Therapeutic Effects and Mechanisms of Herbal Medicines for Treating Polycystic Ovary Syndrome: A Review

Background Polycystic ovary syndrome (PCOS) is one of the most common disorders of endocrinology in reproductive-age women. In this study, we reviewed data on the effects and underlying mechanisms of herbal medicines used in the treatment of PCOS in laboratory studies. Methods Articles published in English up to June 30, 2018 were searched in Medline and EMBASE. We extracted data regarding herbal intervention; target cell (or animal model) usage; method of herbal extraction; route of administration; dosage and periods; and outcomes of the compounds isolated from herbs, individual herbal extracts, and herbal formula decoctions. We summarized the actions and the mechanisms underlying the beneficial effects of herbal medicines on PCOS. Results A total of 27 studies involving 22 herbal medicines reported their efficacy on PCOS. The herbal interventions in the 27 studies comprised four compounds isolated from herbs (6 studies), nine individual herbal extracts (11 studies), and nine herbal formula decoctions (10 studies). Herbal medicines normalized female hormones, diminished male hormones, recovered the estrous cycle, ameliorated insulin resistance, and improved lipid metabolism in PCOS. The mechanisms underlying the beneficial effects of herbal medicines on PCOS were found to be associated with anti-inflammation, anti-oxidative stress, inhibition of autophagy and/or apoptosis, and ovarian nerve growth factor reduction. Conclusions Herbal medicines are thought to be promising resources in the development of effective therapeutic agents for PCOS. Further studies that include methodological quality assessment and quantitative synthesis of outcomes are recommended.


INTRODUCTION
Polycystic ovary syndrome (PCOS) is one of the most common disorders of endocrinology in women of reproductive age. PCOS is diagnosed by confirming the presence of two of the following: oligo-and/or anovulation, clinical and/or biochemical hyperandrogenism, and ultrasound features of polycystic ovaries, with the exclusion of other etiologies (Fr and Tarlatzis, 2004). The prevalence rates of PCOS depend on the diagnostic criteria used, but they can be up to 18% when using the Rotterdam diagnostic criteria (Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group, 2004). Hyperandrogenism is found in 60-80% of women with PCOS (Azziz et al., 2006). The major clinical or biochemical features of hyperandrogenism are acne, hirsutism, alopecia, and seborrheic dermatitis; elevated testosterone, androstenedione, and dehydroepiandrosterone sulfate levels; and decreased sex hormone binding globulin (SHBG) levels. The syndrome not only presents with reproductive manifestations but also has metabolic implications including insulin resistance, dyslipidemia, obesity, type 2 diabetes, and systemic inflammation (Deugarte et al., 2005;Sartor and Dickey, 2005;Escobar-Morreale et al., 2011).
While the first-line treatment for ovulation induction in women with PCOS is clomiphene citrate administration, the antiestrogenic effects of clomiphene citrate on the endometrium and cervical mucus are thought to cause a low conception rate of 20% (Gonen and Casper, 1990). Clomiphene citrate is also associated with a number of side effects including hot flushes, breast discomfort, abdominal distention, nausea, vomiting, nervousness, headache, hair loss, and disturbed vision (Legro et al., 2007a). Recent studies have investigated the role of metformin as an insulin-sensitizing agent, and although its use is increasing, the understanding of its mechanism is incomplete (Legro et al., 2007b). Moreover, it can cause the development of multiple follicles, along with a risk of ovarian hyperstimulation, multiple pregnancies, and congenital malformations such as neural tube defects, thereby leading to potentially unsatisfactory treatment outcomes.
Since PCOS is defined as a multifactorial metabolic-endocrine disorder (Laven et al., 2002), lifestyle and diet, and the Mediterranean diet in particular, play a relevant role, alongside pharmacological treatment (Barrea et al., 2019). Recent studies have suggested that complementary and alternative treatments, including herbal medicines and acupuncture, may alleviate PCOS symptoms, but evidence of their efficacy and safety is insufficient. Therefore, novel treatment strategies incorporating complementary and alternative therapies need to be investigated to optimize the treatment of PCOS. In this study, we reviewed data on the effects and underlying mechanisms of herbal medicines used in the treatment of PCOS model in laboratory studies. The inclusion criteria of our review included the following:

METHODS
1. In vitro and in vivo studies that assessed the potential effects of herbal medicines on PCOS model 2. Research on the compounds isolated from herbs, individual herbal extracts, or herbal formula decoctions 3. Articles written in English The exclusion criteria were as follows: 1. Clinical trials of herbal medicines for PCOS 2. Review articles 3. Articles that did not describe the components of the herbal medicine; however, this was allowed in the case of patented herbal medicines Among the retrieved studies, after removing the duplicates, the titles and abstracts were reviewed to find potentially relevant articles. Then, the full-texts of screened articles were reviewed to confirm that they met our inclusion criteria.
We extracted data regarding herbal intervention; target cell (or animal model) usage; method of herbal extraction: route of administration; dosage and periods; and outcomes of the compounds isolated from herbs, individual herbal extracts, and herbal formula decoctions. Based on those data, we summarized the actions and the mechanisms underlying the beneficial effects of herbal medicines on PCOS model.

Study Characteristics
In the present review, we included a total of 27 studies involving 22 herbal medicines that reported their efficacy on PCOS model. We identified two in vitro studies, 22 in vivo studies, and three studies with both in vitro and in vivo experiments. The herbal interventions in the 27 studies comprised four compounds isolated from herbs (6 studies), nine individual herbal extracts (11 studies), and nine herbal formula decoctions (10 studies). A flow diagram of the article selection process is shown in Figure 1.

Compounds Isolated From Herbs
Three studies revealed the effects of Cryptotanshinone: it reduced ovarian weight and body weight (Yang et al., 2011;Yu et al., 2014;Xia et al., 2017), the level of luteinising hormone (LH) and the LH/ follicle-stimulating hormone (FSH) ratio (Yu et al., 2014;Xia et al., 2017), and the serum insulin and glucose levels (Yang et al., 2011;Yu et al., 2014). Iridoid (genipin, geniposide, and geniposidic acid) reduced the messenger RNA (mRNA) expressions of interleukin (IL)-1b, IL-6, IL-10, and inducible nitric oxide synthase (iNOS), and the over-secretion of nitrite (Zuo et al., 2017). Total saponins from Korean red ginseng (Panax ginseng C. A. Meyer) reduced the number of cystic follicles and the protein expression of nerve growth factor (NGF) in ovaries of Sprague Dawley (SD) female rats (Pak et al., 2005). Quercetin-treated female Wistar rats showed decreased levels of IL-1b, IL-6, and tumor necrosis factor (TNF)-a, and decreased insulin resistance . The data are summarized in Table 1.

The Mechanisms of Action for PCOS
The mechanisms underlying the beneficial effects of herbal medicines on PCOS model were as follows: the alleviation of inflammation (Rezvanfar et al., 2012;Jang et al., 2014;Lee et al., 2016;Wang et al., 2017;Zuo et al., 2017) and/or oxidative stress, (Rezvanfar et al., 2012;Zuo et al., 2017), the inhibition of autophagy and/or apoptosis (Lee et al., 2016;Sun et al., 2017;Xing et al., 2017), and the reduction of the ovarian NGF (Lee et al., 2003;Pak et al., 2005;Kim et al., 2009;Pak et al., 2009;Jung et al., 2011). The mechanisms of action of each in PCOS model and relevant outcomes are shown in Table 5.

