Microtensile Bond Strength of Resin-Modified Glass Ionomer Cement to Sound and Artificial Caries-Affected Root Dentin With Different Conditioning.

In this laboratory study, the microtensile bond strengths (µTBS) of resin-modified glass ionomer cement (RM-GIC) to sound and artificial caries-affected bovine root dentin (ACAD) using three different conditioning agents were evaluated after 24 hours and three months. The fractured interface was examined with a scanning electron microscope (SEM). Specimens were created on bovine root dentin that was embedded in epoxy resin. For the ACAD specimens, artificial carious lesions were created. The RM-GIC (Fuji II LC) was applied either directly (no treatment), after application of self conditioner, cavity conditioner, or 17% ethylenediamine tetraacetic acid (EDTA) applied for 60 seconds, on sound dentin and ACAD, then light cured. They were stored in artificial saliva for 24 hours or three months. Following this, the specimens were cut into sticks for the µTBS test, and the failure mode of the debonded specimens was examined by using SEM. Pretest failures were excluded from the statistical analysis of the µTBS values because of their high incidence in some groups. Results showed that the µTBS values were significantly affected by the dentin substrate as well as the conditioning agent. Self conditioner provided the highest and most stable µTBS values, while cavity conditioner showed stable µTBS values on sound dentin. Both self conditioner and cavity conditioner had significantly higher µTBS values than the no treatment groups. EDTA conditioning reduced the µTBS after three months to sound dentin, while it showed 100% pretest failure with ACAD for both storage periods.

Phosphoric acid-etching promotes bond strength and formation of acid-base resistant zone on enamel.

This study examined the effect of phosphoric acid (PA) etching on the bond strength and acid-base resistant zone (ABRZ) formation of a two-step self-etching adhesive (SEA) system to enamel. An etch-and-rinse adhesive (EAR) system Single Bond (SB) and a two-step SEA system Clearfil SE Bond (SE) were used. Human teeth were randomly divided into four groups according to different adhesive treatments: 1) SB; 2) SE; 3) 35% PA etching→SE primer→SE adhesive (PA/SEp+a); (4) 35% PA etching→SE adhesive (PA/SEa). Microshear bond strength to enamel was measured and then statistically analyzed using one-way analysis of variance and the Tukey honestly significant difference test. The failure mode was recorded and analyzed by χ( 2 ) test. The etching pattern of the enamel surface was observed with scanning electron microscope (SEM). The bonded interface was exposed to a demineralizing solution (pH=4.5) for 4.5 hours and then 5% sodium hypochlorite with ultrasonication for 30 minutes. After argon-ion etching, the interfacial ultrastructure was observed using SEM. The microshear bond strength to enamel of the SE group was significantly lower (p<0.05) than that of the three PA-etched groups, although the latter three were not significantly different from one another. The ABRZ was detected in all the groups. In morphological observation, the ABRZ in the three PA-etched groups were obviously thicker compared with the SE group with an irregular wave-shaped edge.

Multiple injections of anti-mouse ß2glycoprotein 1 antibody induce FcRγ-dependent fetal growth restriction (FGR) in mice.

OBJECTIVES: The antiphospholipid syndrome (APS) is characterized by the presence of circulating antiphospholipid antibodies (aPLs), is a leading cause for thromboembolic events, repeated miscarriage, fetal loss and is a major risk factor for fetal growth restriction (FGR) and preeclampsia. In human, anti-ß2 glycoprotein I (aß2GPI) antibody is one of the aPLs and considered to be a specific and important marker for APS. However, pathophysiological changes induced by aß2GPI antibodies in FGR are largely unknown. METHODS: In the present study, we developed a murine FGR model induced by multiple injections of WBCAL-1, a well-characterized mouse aß2GPI monoclonal antibody. RESULTS: Administration of WBCAL-1, but not the isotype control antibody and saline, into pregnant mice specifically decreased the size of fetuses and placentas without affecting the number of delivered pups. Also, a significant increase in urinary albumin and electron microscopic changes, such as splitting layers of basal membranes in the placental labyrinth and rearrangement of pores in glomerular endothelial cells, were observed in WBCAL-1 treated mice. WBCAL-1 injection did not induce any changes in blood pressure and typical parameters of blood thromboembolic symptoms. Furthermore, FcRγ deficiency protected the fetuses from aß2GPI antibody-induced injuries. CONCLUSIONS: Our present findings suggest that proteinuria is a symptom associated with APS-related FGR with placental and renal tissue injuries, and that FcRγ might be a molecular target for prevention of aß2GPI antibody-mediated obstetrical pathologies.

