Zhuola, Chunhui LIU

Inner Mongolia Medical University, Hohhot 010010, China

Abstract In this paper, by summarizing the literature in recent years, it is considered that the chemical constituents of S. cuscutae are mainly flavonoids. The main pharmacological effects include improving osteoporosis, improving ovarian function, improving drug-induced reproductive toxicity, regulating spermatogenic cell apoptosis, resisting liver fibrosis, resisting inflammation, improving cardiovascular function, protecting fetus, protecting synapses and so on. By summarizing the chemical constituents and pharmacological effects of S. cuscutae, this study aims to better study the mechanism of anti-osteoporosis based on the fact that S. cuscutae has the function of tonifying liver and kidney.

Key words Semen Cuscutae, Flavonoids from S. cuscutae, Pharmacological effect, Tonifying liver and kidney

1 Introduction

Semen Cuscutae is the dried mature seed ofCuscutaaustralisR. Br. orCuscutachinensisLam. It is recorded in the Classic ofShennongMateriaMedicaand is listed as the top grade product.S.cuscutae, also known as dodder, is acrid in taste and neutral in nature. In theClassicofShennongMateriaMedica, it is believed that this traditional Chinese medicine has the characteristics of healing injury, making up for deficiency, increasing strength and nourishing body. It can be mashed into juice to treat skin diseases, and it can be used for a long time to improve eyesight and prolong life. In the 13th Five-Year Plan textbook Traditional Chinese Medicine, it is considered that this traditional Chinese medicine has the effects of securing essence and reducing urination, tonifying liver and kidney, improving eyesight, calming fetus and relieving diarrhea. External use of it can eliminate wind and freckle. It is often used for treating liver and kidney deficiency, impotence, spermatorrhea, frequent enuresis, vaginal bleeding during pregnancy, threatened abortion, soreness of the waist and the knees, and nystagmus with tinnitus. It can be used to treat vitiligo externally. As a commonly used clinical traditional Chinese medicine,S.cuscutaeis found in Zuogui pills, Yougui pills, Suoyang Gujing pills and other commonly used drugs.

2 Main chemical constituents

Wu[2]believes that theS.cuscutaementioned in theClassicofShennongMateriaMedicashould belong toCuscutachinensisLam., which is nearly spherical, ovoid, yellow or brown, and symmetrical on both sides of surface. The hilum is small, located at the top of the seed, and the micropyle is short. Nowadays, the sources of commercialS.cuscutaemedicine includeCuscutaaustralis,S.cuscutaeandC.japonica. Wang[3]studied the chemical constituents ofS.cuscutaeand found thatS.cuscutaeandCuscutaaustraliscontained 185 kinds of phytochemical components including 41 kinds of flavonoids, 18 kinds of phenolic acids, 4 kinds of alkaloids, 29 kinds of lignans, 10 kinds of volatile components, 18 kinds of fatty acids, and 15 kinds of amino acids.S.cuscutaemainly contained flavonoids such as lycopene, kaempferol, quercetin; steroids such as β-sitosterol, stigmasterol, β-sitosterol-3-O-β-D-Xylopyranoside; terpenoids such as diterpene glucoside, australiside A; volatile oil components such as 2-pentylfuran, dodecane; alkaloids such as tryptophan derivatives, methylcytisine; lignans such as cuscutoside A ,B; polysaccharides such as heteropolysaccharide CHC-1, H3, CS-A[4-5]. In addition, some studies[6-7]showed that salt treatment could increase the content of quercetin and total flavonoids and improve their biological activity.

