Betulin and Its Derivatives As Novel Compounds with Different

Biotechnology Advances 38 (2020) 107409

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Biotechnology Advances

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Research review paper Betulin and its derivatives as novel compounds with diﬀerent pharmacological eﬀects T

Shayan Amiria,1, Sanaz Dastghaibb,1, Mazaher Ahmadic,1, Parvaneh Mehrbodd, Forough Khademe, Hamid Behroujb, Mohamad-Reza Aghanoorif, Filip Machajg, Mahdi Ghamsaric, Jakub Rosikg, Andrzej Hudeckih, Abbas Afkhamic, Mohammad Hashemii, ⁎ Marek J. Losj,k, Pooneh Mokarramb,2, Tayyebeh Madrakianc,2, Saeid Ghavamia,l,m,n, ,2 a Department of Human Anatomy and Cell Science, Rady College of Medicine, Max Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Canada b Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Analytical Chemistry, Faculty of Chemistry, Bu-Ali Sina University, Hamedan, Iran d Inﬂuenza and Respiratory Viruses Department, Pasteur Institute of IRAN, Tehran, Iran e Department of Immunology, Rady College of Medicine, Max Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, Canada f Division of Neurodegenerative Disorders, St Boniface Hospital Albrechtsen Research Centre, Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, MB, Canada g Department of Pathology, Pomeranian Medical University, ul. Unii Lubelskiej 1, 71-344 Szczecin, Poland h Institue of Non-Ferrous Metals, ul. Sowińskiego 5, 44-100 Gliwice, Poland i Department of Clinical Biochemistry, Zahedan University of Medical Science, Zahedan, Iran j Biotechnology Center, Silesian University of Technology, ul Bolesława Krzywoustego 8, Gliwice, Poland k Linkocare Life Sciences AB, Teknikringen 10, Plan 3, 583 30 Linköping, Sweden l Biology of Breathing Theme, Children Hospital Research Institute of Manitoba, University of Manitoba, Winnipeg, Canada m Health Policy Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran n Research Institute of Oncology and Hematology, CancerCare Manitoba, University of Manitoba, Winnipeg, Canada

ABSTRACT

Betulin (B) and Betulinic acid (BA) are natural pentacyclic lupane-structure triterpenoids which possess a wide range of pharmacological activities. Recent evidence indicates that B and BA have several properties useful for the treatment of metabolic disorders, infectious diseases, cardiovascular disorders, and neurological disorders. In the current review, we discuss B and BA structures and derivatives and then comprehensively explain their pharmacological effects in relation to various diseases. We also explain antiviral, antibacterial and anti-cancer effects of B and BA. Finally, we discuss the delivery methods, in which these compounds most effectively target different systems.

1. An introduction to betulinic acid (B) (Saneja et al., 2018). BA and B are mainly isolated from the various parts of Betulaceae, Platanaceae, Dilleniaceae, Rhamnaceae, Rosaceae, In recent years, natural compounds have gained attention as effec- Fagaceae families (Cichewicz and Kouzi, 2004; Moghaddam et al., tive therapeutic agents for the treatment of many diseases. Triterpenes 2012). Historically, BA was discovered in the extract of Gratiola offici- and their functionalized forms, triterpenoids, are the largest family of nalis (Plantaginaceae family) in 1902 (Retzlaff, 1902). In 1976, Trum- natural compounds, which have been investigated for their therapeutic bull et al. reported the first biological activity of BA as an anticancer effects (Gershenzon and Dudareva, 2007; Sharma et al., 2018). Betu- agent against lymphocytic leukemia P388 cells (Trumbull et al., 1976). linic acid (3β, hydroxyl-lup-20(29)-en-28-oic acid) (BA) is a triterpe- Since then, there have been numerous reports on the biological activ- noid of lupane-structured pentacyclic triterpene. It can be isolated from ities of B and BA and their synthetic derivatives, which have been many plants (Fig. 1A), mainly the bark of Betulaceae family. It can also summarized in Table 1. Furthermore, there have been many reports on be synthesized by the oxidation of betulin (lup-20(29)-ene-3β, 28-diol) the methods for the preparation of new formulations and new

⁎ Corresponding author at: Department of Human Anatomy and Cell Science, Rady Faculty of Health Sciences, Max Rady College of Medicine, University of Manitoba, Winnipeg, Canada. E-mail address: [email protected] (S. Ghavami). 1 These authors have equal ﬁrst co-authorship 2 These authors have equal senior authorship https://doi.org/10.1016/j.biotechadv.2019.06.008 Received 30 December 2018; Received in revised form 30 May 2019 Available online 18 June 2019 0734-9750/ © 2019 Elsevier Inc. All rights reserved. S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

plenty of pine trees parts. Three adjoining terpene units form a class of chemical compound known as Triterpenes (Csuk, 2012, 2014; Zhang et al., 2015a). Usually, triterpenes occur in their free form or as gly- cosides. Abundant numbers of these compounds adopt tetracyclic and pentacyclic structures. Gammaceranes, hopanes, lupanes, oleananes, ursanes are a few of pentacyclic triterpenes subclasses, in which are allocated based on the chemical structures of their carbon skeletons. Triterpenoids are functionalized triterpenes, which can be synthesized through the cyclization of the squalene intermediate with six isoprene units from isopentenyl pyrophosphate (Csuk, 2012). Betulin (lup- 20(29)-ene-3b, 28 diol) (B) and betulinic acid (BA) (3β-hydroxy-lup- 20(29)-en-28-oic acid) are lupane structured pentacyclic triterpenoids. The structures of B and BA are shown in Fig. 2. The existence of ﬁve- membered E ring and isopropylidene group in the molecule are common structural features of the lupane skeleton (Zhang et al., 2015a).

2.1. Extraction and isolation of betulin and betulinic acid

Betulin was first observed by Lowitz in 1788, from the sublimation products of birch bark. Betulinic acid, the oxidation product of B, was first discovered by Retzlaff in 1902 and was described as an unknown compound extracted from Gratiola officinalis (Plantaginaceae family), which further named as BA and identified as the oxidation product of B by Robertson and colleagues in 1939 (Csuk, 2012). Betulin and BA are pentacyclic triterpene natural products that are observed as secondary metabolites in > 200 different types of plants (Hayek et al., 1989; Jäger et al., 2009; Jonnalagadda et al., 2017). Birch bark is the main source of B and BA and is divided into two clearly external and internal distinguished parts. The outer part of the birch bark is especially rich in the extractives. The bark of white birch trees contains different levels of B (10–35%) depending on the age of the Fig. 1. Family of plants used for the isolation of betulin and betulinic acid (A), tree, site of ground and conditions, birch species and some other factors and the total number of publications on BA and its derivatives as therapeutic (Eckerman, 1985; Ekman, 1983; Tolstikov et al., 2005). Along with agents (B), from 2010 to September 2018 (extracted from Scopus). these two extracts of the bark of these species, other B derivatives like betulinic aldehyde, methyl ester of betulinic acid, betulonic aldehyde, derivatives of B and BA to improve their therapeutic efficacy in in vitro betulonic acid (BE), and triterpenes of oleanane and ursane series could and in vivo models via enhancing their poor water solubility and se- also be found. The extracts of the internal part of the bark are the source lectivity in targeting the desired cell lines. of phenolic compounds although small amounts of B can also be found There have been many reports on B and BA and their derivatives as (Tolstikov et al., 2005). therapeutic agents, which have been highlighted in Fig. 1B. Researchers Generally, liquid-solid extraction with various organic solvents such have investigated different biological aspects of B and BA such as new as ethanol, methanol, n-hexane, ethyl acetate or methanol/di- formulations for improving their bioavailability, targeted drug delivery, chloromethane followed by separation by chromatography is the main designing new derivatives of B and BA with improved therapeutic ef- procedure of BA and its derivatives isolation (Alakurtti et al., 2006; ficacies, and new methods for the isolation of B and BA from plants Alder et al., 2016; Eckerman, 1985; Ekman, 1983; Grazhdannikov et al., resources. 2018; Kuck-Tack et al., 2010; Lai et al., 2012; Li et al., 2012; Mahmoud In the current review, a comprehensive update on B and BA and et al., 2005; Mencherini et al., 2011; Nyasse et al., 2009; Krasutsky their derivatives will be presented. We will review the chemistry and et al., 2012; Popov et al., 2017; Prat et al., 2016; Šiman et al., 2016; methods for the extraction of B and BA, pharmacological properties, Tolstikov et al., 2005). The effect of extractant type on the extraction of mechanisms of action, and advances in the B and BA delivery methods B present in birch bark depends on the particle size of the raw material including its targeted drug delivery strategies. We will also discuss the used. In small particle sizes (0.15–1.5 mm), the extraction is almost effect of the structural modifications on biological and therapeutic ef- independent of the extractant type, but with the raw material having a ficacy of B and BA against different infectious and non-infectious dis- larger particle size (0.8–4.0 mm), the rate and completeness of the ex- eases. Furthermore, current review will summarize suggested me- traction depend on the type of the extracting agent (Tolstikov et al., chanisms of actions and molecular targets of B and BA and their 2005). Activation of the birch bark with water vapor in the presence of derivatives in the treatment of different infectious diseases (viral dis- NaOH leads to reduction in extraction time and improvement in the eases) emphasizing HIV and microbial diseases in addition to non-in- extraction efficiency (Tolstikov et al., 2005). Isolation and purification fectious diseases emphasizing different types of cancers. of BA derivatives can be achieved by crystallization in methanol as a consequence their moderate polarity (Nyasse et al., 2009). Due to the 2. Chemistry of betulin and betulinic acid relatively high level of B existing in many plant species, researchers attend more on the extraction process than on the synthesis of B (Kuck- Isopentenyl pyrophosphate oligomers comprise one of the extended Tack et al., 2010; Lugemwa, 2012). assortments of plant-derived natural product with over 30,000 mem- A mixture solvent system consisting of ethyl acetate, ethyl alcohol bers. These compounds are known as “Terpene” which originates from and water, in a volume ratio of 4.5/4.5/1 was developed and used to turpentine, the so-called “resin of pine trees”- a viscous pleasantly extract B from white birch bark at room temperature (Lugemwa, 2012). smelling balsam flowing upon carving the bark and the new wood of One step extraction of B with ethanol from birch bark was investigated

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Table 1 The summary of recent reports on biological activities of B, BA and its derivatives.

Compound Cell line/species Animal Eﬀect Reference

B HCT116, HT29 – Anticancer (colon) Zhou et al. (2018) BA H460 – Anticancer (lung) Zhan et al. (2018) BA Hep-G2 Rat Anticancer (hepatic) Kumar et al. (2018b) 1,2,4-Oxadiazole amide derivatives of BA Colo-205, Hep-G2, HeLa – Anticancer (colon, liver, cervical) Krishna et al. (2018) Indole derivatives of B Colo-205, HCT-116 Anticancer (colon) Khusnutdinova et al. (2018) BA HPV16 – Anticancer (cervical) Goswami et al. (2018) BA MCF-7, MDA-MB-231, 4 T1 Mice Anticancer (breast) Zeng et al. (2018) B A549, H1264, Calu-6 Rat Anticancer (lung) So et al. (2018) Picolyl amides derivatives of BA G-361, MCF-7, HeLa, CEM – Anticancer Bildziukevich et al. (2018) BA HeLa – Anticancer (cervical) Xu et al. (2017a) BA HUH7, PLC/PRF/5 Mice Anticancer (liver) Xu et al. (2017b) Triphenylphosphonium derivatives of B MCF-7, PC-3, MCF-7/Vinb, human skin – Anticancer toward MCF-7/Vinb cells Tsepaeva et al. (2017) fibroblast (HSF) BA KB Mice Anticancer (head and neck) Shen et al. (2017) A glycosylated derivative of BA U87-MG Mice Anticancer (glioblastoma) Huo et al. (2017) Benzylidene derivative of BA A-549, PC-3, HCT 116, MCF-7, MIA PaCa-2 – Anticancer (lung, prostate, colon, Gupta et al. (2017) breast, pancreatic) Ester derivatives of B Me-45 – Anticancer (melanoma) Drąg-Zalesińska et al. (2017) B SGC7901 – Anticancer (gastric) Li et al. (2016b) Azole derivatives of B A549, Hep-G2, HCT 116, MS, RDTE32 Mice Anticancer Grishko et al. (2017) Ester derivatives of BA-1,2,4-oxadiazoles Colo-205, Hep-G2, HeLa – Anticancer (colorectal, liver, cervical) Challa et al. (2016) acetylenic derivatives of betulonic and BA SW707, P388 – Anticancer (breast, leukemia) Bebenek et al. (2016) BA HeLa, A549, MCF-7, RKO, Co18 – Anticancer Potze et al. (2016) BA MCF-7 – Anticancer (breast) Foo et al. (2015) 1,2,3-Triazole derivatives of BA HT-29 – Anticancer (colon) Chakraborty et al. (2015) NO-releasing derivatives of BA B16, Hep-G2 – Anticancer Liu et al. (2015b) BA MCF-7, T47D – Anticancer (breast) Tiwari et al. (2014) Naphthol derivative of BA HL-60, THP-1, MCF-7 Mice Anticancer (leukemia, breast) Majeed et al. (2014) BA B16F10 – Anticancer (melanoma) Jin et al. (2014) BA A375 – Anticancer (melanoma) Gheorgheosu et al. (2013) BA Hep-G2 Mice Anticancer (liver) Quan et al. (2013) Hydroxypropargylamines derivative of BA SW1736, MCF-7, LIPO, DLD-1, A549, A2780, – Anticancer Csuk et al. (2013a) A253, 8505C, 518A2, NiH 3 T3 3-O-propargylated BA and its 1,2,3- HL-60, MCF-7, SF-295, THP-1, HEP-2, A-549, – Anticancer (leukemia, breast, Majeed et al. (2013) triazoles HCT-15, PC-3, DU-145 neuroblastoma, liver, lung carcinoma, colon, prostate) Hydroxypropargylamines derivative of B SW1736, MCF-7, LIPO, DLD-1, A549, A2780, – Anticancer Csuk et al. (2013b) A253, 8505C, 518A2, NiH 3 T3 BA MDA-MB-453, BT474 Mice Anticancer (breast) Liu et al. (2012) BA A549, H1299, HeLa Mice Anticancer (lung, cervical) Hsu et al. (2012) BA MDA-MB-231, MDA-MB-435, BT474, MDA- Mice Anticancer (breast) Mertens-Talcott et al. MB-468, MDA-MB-453, HS-578 T (2013) BA KM3 – Anticancer (myeloma) Yang et al. (2012) RS01, RS02 and RS03 derivatives of BA Hep-G2, HeLa, Jurkat, Chang – Anticancer (liver) Santos et al. (2011) Ionic derivatives of BA A375, SH-SY5Y, MCF-7 – Anticancer (melanoma, neuroblastoma, Suresh et al. (2012) breast) BA HeLa – Anticancer (cervical) Srivastava et al. (2010) Carbamate and N-acylheterocyclic Hep-G2, Jurkat, HeLa, HT-29, PC-3 – Anticancer (carcinoma, leukemia, Santos et al. (2010) derivatives of B and BA cervical, colon, prostate) BA K562 – Anticancer (leukemia) Wu et al. (2010) BA U937, HL60, K562 – Anticancer (leukemia) Kumar et al. (2010) α- and β- D-glucopyranose B anomers 8505C, SW1736, A253, FaDu, A431, A2780, – Anticancer (thyroid, head and neck, Kommera et al. (2010a) DLD-1, HCT-8, HCT-116, HT-29, SW480, cervical, ovarian, colon, breast, MCF-7, 518A2, A549 melanoma, lung) γ-Butyrolactones and butenolides 518A2, A431, A253, FADU, A549, A2780, – Anticancer Csuk et al. (2010c) derivatives of B DLD-1, HCT-8, HCT-116, HT-29, SW480, 8505C, SW1736, MCF-7, Lipo BA and B A431, MCF-7, HeLa – Anticancer Şoica et al. (2012) BA RKO, SW480 Mice Anticancer (colon) Chintharlapalli et al. (2011) BA Hep-G2, A549, HL-60, MCF-7, HCT-116, PC- – Anti-inflammatory, anticancer Karan et al. (2018) 3, HeLa B RAW264.7 murine macrophage Mice Anti-inflammatory Ci et al. (2017) B, BA, and 16 semisynthetic B derivatives Murine J774 macrophages Mice Anti-inflammatory Laavola et al. (2016) B – Mice Anti-inflammatory Chunhua et al. (2017) BA – Murine Anti-inflammatory Lingaraju et al. (2015) BA RAW264.7 murine macrophages – Anti-inflammatory Peng et al. (2015) B RAW 264.7 mouse macrophage Mice Anti-inflammatory Wu et al. (2014) BA – Rat Anti-inflammatory Nader and Baraka (2012) BA – Mice Anti-inflammatory Viji et al. (2011) (continued on next page)

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Table 1 (continued)

Compound Cell line/species Animal Eﬀect Reference

BA – Mice Anti-inflammatory Tsai et al. (2011) BA BV2 microglia – Anti-inflammatory Lee et al. (2011) 3′-Azido-3′-deoxythy- midine derivatives of MT-4 – Anti-HIV-1 maturation Xiong et al. (2010) B C-28 amine-based derivatives of BA – Rat Anti-HIV-1 maturation Chen et al. (2018) Fluorinated derivatives of BA MT4 – Anti-HIV-1 maturation Li et al. (2016a) C-28 modified derivatives of BA HIV-1 NL4-3, NL4-3/V370A – Anti-Bevirimat- resistant HIV-1 Dang et al. (2013) maturation C-28 modified derivatives of BA NL4-3, V370A, ΔT371, ΔV370 Anti-Bevirimat- resistant HIV-1 Dang et al. (2012) maturation Ionic derivatives of BA HIV-1 protease – Anti-HIV-1 protease Zhao et al. (2012) Ionic derivatives of BA Herpes simplex virus type-2 (HSV-2) – Antimicrobial Visalli et al. (2015) Piperazine derivatives of BA Plasmodium falciparum, 3D7 – Antimicrobial (antimalarial) Silva et al. (2015) B and its derivatives Chlamydia pneumoniae – Antimicrobial Salin et al. (2010) Disuccinyl derivatives of B Leishmania donovani Mice Antimicrobial Chowdhury et al. (2014) Heterocyclic derivatives of B Leishmania donovani, THP-1 – Antimicrobial Alakurtti et al. (2010) BA Leishmania donovani Mice Antimicrobial Halder et al. (2018) BA LLC-MK2 – Antimicrobial (anti-trypanosoma cruzi) Sousa et al. (2017) Amide derivatives of BA LLC-MK2 – Antimicrobial (anti-trypanosoma cruzi) Meira et al. (2016) Pyridine-ester derivatives of BA HMVII, HeLa, Vero – Antimicrobial (anti-trichomonas Hubner et al. (2016) vaginalis) Triphenylphosphonium derivatives of B – Mice Antimicrobial (anti-Schistosoma Spivak et al. (2014) and BA mansoni) B – Rat Antimicrobial (anti-streptococcus Viszwapriya et al. (2017) mutans) BA – Rat Antimicrobial (anti-streptococcus Adeleke and Adaramoye mutans) (2017) BA – Rainbow Cortisol-reducing properties Mullally et al. (2017) trout BA HSC-T6 Rat Anti-fibrotic Wan et al. (2012) B and BA CFSC-2G – Anti-fibrotic Szuster-Ciesielska et al. (2011)

energy and results in highly pure product (Ferreira et al., 2013). Further studies showed the recovery of BA from plane tree bark using different liquid solvent-based extraction methods, namely solid-liquid extraction, ultrasound-assisted extraction, pressurized liquid extraction and su- percritical fluid extraction. BA recovery and solvent consumption of each method were evaluated in details (Pinilla et al., 2014). Subcritical water extraction is a candidate to take the place of organic solvents used in the extraction of weak-polar and non-polar natural compounds. Extraction of BA from birch bark was carried out using subcritical water extraction. Under optimal conditions (extraction time of 27.37 min, extraction temperature at 184.52 °C and a solvent/solid ratio of 59.60 ml/g) the maximum BA yield was 28.03 mg per 10 g birch bark (Liu et al., 2015a). The extraction of B from birch bark utilizing two different methods (i.e. reflux boiling and pressurized liquid extraction) was investigated recently (Fridén et al., 2016). The results showed that reflux boiling method had a higher yield, better selectivity, and less solvent consumption than pressurized liquid extraction method. Therefore, pressurized liquid extraction is likely a more energy-demanding and expensive process. In 2017, another study proposed a fermentation method using Saccharomyces cerevisiae for the Fig. 2. Chemical structure of betulin (A, B and C) and betulinic acid (D). production of BA and its derivatives based on the purification by solid- liquid extraction using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acids (Czarnotta et al., by Kuznetsova et al. The content of B in the products was 74–75% or 2017). Also, another research investigated Ultrasonic Assisted-Reflux 85–89% depending on the presence of sodium or potassium hydroxide, Synergistic Extraction (UARSE) for the isolation of camptothecin and respectively (Kuznetsova et al., 2014). Previous studies compared the BA from Camptotheca acuminata Decne fruits (Nyssaceae family). The ability of four types of extraction methods comprising solid-liquid ex- extraction time using UARSE was only 24 min, 80%, and 60% less than traction, stirring extraction, Soxhlet extraction, ultrasonic extraction that for Heating Reflux Extraction (HRE) and Ultrasonic Assisted Ex- and microwave-assisted extraction for the extraction of BA from the traction (UAE), respectively. The UARSE method has provided higher bark of Ziziphus jujuba from buckthorn family (Rhamnaceae). Hence, the extraction yields than both HRE and UAE suggesting that it is an ef- extraction efficiency and the substantial saving of time by microwave fective method for extracting camptothecin and BA from Camptotheca assisted extraction was reported more capable than the other extraction acuminata Decne (Li et al., 2017). A recent study reported a green techniques (Dubey and Goel, 2013). Another study reported a method, method based on the extraction of B from outer birch bark using wet which was based on the microwave-assisted extraction using limonene ethyl acetate under reflux boiling and microwave assisted conditions for the isolation of B from birch outer bark. This method saves time and

