Open access peer-reviewed chapter

Lupan-Skeleton Pentacyclic Triterpenes with Activity against Skin Cancer: Preclinical Trials Evolution

Written By

Codruţa Şoica, Diana Antal, Florina Andrica, Roxana Băbuţa, Alina Moacă, Florina Ardelean, Roxana Ghiulai, Stefana Avram, Corina Danciu, Dorina Coricovac, Cristina Dehelean and Virgil Păunescu

Submitted: 19 February 2017 Reviewed: 31 March 2017 Published: 12 July 2017

DOI: 10.5772/intechopen.68908

From the Edited Volume

Unique Aspects of Anti-cancer Drug Development

Edited by Jolanta Natalia Latosinska and Magdalena Latosinska

Chapter metrics overview

2,006 Chapter Downloads

View Full Metrics

Abstract

Skin cancer is an increasingly frequent pathology, with a dangerous high percentage of malignant melanoma. The use of synthetic chemotherapy raises the problem of severe adverse effects and the development of resistance to treatment. Therefore, the use of natural therapies became the focus of numerous research groups due to their high efficacy and lower systemic adverse effects. Among natural products evaluated as therapeutical agents against skin cancer, betulinic acid was emphasized as a highly selective anti-melanoma agent and is currently undergoing phase II clinical trials as topical application. Several other pentacyclic triterpenes exhibit antiproliferative activities. This chapter aims to present the latest main discoveries in the class of pentacyclic triterenes with antitumor effect and the evolution of their preclinical trials. Furthermore, it includes reports on plant sources containing pentacyclic triterpenes, as well as the main possibilities of their water solubilization and cancer cell targeting. A review on recent data regarding mechanisms of action at cellular and molecular levels complements information on the outstanding medicinal potential of these compounds.

Keywords

  • pentacyclic triterpenes
  • betulinic acid
  • lupane
  • preclinic
  • mechanism of action

1. Introduction

Skin cancer represents one of the most frequent cancers with an increasing incidence over the past decades [1]. Malignant melanoma, squamous cell carcinoma and basal cell carcinoma represent 98% of all skin cancers [2]. Malignant melanoma determines a higher mortality compared to nonmelanoma skin cancers, being responsible of 75% of skin cancer deaths [2, 3]. Sun exposure is one of the major risk factors, but it influences differently the types of skin cancer. Squamous cell carcinoma is more often related to chronic sun exposure, while malignant melanoma is caused by intermittent sun exposure and overexposure in childhood [4].

Nonmelanoma skin cancer is considered to have the highest incidence of all cancers and occurs more frequently in people with white skin [5]. However, 232,000 new cases of malignant melanoma were diagnosed in 2012, with the highest incidence in Australia. The number of deaths due to this type of skin cancer was 55,000 worldwide in the same year [6]. Malignant melanoma cases tripled in the last 30 years in the United States and Europe [1]. According to World Health Organization [7], each year occur 132,000 cases of melanoma skin cancers worldwide. The increasing incidence is associated with an increase of treatment costs. This aspect underscores the important role of prevention and early detection efforts for this type of cancer [8].

Even though numerous efforts were made for finding effective treatments in melanoma, prognosis for these patients remains unsatisfactory. The standard treatment in early stages is represented by surgical excision, followed by an adjuvant therapy or enrollment in a clinical trial [9]. An early detection of melanoma and a proper treatment increase the chances for cure. Surgery, chemotherapy, immunotherapy or radiation therapy can be used for the treatment [10]. Interferon-α (IFN-α) is used as an adjuvant therapy in patients with high-risk cutaneous melanoma, improving mainly disease-free survival but also overall survival, though not without side effects [11]. An improvement of survival in patients with stage III melanoma has been noticed for ipilimumab therapy. This monoclonal antibody increases the immune response and is approved for treatment in advanced melanoma, but also causes gastrointestinal, endocrine and hepatic adverse effects [12]. New drugs have also been used in the treatment of patients with inoperable or stage IV cutaneous malignant melanoma. The BRAF inhibitors vemurafenib and dabrafenib, the anti-PD1 (programmed death 1) antibodies pembrolizumab and nivolumab or the MEK (mitogen-activated protein kinase) inhibitors trametinib and cobimetinib are new agents proposed in melanoma therapy [13]. Associations of BRAF inhibitors (dabrafenib) and MEK inhibitors (trametinib) have also been evaluated in order to improve overall survival and to delay the appearance of drug resistance in patients with metastatic melanoma with BRAF mutations [14].

In nonmelanoma skin cancer, the therapy is different depending on the severity of the tumor. Standard excision or Mohs micrographic surgery (MMS), radiotherapy, photodynamic therapy (PDT), cryosurgery and topical treatment with imiquimod or 5-fluorouracil are employed in the management of this type of skin cancer [15].

Despite the numerous studies and the advances in targeted therapy and immunotherapy, the treatment options in melanoma are limited [9]. The main inconveniences of chemotherapeutic agents currently used in skin cancer are the severe side effects and the multi-drug resistance [16].

Due to these disadvantages of conventional therapies, new alternatives have been investigated in order to find compounds that can serve for the synthesis of new drugs [16]. Plant-derived compounds are intensively studied as anticancer agents, many studies being performed to evaluate their properties in different types of cancer, including skin cancer [17].

Advertisement

2. Plant sources of pentacyclic triterpenes with lupane scaffold

Plants have gained over the time an important place in the prevention and treatment of various medical conditions. Extracts from plants were obtained since ancient times following simple procedures and used as teas, potions and ointments in an attempt to alleviate pain and to cure diseases. Natural sources of drugs remain an important branch in pharmaceutical drug discovery and therapeutic implementation. Combinatorial chemistry as an initial source of information was unable to offer the expected amount of final products, but is considered a tool for preliminary analysis of new drugs even on cancer treatment. Several groups of researchers provided routes to refine and improve the skeleton of natural compound and to prepare novel active agents [18]. Links between natural sources, synthetic chemistry and knowledge about genetic analysis of microbes are new trends in preclinical evaluations [18, 19]. Drugs derived from nature may be fall in one of the following categories: natural product botanical, derived from natural product or made by total synthesis but with the specification that the pharmacophore is in relation with a natural product [18].

During the last decades, natural remedies are engaged in an unprecedented evolution aimed at an increased efficacy. The development of sophisticated technologies in the fields of phytochemistry, drug formulation and pharmacology, as well as the focus on the mechanism of action on a cellular and molecular level, enables the obtainment of highly efficient drugs from plants.

One of the numerous categories of plant phytochemicals are triterpenes (Figures 13). So far, over 20,000 triterpenes have been isolated from the plant kingdom. They include a variety of structural subtypes: squalene, lanostane, dammarane, tetranortriterpenoids, lupane, oleanane, ursane, hopane and other [20, 21]. Pentacyclic triterpenes, their natural sources and biological effects are presented in Table 1.

SubstancePlant (family)Plant partStudy/effectReference
BAZiziphus mauritiana Lam. (Rhamnaceae)Stem barkIn vitro—inhibitory effect on (MEL-1, -2, -3, -4) cells; apoptotic effect on MEL-2 cells
In vivo—antitumor effect on athymic mice injected with MEL-2 cells
[22]
BE, LUBetula x caerulea Blanch., Betula cordifolia Regel, Betula papyrifera Marsh., Betula populvolia Marsh. (Betulaceae)BarkN/A[23]
BA, LU, BE, UA, OASyzygium formosanum Hay. Mori (Myrtaceae)LeavesN/A[24]
BE, BA, UADiospyros leucomelas Poir. (Ebenaceae)LeavesIn vivo—anti-inflammatory activity on Swiss mice, for induced ear edema and induced paw edema[25]
OARosa canina L. (Rosaceae)Rose hip, powderIn vitro—immunomodulatory activity on Mono Mac 6, obtained when a mixture of OA, BA and UA was used[26]
BARosmarinus officinalis L. (Labiatae)Stems and leavesIn vivo—antidepressant-like effect in the TST, for Swiss mice; anti-immobility effect[27]
BE, BABetula pendula Roth, syn. Betula verrucosa (Betulaceae)BarkIn vitro—cytotoxic effect in EPG85-257 and EPP85-181 cells line[28]
A, BA, BE, LU, UA, OALigustrum pricei Hayata, Ligustrum sinense Lour., Ligustrum lucidum W.T.Aiton (Oleaceae)LeavesIn vivo—analgesic and anti-inflammatory effect on Sprague Dawley rats[29]
OA, BAViscum album L. (Santalaceae) – harvested from Malus domestica Borkh.SproutIn vitro—cytotoxic and apoptotic effect on B16.F10 cells[30]
OA, BAViscum album L. (Santalaceae) – harvested from Malus domestica Borkh.SproutIn vivo—antiapoptotic, antiproliferative effect on C57BL/6NCrL mice injected with B16.F10[31]
BE, UAMyrica cerifera L. (Myricaceae)BarkIn vitro—cytotoxic activity against HL60, A549 and SK-BR-3 cell lines[32]
LUTaraxacum sp. Dandelion (Asteraceae)RootIn vitro—cytostatic, not cytotoxic effect on B16 2F2 cells; inhibition of cells proliferation by differentiation[33]
LULactuca indica L. (Asteraceae)N/AIn vivo—prevents local tumor progression, distant metastasis in dogs with COMM
Dogs: two miniature Dachshunds, two Beagles, two miniature Schnauzers, one Golden Retriever, one Labrador Retriever, one American Cocker Spaniel, one Cavalier King Charles Spaniel and 1 mixed-breed dog
[34]
LULactuca indica L. (Asteraceae)N/AIn vivo—tumor growth suppression and induced cell cycle arrest in C57BL/6 mice injected with B16 2F2 cells[35]
LUBombax ceiba L. (Malvaceae)Stem BarkIn vitro—antiangiogenic effect on SK-MEL-2, A549 and B16-F10 cell lines[36]
BA, OAPaeonia rockii ssp. rockii T.Hong & J.J.Li (Paeoniaceae)RootIn vitro—antiapoptotic effect induced selectively in the M-14 cell line[37]
UASalvia officinalis L. (Lamiaceae)N/AIn vivo—antiprotease and antimetastatic effects on C57BL/6N mice injected with B16 cells[38]
BAAvicennia officinalis L. (Acanthaceae)LeavesIn vivo—anti-inflammatory effect on rats[39]
BA, UA, MABridelia cambodiana Gagnep. (Phyllanthaceae)Whole plantIn vitro—cytotoxic effect against HL60 and LCC cell lines[40]

Table 1.

