Research Progress on the Biological Activities and Mechanisms of Action of Betulinic Acid and Its Derivatives

Abstract: Betulinic acid is a naturally occurring pentacyclic triterpenoid, typically isolated from birch. Betula platyphylla It is isolated from tree bark and has also been found in other plants, where it exists in the form of free glycosides and glycosyl derivatives. Chemical modification of its structure can yield a wide variety of derivatives. Studies have demonstrated that betulinic acid and its derivatives exhibit significant biological activities, including antitumor, antiviral, anti-inflammatory and analgesic effects, as well as the ability to inhibit neuronal and vascular damage and to treat other common diseases. This review summarizes the types, biological activities, and mechanisms of action of betulinic acid and its derivatives, with the aim of providing a theoretical basis for future research and applications.

White Birch Betula platyphylla Suk. is a deciduous tree belonging to the genus Betula in the family Betulaceae. The triterpenoids of white birch mainly include betulinic acid and betulin, which are tetracyclic triterpenoids derived from the bark of the white birch tree. ^[1] Birch triterpenoids exhibit a broad spectrum of biological activities and can be applied in antibacterial, antiviral, antitumor, lipid-modulating, choleretic, and hepatoprotective therapies. ^[2-4] . In 1995, Pisha et al. ^[5] This study represents the first report that betulinic acid selectively inhibits the growth of melanoma cells. As a natural compound, betulinic acid exhibits unique pharmacological properties in anti-tumor and anti-human immunodeficiency virus (HIV) activities, distinct from those of conventional drugs. It demonstrates superior target specificity, enhanced biological activity, and potent tumor-suppressive effects, with no adverse reactions observed. ^[3-4] . In-depth research on the antitumor effects of betulinic acid has revealed that it exerts its antitumor activity by triggering programmed cell death, or apoptosis. This induction may occur through direct action on cellular mitochondria, independent of p53 protein accumulation and independent of the death receptor system CD95. Unlike other cytotoxic agents, betulinic acid exhibits a high degree of target specificity. ^[6] Moreover, the mechanisms of action and activities vary among different tumor cell lines. ^[7-9] Betulinic acid and betulin exhibit numerous active sites; positions C-3, C-19, and C-28 are closely associated with biological activity, and even minor modifications to the carbon skeleton can lead to changes in biological activity. ^[4,9-14] Therefore, betulinic acid and its derivatives have emerged as novel drugs with tremendous development potential. This review summarizes the various types of betulinic acid and its derivatives, as well as their biological activities and mechanisms of action in anti-tumor, antiviral, anti-inflammatory, and other common disease contexts.

1 Types of Betulinic Acid and Its Derivatives

From a chemical-structural perspective, betulinic acid is an isoprenoid polymer, with mevalonic acid—not isoprene—as the most critical precursor in its biosynthetic pathway. Consequently, as a derivative of mevalonic acid, betulinic acid, like betulin, retains the tetracyclic lupane skeleton, as illustrated in Figure 1. Structural modifications in betulin and its derivatives predominantly occur at positions C-2, C-3, C-17, C-20, C-23, and C-28, as well as at the double-bond position of the propenyl group. Studies on the structure–activity relationships of betulinic acid and its derivatives have revealed that the C-3, C-19, and C-28 sites are closely associated with biological activity; structural modifications at these sites can not only enhance compound solubility and bioavailability but also reduce toxicity. For instance, chemical modifications such as esterification or acylation at these three positions yield new compounds that exhibit enhanced biological activity, reduced adverse effects, and improved water solubility compared with the parent compound. In contrast, modifications at other sites—such as bromination or hydroxylation at C-2, formation of a double bond between C-1 and C-2, or between C-2 and C-3—generally do not improve activity; moreover, nearly all direct modifications to the A ring, except those at C-3, result in decreased or even complete loss of biological activity. ^[4] The chemical structures of betulinic acid and some of its biologically active derivatives are shown in Figure 1.

