Scopadulcic Acid B Research Articles

Functional Characterization of Three Diterpene Synthases Responsible for Tetracyclic Diterpene Biosynthesis in Scoparia dulcis

Scoparia dulcis produces unique biologically active diterpenoids such as scopadulcic acid B (SDB). They are biosynthesized from geranylgeranyl diphosphate (GGPP) via syn-copalyl diphosphate (syn-CPP) and scopadulanol as an important key intermediate. In this paper, we functionally characterized three diterpene synthases, SdCPS2, SdKSL1 and SdKSL2, from S. dulcis. The SdCPS2 catalyzed a cyclization reaction from GGPP to syn-CPP, and SdKSL1 did from syn-CPP to scopadulan-13α-ol. On the other hand, SdKSL2 was found to incorporate a non-sense mutation at 682. Therefore, we mutated the nucleotide residue from A to G in SdKSL2 to produce SdKSL2mut, and it was able to recover the catalytic function from syn-CPP to syn-aphidicol-16-ene, the precursor to scopadulin. From our results, SdCPS2 and SdKSL1 might be important key players for SDB biosynthesis in S. dulcis.

Optimization of a Liquid Culture System for Shoot Regeneration and Achieving an Enriched Level of Scopadulcic Acid B in the Leaf Organ Cultures of Scoparia dulcis L. by Response Surface Methodology

Response surface methodology (RSM) approach was utilized in the present investigation to optimize the constituents of Murashige and Skoog’s (MS) liquid medium to enrich the scopadulcic acid B (SDB) content in Scoparia dulcis L. RSM approach using the central composite design (CCD) was employed to identify the precise concentration of growth regulators of medium and substantiated in shake-flask cultivation. CCD-RSM model revealed a strong agreement, with a predicted coefficient of determination (R2) values of 0.881 and 0.872 for the shoot regeneration percentage (Y1) and the average number of shoots per explant (Y2), respectively. RSM predicted augmented conditions of MS liquid medium considerably influence the shoot proliferation (Y1 and Y2) in the leaf explants of S. duclis. The medium was fortified with 3.59 μM kinetin (KN; X1), 6.00 μM 6-benzylaminopurine (BAP; X2), 3.93 μM indole-3-acetic acid (IAA; X3), and 25.84 g L−1 of sucrose (X4). The present investigation conquered a maximal response of Y1 and Y2 with 91.28 ± 3.85 and 82.26 ± 2.13, respectively. The experimentally detected values are in close agreement with the predicted values of 90.07 and 79.70, respectively. This proposed that the developed design using CCD had efficacy in the optimization of medium components. The enhanced level of SDB, 9.89 ± 0.98 mg g−1 FW (ca. 5-fold), in the plantlets grown on liquid medium was significantly greater than those accomplished in the other tested plant tissues, comprises of field-grown parent plant leaves and in vitro developed callus and micropropagated plants on MS solid medium. For the first time, the methodology was developed effectively using RSM to promote the shoot proliferation and enhance the SDB production in the leaf organ culture of S. dulcis on MS liquid medium.

Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis

Scoparia dulcis biosynthesize bioactive diterpenes, such as scopadulcic acid B (SDB), which are known for their unique molecular skeleton. Although the biosynthesis of bioactive diterpenes is catalyzed by a sequence of class II and class I diterpene synthases (diTPSs), the mechanisms underlying this process are yet to be fully identified. To elucidate these biosynthetic machinery, we performed a high-throughput RNA-seq analysis, and de novo assembly of clean reads revealed 46,332 unique transcripts and 40,503 two unigenes. We found diTPSs genes including a putative syn-copalyl diphosphate synthase (SdCPS2) and two kaurene synthase-like (SdKSLs) genes. Besides them, total 79 full-length of cytochrome P450 (CYP450) genes were also discovered. The expression analyses showed selected CYP450s associated with their expression pattern of SdCPS2 and SdKSL1, suggesting that CYP450 candidates involved diterpene modification. SdCPS2 represents the first predicted gene to produce syn-copalyl diphosphate in dicots. In addition, SdKSL1 potentially contributes to the SDB biosynthetic pathway. Therefore, these identified genes associated with diterpene biosynthesis lead to the development of genetic engineering focus on diterpene metabolism in S. dulcis.

