In vitro cytotoxicity of cyclodextrin-bonded birch bark extract.

Triterpenoids from birch bark, like betulin, seem to have an anticancer potential which needs to be further investigated. Aim of this study was first to explore whether a cyclodextrin-solubilised triterpenoid extract (STE) from birch bark induces selective cytotoxic effects in primary liver cancer cells compared to healthy human hepatocytes. Second, selective cytotoxicity against several tumour cell lines should be analysed. For this purpose, human liver cancer cells derived from mouse xenografts (LIXF 575), healthy human hepatocytes, and 42 different human tumour cell lines were incubated with different concentrations of STE corresponding to 4.3 µM - 137.5 µM betulin (BE). Cytotoxicity was tested with the WST-1 cell proliferation assay, apoptosis with caspase 3/7-activity, and necrosis was determined by the propidiumiodid uptake assay. The pathway of cytotoxic effects was further investigated by immunoblotting of apoptosis inducing factor (AIF) and p53. The monolayer assay was used to analyse selectivity of STE towards different tumour cell lines. STE significantly (p < 0.001) reduced viability and induced apoptosis of LIXF cells in low concentrations corresponding to 8.6 µM BE, while human hepatocytes were affected only in concentrations ≥ 68.8 µM. Cell death occurred in a p53 independent manner, and AIF was not involved. The mean IC50 in the 42 tumour cell lines corresponded to 4.3 µM BE and ranged from 2.05 µM to 8.95 µM BE content. Selectivity was, therefore, rather low. In conclusion, STE exhibits in low concentrations cytotoxicity in a broad spectrum of primary cancer cells and cancer cell lines, which is, at least in LIXF cells, induced by caspase 3/7 mediated apoptosis. STE is far less toxic in hepatocytes. The anticancer potential of STE should be further characterised and also investigated in animal models.

Topical treatment of necrotising herpes zoster with betulin from birch bark.

Betulin, a pentacyclic triterpene, is the main constituent of the outer bark of birches (Betula alba). In recent years, anti-microbial, anti-inflammatory and differentiation-promoting effects of betulin have been described. A betulin-based emulsion without preservatives and detergent emulsifiers can be prepared from birch bark extract. We report the successful treatment with betulin emulsion of a severe necrotising herpes zoster in an immunosuppressed patient who had not responded to a conventional topical treatment. The betulin emulsion was directly applied to the wounds without causing any side effects. The presented case report demonstrates impressive skin tolerance and wound-healing properties of the betulin emulsion. These should be further evaluated.

Triterpenes promote keratinocyte differentiation in vitro, ex vivo and in vivo: a role for the transient receptor potential canonical (subtype) 6.

It has been shown recently that triterpenes inhibit cancer cell growth of various cell types in vitro. In this work, the effect of highly purified triterpenes (TE) with betulin as the major compound (>80% w/w) on cell proliferation, apoptosis, and differentiation of human keratinocytes was analyzed in vitro, ex vivo, and in vivo. In vitro, TE increased calcium influx into primary keratinocytes and upregulated various differentiation markers including keratin 10. TE also specifically increased the expression of the non-selective transient receptor potential canonical (subtype) 6 (TRPC6) in keratinocytes, and knocking down TRPC6 inhibited keratin 10 upregulation. Ex vivo, in human skin explants TE induced the expression of TRPC6 in the epidermis and increased DNA fragmentation of terminally differentiating keratinocytes. Topical treatment with TE of actinic keratoses, that represent in situ squamous cell carcinomas with disturbed epithelial differentiation, resulted in downgrading of aberrant Ki67 expression and upregulation of keratin 10 in vivo. Our data indicate that TE promotes keratinocyte differentiation in vitro and in vivo. This effect seems to be mediated at least in part by TRPC6.

Pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in cancer therapy.

