Chinese Journal of Natural Medicines 2014, 12(5): 0321−0334

Chinese Journal of Natural Medicines

Yang-tonifying traditional Chinese medicinal plants and their potential phytoandrogenic activity Munyangaju Jose Edouard 1, MIAO Lin 1, FAN Guan-Wei 1, Barnabas Bessem Orang Ojong 1, ZHEN Hu 1, ZHANG Ju 2, GAO Xiu-Mei 1, ZHU Yan 1, 3* 1

Tianjin State Key Laboratory of Modern Chinese Medicine and Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China;

2

College of Life Sciences and State Key Laboratory of Medicinal Chemical Biology, Nankai University, Tianjin 300071, China;

3

Molecular Cardiology Research Institute, Tufts Medical Center, 750 Washington St., Boston, MA 02111, USA Available online 20 May 2014

[ABSTRACT] The concept of phytoandrogens, plants that contain androgens or those that stimulate androgenic activity in men, is relatively new. In traditional Chinese medicine a number of phytoandrogens are classified in medicinal plant restoratives for reinforcing yang, and they find their application in the treatment of the kidney yang deficiency diseases. In this review, the phytoandrogens used in traditional Chinese medicine are listed, and their proven applications in the treatment of kidney yang deficiency diseases, such as sexual disorders, cancer, and obesity and associated metabolic syndromes are presented. As a background, the mechanism of action of androgens, their synthesis and metabolism, the interrelations of androgens and estrogens, as well as the state of art methods to detect and analyze these hormonal activities in medicinal plants are discussed. [KEY WORDS] Phytoandrogens; Yang-tonifying traditional Chinese herbs; Androgens; Kidney yang deficiency

[CLC Number] R965

[Document code] A

[Article ID] 2095-6975(2014)05-0321-14

Introduction While work on phytoestrogens, plants that contain female hormones is fairly advanced and widely known, the concept of phytoandrogens, meaning plants that contain androgens or those that stimulate androgenic activity in men is relatively new. Phytoestrogens have a much deeper history, and most clinicians have some idea of their existence [1]. Phytoandrogens do the same thing that phytoestrogens do, except that they supply androgens instead of estrogens for men. Phytoandrogens increase the body’s level of free testosterone, and they shift the body’s ratio balance androgen/estrogens towards the androgen

[Received on] 16-Aug.-2013 [Research funding] This project was supported by the Doctoral Fund of the Ministry of Education of China (RFDP No. 20111210110007), the Specialized Research Fund for the Doctoral Program of Higher Education (SRFDP No. 20111210120001), and the State Key Development Program for Basic Research of China (973 program, No. 2012CB723500) [*Corresponding author] ZHU Yan: Prof., E-mail: yanzhu.harvard@ gmail.com These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved

side of the equation. Androgens are steroid hormones which are necessary for normal male phenotype expression, including the outward development of secondary sex characteristics and the initiation and maintenance of spermatogenesis [2]. Conversely, over- active sex steroid (androgen and estrogen) receptors have been linked to increased risks of hormone-sensitive tumors such as prostate and breast cancers. Availability and binding of cognate ligands to the ligand-binding domain (LBD) of the sex steroid receptors are required for the proportionate expression of specific genes responsible for such sex hormone-mediated processes [3]. Estrogens and androgens are linked in many different ways. As testosterone plays a pivotal role in human (both male and female) physiology such as skeletal muscle development, bone density, fertility, and sex drive [4], estrogens also occur in females, and are present in males though generally at much lower levels, where they have a central role in male reproduction [5]. On the hand, a number of phytoandrogens have been reported to manifest androgenic and estrogenic activity by activating AR, ERα, or ERβ. It is important to know the extent to which the androgenicity and estrogenicity of these medicinal plants has been evaluated in order to maximize their benefit in clinical applications. In traditional Chinese medicine, a number of phytoandrogens are classified in medicinal plant restoratives for reinforcing yang, and they find their applications in the treatment of yang

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deficiency diseases which are characterized by metabolic disorders of body fluid, and physiological dysfunctions which are caused by intrinsic factors (e.g., aging and inherent defects) or extrinsic factors (e.g., over-fatigue and sexual overstrain) [6]. Here, a short overview of the androgens and androgen receptor roles in the human will be presented. Afterwards, kidney yang tonifying Chinese medicinal plants are discussed and their proven clinical applications in the treatment of kidney yang deficiency described. This review is a compilation of different data and information, retrieved from a variety of publications. Having a specific focus on the kidney yang tonifying traditional Chinese medicinal plants, multiple searches were performed in Pubmed using the search terms such as: kidney yang tonifying Chinese herbs, kidney yang deficiency, phytoandrogens, and androgens. By focusing this review, the potentially relevant sources identified were reduced from 755 articles to less than 100 articles. By associating either androgens or testosterone with the traditional Chinese medicinal plant of interest, further articles were found. In addition, interesting references listed in the published papers were reviewed. Androgens Androgens or male sex hormones are a class of steroid

hormones produced in the male by testicular Leydig cells, in the female by ovarian theca cells, and in both sexes by zona reticularis of the adrenal cortex [7]. Structurally they all belong to the 19-carbon androstane series of steroids. They are mainly responsible for male sexual differentiation, development, and maintenance of secondary male characteristics, and for the initiation and maintenance of spermatogenesis [8]. The vast number of molecules with biological androgen activity is always based on their ability to bind to and activate the androgen receptor (AR), a ligand-activated transcription factor, and to induce androgen-specific effects on gene expression. In addition, androgens can elicit rapid responses that have been termed ‘non-genomic’ to distinguish them from the direct effects of the AR on gene expression [9]. Androgen synthesis In the human male embryo, the testes begin to secrete androgens at nine weeks of gestation. The most important circulating androgen of gonadal origin is testosterone (T) which is mainly synthesized from cholesterol through a pregnenolone intermediate in Leydig cells of the testes. It is also synthesized in the adrenal cortex, liver, and ovaries in women [10] (Fig. 1).