4 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

Table 2 existing substitution at C-3 the cytotoxicity activity of the product de- Diﬀerent synthesis methods of BA from B precursor. creases; therefore, these ﬁndings declare that there is a size limitation at

Method Reagents and conditions Yield Ref. C-3 (Csuk et al., 2010a; Csuk et al., 2010b; Csuk et al., 2010c; Santos et al., 2009). Transformation of C-3 hydroxyl to various functional 1 1. CrO3 on SiO2 (1:10) 54% Pichette et al. (2004) groups including ketone, oxime, oxime ether, hydrazone, ester, sulfo- 2. KMnO4 (0 °C) nate, amino, alkyne and picolyl amide have been carried out and the 2 1. TEMPO, 1.1 A, 3.4 V, – Holy et al. (2010), Hugues (50 °C) Menard et al. (2006) resulting products exhibited superior cytotoxicity compared to the

2. NaClO2,Na2HPO4, t-BuOH precursor (Abdel Bar et al., 2009; Baratto et al., 2013; Bildziukevich 3 1. TMSCl, 1H-imidazole 64% Barthel et al. (2008) et al., 2018; Csuk et al., 2010c; Mar et al., 2010; Mukherjee et al., 2004; 2. Oxalyl chloride, DMSO Thibeault et al., 2007). Enhancement of hydrosolubility and cytotoxi- − ( 78 °C) city of BA derivatives were observed by the introduction of Boc-pro- 3. NaMnO4, NaH2PO4 tected L-lysine and polar sugar moieties at C-3 position (Ahmad et al., 41.K2Cr2O4 (ultrasound, 92% Mel'nikova et al. (2012) 20 kHz) 2010; Thibeault et al., 2007). Preparation of C-3 glucoside was carried 2. NaBH4 out and the product possessed no conspicuous cytotoxicity against – 51.O2, Pd(AcO)2 (80 °C) Jukka Tulisalo et al. (2013) fi μ − normal skin broblasts WS1 (IC50 >75 M) (Thibeault et al., 2007). 2. NaClO ,BuN+.HSO 2 4 4 An accession of cytotoxicity toward many tumor cell lines was observed 3. NaBH4 by derivatization of BA C-3 by α- and β-anomers of D-glucopyranose, whereas the corresponding per-acetates exhibited much less toxicity and processing the primary extracts with stable monoterpenes, limo- (Csuk et al., 2010c; Kommera et al., 2010a; Kommera et al., 2010b; nene, pinane and hydrogenated turpentine oil ensured separation of Kommera et al., 2010c; Zhao and Yan, 2009). pure B (95–97%) with high yields (75–82%) and the effective recycling Allylic halogenation of C-20 alkene led to the constitution of allyl of extractants (81–87%) (Grazhdannikov et al., 2018). The quantities of halides, which could be converted to various derivatives including BA found in the natural sources are quite insufficient and therefore amines, sulfides, ammonium salts and phosphonium salts upon nu- synthesize of BA from B is a convenient way to deal with this problem cleophilic substitution using secondary amines, thiols, tertiary amines (Chen et al., 2009a; Chen et al., 2009b; Cichewicz and Kouzi, 2004; and phosphines (Biedermann et al., 2010; Bodrikov et al., 2013a, Csuk et al., 2006; Eiznhamer and Xu, 2004; Kim et al., 1997; Liu et al., 2013b; Holy et al., 2010; Kim et al., 2001; Qian et al., 2011; Spivak 2011a). A few improved semi-synthesis methods of BA from B are re- et al., 2014; Suresh et al., 2012; Tolmacheva et al., 2005; Uzenkova presented in Table 2. et al., 2005). Aforementioned amine and ammonium derivatives have exhibited potent cytotoxicity as proteasome inhibiting anticancer agents; however, potent cytotoxic properties of other amine derivatives 2.2. Betulin and betulinic acid derivatization are due to the disruption of the mitochondrial structure and function (Holy et al., 2010; Qian et al., 2011). Allylic trifluoromethylation of Low water solubility and lipophilic characteristic of B and most of betulin diacetate C-20 alkene has also been reported. The resulting the B derivatives have a consequential role in interpreting the bioac- trifluoromethyl-betulin was further hydrolyzed to BA using sodium tivity assay results of cell cultures. Limited solubility of B derivatives in hydroxide followed by the reduction of the keto form to alcohol. Then, aqueous media has become one of the important challenges associated C-3 alcohol was esterified by dimethyl succinic anhydride to form the with the development of B analogs as therapeutic agents (Ali-Seyed final product showing potent anti-HIV activity with 100 nM EC50 value et al., 2016; Dehaen et al., 2011; Jonnalagadda et al., 2013; Periasamy (Li et al., 2016a). et al., 2014; Tolstikov et al., 2005; Yogeeswari and Sriram, 2005; Zhang Many researchers have contributed to the derivatization of B and BA et al., 2015a). Due to the high order of BA safety, lots of structural at the C-28 position. Oxidization of C-28 hydroxyl group to the corre- modifications have been investigated to improve its potency and effi- sponding betulinic aldehyde leads to an accession of cytotoxic activity cacy as a result of improvement of its solubility profile. Accordingly, although the further oxidation to BA leads to a less pronounced effect unraveling of this issue has been investigated by the researchers mostly on cytotoxicity (Alakurtti et al., 2006; Barthel et al., 2008; Csuk et al., based on derivatization of the alcohol group on C-3, C-28 positions in B 2006). Cytotoxic profile of methyl betulinate is approximate as the or the C-28 acid moiety in BA (Fig. 2) as esters, amides, carbamates, same of betulinic aldehyde (Alakurtti et al., 2006; Hata et al., 2003). ureas, hydroxamates, sulfates, sulfobetaines and phosphates (Table 1). These results clearly illustrated that the carbonyl group at C-28 seems Several B and BA derivatives including uracil esters, glyceryl esters, to be effective for the cytotoxic activity, though the corresponding alkyl saponins and amino acid coupled amides exhibited enhancement effects esters lead to a significantly lower cytotoxic activity. Thiocarboxylic on precursor solubility (Alakurtti et al., 2006; Ali-Seyed et al., 2016; acid and methyl ester derivatives of C-28 were obtained by Lawesson Dehaen et al., 2011; Jonnalagadda et al., 2013; Periasamy et al., 2014; reagent and diazomethane, respectively (Abdel Bar et al., 2009; Csuk Tolstikov et al., 2005; Yogeeswari and Sriram, 2005; Zhang et al., et al., 2010c; Mar et al., 2010; Thibeault et al., 2007). Transformation 2015a). As discussed above, the aforementioned derivatization techni- of BA to its corresponding quaternary ammonium salt at C-28 position ques are employed for the purpose of amelioration of anticancer effect led to the enhancement of hydrosolubility and cytotoxicity and pharmacokinetic properties of B and BA (Kim et al., 1997; Nag and (Biedermann et al., 2010). Betulin bearing L-glycine at C-28 position

Bose, 1989). However, over the past decade, a vast number of chemical showed unsatisfactory cytotoxic activity with a high IC50 value of manipulations of BA have been carried out using reactions at C-2, C-3, (> 50 μM). The C-28 amino acid conjugates exhibited enhanced cyto- C-19 and C-28 and modifications of the skeleton (rings A and E, Fig. 2) toxicity (Biedermann et al., 2010). Carbamate derivatives of B exhibited with various functional groups with the aim of exploration of antitumor selective and superior cytotoxic activity on several tumor cell lines. effects of consequent derivatives. In addition, some of these derivatives Accordingly, the reaction product of ethyl isocyanate in chloroform have been developed as twin drugs or prodrugs. with C-28 acetylated betulin showed two or eight times more cyto- In recent years, comprehensive contributions have been carried out toxicity than BA (Csuk et al., 2010c; Kommera et al., 2010a; Kommera with the aim of discovery of the structural features of BA and its natural et al., 2010c). Cyanoethoxybetulin, 1,2,4-oxadiazole, bis-cyanoethox- analog B responsible for improving their antitumor activity and phar- ybetulin and aminopropoxytriterpenoids are a few subgroups of betulin macokinetic properties. Previous findings declare that C-3 modified BA C-28 highlighted derivatives which have also been synthesized on the derivatives have superior in vivo antitumor efficacy in comparison with basis of Michael addition method. Also, promising anticancer activity the bare BA in vivo against human cancer. By increasing the size of the against 60-panel human tumor cell line was observed for

5 .Aii tal. et Amiri, S. Table 3 B and BA prodrugs, twin drugs and hybrid derivatives synthesis information.

Precursor structure Derivative type Reagents and conditions Product structure Ref.

Prodrug 1. Methyl 2,3,4-tri-O-acetyl-1-bromo-D-glucopyranuronate,K2CO3,TBAB Gauthier et al. (2009) reﬂux 2. KOH

Prodrug Multiarm-polyethylene glycol,EDC-DMAP Gauthier et al. (2009)

Twin drug Ac2O, reﬂux Urban et al. (2004) (Dimer) 6

Twin drug 1. Ac2O, imidazole Pettit et al. (2014) (Dimer) 2. PDC + − 3. PhN (CH3)3Br 4. NaN3 5. Ph3P, H2O 6. PTSA,EtOH

Hybrid ASC, DCC or EDCI, DMAP Horwedel et al. (2010) Biotechnology Advances38(2020)107409

Hybrid 1. Ac2O Horwedel et al. 2. ASC, DCC or EDCI, DMAP (2010)

Hybrid 1. HC ≡ C-CH2Br, Cs2CO3 Dang Thi et al. 2. AZT, CUIC t-BuOH (2014) (continued on next page) .Aii tal. et Amiri, S. Table 3 (continued)

Precursor structure Derivative type Reagents and conditions Product structure Ref.

Hybrid 1. Ac2O Emmerich et al. 2. Oxalyl chloride, Et3N (2014) 3. 1,3-Bis(tert-butylcarboxy-amino)2-propanol 4. TFA 1. 5.Pt(DMSO)2Cl2

Hybrid 1. Ac2O Emmerich et al. 2. Oxalyl chloride, Et3N (2014) 3. Diethylenetriamine 4. Pt(DMSO)2Cl2 2. 5.LiCl/H2O 7 Biotechnology Advances38(2020)107409 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409 aminopropoxytriterpenoids (Antimonova et al., 2011; Giniyatyllina recent literatures (Please see; Ríos and Máñez, 2017, and Alakurtti et al., 2015). Betulinic acid C-28-1,2,4-oxadiazole amide derivatives et al., 2006). From the toxicological point of view, BA is considered as a with high cytotoxicity activity against three human cancer cell lines safe compound with low toxicity when tested in both in vitro and in vivo (Colo-205, Hep-G2, HeLa) have been synthesized recently (Krishna model systems. In this regard, previous in vitro studies have shown that et al., 2018). cytotoxic effects of BA specifically engage cancer cells into death and The conversion of betulonate as a precursor to various derivatives have slight cytotoxic effects on normal cells (Kessler et al., 2007; Zuco like hemisuccinate analog, succinic acid hemiester, C-2-hydro- et al., 2002). Similarly, toxicity assessment of BA in animals revealed no xybetulonic acid ester, 2,2-difluoro and 2,2-dibromo derivatives was sign of toxicity in rats (400 mg/kg, intraperitoneal, i.p.) and mice achieved based on the halogenetion/trifluoromethylation method. The (500 mg/kg, sub-chronic administration, i.p.) (Cichewicz and Kouzi, 2,2-difluoro and 2,2-dibromo derivatives exhibited promising cyto- 2004; Pisha et al., 1995). Unfortunately, despite numerous reports on toxicity against Leukemia CEM cancer lines (Borkova et al., 2015; Li the benefits and therapeutic properties of BA and its derivatives, there et al., 2016a; Urban et al., 2012). are few human studies and clinical trials in the literature about the effects of these compounds on humans and we summarized these stu- 2.3. Betulin and betulinic acid prodrug and twin drug derivatives dies in Section 3.12. In the current section, we review recent studies on the novel pharmacological properties of these compounds, which in- One of the widely applicable drug derivatives are prodrugs, which clude: 1) antidiabetic effects, 2) antidyslipidemic effects, 3) cardio- are medications with little or no pharmacological activity in their own vascular effects, 4) anxiolytic and antidepressant effects, 5) effects on right but after administration, are metabolized into pharmacologically neurodegeneration, 6) anti-inflammatory activity, 7) antiviral effects, active drugs (Rautio et al., 2018). Owing to the success of prodrugs, a 8) antimicrobial effects, 9) anticancer effects, and 10) human studies. few research initiatives have been undertaken to the synthesis of B prodrugs. The 28-O-β-D-glucuronide betulinic acid derivative and C-3 multiarm-polyethylene glycol derivative of BA possessed prodrug 3.2. Pharmacokinetics of betulinic acid properties. As a result of treatment with β-D-glucuronidase after 24 h approximately 75% of BA was released from glucuronide prodrug. Several studies have reported the pharmacokinetics of BA and its Polyethylene glycolated derivative exhibited excellent hydrosolubility derivatives in different model systems. It is important to note that and accordingly high therapeutic index (Dai et al., 2014; Gauthier et al., various derivatives of B and BA have different pharmacokinetic fea- 2009). Dimerization of BA and B has also been carried out. The results tures. Using WinNonlin pharmacokinetic software package, Udeani and revealed that the dimers of eventuate exhibited minor cytotoxicity colleagues determined pharmacokinetics and tissue distribution of BA compared to the precursors. However, with the purpose of obtaining in CD-1 mice. The results of their study showed that serum con- minor IC50 values, a combination of cytotoxic agents such as artesunic centration of BA peaked 9–14 min following the administration of drug succinic acid (ASC), azidothymidine (AZT), cisplatin fragment (PtCl2) (500 mg/kg, i.p.) with the distribution volume of 108 L/kg, half-life of and B and BA precursors has been carried out (Dang Thi et al., 2014; 11.8 h, and clearance rate of 13.5 L/kg/h (Udeani et al., 1999). Another Emmerich et al., 2014; Horwedel et al., 2010). A brief description of the study used electrospray liquid chromatography/mass spectrometry to synthesis steps of B and BA prodrug and twin drug derivatives is pro- quantify the binding of BA to serum proteins. They found that BA has a vided in Table 3. high protein-binding capacity (> 99%) following the administration of drug to mice (100 mg/kg, i.p.) and rats (1 g/kg, dermal) (Cheng et al., 3. Pharmacological properties of betulin and betulinic acid 2003). Previous research also demonstrated the tissue concentration of BA 24 h following an acute administration (500 mg/kg, i.p.) in mice, 3.1. An overview to betulin and betulinic acid pharmacology and reported that higher concentrations of BA distributed in fat and ovary tissues, while brain and heart tissues had the lowest concentra- As mentioned earlier, B is a pentacyclic triterpenoid, which is tions (Udeani et al., 1999). abundant in a wide variety of plants, namely in the barks of birch tree Interestingly, 4-nitrobenzyl-oximino betulinic acid (a derivative of (Betula sp., Betulaceae). Considering the chemical structure of B, it is BA with structural modification on C-3 position) has different phar- obvious that B and most of its derivatives are lipophilic compounds due macokinetics and shows stronger antitumor activity in comparison with to their lupane skeleton. BA is the most studied derivative of B and BA (Rajendran et al., 2008). Another example for the altered phar- possesses a wide spectrum of pharmacological properties, but its poor amacokinetics of BA derivatives is Bevirimat [3-O-(3′,3′-di- water solubility restricts its efficacy and potential as an appropriate methylsuccinyl) betulinic acid], which has been recently introduced as pharmacological agent (Alakurtti et al., 2006; Santos et al., 2009). To a new anti-retrovirus agent with significant anti-HIV properties (Smith address this issue, many structure-activity relationship (SAR) studies et al., 2007). Also, a detailed review article by Saneja and colleagues revealed that structural modifications of B and BA are suitable approach discussed that how different types of nanoformulations, such as carbon to synthesize more hydrophilic derivatives. In this context, most of the nanotubes (CNTs), liposomes, nanoemulsions, and other modifications SAR studies on B focused at positions C-28 (primary hydroxy group), C- have been employed to improve the therapeutic impact of B and BA 3 (secondary hydroxy group), and C-20, which the latter is an alkene through altering its pharmacokinetics (Saneja et al., 2018). Focusing on moiety. A comprehensive review article by Alakurtti et al. argued that biotransformation of BA, in vivo studies showed that biotransformation structural variety among BA derivatives is associated with their dif- of BA occurs on C-6, C-23 and C-24 positions and free carboxylic acid ferent pharmacological properties (Alakurtti et al., 2006). Recently, group on the C-28 position is the substrate for human CYP2C9 researchers applied biotechnological techniques in order to make novel (Cichewicz and Kouzi, 2004). Using UHPLC-Q-TOF-MS/MS system, a formulations of BA, which have more tissue-specificity and efficacy. For recent study demonstrated that rats that were treated with B (100 mg/ example, these studies applied phospholipid nanosome formats, spray kg, orally), had 62 metabolites in the urine, bile and plasma samples. dried muco-adhesive microparticle formulations, polyvinylpyrrolidone- Authors of this paper concluded that biotransformation of B is per- betulinic acid complex, nanosystem-entrapped betulin, liposomes, na- formed via demethylation, dehydroxylation, deoxidization, and dehy- noemulsions and other modifications to B and BA to make more phar- dration (Zhang et al., 2018b). Collectively, BA has low toxicity with few macologically efficient compounds (Castor, 2005; Csuk, 2014; Godugu side-effects, and has attracted attentions as a promising therapeutic et al., 2014; Pozharitskaya et al., 2017; Son et al., 1998). compound, but its low aquatic solubility limited its use, and further BA and its derivatives have an array of pharmacological properties, studies should be done to address this issue. which some of them have been extensively studied and reviewed in the

