Gymnadenia conopsea (L.) R. Br.: A Systemic Review of the Ethnobotany, Phytochemistry, and Pharmacology of an Important Asian Folk Medicine

Gymnadenia conopsea (L.) R. Br. (Orchidaceae) is a perennial herbaceous orchid plant that grows widely throughout Europe and in temperate and subtropical zones of Asia. In China, its tuber has been used in traditional Chinese medicines, Tibetan medicines, Mongolian medicines and other ethnic medicines, and taken to treat numerous health conditions. The present paper provides a review of the traditional uses, phytochemistry, biological activities, and toxicology to highlight the future prospects of the plant. More than 120 chemical compounds have been isolated, and the primary components are glucosides, dihydrostilbenes, phenanthrenes, aromatic compounds, and other compounds. G. conopsea and its active constituents possess broad pharmacological properties, such as the tonifying effect, anti-oxidative activity, anti-viral activity, immunoregulatory, antianaphylaxis, antigastric ulcer, sedative, and hypnotic activities, etc. However, overexploitation combined with the habitat destruction has resulted in the rapid decrease of the resources of this plant, and the sustainable use of G. conopsea is necessary to study. Meanwhile, the toxicity of this plant had not been comprehensively studied, and the active constituents and the mechanisms of action of the tuber were still unclear. Further, studies on G. conopsea should lead to the development of scientific quality control and new drugs and therapies for various diseases; thus, its use and development require additional investigation.


INTRODUCTION
Gymnadenia conopsea (L.) R. Br. (Orchidaceae) is a perennial herbaceous flowering plant that is distributed from 200 to 4700 m altitude throughout northern Europe, including England, Ireland, Russia, etc., and temperate and subtropical zones in Asian countries, including Nepal, China, Japan, and the Korean peninsula (Commission of Flora Reipublicae Popularis Sinicae, 2004; http://frps.eflora.cn/frps). For thousands of years, due to the prominent effects on invigorating the spleen, nourishing the lungs and blood, regenerating body fluid, and controlling bleeding with astringents, the tubers of G. conopsea was ascribed as a reinforcing agent of traditional medicines in China. It has been primarily used to treat kidney asthenia, cough, and dyspnea induced by lung asthenia, consumption diseases, neurasthenia, chronic diarrhea, morbid leucorrhea, chronic hepatitis, and other diseases in some Asian countries (Chinese Materia Editorial Committee, State Chinese Medicine Administration Bureau, 2002). Because the contour of the tuber is similar to the palm of the human hand, the tuber was given the Chinese name Shou Zhang Shen, meaning "ginseng likes palm hands" (Figure 1). In 1977, the tuber of G. conopsea was listed in the Pharmacopeia of the People's Republic of China (Committee for the Pharmacopoeia of P.R. China, 1977). Now, it is widely used as a folk medicine and traditional health food by Tibetans, Mongolians, the Han people, and other ethnic groups in China.
Because of the marked therapeutic effects and nutritional actions, researchers have widely investigated the properties of the tuber of G. conopsea Modern pharmacological studies have shown that it possesses broad pharmacological properties and can be used in the following treatments: tonifying effect, antioxidative, anti-viral, gastric ulcer prevention, anti-aging, immunoregulatory, antianaphylaxis, sedative, hypnotic, etc. Most of these actions have closely matched traditional uses. The chemical compounds from this plant have also been extensively studied, and glucosides, toluylenes, dihydrostilbenes, phenanthrenes, aromatic compounds, and other compounds have been isolated and identified.
In this review, advances in the ethnobotanical, phytochemical, biological and pharmacological activities, and toxicology of G. conopsea are presented and critical assessment. And the data supports its use and exploitation in new drugs.