グァラニーインディオの伝承薬の調査と<i>Scoparia dulcis</i>由来ジテルペン類に関する研究

In interviews on the traditional herbal medicines of Tupi-Guarany Indians at the herbal market of Asuncion and questionnaire from their users, it was clarified that various useful medicinal plants are available in Paraguay and most of them are generally used without drying. In the search for bioactive substances from those plants, a β-glucuronidase-inhibitory diterpene called scoparic acid A (SA) was isolated from Scoparia dulcis L. together with scoparic acid B, scoparic acid C, and the aphidicolin-like tetracyclic diterpenes scopadulcic acid A (SDA) and scopadulcic acid B (SDB). HPLC analysis of diterpenes in the individual plants of Paraguayan and Asian S. dulcis revealed the presence of three chemotypes based on major component, i.e., SA type, SDB type, and SDX type containing mainly scopadiol and scopadulciol (SDC). SA and SDB were elucidated to be mainly biosynthesized in the leaves via 2-C-methyl-D-erythritol- 4-phosphate pathway, and a leaf organ culture system containing methyl jasmonate 10 µM was found to enhance the production of diterpenes by activation of Ca-signal transduction systems such as calmodulin and protein kinase C. On the other hand, SDB and SDC were found to show multifaceted pharmacological effects such as inhibitory effects on gastric acid excretion, bone resorption, replication of herpes simplex virus type 1 (HSV-1), etc. In addition, SDC was suggested to be applicable to cancer gene therapy using ganciclovir or acyclovir and the HSV-1 thymidine kinase gene called the suicide gene.

Production of scopadulcic acid B from Scoparia dulcis Linn. using a Luffa sponge bioreactor

Production of Scopadulcic acid B (SDB), a diterpene having antiviral and anti-tumour activity, was accomplished by immobilizing suspension culture-derived cells of Scoparia dulcis on Luffa sponge matrix. The yield of SDB in shake flask cultures was 50.85 mg/g of cells after 30 days of incubation while, in Luffa sponge inoculated columns SDB production was scaled up to 350.57 mg/g of cells by the 19th day of the first batch operation. The trend was maintained up to the 22nd day and the half life period of the reactor was 10 days. The bioreactor with Luffa sponge is a novel system and can be exploited for the production of many pharmaceutically active natural compounds effectively.

Methyl jasmonate influences secondary metabolism and protein expression in Scoparia dulcis

Scoparia dulcis L. (Scrophulariaceae) is a perennial herb distributed in tropical and subtropical regions, and has been used as a folk medicine. This plant was found to produce bicyclic and tetracyclic diterpenes such as scoparic acid A (SA) and scopadulcic acid B (SDB). Our previous studies indicated that SDB production was enhanced by administration of 10µM of methyl jasmonate (MeJA) in leaf organ culture system [1]. Furthermore, calcium signalling was suggested to be involved in the stimulation by MeJA [2].

Involvement of 2-C-Methyl-D-erythritol-4-phosphate Pathway in Biosynthesis of Aphidicolin-Like Tetracyclic Diterpene of Scoparia dulcis

Specific inhibitors of the MVA pathway (pravastatin) and the MEP pathway (fosmidomycin) were used to interfere with the biosynthetic flux which leads to the production of aphidicolin-like diterpene in leaf organ cultures of Scoparia dulcis. Treatment of leaf organs with fosmidomycin resulted in dose dependent inhibition of chlorophylls, carotenoids, scopadulcic acid B (SDB) and phytol production, and no effect on sterol production was observed. In response to the pravastatin treatment, a significant decrease in sterol and perturbation of SDB production was observed.

Selective Enhancement of Scopadulcic Acid B Production in the Cultured Tissues of Scoparia dulcis by Methyl Jasmonate

The effects of methyl jasmonate (MeJA) on isoprenoid production were evaluated in cultured tissues of Scoparia dulcis. It was found that MeJA suppressed the accumulation of chlorophylls, carotenoids, phytol and beta-sitosterol in the tissues. MeJA, however, remarkably enhanced the production of scopadulcic acid B (SDB), with 10 microM being optimal observed concentration for stimulation of SDB production. The maximum concentration of SDB was observed 6 d after MeJA treatment.

Stimulation of calcium signal transduction results in enhancement of production of scopadulcic acid B by methyl jasmonate in the cultured tissues of Scoparia dulcis

Signal transduction events which are involved in methyl jasmonate (MeJA)-enhanced production of the tetracyclic diterpene, scopadulcic acid B (SDB), were investigated in leaf organ cultures of Scoparia dulcis. Pretreatment of leaf organ cultures with Ca2+-channel blocker, verapamil, resulted in a dose-dependent inhibition of MeJA enhanced SDB production. Treatments with Ca2+ ionophore, A23187, stimulated SDB production in the absence of MeJA. In addition, preincubation of the leaf organ cultures with calmodulin (CaM) antagonist, trifluoperazine (TFP), diminished MeJA enhanced SDB production. MeJA-enhanced production of SDB was suppressed by addition of protein kinase inhibitors such as staurosporine and 2,6-diamino-N-([1-(1-oxotridecyl)-2-piperdinyl]methyl)hexanamide (NPC-15437). An activator of protein kinase C, 1-oleoyl-2-acetyl-sn-glycerol (OAG), stimulated this production in the absence of MeJA. These results indicate that Ca2+ and CaM mediates signaling events leading to enhancement of SDB production upon MeJA treatment. It was also suggested that phosphorylation of protein by protein kinases might be involved in this signal pathway.

