Herba Cistanche (Rou Cong-Rong): One of the Best Pharmaceutical Gifts of Traditional Chinese Medicine

Cistanche species, known as Rou Cong-Rong in Chinese, are an endangered wild species and are mainly distributed in the arid lands and warm deserts of northwestern China. Within Traditional Chinese Medicine (TCM), Herba Cistanche is applied as a tonic and/or in a formula for chronic renal disease, impotence, female infertility, morbid leucorrhea, profuse metrorrhagia, and senile constipation. The chemical constituents of Herba Cistanche mainly consist of volatile oils, non-volatile phenylethanoid glycosides (PhGs), iridoids, lignans, alditols, oligosaccharides, and polysaccharides. There have been an increasing number of studies focusing on its bio-activities, including antioxidation, neuroprotection, and antiaging. The objective of this review is to introduce this herb to the world. Its taxonomy, distribution, and corresponding biological functions and molecular mechanisms are addressed in this review.

FIGURE 1 | Timeline of Herba Cistanche records in the traditional Chinese herb classic texts. The uses of Herba Cistanche have evolved over nearly 2,000 years. Herba Cistanche was described in the oldest surviving herb classic, Shennong Bencao Jing (approximately 100 A.D.). Since then, it has been described in many famous traditional Chinese herb classics spanning different dynasties, including Yaoxinglun, The Compendium of Materia Medica (Bencaogangmu), and Benjingfengyuan. It was the most frequently prescribed drug against chronic renal disease in China for successive dynasties. In 2000 and 2005, C. deserticola was reordered in the Chinese Pharmacopoeia (2000), and C. tubulosa was added to the Chinese Pharmacopoeia (2005) as an alternative. Products manufactured in China that are made from Herba Cistanche (e.g., its extracts) or that include the herb (e.g., prepared formulas) are not restricted.
impotence, female infertility, morbid leucorrhea, profuse metrorrhagia, and senile constipation (Zhang et al., 2005). In 2000 and 2005, respectively, C. tubulosa, and C. deserticola were indexed in the Chinese Pharmacopeia (Pharmacopoeia, 2000) (Figure 1). C. tubulosa is offered as an alternative for C. deserticola because of its similar chemical constituents and pharmacological activities and its abundance (Pharmacopoeia, 2005). Other species of this genus, e.g., C. salsa and C. sinensis, are also used as alternatives in some areas. Cistanche is considered a new cultivated plant in several regions in northwestern China, where the rainfall is low and soil desertification is severe.
Since the 1980s, researchers have been interested in Herba Cistanche. A chemical analysis of Herba Cistanche revealed that essential oils, phenylethanoid glycosides (PhGs), iridoids, lignans, alditols, oligosaccharides, cistanosides, and polysaccharides were the main constituents (Jin and Zhang, 1994). Herba Cistanche extracts are pharmacologically active, with a range of functions that include improving chronic renal disease and senile constipation, increasing learning/memorizing ability, treating Alzheimer's disease (AD), and improving immunity (Snytnikova et al., 2012;Zhang et al., 2012;Guo et al., 2013;Li et al., 2013a;Nan et al., 2013). Most pharmaceutical companies are hoping to find the next 'miracle' drug, such as artemisinin (qinghaosu), an antimalarial drug that is extracted from the medicinal plant sweet wormwood and has saved millions of lives. The other classic example of a modern medicine that originated in TCM is arsenic trioxide, which was approved by the US Food and Drug Administration (FDA) to treat leukemia in 2000. The present overview focuses on the progress of the study of chemical constituents of Herba Cistanche and some of its relevant pharmacological activities.

Volatile Compounds
In essential oil of the Herba Cistanche, alkanes, alcohols, aldehydes, and heterocyclics were detected, and palmitic acid, linoleic acid, 14-methylpentadecanoate, ethyl palmitate, and 2,5,6-trimethyloctane were identified. The total oils of C. tubulosa mainly consist of palmitic acid and linoleic acid, while the essential oil of C. salsa includes alkanes, alcohols, aldehydes, and some heterocyclic compounds (Jiang and Tu, 2009). The volatile compounds of Herba Cistanche, like the essential oils, commonly can be extracted by steam distillation or lipophilic organic solvent. By extracting C. deserticola with petroleum ether and then analyzing the sample on a GC-MS, 25 volatile compounds were identified, and the 3 richest constituents in the petroleum ether extract are methyl 14-methylpentadecanoate (13.61%), ethyl palmitate (12.39%), and 2,5,6-trimethyloctane (7.60%) (Jiang and Tu, 2009).