Bioactivity of various plant products containing pentacyclic triterpenes.

BA (betulinic acid); BE (betulin); LU (lupeol); UA (ursolic acid); OA (oleanolic acid); MA (maslinic acid); N/A (not applicable); MEL-1, -2, -3, -4 (human melanoma cell line); Mono Mac 6 (human monocytic cell line); TST (tail suspension test); EPG85-257 (human gastric carcinoma cell line); EPP85-181 (human pancreatic carcinoma cell line); B16.F10 (murine melanoma cell line); HL60 (human promyelocytic leukemia cell line); A549 (human lung carcinoma cell line); SK-BR-3 (human breast cancer cell line); B16 2F2 (mouse melanoma derived subclone with high differentiation capability); COMM (canine oral malignant melanoma); SK-MEL-2 (human melanoma cell line); M-14 (human melanoma cell line); B16 (mouse melanoma cell line derived from spontaneous skin tumor); LLC (mouse lewis lung carcinoma).

Figure 1.

Triterpene structures (a) lupane; (b) oleanane; (c) ursane; (d) hopane; (e) lanostane; (f) dammarane and (g) quassin.

Figure 2.

Lupan skeleton triterpenes (a) betulinic acid; (b) betulin and (c) lupeol.

Figure 3.

Other triterpenes (a) ursolic acid; (b) oleanolic acid and (c) maslinic acid.

Among plant sources containing lupan-skeleton pentacyclic triterpenes (Figure 2), birch bark has received particular attention due to its high content in these substances, its well-proven application and uses over the time [41]. Currently, it is acknowledged that the outer birch bark is a rich source of pentacyclic triterpenes, which include: betulin (B lup-20 (29)-ene-3β, 28-diol), betulinic acid (BA, 3β acid, hydroxy-lup-20 (29)-en-28-oic) and lupeol (L, Lup-20 (29)-en-3-ol). The development of birch bark extracts, their applications and bioactivity has comprehensively been reviewed [42]. By analyzing birch bark extract, it was shown that betulin is present in the highest amount, while betulinic acid content is lower. However, it is possible that plants from different geographical regions present a variable content in pentacyclic triterpene, which requires a rigorous analysis of the content [43]. Differences between barks of birch species regard the content in: betulin, betulinic acid, betulinic aldehyde, lupeol, oleanolic acid, oleanolic acid 3-acetate, betulin 3-caffeate, erythrodiol and other.

Advertisement

3. Obtainment of lupane-skeleton triterpenes with efficacy in skin cancer

Pentacyclic triterpenes from plants are secondary metabolites with high lipophilicity. Therefore, they are mainly located in hydrophobic histological structures. In the cork of trees, which represents the outer tissue of the secondary bark, triterpenes are associated with suberin; a well-known example is birch bark [44]. Triterpenes are as well components of cuticular and epicuticular waxes covering leaves [45] and fruits [46].

Betulin was isolated for the first time in the 1788 by Lowitz [47] from birch cork. The elucidation of its structure was performed by only in 1953 by Guider et al. [48]. Additional plant sources for betulin include hornbeam (Carpinus betulus L) and hazel (Corylus avellana L.), plants which are phylogenetically closely related to birch [49]. Betulinic acid, a triterpene of major therapeutic relevance, was isolated under the name of “graciolon” from Gratiola officinalis [50] and recognized as such only 40 years later [51]. “Platanolic acid,” isolated from Platanus acerifolia bark [52], proved later to be betulinic acid as well [53]. Furthermore, betulinic acid could be obtained from an alcoholic extract of Cornus florida L. bark [54]. Ko and co-workers [55] used mistletoe (Viscum album) to obtain an ethanol extract enriched in triterpenes, including betulinic acid and botulin.

The obtainment of triterpenes from the plant matrices employs as a first step extraction with organic solvents such as methanol or ethanol [56]. Other solvents are chloroform, dichloromethane, ethyl acetate, petroleum ether or various mixtures thereof, in accordance with the low polarity of these phytocompounds. Recovery procedures may include Soxhlet extraction, maceration and ultrasound-assisted processes [57]. In order to progressively enrich/isolate triterpenes, the usual phytochemical approaches are employed: partition among solvents of increasing polarity, column chromatography on silica gel, countercurrent chromatography and preparative chromatography. Triterpene acids are extracted after alkalinization with sodium hydroxide [57] or calcium hydroxyde [58]. Pure betulin was prepared from a crude mixture using a chromatographic column with silica gel as a stationary phase and a mixture of hexane and ethyl acetate as eluent, followed by recrystallization from 75% ethyl alcohol [59]. An effective preparation of crystalline betulin (99% purity) from birch bark is clearly described in a recent work, following the steps to remove betulinic acid and lupeol. Additionally, the authors demonstrate the obvious relationship between the cytotoxic activity of betulin and its purity [58]. The analytic determination of triterpenes in samples is performed by reverse-phase HPTLC and gas chromatography, coupled with the detection using mass spectrometry detection, is a widely used method for analysis of betulin and other triterpenes in samples. High-performance thin-layer chromatography (HPTLC) is a valuable straightforward tool for the visualization of impurities [60].

Betulinic acid received high attention due to its properties to inhibit the growth of cancer cell lines, without being cytotoxic to normal cells. In plant materials used as sources of triterpenes such as birch, the content in betulinic acid is much lower than that in betulin. For this reason, various attempts have been made to obtain betulinic acid, using betulin as a starting point. In the study of Melnikova and co-workers [61], the most intense catalytic activity was noticed for aluminum salts, which also have a selective activity. The reaction proceeds via the intermediate betulonic acid, purified by recrystallization. Betulonic acid is reduced with NaBH4 (THF or isopropanol) at room temperature to obtain a mixture of 3α-betulinic acid and betulinic acid-3β [61].

Enzymatic transformations, privileged for their simplicity, eco-friendliness and safety, are currently a mainstay in the obtainment of drugs. In an enzymatic approach, the fungus Armillaria luteo-virens Sacc ZJUQH100-6 was employed in the biotransformation of betulin into betulinic acid [62]. Optimization of the obtainment was monitored by variation of parameters like pH, glucose, betulin content, addition of tween 80 and stage of inoculation; the presence of the surfactant had a significant impact on the yield of the biotransformation.

While betulin is readily available from birch and other plants, its anticancer activity is only moderate. Being an accessible starting point for derivatizations, betulin has been the subject of many researches, aiming to obtain compounds with enhanced anticancer activities [63]. The acetylated derivates were tested for antiproliferative effect on several cell lines: colorectal adenocarcinoma, leukemia and breast cancer. By esterificating betulin with propionic acid in dichloromethane solution in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, derivatives: 28-O-propynoylbetulin and 3.28-A, IB, dipropynoylbetulin were obtained. Column chromatography was employed in order to obtain the pure components with a yield of 60% in the case of the first derivative and 12% in the second. The reaction of betulin with propargyl chloroformate and 3-butyl-1-yl chloroformate in benzene, in the presence of pyridine, resulted in the formation of a mixture of 28-O-propargyloxycarbonylbetulin monoesters and 28-O- (3-butynyloxycarbonyl) toxin and di-3,28-A sheep-di (pro-pargyloxycarbonyl) 3.28-A toxin and sheep-di (3-butyn-yloxycarbonyl) toxin. The resulting mixture was separated by column chromatography; thus, pure components were obtained in a 64–69% yield for monoesters and 23–27% in the case of diesters [64].

Advertisement

4. Advanced formulation of lupane triterpenes

The most challenging aspect of the biomedical use of lupane triterpenes is their low water solubility which subsequently causes poor bioavailability [65]; so far, several delivery systems have been developed in order to achieve superior pharmacokinetic outcomes. The current subchapter aims to review the most recent and promising delivery options for betulin, betulinic acid and lupeol.

The first step in the attempt to modulate the aqueous solubility of an insoluble compound relies in its convenient derivatization with water-soluble partners; such an attempt was conducted by Drag-Zalesinska and co-workers [66], who prepared mono- and diesters of betulin and betulinic acid with amino acids. All esters revealed higher water solubilities and significant cytotoxic activity via apoptosis induction; the type of ester as well as the type of the amino acid side chain strongly influences the biological effect of the respective compounds [66]. C(2)-propargyl-substituted pentacyclic triterpenoids conjugated with 1,2,3-triazole glucopyranosides were synthesized via “click” chemistry in order to achieve optimized water solubility as well as pharmacokinetic and pharmacological properties [67].

Cyclodextrin (CD) complexation represents an attractive solution to increase the aqueous solubility of numerous compounds; through their hydrophobic interior and hydrophilic surface, cyclodextrins are able to accommodate various lipophilic guest molecules which can thus be water-solubilized. According to molecular studies [68], the bulky structures of betulinic acid and betulin fit best inside the cavity of γ-CD and its semisynthetic derivatives, such as hydroxypropyl-γ-CD (HPGCD). As a result of HPGCD complexation, a 14-fold increased water solubility was reported for BA accompanied by superior biological activity [69], i.e., strong antiangiogenic and antitumor effect [70, 71]. Similar outcomes were achieved in terms of anti-melanoma activity tested on B16 cell line (murine melanoma) [72]. Fontanay et al. conducted a study on the inclusion of hydroxy pentacyclic triterpenoid acids, including BA, inside native γ-CD [73]; the physicochemical analysis revealed the formation of a 1:1 complex with a significantly improved aqueous solubility.

β-CD derivatives also served as complexation partners for BA, and its inclusion in the cyclodextrin hydrophilic matrix led to significantly improved dissolution rate and, subsequently, antiproliferative in vitro activity against MCF7 (breast cancer) cell line [74]. The same tumor cell line was involved in the study of the biological activity of betulinic acid accommodated inside native β-CD [75]; a dose-dependent antiproliferative activity was reported, through mitochondria-mediated apoptosis induction and G2/M cell cycle arrest.