Today cancer treatment is not only a question of eliminating cancer cells by induction of cell death. New therapeutic strategies also include targeting the tumour microenvironment, avoiding angiogenesis, modulating the immune response or the chronic inflammation that is often associated with cancer. Furthermore, the induction of redifferentiation of dedifferentiated cancer cells is an interesting aspect in developing new therapy strategies. Plants provide a broad spectrum of potential drug substances for cancer therapy with multifaceted effects and targets. Pentacyclic triterpenes are one group of promising secondary plant metabolites. This review summarizes the potential of triterpenes belonging to the lupane, oleanane or ursane group, to treat cancer by different modes of action. Since Pisha et al. reported in 1995 that betulinic acid is a highly promising anticancer drug after inducing apoptosis in melanoma cell lines in vitro and in vivo, experimental work focused on the apoptosis inducing mechanisms of betulinic acid and other triterpenes. The antitumour effects were subsequently confirmed in a series of cancer cell lines from other origins, for example breast, colon, lung and neuroblastoma. In addition, in the last decade many studies have shown further effects that justify the expectation that triterpenes are useful to treat cancer by several modes of action. Thus, triterpene acids are known mainly for their antiangiogenic effects as well as their differentiation inducing effects. In particular, lupane-type triterpenes, such as betulin, betulinic acid and lupeol, display anti-inflammatory activities which often accompany immune modulation. Triterpene acids as well as triterpene monoalcohols and diols also show an antioxidative potential. The pharmacological potential of triterpenes of the lupane, oleanane or ursane type for cancer treatment seems high; although up to now no clinical trial has been published using these triterpenes in cancer therapy. They provide a multitarget potential for coping with new cancer strategies. Whether this is an effective approach for cancer treatment has to be proven. Because various triterpenes are an increasingly promising group of plant metabolites, the utilisation of different plants as their sources is of interest. Parts of plants, for example birch bark, rosemary leaves, apple peel and mistletoe shoots are rich in triterpenes and provide different triterpene compositions.

Pentacyclic triterpene distribution in various plants - rich sources for a new group of multi-potent plant extracts.

Pentacyclic triterpenes are secondary plant metabolites widespread in fruit peel, leaves and stem bark. In particular the lupane-, oleanane-, and ursane triterpenes display various pharmacological effects while being devoid of prominent toxicity. Therefore, these triterpenes are promising leading compounds for the development of new multi-targeting bioactive agents. Screening of 39 plant materials identified triterpene rich (> 0.1% dry matter) plant parts. Plant materials with high triterpene concentrations were then used to obtain dry extracts by accelerated solvent extraction resulting in a triterpene content of 50 - 90%. Depending on the plant material, betulin (birch bark), betulinic acid (plane bark), oleanolic acid (olive leaves, olive pomace, mistletoe sprouts, clove flowers), ursolic acid (apple pomace) or an equal mixture of the three triterpene acids (rosemary leaves) are the main components of these dry extracts. They are quantitatively characterised plant extracts supplying a high concentration of actives and therefore can be used for development of phytopharmaceutical formulations.

Treatment of actinic keratoses with a novel betulin-based oleogel. A prospective, randomized, comparative pilot study.

BACKGROUND: Actinic keratoses (AK) are squamous cell carcinomas in situ and require treatment. Betulin-based oleogel prepared from a standardized triterpene dry extract from birch bark represents a new topical agent with anti-inflammatory and anti-tumor potential. PATIENTS AND METHODS: In the prospective, randomized, monocentric phase 2a study 45 patients with < 10 AK were included and randomly assigned to one of the three treatment groups. Intervention consisted of topical betulin-based oleogel twice daily versus cryotherapy with liquid nitrogen versus the combination of cryotherapy with topical betulin-based oleogel. Treatment response was assessed clinically after three months. The clinical response was graded into complete clearing (100 %), therapy responders (> 75 % clearing of the lesions) and non-responders (< 75 % clearing). Additionally, punch biopsies were obtained from some patients before and at the end of treatment. RESULTS: Therapy with betulin-based oleogel was well tolerated.Three patients discontinued therapy because of personal reasons. After three months, the 100% (and > 75%) clearing rates of the lesions were as follows: 64% (86%) with betulin-based oleogel (n = 14),79% (93%) with cryotherapy (n = 14),and 71% (71%) with the combined therapy (n = 14). Histological analysis of biopsies taken before and after treatment (n = 8) showed a reduced degree of dysplasia in the epidermis in all study arms. CONCLUSIONS: Betulin-based oleogel seems to be an effective novel approach in the topical treatment of actinic keratoses. However,the clinical and histological findings of the present pilot study have to be verified against placebo with larger case numbers.

A preliminary pharmacokinetic study of betulin, the main pentacyclic triterpene from extract of outer bark of birch (Betulae alba cortex).