3 Pharmacological effect

3.1 Improving osteoporosisThe researchers used the rat model with osteoporosis induced by ovariectomy as the research object to explore the effect ofS.cuscutaeon osteoporosis. The alcohol extract ofS.cuscutaecould increase the uterine index of rats, which was related to the estrogenic effect of the alcohol extract ofS.cuscutae. It could increase the bone mineral density and improve the trabecular morphology of rats. The preventive and therapeutic effect might be caused by down-regulating the expression of miR-21-5p and up-regulating the level of FasL protein[8]. Lietal.[9]set up model group, sham group, vitamin D3group, high, medium and low dose groups ofS.cuscutaeflavonoids to study the effects of flavonoids fromS.cuscutaeon the related indexes of ovariectomized rats with osteoporosis. The results showed that compared with the model group, the medium and high dose groups ofS.cuscutaeflavonoids and vitamin D3group could enhance the expression of calcium binding protein CaBP-D9K mRNA in intestinal mucosa, increase the bone mineral density of femur, the content of 1, 25(OH)2D3in kidney tissue, and the difference was significant. However, there was no significant difference between medium and high dose ofS.cuscutaeflavonoids and vitamin D3group. Guo’s experiment[10]showed that after 12 weeks of administration of flavonoids fromS.cuscutae, compared with the model group, the bone mineral density, bone mass index and serum ALP level of rats treated with flavonoids fromS.cuscutaeincreased, suggesting that flavonoids fromS.cuscutaehad bone protective effect and improved steroid-type osteoporosis in rats, but the changes of Ca and P levels were not obvious, suggesting that the inhibitory effect on bone resorption was not significant. After establishing the model, Zhaoetal.[11]found that after 12 weeks of intragastric administration of flavonoids fromS.cuscutae, compared with the model group, flavonoids fromS.cuscutaecould increase the bone mineral density of lumbar vertebrae, the levels of β-catenin and OPG in serum, and decrease the levels of RANKL and CCK1. It suggested that flavonoids fromS.cuscutaecould improve the level of bone metabolism, and its mechanism might be associated with the related factors regulating Wnt/β-catenin pathway.

3.2 Improving ovarian functionTotal flavonoids fromS.cuscutaecan correct the abnormal state of hypothalamus-pituitary-gonad axis by improving the secretion of serum testosterone, estradiol, luteinizing hormone and follicle-stimulating hormone in rat model of polycystic ovary syndrome, and inhibit abnormal apoptosis of ovarian granulosa cells and other mechanisms to play a protective role in rats with polycystic ovary syndrome[12-13]. Wangetal.[14]established a rat model of premature ovarian failure by intraperitoneal injection of cyclophosphamide combined with intradermal injection of busulfanum. After the model was successfully established, the total flavonoids fromS.cuscutaewere intragastrically administered for 10 d. The results showed that the estrogenic effect of TFSC could significantly restore the estrous cycle and ovarian endocrine function of rats with premature ovarian failure, promote follicular development and granulosa cell proliferation, and thus restore ovarian function. Zhuetal.[15]took the model rats of kidney deficiency and ovulation disorder established by hydroxyurea as the research object, the modeling and drug intervention were carried out at the same time, and the drug was stopped after 8 d of administration. The observation index showed that the proportion of mature follicles, ovarian index, uterine index, follicle-stimulating hormone and serum estradiol levels inS.cuscutaeandS.cuscutaeflavonoids groups were significantly higher than those in the model group. This showed thatS.cuscutaecould improve the kidney deficiency and ovulation disorder caused by hydroxyurea, and its main effective component might be flavonoids fromS.cuscutae. Flavonoids fromS.cuscutaecould improve the damage to uterus and ovary caused by bisphenol A. The experimental study showed that the structure of uterus and ovary of pregnant rats in blank control group andS.cuscutaeflavonoids + bisphenol A group were intact and clear, while in bisphenol A group, the endometrial epithelium was exfoliated, the boundary of ovarian tissue was blurred, and inflammatory cell infiltration could be seen. Compared with the blank group, steroid acute regulatory protein (StAR) and anti-mullerian hormone (AMH) were increased, cholesterol side-chain cleavage enzyme (CYP11a1) and tyrosine kinase ligand (Kitlg) were decreased in bisphenol A group. Compared with bisphenol A group, the levels of ovarian AMH and StAR inS.cuscutaeflavonoids + bisphenol A group decreased, while the levels of Kitlg and CYP11a1 increased. It suggested that flavonoids fromS.cuscutaecould reversely regulate the effect of bisphenol A on reproduction related proteins in pregnant rats[16].