An important parameter in the cyclodextrin inclusion process is the stability constant of the final complex, its value giving the measure of the potential use of the complex as biologically active agent; significantly high stability constants were achieved for both betulin and betulinic acid in complex with newly synthesized hydrophilic γ-CD derivatives [76]. Such a high stability constant characterizes a strong interaction between the host- and the guest molecule, thus enabling the delivery of the active drug at the target site in the absence of systemic adverse effects. Both complexes, with betulin and betulinic acid, respectively, were in vitro and in vivo tested, revealing moderate in vitro antiproliferative activity; however, in vivo results on murine models showed a significant decline in tumor size and volume [77, 78].

The inclusion of betulin inside HPGCD led to optimized outcomes in terms of bioavailability and antiproliferative activity [79, 80]; similar results were reported for betulin complexation with hydrophilic β-CD derivatives that caused stronger inhibitory activity against MCF7 (breast cancer) cell line than pure betulin [74].

Lupeol was also subjected to inclusion inside γ-CD by kneading in a 1:2 molar ratio; the complex revealed optimized antiproliferative and antiangiogenic activities compared to the pure drug [81].

The use of triterpenes as mixtures such as total extract of birch outer bark may trigger simultaneously various mechanisms of apoptosis induction and therefore result in an additive or synergistic effect. Hertrampf et al. [82] used HPBCD as solubilizer for birch total extract; a series of dilutions were prepared using the main ingredient, betulin, as a reference to calculate concentrations. The study reported the multivalent cytotoxic activity of the birch bark at lower concentrations than previously used presumably due to a higher bioavailability of triterpenes provided by cyclodextrin solubilization; moreover, a synergistic effect was suggested. Triterpene-rich mistletoe extract (6.9% BA) was solubilized by Strüh et al. [30] by using HPBCD and tested against B16F10 melanoma cell line; a dose-dependent reduction of cellular ATP was reported accompanied by high cytotoxicity due to DNA fragmentation. The research was continued by in vivo studies on C57BL/6 mice bearing B16F10 subcutaneous melanoma, revealing an increased antitumor effect and a prolonged mice survival [31].

An alternative research direction was the preparation of cyclodextrin conjugates instead of inclusion complexes; “click chemistry” was involved in the synthesis of triazole-bridged conjugates between β-CD and pentacyclic triterpenes [83]. All bioconjugates showed higher hydrophilicity than the parent compound, and several conjugates displayed significant cytotoxicity on various cancer cell lines; in addition, the cyclodextrin conjugation led to the disappearance of haemolytic toxicity. The authors continued their research by synthesizing α-CD conjugates with several pentacyclic triterpenes including BA [84]; all conjugates exhibited lower hydrophobicity than the parent molecules accompanied by significant anti-HCV (hepatitis C virus) entry activity.

An excellent review was published in 2016 by Lima et al. [85], describing the main attempts to use cyclodextrins as nanocarriers for various terpenes; the authors concluded that cyclodextrins are feasible tools in improving the pharmacological profile of terpenes, limited mainly by the scarce pharmacokinetic and clinical studies.

Liposomes are small vesicles displaying one or more phospholipidic layers and an aqueous core [86] that may incorporate both lipophilic and hydrophilic compounds [87, 88]. Betulinic acid was trapped inside large liposomes by Mullauer et al. [89] and administered to mice bearing experimental models of colon (SW480) and lung (A549) cancer; no systemic adverse effects were reported following parenteral (i.v.) and oral administration. Similar studies reported liposomal and proliposomal formulations with BA with 95% yield of the incorporation process [90]. Phospholipidic nanosomes prepared by means of supercritical fluids were used to entrap BA in order to increase its efficacy as antiviral agent [91, 92]. Several betulin derivatives such as 28-acetylenic derivatives [93] and pyrazoles and 1,2,3-triazole derivatives [94] were synthesized and formulated as liposomes; the nanoformulations exhibited strong apoptotic activity due to both higher biological effect of the active compound and optimized delivery. PEG-ylated BA liposomes were obtained by Liu et al. in 2016, entrapping BA in the lipid bilayer of the liposomes by the ethanol injection technique [95]; the hydrophilic outer PEG layer ensured improved sustained release and antitumor effect compared to free BA or BA liposomes.

Another attractive option in drug delivery is the use of micro- and nanoemulsions; a nanoemulsion containing BA was prepared using flax-seed oil as lipophilic phase and the high-pressure homogenization method [96]; the in vivo testing on the chorioallantoic membrane (CAM assay) revealed a significant antiangiogenic activity. The same procedure was applied for betulin nanoemulsion, followed by in vivo testing by CAM assay and experimental murine cancer model; the study reported strong anti-inflammatory, antiproliferative and antiangiogenic activities of the incorporated betulin as well as its potential benefits in inhibiting metastasis [97]. An oil-in-water nanoemulsion with BA was prepared through the use of phospholipase-catalyzed modified phosphatidylcholine as emulsifier in an ultrasound device; various factors such as composition, ultrasound amplitude, temperature and pH significantly influenced nanoparticle size and stability [98].

A different approach consists in the administration of betulin via the nasal route; in order to avoid mouth sedimentation of betulin particles, the solvent exchange method was used to limit particle sizes to nanoscale, thus leading to higher bioavailability of betulin in the lower respiratory tract [99].

Water solubility may be increased through grinding with hydrophilic polymers (i.e., polyvinylpyrrolidone, polyethylene glycol, arabinogalactan) [100, 101]; solid dispersions of BA with various hydrophilic polymers (i.e., Soluplus, HPMCAS-HF, Kollidon VA64, Kollidon K90, Eudragit RLPO) in 1:4 (w/w) ratio were prepared and analyzed by Yu et al. [102] in 2014, revealing a great potential to increase BA water solubility. Moreover, hydrophilic bioconjugates can be synthesized between active drugs and hydrophilic polymers. BA-monomethoxy polyethylene glycol (mPEG) conjugate was synthesized by covalent bonding of the carboxyl moiety of BA and the amine groups of mPEG [103]; the conjugate exhibited cytotoxicity through cell apoptosis on hepatic cancer cells (Hep3B, Huh7) as well as in vivo antitumor efficacy in Ehrlich ascites tumor (EAT) model while lacking any sign of biochemical and histological toxicity. A step further was represented by the use of multiarm-PEGs as conjugation partner which offer a high density of functional groups; through the formation of an ester bond, BA was linked to eight-arm PEG (8arm-PEG) and then to a targeting molecule (folate) followed by the self-assembly into nanoparticles [104]. A second anticancer drug, hydroxycamptothecin, was added by nanoprecipitation; the ensemble achieved a dramatically increased cytotoxicity, prolonged blood circulation, enhanced tumor targeting and lower systemic toxicity than the free drugs; in addition, a synergistic antitumor efficacy was reported [104]. BA also shows the ability to self-assemble into nano- and microfibers with antileukemic efficacy and cytoprotective activity as well [105].

Biodegradable polymeric nanospheres based on poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) were prepared by nanoprecipitation to incorporate 40% BA [106]; the study reported an increased cytotoxicity and lower IC50 value compared to the pure drug. PLGA was used as building material by interfacial deposition for nanocapsules that efficiently entrapped lupeol [107]. Polymer matrixes can be involved in regional chemotherapy, an approach that avoids systemic adverse effects [108] and allows the controlled release of the pure drug; betulin was incorporated as model compound in such a matrix (poly(3,4-ethylenedioxythiophene), its release being conducted by passive or active mode. The novel formulation exhibited efficient cytotoxic activity against KB and MCF7 cancer cell lines.

BA conjugates with carboxyl-functionalized single-walled carbon nanotubes were synthesized via π–π stacking interaction, leading to a 20% loading of the active drug [109]; following physicochemical and biological analysis, the authors reported the controlled, prolonged release of the drug, with no sign of toxicity on normal fibroblasts and significant cytotoxicity against A549 (lung cancer) cell line. The research continued by coating the nanotubes with four biopolymers: tween 20, tween 80, polyethylene glycol and chitosan in order to further improve biocompatibility [110]; the procedure induced sustained and prolonged release compared to the uncoated nanotubes, while cytotoxicity depended on the chosen biopolymer.

Metallic nanoparticles were also used as nanocarriers for pentacyclic triterpenes; as an example, magnetic nanoparticles coated with chitosan were loaded with BA and exhibited a pseudo-second-order kinetic model release of the active drug [111]; the nanoparticles were cytotoxic on MCF7 cells in a dose-dependent manner while lacking toxicity against normal mouse fibroblast cells. Silver nanoparticles coated with BA were involved in the in vitro testing on a panel of cancer cell lines, including A375 (murine melanoma) [112]; the new formulation revealed strong antiproliferative and antimigratory activity, in particular against melanoma cells.

Advertisement

5. Innovative approaches in preclinical evaluations of pentacyclic triterpenes of the lupane series in skin cancer

Preclinical trials are important in the initial evaluation of new drugs, formulations or specific new design of pathology. They are complex processes with an uncertain ending, as just a reduced percent of evaluations lead to a market product. It is estimated that around 90% of tested drugs are not launched to the market [113]. Despite intense of basic research, the actual delivery of accepted drugs is scarce.