2 Antitumor Effects and Mechanisms of Betulinic Acid and Its Derivatives

Malignant tumors, one of the five leading causes of death worldwide, pose a serious threat to human life and health, making the development of antitumor drugs a prominent focus in contemporary drug discovery. Studies have demonstrated that betulinic acid exerts a significant inhibitory effect on the in vitro proliferation of human cervical cancer HeLa cells, human hepatocellular carcinoma SMMC-7721 cells, and human gastric cancer SGC-7901 cells. ^[7] Moreover, betulinic acid exhibits stronger antitumor activity than betulin. It exerts inhibitory effects on the proliferation of a wide range of tumor cell lines, including melanoma, hepatocellular carcinoma, prostate cancer, neurotumors, head and neck tumors, leukemia, human cervical cancer, small-cell lung cancer, breast cancer, nasopharyngeal carcinoma, lymphoma, and colorectal cancer, while showing no cytotoxic effect on normal cells. ^[8] , the National Cancer Institute (NCI) has designated it as a Rapid Access to Intervention in Development (RAID) project. ^[9] . Betulinic acid derivatives also exhibit antitumor properties, as reported by Huo et al. ^[10] In 2017, a glucosylated derivative of betulinic acid, designated B10, was obtained via chemical transformation. B10 inhibits glioma cell proliferation by suppressing the acetylation of SIRT1, a member of the silent information regulator (Sir2) family, and upregulating Bim and PUMA, thereby inducing mitochondrial dysfunction and activating apoptosis. Modification of B10 can alter its cytotoxicity and water solubility without compromising its selectivity. In cancer therapy, the primary sites for modification of betulinic acid are C-3, C-19, and C-28. ^[11] The main anticancer mechanisms of betulinic acid and its derivatives include the following aspects:

2.1 Cytotoxic effects on tumor cells

Betulinic acid inhibits aerobic glycolysis in tumor cells by modulating the Cav-1/NF-κB/c-Myc signaling pathway. ^[12] ; It induces DNA strand breaks in tumor cells, leading to morphological changes, inhibits matrix metalloproteinases (MMPs), and generates reactive oxygen species (ROS) to achieve the goal of killing tumor cells. ^[13] ; Derivatives of betulinic acid conjugated with quinazolines exhibit selective cytotoxicity against CCRF-CEM leukemia cells. ^[14] ; Betulinic acid acetate exhibits cytotoxicity against the human melanoma G361 cell line. ^[15] 。

2.2 Blocking the cell cycle of tumor cells

Betulinic acid can block tumor cell G ₂ /M phase and G ₀/G₁ The cell cycle of the phase ^[16] Both betulinic acid and betulin can induce cell cycle arrest in the canine T-cell lymphoma CL-1 and canine osteosarcoma D-17 cell lines at the S phase, and in the canine B-cell lymphoma CLBL-1 cell line at the G phase. ₀/G₁ Period ^[17] ; Betulinic acid, in combination with tyrosine kinase inhibitors (TKIs), can downregulate the protein expression levels of cell cycle–related proteins p-Rb, thymidylate synthase, and cyclin-dependent kinases in H1975 lung cancer cells, thereby inducing cell cycle arrest at the S phase. ^[18] Therefore, it can be inferred that the inhibitory effect of betulinic acid on the cell cycle of tumor cells is associated with the suppression of protein-level expression of cell-cycle–related genes.

2.3 Targeted induction of tumor cell apoptosis and autophagy

A common mechanism by which betulinic acid induces apoptosis is through direct modulation of the mitochondrial apoptotic pathway. Betulinic acid can alter mitochondrial membrane permeability, leading to the release of cytochrome c and apoptosis-inducing factor (AIF) from the mitochondria into the cytoplasm, thereby activating downstream caspase family proteins and initiating the caspase-mediated apoptotic cascade, ultimately promoting tumor cell apoptosis. ^[9] ; or induce autophagy to trigger the death of multiple cancer cell types ^[8,19] 。

Betulinic acid in combination with certain antitumor agents can induce apoptosis in U266 multiple myeloma cells, a mechanism that may be associated with the downregulation of Survivin and Bcl-2 and the upregulation of cyto-c and Bax following combined treatment. ^[20] Betulinic acid in combination with EGFR TKIs enhances Sub-G1 accumulation, inhibits cell cycle–related proteins, triggers the expression of apoptosis- and autophagy-related proteins, and induces mitochondrial membrane potential loss, thereby inducing cell apoptosis. ^[18] ; The baijiac acid–ursolic acid conjugate (with higher toxicity than baijiac acid) can induce mitochondria-targeted apoptosis in human breast cancer MCF-7 cells, human colon cancer HCT-116 cells, and human neuroblastoma TET21N cell lines. ^[21] ; The birch triterpene–piperazine–rhodamine B polymer can also inhibit the growth of human ovarian cancer A2780 cells by inducing mitochondrial apoptosis. ^[22] Moreover, betulinol can enhance GRP78-mediated endoplasmic reticulum stress responses, thereby activating protein kinase R and inositol-requiring enzyme 1 and other components to initiate apoptotic signaling pathways, ultimately inducing apoptosis in breast cancer MCF-7 and MDA-MB-231 cells. ^[23] The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is a critical pro-survival pathway in cells, and betulinol can inhibit the proliferation of cervical cancer xenografts in mice by suppressing the PI3K/AKT pathway. ^[24] Betulinic acid induces apoptosis or autophagy in tumor cells by activating the mitochondrial apoptotic pathway and inhibiting nuclear factor kappa-B (NF-κB), among other mechanisms. ^[25] , see Figure 2.

