Research Progress on Pharmacological Effects of Betulin

Shuxin LI, Chenghao JIN

1. College of Forestry, Northeast Forestry University, Harbin 150040, China; 2. College of Life Science and Biotechnology, Heilongjiang Bayi Agricultural University, Daqing 163319, China

Abstract Betulin is main component of triterpenoids in bark extract of Betula platyphylla, and has antibacterial, antiviral, liver protecting, cholagogic, antitumorus and other functions. This paper reviews the pharmacological effects and mechanisms of betulin.

Key words Betulin, Pharmacological effect, Alcoholic liver injury, Reperfusion injury after myocardial ischemia, Antitumorus effect

1 Introduction

BetulaplatyphyllaSuk. is the plant fromBetulaLinn. ofBetulaceae. Its bark components are complex, and its pharmacological activities are diverse. Betulin is widely found in wild jujube kernel,Punicagranatumbark,B.platyphyllabark and other substances[1], and belongs to lupane triterpenoids. Betulin has good pharmacological effects in protecting myocardial cells, anti-radiation, anti-fatigue, anti-hypoxia, anti-inflammatory and anti-cancer[2-6]. In this paper, by consulting and sorting out relevant literature published at home and abroad, the pharmacological effects and mechanisms of betulin are reviewed.

2 Protective effects on alcoholic liver injury

Liver is the main organ of alcohol metabolism, and excessive drinking may lead to varying degrees of liver injury. Alcoholic liver disease is a liver lesion caused by long-term drinking. It contains alcoholic fatty liver (AFL), alcoholic hepatitis (AH), alcoholic fibrosis (AF), and alcoholic cirrhosis (AC). With the economic growth and the change of people’s dietary structure in China, the incidence rate and mortality of alcoholic liver disease are on the rise, second only to viral hepatitis. The pathogenesis of alcoholic liver disease is complex. In the process of liver metabolism, ethanol will produce free radicals, which will destroy cell membranes and organelles, inactivate enzymes that scavenge free radicals (such as SOD, GSH-Px, GST), and eventually lead to liver cell damage and liver function damage.

The research by Zhang Guiyingetal.[7]found that in the antioxidant system of human body, substances represented by glutathione (GSH) and malondialdehyde (MDA) can resist alcohol damage to the liver. The content of GSH represents the antioxidant capacity of the liver when it is damaged, and the content of MDA reflects the rate and intensity of lipid peroxidation in organism and the degree of damage to enzymes in the respiratory chain. The research by Di Yingboetal.[8]showed that after rats fed with alcohol were treated by 40 mg/(kg·d) of betulin, the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and glutamyltransferase (GGT) in rat serum were significantly higher than those in model group. It illustrated that betulin could protect alcoholic liver injury by increasing enzyme activity, scavenging free radicals and protecting the integrity of cell membrane and organelles. The research by Di Yingboetal.[8]found that after betulin treatment, the contents of MDA, free radicals and other substances in liver tissue decreased, while the content of GSH increased. It showed that betulin can also inhibit antioxidant reaction. The research by Wu Guangjianetal.[3]found that after rats fed with alcohol were treated by betulin, the content of triglyceride in rat liver serum decreased. It illustrated that betulin can protect liver tissue by reducing the content of triglyceride and inhibiting the formation of fatty liver.

3 Protective effects on injured myocardial cells

3.1 Inhibition of myocardial cell inflammationMyocardial ischemia-reperfusion injury (MIRI) is a kind of phenomenon that myocardial cell’s function is hindered or structure is damaged after resuming perfusion due to long-term ischemia of myocardial tissue, and it will further induce inflammatory reaction during subsequent blood perfusion. The mechanism of myocardial cell inflammation is very extensive, and the stimulation of a variety of factors can promote the secretion of proinflammatory cytokines, leading to the activation of NF-κB conduction signal. It eventually leads to the aggravation of inflammation and further damage of myocardial cells.