During the last two decades triterpenes have attracted attention because of their pharmacological potential. Triterpene extract (TE) from outer bark of birch consisting mainly of betulin is able to form an oleogel which was successfully tested in the treatment of actinic keratosis. Some aspects of TE in vitro pharmacology are already known. Now we show preliminary pharmacokinetics of betulin and results of a subchronic toxicity study of TE in rats and dogs. Because of poor aqueous solubility of the TE-triterpenes (< 0.1 microg/mL respectively), for pharmacokinetic studies it was suspended in sesame oil (rats, i.p.) and PEG 400 / 0.9 % NaCl (dogs, s.c.). I.p. administered, betulin, the main component of TE, shows time dependency over a period of 4 h and reaches a dose-independent serum level of 0.13 microg/mL. Dose dependency was observed with s.c. administration. At 300 mg/kg a maximum plasma concentration of 0.33 microg/mL betulin was detected after 28 daily applications. The subchronic toxicity study showed no toxicity of TE in rats (i.p.) and dogs (s.c.). In conclusion, triterpene extract from birch bark is safe, its betulin is bioavailable and in addition to published triterpene biological activities TE provides high potential for further pharmaceutical and pharmacological research.

Innate and adaptive immune responses in allergic contact dermatitis and autoimmune skin diseases.

Allergic contact dermatitis is induced by chemicals or metal ions. A hallmark of this T cell mediated skin disease is the activation of the innate immune system by contact allergens. This immune response results in inflammation and is a prerequisite for the activation of the adaptive immune system with tissue-specific migration of effector and regulatory T cells. Recent studies have begun to address in detail the innate immune cells as well as the innate receptors on these cells and the associated signaling pathways which lead to skin inflammation. We review here recent findings regarding innate and adaptive immune responses and immune regulation of contact dermatitis and other skin diseases as well as recent developments towards an in vitro assessment of the allergenic potential of chemicals. The elucidation of the innate inflammatory pathways, cellular components and mediators will help to identify new drug targets for more efficient treatment of allergic contact dermatitis and hopefully also for its prevention.

Physical, chemical and pharmacological characterization of a new oleogel-forming triterpene extract from the outer bark of birch (betulae cortex).

Triterpenes are biologically active secondary plant substances that display antimicrobial, hepatoprotective and anti-inflammatory effects. However, the poor solubility of triterpenes in both polar and non-polar solvents as well as expensive purification procedures have prevented the large-scale isolation of these compounds for medicinal purposes. Here, we describe a novel quantitative extraction method of triterpenes from the outer bark of birch (Betula species) in which betulin, a lupan triterpene, predominates. The resulting highly purified triterpene extract (TE) in the form of a dry powder contains betulin as the major compound, but also betulinic acid, lupeol, erythrodiol and oleanolic acid. We have found that this TE is able to form an oleogel, thus providing an opportunity for the topical application of pharmacologically relevant amounts of triterpenes. Furthermore, we have investigated the TE in comparison to its major isolated compounds in cell culture experiments with human immortalized keratinocytes and skin cancer cells. We could demonstrate dose-dependent cytotoxic and apoptosis-inducing effects of TE and betulin. These experimental data support the notion from a previous clinical study that TE from the outer bark of birch might represent a new tool for the topical treatment of skin cancer and skin cancer precursors like actinic keratoses.

[Treatment of actinic keratoses with birch bark extract: a pilot study]. / Behandlung aktinischer Keratosen mit Birkenkorkextrakt: Eine Pilotstudie.

BACKGROUND: Birch bark contains a variety of apoptosis-inducing and anti-inflammatory substances such as betulinic acid, betulin, oleanolic acid and lupeol. Therefore, birch bark extract may be effective in the treatment of actinic keratoses. To address this issue, a pilot study using a standardized birch bark ointment was performed. METHODS: Twenty-eight patients with actinic keratoses were enrolled in this prospective, non-randomized pilot study. Fourteen patients were treated with birch bark ointment only; fourteen patients received a combination therapy with cryotherapy and birch bark ointment. Treatment response was assessed clinically after two months. RESULTS: Clearing of more than 75 % of the lesions was seen in 79 % of the patients treated with birch bark ointment monotherapy. The response rate of the combined treatment modality was 93 %. Therapy with birch bark ointment was well tolerated. CONCLUSION: In this pilot study, a standardized birch bark extract was effective in the treatment of actinic keratoses. This therapy is easy to perform and it has no side effects. Birch bark ointment may be a new therapeutic option for actinic keratoses.