Fig. 1 Testosterone synthesis[2]. Abbreviations: P450scc, cholesterol side-chain cleavage enzyme; HSD, hydroxy steroid dehydrogenase

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The rate-limiting step in testosterone synthesis, cholesterol side chain cleavage by P450scc, is regulated by luteinizing hormone (LH) from the pituitary, which is controlled by gonadotropin releasing hormone (GnRH) from the hypothalamus and the feedback regulation of testosterone at both the pituitary and hypothalamus levels [7]. In addition, adrenal androgens, such as androstenedione, dehydroepiandrosterone (DHEA), and its sulfate conjugates, are secreted by the adrenal cortex. They are the most abundantly circulating adrenal steroids in the blood; although not as potent as testosterone, adrenal androgens also contribute to other androgenic effects in the body [11]. In prostate cells, testosterone can then be converted to the more potent dihydrotestosterone (DHT) by the 5α-reductase (SRD5A1, SRD5A 2, and SRD5A 3) [12]. Steroid hormone-binding globulin (SHBG) is the circulating steroid-binding protein produced by the liver that binds testosterone with high affinity. It is an important regulator of androgen homeostasis and functions as a modulator of androgen delivery to the tissues. Serum SHBG levels are primarily regulated by sex steroids and thyroxine [13]. Testosterone is highly bound to plasma proteins, with 60% sequestered with high affinity to sex hormone-binding globulin (SHBG), while almost 40% is bound with low affinity to albumin, leaving only about 2% as free, unbound hormone [9]. Synthesis of SHBG and T is a highly correlative biochemical process. Hyperinsulinism, associated with adiposity, suppresses synthesis of SHBG and thus, levels of circulating T. Furthermore, insulin and leptin exert suppressive effects on testicular steroidogenesis and may contribute to further disruption of pulse amplitude of luteinizing hormone (LH) diminishing stimulation of the testicular steroidogenesis [14]. Testosterone metabolism Testosterone can be metabolized in either its target tissues or the liver. In androgen target tissues, testosterone can be converted to physiologically active metabolites. In the prostate gland, skin, and liver, testosterone is reduced to 5α-dihydrotestosterone (DHT) the most potent endogenous androgen, by 5α-reductase (type 1 or type 2) in the presence of NADPH [15]. On the other hand, a small amount of testosterone (0.3%) can also be converted to estradiol by aromatase through the cleavage of the C19 methyl group and aromatization of ring A, which mainly occurs in adipose tissue. This process also occurs in the brain, liver, and the ovaries of women [16]. In men, approximately 80% of the circulating estrogen arises from aromatization of testosterone in the adipose tissue, with the other 20% secreted by the Leydig cells in the testes. Both 5α-reduction and aromatization are irreversible processes [17]. Besides these pathways, testosterone can also be further inactivated in the liver through reduction and oxidation, followed by glucuronidation and renal excretion. It can be metabolized to androstenedione through oxidation of the 17β-OH group and androstanedione with 5α-reduction of ring A [2] (Fig. 2).

AR receptor mechanism of action Androgen receptor AR (NR3C4) is a ligand-dependent transcription factor and belongs to the family of nuclear hormone receptors which has 48 members in human[18]. Like other steroid receptors, AR is a soluble protein that functions as an intracellular transcriptional factor. This receptor’s function is regulated by the binding of androgens, which initiates sequential conformational changes of the receptor that affect receptor–protein interactions and receptor–DNA interactions [2] . Nuclear hormone receptor ligands enter cells by diffusion, where they bind to their cognate steroid receptors. Five major types of steroid receptors are known: those for estrogens, androgens, progestagens, glucocorticoids, and mineralocorticoids [19]. Before ligand binding, many steroid hormone receptors reside in the cytoplasm, where they are sequestered by binding to chaperone molecules such as heat shock proteins. The entry of ligands into the cell results in receptor binding causing a conformational change that releases heat shock proteins and allows the steroid hormone receptor to be translocated into the nucleus [20]. Although studies with AR in some experimental systems suggest that before ligand binding the receptor is cytoplasmic, it is possible that endogenous AR may in fact reside largely in the nucleus [21]. Translocated AR binds to the androgen response element (ARE), located in the promoter or enhancer region of AR gene targets. Recruitment of other transcription co-regulators (including co-activators and co-repressors) and transcriptional machinery further ensures the transactivation of AR-regulated gene expression [22] (Fig. 3). The specific mechanism of AR activation has grown increasingly complicated over the past decade from what was simply considered a ligand-receptor activation pathway in a much more complex system. Studies have provided insight into AR association with co-regulators involved in transcription initiation and on intramolecular interactions of the AR protein during activation. Several diseases, such as androgen insensitivity syndrome (AIS), prostate cancer, and spinal bulbar muscular atrophy (SBMA), have been shown to be associated with transactivation of the AR through a number of non-mutually-exclusive mechanisms. These include increased expression of the receptor, generation of mutations in the receptor, which can change its canonical regulation, and altered action of co-regulatory proteins, through cross talk with other signaling pathways, or through the action of constitutively active splice variants [11, 18, 22]. Interrelations between androgens and estrogens Estrogens belong to the sex hormones that regulate the female reproductive cycle and developmental processes, maintain pregnancy, and prepare the mammary glands for lactation. They are also responsible for the development and maintenance of feminine secondary sex characteristics [5]. Estrogens are also key regulators of growth, differentiation, and the physiological functions of other target tissues, including the male reproductive tracts, skeletal, nervous,

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Fig. 2 Testosterone metabolism UDP-glucuronosyltransferase

[2, 17]

: Abbreviations are as follows: G, glucuronide; HSD, hydroxy steroid dehydrogenase; UGT,

Fig. 3 Basic mechanism of the androgen receptor action through the classical genomic and rapid non-genomic pathways [7, 18]. Abbreviations: HSP, heat shock protein; AN, antagonist; A, agonist; AR, androgen receptor; MAPK, mitogen activated protein kinase; PIPK, phosphatidyl inositol phosphate kinase; TK, tyrosine kinase