Non-Volatile Compounds
Among the non-volatile compounds of Herba Cistanche, more than 100 have currently been isolated and identified. These compounds are mostly PhGs, iridoids, lignans, alditols, oligosaccharides, and polysaccharides. As an important class of the compounds that constitute Herba Cistanche, PhGs have been well studied ( Table 1). To date, 34 PhG compounds have been successfully isolated from Herba Cistanche, including 22 disaccharide glycosides, 10 trisaccharide glycosides, and 2 monosaccharide glycosides. The empirical structural features of PhGs are as follows.
(1) For disaccharide glycosides, the sugar moiety consists of glucose and rhamnose connected by a Glc (3 → 1) Rha linkage; the glucose commonly links directly to an aglycone, and a coumaroyl or caffeoyl is usually located at the C4 or C6 position. (2) For trisaccharide glycosides, there is another glucose or rhamnose at the C6 position of the inside glucose. Recently, Li et al. (2015) completed the first deep transcriptome sequencing of the fleshy stem of C. deserticola by RNA-seq and identified some key enzyme genes and pathways that are involved in the biosynthesis of lignin and PhGs, which provides valuable information for this medical plant.
Frontiers in Pharmacology | www.frontiersin.org glycosides have been isolated from C. deserticola and C. tubulosa, respectively. Only 2 alkaloids, betaine and N,N-dimethyl glycine methyl ester, have been isolated from Herba Cistanche (Jiang and Tu, 2009). Small amounts of other compounds, such as phenolic glycosides, sterols or their glycosides, fatty acids, amino acids, and trace elements, are also present in Herba Cistanche (Snytnikova et al., 2012).

PHARMACOLOGICAL ACTIVITIES
Based on the neuroprotection, immune-enhancement, and sexual health properties of Herba Cistanche, we had a discussion in the paper (Supplementary Figure S1).

Improvement of Brain Function
When considering learning and memory, three levels of mechanisms are involved: (1) the ability to acquire memory, i.e., learning ability; (2) the ability to store memory, i.e., consolidation; and (3) the ability to recall memorized information (Choi et al., 2011). The C. tubulosa extract was confirmed to significantly improve these mechanisms by preventing brain neuron apoptosis through the expression of apoptosis-related factors and neurotrophic factors in MES23.5 cells (Lin et al., 2013). C. tubulosa extract, containing rich echinacoside, and acteoside, can alleviate the cognitive dysfunction caused by Aβ 1−42 through blocking amyloid deposition, reversing cholinergic, and hippocampal dopaminergic neuronal function in AD-like rat model (Wu et al., 2014). One of the PhGs, echinacoside, is typically known as the main phenolic component in the roots of Echinacea angustifolia, which is widely used in Europe and North America for its immunoregulation properties. A recent study determined that echinacoside can rescue human fibroblasts (SHSY5Y) from TNFα-induced apoptosis. The results indicated that echinacoside protects the damaged fibroblasts by regulating the reactive oxygen species level in fibroblasts and the activation of caspase-3 (Zhao et al., 2010). In vitro and in vivo experiments have confirmed that individual PhGs can inhibit the apoptosis of neuronal cells induced by various chemicals (Tian and Pu, 2005;Geng et al., 2007). Therefore, the individual PhGs could be attractive candidates against some typical neurodegenerative disorders, such as dementia or Parkinson's disease (PD).
Moreover, an open-label, non-placebo-controlled study on C. tubulosa glycoside capsules (Memoregain R ) demonstrates that the drug has a potential to be a possible treatment option for mild to moderate AD, and all adverse reactions were mild. (Guo et al., 2013).
Similarly, the C. salsa extract can accelerate the proliferation of fibroblasts and promote the production of neurons by accelerating the growth of the neurites. It also has some properties as follows: prevent damage caused by cerebral ischemiareperfusion; protect against apoptosis of the CA1 region of hippocampus (Wang et al., 2004); and increase the amount of neurotransmitters, such as dopamine (DA), noradrenaline (NA), and serotonin (5-HT), in the rat brain (Chen et al., 2007;Choi et al., 2011;Zhong et al., 2012). The acteosiderich fraction of C. salsa extract can inhibit reactive oxygen species, prevent DNA damage, enhance superoxide dismutase (SOD) activity and prevent lipid peroxidation (Lin et al., 2002;Deng et al., 2004b;He et al., 2009a). Because acteoside has an extremely strong antioxidative effect that is 15 times stronger than resveratrol and five times stronger than vitamin C (Chiou et al., 2004).

