Six isoflavones derived from Millettia griffoniana namely, 4’-methoxy-7-O-[(E)-3-methyl-7hydroxymethyl-2,6 octadienyl]isoflavone (7-O-DHF), Griffonianone C (Griff C), 7-O-geranylformononetin (7-O-GF), 3’,4’-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone (7-O-GISO), Griffonianone E (Griff E), 4’-O-geranylisoliquiritigenin (4-O-GIQ) were tested for potential oestrogenic activities in three different oestrogen receptor alpha (ERα) dependent assays, namely a recombinant yeast assay, a reporter gene assay based on stably transfected MCF-7 cells (MVLN cells) and the induction of alkaline phosphatase in Ishikawa cells. The oestrogenic activities of isoflavones from Millettia griffoniana could be completely suppressed by the pure oestrogen antagonist, fulvestrant. The expression of Ki-67, proliferating cell nuclear antigen (PCNA) and cyclin D1 (CD1) mRNA used as indicator of cell proliferation in MCF-7 cells was assayed. Based on these in vitro results, Griff C was further tested in vivo. The main objective of this part of the work was to study the mechanistic basis of the oestrogenicity Three different doses of Griff C (2, 10, or 20 mg/kg BW) of Griff C in ovariectomised Wistar rats. 17β-oestradiol (E2: 10 µg/kg BW) was used as positive control. They were treated daily for three consecutive days and sacrificed 24 hours after receiving the last dose. The whole uterus was removed and weight. Liver and vena cava fragments were also collected and stored together with uteri in liquid nitrogen for subsequent real-time PCR to evaluate the effects of Griff C on the regulation of some relevant oestrogen–responsive genes in the uterus, the liver and the vena cava. The role of Griff C in apoptosis or in cell survival, through mediation of the phosphatidylinositol 3 kinase-Akt (PI3K-Akt) signaling pathway, was also investigated. Western blot analysis revealed that Griff C slightly increased the phosphorylation of Akt at its serine 473 residue. In this work, oestrogenic properties of the isoflavones derived from Millettia griffoniana are described using reporter gene assays and the oestrogen-inducible alkaline phosphatase Ishikawa model for the first time. These in vitro data were verified in vivo showing the regulation of the expression of various relevant oestrogen-responsive genes by Griff C. The spectrum of its activity was clearly similar to that of 17β-oestradiol on uterine hepatic and vena cava tissues of ovariectomised rats except for the proliferative response. However Griff C remained 100 to 1000 times less effective than oestradiol. These findings confirmed that some of the biological effects attributed to Millettia griffoniana are closely related to oestrogen-mediated action.

The tropical hardwood forests of Mozambique are among its most important natural resources. Long-term sustainable management of these resources will require proper forest management, which depends on understanding the growth rates and the life history of important commercial species as well as the impacts of natural forces (e.g., climate variability) and human management. This study analyzes radial growth rate dynamics and climate-growth relationships of Millettia stuhlmannii and examines its dendrochronological potential. This tree locally known as Panga-panga or Jambirre is one of the most important timber species in Mozambique. Ranked as a first class commercial timber in Mozambique, it is frequently harvested in an unsustainable way and sustainable management of the species is urgently needed for the continued utilization of this resource. Five different methods demonstrate that the semi-ring porous tree rings of M. stuhlmannii are annual: (1) Ring structure and anatomy; (2) Successful cross-dating within and between trees; (3) Ring counting in trees with known age (young trees collected from an experimental "plantation") (4) Cambial wounding and (5) Correlation between ring width and climate data. Through these methods, M. stuhlmannii trees showed distinct reaction to pinning, adding one annual ring after one year. Cross dating of annual ring width growth was successful within and among selected M. stuhlmannii trees, which indicates that this species forms annual rings and that growth responds to an external climate variability. M. stuhlmannii annual growth ring boundaries were characterized by alternating patterns of parenchyma and fibre vessels and marginal parenchyma. Precipitation during previous December (r= 0.30; p<0.05), current February (r=0.30; p<0.05) and the entire rainy season (NDJFM; r=0.43, p<0.01) over a long period (1900-1996) showed a significant influence on Panga-panga tree ring growth. Declining rainfall has caused a growth increment decrease since 1940. The results of this study show that the mean annual increment of M. stuhlmannii is 0.51 cm/year and it takes about 75 years for an average M. stuhlmannii tree to reach the minimum lawful cutting diameter of 40 cm DBH (diameter at breast height). Temporal differences in movement through increasing diameter classes are large among and within classes. The median time necessary for trees to grow into the next diameter class was not statistically significant (Kruskal-Wallis chi-squared = 9.568, p>0.001). The relationship between stem diameter and percentage of heartwood is significantly high (R2 = 0.9701, p < 0.0001) and results suggest that from 33cm diameters on, the HW% remain stable. Partial correlation coefficients show that significant effects on growth to minimum cutting diameter occur while stems move through the 20-30 cm DBH class. This indicates the specific sizes at which silviculture treatments have to be started in order to maximize the productivity of this species. Correlation analyses revealed that heartwood width (HW) is positively correlated with total stem diameter (TSD), cambial Age (Ac), number of rings in heartwood (HWR), heartwood area (HWA), Total stem diameter area (TSDA) and Mean annual increment (MAI). This study suggests that further studies to improve diameter growth rate models as well as volume increment models need to be carried out. Strong correlation with precipitation during the rainy season suggests that this species is potentially useful for future climate reconstruction studies in Mozambique.