BOTANICAL DESCRIPTION
Commission of Flora Reipublicae Popularis Sinica According to the description by Meekers et al. (2012) and the Commission of Flora Reipublicae Popularis Sinicae (2004), Gymnadenia conopsea (L.) R. Br. is an apolycarpic, perennial, terrestrial, and fragrant orchid herb that belongs to the Gymnadenia genus of Orchidaceae family, and distributs at forests, grasslands, and waterlogged meadows from 200 to 4700 m altitude. It has about 69 synonyms of this species, but only Gymnadenia conopsea (L.) R. Br. is an accepted and approved name in the World. The stem is 20-60 cm, erect, slim, terete, or angled above and leafy with 2-3 brown membranous sheaths at the base. The leaves are green with dimensions of 5.5-15 × 1-2.5 cm, and the lower leaves are erect to slightly spreading, more, or less narrowly oblong-lanceolate or linear-lanceolate, obtuse to subacute, and slightly hooded at the apex, entire, keeled, and folded and have 1-2 or more veins on each side of the midrib; the upper 2-3 leaves are smaller, lanceolate or bract-like, and taper to a fine point. The bracts are green and usually tend toward violet at the edges, and they are lanceolate and taper to a fine point at the apex. The raceme is 5.5-15 cm long and ranges in color from pale pink to lilac (rarely white or bright magenta), and it is strongly scented with a flowering season from July to August. Inflorescence 11-26 cm, slender; peduncle with one to a few scattered, lanceolate bracts 1.5-6 cm; rachis 4-12 cm, densely many flowered; floral bracts lanceolate, often longer than ovary and flower, apex long acuminate-caudate. And flowers fragrant, pink, rarely pinkish white; ovary 5-8 mm including pedicel. Dorsal sepal broadly elliptic to broadly ovate-elliptic; lateral sepals reflexed, obliquely ovate, 4-5.5 × 3-4 mm, 3-veined, margin revolute, apex acute. Petals obliquely ovate-triangular, 3veined, apex acute; lip spreading, broadly cuneate-obovate. There are two tubers with dimensions of 14-30 × 7.5-24 mm; the tubers are palmate lobed with thick segments that are tapering and obtuse, pressed together, and split halfway to the base into 3-6 lobes. The short and thick roots are sparse and grow horizontally or even toward the soil surface. The fruit has dimensions of 8.6-9.3 × 2.6-2.7 mm, and they are erect with six ribs. The seeds have dimensions of 0.3 × 0.1 mm and are produced in large numbers (http://www.theplantlist.org 1 ; www.efloras.org 2 ; Figure 1).