Enamel lesion parameter correlations between polychromatic micro-CT and TMR.

Transverse microradiography (TMR) is considered as the gold standard technique for the evaluation of enamel lesions. Micro-computed tomography (µCT) has the advantage of non-destructive measurements, but the beam-hardening effect with polychromatic x-rays is a major drawback. To date, no study has validated µCT against TMR. The objective of this study was to validate µCT measurements of enamel lesions under various x-ray conditions and software beam-hardening correction (BHC) against TMR. Human molars with natural white-spot lesions were scanned for 5 min by µCT at 100 kV in different conditions: 50 µA (0.5-mm Al filter), 165 µA (0.5-mm Al/0.3-mm Cu), and 200 µA (0.5-mm Al/0.4-mm Cu), with or without BHC. Grayscale values were converted into mineral density values using phantoms. Thin sections at the same positions were then prepared for TMR. Lesion depth (LD; µm) and mineral loss (ΔZ; vol%µm) were compared between µCT and TMR by Pearson's correlations. µCT measurements correlated well with TMR under all conditions (p < 0.001, r > 0.86 for LD and ΔZ), except for 0.5-mm Al without BHC (p > 0.05). Even without BHC, combined Al/Cu filters successfully reduced the beam-hardening effect. µCT can be used as a non-destructive alternative to TMR with comparable parameters for the study of enamel lesions.

Clinical guidelines for treating caries in adults following a minimal intervention policy--evidence and consensus based report.

OBJECTIVES: In 2002, FDI (World Dental Federation) published a policy advocating that caries be treated by minimal intervention (MI). This MI policy has been accepted worldwide and is taught in universities. But acceptance in general dental practice has been slower, especially in Japan where healthcare payment and practice favour drilling and filling. To help disseminate this MI policy into general practice, the Japanese Society of Conservative Dentistry developed an evidence-based clinical guideline for restoring carious permanent teeth in adult patients. METHODS: The guideline was developed by a committee of nine university clinicians and a librarian. The committee selected the most frequent clinical questions in treating caries and used electronic databases to search and assess the best scientific evidence for each. Members then added their clinical experience and discussed to reach consensus on each question on treating caries with MI policy. Graded recommendations and guidance were made for each clinical question. The provisional guideline was strengthened after review and discussion with university researchers and general practitioners. RESULTS: The guideline addresses the 16 most frequent clinical questions in treating adult caries, including restorative methods and how to tackle root caries. Recommendations for treatment using MI policy were developed using the best scientific evidence and consensus of experienced clinicians. CLINICAL SIGNIFICANCE: The guideline offers a practical expert view of treating caries with the MI policy that incorporates the best scientific evidence, the latest techniques, the most preferable materials and the general consensus of expert clinicians.

Effects of CPP-ACP with sodium fluoride on inhibition of bovine enamel demineralization: a quantitative assessment using micro-computed tomography.

OBJECTIVES: The present study evaluated the effects of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) and CPP-ACP with 900 ppm fluoride (CPP-ACPF) pastes on inhibition of enamel demineralization over time, using polychromatic micro-computed tomography (micro-CT). METHODS: Enamel blocks were prepared from bovine teeth. The specimens were each treated by one of the following agents, 30 min daily for 7 days: deionized water (negative control); CPP-ACP paste; CPP-ACPF paste; and NaF solutions (positive controls) (90, 900, and 9000 ppm F). After treatment, the specimens were immersed in a demineralizing solution (pH 4.5) for 24, 72, and 120 h. Mean mineral loss (ML) and lesion depth (LD) after each period were determined from mineral density profiles obtained using micro-CT. RESULTS: ML values in all the treatment groups were significantly smaller than those in the control group after 72 and 120 h of demineralization (p < 0.05, two-way ANOVA and t-test with Bonferroni correction). ML values in CPP-ACPF and NaF solution groups were significantly smaller compared to CPP-ACP group after 72 h (p < 0.05). LD values in the CPP-ACPF and all the NaF solutions groups were significantly smaller compared to the control group after 120 h (p < 0.05). The 9000 ppm F group showed the lowest nominal ML and LD values. CONCLUSIONS: The application of CPP-ACP or CPP-ACPF pastes to sound enamel surfaces resulted in inhibition of enamel demineralization, and a better effect was noted for the latter paste. Quantitative assessment using polychromatic micro-CT demonstrated to be useful for detecting mineral density changes.