In the research on pretreatment of myocardial tissue of rat by 100 mg/L of betulin, Zhang Siyanetal.[5]found that betulin can improve the activities of creatine phosphokinase (CK) and lactate dehydrogenase (LDH). It showed that betulin pretreatment can reduce the myocardial inflammation in rats and protect rat cardiomyocytes. Additionally, Zhang Siyanetal.[5]used betulin to treat AC16 cardiomyocyte, and found that betulin activated the expression of suppressor of cytokine signal transduction (SOCS3) and anti apoptotic protein (Bcl-xL), and made the phosphorylation level of intracellular signal transducer and activator of transcription 3 (STAT3) increase. STAT3 is a member of STAT family, and could respond to a variety of cytokines, growth factors and other signals, and activate the expression of downstream genes. It also participates in a variety of physiological and pathological activities of the heart, such as myocardial cell survival and myocardial angiogenesis. If STAT3 lacks, it could induce myocardial cell inflammation, and increase incidence rate of heart failure. The above experimental results show that betulin can activate STAT3 signaling pathway and NF-κB signaling pathway and the expression of its related factors, and inhibit the inflammation of AC16 cardiomyocyte.

3.2 Inhibition of myocardial autophagyAutophagy is a degradation pathway that plays an important role in individual survival and development and body homeostasis. Cells play a key role in immunity, tumor, inflammation and cardiovascular disease by removing damaged organelles and abnormal proteins and regulating the balance of material metabolism. Advanced glycation end products (AGEs) are a major product of vascular complications of diabetes, and AGEs and its receptors interact to cause oxidative stress in vivo, promote inflammatory response, damage tissue cells, and then lead to the increase of autophagy level.

When studying the effect of betulin on autophagy of H9C2 cardiomyocytes induced by AGEs, Luo Pingetal.[9]found when myocardium was damaged, the enzyme concentration of myocardial zymogram series would increase to varying degrees. So myocardial zymogram is often used to detect the damage of myocardial cells. Mb, CK-MB and cTnI in myocardial enzymogram are common markers of myocardial injury. Beclin1, P62 and LC3 are three important proteins in autophagy process. Beclin1 is homologous gene of autophagy related genes in yeast. In the process of autophagy, Beclin1 forms a complex with other autophagy related genes to regulate the initial stage of autophagy. P62 forms a complex with LC3 and is degraded during autophagy. Therefore, LC3II/LC3I and P62 content could mark the occurrence of autophagy.

By comparing H9C2 cell treated by betulin and AGEs with H9C2 cell treated by AGEs separately, Luo Pingetal.[9]found H9C2 cell proliferation after treated by betulin and AGEs decreased, and the expression of Mb, CK-MB and cTnI rose. In autophagy process of H9C2 cell processed by betulin and AGEs, Beclin1protein expression increased, and P62 protein expression declined, while down regulation of LC3II protein expression level and up regulation of LC3I protein expression level made LC3II/LC3I decline significantly. Above results show that betulin could inhibit myocardial autophagy induced by AGEs, and alleviate myocardial cell damage.

4 Antitumorus effects

Cancer is a disease that seriously threatens human health and safety. In the ranking of all disease mortality, it ranks second only to cardiovascular and cerebrovascular diseases. The number of cancer deaths in China is as high as 1.3 million every year. At present, cancer treatment is still mainly surgery and chemotherapy. Although chemotherapy can prolong the survival time of patients for a certain time, it has many problems, such as strong adverse reactions, and large toxic and side effects, which seriously affect the quality of life of patients after surgery. Therefore, it is urgent to develop an efficient and safe natural anticancer drug. Studies have found that betulin has the advantages of good efficacy, safety, non toxicity and low price in anti-cancer. It can effectively inhibit the proliferation of a variety of cancer cells and induce cancer cell apoptosis.

4.1 Inhibitory effects on esophageal cancer cellsEsophageal cancer is a common digestive tract disease and one of the malignant tumors with the highest incidence rate and mortality in China. The number of deaths due to esophageal cancer is as high as 150 000 every year. Because the early symptoms of esophageal cancer are not obvious, and the diagnostic methods have certain limitations, most of esophageal cancer is in the middle and late stage when it is diagnosed.

Cai Weijiaetal.[10]used betulin of different concentrations to treat esophageal cancer EC109 cell, and measured cell growth inhibition rate. It was found that inhibition rate of EC109 cell growth increased with the increase of betulin concentration. The morphology of EC109 cell processed by betulin changed obviously. The cells swelled, and the cell boundary was unclear. Moreover, the higher the betulin concentration, the more obvious the morphological changes of cells, and even cells fell off in pieces. It is proved that betulin has good growth inhibition and killing effects on esophageal cancer cells.