TRADITIONAL USES
G. conopsea is widely distributed in northern Europe and certain Asian countries. Like some TCM, G. conopsea is not used in folk medicine, and just has been considered a fragrant orchid plant in some European countries. So, studies on the ethnopharmacology and clinical uses of the plant have been mainly focused on Asian countries, such as China, Nepal, and Japan.
In China, G. conopsea is primarily distributed in Heilongjiang, Jilin, Liaoning, Hebei, Shanxi, Shangxi, Gansu, Sichuan, and Yunnan Provinces and Inner Mongolia and Xizang Autonomous Regions at altitudes of 265-4700 m (Commission of Flora Reipublicae Popularis Sinicae, 2004). The tuber has been employed as a reinforcing agent of folk medicines and widely used as traditional Chinese medicine (Han national medicine), Tibetan medicine, Mongolian medicine, Baiyao (the Bai national medicine), Chaoyao (the Korean national medicine), and Naxiyao (the Naxi national medicine) to treat various diseases in China. The tuber has also been employed as a health care product with other medicines or food to improve the body and prevent illness, and could be made to tincture and galenical to treat impotence and the bronchial asthma in China and Russia, respectively (Mamedov and Craker, 2001;Matsuda et al., 2004;Gutierrez, 2010). But at the same time, because of the rare resource of this plant, the tuber of Gymnadenia crassinervis, Coeloglossum viride var. bracteatum, and Spiranthea lancea have been used as substitutes for G. conopsea in certain regions, such as Tibet region (Xie et al., 2005;Zi, 2008;Xue et al., 2009).
As a traditional Tibetan medicine, the G. conopsea tuber has been widely used in the Tibetan region to treat lung disease and weakening by invigorating the kidney and moisturizing the lungs. The plant is known as "Wangla, " and it has been recorded in the "Sibuyidian, " the classical book of Tibetan medicine, since the eighth century. In Tibetan medicine, it could be used as a single medicine or as one composition mixed with other medicines to treat diseases. For example, after grinding to powder, it (30 g) concoction with bee honey (40 g), Rhizome Gastrodiae (30 g), Radix Phlomii (30 g), Herb Drosera peltat (30 g), and Rhododendron parvifolium (30 g) could be used to treat impotence, spermatorrhea, anemia, and insomnia for 3 g twice per day (Chinese Materia Editorial Committee, State Chinese Medicine Administration Bureau, 2002). And according to the database of Tibetan prescriptions, out of 4500 traditional prescriptions in Tibet Autonomous Region, the G. conopsea tuber was used 104 times (rate of 2.3%), and 33 prescriptions were used to invigorate the body, strengthen the Yang, and lengthen human life; 26 prescriptions were used to treat kidney diseases; 12 prescriptions were used to treat gout and arthromyodynia diseases; 11 prescriptions were applied to treat lung disease; 7 prescriptions were used to treat eye diseases; and other prescriptions were used to treat parasitic diseases and additional diseases (Ji et al., 2009;Xue et al., 2009). Currently, the tuber of G. conopsea is combined with other medicines in various preparations to treat a number of diseases. Five preparations have been listed in the Chinese Pharmacopeia and approved by the State Food and Drug Administration of China of the People's Republic of China. Medicines such as "Shi Wei Shou Shen Powder" and "Fu Fang Shou Shen Wan" have been widely used to treat kidney asthenia, impotence and spermatorrhea, among other disorders (http://www.sfda.gov.cn, 2014; Table 1). Except the above effects on the clinic, the tuber of G. conopsea also could be used to treat hepatitis B by folk doctors only in the Tibetan region (Chinese Materia Editorial Committee, State Chinese Medicine Administration Bureau, 2002). At the same time, it has also been used as a common food item by local people in the Tibetan region, where it is usually cooked with vegetables and rice. For example, the tuber is used as an important ingredient in the famous dish "Shiguo Ji" (chicken cooked in a stone hotpot).
In the traditional Mongolian medicine, the tuber of G. conopsea is named "Erihaoteng" and has been historically recorded by many classical folk medicine books of the Inner Mongolia Autonomous Region. In additional, it has been widely used to treat kidney asthenia, lumbago and leg pain, light scurvy, spermatorrhea, and impotence (Gege et al., 2013). The tuber of G. conopsea and other traditional medicines have been combined into different preparations to treat various diseases. For example, the tuber and an additional 36 medicines are known as Shouzhangsheng-37 pills, which are used to treat kidney cold and asthenia, edema, tinnitus, spermatorrhea, impotence, stomach diseases, dyspepsia, and other diseases, and this preparation has been approved by the State Administration of Traditional Chinese Medicine of the People's Republic of China (http://www.sfda.gov.cn, 2014; (Si and Liu, 2013); Table 1). Meanwhile, Wu et al. (2014) reported that in clinical practice of Mongolian medicine, after administrating orally two Shenzhujin pills (the tuber is the primary component) for three times per day for 21 days, the kidney deficiency of one patient (65 years) have been cured. Furthermore, the tuber of G. conopsea is also used in Korean national medicine, Bai national medicine, and Naxi national medicine, where it is known as Yinyang Cao, Foshousheng, and Kaishelabei, respectively. However, it has been used as a reinforcing agent within all of these traditional medicines and used to treat similar diseases as previously indicated, such as invigorating the body, strengthening the Yang, etc. (Jia and Li, 2005). But up to now, except the experience-based uses of this plant, the relevant evidence-based clinical uses and the safety data scientifically are very rare and should be investigated further.

PHYTOCHEMISTRY
Approximately 129 compounds have been isolated and identified from G. conopsea to date, and most of compounds isolated from the tuber. Forty-nine glucosides compounds (one of the most important components) were identified, which could be divided into benzylester glucosides and other glucosides. Meanwhile, dihydrostilbenes, phenanthrenes, aromatic compounds, polysaccharides, and other compounds were also isolated and reported (Table 2; Figure 2). The different chemical compositions of G. conopsea provide the foundation for its different pharmacology activities.

Glucosides
As one of the most important components, glucosides are widely studied and separated from the tuber of G. conopsea. According to the structural conformation of the glucosides, they have been divided into benzylester glucosides and other glucosides.