8 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

3.3. Antidiabetic effects of betulinic acid Preclinical studies have shown that BA has strong protective effects against diabetes comorbidities such as diabetes-associated cardiovas- Diabetes mellitus is a chronic metabolic disorder affecting 422 mil- cular disease, hepatic disease and nephropathies. In this context, evi- lion people in the world (9% of world population in 2014), and is ac- dence showed that BA exerts its protective effects against diabetic ne- companied by serious debilitating comorbidities such as nephropathy, phropathy through several mechanisms including: 1) inhibition of obesity, cardiovascular and hepatic disorders (WHO, 2016; Winer and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) Sowers, 2004). Increasing lines of research indicate that BA not only signalling and decreasing the inflammation, 2) decreasing serum crea- has significant antidiabetic properties, but exhibit protective effects tinine and blood urea and increasing serum albumin, 3) increasing against comorbidities of diabetes (Silva et al., 2016; Vinayagam et al., antioxidant capacity and decreasing the pro-inflammatory cytokines in 2017). However, these effects are not limited to BA and other tri- kidney tissue, and 4) inhibition of cell proliferation and fibronectin terpenes such as ursolic acid, oleanolic acid, and pachymic acid possess accumulation in glomerular mesangial cells (Ahangarpour et al., 2016; the same protective properties (Silva et al., 2016). Betulinic acid and Li et al., 2016b; Wang et al., 2016c ; Xie et al., 2017). In the case of some other triterpenes exert their antidiabetic effects through a variety diabetes-associated hepatic disorders, BA exerts its hepato-protective of mechanisms. Hypoglycemic effects of these compounds are achieved effects through several mechanisms including: 1) decreasing the lipo- by reducing the absorption and uptake of glucose, reduction of gluco- genesis and intracellular lipid storage, 2) enhancing lipolysis, and 3) neogenesis, enhancement of glycogen synthesis, activation of TGR5 antioxidant and anti-inflammatory effects in experimental model of receptors, improving the insulin resistance, anti-inflammatory and an- diabetes (Ahangarpour et al., 2016; Oh et al., 2017; Quan et al., 2013). tioxidant effects and insulin secretagogue effects (Castro et al., 2018; It is important to note that BA has several protective effects against liver Genet et al., 2009; Kim et al., 2013; Silva et al., 2016; Tang et al., diseases (such as steatohepatitis) that are beyond the scope of this re- 2011). In this regard, previous studies revealed that BA inhibits the view and were reported elsewhere in the literature (Bai et al., 2016; enzymatic activity of pancreatic and plasma α-amylase and α-glucosi- Domitrović and Potočnjak, 2016; Yao et al., 2009). In addition, pro- dase in animal models of diabetes and reduced blood glucose levels tective effects of BA against cardiovascular comorbidities of diabetes through the inhibition of glucose absorption (Birgani et al., 2018; de will be discussed in this section later. Collectively, BA employs several Melo et al., 2009; Ding et al., 2018). It is well known that type 2 dia- molecular mechanisms, through which it exerts its unique and sig- betes is associated with increased gluconeogenesis, glycogenolysis, and nificant antidiabetic effects. decreased glycogenesis (Hundal et al., 2000). It has been shown that BA is able to reduce glycogenolysis and enhance glycogen synthesis via 3.4. Antidyslipidemic effects of betulinic acid inhibition of glycogen phosphorylase (Wen et al., 2008). In addition, evidence has shown that naturally occurring pentacyclic triterpenes Ample evidence indicates that triterpenes (including BA) have (including B and BA) increase glucose uptake and glycogen synthesis in strong antidyslipidemic effects beside their antidiabetic properties. In an insulin-resistant human hepatoma cell line (Hep-G2) by a me- this context, previous research showed that chronic administration of chanism, through which AMP-activated protein kinase (AMPK) inhibits BA 1) decreases the plasma levels of cholesterol and triglyceride, 2) gluconeogenesis by increasing phosphorylation of glycogen synthase reduces body weights and abdominal fat accumulation, 3) increases kinase (GSK) 3β (Tuan et al., 2009). The activation of AMPK by BA is insulin and leptin levels, and 4) decreases ghrelin levels in obese mice associated with the down-regulation of some genes that control gluco- treated with high fat diet (de Melo et al., 2009). Further, BA was shown neogenesis such as glucose-6-phosphatase and phosphoenolpyruvate to reduce the plasma triglyceride following administration of the lipid carboxykinase in the hepatocytes (Kim et al., 2013). Further, BA-in- emulsion in rats, and exhibits anti-obesity effects through increasing duced AMPK activation leads to the increased expression of both glu- lipolysis by the inhibition of cAMP-dependent phosphodiesterase (Kim cose transporter 1 and 2 in fibroblasts (Heiss et al., 2014). Abnormal et al., 2012). In addition, previous studies revealed that BA hinders function of beta cells and insulin resistance contribute to the patho- pancreatic lipase and cholesterol acyltransferase-2 (hACAT-2), which genesis of type 2 diabetes (Kahn, 2003). Experimental evidence in- leads to a decrease in lipid absorption from the intestine (Kim et al., dicates that BA not only increases insulin sensitivity, but also improves 2012; Lee et al., 2006). Interestingly, BA decreases the triglyceride insulin secretion and synthesis through a variety of mechanisms (Silva accumulation in both Hep-G2 cells (in vitro) and liver of obese mice (in et al., 2016). In this context, previous studies revealed that BA hinders vivo) by inhibiting the activity of sterol regulatory element-binding the activity of protein tyrosine phosphatase 1B (PTPe1B), which per se protein-1c (SREBP-1c) activity (Quan et al., 2013). Further research inhibits PI3K/Akt signalling pathway and consequently induces insulin revealed that BA exerts its antilipogenic effects in adipocytes by in- resistance. Thus, BA increases insulin sensitivity through improved creasing the phosphorylation of acetyl CoA carboxylase, decreasing PI3K/Akt and acceleration of glucose uptake by peripheral tissues such fatty acid synthase, stearoyl CoA desaturase as well as matured SREBP1 as skeletal muscles (Choi et al., 2009; Jiang et al., 2012; Wang et al., (Kim and Go, 2017). Using streptozotocin–nicotinamide-induced dia- 2015a). In addition, BA acts as a selective agonist for the TGR5 re- betic mice, a recent study showed that BA induced a decrease in the ceptors, which recognize bile acids as ligands and play a pivotal role in plasma levels of leptin, alanine-aminotransferase (ALT), aspartate- energy hemostasis through attenuating the insulin resistance (Genet aminotransferase (AST), alkaline phosphatase (ALP), low density lipo- et al., 2009; Trauner et al., 2010). Increased oxidative stress and im- protein cholesterol (LDL-C), triglyceride, cholesterol and an increase in mune-inflammatory signalling contribute to the pathogenesis of meta- adiponectin and high density lipoprotein cholesterol HDL levels in bolic disorders including type 2 diabetes. It has been shown that tri- diabetic mice (Ahangarpour et al., 2016). Overall, BA induced its anti- terpenes (including BA) possess potent antioxidant and anti- obesity eff ects by 1) the regulation of hormones that control food in- inflammatory properties, and are able to alleviate oxidative stress and take, 2) adipogenesis, lipogenesis and lipolysis, and 3) lipid absorption. immune-inflammatory responses in animal models of diabetes and However, these results were obtained from animal studies and there is protect β-cells against apoptosis (Keane et al., 2015; Ko et al., 2016; no clinical data on humans confirming these advantageous effects of BA Ríos and Máñez, 2017). Recent evidence demonstrated that BA has on lipid hemostasis. antagonistic effects on peroxisome proliferator-activated receptor gamma (PPARγ), which mediates its protective effects on insulin re- 3.5. Cardiovascular effects of betulinic acid sistance and type 2 diabetes (Brusotti et al., 2017; Ko et al., 2016). Further, evidence showed that insulinogenic effects of BA are mediated Since protective effects of pentacyclic triterpenes on cardiovascular through ATP-dependent potassium channels as well as chloride chan- system have been already reviewed in the literature, here we discuss nels in rat pancreatic cells (Castro et al., 2018). the recent studies on the effects of BA in particular (Sheng and Sun,

9 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

2011). Ample evidence indicates that BA has advantageous pharma- research revealed that antihypertensive effects of BA are associated cological effects on cardiovascular system through different mechan- with amelioration of endothelium-dependent relation and reduced isms, which include: 1) enhancing the expression of endothelial NOS oxidative stress (decreasing ROS and increasing SOD levels) (Fu et al., (eNOS), 2) reducing platelet aggregation, 3) inhibition of macrophage 2011; Tabassum and Ahmad, 2011). activation, and 4) showing anti-apoptotic and antioxidant effects. Pre- vious experimental investigations showed that BA attenuates the de- 3.6. Anxiolytic and antidepressant effects of betulinic acid velopment of atherosclerosis in the animal models of diabetes through different mechanisms. Using diabetic apolipoprotein-E gene knockout Anxiety and depressive disorders are among most prevalent and mice, Yoon et al. reported that BA decreased atherosclerotic lesions and debilitating mental health issues in the world and frequently co-occur in improved eNOS expression, which resulted in the inhibition of in- patients (Hirschfeld, 2001; Murray et al., 1996). Continuous use of tracellular adhesion molecule 1 and endothelin 1 expression (Yoon antidepressants and anxiolytics is accompanied by a variety of side et al., 2017). Another study used high-fat diet-induced diabetes in mice effects in patients. Increasing lines of evidence indicate that com- and showed that BA inhibited SREBP pathway, which plays a pivotal plementary and alternative medicine have gained attention for the role in the synthesis of cholesterol, fatty acid, and triglyceride in treatment of anxiety and depressive disorders (Farzaei et al., 2016; mammals. Inhibition of SREBP in this study was associated with im- Nabavi et al., 2017; van der Watt et al., 2008). It has been reported that proved lipid profile in animals as well as decreased lesions in the aortic triterpenes (including BA) have therapeutic and protective effects arch and thoracic aorta. Also, authors revealed that BA treatment led to against several brain disorders such as anxiety, depression, cognitive a significant decrease in the accumulation of macrophages and in- deficits and seizures (Manayi et al., 2016). Animal studies showed that creased level of smooth muscle cell marker (SMC-actin) in vascular B and BA exert their anxiolytic and anticonvulsant effects through the tissue (Tang et al., 2011). activation of γ-aminobutyric acid A receptors (GABAAR). GABAergic Endothelial NOS is a crucial enzyme in the regulation of vascular system is the main inhibitory neurotransmission in the brain that at- endothelial function, and NO produced by eNOS has protective effects tenuates the neural excitability in the central nervous system (CNS) of on vasculature. Thus, eNOS is considered as one of the pharmacological mammals (Muceniece et al., 2008; Mullally et al., 2014; Puniani et al., targets in the treatment of cardiovascular disorders (Gkaliagkousi and 2015; Verma et al., 2016). In addition to experimental studies, it has Ferro, 2011). Evidence showed that BA enhanced eNOS expression and been reported that the extract of Souroubea spp. (Marcgraviaceae family) NO production by increasing the intracellular calcium levels, which and Platanus spp. (Platanaceae family), which contains methyl-betulinic leads to the phosphorylation of eNOS at Ser1177 (Jin et al., 2016). acid (as the main pharmacologic compound), possess anxiolytic effects Another study revealed that BA induces eNOS through the activation of and currently is in clinical trial for the treatment of anxiety in Canada estrogen receptor and its downstream signalling through the PI3K/Akt (Liu et al., 2017a). pathway (Hohmann et al., 2016). Also, an in vitro study showed that BA In addition, recent investigations showed that BA has antidepressant increased eNOS mRNA and protein expression in HUVEC and EA.hy effects in animal models of depression (Chen et al., 2017; Liang et al., 926 cells and decreased the expression of NADPH oxidase (Steinkamp- 2016; Machado et al., 2013; Verma et al., 2016). These reports de- Fenske et al., 2007). monstrated that BA exhibits its antidepressant effects by different me- Vascular thrombosis is known as an important risk factor for the chanisms. For example, Chen and colleagues showed that BA produces development of a wide variety of cardiovascular disorders and platelets strong anti-inflammatory effects in mice exposed to unpredictable aggregation is involved in thrombosis formation (von Hundelshausen chronic mild stress. Antidepressant effects of BA in this animal model and Weber, 2007). Evidence indicates that BA inhibited human platelet were accompanied by a significant decrease in the protein expression of aggregation by the reduced expression of P-selectin (Tzakos et al., NF-κB and pro-inflammatory cytokines such as interleukin-1β and 2012). Similarly, BA and 3β-acetoxybetulinic acid inhibited rat platelet tumor necrosis factor (TNF)-alpha in the hippocampal formation of aggregation induced by thrombin, adenosine diphosphate (ADP) and animals (Chen et al., 2017). It is interesting to note that immune-in- epinephrine, and effects were similar to aspirin (Habila et al., 2013). flammatory pathways play a pivotal role in the pathobiology of de- Thus, BA protects cardiovascular system by hindering the platelets pression (Berk et al., 2013; Maes et al., 2009). Experimental and clinical aggregation and thrombosis formation. Betulinic acid was also reported studies have confirmed the efficacy of pharmacological (such as anti- to attenuate the atherosclerosis through reducing macrophage in- depressants and natural products) and non-pharmacological (such as flammation and accumulation by reducing the expression of lipoprotein meditation and regular exercise) therapies that produce antioxidant and lipase and oxidative stress (Peng et al., 2015; Yoon et al., 2017). In anti-inflammatory effects in the treatment of depression (Amini-Khoei addition, BA was shown to reduce the proliferation and migration of et al., 2017; Haj-Mirzaian et al., 2016; Kiecolt-Glaser et al., 2010; endothelial smooth cells following exposure to high concentrations of Lucassen et al., 2010). In addition, it has been shown that BA has glucose (Yoon et al., 2010). Previous studies have also shown that BA protective effects against the negative impact of glucocorticoids in an- has protective effects on cardiac cells and tissue as well. Xia and col- imals. Glucocorticoids and hypothalamic-pituitary-adrenal (HPA) axis leagues administered BA to rats exposed to the myocardial ischemic- dysfunction play a critical role in the pathogenesis of depression and BA reperfusion injury and found that BA improved cardiac physiological has been shown to have protective effects against the negative effects of function and decreased lactate dehydrogenase as well as creatine kinase glucocorticoids (Mullally et al., 2017; Pariante and Lightman, 2008; Yi activities. Also, BA showed anti-apoptotic effect by the regulation of et al., 2015). These studies showed that protective effects of BA against BAX and BCL-2 proteins (Xia et al., 2014). Using an in vitro model glucocorticoids are mediated through anti-apoptotic and antioxidant system, another research showed that BA exerts its protective effects properties of the compound (Yi et al., 2015; Yi et al., 2016). Collec- through enhancement of nuclear factor erythroid 2-like (Nrf2)- heme tively, BA-induced anxiolytic and antidepressant effects in animal oxygenase-1 pathway (Wang et al., 2018). Further, anti-inflammatory models are mediated through antioxidant, anti-inflammatory, anti- effects of B through the activation of signal transducer and activator of apoptotic and GABAergic properties. transcription 3 (STAT3) pathway has been shown to protect cardiac cells (Zhang et al., 2015b). 3.7. Effects of betulinic acid on neurodegeneration Hypertension is a chronic medical condition, which is associated with the increased blood pressure in the arteries. Many people use Neurodegenerative disorders (such as Alzheimer's disease and medicinal plants and herbs such as Zizyphi Spinosi semen, which possess Parkinson's disease) are a range of pathological conditions, which are antihypertensive properties. It is important to note that BA is the main defined by abnormal accumulation of specific proteins in neurons and active constituent of dried seed of Zizyphus jujuba var. spinosa. Recent are associated with progressive neurodegeneration, neurological and

10 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409 cognitive dysfunctions (Jucker and Walker, 2011). Recent evidence and neurodegenerative disorders (Lucas and Maes, 2013). Toll-like re- indicates that BA has protective effects against neurodegeneration and ceptors are known as pattern recognition receptors, which recognize neuronal damage in different animal models of Alzheimer's disease. pathogen-associated molecular patterns (PAMPs) such as lipopoly- Using intracerebroventricular injection of streptozotocin, as an animal saccharide (LPS) as well as damage-associated molecular patterns model of Alzheimer's disease, previous studies showed that BA exhibits (DAMPs) (Akira and Takeda, 2004). Many studies on the anti-in- protective effects by: 1) attenuating the neurobehavioral and cognitive flammatory effects of BA have shown that this compound exerts its dysfunction, 2) decreasing the hippocampal pro-inflammatory cyto- effects through the inhibition of TLR-4/MyD88/NF-κB signalling. For kines, 3) reducing oxidative and nitrosative stress, 4) normalizing example, many studies used animal models, in which TLR-4 was di- acetylcholinesterase activity, 5) restoring the neurotransmission bal- rectly activated by LPS or polymicrobial sepsis, and showed the anti- ance, 6) normalizing long term potential (LTP) parameters, and 7) de- inflammatory effects of BA in different organs such as lungs (decrease in creasing the histological damage to hippocampus (Kaundal et al., TNF-α, TGF-β1 and inducible NOS) (Lingaraju et al., 2015; Nader and 2018a; Navabi et al., 2018). Interestingly, similar protective effects of Baraka, 2012), kidneys (decrease in NF-κB, TNF-α, IL-6 and IL-10), BA were reported against cognitive decline in a mouse model of dia- macrophages (inhibition of IκB phosphorylation, p65 phosphorylation, betes induced by streptozotocin injection (Ma and Long, 2016). In ad- and NF-κB transcription) (Lingaraju et al., 2015; Zhao et al., 2013), dition, a recent study revealed that synthetic analogues of BA reduces human peripheral blood mononuclear cells (reduction in COX-2, pros- the formation of amyloid β-protein (Aβ) through disruption the enzy- taglandin E2, and IkBa phosphorylation) (Viji et al., 2011), and CNS matic activity of APP-cleaving enzyme 1 (BACE1) and γ-secretase (increase in M2 (anti-inflammatory) and decrease in M1 (pro-in- (Zhang et al., 2017a). Similarly, another study showed that BA rapidly flammatory) microglia) (Li et al., 2018). Interestingly, BA has been promotes amyloid- β fibril formation in vitro and disrupts the ag- shown to attenuate the inflammatory responses induced by TLR-4/NF- gregation of the Aβ (Planchard et al., 2012). Betulinic acid has been κB signalling in a variety of animal models of disease such as diabetes- shown to enhance the cognitive ability and protect neurons in animal induced renal fibrosis (Wang et al., 2016c), chemical-induced hapatic model of scopolamine-induced amnesia through: 1) increasing the fibrosis and fatty liver (Wan et al., 2013; Wan et al., 2012) and chronic phosphorylation of cyclic AMP response element-binding protein stress-induced depression (Chen et al., 2017). (CREB), 2) increasing the expression of brain-derived neurotrophic Increased oxidative stress, namely reactive oxygen species (ROS) factor (BDNF) in the cortical and hippocampal regions in mice brain, 3) production is associated with the formation of DAMPs and inflamma- protecting hippocampal HT22 cells against glutamate toxicity, 4) im- somes, which consequently initiate the inflammatory pathways through proving memory and learning, and 5) promoting antioxidant capacity TLR signalling and maturation of IL-1β, respectively (Gill et al., 2010; (Cho et al., 2016; Lee et al., 2012). Also, another research showed that Schroder and Tschopp, 2010). Many studies have shown that BA has BA acts as a phosphodiesterase inhibitor and restores hippocampal potent protective effects against nitrosative and oxidative stress cAMP/cGMP pool and BDNF levels, improves cerebral blood flow and (Lingaraju et al., 2015; Szuster-Ciesielska et al., 2011). Betulinic acid restores memory deficits in a rat model of dementia (Kaundal et al., reduces the expression of inducible nitric oxide synthase (iNOS) and

2018b). Further, BA showed protective effects against Aβ1–42 – induced nitric oxide (NO), which the latter inhibits mitochondrial function and memory impairment by increasing CREB phosphorylation, BDNF ex- produces oxidative stress and free radicals such as peroxynitrite pression and antioxidant capacity in mice (Cho et al., 2014). In the case (Clementi et al., 1998; Nader and Baraka, 2012). Also, BA inhibits the of Parkinson's disease, a recent study showed that BA possesses neu- activity of NADPH oxidase, which is a source of ROS production and roprotective effects in pharmacological and transgenic Caenorhabditis oxidative stress (Lu et al., 2017). Further, protective effects of BA elegans models of Parkinson's disease. Authors of this paper found that against oxidative damage are through improving antioxidant capacity BA enhances the proteasomes activity by increasing Rpn1 expression (increasing glutathione (GSH) and reducing lipid peroxidation) anti- and reducing the transcription of the apoptosis pathway gene, Egl-1.In oxidant enzymes (increasing SOD and GSH peroxidase) (Nader and addition, they found that BA reduces α-synuclein accumulation and Baraka, 2012; Yi et al., 2014). Interestingly, it has been shown that BA improves behavior of C. elegans in both transgenic and pharmacological hinders the formation of NLRP3 inflammasomes by the activation of models (Tsai et al., 2017). Collectively, the results of these studies TGR5 receptors (Guo et al., 2016). Thus, BA exhibits its anti-in- suggest that BA has neuroprotective effects against the negative effects flammatory effects by decreasing oxidative and nitrosative stress, of proteinopathies in the brain through different mechanisms. boosting redox system and inhibition of inflammasomes formation. Previous studies revealed that BA has steroidal mode of action (del 3.8. Anti-inflammatory activity of betulinic acid Carmen Recio et al., 1995; Mukherjee et al., 1997). These studies showed that BA produced anti-inflammatory effects against carra- Inflammation is observed in many pathological conditions in hu- geenan and serotonin-induced inflammation in animals, and these ef- mans and there are several classes of anti-inflammatory drugs used for fects were reversed by progesterone, actinomycin D, and cycloheximide different therapeutic purposes in the clinic. Recently, anti-inflammatory suggesting a steroidal mode of action for BA (del Carmen Recio et al., properties of natural products have gained attention and evidence. It 1995; Mukherjee et al., 1997). Finally, BA has been reported to inhibit indicates that many natural compounds possess anti-inflammatory cyclooxygenase pathway and attenuates the inflammation in response properties. Among these compounds, anti-inflammatory effects of tri- to different kinds of stimuli by reducing the synthesis of prostaglandins terpenes, namely B and BA, have been extensively reviewed and re- (such as prostaglandin E2) (Lin et al., 2015a; Takada and Aggarwal, ported in the literature (Alakurtti et al., 2006; del Carmen Recio et al., 2003; Viji et al., 2011). Collectively, evidence in the literature indicates 1995; Mukherjee et al., 1997; Ríos and Máñez, 2017). Anti-in- BA induces its potent anti-inflammatory effects through different me- flammatory effects of BA have been studied in many different animal chanisms, but application of BA for clinical use requires extensive models of disease. Betulinic acid exhibits its anti-inflammatory re- clinical investigations. sponses through different mechanisms such as: 1) inhibition of TLR4 and NF-κB signalling, 2) potent anti-oxidative and anti-notrosative ef- 3.9. Antiviral activity of betulin and its mechanism of action fects, 3) exerting steroid-like effects, and 4) inhibition of cyclooxygenase pathway. 3.9.1. Anti-HIV activity of betulin and its derivatives Toll-like receptor 4 is an innate immune system receptor, whose HIV (Human Immunodeficiency Virus) is an established cause of activation is accompanied by the initiation of cascades of inflammatory Acquired Immunodeficiency Syndrome (AIDS), which leads to a gra- pathways, which underlie the pathophysiology of a vast range of human dual destruction of the autoimmune system, especially by significantly diseases such as cardiovascular disorders, depression, diabetes, obesity, damaging and reducing the population of CD4+ T cells. With 37

11 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409 million people suﬀering from HIV and 1.8 million newly infected every 1996). This study also evaluated 3-alkylamido-3-deoxy-betulinic acids year, it remains one of the most widespread viral diseases (WHO, for their anti-HIV activity. While 3α-diglycorylamide-3-deoxy-betulinic