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cardiovascular, digestive and immune systems [23]. They are synthesized in the ovaries, testes, and adrenal glands, and are also active metabolites of testosterone [5]. The fundamental components of the estrogen signaling system are the ligand, 17β-estradiol (E2), and the ERs, ERα and ERβ, two genetically distinct receptors [24]. E2 regulates human physiology by diffusion through the plasma membrane of target cells and signaling through intra-cellular hormone-specific estrogen receptors (ERs). Two distinct types of signaling can be mediated, often referred to as genomic and non-genomic, or non-genotropic, pathways [25]. Estrogens and androgens are linked in many different ways. An increasing amount of evidence points to important roles for estrogen receptors in prostate carcinogenesis and progression. Of the two estrogen receptors, estrogen receptor β is the most prominent within the prostate gland. Moreover, estrogen receptor β is the main target for phytoestrogens [26]. Narita et al, in their publication in 2010 support the hypothesis that estrogens, through both alpha- and beta-receptor variants mediate the AR signaling pathway [27]. Leo et al conducted a study on the expression of androgen receptors in breast cancer. Of the 189 total cases, 151 (80%) were positive and 38 (20%) were negative for AR. The majority of ERβ tumors were also positive for AR. Only 8 of the 151 ERβ cases were negative for AR. Therefore, AR expression in breast carcinomas appears to be a favorable prognostic factor. Its expression in ER-negative tumors can be exploited for an additional targeted therapy to be combined with aromatase inhibitors for maximum benefit [28]. As women age, their levels of circulating testosterone gradually decline, and affect their vital energy and feeling of "well-being", with other impacts such as the loss of muscle tone and strength, and sexual dysfunction in menopausal women [4]. Treatment with methyltestosterone (MT), a synthetic testosterone, alone or combined with hormone replacement therapy, has provided benefits for women in menopause with sexual dysfunction [29].

Methods of Detection of the Androgenic and Estrogenic Activity of the Medicinal Plants The identification of nuclear receptor–mediated endocrine activities is important in a variety of fields, ranging from pharmacological and clinical screening, to food safety, toxicological monitoring, and risk assessment. Traditionally, animal studies, such as the Hershberger and Allen-Doisy tests, are used for the assessment of androgenic and estrogenic potencies, respectively. To allow rapid analysis of the activities of new chemicals, food additives, and pharmaceutical compounds, high-throughput screening strategies have been developed [19]. Androgen levels are measured in current clinical practice almost exclusively by immunoassays based on an antibody’s ability to recognize the specific structure of a steroid molecule. The assays have variable specificity and sensitivity and the

immunoreactivity detected does not necessarily correlate with the overall androgenic bioactivity in the sample. This is because the basis of antigen recognition is structural, not functional, and the antibody does not have the same binding specificity as AR. An alternative to immunoassays is gas chromatography-mass spectrometry (GC-MS), a method that is very specific, but more labor-intensive and expensive for routine clinical use. Furthermore, although it measures accurately molecules with a specific chemical structure, it does not monitor their bioactivity [30]. The first hormone assays were based on the measurement of hormonal effects in vivo. The so-called Hershberger assay, widely used in drug and chemical testing, measures androgenic and anti-androgenic activity in castrated mice or rats by monitoring the weight response of their androgen-dependent tissues, such as the levator ani muscle, prostate, or seminal vesicles. These assays are insensitive and labor-intensive, but their functional relevance is good. The higher the androgenic dose of a sample, the higher is the weight gain of the organs [7] . These tests are based on the mechanism of action of compounds, and are able to measure activation or inhibition of specific cellular pathways. The mechanism of action of steroid hormones is well established, and opened opportunities for mechanism-based assays. The in vitro assays using primary cells or cell lines, often after genetic modification, are a novel alternative for in vivo bioassays. Their main advantages over in vivo assays are their high sensitivity and specificity, larger capacity, and better cost-effectiveness. However, the data generated by in vitro assays have limited value in terms of risk assessment, since they cannot fully replicate the complex differences between species. They are, for example, unable to monitor the metabolism of test substances, which is an important determinant of their activity in vivo [19]. Owing to the above limitations, in vitro assays may never totally replace in vivo bioassays, however, they can be good predictors of in vivo androgenic or estrogenic activity of a range of compounds, allowing prescreening and/or possible reduction of animal studies. Receptor binding assays identify compounds, including hormones, that have the potential to bind specifically to receptors. Briefly, the hormone standard or unknown sample is incubated with labeled (usually radiolabeled) ligand hormone in the presence of a receptor preparation. The extent to which the unknown sample replaces binding of the labeled hormone at the receptor correlates with its bioactivity. It is difficult to differentiate between agonists and antagonists in a binding assay, and both classes of compounds will therefore be referred as ligands [31]. The commonly used in vitro assays are presented here: Cell proliferation assays This is one of the simplest and most sensitive assays available for estrogenic compounds. In this assay, using an in vitro system with MCF-7 cells, termed the E-SCREEN assay,

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the ability of a test substance to stimulate cell proliferation is measured [7]. Only a few cell proliferation assays have been developed for androgens, and most of them use the human prostate cell line LNCaP-FGC developed in the early 1980s. LNCaP-FGC cells express a high level of the gain-of-function with the mutated form of AR, in which AR is activated by androgen and also by progesterone, estradiol, and anti-androgens. This assay is therefore not totally specific for androgens. However, although this cell line cannot be used for the specific screening of androgens and anti-androgens, it has been used extensively to study androgen effects on cell proliferation and gene expression [32]. MCF-7 cells stably transfected with human AR have been developed into an androgen-dependent cell proliferation assay, A-SCREEN, which is used for the screening of environmental chemicals [7]. Reporter gene assays These assays measure the level of expression of an androgen-dependent reporter gene in response to hormonal stimulation. They are the only method adapted for the measurement of androgen bioactivity in human serum. In these reporter gene assays, DNA sequences containing specific hormone-responsive elements are linked to the gene of an easily measurable protein (a reporter gene, such as firefly luciferase). When stably introduced into a cell line expressing the cognate receptor, or by double transfection with a receptor of interest, a specific reporter cell line is generated allowing for the large scale screening of compounds [7, 19]. Chromatin immunoprecipitation (ChIP) assays Chromatin is composed of DNA, proteins, and RNA. The chromatin structure is dynamic, and it controls gene expression, cell division, and DNA repair in response to extracellular signals. Chromatin is one of the most intensely studied structures in biology, and chromatin immunoprecipitation assays (ChIP) have proved to be a powerful means to investigate a host of DNA-dependent processes [33]. ChIP assays can be used to determine whether a transcription factor interacts with a candidate target gene, and are used with equal frequency to monitor the presence of histones with post-translational modifications at specific genomic locations [34-35]. While numerous studies have identified a clear connection between AR binding and the expression of target genes for a limited number of loci, they cannot discern direct AR targets from secondary transcriptional events. To identify the direct transcriptional targets of the AR, Massie et al used chromatin immunoprecipitation (ChIP) with on-array detection (ChIP-chip) in the androgen-responsive PrCa cell line LNCaP [36]. Li et al, used the chromatin immunoprecipitation microarray analysis to map AR occupied regions (ARORs) and histone H3 acetylation (AcH3) loci of a human genomic DNA, in order to have insights into the genomic sequences that are bound by the AR. With this technique, they have been able to show that many AR binding sites were not associated with nearby gene expression, despite the fact that they had