Effects of root dentin surface coating with all-in-one adhesive materials on biofilm adherence.

OBJECTIVES: Sealing of exposed root dentinal surfaces with adhesive materials is expected to be a promising approach for preventing root dentin caries. The aim of this study was to evaluate the effects of surface coating with all-in-one adhesives on inhibiting Streptococcus mutans biofilm attachment. MATERIALS AND METHODS: Bovine root dentin was cut and ground with #600-grit SiC paper. Each of the three all-in-one adhesives, Hybrid Bond (HB), Reactmer Bond (RB) or Shake One (SO) was single-coated on the dentin surfaces according to the manufacturers' instructions. The dentin surface without coating served as the control. The surface roughness of the coated and non-coated dentin surfaces was recorded by means of laser microscope measurements. S. mutans artificial biofilms were then grown on the surface of each specimen in a microcosm for 20h. The amounts of bacteria and water insoluble glucan in the retained biofilm on the surface of the specimens were measured. All numerical data were statistically analyzed using one-way ANOVA & Tukey's HSD (p<0.05). RESULTS: All of the coated groups showed significantly lower susceptibility to biofilm attachment compared with the non-coated root dentin (p<0.05). Among the coated groups, SO showed the lowest susceptibility for biofilm formation. CONCLUSIONS: Three all-in-one adhesive materials could be effective for root surface coating. A fluoride-releasing adhesive, SO demonstrated the strongest potentiality in resisting biofilm formation.

Bonding of acid-etch and self-etch adhesives to human fluorosed dentine.

OBJECTIVE: The objective of our laboratory study was to determine the bonding efficacy of two-step self-etching primer system (Clearfil SE Bond), all-in-one adhesive (Clearfil Tri S Bond) and acid-etching system (Single Bond) to human fluorosed dentine. METHODS: Forty-eight human molars were grouped according to modified Thylstrup-Fejerskov index (TFI) into normal (N, TFI 0), mild fluorosis (ML, TFI 1-3) and moderate fluorosis (MD, TFI 4-6). Superficial dentine was exposed and ground with 600-grit silicon carbide paper. Teeth of each group were randomly divided into three sub-groups. Each sub-group was treated with one of the three bonding systems. A resin composite was built up on the bonded surfaces and stored in water at 37 degrees C for 24h. They were serially sectioned in buccal-lingual direction into 0.7-1.0mm thick slabs. Slices were trimmed for microtensile bond test and stressed in tension at a crosshead speed of 1mm/min. The data were analyzed with two-way ANOVA (p=0.05). The adhesive/dentine interfaces of the bonded specimens were examined by a FE-SEM. RESULTS: Significantly higher microtensile bond strength (microTBS) was found with Clearfil SE Bond to mild fluorosed dentine than did Single Bond and Clearfil Tri S Bond. Lower microTBS were found for Single Bond (not significant) and Clearfil Tri S Bond than Clearfil SE Bond with moderately fluorosed dentine. Thickness of the hybrid layer produced by Clearfil SE Bond in mild and moderately fluorosed groups was less than with normal dentine. CONCLUSION: Two-step self-etching system, Clearfil SE Bond, showed a higher bonding performance to fluorosed dentine than Single Bond and Tri S Bond.

Caries susceptibility of human fluorosed enamel and dentine.

OBJECTIVE: Objective of our laboratory study was to determine the impact of dental fluorosis severity on the formation of caries in the human enamel and dentine. MATERIALS AND METHODS: Thirty-three human molars were grouped according to modified Thylstrup-Fejerskov index (TFI) into normal (N, TFI 0), mild fluorosis (ML, TFI 1-3) and moderate fluorosis (MD, TFI 4-6). Three mesio-distal sections were made in corono-apical axis of the tooth, giving enamel and dentine samples. They were embedded in an epoxy resin, and polished. Half of the polished surface was covered with an acid resistant varnish and immersed in standard acidified buffer solution (pH 4.5) for 48 h to create artificial caries lesions. They were treated with 5% NaOCl for 45 min and sectioned longitudinally along the center into two halves. Cut surfaces were polished and observed under a confocal laser scanning microscope for depth of demineralization. Morphology of the demineralized zones was observed under a field emission scanning electron microscope (FE-SEM). Data were analyzed using one-way ANOVA and Sheffe test (p=0.05). RESULTS: Statistically significant difference in depth of demineralization was found between N and MD groups (p=0.046) in the enamel, and between N and ML (p=0.002), N and MD (p<0.001), ML and MD (p=0.029) in dentine. FE-SEM observation of the normal enamel showed direct dissolution with large fissures. Spongy appearance of intertubular dentine gradually disappeared from N to MD. CONCLUSIONS: Moderately fluorosed enamel showed a significant caries resistance. In contrast, mild and moderately fluorosed dentine was significantly caries susceptible in vitro.

