Rubus parvifolius L. is a deciduous small shrub of the genus Rubus of the Rosaceae family, also known as Snake Bubble, Rhizome, March Bubble, Red Plum Flower, etc., which are widely distributed and rich in resources. Its roots, stems, leaves, flowers, and fruits can all be used in medicine, and the traditional Chinese medicine theory believes that it has the functions of hemostasis and blood activation, blood stasis and pain, heat clearing and detoxification, etc., and can be taken internally and externally [1]. Chemical composition studies have shown that strawberries contain flavonoids, triterpenoids, and triterpenoid saponins [2], as well as diterpenoids [3]. Among them, triterpenoids have diverse biological activities and have anti-tumor, neuroprotective and other pharmacological effects[4]. In addition, studies have confirmed that it also has antioxidant, hepatoprotective, and anti-inflammatory effects [5].
Saponins are carbohydrate-conjugated natural compounds with a variety of biological activities, and their pharmacological effects such as anticancer, antioxidant, and antibacterial effects are clear [6], and studies have shown that saponins play an important role in anti-cerebral ischemia-reperfusion injury, inhibition of oxidative stress, and anti-neuroinflammation [7]. However, there is still a lack of systematic review of the pharmacological effects and mechanism of action of saponins. In addition, many of the benefits of the berry (such as antioxidant effects) are also closely related to the flavonoids it contains. In this paper, the application and research status of Strawberry extract and its effective parts in anti-tumor, anti-cerebral ischemia, anti-inflammatory, anti-oxidative stress, anti-liver injury, anti-bone resorption, anti-bacterial infection, and anti-fatigue are reviewed, and the relevant mechanism of action is elaborated, aiming to provide reference and theoretical basis for further research and development and application of Strawberry in drugs and health foods.
1. Antitumor effect
According to the World Health Organization (WHO) in 2019, cancer is the first or second leading cause of death before the age of 70 in 112 out of 183 countries, and cancer is becoming increasingly prominent as the leading cause of death [8].
Despite the rapid development of modern medicine, many anti-tumor chemical drugs have been developed and applied in clinical practice, but most of them face problems such as limited efficacy and many adverse reactions. Traditional Chinese medicine has a long history of anti-tumor activities, and its active ingredients have the characteristics of multi-target effects, which have the unique advantages of good efficacy, small adverse reactions, and high safety. Studies have shown that the anti-tumor effect of Strawberry extract has shown anti-tumor effects on human malignant melanoma, leukemia and liver cancer in in vitro and in vivo experiments, specifically by inhibiting the growth, invasion and migration of tumor cells and inducing apoptosis of tumor cells.
1.1 Anti-melanoma
Cao et al. [9] used in vitro experiments to detect the antiproliferative activity of total saponins (TSRP) on human A375 cells, and found that the longer the treatment time, the better the antiproliferative effect of TSRP. The results of in vitro invasion and migration of human A375 cell lines showed that different concentrations of TSRP (30, 60, and 120 μg·mL-1) significantly inhibited tumor cell invasion and migration to different degrees compared with the vehicle control group, especially the effect of high doses.
In vivo experiments, human malignant melanoma cell line A375 was inoculated into the left axillary of BALB/c nude mice, and the tumors grew to 100 mm3 after 10 days of inoculation, and the xenograft mice were randomly divided into 5 groups, namely vehicle control group, cyclophosphamide group (CTX, 20 mg·kg-1), TSRP 25, 50, and 100 mg·kg-1 groups, with 12 mice in each group. All mice were given IP-related drugs in a volume of 10 mL·kg-1·d-1 for 14 days. The results showed that TSRP had significant anti-tumor effects on A375 xenografts, and was dose-correlated. TSRP significantly reduced malignant melanoma metastases in all dose groups, especially high-dose TSRP (100 mg·kg-1). The results showed that TSRP had a strong inhibitory effect on malignant melanoma metastasis.
1.2 Anti-leukemia
Xu Xiaofeng et al. [10] transplanted human chronic myelinogenous leukemia cells K562 into the subcutaneous dorsal of 4~6-week-old BALB/c nude mice, and the nodules were about 2 mm×2 mm after inoculation, and the ig administration was started, and the nude mice in the high, medium and low dose groups of Maoberry decoction were dosed with 0.5 mL of ig per day at concentrations of 2, 1 and 0.5 g·mL-1, respectively, the cytarabine (Ara-c) group was administered at 40 mg·kg-1 ip per mouse per day, and the control group was given the same amount of normal saline per day. Administered for 14 days. The results showed that the tumor weight of nude mice in the low-, medium-, and high-dose Maoberry decoction groups was significantly reduced compared with the control group, and the tumor inhibition rate was dose-related. The pathological examination of the tumor block showed multiple necrotic foci, tumor cell swelling and rupture, nucleus lysis and disappearance, etc., indicating that the decoction of Maoberry had a strong effect on inhibiting the growth of leukemia cells in vivo. In addition, different concentrations of drug-containing serum and total saponins of Strawberry could inhibit the colony formation of K562 cell lines, and the inhibitory effect was dose-related.