2017). The introduction of readily available anti-HIV therapies, such as acid proved to be effective against HIV (EC50: 0.24 μM), the replace- HAART – involving the use of reverse transcriptase and protease in- ment of C-3 esters with an amido group resulted in less active deriva- hibitors, has resulted in a dramatic improvement in the quality of life tives, indicating that the ester group at C-3 is essential for the potent and life-expectancy of those affected. anti-HIV activity (Kashiwada et al., 2000). Although the HIV replication can now be managed using drugs, Due to impressive anti-HIV activity of 3-O-(3,3-dimethylsuccinyl) modern forms of therapy do not offer the possibility of fully curing HIV derivatives of BA, researchers examined the previously unknown me- infection. What is more, HAART subjects the HIV-infected patient to chanism of its function. Previous research demonstrated that 3-O-(3,3- various side-effects such as nausea, anxiety or insomnia (Ammassari dimethylsuccinyl) BA (PA-457, DSB, Bevirimat, BVM) inhibits replica- et al., 2001). In order to combat these adversities and address the tion of wild-type and drug-resistant HIV-1 by disrupting a late step in problem of drug resistance (Wensing et al., 2016), scientists must Gag processing – precisely the conversion of the capsid precursor (p25) search for new and more effective forms of therapy. to mature capsid protein (p24), which results in disruption of viral In the search of new, viable and easy accessible HIV treatment(s), maturation. Furthermore, p25 cleavage site mutations were proven to scientists have turned their attention toward natural products, biolo- be responsible for the resistance to PA-457 (Li et al., 2003). In order to gically active compounds and their derivatives. Having played a better understand the mechanism of PA-457 and potential resistance to dominant role in traditional medicine systems, pentacyclic triterpe- it, a study examined the efficacy of PA-457 on a panel of mutant viruses noids have emerged as a potential field for HIV drug discovery. Based with point deletions in the CA-SPA1 cleavage domain. Amino acid re- on the promising pharmacological properties, such as cytotoxicity, sidues in the N-terminal half of SP1 were deemed detrimental for PA- antimicrobial and anti-inflammatory effects, triterpene acids became 457 activity. It further supported the thesis that the CA-SP1 cleavage is the subject of rigorous trials. Betulinic acid, along with dihy- inhibited by PA-457 (Li et al., 2006). The properties of bevirimat were drobetulinic acid demonstrated anti-HIV effect in H9 lymphocyte cells later studied in dose-finding trials that aimed to assess its safety. Mul- and exhibited IC50 value of 1.4 μM(Fujioka et al., 1994). The study tiple short-term pharmacokinetic studies in healthy volunteers con- determined that C-17 carboxylic acid group, as well as C-19 sub- firmed that bevirimat was well tolerated (Martin et al., 2007a; Martin stituents enhanced the anti-HIV activity (Fujioka et al., 1994). In an et al., 2007b). Maximal viral load reduction (VLR) of 0.72 log10, was effort to augment the anti-HIV properties, chemists synthesized position observed at 150 and 250 mg doses. No treatment-related adverse ef- C-3- and C-28-modified derivatives of this compound (Hashimoto et al., fects, toxicities or significant changes in CD4+ T cells were reported 1997; Kashiwada et al., 1996). While the modifications resulted in an (Martin et al., 2007b). An in vitro study of PA-457 proved that it is increase of antiviral activity, two distinct potential mechanisms of ac- effective against both wild-type and drug-resistant HIV-1 isolates. PA- tion were revealed. In cell cultures, BA derivatives that were modified 457 also exhibited synergy or additivity when combined with approved at the C-28 position seemed to inhibit HIV-1 cell entry (Mayaux et al., HIV drugs. When co-administered with ritonavir, a moderate decrease 1994). In comparison, later stages of the viral cycle, such as maturation, of bevirimat plasma concentration was observed. A combined therapy were the target of C3-altered derivatives (Kanamoto et al., 2001). In the of atazanavir and bevirimat resulted in minimal pharmacokinetic dif- study by Mayaux et al. the anti-HIV potency of amide derivatives of BA ferences (Martin et al., 2008). was investigated. An 11-aminoundecanoic lateral chain compound The second phase clinical trials demonstrated that a significant (RP70034) was found to be 10-fold more potent against HIV-1. This portion of patients suffering from HIV-1 do not respond accordingly to discovery led to the synthesis of RPR103611, with IC50 values within bevirimat treatment, which ultimately led to the discontinuation of the the range of 40 and 100 nM against HIV-1 (SI > 100) but no activity studies (Wainberg and Albert, 2010). It was reported that CA-SP1 against HIV-2. The compound did not affect viral binding to CD4+ T cleavage site point mutations, naturally occurring polymorphisms at cells, rather inhibited syncytium formation between HIV-1-infected and positions 6, 7, and 8 in Gag spacer peptide 1, were associated with uninfected CD4 cells. The researchers speculated that RPR103611 various level of susceptibility to bevirimat (Van Baelen et al., 2009). blocks the post-binding step of viral infection by targeting HIV-1 en- Among others, SP1-V7A mutation was responsible for high-level BVM velope gp120/gp41 (Mayaux et al., 1994). The observation was later resistance, whereas SP1-V7M and T8Δ mutations were accountable for confirmed as HIV-1 strains carrying mutations in gp41 envelope gly- intermediate levels of BVM resistance (Adamson et al., 2010). Poly- coprotein were resistant to RPR103611 (Labrosse et al., 1997). morphic variation at V370 was identified as crucial to maintain anti- In order to reinforce the success of PRP103611, its novel isomer; HIV activity (Dang et al., 2013). A study by Verheyen et al. reported IC9564 was synthesized. Among the derivatives, the two most pro- that 30% of patients were affected by HIV strains with at least one mising ones exhibited EC50 values of 0.40 and 0.33 μM. The results of mutation associated with reduced susceptibility to bevirimat (H358Y, the trial studies suggested that both of the newly acquired compounds L363 M, Q369H, V370A/M/del and T371del), whereas in HIV isolates were fusion inhibitors (Sun et al., 2002). It is important to note that in conjunction with protease inhibitor resistance, the incidence of HIV-1 gp120 is crucial for the anti-HIV-1 activity of IC9564, as gp120 bevirimat resistance mutations reached 45% (Verheyen et al., 2010). mutations altered its anti-HIV function (Holz-Smith et al., 2001). In an In order to overcome the adversities, the potential ways of enhan- attempt to further enhance the anti-HIV activity, researchers synthe- cing anti-HIV activity were investigated. The C-3 benzoic acid-modified sized ω-undecanoic acids and amides by modifying the C17-COOH and triterpenoids were altered at the C-28 position of the triterpenoid core. C30-methyl groups of BA. Most of the modifications on the triterpene While two compounds (dimethylaminoethyl amides) improved the ac- skeleton resulted in either diminished or complete loss of anti-HIV activity against resistant strains of HIV-1, as shown by an increase in tivity. The 3b-hydroxyl functionality and C-28 amide were found as plasma concentration measured 24 h after dosing, their insufficient ef- detrimental to the properties of the compound and any deviations led to ficacy did not allow them to progress to a more advanced stage of a reduction in activity (Evers et al., 1996; Soler et al., 1996). In another preclinical trials (Swidorski et al., 2016). study the effects of modification on the C-3 hydroxyl group in betulinic Building up on those findings, a second-generation of HIV-1 ma- and dihydro betulinic acids was investigated. The 3-O-(3,3-di- turation inhibitors was synthesized. Through a process of optimization methylsuccinyl) derivative displayed impressive anti-HIV-1 activity by using a panel of engineered viruses containing susceptibility-redu- −4 with IC50 value of 3.5 × 10 μM. Other derivatives, such as the 3,28- cing polymorphic changes in Gag, researchers identified BMS-955176 O-di-3,3-dimethylglutaryl ester of betulin (BE) or 3-O-(3,3-di- (Nowicka-Sans et al., 2016; Regueiro-Ren et al., 2016). During its de- methylsuccinyl) derivative of oleanolic acid exhibited IC50 values of velopment, in addition to maintaining activity against key polymorphic − − 6.6 × 10 4 μM and 8.6 × 10 4 μM, respectively (Kashiwada et al., variations, such as V370A, a strong emphasis was placed on decreasing

12 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409 the binding to human serum components in order to increase the po- exhibited strong antiviral properties – 98% cytopathic effect reduction tency (Nowicka-Sans et al., 2016). BMS-955176 presented median EC50 at the concentration of 50 μM and 30% at 10 μM and was not toxic to of 21 nM toward a library of subtype B clinical isolates. An in vitro A549 cells. In an in vivo model, intraperitoneal administration of BA measurement revealed 86% human serum binding of BMS-955176 and oseltamivir daily for 7 days after influenza virus infection resulted (Regueiro-Ren et al., 2016). in neither body weight loss restoration nor inhibition of viral replica- Two parallel phase IIb studies – Strategy-confirming Study of BMS- tion. However, the therapy was effective in reducing the inflammation 955176 to Treat HIV-1 Infected Treatment-experienced Adults and lung tissue destruction caused by influenza virus (Hong et al., (NCT02386098) and Dose-finding Study of BMS-955176 to treat HIV-1 2015). Another study on the potential anti-influenza activity of penta- Infected Treatment-naive Adults (NCT02415595) aimed to find a safe cyclic triterpenoids showed that an echinocystic acid-galactose con- and tolerable dose of BMS-955176 and evaluate its clinical efficacy in jugate and its analog were effective entry inhibitors against influenza combination with atazanavir and dolutegravir. However, the studies A/WSN/33 virus (EC50 = 5.0 and 14.2 μM). Based on the positive re- had to be terminated early due to gastrointestinal intolerability and sults acquired in the previous studies and the knowledge of in vitro treatment-emergent resistance. antiviral activity of vitamin C, they synthesized L-ascorbic acid-con- A new class of betulin-derived α-keto amide, GSK8999, was iden- jugated pentacyclic triterpene derivatives and evaluated their anti-in- tified as HIV-1 maturation inhibitor. Through lead optimization it was fluenza activity. The most considerable antiviral properties were dis- identified with IC50 values of 17, 23, 25 and 8 nM for wild type, Q369H, played by 2,3-O,O-dibenzyl-6-deoxy-L-ascorbic acid-betulinic acid V370A and T371A, respectively. It was potent against 57 of 62 isolates conjugate (compound 30, EC50 of 8.7 μM) with no toxicity against un- demonstrating an improvement against gag polymorphisms with the infected cells. The 2,3-O,O-dibenzyl substitution of L-ascorbic acid was mechanism of inhibition of the proteolytic cleavage of CA-SP1 (Tang found to be a key to antiviral properties, as all synthetic analogs with et al., 2017). this structural feature expressed high activity (Wang et al., 2016b). The compound 30 inhibited the hemagglutination of chicken red blood cells 3.9.2. The activity of betulin and its derivatives against common pathogenic by influenza virus, by occupying the binding pocket of hemagglutinin viruses for sialic acid receptor (Yu et al., 2014). The results of the 2016 study Although majority of studies on antiviral properties of BA, along confirmed that triterpenes act in the early stage of influenza virus re- with its derivatives and various other triterpenes are focused on anti- plication cycle – entry and/or early replication, without affecting HIV properties, multiple trials have been carried out to assess their genome replication (translation or virion assembly) (Xiao et al., 2016). effectiveness against a panel of other viruses such as herpes simplex Follow-up studies on synthetic compounds that could block influenza virus (HSV), human papilloma virus (HPV), influenza virus and re- virus entry by interrupting hemagglutinin binding resulted in the de- spiratory syncytial virus (RSV). velopment of BA conjugates. Pentacyclic triterpene-functionalized per- In this regard, in vitro anti-HPV activity of derivatives of BE and O-methylated CD conjugates, synthesized by Tian et al presented pro- ursolic acid were assessed using modified triterpenoids against human minent anti-influenza activity, with maximum IC50 of 4.7 μM and papilloma virus type 11. For most of them, selectivity index ranged SI > 15 (Tian et al., 2017). Another research investigated the anti- from 10 (some cellular toxicity) to 35 (no cellular toxicity) (Kazakova influenza activity of the extract from the bark of Alnus japonica (Betu- et al., 2010). The highest activity was presented by 3β,28-di-O-nicoti- laceae family). One compound exerted strong effect against KBNP-0028 noylbetulin (SI = 35), a compound with previously described hepato- which was identified as betulinic aldehyde (EC50 = 12.5 μg/ml; protective, anti-ulcer and anti-HIV activity (Flekhter et al., 2002). SI = 1.9). Betulinic aldehyde, however, was toxic to chicken embryo Furthermore, 3β,28-dihydroxy-29-norlup-20(30)-yne demonstrated fibroblast cells, which significantly limited its potential therapeutic anti-HCV activity (EC50 = 1.32 μM; SI50 = 9.4) with little toxicity. value (Tung et al., 2010). While most of the compounds were inactive against respiratory viruses, An in vitro evaluation of antiviral activity of position C-3- and C-28- 28-O-methoxycynnamoylbetulin displayed anti-influenza properties modified derivatives of B and BA revealed high efficacy against viruses

(EC50 =2μM; SI > 100) (Kazakova et al., 2010). Betulin also ex- like inﬂuenza A and herpes simplex virus type 1, when tested in cell hibited anti-HSV-1 and anti-HSV-2 activity in vitro. Fifty percent ef- cultures with inﬂuenza A/FPV/Rostock/34 (H7N1) and HSV-1 (strain fective concentrations reached the values of 0.40 and 4.15 μg/ml, for 1C) viruses. The 3-oxime of BE showed remarkable activity against

HSV-1 and 2, respectively. A synergistic eﬀect between BE and acy- inﬂuenza A virus (EC50 = 2.2 μM; MNTC/EC50 = 392.2). The 28-N′- clovir was also evaluated. For HSV-1, in higher concentrations alkylureides and amides presented strong anti-HSV-1 activity, with

(> 0.068 μg/ml for acyclovir and > 0.4 μg/ml for BE), the duo ex- EC50 in the range of 0.2 to 2.2. An observation was made that the inhibited strong or moderate antiviral effects, while in lower concentra- troduction of CONH functional group resulted in a gain of anti-HIV tions an additive effect was observed. For HSV-2, however, the sy- activity. Similarly, C-28 substitution by CONH2-group enhanced anti- nergistic effect was only present at higher concentrations of the influenza activity and the presence of NHCONH group resulted in po- compounds (0.45 μg/ml acyclovir plus 8.4 μg/ml of BE) (Gong et al., tent anti-HSV-1 activity. Worth noting was the fact that both B and BA 2004). The mechanism of action remained unclear, although previous were ineffective against influenza A virus and expressed low activity trials suggested the disruption of HSV replication cycle. In another against HSV (Baltina et al., 2003). study, investigators synthetized nitrogen-containing derivatives of be- A recent study modified few compounds including B and BA at C-3, tulinic and betulonic acids and assessed in vitro antiviral activity of the C-20 and C-28 of the lupane core in several derivatives and tested their modified compounds against a panel of viruses – influenza A virus, in vitro anti-influenza activity against type A (H1N1, H3N2 and H5N1) HSV-1, enteric cytopathic human orphan (ECHO)6 and HIV-1. Betu- viruses. Betulonic acid 2-aminopyridinylamide was the most active lonic acid 3-oxime demonstrated the highest antiviral activity against against H1N1, H3N2 and H5N1 with SI > 20, 1.8 and 5.3, respec- influenza A virus (EC50 = 2.17 μM; MTC/EC50 = 392.16). The study tively. The betulonic acid methoxy- and nitro-substituted benzalhy- revealed that four compounds could inhibit the replication of HSV-1. drazides showed selectivity only against H5N1, while lupeol and 3- Derivatives of BE exerted low anti-ECHO6 activity, with the most active oximino-betulonic acid benzalhydrazides were active against three compounds presented in Table 4. One compound, 3β-Hydroxylup- types of Flu A. They showed that triterpenoids with a long-chain sub- 20(29)-en-28-oic acid hydrazide, showed pronounced anti-HIV activity stituent at C-17 containing amide or hydrazide bonds with aromatic or

(EC50 = 0.23 μM; MTC/EC50 = 20) (Flekhter et al., 2003). heterocyclic fragments exert positive influence on the activity Anti-influenza properties of BA, isolated from Jujube tree, were (Smirnova and Kazakova, 2018). evaluated on influenza A/PR/8 virus-infected A549 human lung ade- In order to improve the biological activity of BA, Visalli et al. syn- nocarcinoma epithelial cell line and C57BL/6 mice. Betulinic acid thesized its ionic derivatives. Although the derivatives exhibited human

13 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

Table 4

Summarizing the properties of BA and its derivatives against common pathogenic viruses, with respect to their antiviral attributes, such as EC50,IC50 and SI values.

Reference Virus Compound Antiviral attribute

Flekhter et al. (2003) Inﬂuenza A virus Betulonic acid 3-oxime EC50 = 2.17 μg/ml

Betulinic acid 3-oxime hydrazide EC50 = 2.04 μg/ml

HSV-1 3-ketobetulinic (betulonic) acid EC50 = 2.50 μg/ml

3b-Hydroxylup-20(29)-en-28-oic acid hydrazide EC50 = 9.96 μg/ml

N′-(4-Chlorobenzal)hydrazide of 3b-hydroxylup-20(29)-en-28-oic acid EC50 = 5.16 μg/ml

N′-(Benzal)hydrazide of lup-20(29)-en-28-oic acid 3-oxime EC50 = 7.70 μg/ml

ECHO6 3-ketobetulinic (betulonic) acid EC50 = 73.32 μg/ml

Betulonic acid 3-oxime EC50 = 4.19 μg/ml

N′-(4-Chlorobenzal)hydrazide of 3β-hydroxylup-20(29)-en-28-oic acid EC50 = 50.73 μg/ml

HIV 3β-Hydroxylup-20(29)-en-28-oic acid hydrazide EC50 = 0.23 μM

Baltina et al. (2003) Inﬂuenza A FPV/Rostock/34 (H7N1) Betulonic acid 3-oxime EC50 = 2.2 μM

Amide derivative of betulonic acid chloride EC50 = 0.7 μM

HSV-1 28-N′- alkylureide derivatives of betulonic acid isocyanates EC50 = 0.2 μM

strain 1C EC50 = 2.2 μM

EC50 = 0.6 μM

EC50 = 1.4 μM

Gong et al. (2004) HSV-1 Betulin EC50 = 0.40 μg/ml

HSV-2 Betulin EC50 = 4.15 μg/ml Kazakova et al. (2010) HPV-11 3β,28-di-O-nicotinoylbetulin SI = 35

EC50 = n/d

HCV 3β,28-Dihydroxy-29-norlup-20(30)-yne SI50 = 9.4

EC50 = 1.32 Inﬂuenza A virus (H1N1) 28-O-methoxycynnamoylbetulin SI > 100

EC50 =2

Tung et al. (2010) Inﬂuenza KBNP-0028 Betulinic aldehyde EC50 = 12.5 SI = 1.9

Heidary et al. (2014) HSV-1 Betulin IC50 = 0.32 μg/ml

Betulinic acid IC50 = 0.32 μg/ml

Hong et al. (2015) Inﬂuenza A/PR/8 Betulinic acid EC50 = n/d

Visalli et al. (2015) HSV-2 Betulinic acid IC50 = 1.6 μM

Cholinium salt of betulinic acid-glycine IC50 = 0.6 μM

Cholinium betulinate IC50 = 0.9 μM Álvarez et al. (2015) HSV Four lupene-related pentacyclic triterpenes SI = 7.7–201.9

Inﬂuenza A/WSN/33 2,3-O,O-dibenzyl-6-deoxy-L-ascorbic acid-betulinic acid conjugate EC50 = 8.7 μM

Tian et al. (2017) Inﬂuenza virus Pentacyclic triterpene-functionalized per-O-methylated CD conjugates IC50 = 4.7 μM SI > 15 Smirnova and Kazakova (2018) H1N1 Betulonic acid 2-aminopyridinylamide SI > 20 H3N2 SI > 1.8 H5N1 SI > 5.3 H5N1 Betulonic acid methoxy- and nitro-substituted Benzalhydrazides – H1N1, H3N2 and H5N1 Lupeol and 3-oximino-betulonic acid benzalhydrazides –

Karagöz et al. (2019) HCMV Artesunic acid-betulin hybrid 11 EC50 = 0.88 μM

Artesunic acid-betulinic acid hybrid 12 EC50 = 0.24 μM serum albumin binding properties similar to those of modern anti-HIV antioxidant or anti-inflammatory properties, but also act as a hepato- drugs, they did not inhibit HIV reverse transcriptase – only a slight protective agents (Szuster-Ciesielska and Kandefer-Szerszeń, 2005). inhibition was noted for one of the agents. Three compounds inhibited Another study showed that BE (extracted from Birch bark) decreases HSV-2 replication on a level comparable to acyclovir, while main- the antigenic and infective activity of HCV in vitro and in vivo (Nosik taining minimal cellular toxicity. The IC50 values were 1.6 μM for BA, et al., 2005). Hepatitis C virus infection induces COX-2 over-expression 0.6 μM for cholinium salt of betulinic acid-glycine and 0.9 μM for through the NF-κB signalling pathway, resulting in inflammation and cholinium betulinate (Visalli et al., 2015). The underlying mechanism proliferation of hepatic cells (Waris and Siddiqui, 2005). COX-2 is in- of anti-HSV-2 activity was not determined in the study. In order to volved in the conversion of arachidonic acid to prostaglandins (PG), discover the molecular mechanism of anti-HSV activity, another re- whose function is to induce the cellular proliferation and angiogenesis. search team extracted triterpenes from birch bark. B, lupeol and BA all Studies have demonstrated that the level of COX-2 is elevated among displayed antiviral activity with IC50 ranging from 0.2 to 0.5 μg/ml HCV-infected patients (Waris and Siddiqui, 2005). Abnormal COX-2 concentrations. While the addition of these agents to HSV-infected cells expression has been suggested to be highly associated with carcino- had little to no effect on virus replication, exposing viruses to birch bark genesis (Sobolewski et al., 2010). It has been proposed that inhibition of extract significantly affected virus multiplication, both in acyclovir- COX-2 expression suppresses HCV replication (Chen et al., 2013; Lin sensitive and acyclovir-resistant strains (Heidary Navid et al., 2014). et al., 2015a). Data implies that a drug, which interrupts COX-2 sig- The results of the study suggest that the antiviral activity of pentacyclic nalling pathway could be potentially used for HCV treatment with a triterpenes is mediated by direct drug-virus interactions. Further, an- preventive role in HCV-related diseases (Chen et al., 2013). A study by other study investigated the potential for cyanoethylation of ring A of Lin et al. investigated the effect of BA, extracted from Avicenna marina, triterpenoids and their A-seco derivatives prepared from allobetulone on the replication of HCV and the mechanism of action of BA-mediated and methyl betulonate. The inhibitory activity of triterpenoids with a anti-HCV activity in human hepatoma cells. Betulinic acid significantly cyanoethyl moiety on C-1, C-2, and C-3 and derivatives with two cya- reduced HCV RNA levels with no cytotoxic effects. The study further noethyl groups on ring A was studied against HSV-1 in vitro (Galaiko revealed a synergistic effect of BA against HCV replication in combi- et al., 2018). nation with either IFN-α, the NS3/4A protease inhibitor telaprevir, or Previous studies have demonstrated that BE and BA not only exert the NS5B polymerase inhibitor sofosbuvir (Lin et al., 2015a). A trial