Six isoflavones derived from Millettia griffoniana namely, 4’-methoxy-7-O-[(E)-3-methyl-7hydroxymethyl-2,6 octadienyl]isoflavone (7-O-DHF), Griffonianone C (Griff C), 7-O-geranylformononetin (7-O-GF), 3’,4’-dihydroxy-7-O-[(E)-3,7-dimethyl-2,6-octadienyl]isoflavone (7-O-GISO), Griffonianone E (Griff E), 4’-O-geranylisoliquiritigenin (4-O-GIQ) were tested for potential oestrogenic activities in three different oestrogen receptor alpha (ERα) dependent assays, namely a recombinant yeast assay, a reporter gene assay based on stably transfected MCF-7 cells (MVLN cells) and the induction of alkaline phosphatase in Ishikawa cells. The oestrogenic activities of isoflavones from Millettia griffoniana could be completely suppressed by the pure oestrogen antagonist, fulvestrant. The expression of Ki-67, proliferating cell nuclear antigen (PCNA) and cyclin D1 (CD1) mRNA used as indicator of cell proliferation in MCF-7 cells was assayed. Based on these in vitro results, Griff C was further tested in vivo. The main objective of this part of the work was to study the mechanistic basis of the oestrogenicity Three different doses of Griff C (2, 10, or 20 mg/kg BW) of Griff C in ovariectomised Wistar rats. 17β-oestradiol (E2: 10 µg/kg BW) was used as positive control. They were treated daily for three consecutive days and sacrificed 24 hours after receiving the last dose. The whole uterus was removed and weight. Liver and vena cava fragments were also collected and stored together with uteri in liquid nitrogen for subsequent real-time PCR to evaluate the effects of Griff C on the regulation of some relevant oestrogen–responsive genes in the uterus, the liver and the vena cava. The role of Griff C in apoptosis or in cell survival, through mediation of the phosphatidylinositol 3 kinase-Akt (PI3K-Akt) signaling pathway, was also investigated. Western blot analysis revealed that Griff C slightly increased the phosphorylation of Akt at its serine 473 residue. In this work, oestrogenic properties of the isoflavones derived from Millettia griffoniana are described using reporter gene assays and the oestrogen-inducible alkaline phosphatase Ishikawa model for the first time. These in vitro data were verified in vivo showing the regulation of the expression of various relevant oestrogen-responsive genes by Griff C. The spectrum of its activity was clearly similar to that of 17β-oestradiol on uterine hepatic and vena cava tissues of ovariectomised rats except for the proliferative response. However Griff C remained 100 to 1000 times less effective than oestradiol. These findings confirmed that some of the biological effects attributed to Millettia griffoniana are closely related to oestrogen-mediated action.

Diss. (sammanfattning) Umeå : Sveriges lantbruksuniv., 2003. / Härtill 4 uppsatser.