2.4  Inducing tumor cell differentiation

Betulinic acid inhibits pancreatic cancer stem cell formation by activating the AMP-activated protein kinase (AMPK) signaling pathway, thereby suppressing pancreatic cancer cell proliferation and tumorigenesis. It also suppresses epithelial–mesenchymal transition (EMT) and induces tumor cell differentiation by downregulating the expression of three pluripotency factors: the stem cell transcription factor SRY-Box2, Octamer-binding protein 4, and the embryonic stem cell–specific protein Nanog. ^[26] 。

2.5 Inhibits tumor cell migration, invasion, and other effects

Betulinic acid can be combined with other substances to inhibit the migration and invasion of tumor cells; for example, a paclitaxel–betulinic acid mixed nanosuspension can block the G phase of the MCF7 breast cancer cell line. ₀/G₁ During the cell cycle, it induces apoptosis and inhibits cell migration; betulinic acid–sulfonate ester conjugates can trigger caspase-mediated apoptosis, potentially by inhibiting carbonic anhydrase IX (a common target in certain tumor cells), thereby suppressing breast cancer cell migration and invasion; furthermore, they can attenuate tumor angiogenesis via the vascular endothelial growth factor Sp1/VEGF signaling pathway. ^[27-29] 。

2.6 Enhance the body's immune function

In vitro experiments demonstrated that γδ T cells induced by betulinic acid exhibited a significantly higher secretion of interferon-γ compared with the control group, and their cytotoxic activity against pancreatic cancer cells was also markedly enhanced. ^[30] Betulinic acid can promote the secretion of tumor necrosis factor (TNF) by macrophages and splenocytes, enhance the cytotoxic activity of macrophages, and improve the body’s nonspecific immune function, thereby exerting its targeted cytotoxic effect on tumor cells. ^[31] 。

Betulinic acid can overcome multidrug resistance (MDR phenotype), possibly by inhibiting the activity of the autocrine motor factor receptor (AMFR). ^[32] Furthermore, it can upregulate the expression of the tumor suppressor PTEN protein by downregulating Sp1 and inducing Sp1 ubiquitination, thereby enhancing the radiosensitivity of oral squamous cell carcinoma (OSCC) cells and facilitating their treatment in combination with radiotherapy. ^[33] ; It can also inhibit multiple deubiquitinating enzymes, thereby reducing the stability and mRNA expression of the androgen receptor protein in prostate cancer cells, which in turn decreases prostate cancer resistance to enzalutamide and facilitates the treatment of prostate cancer. ^[34] 。

3 Antiviral Effects and Mechanisms of Betulinic Acid and Its Derivatives

Viruses are a class of primitive, living entities that exhibit biological characteristics, can replicate themselves, and are obligate intracellular parasites; 70% to 80% of human infectious diseases are caused by viruses. ^[35] , common diseases such as acquired immunodeficiency syndrome (AIDS), influenza, hepatitis B and C, and herpes zoster. Since the early 20th century, scientists have successively discovered that infections caused by certain viruses can induce tumorigenesis; these viruses are referred to as RNA tumor viruses. ^[36] Moreover, birch triterpenoids exhibit pharmacological activities that inhibit a variety of viruses and alleviate disease symptoms caused by these viral infections. ^[37] 。

Current antiviral drugs typically exert their therapeutic effects by targeting specific stages of viral replication. Based on differences in nucleic acid type, viruses are classified as RNA viruses (single- or double-stranded) and DNA viruses (single- or double-stranded), and the molecular targets for drug inhibition vary accordingly depending on the virus’s genetic material. For DNA viruses, a unique target is DNA polymerase; examples include acyclovir and ganciclovir, which inhibit herpes zoster virus, human herpesviruses, and herpes simplex viruses types 1 and 2. In contrast, retroviruses with RNA as their genetic material have reverse transcriptase as a distinctive target; commonly used agents such as zidovudine and lamivudine inhibit HIV. ^[38] Moreover, betulinic acid and its derivatives exhibit inhibitory activity against several common DNA and RNA viruses.