14 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409 which enrolled 42 patients with serologically confirmed chronic HCV would further amplify the problem. MDR bacteria are responsible for took a closer look at the potential hepatoprotective effects of BE and BA both health-care and community-associated infections. Some of the in humans. Following 12 weeks of birch bark extract (containing 75% B most common MDR bacteria include: Methicillin-resistant Staphylo- and 3.5% BA) treatment, significant differences in alanine transaminase coccus aureus (MRSA), vancomycin-resistant Enterococci (VRE), multi- (ALT) and HCV RNA levels were observed. In addition, fatigue, ab- drug resistant Pseudomonas aeruginosa (MDRPA) and many En- dominal discomfort and dyspepsia were significantly reduced in parti- terobacteriaceae e.g. carbapenem-resistant Klebsiella pneumoniae (CRKP) cipants (Shikov et al., 2011). Despite positive results of the trial, no and extended spectrum β-lactamases (ESBL) producing Escherichia coli. long-reaching clinically relevant conclusions could be made, as there MRSA is the most prevalent trigger of community-associated MDR was no control group. bacterial infection, but in the next 10 years, resistant to almost every β- Betulinic acid extracted from Pulsatilla chinensis (Ranunculaceae fa- lactam antibiotic by E. coli will possibly become an even bigger threat mily) was found to inhibit the replication of hepatitis B virus (HBV) in to the public health (van Duin and Paterson, 2016). transgenic mice by down-regulating the expression of manganese su- There are plenty of common antibacterial mechanisms used by an- peroxide dismutase (SOD2), in turn causing ROS generation and mi- tibiotics. The most frequently used mechanisms are blocking the cell tochondrial dysfunction (Yao et al., 2009). The study showed that BA- wall formation (β-lactam antibiotics) and inhibiting proteins (ami- induced dephosphorylation of cAMP-response element-binding (CREB) noglycosides) or DNAs (quinolone antibiotics) biosynthesis. The studies protein at Ser133, resulted in repression of SOD2 expression. Inhibition suggest that B and its derivatives may inhibit bacterial growth by dis- of HBV replication by BA was blocked by SOD2 overexpression. The turbing more than one metabolic pathway. The cell membrane plays SOD2 knockdown validated the inhibition, pointing to modulation of multiple functions, like separating inside area from the outside en- the balance of mitochondrial redox as the underlying mechanism of vironment, receiving stimuli and enabling the exchange of necessary action. The suppressed SOD2 expression with subsequent ROS genera- substances for survival. Targeting cell membrane may disturb these tion followed by a decline in HBV replication suggests that BA is a important activities. As, cytoplasmic membrane is composed of phos- potent anti-HBV candidate (Yao et al., 2009). pholipid bilayer, transport channels, integral and surface proteins, li- A series of novel triazoles were prepared by the 1,3-dipolar cy- pophilic compounds like pentacyclic triterpenoid may disrupt mem- cloaddition reaction between the alkyne derivatives of BE and organic brane stability and stop cell growth (Chung et al., 2011; Cowan, 1999; azides. The compounds showed inhibitory effect on enteric cytopathic Kuete et al., 2007). This mechanism leads to the lysis of bacterial cell bovine orphan (ECBO) virus and human adenovirus C serotype 5 (Shah et al., 2004). (HAdV-5) added after the adsorption period. The transformation of Targeting the cell membrane is not the only proposed antibacterial acetylenic moiety to triazole ring was necessary to demonstrate anti- mechanism of B and its derivatives. Another hypothesis is the inhibition viral activity. The most active compound was betulin 6 h, which con- of DNA and macromolecular synthesis in Gram-positive bacteria tained a 3-hydroxypropyl substituted 1,2,3-triazole ring (Bębenek et al., (Chung et al., 2014; Chung et al., 2011). Possibly BA affects ABC 2017). transporters, ribosomes (Chung et al., 2014) and host defense me- For hRSV infection, the only promising drug is ribavirin, which is chanism by restoring antioxidants (Lingaraju et al., 2015). The simila- used in severe cases. The triterpenes and their derivatives are promising rities between triterpenoids and other compounds with supposed anti- alternatives in this regard. A study reported the effects of BE, BA and bacterial activity mechanism lead to a hypothesis that triterpenoids like ursolic acid (UA) against A549 cells infected by hRSV. The triterpene di-terpenes can inhibit bacterial efflux pumps (Rao et al., 2018). The esterification at the hydroxyl group resulted in 17 derivatives. The 3,28- lack of data makes it difficult to point an exact antibacterial mechanism di-O-acetylbetulin derivative showed the best results for cell viability for B, but it is possible to predict which part of B compound may be and a suitable alternative for a new compound against hRSV (Santos da responsible for its antibacterial activity. For example, a B derivative Silva et al., 2018). with lack of hydroxyl group in position 3 did not exert antimicrobial A series of B/BE-based dimer/hybrid compounds carrying ferrocene activity against E. coli and S. aureus (Silva et al., 2012). and/or artesunic acid moieties were designed and synthesized de novo and analyzed in vitro against human cytomegalovirus (HCMV). From 3.10.1. Betulin and its derivatives against common MDM bacteria this series, the BA/BE and artesunic acid-betulin hybrid 11 and arte- Toxins like heat-labile enterotoxin (LT) produced by enterotoxigenic sunic acid-betulinic acid hybrid 12 showed the most potent activities E. coli (ETEC) and cholera toxin (CT) are responsible for the cholera-like against HCMV with low EC50 values of 0.88 and 0.24 μM, respectively. diarrhea, which is able to connect to monosialotetrahexosylganglioside These compounds are suggested on the basis of putatively complex (GM1) in the intestine. This leads to continuous stimulation of adeny- modes of antimicrobial action (Karagöz et al., 2019). late cyclase, elevation of cyclic adenosine monophosphate (cAMP) and then electrolyte and water loss. In this regard, Chen et al., aimed to 3.10. Antibacterial mechanisms of betulin and its derivatives verify Chaenomeles speciosa (Rosaceae family) therapeutic attributes which contains BA, for the treatment of diarrhea. There are two me- The first substances which were used to fight against bacteria were chanisms of stopping diarrhea by either minimizing the interaction produced by molds i.e. Penicilium notatum. Over the years, people have between the toxin and its receptor or killing the microorganisms which looked for the antibiotics among products of bacteria metabolism. produce the toxin. Betulinic acid was shown to block LT binding to GM1 Currently, more and more compounds are produced or processed in and prevent the effects of LT-GM1 connection, which may stop diar- laboratories. Nowadays, despite exploring all the aforementioned rhea. Carboxylic groups of triterpenoids were predicted to be the ele- sources, the problem of multi drug resistance (MDR) is growing and ments responsible for this binding. This data suggested that BA may be people may find the solution in the territory of plants. Infectious dis- useful for diarrhea treatment (Chen et al., 2007). eases caused by MDR bacteria are becoming an increasingly major In another study, BA isolated from Dendrobenthamia capitata problem. According to the Infectious Diseases Society of America (Cornaceae family) presented anti-MRSA activity, with MIC of 62.5 mg/ (IDSA) “they are one of the greatest threats to human health world- ml (Table 5)(Zuo et al., 2008). Further, results of another study found wide” (Spellberg et al., 2011). The MDR bacterial infections are much that pentacyclic triterpenoids are active against MRSA, but their MICs worse than antibiotic susceptible ones (Bodi et al., 2001; Vardakas were higher than vancomycin. They verified the synergistic effects of et al., 2013). One of the ways to fight against these bacteria is to treat combinations of α-amyrin, BA, betulinaldehyde, methicilin and van- patients with novel antibiotics or combinations of antibiotics from comycin. The fractional inhibitory concentration (FIC) ≤ 0.5, which different categories. Unfortunately, this strategy could lead to surge of proves the synergistic effects of combinations, was found for combi- resistance even against so called “antibiotics of last resort”, which nations of: α-amyrin-BA, BA-methicilin and betulinaldehyde-

15 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

Table 5 B, BA and its derivatives antibacterial activity according to the recent studies (MIC).

Reference S. aureus E. coli E. aerogenes K. pneumoniae P. aeruginosa Enterococcus spp. S. pneumoniae B. subtilis

Chandramu et al. (2003) Inactive Less active Woldemichael et al. (2003) > 128 μg/ml > 128 μg/ml > 128 μg/ml

Chen et al. (2007) IC50 = 0.5 mM Horiuchi et al. (2007) > 128 μg/ml > 128 μg/ml > 128 μg/ml Zuo et al. (2008) 62.5 mg/ml Fontanay et al. (2008) > 256 μg/ml > 256 μg/ml > 256 μg/ml > 256 μg/ml Kuete et al. (2011) > 256 μg/ml > 256 μg/ml > 256 μg/ml > 256 μg/ml Wang et al. (2016a) > 128 μg/ml > 128 μg/ml > 128 μg/ml > 128 μg/ml Oloyede et al. (2017) 64–256 μg/ml 256–1024 μg/ml 256–512 μg/ml Bębenek et al. (2017) 1.95 μg/ml 0.95 μg/ml Kamtcha et al. (2018) >10μg/ml > 10 μg/ml > 10 μg/ml > 10 μg/ml Rajkumari et al. (2018) 250 μg/ml

Table 6 Common drugs and betulin derivatives activity against M. tuberculosis (MIC).

References BA epi-BA Estriﬁcated in position 3 with cinnamic Betulinaldehyde A-azepano-28- Kanamycin Rifampicin acid p-coumaroyl analogue BA cinnamoyloxybetulin

Wachter et al. (1999) 32 μM64μM Tanachatchairatana et al. 50 μg/ml 6.25 μg/ml (2008) Suksamrarn et al. (2006) 25 μg/ml 25 μg/ml 2.5 μg/ml 0.004 μg/ml Theerachayanan et al. (2007) 25 μg/ml Fomogne-Fodjo et al. (2017) 15 μg/ml Medvedeva et al. (2018) 2 mM against MTB H37Rv. 1–4 mM against SDR strains

BA: betulinic acid. methicilin. Such combinations could be considered as promising op- extracted B from the stem bark of Salacia staudtiana (Celastraceae fa- tions for MDR bacterial infections treatment (Chung et al., 2011). mily) along with some other compounds. The fraction SB3-1b contained Some other studies presented different results (Table 6). It was B and showed in vitro antibacterial activities against a panel of standard proven that BA inhibits the growth of Bacillus subtilis, but this effect was Gram-positive and Gram-negative pathogenic strains of bacteria with not significant in comparison with currently used drugs. No inhibition MIC values ˃ 10 (Table 7)(Kamtcha et al., 2018). was found by BA against E. coli growth (Chandramu et al., 2003). Re- Despite intensive antibiotic therapies, P. aeruginosa is still an im- searchers tested BA extracted from Caesalpinia paraguariensis (Fabaceae portant cause of nosocomial infections and mortality in Cystic Fibrosis family) against several bacteria: B. subtilis, methicillin-sensitive and patients. The Quorum sensing (QS) system, a cell-density-dependent -resistant S. aureus, and E. coli. Betulinic acid was found to be inactive phenomenon, is closely related to P. aeruginosa pathogenicity. BE and against the tested organisms with MICs > 128 μg/ml (Woldemichael BA exhibited significant attenuation in production of QS-regulated et al., 2003). Another study showed lack of antibacterial activity virulence factors and biofilm formation in P. aeruginosa, at the sub-MIC (MIC > 128 μg/ml) of BA and BE against MRSA, VRE and Streptococcus concentration (125 μg/ml). In addition, molecular docking analysis pneumoniae (Horiuchi et al., 2007). In an extensive study verifying the revealed that BE and BA can act as a strong competitive inhibitor for QS hypothesis of antibacterial activity of BA, the substance was found in- receptors; LasR and RhlR. The findings suggested that BE and BA can active (MIC ≥256 μg/ml) against E. coli, S. aureus, Enterococcus faecalis, serve as potential anti-infectivity against P. aeruginosa (Rajkumari et al., Enterococcus faecium (vanB gene), P. aeruginosa. The authors tried to 2018). explain this lack of activity as a result of similarity of the BA to the compounds naturally present in the bacterial membrane (Fontanay 3.10.2. Betulin and its derivatives against helicobacter pylori et al., 2008). Another research did not find any antibacterial activity of H. pylori is able to survive in the stomach owing to different me- BA either. The MICs against E. coli, Enterobacter aerogenes, K. pneumo- chanisms like production of urease, an enzyme that catalyzes the hy- μ niae and P. aeruginosa were higher than 256 g/ml (Kuete et al., 2011). drolysis of urea into CO2 and NH3. The reaction raises the pH in the Six pentacyclic triterpenoids were isolated from the leaves of Alstonia stomach and makes the environment suitable for H. pylori survival scholaris (Apocynaceae family), an important medicinal plant in Chinese (Kusters et al., 2006). Eradication of this microbe is a long-term process, medicine. BA and B with MIC values > 128 against different bacteria which requires polytherapy composed of antibiotics and proton pump were included. The ability of pentacyclic triterpenoid to enhance the inhibitors. The problem of drug resistance in H. pylori population en- activity of β-lactams could constitute a valuable group of therapeutic couraged researchers to look for new anti-ulcer substances among agents in the future (Wang et al., 2016a). natural compounds (Chen et al., 2007). Life-threatening gastric diseases Contribution of oxidative stress in the bacterial lethality activities of are a serious threat to public health in Asia. Natural recipes coming B and BA were investigated against bacteria. The MICs of B and BA from this continent may become a new way of fighting H. pylori and its against E. coli, P. aeruginosa and S. aureus were obtained 1024 and associated illnesses. 256 μg/ml; 512 and 256 μg/ml; 256 and 256 μg/ml, respectively Betulinic acid found in the extract of Forsynthia suspense (Oleaceae (Oloyede et al., 2017). A series of novel triazoles were prepared by the family) decreased the urease activity by 43.5% in the concentration of 1,3-dipolar cycloaddition reaction between the alkyne derivatives of B 10 mg/ml (Shin et al., 2009). Betulinic acid would be able to stop the and organic azides. The MIC of triterpene derivative (betulin 5e) growth and reproduction of H. pylori. The mechanism of anti-urease against E. coli and K. pneumonia were obtained at 1.95 and 0.95 μg/ml, activity is probably due to the binding between carbonyl oxygen of BA respectively (Bębenek et al., 2017). In another study, researchers and nickel atom in the active site of the enzyme, which makes the

16 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

enzyme ineﬃcient (Shin et al., 2009).

M 3.10.3. Betulin and its derivatives against Mycobacterium tuberculosis μ The International Cooperative Biodiversity Group (ICBG) project “Bioactive Agents from Dryland Biodiversity of Latin America” col- lected extracts from numerous plants in South America and Mexico which contained B and its derivatives. These extracts inhibited M. tu-

M>20 berculosis growth (Timmermann, 2001; Wachter et al., 1999). It was μ discovered that B and BA antimycobacterial activity against M. tuberculosis H37Rv (ATCC 27294) was only slightly inﬂuenced by the presence of hydroxyl group in the position 3, either in β position [BA, MIC = 32 μM] or α position [epi-BA, MIC = 64 μM] (Wachter et al., M>20 μ 1999). The esteriﬁed compound in position 3 with cinnamic acid p-

11.8 coumaroyl analogue BA had even better activity (MIC = 6.25 μg/ml) against M. tuberculosis H37Ra compared to BA – (MIC = 50 μg/ml) (Tanachatchairatana et al., 2008). The B derivatives were compared to well-known antibiotics regarding their anti-mycobacterial activity. The outcome was highlighted in Table 6. It was found that betulinaldehyde and BA presented antimycobacterial activity, but their MICs (both of them 25 μg/ml) were 10

g/ml μ

μ times higher than kanamycin MIC (2.5 g/ml) and 6250 times higher

3.7 than rifampicin MIC (4 ng/ml) (Suksamrarn et al., 2006; Theerachayanan et al., 2007). Among several compounds isolated from the extract of Tetracera potatoria (Dilleniaceae family), BA and BE were identiﬁed as promising anti-mycobacterial agents against M. smegmatis and M. aurum (MIC 15 μg/ml), which are used as model microorgan- g/ml

μ isms for more pathogenic strain M. tuberculosis.(Fomogne-Fodjo et al.,

3.0 2017). In a recent study, the synthesis and modification of the in vitro antitubercular activities of lupane, oleanane and ursane based on type A- azepanes was evaluated. Modification of azepanes at NH group and atoms С-12, C-20, C-28 and C-29 of triterpenic core led to the derivatives with oxo, epoxy, aminopropyl, oximino and acyl substituents. Chemical modifications of lupane and oleanane A-azepano-28-hydroxy- derivatives didn't lead to more active compounds. The follow-up testing revealed A-azepano-28-cinnamoyloxybetulin as a leader compound -p-coumaroyl alphitolic acid Zizyberenalic acid 3-O-vanillylceanothic acid BA acetate Betulonic acid BA methyl ester

g/ml with MIC 2 mM against MTB H37Rv and MICs 4, 1 and 1 mM against E μ INH, RIF and OFX single drug resistant (SDR) strains, respectively 2-O- 0.9 (Medvedeva et al., 2018). g/ml

μ 3.11. Anti-cancer properties of betulinic acid

The occurrence of cancer and its mortality are rapidly increasing M

μ worldwide. Various reasons are involved in cancer incidence such as aging, increase in population growth, lifestyle, and alterations in the B prevalence and distribution of cancer risk factors. According to the World Health Organization (WHO) estimates in 2015, cancer is one of M>20 g/ml inactive the major causes of death before the age 70 years. It has been estimated μ μ g/ml Samvisterin 7.5 μ M

μ that 18.1 million new cancer cases would be diagnosed and 9.6 million 1 1.7 22.5 19.6 cancer deaths would occur in 2018. Lung cancer is the most commonly

). diagnosed cancer (11.6% of the total cases) in both sexes, which ac- 50 counts for 18.4% of cancer deaths. Moreover, the prevalence of female (IC breast cancer, prostate cancer, and colorectal cancer are 11.6%, 7.1%, g/ml μ and 6.1%, respectively. However, the mortality of colorectal cancer, Betulinaldehyde BA 6.25 stomach cancer, and liver cancer are 9.2%, 8.2%, and 8.2%, respectively (Bray, 2014; Bray et al., 2018). P. falciparum Natural compounds possess promising therapeutic properties and have been used for the treatment of a variety of diseases for centuries (Newman et al., 2003). In the recent decades, using natural compounds for the therapeutic approaches has gained attentions due to the in- efficacy of chemical and synthetic medications. Extensive research was conducted to examine the efficacy of therapeutic agents derived from plants against cancers, and outstanding results suggest that many natural compounds have potent anticancer effects (Sisodiya, 2013). In this regard, National Cancer Institute (NCI) conducted a systematic Silva et al. (2015) Banzouzi et al. (2015) Dominguez-Carmona et al. (2010) Suksamrarn et al. (2006) Theerachayanan et al. (2007) Steele et al. (1999) References

Table 7 BE derivatives activity against BA: betulinic acid, B: betulin. screening of 2500 plant extracts and found that BA exhibits potent