The classical route of a tested compound in preclinical trials includes in vitro tests followed by in vivo tests on animals [113]. These tests may be conceived in a variety of ways and are constantly improved and diversified. The mainstay of preclinical tests regarding a potential efficacy against skin cancer types is in vitro tests using different types of cell lines. In this regard, lupane triterpenes were tested on human melanoma (G361, SK-MEL-28, MEL-2, SK-MEL2, A375), mouse melanoma (B16-F1, B16 2F2) and human skin epidermoid carcinoma A431. In case of betulin, the latter showed a particularly high sensitivity (with a IC50 value below 10µM), while various types of human melanoma cell lines may display a high variation range of the sensitivity to betulin, with differences in IC50 values of one order of magnitude, from 12.4 to over 250 µM [114]. For betulinic acid, IC50 was 154 µM when tested on A375 melanoma [115], and 70 µM when tested on B16-F10 murine melanoma [116]. Information of particular relevance for the actual clinical utilization as anticancer agent comes from comparative data on the cytotoxicity against normal cells and cancer cell lines. Betulin, for example, is more cytotoxic against cancerous cells than nontumoral ones [114]. Further steps in preclinical evaluation are the investigation of the mechanism of action; lupeol, betulin, betulinic acid and their semi-synthetic derivatives have so far shown significant effects on apoptosis and cell cycle regulation [117, 118]. As inflammation is an important player in the pathogenesis of cancer, the anti-inflammatory effects and mechanisms are as well explored to give a correct picture of the antitumoral potential [117]. Furthermore, it is important to explore the antimigratory potential of natural products, as it has a seminal importance for malignant melanoma—a cancer type with a high invasiveness [119]. A global approach to relevant preclinical tests regarding triterpenes should thus be multi-component. In this regard, our workgroup has established an efficient battery of tests for aimed at establishing the potential of natural triterpenes with anticancer/anti-inflammatory activity as agents against skin cancer. The module of preclinical evaluations includes as follows:

  • Step 1: in vitro tests on normal cells (e.g., HaCat) comparing with specific pathological tests; evaluation of cytotoxic activity;

  • Step 2: in vitro evaluations concerning the impact on apoptosis, and observations of specific markers via DAPI/HOPI staining, and evaluation of Annexin V, caspases and other cellular markers [120];

  • Step 3: in vivo embryonated egg membrane assay for toxicological evaluation (HET CAM assay) and investigations of the potential to affect angiogenesis; future aspects include cultivation of cancer cells on embryonated eggs and direct research of the therapeutic potential;

  • Step 4: in vivo tests including a large number of experimental protocols: photochemical model, inoculation of murine cells, xenograft of human pathological cells on adequate mouse hosts and correlations with therapeutical surveillance. Furthermore, histopathological evaluations and immuno-histochemical assays are correlated with innovative approaches like RAMAN skin evaluation, noninvasive methods for skin quality and surface damage characterization.

Additional determinations could require selection of cells from a primary experimental tumor, cultivation of cells and evaluation of compounds, PET animal observations and other methods applied for a detailed pathologic surveillance of drugs.

Advertisement

6. Pentacyclic triterpenes: mechanism of action at cellular and molecular level

Apoptosis is a programmed cell death consisting in morphological changes including cell shrinkage, nuclear condensation, chromosomal DNA fragmentation, plasma membrane blebbing and caspase activation [121]. In this regard, apoptosis is considered a crucial physiological process in tumor clearance, being a major target for anticancer drugs [122]. The molecular mechanisms of apoptosis can include extrinsic and intrinsic pathways. The extrinsic pathway of apoptosis is initiated by external signals, which can activate TNF/Fas-receptor, which in turn activates procaspase-8 [123]. The activated caspase-8 is involved into the caspase-3, -6 and -7 cascade activation [124]. Caspases are important cellular enzymes synthesized as inactive zymogens, which can be activated into their active tetrameric forms by various apoptotic signals [124]. The activation of caspase-3, -6 and -7 leads to cell death not only by breaking down the cytoskeleton, but also the nucleus.

The intrinsic pathway of apoptosis, known as the mitochondrial pathway, is initiated by internal stimuli which can activate the proapoptotic genes from the outer membrane of mitochondria. Bcl-2 family proteins (Bax, Bak, Bcl-xs) are important proapoptotic genes involved in permeabilization of the mitochondrial membrane in order to release cytochrome c in cytosol, where it binds to the caspase-activating protein apoptotic protease activating factor-1 (Apaf-1) and with the procaspase-9, transforming into an apoptosome [124]. The apoptosome releases the activated form of caspase-9, which is also involved into the caspase-3, -6 and -7 activations, which lead to cell death [123].

Previous evidences showed that the pentacyclic triterpens, especially betulin, betulinic acid, lupeol and ursolic acid, have induced apoptosis in different types of cancer cells via activation of the mitochondrial pathway and not to the death receptor pathway (extrinsic way) [125, 126]. These data have been supported by Drag-Zalesinska et al. [118] study in which betulin and betulinic acid proved to induce apoptosis in human metastatic melanoma cells (Me-45) by releasing cytochrome c or the apoptosis inducing factor (AIF) through the mitochondrial membrane. Liu et al. [122] have also demonstrated that betulinic acid, as well as betulin, could kill CNE2 cells through the mitochondrial pathway. Betulinic acid induced the DNA fragmentation, caspase activation and cytochrome c release but independent of Bax proteins [115, 122]. Moreover, betulinic acid has been also involved in activation of nuclear factor kappa B (NF-κB) responsible for apoptosis in various types of cancer cells [127].

The increased production of reactive oxygen species (ROS) caused by betulin and betulinic acid stimulation [128, 129] has been considered a stress factor involved in the depolarization of mitochondrial membrane [130]. Furthermore, both calcium overload and ATP depletion were additional stress factors responsible for increasing the permeability of the inner mitochondrial membrane through formation of nonspecific pores [116]. For instance, the dimethylaminopyridine triterpenoid derivatives have also caused the depolarization of the mitochondrial membrane in situ, in order to increase the permeability transition pore [131].

Unlike the previous data, the study of Şoica et al. [77] on B164A5 murine melanoma cells and on a mouse melanoma model showed that BE and its derivatives had no effect on caspase-2 regulation, the apoptotic mechanism of betulin being suggested to be probably through the transformation of BE into betulinic acid inside the cells.

According to the study of Muceniece et al. [132] in vitro, betulin had a mimetic effect on melanocortin (MC) receptor, especially on MC-1 subtype. This observation has been also supported by Şoica et al. [77] study in which botulin had revealed strong inhibitory effects on B64A5 murine melanoma cells, by binding to the melanocortin receptors. Betulin has been not involved by itself in stimulation of cAMP generation, but it acted as a weak antagonist on alpha-melanocyte-stimulating hormone (alpha-MSH)-induced cAMP accumulation in B16-F1 mouse melanoma cells [132].

In vitro and in vivo studies have also revealed that the birch bark extract and betulin have significantly increased the expression of PARP-1 in melanoma cells [118], exhibiting interferon-inducing activity [133].

According to Zhang et al. [126] study, betulinic acid induced apoptosis by suppressing the cyclic AMP-dependent transcription factor ATF-3 and NF-κB pathways and decreasing the expression of topoisomerase I, p53 and lamin B1. On one hand, earlier studies indicated that betulinic acid had induced apoptosis of cells due to the p53 pathways [134]. This conclusion has been also supported by Tiwari et al. [135] study, in which BA proved a dose-dependent apoptotic effect on both p53 mutant and wild-type cells probably because of its involvement in p53-independent apoptotic pathway. On the other hand, a recent study has shown that the apoptotic effect of betulinic acid in human metastatic melanoma cells (Me-45) had been independent of p53-apoptotic pathway [118]. The presumable mechanisms of action of betulinic acid and betulin in skin cancer are depicted in Figure 4.

Figure 4.

The mechanism of action of betulinic acid and betulin in skin cancer.

Lupeol is a complex multitarget phytochemical, being involved in controlling IL-1 receptor-associated kinase-mediated toll-like receptor 4 (IRAK-TLR4), Bcl-2 family, nuclear factor kappa B (NF-κB), phosphatidylinositol-3-kinase (PI3-K)/Akt and Wnt/β-catenin signaling pathways [136]. According to the Tarapore et al. study, the anticarcinogenic effect of lupeol has been related to the Wnt/β-catenin signaling pathway. That study has revealed that lupeol caused a dose-dependent decrease in Wnt target genes in Mel 1011 cells. Moreover, there has been also observed a decrease of nuclear β-catenin expression, associated with an enhancement of plasmatic β-catenin expression in melanoma cells (Mel 928 and Mel 1241). Consequently, lupeol has been involved in blocking the movement of β-catenin between cytoplasm and nucleus [137].

An in vivo study on Swiss Albino mice showed that lupeol exerted apoptotic effects through the enhancement of bax and caspase-3 genes expression and downregulation of bcl-2 antiapoptotic genes [138].

Unlike botulin and betulinic acid, lupeol has also induced apoptosis via extrinsic pathway by enhancing the expression of FADD protein and Fas receptors [127].

Ursolic acid has strongly increased the IR-induced apoptotic effect in various types of cancer cells, likely DU145, CT26 and B16F10, playing a major role in DNA fragmentation, mitochondrial dysfunction and apoptotic marker modulation [139]. Moreover, ursolic acid has induced apoptosis in M4Beu cells human melanoma through intrinsic pathway by enhancing the caspase-3 activity in a dose-dependent manner, correlated with a low caspase-9 activity [140]. Ursolic acid has also proved to act as an inhibitor of the endogenous reverse transcriptase (RT) activity in the following tumor cells: melanoma (A375), glioblastoma (U87) and thyroid anaplastic carcinoma (ARO), as well as on nontransformed human fibroblast cell line (WI-38), exhibiting strong antiproliferative effects [141].

The mechanism of apoptosis induced by pentacyclic triterpens is not fully understood, although, according to the previous studies, we can conclude that these triterpens exhibited strong apoptotic effects, especially via intrinsic pathway, being involved in increasing the permeability of inner mitochondrial membrane, activation of caspase-9 and 3, as well as cell death.

Advertisement

7. Conclusion

Pentacyclic triterpenes represent an important issue in the field of antiskin cancer formulations; nowadays, the researches focus on the development of nanoformulations that provide multiple advantages over the classical pharmaceutical formulations, including the possibility of being decorated with targeting moieties that significantly improve the antiproliferative activity of the loaded active drug. Different mechanisms of action have been identified so far at cellular and molecular level, in particular for betulinic acid; however, future studies are needed in order to fully comprehend the intimate details of the anticancer treatment with pentacyclic triterpenes and formulations thereof.

Advertisement

Acknowledgments

This work was supported by a grant financed by the University of Medicine and Pharmacy “Victor Babes” Timisoara (Grant PIII-C4-PCFI-2016/2017-03, acronym NANOCEL to C.S. and V.P.).