intrinsic positive regulatory activity in chromatin-independent assays. They highlighted that AR occupancy and associated histone acetylation are both necessary requirements for positive regulation [35]. There are mainly two types of ChIP, primarily differing in the starting chromatin preparation. The first uses reversibly cross-linked chromatin sheared by sonication called crosslinked ChIP (XChIP) [34], while the second called Native ChIP (NChIP), uses native chromatin sheared by micrococcal nuclease digestion [37]. The traditional method has several limitations, it takes several days to complete, requires DNA precipitations, which is time-consuming and tedious, and involves multiple tube transfers with risks of sample loss and contamination. Techniques using ChIP have been developed and refined, to make them work faster and more efficiently. A typical ChIP assay usually takes 4–5 days, and requires at least 106–107 cells. Presently, new techniques on ChIP could be achieved with as few as 100-1000 cells and be completed within one day. Some of those new techniques include the Fast ChIP assay (qChIP), and the Quick and quantitative ChIP (Q2ChIP) assay [33, 38].

Phytoandrogens The actions of phytoandrogens may broadly be divided into agonists and antagonists. An androgen agonist works with androgens thereby supporting, restoring, enhancing, or substituting for one or more of its functions. An androgen antagonist works against androgens, diminishing, quenching, or blocking one or more of the effects of androgens. Based on the way in which they exert their actions, the traditional perception of phytotherapeutic agents as only “phytohormones” is expanding to include phytohormonogenics and functional mimetics of hormones. The mechanism of action of different classes of phytoandrogens suggest three primary classes; cognate or true phytoandrogens, phytoandrogen-ogenic, and functional mimetics of androgens [39-40]. Cognate phytoandrogens are plant constituents with androgen-like structures. They possess weak hormone activity and may bind to androgen receptors resulting in the same type of response that the hormone would cause. Even though the type of response is the same, the intensity may be weaker, and/or the duration of the response (retention time) may be different. Protodioscin is a steroidal saponin from the aerial parts of Tribulus terrestris, and is considered in traditional medicine to be a reproductive tonic. As a result of its particular steroidal structure it has androgen-mimetic action, binding and activating the receptor of testosterone [41]. Eucommia ulmoides cortical extract also demonstrated androgenic activities by binding the androgen receptor and weakly activating AR transactivational function in a dose-dependent manner [3]. This class of metabolite also includes a clinically valuable group of “phytoantiandrogens”. These compounds bind to androgen receptors, although they exert an antagonistic functional response [3, 40].

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The class of phytoandrogen-ogenic compounds within the phytohormonogenics for androgens contains plants that augment the ability of the body to generate androgens. These plants may have a direct effect on the target tissue, thereby increasing hormone production within specific endocrine tissue, or they may have an effect on the hypothalamic-pituitary-gonadal axis, increasing gonadotrophic androgens or functions (e.g., the case of Suo Yang) [42]. The plants may also act by interference with the breakdown of androgens into estrogens, or their binding to SHBG or albumin (e.g., nettle root) [1]. The functional mimetics of androgens can cause the same physiological response of the hormone they are mimicking. They do not need to bind to an androgen receptor to cause a similar functional response as the androgens. These phytocrines may mimic one or more functions of androgens and may also be considered as functional agonists. For illustration, damiana (Turnera diffusa), which is widely used in traditional medicine as an aphrodisiac, has the ability to enhance engorgement of erectile tissue through its vasodilatory abilities due to the release of nitric oxide. This suggests that damiana is a functional mimetic of testosterone [41]. Icariin from Epimedii species is another good example; its testosterone mimetic properties, are believed to occur through the inhibition of phosphodiesterase type 5 (PDE5), an enzyme that breaks down cyclic guanosine monophosphate (cGMP) and produce a negative impact on penile erection [43]. Beside the three classes described above, some substances, which were not originally phytoandrogens, are reported to have effects similar to testosterone in animals. Genistein and daidzein, two isoflavones present in soy, are also classified as phytoestrogens since they are plant-derived non-steroidal compounds with estrogen-like biological activity [44]. The study of Maggiolini et al. (2002), on the effects of genistein and quercetin, another phytoestrogen from soybeans, demonstrated that genistein and quercetin at low concentrations induce the proliferation of LNCaP cells, acting as agonists for the mutant AR T877A. They are able to activate the receptor, inducing its nuclear accumulation in LNCaP cells, autoregulate AR mRNA and protein levels, modulate the expression of androgen target genes, such as PSA (prostate specific antigen) and PAP (prostatic acid phosphatase), and induce LNCaP cell proliferation as a counterpart of the aforementioned action. At concentrations eliciting transcriptional activity, both genistein and quercetin stimulate LNCaP cell growth, whereas at high levels, they become cytotoxic independently of AR expression [45]. In 2007, Chen et al. established an assay to assess the androgenicity of daidzein using androgen receptor (AR) cofactors to modulate the AR transcriptional activity. The dual luciferase data showed that daidzein can enhance androgenic effects in AR negative PC-3 cells co-transfected with AR and AR cofactors. In AR and ARA70 positive LNCaP cells, daidzein can enhance ARA55-mediated induction of AR transcriptional activity [46].