Polysaccharides
In 2009,  studied the four extraction and purification methods for the polysaccharides of G. conopsea. The results showed that the optimum parameters for solvent extraction were 80 • C for 2.5 h, a ratio of material to water of 1:30 with three repetitions, which yielded a polysaccharide extraction rate of up to 11.83%. The microwave extraction method were 250 W for 20 min, a ratio of material to water of 1:30 with one repetition, and the extraction rate was up to 13.56%. The ultrasound extraction method was 240 W, 60 • C for 10 min, and three repetitions, and the extraction rate was 15.24%. Finally, the parameters for cellulose extraction were to add cellulose at 1% of the tuber concentration for 4 h at 50 • C and Ph 4.8, and the extraction rate was 16.58%. Thus, the cellulose method is the best among the four methods. Meanwhile, vapor phase chromatography was adopted to analyze the monosaccharides from the polysaccharides, and the results showed that the polysaccharides are composed of arabinose (110), xylose (111), mannitose (112), galactose (113), and glucose (114) (Sun et al., 2010). And in 2014, the water soluble polysaccharides from the tuber of G. conopsea collected at seven regions in China were investigated and compared using high performance size exclusion chromatography coupled with multi-angle laser light scattering/refractive index detector (HPSEC-MALLS/RID) and saccharide mapping based on polysaccharide analysis by carbohydrate gel electrophoresis (PACE), respectively. The results showed that the weight-average molecular weight (M w ) and the radius of gyration (<S 2 >z 1/2 ) of polysaccharides were ranging from 4.46 × 105 to 7.41 × 105 Da and 73.3-94.2 nm, respectively. And the exponent (v) values of <S 2 >z 1/2 = kM v w were ranging from 0.36 to 0.42, which indicated that α-1,4and β-1,3(4)-glucosidic, α-1,5-arabinosidic, β-1,4-mannosidic and α-1,4-D-galactosiduronic linkages existed in globular polysaccharides. The further studies results that the nitric oxide released from RAW 264.7 cells induced by polysaccharides were significantly affected by their α-1,5-arabinosidic and β-1,3(4)-glucosidic, especially α-1,4-D-galactosiduronic and β-1,4mannosidic linkages (Lin et al., 2015).

PHARMACOLOGY Tonifying Effect and Anti-Fatigue Activity
In China, the G. conopsea tuber has been used as a reinforcing agent for traditional medicines, and it has historically been used to treat kidney asthenia, lung asthenia, consumption diseases, neurasthenia, impotence, spermatorrhea, and other diseases. According to the theory of traditional Chinese medicine, kidney asthenia is related to weakness in the body. Thus, the tonifying activity (kidney-yang-tonifying activity) was the primary subject of study in China. The results showed that after administering orally 2 and 1 g/kg the tuber for 10 days, the medicine relieved the symptoms in mice with yang deficiencies that had been induced by hydrocortisone (25 mg/kg, i.g.), increased the body weight of kidney-yang-deficient mice (7.82 and 5.56 g, P > 0.05), prolonged the retention time on the rotating bar (19.57 ± 10.21 min, P < 0.01, and 5.90 ± 2.47 min, P < 0.05) and increased the kidney indexes of treated mice compared with those of the model group. And the increased weight and time on the rotating bar for the untreated control groups were 8.71 g (P > 0.05) and 24.20 ± 13.95 min (P < 0.01), respectively. Further, study showed that the tubers could also increase DNA synthesis in the spleen (48.89 ± 18.68 and 46.63 ± 13.80 µg/g, P < 0.01), kidneys (14.26 ± 5.34 and 5.94 ± 4.88 µg/g, P > 0.05), and liver (42.94 ± 13.95 µg/g and 42.02 ± 17.69, P < 0.05) of mice with yang deficiencies that were induced by hydrocortisone compared with the mice in the model group (27.25 ± 13.18 µg/g, P < 0.01, 4.90 ± 4.82 µg/g, P > 0.05, and 19.02 ± 15.49 µg/g). These results suggested that the tuber has a significant tonifying effect on the kidney and strengthens the bodies of mice with kidney deficiencies that were induced by hydrocortisone (Lin, 2009).
In 2011, Zhao and Liu (2011) studied the anti-fatigue activity of the tuber of G. conopsea in mice. After administering orally (i.g.) aqueous extracts to mice (40, 20, and 10 g/kg) for 6 days, the swimming times were markedly increased (1441.6, 1357.0, and 1249.9 s, P < 0.01) during the weight-bearing swimming tests in a dose-dependent manner. The swimming times of the control and positive groups (Radix ginseng, 20 g/kg) were 491.1 s and 1685.9 s (P < 0.01). According to the processing method of traditional Mongolia medicine in China, the tuber should be decocted in goat or cow milk before clinical use. After processing with a different method, Jin and Wang (2009) studied and compared the strengthening effects of the tuber of G. conopsea tonics. The results showed that compared with the control group (saline water, 6.38 ± 4.22 min), a goat milk decoction (2 g/kg. i.g.) could significantly increase the swimming time of mice (19.44 ± 12.6 min, P < 0.01); the unprocessed group (2 g/kg) and cow's milk decoction group (2 g/kg) had swimming times of 9.09 ± 9.81 min and 6.43 ± 7.21 min (P > 0.05), respectively, but they did not show significant difference. All of the decoctions could increase the SOD activity and MDA content compared with that of the control group. Thus, the researchers indicated that after processing with goat milk, the tuber have a better effect with the strengthening tonics than unprocessed medicine ( Table 4).