3.1 To HIV Inhibition and its mechanism

AIDS caused by HIV has become one of the major infectious diseases that threaten human health. ^[39] Currently, the well-recognized mechanisms of action against HIV include entry inhibitors, reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors, and maturation inhibitors. At present, the majority of antiretroviral drugs are either reverse transcriptase inhibitors or protease inhibitors, which are associated with drawbacks such as drug resistance. ^[40] Betulinic acid and its derivatives exert their antiviral activity by disrupting the later stages of the viral life cycle—namely, viral entry, replication, and maturation—and are primarily classified as entry and maturation inhibitors. As emerging classes of HIV inhibitors currently under development, betulinic acid’s anti-HIV activity has garnered considerable attention in recent years. ^[41] Studies on the anti-HIV activity of betulinic acid have identified several highly active derivatives; among them, Panacos Pharmaceuticals in the United States has developed the anti-HIV-1 derivative bevirimat (Figure 1), which has achieved success in Phase II clinical trials. ^[42] 。

Studies have shown that the primary active sites of betulinic acid against HIV are the carboxyl group at C-17 and the isopropenyl group at C-19, while differences in its mechanism of action primarily depend on modifications and alterations to the side-chain structure at positions C-3, C-28, and C-30. ^[43-45] The mechanisms of anti-HIV activity of betulinic acid and its derivatives are as follows: ^[37] ：

3.1.1  Entry inhibitors—by blocking or inhibiting the fusion of the virus with the cellular membrane, they prevent RNA viruses from entering cells. Derivatives of betulinic acid, such as RPR103611, inhibit HIV-1 fusion by modulating the molecular signaling between gp120 and gp41, which is essential for triggering receptor-induced conformational changes in gp41. ^[46] The betulinic acid derivative IC9564 (Figure 1) inhibits HIV-1 envelope-mediated membrane fusion by targeting HIV-1 gp120. ^[47] 。

3.1.2 Maturation inhibitors—by inhibiting the production of RNA virus proteins, they suppress viral replication and maturation. Biverma can inhibit HIV replication by blocking the conversion of the capsid precursor protein, capsid spacer peptide 1, into mature capsids, thereby resulting in the production of non-infectious viral particles. ^[48] ; Betulinic acid extracted from the leaves of Syzygium buxifolium can inhibit the replication of HIV-1 in lymphocytes. ^[49] ; It can also inhibit viral maturation by blocking the cleavage of the capsid–intermediate peptide in viral proteins. ^[50] 。

3.1.3 Multifunctional inhibitors: Many betulinic acid derivatives can target multiple molecular pathways to exert antiviral activity against HIV. For example, LH15, a C-28–modified derivative of betulinic acid ( N -[3 -O -(3′,3′-dimethylsuccinyl)-lup-20(29)-en-28-oyl]leucine) and LH55 ( N -[3- O -(3′,3′-dimethylsuccinyl)-lup-20(29)-en-28-oyl]-11-aminoundecanoic acid (Figure 1) can inhibit HIV-1 entry and, by interfering with the processing of P25, also inhibit HIV-1 maturation. ^[51] The feathered-swan-type pentacyclic triterpene LPT12 (Figure 1) primarily inhibits HIV-1 infection by suppressing reverse transcription, viral genome integration, viral transcription, and the synthesis and assembly/maturation of viral proteins; LPT38 (Figure 1) exerts its anti-HIV activity by inhibiting processes such as viral genome integration, viral transcription, and viral protein synthesis; whereas LPT42 (Figure 1) is capable of inhibiting reverse transcription, viral transcription, and viral protein synthesis. ^[52] 。

3.2 Regarding influenza 、 Hepatitis C virus ( HCV ) Wait for common RNA Viral suppression

Betulinic acid and its derivatives also exhibit inhibitory effects against other RNA viruses; for example, betulinic acid can suppress influenza virus replication by inhibiting the cyclooxygenase-2–mediated NF-κB and ERK1/2 signaling pathways. ^[41] ; Both betulinic acid and betulin exhibit antiviral activity against the intestinal ECHO-6 virus. ^[46] ; Betulinol derivative 3,28-di- O - Acetyl betaine exhibits high activity against respiratory syncytial virus in individuals receiving only one therapeutic agent (ribavirin). ^[53] ; The ionic derivatives of betulinic acid (Figure 1) can inhibit the maturation of the murine leukemia virus MuLV protein. ^[54] 。

Based on the stage-specific targets of viral expression within host cells, betulinic acid and its derivatives exert several distinct mechanisms of inhibition against RNA viruses. ^[37] ：