3.3 Improving drug-induced reproductive toxicityTaoetal.[17]took TM3 mouse Leydig cells as the experimental object, first determined the optimal poisoning dose of bisphenol A (BPA) to TM3 Leydig cells, and determined the safe dose of flavonoids fromS.cuscutaeto TM3 cells, and then carried out the experiment. Flavonoids fromS.cuscutaewere given to TM3 cells in advance to produce protective effect, and then bisphenol A was used for poisoning. The results showed that flavonoids fromS.cuscutaecould positively regulate the abnormal secretion of testosterone and flavonoids in mouse Leydig cells caused by bisphenol A. In the experiment of Ma[18], BPA was used to act on pregnant mice, resulting in reproductive organ damage and abnormal expression of reproduction-related genes in offspring. On this basis, it was found that the reproductive toxicity of offspring induced by BPA could be alleviated by administration of flavonoids fromS.cuscutaeevery day on the 1st-7thday of pregnancy. Zhangetal.[19]used high-throughput transcriptome sequencing technique to sequence the RNA of rat spermatogenic cells treated withTripterygiumwilfordiipolycoridetablets and flavonoids fromS.cuscutae. After comparative analysis of statistical sequencing data, it was found that flavonoids fromS.cuscutaecould improve the male reproductive toxicity induced byTripterygiumwilfordiipolycoridetablets. The mechanism may be related to the up-regulation of DNA-like methyltransferase 3 gene (DNMT3L) and intervention of thyroid hormone signal pathway.

3.4 Regulating spermatogenic cell apoptosisFor hydrocortisone-induced oligoasthenospermia rat model[20], after treatment with total flavonoids fromS.cuscutae(TFSC), compared with the model group, the body weight and testicular index of rats in TFSC group were improved, and the number of sperm increased and sperm motility increased. This effect might be related to that TFSC could slow down the effect of hydrocortisone on testicular spermatogenic cell apoptosis, down-regulate the expression of apoptosis genes Fas and Fasl, and inhibit death receptor pathway, thus inhibiting spermatogenic cell apoptosis. Hanetal.[21]selected ICR male mice as experimental subjects and established a mouse cryptorchidism model by subcutaneous injection of 17-β estradiol. After the model was successfully established, the mice were given different concentrations ofS.cuscutaedrinking water for 15 d. The results showed thatS.cuscutaecould promote the recovery of seminiferous epithelial cells in seminiferous tubules, increase the weight of testis, increase the value of testosterone, improve sperm motility and promote sperm production in cryptorchidism model mice. Suetal.[22]used flavonoids fromS.cuscutaeandTripterygiumwilfordiipolycorideto prepare drug-containing serum, which was used for rat testicular spermatogenic cells culturedinvitro. The results showed that flavonoids fromS.cuscutaecould interfere with the division cycle of spermatogenic cells and regulate the apoptosis of spermatogenic cells.

3.5 Resisting hepatic fibrosisZhengetal.[23]found that total flavonoids fromS.cuscutaehad inhibitory effect on human hepatoma HepG2 cells, and the mechanism might be related to the fact that total flavonoids fromS.cuscutaecould up-regulate the expression of tumor suppressor gene PTEN in HepG2 cells, dephosphorylate PIP3, and transform it into PIP2 without Akt activation, thus cutting off a series of physiological changes such as proliferation, migration, invasion and angiogenesis of tumor cells and endothelial cells produced by Akt activation, to stop the development of the tumor.

Baoetal.[24]used microfluidic chip technology to integrate concentration gradient and microvalve structure on the chip to explore the effects of different concentrations of flavonoids fromS.cuscutaeon apoptosis and necrosis of rat hepatic stellate cell line (HSC-T6). The experiment showed that all concentrations of flavonoids fromS.cuscutaecould significantly promote the apoptosis and necrosis of HSC-T6 cells, which confirmed that flavonoids fromS.cuscutaehad the effect of anti-hepatic fibrosis.

3.6 Resisting inflammationYangetal.[25]found that flavonoids fromS.cuscutaehad a protective effect on cerebral ischemia-reperfusion modelinvivo, and the mechanism might be that flavonoids fromS.cuscutaeinhibited inflammatory response, thus reducing the expression of IL-1β, TNF-α and ICAM-1.