DISCUSSION
Our review of the literature published up to June 30, 2018 summarized the findings of in vitro and in vivo studies on the efficacy of herbal medicines for the treatment of PCOS model. A total of four compounds isolated from herbs (6 studies), nine individual herbal extracts (11 studies), and nine herbal formula decoctions (10 studies) were found to have inhibitory effects on PCOS. According to the results reported, herbal medicines normalized female hormones, diminished male hormones, recovered the estrous cycle, ameliorated insulin resistance, and improved lipid metabolism. We found that the potential inhibitory activity of herbal medicines could influence different aspects of PCOS, with the beneficial effects of herbal medicines arising mainly through anti-inflammation, anti-oxidative stress, inhibition of autophagy or apoptosis, and ovarian NGF reduction.

Herbal Medicines Can Inhibit the Inflammatory Conditions of PCOS
Recent studies have further explored the etiology and pathology of PCOS. Scientists found that in the microenvironment of patients with PCOS, mild chronic inflammation is a hallmark of the syndrome (Nestler, 2000). PCOS has been relevant in chronic inflammation (Benson et al., 2008), and macrophages have been its major pathogenesis (Wu et al., 2004). Ovarian macrophages produce cytokines, chemokines, and growth factors in both the normal and the inflammatory processes of the ovary. The macrophages can orchestrate apoptosis and tissue remodeling, which are involved in folliculogenesis, ovulation, and formation of the corpus luteum (Benson et al., 2008). Given the critical role of macrophages in PCOS (Benson et al., 2008), numerous studies have compared cytokine levels in serum and in follicular fluids in PCOS patients. TNF-a and IL-6 levels in serum and in follicular fluids were elevated in non-obese/nondiabetic PCOS patients treated with gonadotrophins (Amato et al., 2003). Macrophage inflammatory protein-1a and MCP-1 were increased in PCOS patients and associated with adiposity (Glintborg et al., 2009). iNOS, cyclooxygenase-2 (COX-2), and transforming growth factor (TGF)-b activity were increased in the ovaries of PCOS patients (Elia et al., 2006;Hatzirodos et al., 2011), and iNOS and COX-2 activity were prevented by metformin administration (Elia et al., 2006). These results suggest that the immune system is relevant to the pathogenesis of PCOS. Therefore new remedies targeting this inflammatory process can be a therapeutic alternative to the current treatment.
In this review, iridoids significantly inhibited IL-1b, IL-6, IL-10, and iNOS expression, thereby inhibiting inflammatory conditions (Zuo et al., 2017). Quercetin also significantly reduced the levels of IL-1b, IL-6, and TNF-a, and decreased nuclear translocation of nuclear factor kappa B (NF-kB) in an insulin-resistant PCOS rat model . Pre-administration of KOK diminished the increased expression of ionized calcium-binding adapter molecule -1 (+) macrophages in the theca cell layer of cysts and the stroma. KOK also increased mRNA expression of CD11b and CD3 in PCOS ovarian tissue. Pre-administration of KOK significantly decreased the increased levels of IL-1b, IL-6, IL-8, TNF-a, MCP-1, and iNOS; and increased the reduced mRNA expression of epidermal growth factor and TGF-b in PCOS ovaries. These results demonstrated that KOK regulates the expression of inflammatory mediators in the dehydroepiandrosterone (DHEA)induced PCOS model (Jang et al., 2014). Inflammatory mediators were also regulated in the endometrium of the uterus by KOK administration, which has been shown to prevent endometrial hyperplasia in PCOS models (Lee et al., 2016). It was also shown that TNF-a in serum and prostaglandin E (PGE) in the ovary were decreased by IMODs (Rezvanfar et al., 2012).
In addition, they play a key role in the pathological processes of female reproduction (Agarwal et al., 2003;Agarwal et al., 2008). Oxidative stress is a condition in which the equilibrium between antioxidant capacity of the body and toxic oxygen-and/or nitrogen-derived products is impaired. Consequently, free radicals are insufficiently detoxified by cellular antioxidants. Oxidative stress plays an important role in the female reproduction (Ruder et al., 2008;Ruder et al., 2009;Vakilian et al., 2009), and there is increasing literature on the effects of