3.2.1 Anti-influenza viral activity—entry inhibitor: The betulinic acid–ascorbic acid conjugate (as shown in Figure 1) exhibits activity against influenza A H1N1. Its mechanism of action likely involves high-affinity binding to the HA protein, thereby sequestering a large amount of HA and blocking the attachment of the influenza virus to host cells (Figure 3). ^[55] 。

3.2.2 Anti-HCV activity: HCV is the causative agent of hepatitis C, and clinical studies have demonstrated that birch bark extract can significantly alleviate symptoms in patients with HCV. ^[56] Betulinic acid inhibits HCV replication in Ava5 replicon cells and in cell-culture–derived infectious HCV particle systems by suppressing the NF-κB– and ERK1/2–mediated COX-2 pathway; moreover, combination therapy with various HCV inhibitors exhibits synergistic antiviral activity against HCV replication. ^[57] 。

3.3 Regarding common DNA Viral suppression

Betulinic acid and its derivatives also exhibit inhibitory activity against DNA viruses. Betulinic acid 3-oxime benzoylhydrazide and betulinic acid hydrazide can inhibit the replication of herpes simplex virus type 1. ^[58] ; Ionic derivatives of betulinic acid (as shown in Figure 1) exhibit antiviral activity against herpes simplex virus type 2. ^[59] Betulinic acid significantly inhibits hepatitis B virus replication by downregulating manganese superoxide dismutase expression, thereby inducing reactive oxygen species production and mitochondrial dysfunction. ^[60] ; Betulinic acid and cediranib can exert synergistic effects by inducing ROS generation and DNA damage, and they significantly inhibit EBV replication in LCL cells infected with the lymphotropic herpesvirus Epstein–Barr virus (EBV). ^[61] ; Artemisinic acid–betulin hybrid and artemisinic acid–betulinic acid hybrid (Figure 1) both exhibit activity against Plasmodium falciparum and human cytomegalovirus, and at low concentrations they are effective even when compared with valganciclovir, the standard anticytomegalovirus drug. ^[62] ; Betulinol derivative 3β,28-di- O - Nicotinoyl betulinol (Figure 1) exhibits high inhibitory activity against human papillomavirus replication. ^[63] 。

4 Anti-inflammatory and Analgesic Effects and Mechanisms of Betulinic Acid and Its Derivatives

NF-κB is a family of nuclear transcription factors that target κB response elements, with diverse functions that include the regulation of the expression of numerous pro-inflammatory cytokine genes. ^[64] ; whereas phospholipase A2 (PLA2) is involved in the pathogenic processes of inflammation and acute injury. ^[65] The primary anti-inflammatory mechanism of birch triterpenoids is mediated by inhibition of the NF-κB signaling pathway, thereby suppressing the gene and protein expression of downstream inflammatory mediators (Figure 4). ^[66] ; or by inhibiting PLA2 activity, enhancing nitric oxide synthase activity to reduce NO production, and promoting ROS generation to induce oxidative stress, thereby achieving anti-inflammatory effects (Figure 5).

Betulin and betulinic acid can inhibit pro-inflammatory processes by exerting anti-PLA2 activity. ^[65] ; Betulinic acid exhibits anti-inflammatory effects in the rat acute foot edema model by reducing NO levels. ^[67] It can markedly suppress the arthritis index, ameliorate joint pathology, reduce toe swelling, improve hemorheology, inhibit synovial cell apoptosis, and restore the negative regulatory effect of relevant cytokines on the ROCK/NF-κB signaling pathway. ^[50] ; The betulinic acid derivative SH479 (Figure 1) suppresses collagen-induced arthritis by modulating T-cell differentiation and cytokine balance. ^[68] ; Betulinic acid can treat type II collagen-induced arthritis by exerting an anti-atherosclerotic effect via a Toll-like receptor 4 (TLR4)-mediated mechanism in synergy with fluvastatin. ^[69] ; It can also treat pneumonia and colitis in mice by reducing levels of inflammatory cytokines, particularly interferon-γ, and alleviate pain. ^[70-71] ; Previous studies have demonstrated that betulinol can significantly inhibit the transcriptional levels of inflammatory genes induced by TNF-α, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1); recent research has shown that betulinic acid, acting as a protease activator, can effectively reduce lipopolysaccharide-induced IL-1β protein levels. ^[72] 。

