In vitro & in vivo assessment of a herbal formula used topically for bone fracture treatment

Aim of the study

A novel topical paste used for fracture healing (FH), consisting of the extracts of six herbs, Radix Dipsaci, Ramulus Sambucus Williamsii, Rhizoma Notoginseng, Flos Carthami, Rhizoma Rhei and Fructus Gardeniae, was developed according to the classical theory of traditional Chinese medicine. This study aimed to determine the effectiveness of this formula, and some of its important chemical components in the promotion of fracture healing. The transdermal transport of FH was also examined.

Materials and methods

The osteogenic, angiogenic and nitric oxide suppressing effects of FH and its important chemical marker components were assessed by using osteoblastosacroma UMR-106 cells, human umbilical vein endothelial cells (HUVEC) and murine macrophage RAW264.7 cells, respectively. The bone healing effects of the FH paste and its transdermal absorption were determined using a rabbit fracture model. The callus sizes, bone specific alkaline phosphatase levels and biomechanical properties of the healed bone were assessed.

Results

FH significantly increased the cell proliferation in UMR-106 and HUVEC cells and inhibited the nitric oxide production in murine macrophage in dose-dependent manner. Its important chemical components asperosaponin VI, ginsenoside Rg1 and emodin were shown to be acting positively in the respective in vitro studies. FH paste significantly improved the bone healing in the rabbit fracture model, as was indicated by the increases in callus size at weeks 2-5, and the elevations in bone specific alkaline phosphatase activities at weeks 5-6. The analysis using LC/MS/MS also showed the presence of important chemical marker components of the FH formula in the plasma after 8 weeks of topical treatment.

Conclusion

This study presents the first scientific evidence of the efficacy of a herbal paste in the promotion of fracture healing. There were evidences of transdermal transport of the chemical components, control the inflammation through nitric oxide inhibition, promotion of angiogenesis, and bone healing in the in vitro tests, as well as in the experimental animal.     

In vitro anti-breast cancer activity of ethanolic extract of Wrightia tomentosa: Role of pro-apoptotic effects of oleanolic acid and urosolic acid

Ethnopharmacological relevance

Wrightia tomentosa Roem. & Schult. (Apocynaceae) is known in the traditional medicine for anti-cancer activity along with other broad indications like snake and scorpion bites, renal complications, menstrual disorders etc. However, the anti-cancer activity of this plant or its constituents has never been studied systematically in any cancer types so far.

Aim of the study

To evaluate the anti-cancer activities of the ethanolic extract of W. tomentosa and identified constituent active molecule(s) against breast cancer.

Material and methods

Powdered leaves of W. tomentosa were extracted with ethanol. The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa were tested for its anti-proliferative and pro-apoptotic effects in breast cancer cells MCF-7 and MDA-MB-231.

Results

The ethanolic extract, subsequent hexane fractions and fraction F-4 of W. tomentosa inhibited the proliferation of human breast cancer cell lines, MCF-7 and MDA-MB-231. The fraction F-4 obtained from hexane fraction inhibited proliferation of MCF-7 and MDA-MB-231 cells in concentration and time dependent manner with IC₅₀ of 50 μg/ml and 30 μg/ml for 24 h, 28 μg/ml and 22 μg/ml for 48 h and 25 μg/ml and 20 μg/ml for 72 h respectively. The fraction F-4 induced G1 cell cycle arrest, reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential and subsequent apoptosis. Apoptosis is indicated in terms of increased Bax/Bcl-2 ratio, enhanced Annexin-V positivity, caspase 8 activation and DNA fragmentation. The active molecule isolated from fraction F-4, oleanolic acid and urosolic acid inhibited cell proliferation of MCF-7 and MDA-MB-231 cells at IC₅₀ value of 7.5 μM and 7.0 μM respectively, whereas there is devoid of significant cell inhibiting activity in non-cancer originated cells, HEK-293. In both MCF-7 and MDA-MB-231, oleanolic acid and urosolic acid induced cell cycle arrest and apoptosis as indicated by significant increase in Annexin-V positive apoptotic cell counts.

Conclusion

Our results suggest that W. tomentosa extracts has significant anti-cancer activity against breast cancer cells due to induction of apoptosis pathway. Olenolic and urosolic acid are important constituent molecules in the extract responsible for anti-cancer activity of W. tomentosa.     

4-Methylthio-butanyl derivatives from the seeds of Raphanus sativus and their biological evaluation on anti-inflammatory and antitumor activities

Ethnopharmacological relevance

Raphanus sativus seeds (Brassicaceae) known as Raphani Semen have long been used as anti-cancer and/or anti-inflammatory agents in Korean traditional medicine. This study was designed to isolate the bioactive constituents from the seed extracts of Raphanus sativus and evaluate their anti-inflammatory and antitumor activities.

Material and methods

Bioassay-guided fractionation and chemical investigation of a methanolic extract of the seeds of Raphanus sativus led to the isolation and identification of seven 4-methylthio-butanyl derivatives. Structural elucidation of the isolated compounds was carried out using 1D and 2D nuclear magnetic resonance (NMR) spectroscopy techniques (¹H, ¹³C, COSY, HMQC and HMBC experiments) and mass spectrometry.

Results

The isolated compounds were characterized as in the following: three new 4-methylthio-butanyl derivatives, sinapoyl desulfoglucoraphenin (1), (E)-5-(methylsulfinyl)pent-4-enoxylimidic acid methyl ester (2), and (S)-5-((methylsulfinyl)methyl)pyrrolidine-2-thione (3), together with four known compounds, 5-(methylsulfinyl)-4-pentenenitrile (4), 5-(methylsulfinyl)-pentanenitrile (5), sulforaphene (6), and sulforaphane (7). Full NMR data assignments of the three known compounds 4–6 were also reported for the first time. We evaluated the anti-neuroinflammatory effect of 1–7 in lipopolysaccharide-stimulated murine microglia BV2 cells. Compound 1 significantly inhibited nitrite oxide production with IC₅₀ values of 45.36 μM. Moreover, it also reduced the protein expression of inducible nitric oxide synthase. All isolates were also evaluated for their antiproliferative activities against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT-15), and all of them showed antiproliferative activity against the HCT-15 cell, with IC₅₀ values of 8.49–23.97 μM.

Conclusions

4-Methylthio-butanyl derivatives were one of the main compositions of Raphanus sativus seeds, and activities demonstrated by the isolated compounds support the ethnopharmacological use of Raphanus sativus seeds (Brassicaceae) as anti-cancer and/or anti-inflammatory agents.