Ge et al. [11] conducted further studies on the mechanism of action. The results showed that the inhibitory effect of SRP on the proliferation of K562 cells was dose-related. Compared with the control group, the apoptosis rate of SRP treatment was significantly increased, the cells exhibited the morphological characteristics of apoptotic cells, and the cleavage of pro-apoptotic proteins, including poly ADP-ribose polymerase (PARP), caspase-3 and caspase-9, was significantly increased. In addition, SRP also inhibited the expression of Bcl-2, a key regulator of the anti-apoptotic family members. SRP treatment also increased the activity of the AMPK and c-Jun N-terminal kinase (JNK) pathways, and inhibited the phosphorylation expression level of STAT3 in K562 cells. Inhibition of the AMPK pathway blocked the activation of JNK by SRP, suggesting that SRP relied on the AMPK pathway to regulate the expression of JNK. In addition, the inhibition of the latter significantly confers resistance to the pro-apoptotic activity of SRP, suggesting that the AMPK pathway is involved in inducing apoptosis. Pretreatment with STAT3 inhibitor also enhanced SRP-induced growth inhibition and apoptosis, which further confirmed the role of STAT3 pathway after SRP treatment.
Xu et al. [12] inoculated K562 cells subcutaneously on the backs of 4~6-week-old male nude mice, and allowed them to grow for 7 days to reach a tumor volume of 50 mm3, and then randomly divided the mice into 5 groups, one of which was given Ara-c 40 mg·kg-1 per day with a daily ig dose of 0.1% dimethyl sulfoxide (PBS) as a control, the positive control group was given Ara-c 40 mg·kg-1 per day, and the other 3 groups were treated with a low dose (20 mg·kg-1) and a medium dose (40 mg· kg-1) and high-dose (100 mg·kg-1) TSRP. The results showed that TSRP had an excellent tumor suppressive effect on K562 cells in a nude mouse xenograft model. Nude mice treated with TSRP have significantly reduced tumor growth rates and tumor quality, and induce apoptosis. Immunohistochemistry assays showed that the Bcl-2 gene was down-regulated, and the phosphorylation levels of eukaryotic initiation factor 4E (eIF4E) and STAT3 were significantly reduced in TSRP-treated cells. At the same time, while effectively inhibiting the proliferation of leukemia cells, TSRP has less toxicity to normal bone marrow hematopoietic stem cells, showing good development prospects.
Xu Xiaofeng et al. [13] showed that TSRP could inhibit the proliferation of K562 and human primary leukemia cells in vitro in a concentration-correlated manner, and TSRP and chemotherapy drugs had a significant synergistic effect within a certain concentration range. The synergy between TSRP and chemotherapy drugs showed obvious tumor cell apoptosis compared with chemotherapy drugs alone, and it mainly induced tumor cell apoptosis through the mitochondrial pathway. Different concentrations (200, 400, 800 mg· L-1) was dose-related in the inhibitory effect of TSRP on the growth of HL-60 cells. After TSRP was intervened with HL-60 cells, there was obvious apoptosis, and the early apoptosis rate increased significantly, showing a dose-correlation. TSRP significantly inhibited the mRNA expression of Bcl-2 and Fas in cells, while the mRNA expression of Bax, Caspase-9, and Caspase-3 was significantly increased, all of which were dose-related[14]. These results indicate that TSRP induces apoptosis in HL-60 cells via the Bcl-2 and Fas pathways in vitro.
1.3 Anti-liver cancer effect
Hu Xiaogang et al. [15] studied the anti-tumor activity of human hepatocellular carcinoma HepG2 cells in vitro, and the results showed that the n-butanol extract, ethyl acetate extract, and petroleum ether extract of Syranberry nigra had the effect of inhibiting tumor growth at the action of 1 mg·mL-1 and 500 μg·mL-1, and showed a clear dose-response relationship, and the results showed that the distribution of anti-tumor active components in n-butanol extract, ethyl acetate extract and petroleum ether extract were all present. Diterpenoids were isolated and identified for the first time from Berryberry.