Efficacy of scopadulcic acid A against Plasmodium falciparum in vitro.

Scoparia dulcis is a perennial herb widely distributed in many tropical countries. It is used as an herbal remedy for gastrointestinal and many other ailments, and in Nicaragua extracts are used to treat malaria. Phytochemical screening has shown that scopadulcic acid A (SDA), scopadulcic acid B (SDB), and semisynthetic analogues are pharmacologically active compounds from S. dulcis. SDB has antiviral activity against Herpes simplex virus type 1, antitumor activity in various human cell lines, and direct inhibitory activity against porcine gastric H(+), K(+)-ATPase. A methyl ester of scopadulcic acid B showed the most potent inhibitory activity against gastric proton pumps of 30 compounds tested in one study. Compounds with antiviral, antifungal, and antitumor activity often show activity against Plasmodium falciparum. In P. falciparum, the plasma membrane and food vacuole have H(+)-ATPases and the acidocalcisome has an H(+)-Ppase. These proton pumps are potential targets for antimalarial therapy and may have their function disrupted by compounds known to inhibit gastric proton pumps. We tested pure SDA and found in vitro activity against P. falciparum with an IC(50) of 27 and 19 microM against the D6 and W2 clones, respectively. The IC(50) against the multidrug-resistant isolate, TM91C235, was 23 microM.

Stimulative effects of lead on bone resorption in organ culture

To clarify whether hypercalcemia after injection of Pb to rats is due to biological bone resorption or physicochemical mineral dissolution, the effect of lead (Pb) on release of previously incorporated 45Ca in organ culture was investigated. Pb at 50 μM and above stimulated the release of 45Ca and hydroxyproline (Hyp). Pb did not stimulate 45Ca release from the bones inactivated by freezing and thawing. Eel calcitonin (ECT), bafilomycin A 1 and scopadulcic acid B (SDB) inhibited Pb-stimulated 45Ca release. These results indicate that Pb-induced 45Ca release is due to osteoclastic bone resorption. Pb-stimulated bone resorption was inhibited by indomethacin and flurbiprofen. Pb stimulated the release of prostaglandin E 2 (PGE 2) from the bones into the media. There was significantly high correlation between 45Ca and PGE 2 release. Pb-induced bone resorption was inferred to be mediated by PGE 2. From these results, it was suggested that hypercalcemia after Pb injection might be caused by biological bone resorption.

Production of diterpenoids by cultured cells from two chemotypes of Scoparia dulcis

Callus and multiple shoot cultures from two chemotypes of Scoparia dulcis were established. A relatively small amount of scoparic acid A (SA) was produced, and the production of scopadulcic acid B (SDB) was strongly repressed in all callus tissues tested. On the other hand, the multiple shoots contained either SA or SDB and produced the same type of diterpene as that produced by the parent plants. These results suggest that SA is an important intermediate in the biosynthesis of SDB and that the production of SDB is related to the differentiation of leaves.

Reversible inhibitions of gastric H+,K(+)-ATPase by scopadulcic acid B and diacetyl scopadol. New biochemical tools of H+,K(+)-ATPase.

Scopadulcic acid B (SA-B), a novel diterpenoid, is a main ingredient of the Paraguayan traditional medicinal herb "Typychá kuratú (Scoparia dulcis L.). SA-B and its debenzoyl derivative, diacetyl scopadol (DAS), specifically inhibit ATP hydrolysis of gastric H+,K(+)-ATPase. Both compounds inhibit the K(+)-dependent dephosphorylation step of the enzyme without any effect on the phosphorylation step. SA-B is a mixed-type inhibitor with respect to the activating cation, K+. SA-B lowers the affinity of H+,K(+)-ATPase to K+ and decreases the maximal velocity of ATP hydrolysis, whereas DAS is an uncompetitive inhibitor with respect to K+. Furthermore, the effects of SA-B and DAS on conformational states of the ATPase were studied by measuring the changes in the fluorescence intensity of the fluorescein isothiocyanate-labeled enzyme. The fluorescence study shows that SA-B primarily binds to the E2K form in the presence of Mg2+ and stabilizes the form and that DAS stabilizes the E2PK form. Therefore, the chemical modification of SA-B, debenzoylation, induced the changes in the pattern of inhibition of H+,K(+)-ATPase. Furthermore, the inhibition mechanisms of SA-B and DAS were different from those of omeprazole, which is an irreversible inhibitor, and SCH 28080, which is a reversible, competitive inhibitor with respect to K+. DAS also inhibited the K(+)-dependent p-nitrophenyl phosphatase activity, and the inhibition was competitive with respect to K+, indicating that the K(+)-dependent p-nitrophenylphosphatase activity does not represent the partial reaction step of H+,K(+)-ATPase.