3.7 Improving cardiovascular systemLiu[26]adopted the model of acute preload reduction in the heart of rats in vivo. Before the establishment of the model, flavonoids fromS.cuscutaewere given by intragastric administration for 7 d. On the 8thday, the experiment was carried out and the effect of flavonoids fromS.cuscutaeon cardiac hemodynamics in rats was evaluated by pressure-volume loop analysis. The results showed that flavonoids fromS.cuscutaecould improve the indexes of cardiac function, enhance the contractility and relaxation of rat heart, enhance the energy utilization efficiency of rat heart, and improve the efficiency of heart in pumping and reflux of blood, so as to ensure the normal operation of blood supply of the body. Wangetal.[27]conducted experiments with subcultured rat thoracic aortic smooth muscle cells. After administration of total flavonoids fromS.cuscutae, they found that total flavonoids fromS.cuscutaecould reduce the number of cell deaths and increase the number of living cells, and compared with the model group, the number of early apoptotic cells decreased significantly, indicating that flavonoids fromS.cuscutaehad a protective effect on cardiovascular system. This mechanism might be due to the decrease of Bax protein and the increase of Bcl-2 protein and C-myc gene expression caused by total flavonoids fromS.cuscutae.

3.8 Protecting the fetusA study[28]took human chorionic trophoblast cells (HTR-8) as the experimental object to explore the mechanism of total flavonoids fromS.cuscutaein preventing abortion. The results showed that total flavonoids fromS.cuscutaecould up-regulate the expression of matrix metalloproteinase-9, enhance the migration and infiltration of extravillous trophoblast cells, and activate Notch, AKT, MAPK signal pathway, thus play a role in preventing abortion. When using this drug, we also need to pay attention to other contents, for example, Xiaetal.[29]used rats and pregnant rats and embryos to explore the potential genotoxicity of the water extract fromS.cuscutae. The results showed that there was no damage to the organ structure and function after oral administration of 80 g/kg water extract fromS.cuscutaein adult SD rats. According to the principle of acute toxicity test, the acute toxicity of the experimental drug was very low. The micronucleus rate of bone marrow cells of pregnant mice in the group of 40 g/kg water extract fromS.cuscutaewas higher than that in the negative control group, suggesting that the water extract fromS.cuscutaehad potential genotoxicity to pregnant mice. Compared with the negative control group, the micronucleus rate of polychromatic erythrocytes in embryonic liver increased in the group of 40 g/kg water extract fromS.cuscutae, suggesting that the water extract fromS.cuscutaemight have some mutagenicity.

3.9 Protecting the nerve synapsePengetal.[30]established a mouse model of endocrine degenerative dementia with an improved method in our laboratory. After the model was successfully established, the total flavonoids fromS.cuscutaewere given by intragastric administration for three months. After the administration, various indexes were detected, and the behavior level and learning ability of mice were evaluated by Morris water maze test. The results showed that the total flavonoids fromS.cuscutaeand estrogen could significantly increase the estrogen level of dementia mice, increase the expression of Bcl-2 protein, inhibit the apoptosis rate of hippocampal neurons, and improve the learning and memory ability of mice. The mechanism of TFSC may be to regulate the function of hypothalamus-pituitary-gonadal axis.

Some researchers[31]used 5xFAD transgenic mice with Alzheimer’s disease as the research object to explore whetherS.cuscutaecould reduce the memory damage and synaptic damage of β-amyloid protein in mice. The results showed thatS.cuscutaecould improve the decline of memory ability and protect synapses. This effect might be achieved by inhibiting the expression of activated caspase-3 and inhibiting the phosphorylation of glycogen synthase kinase-3β and tau proteins.

3.10 Other functionsWang[32]made dysmenorrhea models in rats and mice with estradiol benzoate and estradiol benzoate combined with ice water bath before intragastric administration of total flavonoids fromS.cuscutaefor 3 d. The results showed thatS.cuscutaecould inhibit the spasmodic contraction of uterine smooth muscle, dilate blood vessels, improve the level of prostaglandin in microcirculation and uterine smooth muscle, and thus improve the symptoms of dysmenorrhea.

4 Conclusions

Cuscutae, as a classic traditional Chinese medicine, is widely used in clinic. Studies have shown thatS.cuscutaehas a therapeutic effect on reproductive system diseases, osteoporosis, cardiovascular diseases and liver diseases. In recent years, scholars have also conducted in-depth studies on the chemical constituents and active components ofS.cuscutae, and most studies believe that flavonoids are the main effective components ofS.cuscutae[3]. At present, although great progress has been made in the study of pharmacological effects ofS.cuscutae, the research on its mechanism is still a hot spot. Through a systematic summary of the pharmacological effects ofS.cuscutae, our group hopes to study the anti-osteoporotic effect ofS.cuscutaeunder the guidance of the theory of tonifying the liver and kidney[33].