesian medicinal plants were screened for their antibabesial activity against Babesia gibsoni in vitro. Of these extracts, the fruit of Brucea javanica was the most active in inhibiting parasite growth at a concentration of 10 microg/mL. Bioassay-guided fractionation of the fruit extract of Br. javanica led to the isolation of two new quassinoids, bruceantinol B and bruceine J, and the structures of these compounds were elucidated on the basis of their spectroscopic data and by chemical transformation to known compounds. In addition, the known quassinoids bruceines A-D, bruceantinol, and yadanziolide A were isolated. Antibabesial activities were also examined in vitro, and bruceine A and bruceantinol were shown to be more potent than diminazene aceturate, a drug (IC50 = 103 ng/mL) used clinically against B. gibsoni, with IC50 values of 4 and 12 ng/mL, respectively.