References

  1. 1. Katalinic A, Waldmann A, Weinstock MA, Geller AC, Eisemann N, Greinert R, Volkmer B, Breitbart E. Does skin cancer screening save lives? An observational study comparing trends in melanoma mortality in regions with and without screening. Cancer. 2012;118(21):5395-5402. DOI: 10.1002/cncr.27566
  2. 2. de Vries E, Trakatelli M, Kalabalikis D, Ferrandiz L, Ruiz-de-Casas A, Moreno-Ramirez D, Sotiriadis D, Ioannides D, Aquilina S, Apap C, Micallef R, Scerri L, Ulrich M, Pitkänen S, Saksela O, Altsitsiadis E, Hinrichs B, Magnoni C, Fiorentini C, Majewski S, Ranki A, Stockfleth E, Proby C, EPIDERM Group. Known and potential new risk factors for skin cancer in European populations: A multicentre case-control study. British Journal of Dermatology. 2012;167(Suppl 2):1-13. DOI: 10.1111/j.1365-2133.2012.11081.x
  3. 3. Guy GP Jr, Thomas CC, Thompson T, Watson M, Massetti GM, Richardson LC, Centers for Disease Control and Prevention (CDC). Vital signs: Melanoma incidence and mortality trends and projections—United States, 1982-2030. MMWR: Morbidity and Mortality Weekly Report. 2015;64(21):591-596
  4. 4. Brøndum-Jacobsen P, Nordestgaard BG, Nielsen SF, Benn M. Skin cancer as a marker of sun exposure associates with myocardial infarction, hip fracture and death from any cause. International Journal of Epidemiology. 2013;42(5):1486-1496. DOI: 10.1093/ije/dyt168
  5. 5. Lomas A, Leonardi-Bee J, Bath-Hextall F. A systematic review of worldwide incidence of nonmelanoma skin cancer. British Journal of Dermatology. 2012;166(5):1069-1080. DOI: 10.1111/j.1365-2133.2012.10830.x
  6. 6. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F. Cancer incidence and mortality worldwide: Sources, methods and major patterns in GLOBOCAN 2012. International Journal of Cancer. 2015;136(5):E359-E386. DOI: 10.1002/ijc.29210
  7. 7. World Health Organization. Available from: http://www.who.int/uv/faq/skincancer/en/ [Accessed: February 16, 2017]
  8. 8. Guy GP Jr, Machlin SR, Ekwueme DU, Yabroff KR. Prevalence and costs of skin cancer treatment in the U.S., 2002-2006 and 2007-2011. American Journal of Preventive Medicine. 2015;48(2):183-187. DOI: 10.1016/j.amepre.2014.08.036
  9. 9. Garbe C, Eigentler TK, Keilholz U, Hauschild A, Kirkwood JM. Systematic review of medical treatment in melanoma: Current status and future prospects. Oncologist. 2011;16(1):5-24. DOI: 10.1634/theoncologist.2010-0190
  10. 10. Aris M, Barrio MM. Combining immunotherapy with oncogene-targeted therapy: A new road for melanoma treatment. Frontiers in Immunology. 2015;6:46. DOI: 10.3389/fimmu.2015.00046
  11. 11. Mocellin S, Lens MB, Pasquali S, Pilati P, Chiarion Sileni V. Interferon alpha for the adjuvant treatment of cutaneous melanoma. Cochrane Database of Systematic Reviews. 2013;(6):CD008955. DOI: 10.1002/14651858.CD008955.pub2
  12. 12. Eggermont AM, Chiarion-Sileni V, Grob JJ, Dummer R, Wolchok JD, Schmidt H, Hamid O, Robert C, Ascierto PA, Richards JM, Lebbé C, Ferraresi V, Smylie M, Weber JS, Maio M, Konto C, Hoos A, de Pril V, Gurunath RK, de Schaetzen G, Suciu S, Testori A. Adjuvant ipilimumab versus placebo after complete resection of high-risk stage III melanoma (EORTC 18071): A randomised, double-blind, phase 3 trial. Lancet Oncology. 2015;16(5):522-530. DOI: 10.1016/S1470-2045(15)70122-1
  13. 13. Svedman FC, Pillas D, Taylor A, Kaur M, Linder R, Hansson J. Stage-specific survival and recurrence in patients with cutaneous malignant melanoma in Europe—A systematic review of the literature. Clinical Epidemiology. 2016;8:109-122. DOI: 10.2147/CLEP.S99021
  14. 14. Robert C, Karaszewska B, Schachter J, Rutkowski P, Mackiewicz A, Stroiakovski D, Lichinitser M, Dummer R, Grange F, Mortier L, Chiarion-Sileni V, Drucis K, Krajsova I, Hauschild A, Lorigan P, Wolter P, Long GV, Flaherty K, Nathan P, Ribas A, Martin AM, Sun P, Crist W, Legos J, Rubin SD, Little SM, Schadendorf D. Improved overall survival in melanoma with combined dabrafenib and trametinib. New England Journal of Medicine. 2015;372(1):30-39. DOI: 10.1056/NEJMoa1412690
  15. 15. Samarasinghe V, Madan V. Nonmelanoma skin cancer. Journal of Cutaneous and Aesthetic Surgery. 2012;5(1):3-10. DOI: 10.4103/0974-2077.94323
  16. 16. Chinembiri TN, du Plessis LH, Gerber M, Hamman JH, du Plessis J. Review of natural compounds for potential skin cancer treatment. Molecules. 2014;19(8):11679-11721. DOI: 10.3390/molecules190811679
  17. 17. Wang H, Khor TO, Shu L, Su ZY, Fuentes F, Lee JH, Kong AN. Plants against cancer: A review on natural phytochemicals in preventing and treating cancers and their druggability. Anti-Cancer Agents in Medicinal Chemistry. 2012;12(10):1281-1305
  18. 18. Newman DJ, Cragg GM. Natural products as sources of new drugs over the 30 years from 1981 to 2010. Journal of Natural Products. 2012;75(3):311-335. DOI: 10.1021/np200906s
  19. 19. Mobegi FM, van Hijum SA, Burghout P, Bootsma HJ, de Vries SP, van der Gaast-de Jongh CE, Simonetti E, Langereis JD, Hermans PW, de Jonge MI, Zomer A. From microbial gene essentiality to novel antimicrobial drug targets. BMC Genomics. 2014;15:958. DOI: 10.1186/1471-2164-15-958
  20. 20. Hill RA, Connolly JD. Triterpenoids. Natural Product Reports. 2013;30(7):1028-1065. DOI: 10.1039/c3np70032a
  21. 21. Thimmappa R, Geisler K, Louveau T, O’Maille P, Osbourn A. Triterpene biosynthesis in plants. Annual Review of Plant Biology. 2014;65:225-257. DOI: 10.1146/annurev-arplant-050312-120229
  22. 22. Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, Cordell GA, Beecher CW, Fong HH, Kinghorn AD, Brown DM, Wani MC, Wall ME, Hieken TJ, Das Gupta TK, Pezzuto JM. Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nature Medicine. 1995;1(10):1046-1051. DOI: 10.1038/nm1095-1046
  23. 23. O’Connell MM, Bentley MD, Campbell CS, Cole BJW. Betulin and lupeol in bark from four white-barked birches. Phytochemistry. 1988;27(7):2175-2176. DOI: 10.1016/0031-9422(88)80120-1
  24. 24. Chang CW, Wu TS, Hsieh YS, Kuo SC, Lee Chao PD. Terpenoids of Syzygium formosanum. Journal of Natural Products. 1999;62(2):327-328
  25. 25. Del Carmen Recio M, Giner RM, Manez S, Gueho J, Julien HR, Hostettmann K, Rios JL. Investigations on the steroidal anti-inflammatory activity of triterpenoids from Diospyros leucomelas. Planta Medica. 1995;61(1):9-12. DOI: 10.1055/s-2006-957988
  26. 26. Saaby L, Moesby L, Hansen EW, Christensen SB. Isolation of immunomodulatory triterpene acids from a standardized rose hip powder (Rosa canina L.). Phytotherapy Research. 2011;25(2):195-201. DOI: 10.1002/ptr.3241
  27. 27. Machado DG, Cunha MP, Neis VB, Balen GO, Colla A, Bettio LEB, Oliveira A, Pazini FL, Dalmarco JB, Simionatto EL, Pizzolatti MG, Rodrigues AL. Antidepressant-like effects of fractions, essential oil, carnosol and betulinic acid isolated from Rosmarinus officinalis L. Food Chemistry. 2013;136(2):999-1005. DOI: 10.1016/j.foodchem.2012.09.028
  28. 28. Drag M, Surowiak P, Malgorzata DZ, Dietel M, Lage H, Oleksyszyn J. Comparision of the cytotoxic effects of birch bark extract, betulin and betulinic acid towards human gastric carcinoma and pancreatic carcinoma drug-sensitive and drug-resistant cell lines. Molecules. 2009;14(4):1639-1651. DOI: 10.3390/molecules14041639
  29. 29. Wu CR, Hseu YC, Lien JC, Lin LW, Lin YT, Ching H. Triterpenoid contents and anti-inflammatory properties of the methanol extracts of Ligustrum species leaves. Molecules. 2011;16(1):1-15. DOI: 10.3390/molecules16010001
  30. 30. Strüh CM, Jäger S, Schempp CM, Scheffler A, Martin SF. A novel triterpene extract from mistletoe induces rapid apoptosis in murine B16.F10 melanoma cells. Phytotherapy Research. 2012;26(10):1507-1512. DOI: 10.1002/ptr.4604
  31. 31. Strüh CM, Jäger S, Kersten A, Schempp CM, Scheffler A, Martin SF. Triterpenoids amplify anti-tumoral effects of mistletoe extracts on murine B16.F10 melanoma in vivo. PLoS One. 2013;8(4):e62168. DOI: 10.1371/journal.pone.0062168
  32. 32. Zhang J, Yamada S, Ogihara E, Kurita M, Banno N, Qu W, Feng F, Akihisa T. Biological activities of triterpenoids and phenolic compounds from Myricacerifera bark. Chemistry & Biodiversity. 2016;13(11):1601-1609. DOI: 10.1002/cbdv.201600247
  33. 33. Hata K, Ishikawa K, Hori K, Konishi T. Differentiation-inducing activity of lupeol, a lupane-type triterpene from Chinese dandelion root (Hokouei-kon), on a mouse melanoma cell line. Biological and Pharmaceutical Bulletin. 2000;23(8):962-967. DOI: 10.1248/bpb.23.962