17 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409 anticancer effects (Dzubak et al., 2006; Mullauer et al., 2010; Periasamy potent anti-proliferative ability of compounds (Periasamy et al., 2014; et al., 2014). It has been reported that BA and B selectively affect a Yang et al., 2012). In recent years, BA derivatives such as imidazole wide-range of tumor cell lines both in vitro and in vivo. Interestingly, carboxylic esters (carbamates) and N-acylimidazole have been synthe- many studies showed that BA and B have no cytotoxic effects on normal sized. These new derivatives also show effective cytotoxic activity in cells such as human fibroblasts, peripheral blood lymphoblasts, mela- human Hep-G2, Jurkat, and HeLa cells. The IC50 values of some of these nocytes and astrocytes (Selzer et al., 2002; Wick et al., 1999). For ex- compounds were lower than 2 μM, which is even better than BA alone ample, BA was shown to suppress deubiquitinase activity in prostate (Qian et al., 2011; Santos et al., 2010). cancer cells, but not in corresponding normal cells, which may be as- Currently, several modifications in BA structure have been made to sociated to the overexpression of deubiquitinase in cancerous cells improve and fulfill the potency of this multifunctional compound. (Reiner et al.,2013). Indeed, BA have strong anticancer properties and Evidence indicates that substitution of ionic groups in BA is associated its interesting chemical structure makes it a unique candidate for ap- with better hydrophilicity, minimal changes in the functional and propriate modifications for carrying different effective chemicals in structural integrity, and enhanced inhibitory effects on tumor growth in order to synthesize more effective anticancer drugs (Gupta et al., 2017; many cell lines such as SH-SY5Y (neuroblastoma), MCF7 (breast ade- Saneja et al., 2017b; Willmann et al., 2009). nocarcinoma) and A375 (melanoma) (Liby et al., 2007). Furthermore, it Anticancer effects of BA was initially observed against human has been shown that 23-hydroxyl betulinic acid has inhibitory effects melanoma cells with reported IC50 of 1.5–1.6 μg/ml (Tan et al., 2003; on the proliferation of multiple types of tumor cells such as HeLa, HL-60 Zuco et al., 2002), and then its effects were reported on other cancer (human leukemia cell line), and namely on B16 cells (human mela- cell lines including neuroblastoma (IC50 =14–17 μg/ml), medullo- noma) (Liu et al., 2004). blastoma (IC50 =3–13.5 μg/ml), Ewing's sarcoma, leukemia, brain-tu- BA in comparison with B has more potent inhibitory effects on the mors, glioblastoma (IC50 =2–17 μg/ml), colon carcinoma, ovarian growth of Canine T-cell lymphoma CL-1 (23.50, 27 μM, respectively), B- cancer (IC50 = 1.8–4.5 μg/ml), lung cancer (IC50 = 1.5–4.2 μg/ml), cell lymphoma CLBL-1 (18.2, 28.9 μM, respectively) and osteosarcoma breast, prostate, head and neck squamous cellular carcinoma cells and D17 (18.59, 22.73 μM, respectively) cell lines. Further, it has been de- hepatocellular, renal and cervical cancers (IC50 = 1.8 μg/ml). Collec- monstrated that 23-hydroxy BA induces cell death in hepatocarcinoma tively, the apoptotic and cytotoxic effects of BA and its derivatives are as well as breast cancer cells in a dose-dependent manner (Zhao et al., observed following the administration of 3.8 to 16.4 μg/ml of drugs 2018). A previous research on Helicteres angustifolia (Malvaceae family) without any dependence on p53 levels (Chintharlapalli et al., 2011; Cui showed BA and oleanic acid are able to induce cell death and suppress et al., 2015; Ehrhardt et al., 2004; Fulda et al., 1999; Kessler et al., proliferation in HT-29 colorectal cancer cell line (Su et al., 2017). 2007; Kumar et al., 2018a; Mullauer et al., 2010). Furthermore, BA Recently, co-administration of BA with conventional herbal medi- showed therapeutic effects against hematological (AML) malignancies. cine has gained attention. For instance, TMP (traditional Chinese Previous research revealed that the sensitivity of murine leukemia cell medicine 2,3,5,6-tetramethylpyrazine) has been used for the treatment line (L1210) to BA is pH and time-dependent (Ehrhardt et al., 2004; of cerebrovascular and cardiovascular diseases. Co-administration of Mullauer et al., 2010). Intriguingly, BA also induced toxic effects on TMP with several derivatives of BA such as (3b-Hydroxy-lup-20(29)- isolated primary cells isolated from glioblastoma, leukemia and neu- ene-28-oic acid-3, 5, 6-trimethylpyrazin-2-methyl ester or TBA) re- roblastoma. Evidence indicates that the toxic effect of BA on malignant sulted in cell death in a variety of cancer cell lines such as Hep-G2, HT- cells is ten times more than its effect on normal cells. In addition, 29, Hela, BGC823 and A549 (Xu et al., 2017b). Similarly, co-adminis- achievable in vivo concentration of BA has the ability to stop the colony tration of BA with Ginsenoside Rh2 (extracted from Panax ginseng from formation by tumor cells. These evidence indicates the valuable char- Araliaceae family) showed promising anticancer effects in human cer- acteristic of BA to be used as a strong therapeutic agent against most of vical adenocarcinoma cell (HeLa), human lung cancer (A549) and the frequent cancer cell types (Jung et al., 2007). Interestingly BA human hepatoma (Hep-G2) cells. The latter report also showed that showed no serious adverse effects such as systemic toxicity and weight synergistic effects of combination therapy are associated with upregu- loss, when administered in high doses to animals (Chintharlapalli et al., lation of proteins relevant to apoptosis in cancer cells (An et al., 2013; 2007; Mullauer et al., 2010; Mullauer et al., 2011; Udeani et al., 1999). Li et al., 2011). Another combinational study revealed that Mangostin BA has also shown anticancer effects against melanoma and prostate enhances BA cytotoxicity against HCT 116 human colorectal carcinoma malignancies in vivo. A study by Falamas and colleagues showed BA cells (Aisha et al., 2012). In a study by Liebscher et al., the authors (100 mg/kg) increased the survival time of xenograft mouse model manipulated the structure of BA to improve its water solubility and when compared to the control groups (Falamas et al., 2018). Recently, synthesized two glucopyranoside derivatives (B10 and NVX-207). They intravenous (i.v.) injection of BA to a human adenocarcinoma xenograft also examined their effects on equine melanoma (MelDuWi and Mell- mouse resulted in tumor growth inhibition (Patlolla and Rao, 2012). Jess/HoMelZh) and human melanoma (A375) cell lines. These com- Betulinic acid has poor hydrophilic properties and its half-life is pounds, namely NVX-207, showed high cytotoxicity on both equine short in biological systems, thus researchers have synthesized more melanoma cell lines suggesting the potential therapeutic effects of BA potent derivatives to improve its therapeutic efficacy against cancer not only for human malignancies but also for animals (Liebscher et al., cells (Saneja et al., 2017b). In this regard, a recent study assessed the 2016). Another study explored the e ffects of self-assembled nano-size cytotoxic effects of a series of picolyl amides derivatives of BA in several BA fiber (SABA) on human leukemic cell lines (KG-1A and K562) and cancer cell lines such as G-361 (melanoma). It has been shown that C-3 showed that this compound has better anti-leukemia properties (25 μg/ has a key position in the structure of BA with respect to cytotoxicity ml with 24 h incubation) in comparison with BA (Dash et al., 2015a). effects (Bildziukevich et al., 2018). In addition, monoesters with Further, novel BA derivatives using nitric oxide (NO) donors (nitrates transformation in the hydroxyl group on C-28 with alkynyl group have and furoxams) have the capability of releasing NO and were evaluated the most cytotoxic effect on G-361 cells (You et al., 2003). The most for their antitumor activity. Results of the study showed that furoxan- active derivative of BA against melanoma cancer cell line G-361 is m- based derivatives release larger amounts of NO and have higher anti- substitution in (3β)-3-(4-Oxo-4- (pyridin-n-ylmethyl) amino butanoy- tumor activity than nitrate-based derivatives against Hep-G2 and B16 loxy) lup-20(29)-en-28-oic acid (Orchel et al., 2014). Antitumor activity cell lines (Liu et al., 2015b). of several synthesized BA derivatives with the manipulation of C-28 Several heterocycle-modified BA derivatives that have the linkage of position was reported in several cancer cell lines such as MGC-803 amide or ester are approximately 20 times more potent than the ori-

(IC50 = 2.3 μM), PC3 (IC50 = 4.6 μM), A375 (3.3 μM), Bcap-37 ginal BA compound (IC50 = 1.19 μM). Furthermore, the antitumor (IC50 = 3.6 μM) and A431 (IC50 = 4.3 μM). These results suggest that properties of synthetic saturated N-heterocycle derivatives of BA is 117 the manipulation of C-28 position with amino group in BA results in times stronger than BA (IC50 = 0.33 μM) on MCF-7/ADR- multidrug-

18 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

resistant tumor cell line (Cui et al., 2015). A similar approach examined (cervical) and SK-Hep-1(liver) with IC50 =0–50 μM. Evidence indicates the antitumor effects of 28 (1 N (4-fluoro phenyl)-1H-1, 2, 3-triazol-4- that the combination of B with 5-FU, temozolomide, and etoposide have yl) methyloxy betulinic ester and 2c [(3S)-3-{2-(4-(hydroxymethyl-1H- significant synergistic effects against the drug resistant RCC cells. In this 1,2,3-triazol-1-yl) acetyloxy)-lup-20(29)-en-28-oic acid] as triazole de- regard, a combination of B and etopside significantly increased the rivatives of BA modified at C-28 and C-3. Results showed C-28 deri- cleaved PARP (apoptosis-related proteins) and suppressed MDR1 (the vatives have better cytotoxic effects on HL-60, MiaPaCa-2, PC-3, A549 most ATP binding cassette transporter superfamily involved in drug and HT29 human cell lines when compared to BA (IC50 =5–7 and resistant) in RCC cells. In comparison with untreated RCC cells, B 14.9 μM) (Chakraborty et al., 2015; Khan et al., 2016). (25 μM after 24 h) significantly induced the expression of caspases Tyrosine kinase inhibitors (TKI) (including Gefitinib and Erlotinib) (−3,-7,-8 and-9), truncation of Bid and cleaved PARP in B-treated cells are considered as the first generation targeting chemo-drugs to treat in B-treated suggesting that the sensitization of cancer cells by B is a patients who have lung cancer. It has been shown that a combination of caspase dependent pathway (Yim et al., 2015). BA C30H48O3, (3ß-Hydroxy-lup-20[29]-en-28-oic acid) and TKIs pro- Oral squamous cell carcinoma (OSCC) is the most common cancer of duced promising results (Ko et al., 2018). Over half of the patients who the head and neck in the world. Prevalence of OSCC is increasing and have lung cancer are identified with the mutations in epidermal growth the 5-year survival rate is about 50%. One of the effective non-surgical factor receptor (EGFR) and after about 8 months, they show resistant to treatments for OSCC is radiotherapy, which induces apoptosis and Gefitinib and Erlotinib antitumor effects. It has been shown that a suppresses the cell cycle and progression of tumor cells. Unfortunately, combination of BA and TKIs increases cell death in HCC827 and H1975 radio-resistance is common in the OSCC cases and it is a major obstacle lung cancer cell lines by enhancing the expression of apoptotic and in the treatment of this aggressive tumor. Many signalling pathways are autophagy proteins. The combination therapy with BA not only im- involved in the radio-resistance of OSCC such as NF-κB, STAT3, and proved the efficiency of anticancer therapy, but also decreased the side- specificity factor 1 (Sp1), which its upregulation is a negative prog- effects of chemo-therapy agents (Ko et al., 2018). nostic factor for the survival of patients. It has been reported that Sp1 Photothermal therapy in combination with chemotherapy is one of decreases the expression of PTEN (Phosphatase and tension homolog the best non-invasive models for cancer therapy (Liu et al., 2018). In deleted on chromosome ten) by binding to a specific site of its promotor this context, (BA-Lips@Pd@Au NFs), which is poly-branched AuePd and consequently sensitizes tumor cells to radiotherapy (Kreppel et al., bimetallic nano-flowers coated BA in liposomes, improved the solubi- 2011). lity and bioavailability of BA and exhibited potent synergetic effects Previous studies showed that combination therapy with radiation (tumor inhibition ratio of > 90%) in chemo-photothermal therapy (8 Gy at a dose rate of 2.544 Gy/min) and BA (10 μM) significantly against Hela cells and U14 cancerous mice (Liu et al., 2018). enhances apoptosis in CAL-27 and Tca-83 cells. Betulinic acid abrogates Upregulation of ATP-binding cassette (ABC) drug transporters is a the radio-resistance in OSCC by down-regulating Sp1 and consequently key mechanism in cancer cells resistant to chemotherapy. P-glycopro- down-regulation of PTEN (Yuan et al., 2017). tein (MDR1/ABCB1) and BCRP (ABCG2) are two of ABC drug trans- Recently, researchers synthesized a novel hybrid molecule (BA-78), portes, which expel the drugs outside the cell and decrease the effective which is made of BA and Diazeniumdiolates (NONOates), as a NO amount of chemo-agents in the cells. It has been reported that doxor- donor. In comparison with cisplatin, BA-78 showed better anti-pro- ubicin (5000 ng/ml) and geneticin (800 ng/ml) upregulate P-gp liferation activity (IC50 = 0.89–7.44 μM) in several cell lines such as (MDR1, ABCB1) and ABCG2 in leukemia cells and breast cancer cells. mouse melanoma cells (B16F10), human breast cancer cells (MCF-7), Interestingly, BA was shown to reverse the effect of doxorubicin and human colon cancer cells (HCT-116), human adenocarcinoma alveolar geneticin and exhibited remarkable cytotoxic activity in different cell basal epithelial cells (A549), human embryonic lung fibroblast (Helf) lines (Saeed et al., 2018). The effects of BA on paclitaxel-resistant and human hepatocarcinoma (Hep-G2). Furthermore, BA-78 exhibited human lung cancer cell line (H460) indicated that BA has beneficial anticancer activity by variety of mechanisms such as induction of cytotoxic properties (IC50 =50μM). Authors of this work showed that apoptosis by suppression of BCL-XL, BCL-2 and increasing BAD, BAX, BA exerts its cytotoxic effects by decreasing the mitochondrial mem- cleaved BID, caspase-3 and cytochrome C, and finally through cell cycle brane potential, inducing cell cycle arrest in G2/M phase, down-reg- arrest in G1 phase (Zhang et al., 2018a). ulation of BCL-2 and the up-regulation of BAX, which alters the BAX/ The prevalence of breast cancer is rapidly growing worldwide, and BCL-2 proportion signalling in a dose-dependent manner. Collectively, despite the efforts done to treat breast cancer, chemo-resistance in both BA is an advantageous therapeutic candidate for the treatment of drug- early-stage and advanced tumors remains an unsolved issue. Indeed, resistance in lung cancer in the near future (Zhan et al., 2018; Zheng 40% of diagnosed cases experience recurrence and there is a great need et al., 2010). for a novel effective regime to sensitize cancerous cells to routine Chidamide (CDM, CS055) is a novel benzamide-type histone dea- medication. In this context, BA has attracted the attention of scientists cetylase inhibitor (HDACi), which has been used for its anti-leukemic since its combination with other chemotherapy agents improved the properties against AML by suppression of cell proliferation, induction of cytotoxic effects against cancer cells. For instance, BA combined with cell cycle arrest and apoptosis. Combination therapy with BA and CDM Taxol (paclitaxel) synergistically improved the chemosensitivity of showed synergistic effects in human AML cell lines such as Kasumi-1, Taxol-resistant MCF-7 (IC50 = 18.411 μM, in 48 h), and MDA-MB-231 HL-60 and THP-1. This combination therapy was associated with the (IC50 = 20.465 μM, in 72 h) by inducing cell death, cell cycle arrest, increased expression of super oxide dismutase (SOD) causing over- and interacting with GRP78 (chaperone glucose-regulated protein 78) production of ROS and cell death through induction of DNA damage, (Shoeb, 2006). suppressing hypoxia-inducible factor-1α (HIF1-α) and downstream of Evidence indicates that endoplasmic reticulum activity is increased vascular endothelial growth factor (VEGF). These results suggest that to accelerate proper folding for proteins and intercellular transport the combination of BA and CDM could be a satisfactory product to during the growth of tumor cells, metastasis and upon chemotherapy manage AML in the future (Zhang et al., 2017b). (Hombach-Klonisch et al., 2018). Uncontrolled cell growth is associated Renal cell carcinoma (RCC), a prevalent type of kidney cancer, is with oxidative stress, reduced cellular calcium, and ER stress, and extremely resistant to radiotherapy and other recent chemo-therapy consequently leads to the unfolded protein response (UPR) compensa- protocols such as immune-therapy with cytokines (including interferon tory pathway (Ghavami et al., 2018). Once unfold proteins accumu- α/γ, interleukin-2) with a response rate of < 20%. These cells are also lated, they bind to the main chaperon in ER referring as Ig heavy chain resistant to anticancer drugs such as 5-FU, temozolomide and etopo- binding protein (GRP78/BIP). Then, GRP78 dissociate from the protein side. As mentioned earlier, B has shown cytotoxic effects against several of ER membrane sensors including IRE-1 (inositol requiring enzyme-1), human cancer cell lines such as RCC4 (kidney), AGS (stomach), HeLa ATF-6 (activating transcription factor-6) and PERK (pancreatic ER

19 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

Table 8 Clinical studies on B, BA, and their derivatives.

References Disease Compound Mechanism

Huyke et al. (2009) Randomized, monocentric phase 2a study for the treatment of Betulin-based oleogel Anti-inﬂammation actinic keratosis Anti-tumor Csuk (2014) Phase I and Phase II, NCT00701987 and Phase II, BA Inhibition of tumor formation NCT00346502 for dysplastic nevi with the potential to trans- form into melanoma Csuk (2014) Anti-HIV1 (Phase II multicenter, open-label, randomized, BA-derived bevirimat dimeglumine Inhibition of virus maturation parallel-group in HIV-1 patients)

Liu et al. (2017a) Unpublished data on the anxiolytic effects. Extract of Souroubea spp. and Platanus spp. Activation of GABAA receptors containing BA as the main active compound Schwieger-Briel et al. Dystrophic epidermolysis bullosa phase II pilot trial Betulin-based oleogel Enhanced epithelialization, anti- (2017) inflammatory Scheffler (2019) EU/1/15/1069/001, Treatment of superficial skin wounds and Betulin-based oleogel Accelerated maturation of the barrier, IIa-degree burn wounds Increased migration of skin cells Shikov et al. (2011) Hepatoprotective effects against HCV in chronic hepatitis C Birch bark extract containing 75% B and Reduced HCV RNA, ALT, AST, fatigue and patients 3.5% BA abdominal discomfort kinase (PKR-) like ER kinases) (Ghavami et al., 2014; Hombach- VEGFR2-related angiogenesis and RAS/RAF/ERK pathway. Previous Klonisch et al., 2014; Lee, 2007; Pyrko et al., 2007; Wang et al., 2010). research revealed that combination therapy with of BA and sorafenib Combination therapy of BA and Taxol induces apoptosis through ER showed enhanced and synergistic inhibitory effects on the proliferation stress pathway and blocks the communication between GRP78 and of cancerous cells such as non-small cell lung cancer (NSCLC), A549, PERK. Indeed, BA competitively attaches to GRP78 and dissociates H358 and A427 with different KRAS mutations. The combination of PERK and GRP78, which result in cell death through UPR signalling sorafenib and BA exerts its anticancer effects through several me- cascade (Cai et al., 2018). chanisms such as Akt suppression, which per se promotes ER stress, A recent study examined the cytotoxic effects of a combination of 5- CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) FU, hydrazine sulphate (monoamine oxidase inhibitor) and water-so- overexpression, downregulation of BCL2 expression and overexpression luble betulin-3,28-diphosphate on in vitro and in vivo models of cancer, of BAK. Combinatorial treatments with a low dose of BA and sorafenib and showed this combination therapy promotes antioxidant capacity by showed a potent therapeutic capability for the lung cancer treatment, increasing SOD and LDH and decreasing MDA in a dose-dependent namely because of non-toxic effects on lymphocytes as an adverse effect manner. It is important to note that combination of betulin-3, 28-di- (Kutkowska et al., 2017; Kutkowska et al., 2018; Liu et al., 2014). phosphate with 5-FU and hydrazine sulphate decreased the doses of Another study demonstrated mithramycin A (MIT) and BA down- these cytotoxic compounds in the recommended pharmaceutical com- regulate Sp1, which is involved in several main cascades in cells such as posite more than five times and had better outcomes. Further, this survival, growth and differentiation as well as tumor development and combination therapy was associated with improved health condition of progression. This combination therapy also affected TGF-β, which in- animals such as survival rate, body weight, behavior, reduced tumor duces epithelial to mesenchymal cell transformation in pancreatic size and cardiotoxicity (Vorobyova et al., 2018). tumor cells. In addition, MIT and BA combination therapy was shown to In another study, researchers fabricated a new compound by at- inhibit Sp1 and VEGF at mRNA and protein levels better than being taching dichloroacetate (DCA) to C-3 hydroxyl group of BA and syn- administered alone (Gao et al., 2011). thesized an ester derivative of BA, which has more water solubility and cytotoxic activity in several cancerous cell lines including breast cancer 3.12. Human studies (MCF7, MDA-MB231, MDA-MB468), prostate cancer (DU 145, PC-3), cancer of mouse skin and embryonic fibroblast (B16-F10, NIH/3T3), Despite the high potential of B and BA in the treatment and pre- lung fibroblast (WI-38) as well as in vivo mouse models. Intriguingly, it vention of many diseases, there are few clinical trials with these com- has been shown that combination therapy with B and DCA suppresses pounds. With limited number of human studies, true efficacy, drug proliferation of mentioned cancer cells through inducing apoptosis and interactions, tolerability, and safety of these compounds are not clear. mitochondrial dysfunction (Saha et al., 2015). In addition, combination One of the major limitations in the use of B and BA is their low of TMP, conventional Chinese medicine Rhizoma Chuanxiong, and li- bioavailability. Evidence indicates that plasma concentration after the gustrazine with several BA derivatives (such as TBA, amino-acid/di- oral administration of BA (100 mg/kg) to animals does not exceed 1% peptide derivatives) resulted in significant cell death in several cell lines of the administered dose (Godugu et al., 2014). These results indicate such as Hela, HT-29, Hep-G2, BGC823 and A549. Similar results were that low bioavailability of BA limits the development of promising reported for human cervical adenocarcinoma cell (HeLa), human lung pharmacological agents for clinical use. Many efforts have been made cancer A549 and human hepatoma Hep-G2 cells, when treated with a to improve the bioavailability of B and BA by developing drug delivery combination of BA Ginsenoside Rh2. In another investigation, a com- systems and synthesis of new derivative structures. Despite these lim- bination of BA and 5-FU altered the mitochondrial membrane potential itations, there are clinical studies that used these compounds as pro- and the number of sub-G1 cells, resulted in a significant decreased mising therapeutic agents (Table 8). For example, antiviral effects of BA growth of ovarian carcinoma cells (Xu et al., 2017b). derivatives were discussed in Sections 3.9.1 and 3.9.2 of this review. Doxorubicin is a common drug for treating several forms of cancers Another example is betulin-0leogel (Episalvan), which is recently ap- such as solid tumors and hematological malignancies but its application proved (European Medicines Agency, EU/1/15/1069/001) for the is restricted due to a variety of side effects such as cardiotoxicity, severe treatment of superficial skin wounds and IIa-degree burn wounds in cardiac attack and lymphocytes toxicity. Interestingly, lymphocytes, adults (Frew et al., 2019; Scheffler, 2019). Overall, considering data pre-treated with BA 24 h before doxorubicin, showed significantly from the numerous studies discussed in this review, we expect the de- lower oxidative damage and cell death (Dash et al., 2016; de Melo et al., velopment of new strategies in the future due to the improvement of B 2018). Sorafenib is a multi-targeted kinase inhibitor, which has ther- and BA bioavailability and the efficacy for the treatment of human apeutic effects against a variety of tumors through inhibition of diseases. A summary of clinical studies on B, BA, and their derivatives is