Anti-Oxidant Activity
The anti-oxidant activity of the tuber of G. conopsea was first comprehensive studied in 2006. The results showed that after isolating by Diaion HP-20 column chromatography (reverse-phase silica gel columnchromatography, Chromatorex ODSDM1020T, 100-200 mesh, Fuji Silysia Chemical, Ltd.), the MeOH-eluted fraction showed the radical scavenging activities for DPPH radical (SC 50 = 59.1 µg/ml) and.O − 2 (IC 50 = 13.3 µg/ml), but without the inhibitory activity on xanthine oxidase (IC 50 > 100 µg/ml). Meanwhile, the radical scavenging activities for DPPH radical and . O − 2 exhibiting inhibitory activity on formozan formation and xanthine oxidase of the acetoneeluted fraction were 55.4, 33.2, and 29.7 µg/ml, respectively. But H 2 O-eluted fraction didn't present any activities. Then, the anti-oxidant 13 compounds were studied further, some compounds showed the marked radical scavenging activities for DPPH radical and . O − 2 exhibiting inhibitory activity on formozan formation and xanthine oxidase of 13 compounds (Morikawa et al., 2006b; Table 3). Meanwhile, the study simultaneously showed that at a concentration of 10 −6 mol/L, arctigenin, lappaol A, and lappaol F have anti-oxidative activities that inhibit Fe 2+cystine-induced lipid peroxidation in rat liver microsomes with inhibitory rates of 53, 59, and 52%, respectively, relative to that of vitamin E at 35% .
In 2010, Li et al. (2010) screened and evaluated the aqueous extract, acidic aqueous extract (pH = 3), 60% ethanol extract, 95% ethanol extract, n-butanol extract, n-butanol saturated by aqueous extract and ethyl acetate extracts of the tuber of G. conopsea for their radical scavenging capacity. The results showed that the radical scavenging capacity of the aqueous and acidic aqueous (pH = 3) extracts were weak. The radical scavenging activities (IC 50 ) of the other extracts were 0.1266, 0.4537, 0.3151, and 0.1305 mg /ml. Thus, the investigators indicated that the 95% ethanol extract was the most effective among the six extracts (Table 4).

Anti-Viral Activity
In 2008, Zi (2008) studied the anti-HIV activity of certain compounds from the tuber of G. conopsea at a concentration of 10 −5 mol/L in relation to the drugs zidovudine (10 −7 mol/L, 85.6%) and lamivudine (10 −8 mol/L, 47.4%), and the inhibition rates of these compounds against the VSVC/HIV-luc model in 293 cell lines were 0.6-13.3% (Table 3).
In Tibet, G. conopsea has primarily been used by local people to treat chronic hepatitis B. In 2002, Lu et al. (2002) studied the anti-HBV activity. The results showed that after treating the patient's serum that contained hepatitis B for 4 h, the medicine (0.03, 0.06, and 0.12 mg/L) could inhibit eight hemagglutin units of HBsAg. Thus, they indicated that the tuber has medium anti-HBV activity. In 2003, Kimura et al. (2003) studied the inhibitory effect of Tibetan medicinal plants on viral polymerases. The results showed that at 100 µg/ml, 28 species of 76 medicines presented the anti-RTase activity with the more than 70% inhibition rates. But after adding BSA to these drugs, except the fruit of Terminalia chebula and Areca catechu, other species didn't show the anti-RTase activity, including G. conopsea (Kimura et al., 2003; Table 4).