  34. 34. Yokoe I, Azuma K, Hata K, Mukaiyama T, Goto T, Tsuka T, Imagawa T, Itoh N, Murahata Y, Osaki Y, Minami S, Okamoto Y. Clinical systemic lupeol administration for canine oral malignant melanoma. Molecular and Clinical Oncology. 2015;3(1):89-92. DOI: 10.3892/mco.2014.450
  35. 35. Nitta M, Azuma K, Hata K, Takahashi S, Ogiwara K, Tsuka T, Imagawa T, Yokoe I, Osaki T, Minami S, Okamoto, Y. Systemic and local injections of lupeol inhibit tumor growth in a melanoma-bearing mouse model. Biomedical Reports. 2013;1(4):641-645. DOI: 10.3892/br.2013.116
  36. 36. You YJ, Nam NH, Kim Y, Bae KH, Ahn BZ. Antiangiogenic activity of lupeol from Bombax ceiba. Phytotherapy Research. 2003;17(4):341-344. DOI: 10.1002/ptr.1140
  37. 37. Mencherini T, Picerno P, Festa M, Russo P, Capasso A, Aquino R. Triterpenoid constituents from the roots of Paeonia rockii ssp. rockii. Journal of Natural Products. 2011;74(10):2116-2121. DOI: 10.1021/np200359v
  38. 38. Jedinák A, Mučková M, Košt’álová D, Maliar T, Mašterová I. Antiprotease and antimetastatic activity of ursolic acid isolated from Salvia officinalis. Zeitschrift für Naturforschung. 2006;61:777-782
  39. 39. Sumithra M, Kumar JV, Kancharana VS. Influence of methanolic extract of Avicennia officinalis leaves on acute, subacute and chronic inflammatory models. International Journal of PharmTech Research. 2011;3(2):763-768
  40. 40. Khiev P, Cai XF, Chin Y, Ahn KS, Lee HK, Oh SR. Cytotoxic terpenoids from the methanolic extract of Bridelia cambodiana. Journal of the Korean Society for Applied Biological Chemistry. 2009;52(6):626-631. DOI: 10.3839/jksabc.2009.104
  41. 41. Peyton JL. The Birch: Bright Tree of Life and Legend. Granville, OH, USA: McDonald Woodward Publishing Company; 1994
  42. 42. Krasutsky PA. Birch bark research and development. Natural Product Reports. 2006;23(6):919-942. DOI: 10.1039/b606816b
  43. 43. Cîntă-Pînzaru S, Dehelean CA, Soica C, Culea M, Borcan F. Evaluation and differentiation of the Betulaceae birch bark species and their bioactive triterpene content using analytical FT-vibrational spectroscopy and GC-MS. Chemistry Central Journal. 2012;6(1):67. DOI: 10.1186/1752-153X-6-67
  44. 44. Ekman R. The suberin monomers and triterpenoids from the outer bark of Betula verrucosa. Ehrh. Holzforschung. 1983;37:205-211
  45. 45. Heredia-Guerrero JA, Benítez JJ, Domínguez E, Bayer IS, Cingolani R, Athanassiou A, Heredia A. Infrared and Raman spectroscopic features of plant cuticles: A review. Frontiers in Plant Science. 2014;5:305. DOI: 10.3389/fpls.2014.00305
  46. 46. Szakiel A, Paczkowski C, Pensec F, Bertsch C. Fruit cuticular waxes as a source of biologically active triterpenoids. Phytochemistry Reviews. 2012;11(2-3):263-284
  47. 47. Lowitz JT. Uber eine neue, fast benzoeartige Substanz der Birken [On a novel, nearly benzoin resin like substance of the silver birches]. Chemische Annalen Fur Freunde Der Naturlehre, Arzneygelahrtheit, Haushaltungskunst Und Manufakturen. 1788;2:312-316
  48. 48. Guider JM, Halsall TG, Jones ERH. The chemistry of the triterpenes and related compounds. Part XX. The stereochemistry of ring E of betulin and related compounds. Journal of the Chemical Society (Resumed). 1953:3024-3028. DOI: 10.1039/JR9530003024
  49. 49. Hayek EWH, Jordis U, Moche W, Sauter F. A bicentennial of betulin. Phytochemistry. 1989;28(9):2229-2242. DOI: 10.1016/S0031-9422(00)97961-5
  50. 50. Retzlaff F. Ueber Herba Gratiolae. Archiv der Pharmazie. 1902;240(8):561-568. DOI: 10.1002/ardp.19022400802
  51. 51. Barton DHR, Jones ERH. Optical rotatory power and structure in triterpenoid compounds. Application of the method of molecular rotation differences. Journal of the Chemical Society (Resumed). 1944:659. DOI: 10.1039/jr9440000659
  52. 52. Zellner J, Ziffer D. “Plantanolsaure” aus der Rinde von Platanus orientalis L. Monatshefte für Chemie. 1925;46:323-325
  53. 53. Bruckner V, Kovács J, Koczka I. Occurrence of betulinic acid in the bark of the plane tree. Journal of the Chemical Society. 1948:948-951. DOI: 10.1039/JR9480000948
  54. 54. Robertson A, Soliman G, Owen EC. Polyterpenoid compund. Part I. Betulic acid from Cornus florida, L. Journal of the Chemical Society. 1939:1267-1273. DOI: 10.1039/JR9390001267
  55. 55. Ko BS, Kang S, Moon BR, Ryuk JA, Park S. A 70% ethanol extract of mistletoe rich in betulin, betulinic acid, and oleanolic acid potentiated β-cell function and mass and enhanced hepatic insulin sensitivity. Evidence-Based Complementary and Alternative Medicine. 2016;2016:7836823. DOI: 10.1155/2016/7836823
  56. 56. Goulas V, Manganaris GA. Towards an efficient protocol for the determination of triterpenic acids in olive fruit: A comparative study of drying and extraction methods. Phytochemical Analysis. 2012;23(5):444-449
  57. 57. Belem Lima AM, Siani AC, Nakamura MJ, D’Avila LA. Selective and cost-effective protocol to separate bioactive triterpene acids from plant matrices using alkalinized ethanol: Application to leaves of Myrtaceae species. Pharmacognosy Magazine. 2015,11(43):470-476. DOI: 10.4103/0973-1296.160453
  58. 58. Šiman P, Filipová A, Tichá A, Niang M, Bezrouk A, Havelek R. Effective method of purification of betulin from birch bark: The importance of its purity for scientific and medicinal use. PLoS One. 2016;11(5):e0154933. DOI: 10.1371/journal.pone.0154933
  59. 59. Lugemwa FN. Extraction of betulin, trimyristin, eugenol and carnosic acid using water-organic solvent mixtures. Molecules. 2012;17:9274-9282
  60. 60. Joshi H, Saxena GK, Singh V, Arya E, Singh RP. Phytochemical investigation, isolation and characterization of betulin from bark of Betula utilis. Journal of Pharmacognosy and Phytochemistry. 2013;2:145-151
  61. 61. Melnikova N, Burlova I, Kiseleva T, Klabukova I, Gulenova M, Kislitsin A, Vasin V, Tanaseichuk B. A practical synthesis of betulonic acid using selective oxidation of betulin on aluminium solid support. Molecules. 2012;17:11849-11863
  62. 62. Liu J, Fu ML, Chen QH. Biotransformation optimization of betulin into betulinic acid production catalysed by cultured Armillaria luteo-virens Sacc ZJUQH100-6 cells. Journal of Applied Microbiology. 2011;110(1):90-97
  63. 63. Pettit GR, Melody N, Hempenstall F, Chapuis JC, Groy TL, Williams L. Antineoplastic agents. 595. Structural modifications of betulin and the X-ray crystal structure of an unusual betulin amine dimer. Journal of Natural Products. 2014;77(4):863-872
  64. 64. Boryczka S, Bębenek E, Wietrzyk J, Kempińska K, Jastrzębska M, Kusz J, Nowak M. Synthesis, structure and cytotoxic activity of new acetylenic derivatives of betulin. Molecules. 2013;18:4526-4543
  65. 65. Jäger S, Laszczyk MN, Scheffler A. A preliminary pharmacokinetic study of betulin, the main pentacyclic triterpene from extract of outer bark of birch (Betulae alba cortex). Molecules. 2008;13:3224-3235
  66. 66. Drag-Zalesinska M, Kulbacka J, Saczko J, Wysocka T, Zabel M, Surowiak P, Drag M. Esters of betulin and betulinic acid with amino acids have improved water solubility and are selectively cytotoxic toward cancer cells. Bioorganic & Medicinal Chemistry Letters. 2009;19:4814-4817. DOI: 10.1016/j.bmcl.2009.06.046
  67. 67. Spivak AY, Gubaidullin RR, Galimshina ZR, Nedopekina DA, Odinokov VN. Effective synthesis of novel C(2)-propargyl derivatives of betulinic and ursolic acids and their conjugation with β-d-glucopyranoside azides via click chemistry. Tetrahedron. 2016;72:1249-1256. DOI: 10.1016/j.tet.2016.01.024
  68. 68. Dehelean C, Şoica C, Peev C, Gruia AT, Şeclaman E. Physico-chemical and molecular analysis of antitumoral pentacyclic triterpenes in complexation with gamma-cyclodextrin. Revista de Chimie. 2008;59:887-890
  69. 69. Şoica C, Dehelean C, Peev C, Coneac G, Gruia AT. Complexation with hydroxipropilgamma cyclodextrin of some pentacyclic triterpenes. Characterisation of their binary products. Farmacia. 2008;56:182-190
  70. 70. Dehelean CA, Soica C, Peev C, Ciurlea S, Feflea S, Kasa P Jr. A pharmaco-toxicological evaluation for betulinic acid mixed with hydroxipropilgamma cyclodextrin on in vitro and in vivo models. Farmacia. 2011;59:51-59
  71. 71. Dehelean C, Zupko I, Rethy B, Şoica C, Coneac G, Peev C, Bumbacila B. In vitro analysis of betulinic acid in lower concentrations and its anticancer activity/toxicity by changing the hydrosolubility with hydroxipropilgamma cyclodextrin. Toxicology Letters. 2008;180:S100