2 Neuroprotective effects
Wang et al. [16] used a model of middle cerebral artery occlusion (MCAO) with ischemia-reperfusion injury in rats, with 0.9% sodium chloride solution in both the sham and cerebral ischemia-reperfusion (CIR) groups and TSRP or nimodipine (Nim) in the other groups. The drug was administered once a day for 3 days before molding. At 24 h after MCAO, compared with the CIR group, the neurological deficit was significantly alleviated, the volume of cerebral infarction was significantly reduced, and the cerebral edema was reduced in the TSRP group. The apoptosis rate was significantly reduced at 5, 10 and 20 mg·kg-1 doses of TSRP. In addition, compared with the CIR group, the expression of Bcl-2 protein and mRNA in the TSRP group and Nim group were significantly increased, while the expression of Bax protein in the TSRP 10, 20 mg·kg-1 group and Nim group was significantly decreased, and the expression of Bax mRNA in the TSRP group and Nim group was significantly decreased. Thus, TSRP may prevent ischemia-reperfusion injury by increasing Bcl-2 expression and decreasing Bax expression.
Wang Jisheng et al. [17] conducted a study based on animal experiments, and the results showed that after Ig administration of Gramberry extract to Mice Grassberry for 10 g·kg-1 for 3 days, compared with other groups, n-butanol extract could significantly prolong the bleeding time of tail docking mice, increase the bleeding volume of heparinized mice, prolong the survival time of mice under normal pressure and hypoxia, prolong the gasping time of decapitated mice, and reduce the mortality rate of mice ligated with bilateral common arteries. The above results showed that n-butanol extract had a significant protective effect on cerebral ischemia and hypoxia, and the main effective part of n-butanol extract was saponin.
3 Anti-inflammatory action
Yang Liuqing et al. [18] showed that the high-, medium-, and low-dose groups of different medicinal parts of Strawberry could inhibit the plantar swelling of rats caused by carrageenan to a certain extent, and its effects were as follows: Strawberry leaf extract> root extract> stem extract; Except for the low-dose group, the root, stem and leaf extracts of Grassberry root decreased the mass of cotton ball granuloma in rats (P<0.05), and the effects were as follows: Strawberry leaf extract> root extract > stem extract. The results showed that the roots, stems and leaves of Strawberry had certain anti-inflammatory effects, and the leaf extract of Strawberry had the strongest effect.
4. Antioxidant and hepatoprotective effects
Zheng et al. [19] showed that the total flavonoids in roots, stems and leaves of Strawberry had certain antioxidant capacity, and within a certain concentration range, their effects on scavenging hydroxyl free radicals, superoxide anion ions, organic free radicals and inhibiting malondialdehyde (MDA) were all dose-response relationships. The flavonoid content in the leaves of Strawberry is relatively high, so its antioxidant activity in vitro is relatively high. This antioxidant capacity comes from the ability of its phenolic hydroxyl group to donate hydrogen or electrons, resulting in a redox reaction with reactive oxygen species. In addition, the total flavonoids of Strawberry had a certain inhibitory effect on the spontaneous lipid peroxidation product MDA in rat liver tissue homogenate, so the total flavonoids in Strawberry had a certain resistance to lipid peroxidation, and there was a dose-response relationship with the concentration of total flavonoids.
Gao et al. [20] established a model of acute liver injury by chemical induction of CCl4 injection, in which the ig of the Strawberry extract group was given 20 mg·kg-1 RPL aqueous solution, and the positive control group was given 200 mg·kg-1 biphenyl diester twice a day for 7 days before CCl4 treatment. The results showed that the extract had a significant concentration-dependent DPPH free radical scavenging effect. Mice treated with n-butanol extract of Strawberry can significantly reduce the increase of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels caused by CCl4 poisoning, and also significantly prevent the decrease of superoxide dismutase (SOD) activity and the increase of MDA content in liver tissue. Through histopathological observation, it was found that the treatment of Strawberry extract significantly reduced balloon degeneration hepatocytes, significantly reduced the area of necrosis, and significantly improved the histopathological changes of liver injury, and Strawberry extract had a stronger protective effect than bifendiester in CCl4-induced acute liver injury.
On the other hand, the potent scavenging activity of Strawberry extract against DPPH suggests that Strawberry extract may be a potent free radical scavenger that can ameliorate oxidative stress and inhibit the chain reaction of lipid peroxidation. Therefore, the free radical scavenging activity of Strawberry extract is thought to be responsible for its hepatoprotective effect on CCl4 inducing liver injury in vivo. Phytochemical analysis of n-butanol extract from Radix vulgaris extract by HPLC-MS/MS showed that phenolic compounds may be the main effective site of the extract.