Aphrodisiac Effect
A recent study has demonstrated that an ethanol extract of C. tubulosa could increase the sex hormone levels by inducing testicular steroidogenic enzymes (e.g., CYP11A1, CYP17A1, CYP3A4) . Besides, the gene expression of 3β-hydroxysteroid dehydrogenase (3β-HSD), which is responsible for the synthesis of testosterone, 5α-reductase-2 and aldo-keto reductase (enzymes that are responsible for the synthesis of dihydrotestosterone), can be induced by the C. tubulosa extract, suggesting the positive effect of C. tubulosa extract on male hormone production (Shimoda et al., 2009). The acteoside, which was distilled from C. tubulosa, significantly shortened the latent period of penis erection (p < 0.01), increased the number of germ cells (p < 0.01), and improved pathological changes in the testes . Echinacoside, which is another newly identified PhG and is typically known as the main component of echinacea, is abundant in C. tubulosa and possesses vaso-relaxing activity . The aphrodisiac mechanisms of C. tubulosa might involve the NO-cGMP signal transduction pathway, with increasing cGMP levels in the corpus cavernosum smooth muscle (He et al., 2009b). Pan and Min (2004) indicated that the combined use of Herba Cistanche extract could prevent adrenal cortical atrophy, which is typically caused by using corticosteroids only. Moreover, C. deserticola extract could reverse the reproductive toxicity in mice induced by hydroxyurea (Gu et al., 2013) and glycoside of Leigongteng (Radix et Rhizoma Tripterygii) .
According to TCM, Yang-Qi Kidney-Yang Deficiency Syndrome (KDS-Yang) is caused by insufficient "Yang-Qi" in the kidney. Briefly, Yang-Qi is a TCM term and likely indicates mitochondrion-driven biological activities of the human body in the view of biomedical research (Leong et al., 2015). In H9c2 cardiomyocytes, Herba Cistanche was proven to enhance the mitochondrial respiration and glutathione antioxidant status (Wong and Ko, 2013). Yang-Qi deficiencies in TCM resemble those of chronic fatigue syndrome in Western medicine. KDS-Yang symptoms include soreness and weakness of the waist and knees, cold chills, deafness, and tinnitus. Modern studies showed that damage and functional disorders of the hypothalamic-pituitary-target gland axis, including the adrenal gland, thyroid, and gonad, are the main pathological mechanisms of KDS-Yang . Gong et al. (2008) investigated the intervention effect of C. deserticola (decoction, 10 g/kg·d) in hydrocortisone-induced KDS-Yang model rats and found that C. deserticola extract could increase body weight, autonomic activity, and swimming time while decreasing post-exercise blood lactic acid (LAC) and blood urea nitrogen (BUN). As described in the Compendium of Materia Medica, Herba Cistanche is mild and would not be extremely abrupt or harsh when treating KDS-Yang. Interestingly, KDS-Yang also leads to low and disordered immune function, and thus, there is a close relationship between treating KDS-Yang and improving immune function (Yim and Ko, 2002).