  72. 72. Dehelean CA, Soica C, Muresan A, Tatu C, Aigner Z. Toxicological evaluations for betulinic acid in cyclodextrins complexes on in vitro and in vivo melanoma models. Planta Medica. 2009;75:PE3
  73. 73. Fontanay S, Kedzierewicz F, Duval RE, Clarot I. Physicochemical and thermodynamic characterization of hydroxy pentacyclic triterpenoic acid/γ-cyclodextrin inclusion complexes. Journal of Inclusion Phenomena and Macrocyclic Chemistry. 2012;73:341-347
  74. 74. Şoica CM, Peev CI, Ciurlea S, Ambrus R, Dehelean C. Physico-chemical and toxicological evaluations of betulin and betulinic acid interactions with hydrophilic cyclodextrins. Farmacia. 2010;58:611-619
  75. 75. Sun YF, Song CK, Viernstein H, Unger F, Liang ZS. Apoptosis of human breast cancer cells induced by microencapsulated betulinic acid from sour jujube fruits through the mitochondria transduction pathway. Food Chemistry. 2013;138:1998-2007. DOI: 10.1016/j.foodchem.2012.10.079
  76. 76. Wang HM, Soica C, Wenz G. A comparison investigation on the solubilization of betulin and betulinic acid in cyclodextrin derivatives. Natural Product Communications. 2012;7:289-291
  77. 77. Şoica C, Dehelean C, Danciu C, Wang HM, Wenz G, Ambrus R, Bojin F, Anghel M. Betulin complex in γ-cyclodextrin derivatives: Properties and antineoplasic activities in in vitro and in vivo tumor models. International Journal of Molecular Sciences. 2012;13(11):14992-15011. DOI: 10.3390/ijms131114992
  78. 78. Soica C, Danciu C, Savoiu-Balint G, Borcan F, Ambrus R, Zupko I, Bojin F, Coricovac D, Ciurlea S, Avram S, Dehelean CA, Olariu T, Matusz P. Betulinic acid in complex with a gamma-cyclodextrin derivative decreases proliferation and in vivo tumor development of non-metastatic and metastatic B164A5 cells. International Journal of Molecular Sciences. 2014;15:8235-8255
  79. 79. Dehelean C, Şoica C, Peev C, Ordodi V, Tatu C. Betulinic acid dissolved with PVP dose/effect relationship and its intervention on skin pathology and melanoma. A pharmacotoxicological evaluation. Timişoara Medical Journal. 2008;58(Suppl. 2):345-349
  80. 80. Dehelean C, Şoica C, Ciurlea S, Urşica L, Peev C, Aigner Z. Consequences of increasing betulin hydrosolubility with hydroxipropilgamma cyclodextrin, in vitro analysis of its efficacy/noxious activity. Timişoara Medical Journal. 2008;58(Suppl. 2):341-345
  81. 81. Dehelean C, Soica C, Peev C, Ciurlea S, Coneac G, Cinta-Pinzaru S. Pentacyclic triterpenes interventions in skin pathology/toxicity and treatment: In vitro and in vivo correlations. Bulletin of the University of Agricultural Sciences and Veterinary Medicine. 2008;65:370-375
  82. 82. Hertrampf A, Gründemann C, Jäger S, Laszczyk M, Giesemann T, Huber R. In vitro cytotoxicity of cyclodextrin-bonded birch bark extract. Planta Medica. 2012;78(9):881-889. DOI: 10.1055/s-0031-1298473
  83. 83. Xiao S, Wang Q, Si L, Shi Y, Wang H, Yu F, Zhang Y, Li Y, Zheng Y, Zhang C, Wang C, Zhang L, Zhou D. Synthesis and anti-HCV entry activity studies of β-cyclodextrin-pentacyclic triterpene conjugates. ChemMedChem. 2014;9:1060-1070. DOI: 10.1002/cmdc.201300545
  84. 84. Xiao S, Wang Q, Si L, Zhou X, Zhang Y, Zhang L, Zhou D. Synthesis and biological evaluation of novel pentacyclic triterpene α-cyclodextrin conjugates as HCV entry inhibitors. European Journal of Medicinal Chemistry. 2016;124:1-9. DOI: 10.1016/j.ejmech.2016.08.020
  85. 85. Lima PS, Lucchese AM, Araújo-Filho HG, Menezes PP, Araújo AA, Quintans-Júnior LJ, Quintans JS. Inclusion of terpenes in cyclodextrins: Preparation, characterization and pharmacological approaches. Carbohydrate Polymers. 2016;151:965-987. DOI: 10.1016/j.carbpol.2016.06.040
  86. 86. Chang HI, Cheng MY, Yeh MK. Clinically proven liposome-based drug delivery: Formulation, characterization and therapeutic efficacy. Open Access Scientific Reports. 2012;1:195. DOI: 10.4172/scientific reports.195
  87. 87. Immordino ML, Dosio F, Cattel L. Stealth liposomes: Review of the basic science, rationale, and clinical applications, existing and potential. International Journal of Nanomedicine. 2006;1:297-315
  88. 88. Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nature Reviews Drug Discovery. 2005;4:145-160
  89. 89. Mullauer FB, van Bloois L, Daalhuisen JB, Ten Brink MS, Storm G, Medema JP, Schiffelers RM, Kessler JH. Betulinic acid delivered in liposomes reduces growth of human lung and colon cancers in mice without causing systemic toxicity. Anticancer Drugs. 2011;22:223-233
  90. 90. Khattar D, Kumar M, Mukherjee R, Burman AC, Garg M, Jaggi M, Singh AT, Awasthi A. Proliposomal And Liposomal Compositions Of Poorly Water Soluble Drugs; US Patent 2009/0017105 A1, January 15, 2009, http://www.freepatentsonline.com/y2009/0017105.html
  91. 91. Castor TP. Phospholipid nanosomes. Current Drug Delivery. 2005;2:329-340
  92. 92. Son LB, Kaplun AP, Spilevskiĭ AA, Andiia-Pravdivyĭ IuE, Alekseeva SG, Gribor’ev VB, Shvets VI. Synthesis of betulinic acid from betulin and study of its solubilization using liposomes. Bioorganicheskaia Khimiia. 1998;24:787-793
  93. 93. Csuk R, Barthel A, Kluge R, Ströhl D. Synthesis, cytotoxicity and liposome preparation of 28-acetylenic betulin derivatives. Bioorganic & Medicinal Chemistry. 2010;18:7252-7259
  94. 94. Csuk R, Barthel A, Sczepek R, Siewert B, Schwarz S. Synthesis, encapsulation and antitumor activity of new betulin derivatives. Archiv der Pharmazie. 2011;344:37-49
  95. 95. Liu Y, Gao D, Zhang X, Liu Z, Dai K, Ji B, Wang Q, Luo L. Antitumor drug effect of betulinic acid mediated by polyethylene glycol modified liposomes. Materials Science and Engineering C: Materials for Biological Applications. 2016;64:124-132. DOI: 10.1016/j.msec.2016.03.080
  96. 96. Dehelean CA, Feflea S, Ganta S, Amiji M. Anti-angiogenic effects of betulinic acid administered in nanoemulsion formulation using chorioallantoic membrane assay. Journal of Biomedical Nanotechnology. 2011;7:317-324
  97. 97. Dehelean CA, Feflea S, Gheorgheosu D, Ganta S, Cimpean AM, Muntean D, Amiji MM. Anti-angiogenic and anti-cancer evaluation of betulin nanoemulsion in chicken chorioallantoic membrane and skin carcinoma in Balb/c mice. Journal of Biomedical Nanotechnology. 2013;9:577-589
  98. 98. Cavazos-Garduño A, Ochoa Flores AA, Serrano-Niño JC, Martínez-Sanchez CE, Beristain CI, García HS. Preparation of betulinic acid nanoemulsions stabilized by ω-3 enriched phosphatidylcholine. Ultrasonics Sonochemistry. 2015;24:204-213. DOI: 10.1016/j.ultsonch.2014.12.007
  99. 99. Karlina MV, Pozharitskaya ON, Shikov A, Makarov VG, Mirza S, Miroshnyk I, Hiltunen R. Biopharmaceutical study of nanosystems containing betulin for inhalation administration. Pharmaceutical Chemistry Journal. 2010;44:501-503
  100. 100. Shakhtshneider TP, Kuznetsova SA, Mikhailenko MA, Malyar YN, Skvortsova G, Boldyrev VV. Obtaining of nontoxic betulin composites with polyvinylpyrrolidone and polyethylene glycole. Journal of Siberian Federal University. Chemistry. 2012;1:52-60
  101. 101. Shakhtshneider TP, Kuznetsova SA, Mikhailenko MA, Zamai AS, Malyar YN, Zamai TN, Boldyrev VV. Effect of mechanochemical treatment on physicochemical and antitumor properties of betulin diacetate mixtures with arabinogalactan. Chemistry of Natural Compounds. 2013;49:470-474
  102. 102. Yu M, Ocando JE, Trombetta L, Chatterjee P. Molecular interaction studies of amorphous solid dispersions of the antimelanoma agent betulinic acid. AAPS PharmSciTech. 2015;16:384-397. DOI: 10.1208/s12249-014-0220-x
  103. 103. Saneja A, Sharma L, Dubey RD, Mintoo MJ, Singh A, Kumar A, Sangwan PL, Tasaduq SA, Singh G, Mondhe DM, Gupta PN. Synthesis, characterization and augmented anticancer potential of PEG-betulinic acid conjugate. Materials Science and Engineering: C. 2017;73:616-626
  104. 104. Dai L, Cao X, Liu K-F, Li CX, Zhang GF, Deng LH, Si CL, He J, Lei JD. Self-assembled targeted folate-conjugated eight-arm-polyethylene glycol–betulinic acid nanoparticles for co-delivery of anticancer drugs. Journal of Materials Chemistry B. 2015;3:3754-3766
  105. 105. Dash SK, Chattopadhyay S, Karmakar P, Roy S. Anti-leukemic activity of betulinic acid from bulk to self-assembled structure. BLDE University Journal of Health Sciences. 2016;1:14-19
  106. 106. Li J. Development, characterization and in vivo evaluation of biodegradable nanospheres and nanocapsules [thesis]. Halle: Martin-Luther University Halle-Wittenberg, Germany; 2012