IGFBP1 and Follistatin-like 3 genes are significantly up-regulated in expression profiles of the IUGR placenta.

To date, the clinicopathological features of intrauterine growth restriction (IUGR) are not clearly understood, and no effective therapy has been established for IUGR. This is the first study that uses microarray analysis to identify differentially expressed genes in the IUGR placenta. The expression profiles of a total of 9121 genes were examined by cDNA microarray analysis, using mRNA from an appropriate gestational age (AGA) placenta and an IUGR placenta from discordant dichorionic twins. Up-regulation of the IGFBP1 and Follistatin-like 3 genes was detected in the IUGR placenta, with a balanced differential degree of 20.7+/-1.3 and 13.1+/-2.1, respectively, while the balanced differential degrees of other genes were 2.6 or less. The expressions of the IGFBP1 and Follistatin-like 3 genes in four single IUGR and four AGA placentas were also examined by RT-PCR. Consistent with our data in discordant chorionic twin placentas, three of four IUGR placentas showed up-regulation of the IGFBP1 and all four IUGR placentas showed upregulation of Follistatin-like 3 genes when compared to the AGA placentas. Our results suggest that IGFBP1 and Follistatin-like 3 are highly up-regulated in IUGR in the placenta. IGFBP1 and Follistatin-like 3 are known critical regulators of fetal growth and differentiation. Pathways associated with these genes might be important for the pathogenesis of IUGR.

Detection of acid diffusion through bovine dentine after adhesive application.

AIM: Acidic diffusion through bovine dentine was investigated by measuring pH changes on dentine surfaces after applying three adhesive systems. METHODOLOGY: Coronal incisor bovine dentine discs, 0.5 mm thick, were prepared from dentine close to the pulp chamber. A single-bottle adhesive system-Single Bond, a self-etching primer system-Clearfil SE Bond and an 'all-in-one' adhesive system-AQ Bond were used. The labial dentine surfaces were conditioned as follows: Single Bond groups: (SB-1) 35% phosphoric acid etchant was applied and left in place; (SB-2) the etchant was applied for 15 s and rinsed off for 10 s; (SB-3) application of adhesive agent and light curing following step SB-2; Clearfil SE Bond groups: (SE-1) SE primer was applied for 20 s and dried; (SE-2) application of adhesive agent and light curing following step SE-1; AQ Bond groups: (AQ-1) AQ Bond adhesive was applied for 20 s and dried, applied for additional 5 s and dried again; (AQ-2) light curing following step AQ-1. The pH change on the pulpal dentine surface was measured using a pH-imaging microscope. RESULTS: All the Single Bond groups revealed a lower pH on the pulpal surface (pH 6.25, 6.59 and 6.64 for SB-1, SB-2 and SB-3, respectively) compared with intact dentine. Clearfil SE Bond and AQ Bond groups showed no significant deference in pH value from intact dentine. CONCLUSIONS: Acid diffusion from phosphoric acid etching was observed when placed on 0.5 mm-thick dentine discs; however, there was only limited evidence of acid diffusion from SE primer and AQ Bond.

Squamous metaplasia of Paget's disease.

A patient had triple extramammary Paget's disease of both axillary and genital regions. Right inguinal lymphadenopathy was found 1 year after excision of all the skin lesions. Excisional biopsy of the lymph node demonstrated a mixture of Paget cells and atypical squamoid cells with horn pearls suggestive of keratinization. The squamoid cells were positive for cytokeratin 10, a marker of suprabasal epidermis, and also positive for laminin gamma2 which is often expressed in invasive squamous cell carcinoma. The coexistence of these different cells within the same tumour island suggested that the squamoid cells derived from metaplasia of Paget cells.