Immune-Boosting Effect
Besides its traditional use, the daily consumption of Herba Cistanche is believed to the key of the people with longevity in some region of China and Japan where known for longevity and oasis. Studies shown that both C. salsa extract and C. tubulosa extract can activate lymphoid cells and increase the killed rate of cancer cells (Maruyama et al., 2009). C. deserticola extract can activate the phagocytic function of macrophages in mice and enhance body immunity (Li et al., 2009). Carbohydrates account for a high proportion of the dry mass of Herba Cistanche. The polysaccharides of C. deserticola are closely related to the immunity enhancing and anti-cancer functions (Xu et al., 2011). Galactitol is one of the monosaccharides in Herba Cistanche with laxative activity (Baishun et al., 2003). Individual compounds such as oligosaccharides present an excellent effect on the spleen activity of mice, increase the phagocytotic activity of macrophages and stimulate the proliferation of antibody-producing cells (Maruyama et al., 2008). Acteoside (10 or 50 mg/kg subcutaneously) significantly inhibits hepatic apoptosis, hepatitis and lethality in mice with hepatic apoptosis and liver failure were induced by D-galactosamine (DGalN) and lipopolysaccharide (LPS) (Xiong et al., 1999). Echinacosideenriched extract of C. tubulosa is effective in preventing dextran sulphate sodium (DSS)-induced colitis in mice (Jia et al., 2014). Recently, Zhang et al. (2014) discovered that C. deserticola extract could antagonize immune-related senescence and extend the lifespan in SAM-P8 mice. In Zhang's et al. (2014) study, dietary supplementation with C. deserticola extract can decrease the level of peripheral memory T cells and enhance levels of naive T cells. Additionally, Herba Cistanche aqueous extract was proven to prevent bone loss caused by ovarian hormone deficiency through regulating some bone metabolism related genes (e.g., Smad1, Smad5, TGF-b1, and TIEG1) (Liang et al., 2011(Liang et al., , 2013, and amonoterpene from C. salsa has been identified as an anti-osteoporotic compound (Yamaguchi et al., 1999). Herba Cistanche helps people better understand the synergistic effect mechanisms of the effective components in TCM.

CONCLUSION
Herba Cistanche has been commonly used traditionally for enhancing immunity, sexual health, antioxidation, and neuroprotection, and tonic. Various Herba Cistanche products and its derivative are widely used in modern China. The uses of this drug have evolved over nearly 2,000 years, and the demand for Herba Cistanche has grown rapidly in recent years. After a long-term use, those compounds with proven pharmacological activity, such as acteoside, and echinacoside, deserve more indepth study before they truly could improve patient quality of life. With further study, this important TCM is believed to have profound prospects.

AUTHOR CONTRIBUTIONS
HL drafted the table. LG and JG designed the figure. ZL and CT drafted and revised the manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fphar. 2016.00041 FIGURE S1 | Main biological functions and mechanisms of the active components of Herba Cistanche. The active ingredients isolated from Herba Cistanche have three main biological functions: improvement of brain function, immune-boosting effect, and nourishing aphrodisiac effect. Herba Cistanche extracts are mixtures of multiple active compounds, and a typical traditional Chinese medicine (TCM) recognizes multiple biological targets. Phenylethanoid glycosides (PhGs) have neuroprotective properties in neurodegenerative disease based on their free radical scavenging ability, anti-apoptosis activity, anti-inflammation activity, the increasing amount of brain neurotransmitters and enhanced SOD activity. A modern use of Herba Cistanche in Chinese herbalism is to treat KDS-Yang, which contributes to fertility problems (including impotence and female infertility) because Herba Cistanche is different from other herbs that often have a more vigorous action. Herba Cistanche regulates the hypothalamic-pituitary-gonad (HPG) and hypothalamic-pituitary-adrenal (PHA) axes, which may induce a smooth and balanced sexual vitality effect. Moreover, phenylethanoid oligoglycosides and acylated oligosaccharides from Herba Cistanche have vasorelaxant activity via the NO-cGMP pathway. Based on clinical and experimental data, activation of the HPA axis increases plasma cytokines and acute phase protein concentrations. Thus, it can increase phytohemagglutinin-stimulated lymphocyte proliferation and enhance antibody production in response to T-dependent antigens in spleen cells.