Betulinic acid also exerts analgesic effects by simultaneously modulating NF-κB and calcium channels. Recent studies have shown that BA5-1, a semi-synthetic amide derivative of betulinic acid (Figure 1), is a dual inhibitor of NF-κB and calcineurin, capable of attenuating experimental shock and delayed-type hypersensitivity reactions. ^[73] Betulinic acid derived from desert lavender can inhibit chemotherapy-induced peripheral neuropathy and HIV-associated peripheral sensory neuropathy–induced mechanical allodynia by blocking N-type and T-type calcium channels, suggesting its potential as a non-opioid analgesic. ^[74] 。

5 Betulinic acid and its derivatives inhibit brain neurons. 、 Vascular Injury and Its Mechanisms

5.1 Effects on the Central Nervous System

Betulinic acid and its derivatives exert beneficial effects on the central nervous system, likely through the regulation of certain cytokines and the modulation of neuron-related signaling pathways; however, the underlying mechanisms require further investigation. Machado et al. ^[75] The tail suspension test (TST) has demonstrated that betulinic acid exhibits antidepressant effects in animal models of depression; furthermore, studies have shown that betulinic acid can modulate the neuron-specific cAMP response element-binding protein–brain-derived neurotrophic factor (CREB–BDNF) signaling pathway, which is implicated in depressive disorders and other neurological conditions. ^[76-77] ; Betulinol can suppress epileptic seizures in mice by modulating the signaling pathway mediated by γ-aminobutyric acid (GABAA) receptors. ^[78] Furthermore, betulinic acid can inhibit FAS/FASL–mediated apoptosis in neuronal cells by modulating the levels of inflammatory cytokines and reducing oxidative stress, thereby attenuating neuronal damage in neonatal mice exposed to isoflurane and mitigating isoflurane–induced impairment of cognitive function. ^[79-80] 。

5.2  Regulate vascular injury and inhibit neuronal damage.

Betulinic acid also exerts reparative effects on vascular and neuronal damage by modulating relevant signaling pathways. Nuclear factor erythroid-derived 2–related factor 2 (Nrf2) is a key transcription factor that regulates the expression of endogenous antioxidant genes; betulinic acid, by modulating Nrf2-mediated antioxidant activity, attenuates lipopolysaccharide-induced vascular hyporeactivity in the rat aorta. ^[81] ; Betulinic acid can also significantly inhibit the impaired angiogenesis caused by Atg7 knockdown by activating NF-κB. ^[82] ; It can also treat and prevent early atherosclerosis by alleviating endothelial dysfunction. ^[83] ; Betulinic acid can also prevent cerebral ischemia in mice by downregulating NADPH oxidase NOX4, thereby reducing the risk of ischemic stroke. ^[84] Betulinic acid inhibits OGD/R-induced hippocampal neuronal injury in rats by activating the PI3K/Akt signaling pathway.

5.3 Improving Dementia in the Elderly

Dementia is a syndrome characterized by persistent, acquired impairment of intellectual functioning. The two most common forms of senile dementia today are Alzheimer’s disease and vascular dementia; recent studies have demonstrated that betulinic acid exerts a beneficial effect in ameliorating both of these prevalent types of dementia.

5.3.1   Amelioration of Alzheimer’s Disease: Betulinic acid exerts neuroprotective effects against neurodegeneration and neuronal damage in various animal models of Alzheimer’s disease; it can prevent neurobehavioral deficits induced by Alzheimer’s disease in rats and can also mitigate the impairment of long-term potentiation (LTP) in these rats. ^[85] Studies have shown that betulinic acid exerts protective effects by: attenuating neurobehavioral and cognitive deficits; reducing pro-inflammatory cytokines in the hippocampus; mitigating oxidative and nitrosative stress; normalizing acetylcholinesterase activity; restoring neurotransmitter balance; normalizing long-term potentiation parameters; and reducing histological damage to the hippocampus. ^[25] 。

5.3.2   Improvement of vascular dementia: Betulinic acid can exert synergistic effects with antioxidants and anti-inflammatory agents by modulating the cAMP/cGMP/CREB/BDNF signaling pathway, thereby inhibiting the progression of vascular dementia, increasing cerebral blood flow, attenuating hippocampal damage, and partially restoring cognitive impairment. ^[77] 。

6 Therapeutic Effects and Mechanisms of Betulinic Acid and Its Derivatives in Other Common Diseases

6.1 Protect myocardial cells

Betulinic acid and its derivatives exert cardioprotective effects by modulating cellular signaling pathways. For instance, betulinic acid can mitigate myocardial hypoxia/reoxygenation injury by inducing the Nrf2/HO-1 pathway while inhibiting the p38 and JNK signaling pathways; it also suppresses the TGF-β1/Smad signaling cascade to block the expression of extracellular matrix proteins in cardiac fibroblasts induced by high glucose, thereby ameliorating cardiac fibrosis and protecting cardiac cells. ^[86-87] ; Betulinol can reduce the level of autophagy in AGEs-induced H9C2 cardiomyocytes by inhibiting PI3K/AKT activity. ^[88] ; The betulinic acid derivative BA5-2 (Figure 1) attenuates inflammation and fibrosis in experimental chronic Chagas cardiomyopathy by inducing interleukin-10 production and M2 macrophage polarization.