Mechanisms
Interventions Outcomes Author (  increased oxidative stress markers in infertile females, and on their involvement in the pathophysiology of PCOS (Joo et al., 2010;Mohamadin et al., 2010). PCOS is characterized by chronic inflammation, oxidative stress, and abnormal microRNA expression (Zhao et al., 2015;Zuo et al., 2016). Since it is known that inflammation and oxidative stress are closely linked, elevated oxidative stress usually results from, and leads to, an inflammatory condition (Zuo et al., 2016). It is difficult to separate inflammation from oxidative stress, and it has been proposed in recent years that inflammation and oxidative stress comprise the main foundation of disease occurrence (Duleba and Dokras, 2012). In this review, IMODs (Rezvanfar et al., 2012) and iridoids (Zuo et al., 2017) showed positive effects on oxidative/nitrosative stress, either directly or indirectly, mainly by reducing free radicals and inhibiting inflammatory cytokines in PCOS rats. Administration of IMODs significantly reduced lipid peroxidation (a marker of oxidative stress) and increased superoxide dismutase, catalase and glutathione peroxidase (markers of antioxidant potential) levels in hyperandrogenism-induced PCOS. In addition, peroxynitrite (a marker of nitrosative stress), TNF-a, and PGE levels were significantly reduced by IMODs. Furthermore, these effects of IMODs were consistent with histologic evidence, which showed significant improvement in the microscopic characteristics of folliculogenesis compared with those in the control group (Rezvanfar et al., 2012). The NF-kB signaling system is known as a dominant paradigm for specific signal transduction molecules, gene activation, and regulatory proteins in response to inflammation (Ivanenkov et al., 2011). The iridoids efficiently attenuated the lipopolysaccharide induced elevation of IkB phosphorylation levels, decreased IkB expression levels, and decreased NF-kB P65, indicating that the iridoids exert their antioxidant effects via the NF-kB pathway (Zuo et al., 2017).

Herbal Medicines Can Regulate Apoptosis and/or Autophagy in PCOS
The survival or death of granulosa cells is recognized as a critical factor impacting the fate of follicles (Matsuda et al., 2012). Apoptosis and autophagy are two forms of programmed cell death. Autophagy is the process by which an autophagosome, which is a double-membrane vesicle, carries cytoplasmic material to the lysosome (Mizushima and Komatsu, 2011). It has been reported that both apoptosis and autophagy can be induced in granulosa cells, and that they are involved in the control of follicular development (Choi et al., 2010;Choi et al., 2013). Granulosa cells are recognized as critical players in follicle development. They produce estradiol, insulin-like growth factors, and other cytokines in the ovary and express the receptors for estradiol, LH, and FSH, (Juengel et al., 2006), all of which participate in the regulation of follicle development. Thus, any impairment of the granulosa cells may results in disordered development of follicles.
In the last decade, autophagy-related signaling pathways and their major protein regulators have been identified. The rat microtubule-associated protein 1 light chain 3 is associated with autophagosome membrane processing (Kabeya et al., 2000). Beclin-1 has also been demonstrated to have a critical role in autophagosome formation (Von Hoven et al., 2012). Furthermore, previous studies have identified tumor suppressor p53 (p53) as a dual modulator of autophagy in regulating cell death or survival (Vousden and Ryan, 2009;Zhang et al., 2010). At low energy levels, adenosine monophosphate-activated protein kinase (AMPK), which is activated by p53, is able to activate tuberous sclerosis complex 2, and therefore inhibit the mammalian target of rapamycin (mTOR) activity and increase autophagy (Feng et al., 2007). In this review, p53, which is activated by GZYKF in the nucleus, in turn activated AMPK and sestrin, acting as a feedback in mTOR inhibition, thereby activating autophagy. Autophagy is also modulated by phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathways (Pyo et al., 2012). In this review, XYS alleviated the reduction of phosphorylation of ribosomal protein S6 kinase polypeptide I and Akt, as well as the increase of microtubule-associated protein light chain 3-I to microtubuleassociated protein light chain 3-II conversion both in vivo and in vitro (Sun et al., 2017).
uterine tissue of the sham or the KOK-alone group. However, the number of TUNEL-positive cells was increased in the endometria of DHEA group. The increased number of apoptotic cells was significantly reduced after administration of KOK. These results indicate that pre-administration of KOK inhibited DHEAinduced endometrial malformation by reducing endometrial apoptosis (Lee et al., 2016).