Radix vulgaris root extract (RRE) had a strong scavenging ability to DPPH free radicals, showed strong antioxidant capacity in vitro, and had a high correlation with its total phenol and flavonoid content. RRE has a certain cationic radical scavenging capacity of 2,2'-azino-bis-3-ethylbenzothiazoliline-6-sulfonic acid (ABTS), which is related to its abundant content of phenolic compounds, and the superoxide anion radical scavenging capacity in RRE has the highest correlation with total flavonoid content, but not with total phenol content, indicating that RRE has a certain antioxidant capacity in vitro [21].
As for the anti-hepatitis B virus (HBV) effect of Strawberry, it is consistent with the fact that quite a number of drugs reported in China have inhibition of viral replication in vitro, but have no antiviral effect in vivo. Liu et al. [22] used in vivo experiments in ducks, and the results showed that 3-day-old ducks with hepatitis B virus (DHBV) positive were screened by polymerase chain reaction (PCR) method, and the 3-day-old ducks were screened with 400, 200, and 100 mg·kg-1 groups of Strawberry extract, and lamivudine 50 mg·kg-1 group, and were administered Ig twice a day for 21 days. The results showed that ig lamivudine could significantly inhibit serum DHBV-DNA on 21 days, and DHBV-DNA rebounded significantly after 3 and 7 days of drug withdrawal, while there was no significant difference in serum DHBV-DNA changes at different time points in each dose group of Strawberry extract. Liver pathological examination showed that there was no significant difference between the groups. Therefore, the anti-HBV effect of Berry extract is mainly through its original form, and it loses its anti-HBV effect after metabolism in Ma duck.
5. Anti-bone resorption
Sakai et al. [23] studied the effect of Macrophage extract on bone resorption, and cultured bone marrow macrophages (BMMs) with macrophage colony-stimulating factor (M-CSF) and nuclear factor-κB receptor activating factor ligand (RANKL) for 3 days until multinucleated osteoclasts were formed, and the control group showed many absorption pits, while Macrophage macrophages (BMMs) were cultured with macrophage colony-stimulating factor (M-CSF) and nuclear factor-κB receptor activating factor ligand (RANKL) for 3 days until multinucleated osteoclasts were formed.
The 9.6 μg·mL-1 group significantly and dose-related inhibited the formation of pits, revealing the anti-bone resorption activity of Strawberry extract. Further studies showed that its tannient component, Sanguiin H-6, inhibited the spread of mature osteoclasts and significantly and dose-correlated inhibited pit formation, significantly inhibiting RANKL-induced osteoclast differentiation and bone resorption. Sanguiin H-6 dose-correlated inhibits reactive oxygen species (ROS) production and inhibits phosphorylation of nuclear factor-κB (NF-κB) inhibitory protein α (IκBα) and p38 mitogen-activated protein kinase; Reduced protein levels of activated T cell nuclear factor c1 (NFATc1), cathepsin K, and cellular tyrosine protein kinase (c-Src). In addition, Sanguiin H-6 inhibits NFATc1, nuclear translocation of phosphorylated cell oncogenes fos (c-Fos) and NF-κB in vitro, and tumor necrosis factor α (TNF-α)-mediated osteoclast production in vivo. Therefore, the berry has anti-bone resorptive activity, and its ingredient Sanguiin H-6 can be used for the prevention and treatment of bone diseases associated with excessive osteoclast formation and its bone destruction.
6 Antimicrobial
Liu et al. [24] found that 8 species of Rubus extracts, including Strawberry, had a certain degree of inhibitory effect on the tested species, among which the extracts of Raspberry, Spp. spp., Radix Vulgaris, and S. orientalis had a good antibacterial effect and a wide antibacterial spectrum, and the extract of Grassberry had the best inhibitory effect on Escherichia coli, and the minimum bactericidal concentration (MBC) of Staphylococcus aureus reached 37.5 mg·mL-1. The scavenging ability of the aqueous extract of Rubus spp. to hydroxyl radicals increased with the increase of concentration, and it also had a certain scavenging ability to superoxide aniions.
7 Anti-fatigue
Chen et al. [25] used a weight-bearing swimming experiment in mice to observe the anti-fatigue effect of Grassberry extract, and the results showed that Grassberry extract and its components could effectively increase the time of swimming to exhaustion, indicating that Grassberry extract had anti-fatigue activity. Underlying mechanisms may include delayed accumulation of serum urea nitrogen (SUN) and lactate (LA), reduction of triacylglycerol (TG) levels by increasing fat consumption, increasing hepatic glycogen (HG) and lactate dehydrogenase (LDH) to reduce lactate and ammonia accumulation in muscle, and inhibition of increased immune activation and production of inflammatory cytokines (IL-6) and TNF-α. The comparison of its components showed that the main effective site of the anti-fatigue effect of Strawberry extract was its total saponins.