4.2 Inhibitory effects on gastric cancer cellsGastric cancer is a kind of digestive tract tumor originated from gastric mucosal epithelium. In China, the incidence rate of gastric cancer is higher, and there are obvious regional differences. The incidence rate of gastric cancer in the northwest and eastern coastal areas is higher than that in the south. Due to the change of diet structure and the increase of work pressure, the incidence of gastric cancer tends to be younger at present.

In the research by Zhang Yuetal.[4], it was found that MGC-803 cell activity decreased when 50 μmol/L of betulin was used to treat gastric cancer MGC-803 cell. After different concentrations of betulin was used to treat gastric cancer fcells or 0-4 d, it was found that the number of cell proliferation decreased in varying degrees, and the effect showed dependence of time and dose. It showed that betulin had a good inhibitory effect on the proliferation of gastric cancer cells. After different concentrations of betulin (20 and 50 μmol/L) was used to treat gastric cancer cells for 48 h, Ki67 and PCNA protein expression was detected by Western blotting, and its protein expression level declined. It illustrated that betulin could effectively inhibit Ki67 and PCNA protein expression. The research by Zhang Yuetal.[4]found that the expression level of protein PI3K and p-AKT in gastric cancer cells processed by betulin decreased, further inducing apoptosis of gastric cancer cells. It illustrates that betulin induced apoptosis is related to the inhibition of PI3K/AKT pathway.

4.3 Inhibitory effects on cervical cancer cellsCervical cancer is a common malignant tumor of female reproductive system, which mostly occurs in women aged 50-55, and the incidence rate is increasing year by year. There are about 130 000 new cases of cervical cancer in China every year, ranking second in the cause of female cancer deaths.

MIF is a regulator of innate immunity and is usually highly expressed in invasive cervical cancer. VEGF-C is a highly specific vascular endothelial growth factor, which can promote the metastasis of cervical cancer cells. p53 protein is involved in inhibiting the proliferation and division of tumor cells. Huang Shanetal.[11]used betulin (50, 100 and 200 mg/kg) to treat mice, and measured tumor inhibition rate, thymus index and spleen index of mice. It was found that thymus index, spleen index and apoptosis rate increased in mice. Further, hematoxylin-eosin was used to stain the tumor tissue of mice, and the tumor tissue of mice was observed under the light microscope. It was found that a large number of necrosis occurred in the tumor tissue. By using Western blotting, the expression levels of VEGF-C, MIF and p53 proteins in mouse tumor tissues were detected. It was found that the expression levels of MIF and VEGF-C proteins in tumor tissues decreased, while the expression level of p53 protein increased. It illustrated that betulin ultimately inhibited tumor growth by restraining MIF and VEGF-C protein expression and increasing p53 protein expression.

4.4 Antitumor mechanisminvivoSong Tiantianetal.[2]treated mice by different concentrations of betulin, and determined the effects of betulin on tumor inhibition rate, spleen index, gland index, T lymphocyte transformation function and NK cell killing activity of tumor bearing mice. It was found that after betulin treatment, the transformation function of T lymphocytes in tumor bearing mice was enhanced, and the activity of NK cells was also increased. It illustrates that betulin can activate the immune system and inhibit tumor proliferation by improving T cell transformation function and NK cell killing activity. Additionally, betulin had a tumor growth inhibition rate of 51.31% in tumor bearing mice. It also showed that betulin can well inhibit tumor growth.

5 Conclusions and prospect

Betulin, as a natural compound extracted from birch bark, has the advantages of rich sources and low toxicity. Because of its various pharmacological activities, such as good anti-cancer, treatment of alcoholic liver disease, anti-inflammatory and anti fatigue effects, betulin has become one of the hot spots in the field of traditional Chinese medicine research. In drug development, rich pharmacological activities of betulin avoids the disadvantages of single mechanism of action and easy to produce drug resistance. However, as a new natural compound, betulin has not been fully studied on its molecular mechanism. Therefore, it is necessary to carry out more in-depth research at the cellular and molecular levels, so as to lay a solid theoretical foundation for the research and application development of the pharmacological effects of betulin.