Herbal Medicines Can Reduce the Level of NGF in PCOS
Previous studies have shown that PCOS is associated with abnormal activation of the sympathetic nervous system, resulting in increased catecholaminergic nerves (Semenova, 1969), impaired norepinephrine metabolism (Garcia-Rudaz et al., 1998), and increased activity of sympathetic nerves via the superior ovarian nerve (Lara et al., 1993). The development and function of ovarian sympathetic innervation depend on the ovary for the production of NGF, a target-derived neurotrophin required for peripheral sympathetic system development (Levi-Montalcini, 1987). The neurotrophin family, implicates the NGF receptor and NGF mRNA in ovulation and in the pathophysiology of PCOS (Lara et al., 2000;Stener-Victorin et al., 2003). In rat ovaries, NGF is principally synthesized in the cells of the follicular wall (Dissen et al., 1996), and in PCOS, the activation of NGF may be a factor involved in enhancing norepinephrine outflow to the gland, which is induced by estradiol valerate (EV) (Lara et al., 2000) PCOS exhibits a high intraovarian nerve fiber density that is associated with sympathetic hyperresponsiveness (Stener-Victorin et al., 2003). In this review, the administration of the herbal formulas Changbudodam-Tang and Yongdamsagan-Tang significantly decreased elevated NGF in the ovaries with little effect on brain tissue (Lee et al., 2003). HemoHIM also normalized NGF, lowered the high number of antral follicles, and increased the number of corpora lutea in PCOS. These results are consistent with those of previous studies on the beneficial effects of HemoHIM in the prevention and treatment of PCOS (Pak et al., 2009). In addition, administration of Korean red ginseng extract (Pak et al., 2009;Jung et al., 2011) and Korean red ginseng total saponins (Pak et al., 2005) significantly decreased the expression of NGF protein and NGF mRNA, compared with those in EV-treated ovaries.
This study reviewed the evidence for herbal medicines that may be used to treat PCOS and its associated symptoms, and the findings are intended to add to clinicians' understanding of the mechanisms of action of herbal medicines in PCOS treatment. The main limitation of our study is the heterogeneity of interventions. Furthermore, our study did not perform a quality assessment of each study or a quantitative synthesis of the outcomes. Further studies that include methodological quality assessment and quantitative synthesis of outcomes are warranted.

CONCLUSION
In this review, a total of 27 studies involving 22 herbal medicines exhibited beneficial effects on PCOS. Herbal interventions in the 27 studies comprised four compounds isolated from herbs (6 studies), nine individual herbal extracts (11 studies), and nine herbal formula decoctions (10 studies). Herbal medicines were shown to normalize female hormones, diminish male hormones, recover the estrous cycle, ameliorate insulin resistance, and improve lipid metabolism in PCOS. The mechanisms underlying the beneficial effects of herbal medicines on PCOS are associated with anti-inflammation, anti-oxidative stress, inhibition of autophagy and/or apoptosis, and ovarian NGF reduction. Herbal medicines can be considered as promising resources in the development of effective therapeutic agents for PCOS.

AUTHOR CONTRIBUTIONS
I-HC and KP designed the study. C-YK and KP searched the articles and analyzed the data. KP wrote the manuscript and I-HC revised it. All authors contributed to the article and approved the submitted version.