In Chinese traditional medicine, there are a number of phytoandrogens classified as medicinal plant restoratives for reinforcing yang. This category of medicinal plants is mostly sweet, salty, or pungent in flavor, with the action of warming and nourishing yang in the body [47]. According to TCM theory, kidney yang is the master of yang qi all over the body, therefore nourishing kidney yang is to warm all of the other organs to get rid or treat all the syndromes of yang deficiency in the body [47]. Studies based on Western pharmacology and clinical investigations in China found that patients with kidney yang deficiency develop the following syndromes [6]: Dysfunction of autonomic nervous system, hyperfunction of the parasympathetic nervous system, and hypofunction of the sympathetic system Myocardial infarction Low basal metabolic rate and low pulse and respiratory rates Decreased urinary catecholamine Degeneration or dysfunction of an endocrine gland, such as adrenal glands, pituitary glands , testes , ovaries Anemia, Sheehan’s syndrome, and Addison’s disease; and marked atrophy of the spleen and thymus. Examples of herbs with kidney yang tonifying properties (Table 1) The purpose of the yang tonic herbs is to reinforce the vital functions of the kidney, improve the function of the adrenal glands and the sexual functions, strengthen the bones and muscles, promote normal growth, and improve the immune system. Clinical applications of phytoandrogens Phytoandrogens have multiple clinical applications with the overall function being the direct supply of androgens such as testosterone, stimulation of the body’s production of androgens, or interference with the breakdown of androgens into estrogens, or their binding to SHBG or albumin. They may also work against androgens, diminishing, quenching or blocking one or more of the effects of androgens. In traditional Chinese medicine, their ability to treat the yang deficiency associated diseases by reinforcing the kidney yang, is used to treat several kinds of diseases. Kidney-yang deficiency (KD) is a typical pattern of kidney-disorder induced syndrome, characterized by metabolic disorders of body fluid and physiological dysfunctions which are caused by intrinsic factors (e.g., aging and inherent defects) or extrinsic factors (e.g, over-fatigue and sexual overstrain) [6]. Sexual disorders treatment The Chinese traditional medicinal plants listed as phytoandrogens are clinically used in the treatment of syndromes like impotence, infertility, and erectile dysfunction [56]. They are used to promote the circulation of blood, or strengthen the bones and tendons, to treat lower back pain, nocturnal emission, enuresis, or leucorrhagia. The toothed elliptic leaves and bark of the tree Eucommia ulmoides are used medicinally in herbal pharmacopoeias, such as Kampo (traditional Japanese medicine) and Zhong-Yao (traditional Chinese medicine) for indications such as the relief of back pain, to increase stamina, to strengthen bones and muscles, and to hasten recovery from fatigue. It is noted that these are male hormone-related

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Table 1 List of yang-tonifying Chinese herbs [47] No.

Scientific name

1

Herba epimedii

2

Cortex Eucommia

3

Fructus Psoraleae

4

Cordyceps sinensis

5

Rhizoma curculiginis

6

Cuscuta chinensis Lam., Semen Cuscutae)

7

Fructus Lycii, Lycii barbarum

8

Cornu Cervi Pantotrichum

9

Morinda officinalis

10

Herba Cistanches

Herba Cynomorri, 11 Cynomorium songaricum

12

Radix Dipsaci

13

Rhizoma Cibotii

14

Rhizoma Drynariae

15

Fructus Alpiniae oxyphyllae

16

Semen Juglandis

Chinese name

TCM clinical applications

It has been commonly used in the treatment of cardiovascular diseases and other chronic illnesses, such as infertility, amnesia, asthenia, impotence and senile functional diseases in China for over 2,000 years Nourishes the liver and kidneys, strengthens the bones and Du zhong muscles, and soothe the fetus. Invigorate the kidneys and strengthen yang, consolidate Bu gu zhi essence (jing) and reduce excessive urination. It can warm the spleen and relieve diarrhea. Nourish the lung, strengthen yang and body essence, relieve Dong chong xia cao, cough and dyspnea. It can replenish the vital essence and rou cong dong relieve constipation. It is indicated for kidney yang deficiency manifested as impotence or emission. It is indicated for weakness of kidney yang manifested as xian mao impotence, frigidity, and cold pain in the lower back and knees due to obstruction by invasion of wind-cold dampness. Indicated for kidney yang deficiency manifested as impotu si zi tence, lower back pain, erectile dysfunction, emission, enuresis, or leucorrhagia. Usually used in liver and kidney deficiencies that present gou qi zi with lumbar soreness, sexual dysfunction, dizziness, blurred vision, poor vision, cataracts, and diabetic symptoms Nourish kidney yang, promote the production of blood and essence, strengthen the bones and tendons. It is indicated for the treatment of deficient kidney yang manifested as weakness of the body, aversion to cold, cold extremities, impotence in men, frigidity in women, infertility, urination, soreness and pain in the lower back and knees, dizziness, tinnitus, gradual loss of hearing, and listlessness Indicated for the deficient kidney yang manifested as soreness and weakness in the lower back and knees, impotence, Bajitian premature ejaculation, infertility, frigidity, irregular menstruation, and cold sensation and pain in the lower abdomen. Tonify the kidneys and strengthen yang, moisten the intestines, and move feces. It is indicated for the deficient kidney yang manifested as soreness and weakness in the lower back and knees with fragile tendons and bones, impotence, infertility, frigidity, and constipation due to dryness in the intestines Tonify kidney yang, lubricate intestines, and promote bowel movement. It is used for the treatment of impotence, Suo yang amenorrhea, and infertility. It is also used to treat constipation due to dryness. Tonify the liver and kidneys, promote the circulation of blood, and strengthen the bones and tendons. It is indicated for deficiency of the liver and kidneys manifested as soreXuduan ness and pain in the lower back and knees, or weakness of the legs. Also indicated for menstrual flow, uterine bleeding, and threatened abortion (restless fetus). Tonify liver and kidney, to strengthen bones and tendons, and to expel wind and dampness. It is indicated for defiGouji, ciency of the liver and kidneys manifested as soreness and pain in the lower back and knees and motor impairment. Indicated for deficient kidneys manifested as lower back Gusuibu pain, weakness of the legs, tinnitus, deafness or toothache, and swelling and pain due to external trauma or injury. Warm and tonify the spleen and kidneys, prevent emissions, and stop diarrhea. It is indicated for the invasion of cold in the spleen and kidneys manifested as abdominal pain and Yizhiren vomiting, for kidney deficiency manifested as enuresis and seminal emissions, and for diarrhea and excessive salivation due to spleen deficiency. Tonify the lungs and kidneys, moisten the intestines, and free the bowels. It is indicated for lower back pain and Hutaoren weakness of the legs due to deficient kidneys, cough and asthma due to deficient lungs, and constipation due to dryness in the intestines Yin yang huo