6.2 Lipid-modulating and hepatoprotective effects

Betulinic acid and its derivatives also exert lipid-modulating and hepatoprotective effects. They can prevent ethanol-induced fatty liver; feeding mice a high-fat diet supplemented with betulinic acid significantly inhibits obesity; moreover, betulinic acid can suppress nonalcoholic fatty liver disease and reduce total cholesterol and triglyceride levels. ^[50] Two possible mechanisms by which betulinic acid exerts anti-obesity effects are AMPK activation and inhibition of intestinal cholesterol absorption (Figure 6). ^[89] Regulation of the AMPK-SREBP signaling pathway helps reduce hepatic lipid accumulation; inhibition of human cholesterol acyltransferase-1 (which mediates foam cell formation in macrophages) and human cholesterol acyltransferase-2 (which mediates cholesterol absorption in intestinal epithelial cells) reduces intestinal cholesterol absorption. ^[50] 。

6.3 Antibacterial activity

Betulinic acid and its derivatives exert their antibacterial activity by inhibiting bacterial and fungal biofilms. Both betulinic acid and betulinol can suppress the growth of Pseudomonas aeruginosa populations and reduce biofilm formation. ^[ninety] Betulin, betulinic acid, and ursolic acid induce and enhance ROS production by boosting the activity of the electron transport chain in Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, leading to lipid peroxidation and DNA fragmentation, ultimately resulting in bacterial death. ^[ninety-one] ; Betulinic acid can also inhibit Staphylococcus aureus by inhibiting peptidoglycan synthesis. ^[ninety-two] ; Betulinic acid-amide compounds (Figure 1) against Streptococcus mutans Streptococcus mutans and Bacillus cereus Bacillus cereus It also has an inhibitory effect. ^[15] 。

6.4 Suppressing Diabetes and Its Complications

Diabetes is one of the major diseases that humanity currently faces. Peroxisome proliferator-activated receptors γ (PPARγ) play a crucial role in regulating glucose metabolism and lipid homeostasis. Betulinic acid, as a PPARγ antagonist, can inhibit diabetes by promoting osteogenic differentiation and suppressing adipogenesis. ^[93] Betulinic acid exerts its antidiabetic effects primarily through two mechanisms: (1) inhibition of α-amylase and α-glucosidase, thereby reducing the hydrolysis of polysaccharides and lowering free glucose levels, which in turn decreases carbohydrate absorption; and (2) activation of AMPK, leading to downregulation of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression, while simultaneously enhancing transcellular glucose transport and upregulating the expression of glucose transporter proteins GLUT-1 and GLUT-2, thus promoting glucose uptake and glycogen synthesis (Figure 7). ^[50] Betulinic acid derivatives also inhibit the development of diabetic complications; for example, betulinic acid can ameliorate proteinuria in experimental membranous nephropathy by modulating the Nrf2/NF-κB signaling pathway. ^[94] 。

6.5   Effects on other diseases

Human diseases are highly diverse, and numerous studies in recent years have demonstrated that betulinic acid and its derivatives may exhibit preventive or therapeutic activities against a wide range of conditions; however, these findings still require further validation and investigation. Additionally, reports indicate that betulinic acid derivatives H3, H5, and H7 (Figure 1) can protect human retinal glial MIO-M1 cells from glutamate-induced oxidative stress, suggesting potential preventive or therapeutic effects on human retinal diseases. ^[95] ; Betulinic acid can induce autophagy by activating the protein kinase/extracellular signal-regulated kinase pathway, thereby alleviating hepatic and renal fibrosis. ^[82] ; Betulinol derivative betulinol- L -2,4-Diaminobutyric acid (Figure 1) exhibits a potent ability to stimulate collagen synthesis in human fibroblasts and demonstrates superior water solubility; therefore, betulinol- L -2,4-Diaminobutyric acid is suitable for inclusion in formulations intended to promote wound healing, regenerate oral mucosa, and revitalize the skin. ^[96] ; Betulinic acid and its derivatives also exhibit certain insecticidal activity, particularly against the larvae of Aedes aegypti, and can treat Chagas disease by killing Trypanosoma cruzi and inducing its necrosis. ^[2.97] In addition, betulinic acid can inhibit the toxicity of snake venom metalloproteinases—among the major functional proteins in snake venom—thereby alleviating local tissue damage caused by snake venom. ^[98] ; prevent N -Oxidative Redox Imbalance in Rat Testes Induced by N-Nitrosodimethylamine ^{[ninety-nine]} ; Additionally, studies have shown that the betulinic acid derivative ethyl betulinate (Figure 1) exhibits potent anti-tuberculosis activity. ^[100] Furthermore, betulinic acid can synergistically enhance BMP2-induced bone formation by stimulating the Smad 1/5/8 and p38 signaling pathways, whereas betulin can inhibit RANKL-mediated osteoclast differentiation by downregulating nuclear factor of activated T cells and receptor activator of nuclear factor κB ligand. ^[101] 。