20 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409 listed in Table 8. known protein family that regulates mitochondrial outer membrane permeabilization and consists of anti-apoptotic and pro-apoptotic 4. Mechanism of action of betulinic acid in cancer cells members (Adams and Cory, 2007). The anti-apoptotic members of this family (BCL-2, BCL-XS, BCL-XL and MCL-1) prevent apoptosis either by As mentioned earlier, BA has significant anticancer effects on var- sequestering proforms of death-driving caspases (a complex called the ious cancer cell lines without affecting normal cells. However, the exact apoptosome) or by preventing the release of mitochondrial apoptogenic molecular mechanism of BA remains unknown, and researchers believe factors including cytochrome C and AIF into the cytoplasm (Adams and that BA exerts its effects through different pathways such as: 1) in- Cory, 2007). In contrast, pro-apoptotic factors of this family, (such as duction of apoptosis, 2) regulation of autophagy machinery, 3) inhibi- BAX, BAK, BAD,and NOXA) initiate the release of caspases from death tion of angiogenesis and metastasis, and 4) chemo-sensitization me- inhibitors by heterodimerization or by inducing the leakage of mi- chanisms. Here, we summarize and discuss these proposed mechanisms tochondrial apoptogenic factors through increasing mPT, which con- utilized by BA. sequently leads to caspase activation (Adams and Cory, 2007). Increasing lines of research indicate that BA has a considerable 4.1. Betulinic acid and apoptosis induction potential to regulate the expression levels of BCL-2 family proteins. For instance, BA treatment upregulates pro-apoptotic BAX in glioblastoma, Programmed cell death I, also known as apoptosis, is a conserved neuroblastoma and melanoma cells (Fulda et al., 1997; Wick et al., intrinsic cellular mechanism, which plays critical roles in physiological 1999). Moreover, it induces NOXA, a pro-apoptotic member of the BCL- and pathological conditions (Evan and Vousden, 2001; Ghavami et al., 2 protein family in colon cancer cells (Zhou et al., 2018). However, the 2009a; Moghadam et al., 2018). Apoptosis occurs via two distinct me- expression of BAK and BAD is not affected by BA in melanoma cells chanisms including the extrinsic pathway, which is known as the re- (Selzer et al., 2000). Interestingly, it has also been reported that BA ceptor pathway and intrinsic or mitochondrial pathway (Alizadeh et al., upregulates anti-apoptotic proteins such as MCL-1, BCL-2 and BCL-XS 2017; Fulda and Debatin, 2004). Caspases, as cell death executioners, in melanoma, glioblastoma and neuroblastoma cells, respectively are a group of proteolytic enzymes that play a vital role in the apoptosis (Selzer et al., 2000; Selzer et al., 2002; Thurnher et al., 2003). Besides, pathway (Ghavami et al., 2009b; Ghavami et al., 2005). Once activated, BA produced no significant alterations in the expression levels of BCL- caspases degrade a wide variety of substrates in both cytoplasm and XL in neuroblastoma, glioblastoma or melanoma cells (Fulda et al., nucleus, which results in the characteristic features of apoptotic cells 1997; Selzer et al., 2002; Wick et al., 1999). These findings suggest that (Degterev et al., 2003; Rashedi et al., 2007). The initiation of extrinsic BA has an excellent ability to modulate BCL-2 family proteins in a pathway occurs following the activation of specific receptors (such as context-dependant manner. CD95 (APO-1/Fas) and tumor necrosis factor-related apoptosis-indu- Structural change in the fatty acid composition of cardiolipin, a cing ligand (TRAIL) receptors) by their respective death ligands mitochondrial lipid, is another mechanism that leads to the mitochon- (Iranpour et al., 2016; Reiner et al, 2013; Yeganeh et al., 2013). During drial membrane permeabilization (Zeglinski et al., 2018). The expres- the subsequent events in extrinsic apoptotic pathway, a group of cas- sion of Steroyl-CoA-desaturase (SCD-1), which converts saturated FAs pases known as initiators (such as caspase-8) directly cleave down- to unsaturated FAs, increased in different tumor cells. This enzyme is stream effector caspases such as caspase-3, caspase-7 and caspase-9 vital for those cells that utilize de novo FA synthesis. It has been shown (Adams and Cory, 2007; Alavian et al., 2011). that BA directly alters the level of saturation in cardiolipin. Conse- On the other hand, during intrinsic apoptosis, several molecular quently, the structure of mitochondria in cancer cells face a deep events lead to the leakage of apoptogenic factors from the mitochon- structural change leading to the release of cytochrome c, and eventually drial intermembrane space into the cytosol such as apoptosis inducing induction of apoptosis (Potze et al., 2016). Also, BA alters permeability factor (AIF), second mitochondria derived activator of caspase (Smac)/ of mitochondrial membrane through overproduction of ROS in tumor direct inhibitor of apoptosis protein (IAP), binding protein with Low PI cell lines (Li et al., 2016b; Wang et al., 2017b; Wick et al., 1999). (DIABLO) or omi/high-temperature requirement protein A (HtrA2) Furthermore, antioxidant therapy prior to BA blocks the BA-induced (Ghavami et al., 2008a; Green and Kroemer, 2004; Walczak and apoptosis in cancer cells (Ehrhardt et al., 2004). In addition, research Krammer, 2000). In the cytosol, the formation of the cytochrome c/ revealed that BA treatment induces apoptosis in liver cancer through apoptotic protease activating factor-1 (Apaf-1)/caspase9-containing the extrinsic pathway. TRAIL or APO2 was shown to initiate apoptosis apoptosome complex leads to the activation of caspase-3 (Gerthoffer through the involvement of decoy receptors (DcRs). Co-treatment with et al., 2012; Ghavami et al., 2008b). Under normal conditions, the ef- BA and Apo2 induces p53 activation and significantly upregulates fector caspases are inhibited by IAPs, but the apoptotic process exceeds DcR1, DcR2, FADD and p73, which eventually activate caspase-8 and by the elimination of this inhibition by Smac (Ghavami et al., 2008b; apoptosis. These observations indicate that BA induces apoptosis Potze et al., 2014). through both the extrinsic and intrinsic pathways (Xu et al., 2017b). According to the literature, the anticancer properties of BA are as- Modulation of NF-κB activity is another mechanism that is involved sociated with its ability to activate apoptotic signalling pathways in in the BA-induced apoptosis. In this regard, BA has been shown to in- malignant cells (Please see Fig. 3). Increased mitochondrial perme- duce NF-κB, a central regulator of stress-induced transcriptional acti- ability is a key feature of the intrinsic pathway and BA was shown to vation in a number of cancer cell lines (Kasperczyk et al., 2005). The induce apoptosis via disrupting mitochondrial permeability. Previous activation of NF-κB by BA is accompanied by increased activity of in- research showed that treating isolated mitochondria with BA leads to hibitor of NF-κB(IκBa) kinase (IKK), phosphorylation of IκBα followed loss of mitochondrial membrane potential, which is not affected by a by the degradation of IκBα and nuclear translocation of the NF-κB caspase inhibitor N-benzyloxycarbonyl-ValAla-Asp-fluoromethylketone subunit p65. Interestingly, NF-κB inhibitors (such as IKK inhibitor, (zVAD.fmk) (Fulda et al., 1998). Interestingly, another study by André proteasome inhibitor, antioxidants) abrogate the BA-induced apoptosis. et al. showed that bongkrekic acid, an inhibitor of the permeability Conversely, BA was shown to interfere with NF-κB activation and NF-κB transition pore complex, blocks the effects of BA suggesting that BA regulated gene expression under inflammatory conditions such as ex- targets mitochondrial permeability in cancer cells (André et al., 2002). posure to carcinogens (Takada and Aggarwal, 2003). As mentioned in Furthermore, increased mitochondrial membrane permeability transi- Section 3, these findings suggest the ability of BA to suppress in- tion (mPT) leads to the release of soluble proteins such as cytochrome flammation and to regulate the immune response. Moreover, BA in- C, Smac or AIF from the mitochondria to the cytosol, which causes hibits the nuclear location and nuclear binding of NF-κB/p65 by re- cytosolic caspase activation (caspase 3 and 8) followed by nuclear and ducing the phosphorylation of IκB kinase (IKK)α and I-Kappa-B-alpha DNA fragmentation (Green and Kroemer, 2004). The BCL-2 is a well- (Iκ Bα). This is another possible mechanism to induce apoptosis by BA,

21 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

Fig. 3. Betulinic acid (BA) initiates apoptosis via multiple pathways. They include both extrinsic and intrinsic apoptosis pathways via regulation of caspase-8 activity and also expression of both anti- and pro-apoptotic proteins. BA is also involved in modulation of NF-κB activity and autophagy flux which is solely depended on the context of cell line model. For further details, please refer to the text. BA: Betulinic Acid, FADD: Fas Associated via Death Domain, TRAIL: TNF-Related Apoptosis- Inducing Ligand, NF-κB: Nuclear Factor kappa-light-chain-enhancer of activated B cells, ROS: Reactive Oxygen Species, SCD1: Stearoyl-CoA Desaturase 1, BAX: Bcl-2 Associated X, BAD: Bcl-2 associated Agonist of cell Death, Bcl-2: B-Cell Lymphoma 2, Cyto C: Cytochrome C, AIF: Apoptosis-Inducing Factor, SMAC: Second Mitochondria-derived Activator of Caspases. which is seen in human prostate cancer cells (Shankar et al., 2017). autophagy into a cell death mechanism by concomitant induction of Altogether, these findings suggest that BA can induce apoptosis in a autophagy and inhibition of the autophagic flux (Gonzalez et al., 2012). context-dependent manner through NF-κB activation or inhibition. Besides, evidence indicates that induced mitochondrial damage leads to the activation of autophagy as a survival mechanism. However, under conditions that autophagy cannot reverse the cellular injury (such as 4.2. Betulinic acid and autophagy regulation massive mitochondrial damage or lysosomal exhaustion) further autophagic flux will be prevented. Consequently, the release of cyto- Autophagy is a natural, tightly regulated and dynamic process that chrome C occurs and activates caspases and apoptosis (Potze et al., sequesters damage proteins and organelles by intracellular membrane 2014). structures to degrade and reuse them in starvation or stress conditions, It has been demonstrated that BA inhibits the PI3K–AKT–mTOR which protect and maintain cells in those situations (Klionsky et al., signalling pathway through miR-218 upregulation, which results in the 2016; Lin et al., 2015b; Mokarram et al., 2017; Yeganeh et al., 2015). induction of protective autophagy in colorectal cancer cells (Wang Excessive autophagy may also cause cell death known as type II pro- et al., 2017a). Furthermore, BA-induced autophagy could be dimin- gramed cell death. The PI3K–AKT–mTOR signalling pathway, which is ished by siRNA-mediated p53 knockdown. In contrast, this could be activated in the many types of cancers is the main regulator of autop- decreased by the over-expression of p53. This observation suggests the hagy (Zhai et al., 2014). Recently, studies have shown that autophagy is contribution of p53 in BA-induced autophagy. To achieve the killer involved in BA-induced apoptosis in cancer cells but the exact me- effect, BA reduces the over-accumulation of BA-induced protective chanism of this process is not still clear (Gonzalez et al., 2012; Wang autophagy by degrading wtp53 and mutp53 by miR-502 upregulation et al., 2017a). It has been shown that BA induces apoptosis in KM3 cells and regulation of dual role of p53 in autophagy and apoptosis induction by the activation of caspase-3. However, applying caspase-3 inhibitor, (Wang et al., 2017a). According to these results, BA-induced autophagy Z-DEVD-FMK, to cells not only decreased, but also sensitized cells to could be considered as an alternative mechanism for cell death, namely BA-induced apoptosis suggesting the involvement of an alternative in apoptosis resistant cells. molecular mechanism (Yang et al., 2012). Furthermore, BA treatment enhanced the accumulation of LC3-II and P62 in KM3 cells, indicating the inhibition of autophagic flux and the induction of apoptosis in KM3 4.3. Angiogenesis and metastasis inhibition in response to betulinic acid cells (Yang et al., 2012). Similarly, B10, a semi-synthetic glycosylated derivative of BA, has been shown to induce autophagy and inhibits Angiogenesis and metastasis are among the most important me- autophagic flux by the destabilization of lysosomes. Thus, B10 converts chanisms that facilitate the outgrowth and progression of cancer cells.

22 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

There are several molecular and cellular mechanisms required for the tumor-suppressor gene that inhibits the PI3K-AKT signalling pathway invasiveness of cancer cells. Matrix metalloproteinases are a group of (Maehama and Dixon, 1998). Mutation, deletion or dysfunction of calcium-dependent zinc-containing endopeptidases, which play a role PTEN have been reported in many types of tumors (Li et al., 1997). As in degradation of the extracellular matrix. Increased activity of matrix mentioned earlier, Sp1 was shown to decrease the PTEN expression by metalloproteinases, migratory activity, neovasculogenesis, and con- binding to the specific site on PTEN promoter. Research showed that BA stitutive actin remodelling are examples of such mechanisms involved upregulates PTEN via Sp1 sumoylation, and subsequently increases in cancer cells invasion (Chambers et al., 2002; Sahai, 2007). Hypoxia, radiosensitization in CAL-27and Tca-83 cells (Yuan et al., 2017). a common characteristic of many tumors, stimulates angiogenesis Most drugs used in chemotherapy act through the induction of through the activation of particular transcription factors such as STAT3 apoptosis in tumors and chemo-resistance could be associated with a and hypoxia-inducible factor-1 α (HIF-1α)(Höckel et al., 1996; Jung defect in the apoptosis program (Herr and Debatin, 2001). Further- et al., 2005; Koh et al., 2008). These factors control the expression of more, it has been reported that several chemotherapeutic drugs (such as many genes and their corresponding proteins such as VEGF, BCL-2 and cisplatin, taxol and 5FU) change specific apoptotic pathways. It has cyclin D1, which are involved in the angiogenesis process, anti-apop- been shown that BA facilitates apoptosis in paclitaxel-resistant lung totic activity and proliferation, respectively (Aggarwal et al., 2006). It cancer cell line (H460) by reducing mitochondrial membrane potential has been reported that BA inhibits the expression of HIF-1α and VEGF and regulating the BCL-2/BAX signalling pathway (Zhan et al., 2018). and abrogates angiogenesis in human endometrial cancer cells (Karna Moreover, a recent study showed that a combination of BA with 5FU, et al., 2010). In another study, BA treatment blocked hypoxia-induced irinotecan and oxaliplatin sensitizes drug-resistant colon cancer cells by tyrosine phosphorylation, DNA binding activity, nuclear translocation increasing the expression of PARP and BCL-2 family members. Simi- of STAT, and consequently inhibited angiogenesis in multiple myeloma larly, BA induced ER stress and apoptosis in chemo-resistance breast cells (Pandey et al., 2010). Another study showed that BA suppresses cancer cell lines (MCF-7 and MDA-MB23) through interaction with angiogenesis via inhibiting the binding of HIF-1α and STAT3 to the GRP78 and PERK (Cai et al., 2018). VEGF promoter in PC-3 cells under hypoxic situation (Shin et al., 2011). The hedgehog (Hh) signalling pathway contributes to the main- Moreover, the aryl hydrocarbon receptor (AHR) is a ligand-in- tenance of somatic stem cells and embryonic development at early ducible transcription factor, which acts as either a co-activator or co- stages of life. However, Hh signalling pathway also plays an important repressor by binding to aryl hydrocarbon receptor nuclear translocator role in chemo-resistance (Ingham and McMahon, 2001). It has been (ARNT) (Barouki et al., 2007). It has been shown that AHR over- shown that Hh ligands (Sonic, Desert and Indian) interact with cell expression competitively inhibits the interaction of ARNT and HIF1α, surface receptor patched (PTCH) and reverse the PTCH mediated sup- resulting in the suppression of HIF1α pathway and VEGF production pression of the transmembrane protein smoothened (SMO). As sub- (Fritz et al., 2008). Interestingly, a recent study showed that BA sig- sequent events, SMO activates and translocates the Gli family of the nificantly upregulates the AHR gene through the demethylation of AHR transcription factors (Gli-1, 2 and 3) to the nucleus. These transcription promoter in AML cells and disrupts the interaction between ARNT and factors regulate the expression of genes, which control cell prolifera- HIF1α leading to the decreased expression of VEGF (Zhang et al., tion, differentiation and survival (Ingham and McMahon, 2001; Ruel 2017b). Collectively, these studies highlight the importance of HIF-1α, et al., 2003). It has been reported that sequential treatment of RMS-13 STAT3, and VEGF in angiogenesis and suggest BA as therapeutic agent cells with 5-FU and BA, selectively halt hedgehog signalling and en- that regulates them through different mechanisms. hance chemo-sensitization through inhibition of GLI1, GLI2 and PTCH1 E-cadherin, a member of cadherin superfamily and vimentin, is a in RMS-13 cells (Wang et al., 2015b). The schematic mechanisms of BA- type III intermediate filament protein, which plays a critical role in induced chemo-sensitization have been demonstrated in Fig. 4. cellular adhesion and tumor metastasis (Nijkamp et al., 2011). Evidence showed that BA significantly represses the migration and invasion of 5. Advances in the delivery methods of betulinic acid and its cancer cells through variety of mechanisms such as the 1) down- derivatives regulation of matrix metalloproteinase MMP2, MMP9, and vimentin, and 2) upregulation of tissue inhibitor of metalloproteinase 2 (TIMP2) Betulinic acid and B have low solubility in aqueous medium and and E-cadherin in renal carcinoma cell line (Yang et al., 2018). Fur- effective delivery of these compounds still remains a challenging issue thermore, the Src/FAK/Rac1 signalling pathway plays a pivotal role in (Jonnalagadda et al., 2017; Moodley et al., 2016). The poor aqueous regulating cell migration and invasion through controlling the expres- solubility of BA leads to its poor bioavailability and low systemic cir- sion of many genes such as MMP-2 and MMP-9. In this regard, a recent culation half-time. By far, various strategies have been taken to enhance study showed that BA decreases the expression of MMP-2 and MMP-9 the effective delivery of drugs such as the synthesis of derivatives with while increases the expression of TIMP-2 via inhibition of STAT3 and higher water solubility, reduction of particle size, adduct compounds Src/FAK/Rac1 pathway in 4T1 and MDA-MB-231 cells (Zeng et al., and the use of mixed solvents and surfactants. These strategies always 2018). Evidence showed that accumulation of myeloid-derived sup- face some drawbacks such as the associated toxicity of the materials pressor cells (MDSCs) in the tumors and lungs plays a key role in the used to prepare higher water soluble BA formulations and non-specific development of metastasis. Also, BA has been shown to potently reduce delivery, which subsequently lead to a non-specific distribution of the metastasis by decreasing the infiltration of MDSCs into the tumors and drug (Kim et al., 2010). One of the most interesting strategies which lung (Zeng et al., 2018). Altogether, these evidences suggest that BA has have been utilized to enhance bioavailability of BA is the application of the ability to inhibit metastasis and angiogenesis through controlling a nano/micro-formulations. To enhance the overall bioavailability of BA, variety of genes and pathways. the drug molecules can be effectively encapsulated inside biocompatible/biodegradable carriers or be adsorbed on them and be attached to 4.4. Betulinic acid and chemosensitization mechanisms the carrier through covalent bonds formation. These formulations can extend the drug circulation time and overcome biological barriers (Guo Resistance to chemotherapy is associated with high mortality and Huang, 2011; Li and Huang, 2009; Saneja et al., 2018). Further- among individuals suffering from cancer. Although, there are several more, to increase the selectivity in delivery and subsequently to im- suggested mechanisms which are involved in MDR in cancer cells, prove the therapeutic efficacy, targeted delivery of BA and its deriva- precise mechanisms of chemo-resistance remains unknown. Several tives using special carriers have been investigated (Dai et al., 2015; studies have reported that the PI3K/Akt signalling cascade plays an Dash et al., 2015b; Liu et al., 2016; Lomkova et al., 2016). In this ap- important role in chemo-resistance of cancer cells (Nimptsch et al., proach, the drug is loaded by a carrier, which can selectively deliver the 2015; Zheng et al., 2015). It has been well documented that PTEN is a drug to the desired tissue. The selectivity in delivery can be achieved by