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Phytoandrogenic activity/ kidney yang tonifying activity reference Sze et al. (2010) [48] Yu et al. (2007) [49] Ong et al. (2011) [3] Qingchi and Xiaojing (2002) [50] Min et al. (2008) [51] Guo et al. (2002) [52] Dong and Zhang (2010) [53] Hong et al. (2011) [6] Yang et al. (2008) [54] Qin et al. (2000) [55] Benson et al. 2012 [56] Yu et al. 2007 [57]

Tongming et al. 2011 [58] Aramwit and Wirotsaengthon (2012) [59]

Mengjuan et al. 2012 [60] Peng et al. 1997 [61]

Wu et al. [62]

Liu et al. (2012) [63]

Liu et al. (2012) [64] Niu et al. (2012) [65]

Mai et al. (2012) [66] Wang (2005) [67] Huang et al.( 2011) [68]

Li et al. (2012) [69]

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Continued No

17

Scientific name Semen Astragali complanati

18 Semen Allii tuberose

19

20

Fructus Cnidii

Panax ginseng

Chinese name

Shayuanzi

Jiuzi

Shechuangzi

Renshen

TCM clinical applications Tonify the kidneys and control essence, to nourish the liver, and brighten the eyes. It is indicated for the deficient kidneys manifested as impotence, seminal emissions, premature ejaculation, or leucorrhea, and blurred vision due to deficiency of liver blood. Tonify the liver and kidneys, strengthen yang, and control the essence. It is indicated for deficiency of kidney yang manifested as impotence and cold pain in the lower back and knees, and frequent urination or leukorrhea due to deficient kidneys. Warm the kidneys and strengthen the yang, dispel dampness, and kill worms. It is indicated for genital itching, swollen and painful scrotum, hemorrhoid pain, scabies, weeping skin lesions, and itching lesions. It is also used to treat the turbid white vaginal discharge caused by deficiencies of the spleen and kidney. Lower back pain, impotence and infertility are the other indications for this medicine. This medicinal plant is used to replenish qi, prevent collapse and strengthen yang, tonify the spleen and the lungs, promote body fluids, and relieve thirst. It is also used to calm the heart and soothe the mind. It is commonly used with the kidney yang tonic herbs to treat impotence by tonifying qi and yang.

pharmacological effects. The phytoandrogenic activity of this medicinal plant was mediated by plant triterpenoids binding cognately to the androgen receptor (AR) ligand-binding domain [3]. In the same perspective, the extract from Cynomorium songaricum (Suo Yang) is reported in traditional Chinese medicine to treating sexual dysfunction, infertility, deficient kidney function, and lumbar weakness, as well as for facilitating catharsis, as described in ancient Chinese medical books. A recent study by Liu et al. provides clues as to how C. songaricum could alleviate age-related syndromes, especially in terms of brain function, courtship behavior, and reproductive capacity, when it is provided at a younger age. The study provides some basic evidence supporting clinical treatment of replenishing the vital essence in traditional Chinese medicine [63] . In 2010, Yang et al. described the potent effect of C. songaricum extracts on promoting spermatogenesis in rat testes as mediated through glial cell-derived neurotrophic factor (GDNF) stimulation, supporting the traditional utilization of this medicinal plant for male sexual dysfunction [76]. A study by Cherdshewasart et al in Thailand suggests the use of the phytoandrogen Butea superba for the treatment of erectile dysfunction. The authors believe that B. superba may act primarily by increasing the relaxation capacity of the corpus cavernosum smooth muscles through cAMP phosphodiesterase inhibition, and may also affect the brain, triggering the improvement of the emotional sexual response [77]. Icarrin (ICA) is the major active extract of many Epimedii species, including Herba epimedii and Epimedium sagittatum, also called “Horny Goat Weed”. Both species have been demonstrated to play an important role in the treatment of erectile dysfunction. ICA was demonstrated to exert inhibitory activity against phosphodiesterase type 5 (PDE5) in vitro. Through its mechanism of action, it can

Phytoandrogenic activity/ kidney yang tonifying activity reference Huang et al. (2011) [70]

Wu et al. (2012) [71]

Liu et al. (2010) [72] Yuan et al. (2007) [73]

Aramwit and Wirotsaengthong 2012 [59] Yue et al. (2007) [74] Furukawa et al. (2006) [75]

block the degradative action of phosphodiesterase type 5 on cyclic GMP in the smooth muscle cells lining the blood vessels supplying the corpus cavernosum of the penis [78-81]. Erectile dysfunction (ED) is also caused by the impaired formation and action of nitric oxide (NO). Thus, replenishment of this molecule or intracellular cyclic GMP are expected to be the most promising therapeutic measures for patients with ED. NO is formed from L-arginine through catalysis by NO synthase (NOS) isoforms, neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS). NO from nerves, and possibly endothelia, plays a crucial role in initiating and maintaining an intracavernous pressure increase, penile vasodilatation, and penile erection, that are dependent on cyclic GMP synthesized with the activation of soluble guanylyl cyclase by NO in smooth muscle cells [82]. In 2010, Koizumi et al. showed that icariin isolated from Epimedii herba stimulates the release of NO by AR-dependent activation of eNOS in human umbilical vein endothelial cells (HUVECs). PI3K/Akt and MAPK-ERK kinase (MEK)/ERK1/2 pathways were involved in the phosphorylation of eNOS by icariin [83]. These effects support the use of ICA for the management of sexual disorders. Ginseng, a precious Chinese traditional medicinal plant, has been known clinically used in China for thousands of years. In TCM, ginseng is used as a tonic to invigorate a human’s physical, mental, and sexual capability [74]. An in vitro study using a crude extract of ginseng saponins showed the relaxation effect on rabbit corpus carvernosum smooth muscle, suggesting that some components of ginseng may be nitric oxide donors [59]. This is supported by a study done by Furukawa et al [75] who provide compelling evidence that ginsenoside Re, a main phytosterol of Panax ginseng, activates endothelial NO synthase (eNOS) to release NO. The eNOS