Autoimmune hemolytic anemia as a first manifestation of primary effusion lymphoma.

A 55-year-old man developing transfusion-dependant anemia was diagnosed with autoimmune hemolytic anemia (AIHA). Although he received prednisolone (PSL) (daily 60 mg), his hemoglobin level continued to decrease. After 3 weeks of treatment, he presented with a distension of the abdomen. Cytological examination of ascitic fluid revealed large, immunoblastic lymphocytes with plasmacytoid features and abundant IgM chains on the cellular surface; this was diagnosed as primary effusion lymphoma (PEL). Administration of CHOP (cyclophosphamide, Adriamycin, vincristine, and PSL) chemotherapy elicited regression of ascites as well as recovery of hemoglobin level. We hypothesize that PEL cells generated antibodies against red blood cells, resulting in AIHA resistance to PSL.

Acidity of conventional luting cements and their diffusion through bovine dentine.

AIM: To examine the changes in pH of luting cements and acid diffusion of luting cements through bovine dentine using a pH-imaging microscope (SCHEM-100; Horiba Ltd, Kyoto, Japan). METHODOLOGY: The pH of the surface of three conventional luting cements, glass-ionomer, zinc phosphate and zinc polycarboxylate was measured with SCHEM-100 for 1 month. The acid diffusion from the three luting cements through bovine dentine was investigated by measuring pH changes during the application of each luting cement on the bovine dentine surface. Coronal bovine dentine disks were prepared to thicknesses of 0.50 and 0.25 mm. Each luting cement was placed on the labial dentine surface, and the pH change of the pulpal surface was observed every 3 min for 30 min with SCHEM-100. RESULTS: Glass-ionomer showed the lowest pH values for longer times. Neutralization proceeded furthest in zinc polycarboxylate. The 0.5-mm-thick dentine disks showed no pH change on the pulpal side with all the three cements. The 0.25-mm-thick disks revealed evidence of acid diffusion on the pulpal side of the cemented dentine and significantly lower pH when cemented with glass-ionomer and zinc phosphate than with zinc polycarboxylates. CONCLUSIONS: This study demonstrated that glass-ionomer exhibited a lower setting pH than zinc phosphate and zinc polycarboxylate, and acid diffusions from glass-ionomer and zinc phosphate cements were observed when placed on 0.25-mm-thick dentine disks.

Expression and activation of MAP kinases, ERK1/2, in the human villous trophoblasts.

Mitogen-activated protein kinase (MAP kinase) plays a central role in the signal transduction for diverse cellular responses, such as proliferation, differentiation, stress response and cell death, via activation after binding of growth factors to the respective receptors on the cell membrane. In the human placental tissues, however, little is known about the expression and activation of the classical MAP kinases, extracellular signal-regulated kinase1/2 (ERK1/2). We therefore examined the expression of ERK1/2 in the human chorionic and placental tissues between 5 and 41 weeks of gestation, using Western blotting, immunohistochemistry and in situ hybridization. To explore the activation of ERK1/2 protein, we used an antibody that reacts with both phosphorylated and non-phosphorylated ERK1/2 (total ERK1/2), as well as antibodies that react only with phosphorylated ERK1/2. The expression pattern of phosphorylated ERK1/2 in the trophoblasts was compared with that of various growth factor receptors, such as c-met, IGF-1R, flt-1, EGFR, PDGFR, Bek, and flg. Total ERK1/2 was immunolocalized in the villous cytotrophoblasts (CTs), but not in the syncytiotrophoblasts (STs), throughout pregnancy. In situ hybridization also showed the localization of ERK1 mRNA in the villous CTs. Interestingly, however, phosphorylated ERK1/2 was immunolocalized in the villous CTs only up to 12 weeks of gestation. Western blot also showed the stronger bands of phosphorylated ERK1/2 in the tissues of the first trimester. Among the growth factor receptors, c-met was strongly expressed in the villous CTs during the first trimester, and resembled the expression pattern of phosphorylated ERK1/2. These findings suggest that the MAP kinase pathway is activated in the villous CTs during the first trimester in the human placenta.

In vitro surface analysis of active and arrested dentinal caries using a pH-imaging microscope.