23 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

Fig. 4. Mechanism of chemosensitization of the cells by betulinic acid (BA). It is regulated via different pathways including Gli 1, Gli 2, UPR, and SP1. For further details, please refer to the text. Hh: Hedgehog, PTCH: Protein patched homolog, SMO: Smoothened, Gli: Glioma-associated oncogene homolog (zinc finger protein), PTEN: Phosphatase and tensin homolog, SP1: Specificity Protein 1, PERK: Protein kinase R (PKR)-like Endoplasmic Reticulum Kinase, GRP78: 78 kDa Glucose- Regulated Protein, eIF2a: Eukaryotic translation Initiation Factor 2A, CHOP: CCAAT-enhancer-binding protein Homologous Protein, ER: Endoplasmic Reticulum. targeting certain receptors, which are over-expressed on the desired BA utilizing emulsification and solvent evaporation (Kumar et al., cells or through controlled triggered drug release in the desired tissue. 2018b). To synthesis the carrier, BA and PLGA were dissolved in a Usually, the general structure of this class of carriers involves a tar- volatile organic solvent. Then, the solution was added to an aqueous geting moiety linked to a therapeutic payload via a spacer that often solution of PVA as a stabilizer under sonication followed by evaporation contains cleavable bonds. The targeting moiety can bind to a specific of the organic solvent. The particle size was 257.1 nm with a zeta po- receptor on cells of interest. In the case of different cancerous diseases, tential of −0.170 mV. The cytotoxicity results showed that the syn- the targeting moiety is designed to selectively bind to different over- thesized formulation provided higher inhibition and penetration prop- expressed (relative to normal cells) receptors (Lomkova et al., 2016). erties against Hep-G2 cells compared to bare BA. Furthermore, in vivo The spacer encapsulates and releases the agent into the cells of interest. studies revealed that the prepared formulation had a higher plasma The carrier should be able to selectivity release the encapsulated drug concentration and circulation time than the bare BA, which led to inside of the cells. To this end, the used spacer should contain cleavable higher bioavailability of the drug in the new formulation to treat bonds sensitive to the specific physiological condition of cells of in- cancer. In another study, BA was bonded to mPEG through covalent terest. In the case of cancerous cells, the abnormal physiological con- bond formation using carbodiimide chemistry to enhance the drug so- ditions of the tumors have been used to design different carriers with lubility in water and subsequently its anticancer efficacy (Saneja et al., pH-sensitive and redox-sensitive bonds. 2017b). The results showed that the water solubility of BA in the new formulation was improved by 45.3 folds and also the results demonstrated that the new formulation induced cell apoptosis in Hep3B and 5.1. New formulations of BA and its derivatives Huh7 hepatic cancer cell lines. The intravenous administration of the new formulation revealed that the formulation provided higher anti- New formulations of BA and its derivatives can be divided into tumor activity than the bare BA in mice models. In another study, a new nano/micro-carriers, polymeric carriers, and nano/micro-emulsions. formulation of BA was developed by covalently bonding the drug mo- Table 9 summaries recent formulations of BA and its derivatives used to lecules to multiarm-PEG (Dai et al., 2014). Authors of latter study used enhance the overall bioavailability of the drug. 4- and 8-armed PEG with terminal carboxylate functional groups to load the drug molecules with loading capacities of 3.26–11.81 wt%. 5.1.1. Polymeric carriers The water solubility of the drug in the new formulation was higher than Various polymeric carriers have been prepared to enhance BA and bare BA (290–750 folds), which subsequently led to the higher anti- ffi its derivatives: water solubility and pharmaceutical e cacy utilizing tumor efficacy of the new formulation in vitro and in vivo. The in- biocompatible polymers or natural polymers such as PLGA, PEG, PVA, travenous administration of the new formulation to mice models and chitosan. To this end, the drug mainly has been encapsulated inside showed that the 8-armed-PEG formulation have higher inhibitory effect fi polymeric carriers by emulsi cation and solvent evaporation method or on lung carcinomas tumor growth (on day 20, 72.1–90.7%) than 4- has been bonded to polymeric moieties by covalent bond formation armed-PEG formulation and bare drug. In an attempt toward the (Table 9). In this context, researchers synthesized a polymeric carrier of

24 .Aii tal. et Amiri, S.

Table 9 Various formulations used to enhance the overall bioavailability of B, BA, and their derivatives.

Formulation Drug Method of preparation Main excipients Status of Main outcome Reference investigation

Polymeric carriers BA Emulsification and solvent evaporation PLGA/PVA In vitro/in vivo Enhanced antitumor efficacy and half-life Kumar et al. (2018b) Betulinic Covalent bonding and hydrogelation Chitosan In vitro Chiroptical hydrogels of pH-depending swelling Iftime and Marin aldehyde property were synthesized (2018) BA Covalent bonding mPEG In vitro/in vivo Enhanced solubility (45.3 fold) and antitumor Saneja et al. (2017b) efficacy in vivo BA Emulsification and solvent evaporation PLGA/Lactoferrin In vitro/in vivo Enhanced anti-leishmanial efficacy compared to Halder et al. (2018) BA and PLGA/BA BA Emulsification and solvent evaporation PLGA/mPEG In vitro/in vivo Enhanced circulation time and antitumor efficacy Saneja et al. (2017a) B Electropolymerization of the polymer film and PEDOT In vitro Enhanced B release by applying a potential to the Krukiewicz et al. immobilization of B polymer matrices (2017) BVA/BVB Copolymerization AgNPs/N-vinylpyrrolidone/acrylonitrile In vitro Enhanced Antitumor efficacy Gorbunova et al. (2016) B Emulsification and solvent evaporation PLA or PVA In vitro Enhanced solubility and antitumor efficacy, PLA Yadav et al. (2016) formulation was more efficacious BA Covalent bonding Multiarm-PEG-COOH In vitro/in vivo Enhanced solubility (290–750 fold) and antitumor Dai et al. (2014)

25 efficacy BA Covalent bonding Polyphenol In vitro Enhanced anti-HIV-1 efficacy Liu et al., 2011a,b Nano/micro-emulsions BA Spontaneous nano-emulsification (oil-in-water) Olive oil/Labrasol/Plurol isostearate In vivo Enhanced solubility and hepatoprotective efficacy Harwansh et al. (2017) B High-pressure homogenization (oil-in-water) Flax-seed oil/Egg phosphatidylcholine In vivo Enhanced anti-angiogenic and antitumor efficacy Dehelean et al. (2013) Nano/micro-carriers BA Seed-mediated growth Liposome/Glutathione/Au/Pd In vitro/in vivo Photothermal sensitive BA release and enhanced Liu et al. (2018) antitumor efficacy BA Seed-mediated growth Liposome/Glutathione/Au In vitro/in vivo Photothermal sensitive BA release and enhanced Liu et al. (2017b) antitumor efficacy BA Self-assembly HSA In vitro Enhanced solubility and antitumor efficacy Srivari and Chatterjee (2016) BA Adsorption SWCNT-COOH/Chitosan or tween 20 or In vitro Prolonged BA release of polymer coated SWCNT Tan et al. (2016) tween 80 or PEG compared to the un-coated carrier B Covalent bonding MWCNTs-COOH In vitro Enhanced anti-leishmania parasite efficacy Saudagar and Dubey (2014) BA Co-precipitation β-Cyclodextrin In vitro Enhanced bioavailability Sun et al. (2013) B Antisolvent precipitation – In vitro/in vivo Enhanced dissolution rate (3.12 fold), solubility Zhao et al. (2014) (1.54 fold), and bioavailability (1.21 fold) Biotechnology Advances38(2020)107409

PLGA, poly(lactic-co-glycolic acid); PVA, polyvinyl alcohol; mPEG, Monomethoxy poly(ethylene glycol); MIpGa, 6-O-Methacryloyl-1,2:3,4-di-O-isopropylidene-D-galactopyranose, PEDOT, poly(3,4-ethylenediox- ythiophene); Betulin С-28 esters [lup-20(29)-ene-3,28-diol-28-О-vinylacetate (BVA) and 28-О-vinylbenzoate (BVB)]; AgNPs, silver nanoparticles; PLA, poly-lactide; PVA, polyvinyl alcohol; Multiarm-PEG-COOH, Multiarm-polyethylene glycol carboxylic acid; HIV-1, human immunodeficiency virus type 1; HSA, human serum albumin; SWCNT-COOH, carboxylated single walled carbon nanotubes; PEG, polyethylene glycol; MWCNTs-COOH, carboxylated multi-walled carbon nanotubes. S. Amiri, et al. Biotechnology Advances 38 (2020) 107409 controlled release of B, researchers synthesized an electrodeposited 5.1.3. Nano/micro-emulsions polymeric film, which can release the drug potentially in the desired When a liquid is dispersed in another immiscible liquid, hetero- tissue by applying an electrical stimulus in two different modes of open geneous system of droplets of the emulsion can be obtained. For drug circuit and constant potential (Krukiewicz et al., 2017). To this end, a formulation purposes, the droplets should be small in size and possess conductive polymer (i.e. PEDOT) was electrochemically deposited on a high stability. To obtain small droplets (i.e. nano/micro-emulsions), platinum foil electrode, then the film was developed and the drug was different methods have been developed, but the most widely used oxidatively immobilized. The in vitro cytotoxicity studies of the released method is high-energy oil-in-water emulsification processes such as drug from the polymeric matrix by applying potential against KB and high-pressure homogenization, microfluidization, and ultrasonication MCF-7 cancer cell lines showed that the cytotoxic effect can be sub- (Lee and Norton, 2013; Pinnamaneni et al., 2003; Tang et al., 2013). stantially increased by applying a potential to the polymer matrices. These small droplets can enhance the oral bioavailability of BA and B in However, further studies should be conducted to evaluate the approach an aqueous medium by enhancing water solubility and cell membrane efficacy in vivo. penetration (Table 9). In addition, the drug can be protected against harsh physicochemical conditions and enzymatic degradation (Cavazos-Garduño et al., 2015; Dehelean et al., 2013; Harwansh et al., 5.1.2. Nano/micro-carriers 2017). In this regard, researchers developed a high-pressure homo- Various nano/micro-carriers such as liposomes and carbon nano- genization oil-in-water emulsification process to produce a new B tubes have been utilized to load B and BA to develop new drug for- emulsion (Dehelean et al., 2013). An oil phase of B was prepared in mulations (Table 9). The materials used to prepare drug carriers should flax-seed oil, and an aqueous solution containing egg phosphati- provide high biocompatibility and biodegradability. Ideally, the syn- dylcholine was added to the oil phase under constant stirring. To de- thesized carrier should be able to provide high drug loading effi- crease the droplet size, the mixture was homogenized using a micro- ciencies, prolonged drug release behavior, and selectivity in drug re- fludizer processor at 1000–12000 psi. The in vivo examination of the lease. The selectivity in drug release could be achieved by using new formulation was conducted for the evaluation of anti-angiogenic materials, which are sensitive to the desired tissue conditions such as effects and skin carcinoma for the study of antitumor and anti-in- pH or possess stimuli-sensitive release properties. In this context, Liu flammatory effects, respectively. The results showed that both anti- et al. synthesized a BA carrier based on gold (Au) nanoparticle-coated angiogenic and antitumor efficacies of B were enhanced using the new liposomes, which provide photothermal sensitive BA release with en- formulation. Another team of researchers prepared a BA nano-emulsion hanced antitumor efficacy (Liu et al., 2018). The carrier was synthe- to enhance the drug oral bioavailability and hepatoprotective activity sized using a seed-mediated growth method. To this end, BA was first (Harwansh et al., 2017). To prepare the new formulation, the drug was encapsulated inside glutathione modified liposomes, then palladium first dissolved in olive oil, and the solution was dispersed in an aqueous (Pd) nanoparticles were surface decorated on the liposomes by reduc- solution containing labrasol as a surfactant and plurol isostearate as a tion of Pd ions in the presence of a reducer agent. Subsequently, Pd co-surfactant. The average droplet size was 150.3 ± 0.56 nm with an seeds were utilized for coating of Au nanoparticles as growth promoters average zeta potential of −10.2 ± 0.11 mV. The new formulation to produce flower-like gold crystal on the liposomes. The results showed higher bioavailability and circulation time of the drug in vivo. showed that the entrapment percent of the drug in the carrier with an average diameter of 144.4 nm was 81.63%. The in vivo and in vitro 5.2. Targeted drug delivery of BA and its derivatives evaluation of the carrier release behavior showed that under NIR-irradiation, the hyperthermia of BA delivery significantly improved the To enhance the overall bioavailability of BA and its derivatives and cellular uptake of the carrier, which subsequently enhanced the anti- to improve the therapeutic efficacy of these compounds, new for- tumor efficacy of BA in the new formulation through the synergetic mulations have been prepared using targeted drug delivery approaches effect of chemo-photothermal therapy. In another study, investigators (Dai et al., 2015; Dash et al., 2015b; Liu et al., 2016; Lomkova et al., developed a BA nanocarrier based on adsorption of the drug molecules 2016). In this approach, the drug is loaded by a carrier, which can on oxidized single-wall carbon nanotubes (SWCNT) (Tan et al., 2016). selectively deliver the drug to desired tissue. The selectivity in delivery To decrease SWCNT cytotoxicity and improve poor solubility in phy- can be achieved by targeting certain receptors, which are over-ex- siological medium and to decrease SWCNT agglomeration, the carrier pressed on the desired cells or through controlled triggered drug release was coated with different agents including chitosan, Tween 20, Tween in the desired tissue. Usually, the general structure of this class of 80, and PEG separately. The results showed that all the coated carriers carriers involves a targeting moiety (targeting ligand) linked to a could prolong the drug release compared to the un-coated carrier. The therapeutic payload (encapsulated therapeutic agent) via a spacer in vitro evaluation of the carriers showed that the cytotoxicity of the (encapsulating matrix) that often contains cleavable bonds (Fig. 5). The carriers against mouse embryonic fibroblast cells (3T3) was dependent targeting moiety can be a protein, antibody, peptide, carbohydrate and on the drug release profiles and the type of the coating material and the small molecule, with the ability of selective binding to a specific re- PEG-coated carrier showed the best efficacy. Furthermore, the results ceptor on the cells of interest. The spacer can be a polymer, liposome, showed that the coating agents could significantly reduce SWCNT cy- hydrogel or micelle with the aim of the target therapeutic agent en- totoxicity. Another study suggested a new formulation of B by reducing capsulation and with the ability to release the agent into the cells of the particle size and crystallinity of raw drug powder (Zhao et al., interest. In the case of different cancerous diseases, the targeting moiety 2014). An anti-solvent precipitation method was followed to produce is designed to selectively bind to different over-expressed (relative to drug particles with an average diameter of 110 nm. In this method, normal cells) receptors (Srinivasarao et al., 2015). After tagging the ethanol and water were used as the solvent for B and the anti-solvent, receptors, the carrier can enter the cell by different processes such as respectively. The results showed that the new formulation possesses the most commonly endocytosis. The carrier should be able to selectivity same chemical structure of raw B, but with improved physicochemical release the encapsulated drug inside the cells. To this end, the used properties such as dissolution rate, solubility, and particle size. The in spacer should contain cleavable bonds sensitive to the specific physio- vitro and in vivo evaluations of the new formulation revealed that the logical condition of the cells of interest. In the case of cancerous cells, dissolution rate, solubility and bioavailability of drug were improved by the abnormal physiological conditions of the tumors (e.g. glutathione, 3.12, 1.54, 1.21 folds in comparison to bare B, respectively. Further- pH, temperature, enzyme, etc.) have been used to design different more, the results revealed that the new formulation provides a higher carriers with pH-sensitive and redox-sensitive bonds (Lühmann and hypoglycemic effect in the studied diabetic animals. Meinel, 2016). Researchers synthesized nano-carriers of BA derivatives containing

26 S. Amiri, et al. Biotechnology Advances 38 (2020) 107409

and viral and bacterial infections. All of these vast areas of research are under daily development via meticulous efforts of chemists and biologists. Chemists are focused on changing the structures of these compounds in various stages to fulfill the requests of biologists to improve the effectiveness and potency of these compounds. In the current comprehensive review, we have tried to cover the latest progress and knowledge about B and BA, which are currently widely used in different aspects of biomedicine. We have tried to show the importance of the natural compounds and also the importance of developing new compounds for improvement of the health in modern society. As an example, antiviral properties of B and BA have been investigated against several viral infections in vitro and in vivo. Although further studies are necessary to clarify the detailed antiviral mechanisms of these compounds, it is suggested that they can be introduced as the potential therapeutic agents for treating several important viral infections via di fferent host-virus interaction pathways. Researchers try Fig. 5. The most common structure of a drug carrier for targeted drug delivery. to find B and BA derivatives, whose activity could inhibit bacterial growth and reproduction. Such derivatives would play a role of the last methylated carboxyl groups with pH-sensitive release behavior resort drugs, being a part of antibacterial polytherapy or an alternative (Lomkova et al., 2016). The nano-carrier preparation was based on the to a toxic antibiotic of the last resort. The new possibilities are being self-assembly of BA derivatives of N-(2-hydroxypropyl) methacrylamide searched in the therapy of MDR-TB, XDR-TB, extremely drug resistant (HPMA) copolymer through hydrazone bonds. The results showed that tuberculosis (XXDR-TB) and totally drug resistant TB (TDR-TB). The last at basic medium the copolymer produces self-assembly with the drug in two terms are new ones that have gained popularity among researchers the core. However, at acidic pH, the self-assembly broke out and, fur- and clinicians, who want to describe the strains, whose growth was not thermore, the hydrazone bonds between BA derivatives and the other inhibited by any type of antibiotic. Although antibacterial activities of polymer was broken, which result in the higher exposure of the desired pentacyclic triterpenoids are weaker than of an average antibiotic, a ff cells to the therapeutic agent. The pH-triggered release behavior of the combination of pentacyclic triterpenoid and a di erent antibiotic could nano-carrier enables the controlled release of cytotoxic BA derivatives synergistically enhance the antimicrobial activity and lower MIC of the selectively in the tumor site, which provides benefits such as minimal used antibiotic. This may be a therapeutic option for the patients in- ff damage to the healthy cells and enhancing the drug bioavailability. The fected with e.g. S. aureus resistant to di erent antibiotics. ff BA derivative and the polymeric nano-carrier both exhibited high cy- Regarding anticancer e ects of B, BA and its derivatives it is worth ff totoxicity activity against DLD-1, HT-29 and HeLa carcinoma cell lines to mention that the sensitizing e ects of these compounds on che- ff in vitro. Furthermore, therapeutic agents enhanced tumor accumulation motherapy agent apoptotic e ects are currently very well appreciated in HT-29 xenograft in mice models in vivo. and under investigation in multiple disciplines including improving ff Another research developed a new formulation of BA by covalently apoptotic e ects or targeted delivery of these compounds to cancer bonding the drug molecules to folate-conjugated 8-armed-PEG con- cells. ff taining terminal carboxylate functional groups (Dai et al., 2015). In In recent years many e orts have been focused on targeted delivery addition, another anticancer drug (i.e. hydroxycamptothecin, HCPT) of B and BA via encapsulation in Nano carriers. These approaches could was encapsulated inside the BA carrier by self-assembly of the polymer be a breaking through progress in application of B and BA derivatives in conjugate around HCPT molecules producing a nano-carrier with an future successful clinical trial for cancer, viral infection and systemic average size of ~120 nm for co-delivery of BA and HCPT. The in vitro diseases. Smart targeted deliveries of these compounds could possibly ff ff experiments revealed that the nano-carrier significantly enhanced the reduce some of their side e ects and undesired e ects in other organs. cellular cytotoxicity compared to the bare drugs suggesting there was a fl synergistic effect in the co-delivery of two drugs. The in vivo results Con ict of interest showed that the nano-carrier could prolong the drug circulation time. Furthermore, the results showed that the folate moieties enabled tar- None. geted delivery of both drugs resulting in the higher antitumor efficacy of the drugs in the new formulation with lower damage to the normal Acknowledgments cells. Utilization of folate ligand for targeted drug delivery of BA was also reported (Dash et al., 2015b). A polymeric nano-carrier based on Saeid Ghavami was supported by Research Manitoba New the self-assembly of BA in the presence of PEG was synthesized, and the Investigator Operating grant, Health Science Centre General Operating carrier was modified with folic acid. The results showed that the carrier Grant, NIMAD operating grant and CancerCare Manitoba operating was more stable at acidic pH suggesting facile internalization of folate grants. Pouneh Mokarram, Sanaz Dastghaib and Mazaher Ahmad were receptor over-expressing cells. While the carrier showed high inter- supported by NIMAD operating grant. nalization folate receptor over-expressing K562 cells, it exhibited a lower impact on normal cells with lower expressed folate receptors. References

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