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activation was found to occur through a non-genomic pathway of each of androgen receptor, estrogen receptor, and progesterone receptor, in which c-Src, phosphoinositide 3-kinase, Akt, and eNOS are sequentially activated. This finding might provide scientific evidence for traditional claims that ginseng enhances sexual potency. Cancer treatment Fighting cancer is considered one of the most important areas of research in medicine and immunology. Some phytoandrogens have been explored for their potential role in the treatment of cancer through their ability to penetrate the cell membrane and interfere with particular signal transduction pathways linked to processes such as inflammation, cell differentiation and survival, carcinogenesis, and metastasis. One such crucial pathway involved in the above mentioned processes, is the activation of the nuclear transcription factor kB (NF-κB). Polysaccharides, sterols, lipids, nucleosides, and deoxy-nucleosides from Cordyceps sinensis have shown an antitumor effect by strongly inhibiting IκBα, and by a degradation and a moderate inhibition of IκBα, phosphorylation [84]. A study by Gan et al (2004) on the tumor inhibition activity of Fructus Lycii, the fruit of Lycium barbarum, showed that the polysaccharides from L. barbarum (LBP) can increase interleukin-2 (IL-2) and tumor necrosis factor-α level, two cytokines that can inhibit tumor growth. From the results of this study, LBP could significantly inhibit the growth of transplantable sarcoma S180 in S180-bearing mice [85]. This suggests LBP might also contribute to antitumor activity. Prostate cancer is one of the most common types of cancer in men. Although conventional medicine has greatly improved early diagnosis and treatment options, advanced metastatic prostate cancer remains a serious disease. Modern chemotherapy has had only a limited success in treating metastatic prostate cancer, and this is where TCM can provide a valuable alternative approach to reduce pain and improve the quality of life of patients [86]. Here, the applications of phytoandrogens and yang tonifying medicinal plants in the treatment of prostate cancer are described. Equiguard is a dietary supplement comprised of standardized extracts from nine herbs, respectively, the Epimedium brevicornu Maxim (stem and leaves), Morinda officinalis F.C.How (root), Rosa laevigata Michx. (fruit), Rubus chingii Hu (fruit), Schisandra chinensis (Turz.) Baill. (fruit), Ligustrum lucidum W.T.Aiton (fruit), Cuscuta chinensis Lam. (seed), Psoralea corylifolia L. (fruit), and Astragalus membranaceus Moench. (root). This proprietary product, formulated according to Chinese traditional medicinal concepts, is aimed at restoring harmony in the primordial (original) yin-yang of the kidney, an organ which Chinese medicinal principles consider to be vital for invigorating, as well as maintaining, balance of the entire urological system. As the prostate is an integral component of the urological system, Hsieh et al performed in vitro studies to test the effects of ethanol extracts of Equiguard to modulate prostate growth and gene expression using prostate cancer

cells mimicking the androgen-dependent (AD) and androgen-independent (AI) states of prostate carcinogenesis. The results showed that Equiguard significantly reduced cancer cell growth, induced apoptosis, suppressed expression of the androgen receptor (AR), and lowered intracellular and secreted prostate specific antigen (PSA), and almost completely abolished the colony-forming abilities of prostate cancer cells [87] . The anti- prostatic activities of Equiguard may stem from its complex composition capable of targeting multiple signal transduction and metabolic pathways, and to effectively correct, counteract, or circumvent the impaired or dysfunctional mechanisms accompanying different stages of prostate carcinogenesis. Using Western blot analysis, Lu et al. (2004), showed that treatment caused inhibition of retinoblastoma protein (Rb) phosphorylation, which was accompanied by reduction of cyclins D/E expression, in androgen-proficient and androgen- deficient conditions. Moreover, cells treated with Equiguard and cultured with FBS-supplemented media showed up-regulation of cyclin-dependent kinase inhibitor Kip1/p27, supporting the interpretation that suppression of Rb phosphorylation mediated the observed growth arrest induced by Equiguard under androgen-proficient conditions. In contrast, Equiguard-treated cells cultured in CS-FBS had lowered expression of Kip1/p27, suggesting that different control mechanisms, possibly evoked by changes in cellular microenvironments, contributed to growth suppression by Equiguard [88-89]. Lycium barbarum polysaccharides (LBPs) are important functional constituents in the red-colored fruits of L. barbarum. This is known as Guo Qi Zi, a well-known traditional Chinese medicinal plant commonly known as Goji berry or wolfberry. The in vitro effects of LBP on two cell lines, PC-3 and DU-145, showed that LBP can dose- and time-dependently inhibit the growth of both PC-3 and DU-145 cells by causing the breakage of DNA strands of these cell lines; LBP also markedly induced PC-3 and DU-145 cell apoptosis, with the highest apoptosis rates at 41.5% and 35.5%, respectively, by regulating the expression of Bcl-2 and Bax in both cell lines. The ratio of Bcl-2/Bax protein expression following LBP treatments decreased significantly with a dose-effect relationship. The in vivo effect of LBP on PC-3 cells was assessed in the nude mouse xenograft tumor model with the results indicating that LBP might significantly inhibit PC-3 tumor growth. Both the tumor volume and weight of the LBP treatment group were significantly lower than those of the control group [90]. Cola acuminate, commonly known as Kola nuts, is a medicinal plant well-known in different parts of the world, including Asia and Africa, for its multiple applications in traditional medicine [91-92], and it is documented in various pharmacopoeiae [93]. The efficacy and mechanism in preventing prostate cancer of this medicinal plant are worth mentioning. A study by Solipuram et al (2009), characterized the androgenic and chemopreventive properties of kola nuts using