  107. 107. Silva MAA, Naves LN, Lima EM, Bozinis MCV, Diniz DGA. Development and characterization of lupeol-loaded nanocapsules. In: SINPOSPq: 4th International Symposium of Post-Graduation and Research; 4-6 November 2010; Sao Paulo, Brazil
  108. 108. Krukiewicz K, Cichy M, Ruszkowski P, Turczyn R, Jarosz T, Zak JK, Lapkowski M, Bednarczyk-Cwynar B. Betulin-loaded PEDOT films for regional chemotherapy. Materials Science and Engineering: C. 2017;73:611-615
  109. 109. Tan JM, Karthivashan G, Arulselvan P, Fakurazi S, Hussein MZ. Sustained release and cytotoxicity evaluation of carbon nanotube-mediated drug delivery system for betulinic acid. Journal of Nanomaterials. 2014;2014:862148. DOI: 10.1155/2014/862148
  110. 110. Tan JM, Karthivashan G, Abd Gani S, Fakurazi S, Hussein MZ. Biocompatible polymers coated on carboxylated nanotubes functionalized with betulinic acid for effective drug delivery. Journal of Materials Science: Materials in Medicine. 2016;27:26. DOI: 10.1007/s10856-015-5635-8
  111. 111. Hussein-Al-Ali SH, Arulselvan P, Fakurazi S, Hussein MZ. The in vitro therapeutic activity of betulinic acid nanocomposite on breast cancer cells (MCF-7) and normal fibroblast cell (3T3). Journal of Materials Science. 2014;49:8171-8182. DOI: 10.1007/s10853-014-8526-3
  112. 112. Soica C, Coricovac D, Dehelean C, Pinzaru I, Mioc M, Danciu C, Fulias A, Puiu M, Sitaru C. Nanocarriers as tools in delivering active compounds for immune system related pathologies. Recent Patents on Nanotechnology. 2016;10(2):128-145
  113. 113. Brodniewicz T, Grynkiewicz G. Preclinical drug development. Acta Poloniae Pharmaceutica. 2010;67(6):578-585
  114. 114. Krol SK, Kielbus M, Rivero-Müller AR, Stepulak A. Comprehensive review on betulin as a potential anticancer agent. BioMed Research International. 2015:584189. DOI: 10.1155/2015/58418
  115. 115. Suresh C, Zhao H, Gumbs A, Chetty CS, Bose HS. New ionic derivatives of betulinic acid as highly potent anti-cancer agents. Bioorganic & Medicinal Chemistry Letters. 2012;22(4):1734-1738. DOI: 10.1016/j.bmcl.2011.12.102
  116. 116. Saha S, Ghosh M, Dutta SK. A potent tumoricidal co-drug ‘Bet-CA’—An ester derivative of betulinic acid and dichloroacetate selectively and synergistically kills cancer cells. Scientific Reports. 2015;5:7762. DOI: 10.1038/srep07762
  117. 117. Yadav VR, Prasad S, Sung B, Kannappan R, Aggarwal BB. Targeting inflammatory pathways by triterpenoids for prevention and treatment of cancer. Toxins. 2010;2:2428-2466. DOI: 10.3390/toxins2102428
  118. 118. Drąg-Zalesinska M, Drąg M, Poreba M, Borska S, Kulbacka J, Saczko J. Anticancer properties of ester derivatives of betulin in human metastatic melanoma cells (Me-45). Cancer Cell International. 2017;17:4. DOI: 10.1186/s12935-016-0369-3
  119. 119. AlQathama A, Prieto JM. Natural products with therapeutic potential in melanoma metastasis. Natural Product Reports. 2015;32(8):1170-1182
  120. 120. McIlwain DR, Berger T, Mak TW. Caspase functions in cell death and disease. Cold Spring Harbor Perspectives in Biology. 2013;5(4):a008656. DOI: 10.1101/cshperspect.a008656
  121. 121. Alakurtti S, Makela T, Koskimies S, Yli-Kauhaluoma J. Pharmacological properties of the ubiquitous natural product betulin. European Journal of Pharmaceutical Sciences. 2006;29(1):1-13. DOI: 10.1016/j.ejps.2006.04.006
  122. 122. Liu Y, Luo W. Betulinic acid induces Bax/Bak-independent cytochrome c release in human nasopharyngeal carcinoma cells. Molecules and Cells. 2012;33(5):517-524. DOI: 10.1007/s10059-012-0022-5
  123. 123. Oh SH, Choi JE, Lim SC. Protection of betulin against cadmium-induced apoptosis in hepatoma cells. Toxicology. 2006;220(1):1-12. DOI: 10.1016/j.tox.2005.08.025
  124. 124. Li Y, He K, Huang Y, Zheng D, Gao C, Cui L, Jin YH. Betulin induces mitochondrial cytochrome c release associated apoptosis in human cancer cells. Molecular Carcinogenesis. 2010;49(7):630-640. DOI: 10.1002/mc.20638
  125. 125. Mullauer FB, Kessler JH, Medema JP. Betulin is a potent anti-tumor agent that is enhanced by cholesterol. PLoS One. 2009;4(4):e1. DOI: 10.1371/journal.pone.0005361
  126. 126. Zhang X, Hu J, Chen Y. Betulinic acid and the pharmacological effects of tumor suppression (Review). Molecular Medicine Reports. 2016;14(5):4489-4495. DOI: 10.3892/mmr.2016.5792
  127. 127. Laszczyk MN. Pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in cancer therapy. Planta Medica. 2009;75(15):1549-1560. DOI: 10.1055/s-0029-1186102
  128. 128. Csuk R, Barthel A, Kluge R, Strohl D, Kommera H, Paschke R. Synthesis and biological evaluation of antitumour-active betulin derivatives. Bioorganic & Medicinal Chemistry. 2010;18(3):1344-1355. DOI: 10.1016/j.bmc.2009.12.024
  129. 129. Csuk R, Barthel A, Schwarz S, Kommera H, Paschke R. Synthesis and biological evaluation of antitumor-active gamma-butyrolactone substituted betulin derivatives. Bioorganic & Medicinal Chemistry. 2010;18(7):2549-2558. DOI: 10.1016/j.bmc.2010.02.042
  130. 130. Saudagar P, Dubey VK. Molecular mechanisms of in vitro betulin-induced apoptosis of Leishmania donovani. American Journal of Tropical Medicine and Hygiene. 2014;90(2):354-360. DOI: 10.4269/ajtmh.13-0320
  131. 131. Bernardo TC, Cunha-Oliveira T, Serafim TL, Holy J, Krasutsky D, Kolomitsyna O, Krasutsky P, Moreno AM, Oliveira PJ. Dimethylaminopyridine derivatives of lupane triterpenoids cause mitochondrial disruption and induce the permeability transition. Bioorganic & Medicinal Chemistry. 2013;21(23):7239-7249. DOI: 10.1016/j.bmc.2013.09.066
  132. 132. Muceniece R, Saleniece K, Riekstina U, Krigere L, Tirzitis G, Ancans J. Betulin binds to melanocortin receptors and antagonizes alpha-melanocyte stimulating hormone induced cAMP generation in mouse melanoma cells. Cell Biochemistry & Function. 2007;25(5):591-596. DOI: 10.1002/cbf.1427
  133. 133. Dehelean CA, Soica C, Ledeti I, Aluas M, Zupko I, Galuscan A, Cinta-Pinzaru S, Munteanu M. Study of the betulin enriched birch bark extracts effects on human carcinoma cells and ear inflammation. Chemistry Central Journal. 2012;6(1):137. DOI: 10.1186/1752-153X-6-137
  134. 134. Rosas LV, Cordeiro MS, Campos FR, Nascimento SK, Januario AH, Franca SC, Nomizo A, Toldo MP, Albuquerque S, Pereira PS. In vitro evaluation of the cytotoxic and trypanocidal activities of Ampelozizyphus amazonicus (Rhamnaceae). Brazilian Journal of Medical and Biological Research. 2007;40(5):663-670
  135. 135. Tiwari R, Puthli A, Balakrishnan S, Sapra BK, Mishra KP. Betulinic acid-induced cytotoxicity in human breast tumor cell lines MCF-7 and T47D and its modification by tocopherol. Cancer Investigation. 2014;2(8):402-408. DOI: 10.3109/07357907.2014.933234
  136. 136. Tsai FS, Lin LW, Wu CR. Lupeol and its role in chronic diseases. Advances in Experimental Medicine and Biology. 2016;929:145-175. DOI: 10.1007/978-3-319-41342-6_7
  137. 137. Tarapore RS, Siddiqui IA, Saleem M, Adhami VM, Spiegelman VS, Mukhtar H. Specific targeting of Wnt/beta-catenin signaling in human melanoma cells by a dietary triterpene lupeol. Carcinogenesis. 2010;31(10):1844-1853. DOI: 10.1093/carcin/bgq169
  138. 138. Nigam N, Prasad S, George J, Shukla Y. Lupeol induces p53 and cyclin-B-mediated G2/M arrest and targets apoptosis through activation of caspase in mouse skin. Biochemical and Biophysical Research Communications. 2009;381(2):253-258. DOI: 10.1016/j.bbrc.2009.02.033
  139. 139. Koh SJ, Tak JK, Kim ST, Nam WS, Kim SY, Park KM, Park JW. Sensitization of ionizing radiation-induced apoptosis by ursolic acid. Free Radical Research. 2012;46(3):339-345. DOI: 10.3109/10715762.2012.656101
  140. 140. Duval RE, Harmand PO, Jayat-Vignoles C, Cook-Moreau J, Pinon A, Delage C, Simon A. Differential involvement of mitochondria during ursolic acid-induced apoptotic process in HaCaT and M4Beu cells. Oncology Reports. 2008;19(1):145-149
  141. 141. Bonaccorsi I, Altieri F, Sciamanna I, Oricchio E, Grillo C, Contartese G, Galati EM. Endogenous reverse transcriptase as a mediator of ursolic acid’s anti-proliferative and differentiating effects in human cancer cell lines. Cancer Letters. 2008;263(1):130-139. DOI: 10.1016/j.canlet.2007.12.026

Written By

Codruţa Şoica, Diana Antal, Florina Andrica, Roxana Băbuţa, Alina Moacă, Florina Ardelean, Roxana Ghiulai, Stefana Avram, Corina Danciu, Dorina Coricovac, Cristina Dehelean and Virgil Păunescu

Submitted: 19 February 2017 Reviewed: 31 March 2017 Published: 12 July 2017