This in vitro study evaluated the pH value of active and arrested caries lesions in deep dentinal caries using a pH-imaging microscope (SCHEM-100, HORIBA Ltd, Kyoto, Japan). Buccal-lingual cut sections of extracted human teeth that had either active or arrested dentinal caries lesions were placed on the pH-imaging sensor of the microscope. The pH values were compared statistically by one-way ANOVA and Fisher's PLSD test (p<0.05). In addition, both types of caries lesions were compared with a caries detector solution for this pH-imaging characterization. For both active and arrested lesions in dentin, the lowest pH values in the caries lesions (range from 5.3 to 6.6) were lower than that of intact dentin (range from 6.8 to 7.4). There were statistical differences between the lowest pH value within the active lesion (range from 5.3 to 5.8) and the arrested lesion (range from 6.3 to 6.6) (p<0.05). Although the arrested lesion was unstainable and impermeable to the dye, there was a close relationship between dye staining and pH-imaging characterization within the active lesion from visual inspection.

Evaluation of thermal cycling and mechanical loading on bond strength of a self-etching primer system to dentin.

OBJECTIVE: The aim of this study was to evaluate the in vitro durability and fracture modes of the tooth-resin interface following thermal cycling, fatigue load cycling (FLC) and micro-tensile bond strength testing (MTBS). METHODS: Twenty-four human molars were divided into two groups. In group A, coronal dentin was ground flat with #600-grit SiC paper, Clearfil Linerbond.2V (LB.2V) adhesive was applied and a crown fabricated with Clearfil AP-X resin composite. Samples were loaded in the FLC simulator and thermal cycle device. In group B, Class I cavities were prepared with a diamond bur and the cavity restored with LB.2V/AP-X. Samples were simultaneously subjected to FLC and thermal cycling. After storage of the specimens in water for 1 week, MTBS tests were performed, and fracture modes examined by SEM. RESULTS: For group A, MTBS were approximately 40 MPa with cohesive failures in the bonding resin of all specimens. Neither thermal nor mechanical stressing altered bond strengths on flat dentin surfaces. In group B, MTBS in the unstressed control group was 21 MPa with cohesive failures in the resin. As both thermal and mechanical stresses increased, there was a significant decrease in bond strength (16 MPa, P<0.05) and the fractures were seen in the resin-dentin interface and hybrid layer. SIGNIFICANCE: Surface preparation, C-factor, cavity depth, the dentin substrate and character of the smear layer strongly influence the bond strength values after thermal and fatigue loading. The FLC simulator in combination with MTBS testing provides meaningful in vitro evaluation of dentin bonding durability.

Long-term tensile bond durability of two different 4-META containing resin cements to dentin.

OBJECTIVES: This study was conducted to evaluate the tensile bond durability of two different types of 4-META containing resin cements over a period of 3 years. METHODS: Ten bovine dentin specimens were tested for tensile bond strengths with each of the following materials: Super Bond C&B: unfilled methyl methacrylate (MMA)/polymethyl methacrylate (PMMA) resin cement, MASA Bond (experimental material): filled dimethacrylate resin cement at 1 day, 6 months, 1 and 3 years. The mean bond strengths were compared statistically by two-way ANOVA and Fisher's PLSD test (P<0.05). The mode of failure was classified by SEM observation. Results for the mode of fracture were analyzed using the Mann-Whitney U test. RESULTS: Although there was no statistical difference in mean bond strength between Super Bond C&B and MASA Bond (P>0.05) during the experimental periods, the 1-day bond strengths were significantly greater than those at the other experimental periods except for 6 months (P<0.05). Regarding the fracture modes, at 6 months and 1 year, statistical differences were observed between Super Bond C&B and MASA Bond (P<0.05). SIGNIFICANCE: The bond strengths of both resin cements to dentin significantly decreased after 6 months, and the long-term failure patterns of the 4-META/TBB resin cements showed a marked change.

Triterpenoid saponins from Mollugo spergula.

Two novel triterpenoid saponins belonging to a modified hopane group, spergulin A [3-O-(beta-D-xylopyranosyl 4-sulphate)-spergulagenin A] (1) and spergulin B [3-O-[alpha-rhamnopyranosyl (1-->2)-beta-D-xylopyranosyl]-spergulatriol] (4) were isolated from the aerial part of Mollugo spergula along with spergulacin (2) and spergulacin A (3). Their structures and relative stereochemistry were determined by a combination of 2D-NMR (COSY, TOCSY, HETCOR, NOESY and HMBC) and HR-FAB-MS analysis coupled with strategic chemical and enzymatic transformations.