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receptor androgen dependent (LNCaP), along with the androgen-independent (DU145) prostate tumor cell models to ascertain the androgenic effect of this medicinal extract. Exposure of prostate cells to Biz-2, an ether extract of C. acuminata resulted in growth inhibition, and apoptosis in both cell lines, with a sharp decrease of the PSA in LNCaP. Biz-2 elicited a time- and dose-dependent stimulation of both the protein and mRNA levels of several androgen-regulated genes. Again, Biz-2 was found to be a specific ligand for the AR in that the natural ligand, DHT, and the anti-androgen, flutamide, displaced Biz-2 bound to AR, and inhibited Biz-2-induced transcription and PSA secretion. This study provided evidence that the Biz-2 extract possesses the ability to modulate prostate cancer cell biology in an AR-dependent manner [94]. Various studies present the numerous pharmacological activities of the Panax ginseng. Of several products of its root, red ginseng, having many bioactive ginsenosides, is most popularly used in Korea, and recently has been reported to control the proliferation of cancer cells. The water extract of Korean red ginseng WKRG (daily intraperitoneal injection) prevented prostate overgrowth and epithelial thickening induced by testosterone in rats, and suppressed rat prostate kallikrein-S3. In human prostate cells, WKRG inhibited testosterone-induced cell proliferation, arrested cell cycle by inducing p21 and p27, and induced apoptosis. Testosterone-induced expression of human kallikrein-3 mRNA and activation of androgen receptor (AR) were effectively inhibited by WKRG. 20(S)-Rg3, a major ginsenoside in WKRG, was shown to repress AR activity and to attenuate prostate cell growth during testosterone stimulation. Moreover, 20(S)-Rg3 down-regulated AR by facilitating the degradation of AR protein. WKRG and 20(S)-Rg3 were found to have new pharmacological activities against testosterone-induced prostate overgrowth. Given that red ginseng has been used safely in Asia for 1000 years, red ginseng and 20(S)-Rg3 could be potential therapeutic regimens for treating benign prostatic hyperplasia (BPH) [95-96]. Antioxidative activity Oxidative damage is a critical etiological factor implicated in several chronic human diseases such as diabetes mellitus, cancer, atherosclerosis, arthritis, neurodegenerative diseases, and in the ageing process [97]. Antioxidants have been reported to prevent oxidative damage caused by free radicals, and can be used to treat cardiovascular and anti- inflammatory diseases. A number of phytoandrogens possess antioxidant activity which has been exploited in therapeutics for a very long time. From the viewpoint of traditional Chinese medicine (TCM), Rhizoma Cibotii has the effects of tonifying the liver and the kidney, strengthening bones and tendons, relaxing the meridians, and dispelling wind-dampness. Recently, Mai et al. published a study on the antioxidant activity of Rhizoma Cibotii in vitro, they attributed the pharmacological effects of this plant on its antioxidative activity, which may result from

radical-scavenging and reducing power, attributed to the total phenolics, among which caffeic acid is regarded as one of bioactive compounds [66]. Herba Epimedii is widely used in an effective remedy for cardiovascular diseases, osteoporosis, and for improving sexual and neurological functions, as seen in the previous paragraphs. It also contains antioxidant constituents, such as total flavonoids, icariin, polysaccharides, and vitamin C, which are proven to be effective against oxidative-stress related pathologies (cardiovascular diseases, Alzheimer’s disease, and inflammation) in animal rodent models and in in vitro studies. These antioxidative properties were found to be related to an inductive effect on endogenous free radical scavenging enzymes, such as catalase and glutathione peroxidase, and the inherent electron-donating ability of flavonoids [48]. Obesity and associated metabolic syndromes In addition to its role as a critical steroid hormone in reproductive and sexual functioning, testosterone is an important signaling molecule in regulating energy utilization and multiple cellular metabolic pathways, including nitrogen retention and regulation of adipogenesis [14]. By increasing and maintaining muscle mass and reducing fat mass, it plays an important role in the regulation of the body composition [98]. Androgen deficiency is associated with insulin resistance (IR), type 2 diabetes (T2D), metabolic syndrome (MetS), and increased deposition of visceral fat, which serves as an endocrine organ, producing inflammatory cytokines, and thus promoting endothelial dysfunction and vascular diseases [99]. Testosterone treatment reverses fat accumulation with significant improvement in lean body mass, insulin sensitivity, and biochemical profiles of cardiovascular risk [14]. However, androgen pharmacotherapy with testosterone, dihydrotestosterone (DHT), and anabolic steroids carries many limitations, such as the requirement for parenteral or transdermal formulations, and severe side effects that include hypertension and hyperandrogenism. As such, development of orally bioavailable phytoandrogen agents that can modulate the AR, without the limitations associated with androgen replacement therapy, is critical in enabling the treatment of the obesity-induced testosterone deficiency diseases and the associated metabolic diseases [40].

Conclusions The concept of phytoandrogens is relatively new when compared to that of phytoestrogens. Their characterization, classification, and understanding their mechanism of action could benefit from the advanced techniques applied to phytoestrogen studies. Metabolic conversion and crosstalk may also exist between these two classes of sex hormone mimics. In traditional Chinese medicine, a number of phytoandrogens are classified in restoratives medicinal plants for reinforcing yang with the action of reinforcing the vital functions of the kidney to treat metabolic disorders of the body fluids and the physiological dysfunctions associated with kidney yang defi-

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ciency. Since some phytoandrogens also exhibit estrogenic activity, particular attention must be paid to ensure safety and to achieve maximum benefit in their clinical applications.

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Cite this article as: Munyangaju Jose Edouard, MIAO Lin, FAN Guan-Wei, Barnabas Bessem Orang Ojong, ZHEN Hu, ZHANG Ju, GAO Xiu-Mei, ZHU Yan. Yang-tonifying traditional Chinese medicinal plants and their potential phytoandrogenic activity [J]. Chinese Journal of Natural Medicines, 2014, 12 (5): 321-334

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