8 Other effects
Intestinal ischemia-reperfusion injury caused by microcirculatory blood flow disorders is a common pathological and physiological process after severe trauma, burns, or shock, and can cause severe inflammatory damage to the intestine. The saponins contained in Strawberry can alleviate intestinal injury and reduce intestinal inflammation induced by intestinal ischemia-reperfusion, providing a new treatment for intestinal ischemia-reperfusion injury [26]
9 Conclusion
Ancient books of traditional Chinese medicine have long recorded thatch berries, and their names include snake bubbles, spp. spp., March pour, red plum and so on. The roots, stems and leaves of the strawberry contain flavonoids, diterpenoids, triterpenoids and triterpenoid saponins, and total saponins and flavonoids are the main effective parts. The extract has an inhibitory effect on the proliferation of skin tumors, leukemia and liver cancer cells, and the active substance is saponins, which show a good prospect for drug development due to the low toxicity of saponins in vivo. It has a neuroprotective effect on cerebral ischemia-reperfusion injury, and the active substance is its n-butanol extract, which is the total saponin of Grassberry.
It is able to exert anti-inflammatory effects by reducing inflammation and improve oxidative stress by scavenging free radicals, but its anti-hepatitis B virus effect in the body has not been clearly proven. Grassberry shows good antibacterial ability against Staphylococcus aureus and Escherichia coli. The total saponins of the raspberry exert a certain anti-fatigue effect by reducing the accumulation of lactic acid and ammonia in the muscles, as well as inhibiting the production of related cytokines. The saponins contained in Strawberry can reduce intestinal ischemia-reperfusion-induced intestinal inflammatory response, and provide a new treatment for intestinal ischemia-reperfusion injury.
It is worth noting that medical classics record that it can be used for the treatment of rheumatism and bone pain, but few relevant in vitro and in vivo experiments have been carried out and recorded. Some scholars have confirmed at the molecular level that the specific chemical components in the berry can inhibit the bone resorption activity of osteoclasts, so as to achieve the purpose of treating bone diseases caused by enhanced osteoclast production, such as osteoporosis and rheumatoid arthritis. However, the effective site and mechanism of action have not been elucidated. Expanding the thinking and methods of TCM in the treatment of rheumatism and bone pain has proved to be equally useful in experimental research, and can achieve the expected experimental goals and results. At the same time, it suggests that Cinnamomum has a broad application prospect in the clinical treatment of diseases.
At present, there are still some urgent problems to be solved in the pharmacological effects of Strawberry extract and its effective parts, which are summarized as the following four points: (1) The research data on the treatment and efficacy of Strawberry are not sufficient, and its exact mechanism of action still needs to be elucidated, and the data provided at the molecular level are limited. Previous studies have shown that it has anti-tumor activity against a variety of cancer cells, but its potential pharmacological effects on tumor cells are still unknown, and its specific active components are still not very clear, which also limits the further development and utilization of Strawberry as a potential anticancer drug. (2) The effective part of the neuroprotective effect of Strawberry extract has been proved to be its total saponins, which are currently limited to anti-cerebral ischemia, and there is still room to be explored. (3) Strawberry could exert anti-inflammatory effect by reducing inflammatory reaction, but the active ingredients and mechanism of its action were not clear. (4) There is a lack of clinical research on the clinical effect of the strawberry, and there is also a lack of research on the safety of the strawberry and its effective parts.
Therefore, in the future, it is possible to conduct a comprehensive and systematic screening of pharmacodynamic substances on the effective parts and active ingredients of Strawberry, clarify its pharmacodynamic components, further carry out basic research with the goal of elucidating the mechanism of action of Strawberry and strengthen the development and research of the application of Strawberry, and broaden and deepen the research on the neuroprotective effect of Strawberry extract. In terms of the anti-inflammatory effect of strawberry extract, both internal and external use are taken into account; In terms of safety, considering its potential drug toxicity, the toxicological effects, mechanism of action, and in vivo metabolism of Strawberry and its effective parts were further studied, so as to determine its relatively safe and effective dosage, and further combine it with clinical application.
Source: Zhang Ying, Ma Huiyuan, Jia Yingjie, Kong Fanming, Yi Dan. Research Progress on the Pharmacological Effects of Strawberry Extract and Its Effective Parts [J]. Drug Evaluation Research, 2024, 47(4): 914-920.