7 Conclusion and Outlook

Betulinic acid and its derivatives exhibit a remarkably broad spectrum of pharmacological activities, prompting increasingly in-depth research on these compounds. A thorough understanding of the mechanisms underlying their diverse pharmacological effects reveals that betulinic acid and its derivatives exert distinct actions in different cell types and under varying physiological conditions. For instance, in tumor cells, betulinic acid can induce mitochondria-mediated apoptosis; in mouse models of ischemia-reperfusion injury, it suppresses neuronal apoptosis and damage; and in the regulation of inflammation, it inhibits the production of pro-inflammatory cytokines by suppressing the NF-κB signaling pathway in phagocytes, while simultaneously activating this same pathway to inhibit pathological angiogenesis. These findings underscore the selective pharmacological profiles of betulinic acid and its derivatives, highlighting their substantial therapeutic potential.

In addition to elucidating its pharmacological activities and exploring its mechanisms of action, another key focus of current research is how to obtain larger quantities of natural products. Since the extraction of betulinic acid from plant sources—such as birch bark—is no longer sufficient to meet the needs of both research and further applications, efforts to enhance betulinic acid production have become a major research priority. In this regard, our laboratory has conducted extensive preliminary studies and achieved promising progress: by employing birch cell culture, genetic engineering, environmental regulation, and elicitation with stress-responsive signaling molecules—including methyl jasmonate, nitric oxide, salicylic acid, and endophytic fungi—we have significantly increased the triterpene content in birch cells, raising the total dry weight of triterpenes from less than 1 mg/g to 50–60 mg/g. ^[4,102-103] , thereby achieving the goal of obtaining birch triterpenes from birch cells and tissues. Both abiotic and biotic elicitors can enhance the production of plant secondary metabolites, as reported by Hajati et al. ^[104] In 2018, treatment of birch cells with 0.5 mg/L chlormequat chloride (CCC)—an abiotic elicitor and also an inhibitor of the sterol biosynthesis pathway—resulted in increased production of betulinic acid; Yin et al. ^[103] Overexpression of the bHLH9 transcription factor, which regulates betulinic acid biosynthesis, enhances betulinic acid production.

In recent years, synthetic biology has emerged as a major research focus. Leveraging the advantages of yeast—such as its biosafety profile, well-established genetic engineering tools, and mature cultivation techniques—researchers have been constructing and optimizing metabolic pathways for betulinic acid synthesis in yeast, with the aim of producing this compound. This approach has garnered significant attention and has already yielded notable progress. ^[105-106] In addition to betulinic acid and some of its naturally occurring derivatives, derivatives obtained through chemical modification of its structural active sites have also increasingly become the focus of research. The primary focus of these chemical modification studies is the substitution at the C-3, C-19, and C-28 positions, with the main chemical transformations including amination, esterification, alkylation, and sulfonation. ^[107] Furthermore, in practical applications, betulinic acid is limited by poor water solubility and a short in vivo half-life, which hinder its therapeutic use. The incorporation of nanoscale drug-delivery systems can enhance its water solubility, prolong its half-life, and improve its efficacy. To date, several types of nanoscale delivery systems have been developed, including polymeric nanoparticles, magnetic nanoparticles, liposomes, polymeric conjugates, nanoemulsions, cyclodextrin complexes, and carbon nanotubes. In addition, approaches such as crystal engineering have been employed to co-crystallize other compounds, such as vitamin C, with betulinic acid, thereby modifying its physicochemical properties. ^[108] It is believed that, with the further application of genetic engineering, metabolic engineering, and synthetic biology technologies, birch taraxasterol-based drugs will be efficiently synthesized in the near future and will play a significant role in the treatment of human diseases.

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