Sedative and Hypnotic Activities
In 2009, Lin (2009) studied the sedative and hypnotic effects of the tuber of Gymnadeniae tubers at different dosages. In the sedative test, administering the tuber (2 and 1 g/kg i.g.) for 30 min, it could reduce the spontaneous activity of mice within 5 min (6.63 ± 3.18 and 5.36 ± 3.44) at inhibitory rates of 37.44% (P < 0.05) and 45.60% (P < 0.05), and the control group (normal saline) and positive group (diazepam, 0.004 g/kg) exhibited inhibition of 10.60 ± 5.77 and 3.66 ± 1.47 (an inhibition of 64.21%, P < 0.01), respectively. The tubers could also decrease the frequency of upward-raising motion in mice forelimbs within 5 min, especially for the high dose group at 15.42 ± 11.77 times (P < 0.05). The upward-raising frequency of the control and positive groups were 22.11 ± 6.92 and 9.37 ± 3.65 (P < 0.01), respectively.

Compounds
Effects

Anti-oxidant activity
Anti-oxidative activity inhibiting Fe 2+ -cystine activity induced in rat liver microsomal lipid peroxidation with inhibitory rates of 53, 59, and 52%, compared with that of vitamin E at 35%. 1.00 min, P < 0.01; and 2.56 ± 0.49 min, P < 0.05), and it prolonged their sleeping times (28.22 ± 10.50 min, P < 0.01; and 20.81 ± 9.22 min, P < 0.05) relative to that of the control group (7, 0.92 ± 1.58 and 8.56 ± 15.53 min), which was induced by pentobarbital sodium at less than the threshold dose (45 mg/kg), and the diazepam group (20, 6.35 ± 0.82 and 38.22 ± 3.84; P < 0.01). These results indicated that the tuber of G. conopsea has marked sedative and hypnotic dose-dependent effects, and its mechanism of action requires further research ( Table 4).

Preventing and Treating Gastric Ulcers
With normal saline as the control, the inhibitory effect of the tubers on gastric ulcers was induced by hydrochlorideethanol solution (7.5 ml/kg for 3 days, i.g.). The macroscopic and pathological results showed that at doses of 2 and 1 g/kg, the treatment could relieve the pathology index of gastric ulcers induced by hydrochloride-ethanol solution and decrease the gastric ulcer index (0.05 ± 0.13 cm and 0.22 ± 0.38 cm) at inhibitions of 88.86% (P < 0.01) and 48.88%, respectively. The biochemical test showed that after treatment with Gymnadeniae tubers, the serum (9.30 ± 4.18 nmol/ml, P < 0.01; and 13.71 ± 3.89 nmol/ml) and gastric (1.10 ± 1.19 nmol/ml, P < 0.05; and 0.96 ± 0.69 nmol/ml) MDA contents could be decreased in rats. The gastric ulcer index, MDA content in the serum and gastric ulcer index of the model group were 0.44 ± 0.18 cm, 14.17 ± 4.88 and 3.81 ± 5.35 nmol/ml, respectively. The above results suggest that the tubers exert a degree of inhibition on gastric ulcers that were induced by the hydrochloride-ethanol mixture (Lin, 2009). Jiang et al. (2009) also studied the beneficial effect of the tuber of G. conopsea on acute gastric ulcers in rats that were induced by hydrochloride-ethanol solution (7.5 ml/kg for 3 days). After drug administration orally (2 and 1 g/kg) for 30 min, the gastric ulcer areas were decreased with inhibitions of 88.86 (P < 0.01) and 48.88 (P < 0.01); the inhibition by a commonly prescribed drug (ranitidine, 1.95 g/kg) was 91.12 (P < 0.01). The pathology of the gastric ulcers was markedly improved by these drugs (Table 4).

Anti-Hyperlipidemia Activity
In 2013, Zhang et al. (2013) studied the effects of the tuber of G. conopsea ethanol extracts on the blood lipids and liver function of experimental hyperlipidemia rats. The results showed that after treating the hyperlipidemia model rats that had been induced by high-fat diets with ethanol extracts (at 5, 2.5, and 1.25 g/kg, i.g.), the TC and LDL-C serum contents of the model rats were not improved; however, the TG and HDL-C contents were decreased at 1.35, 0.89, 0.97 mmol/L (P < 0.01), and 0.49 (P < 0.05), 0.53 and 0.49 mmol/L (P < 0.05) compared with the model group (2.43 and 0.64 mmol/L), and the commonly prescribed drug lovastatin also showed markedly decreased TG and HDL-C content (P < 0.01). In addition, the ALT and AST activities in the serum were also inhibited at10.71, 10.82, 8.21 U/L (P < 0.05), 6.97 (P < 0.01), 13.60 (P < 0.05), and 18.02 U/L compared with that of the model group, and the commonly prescribed drug results were 8.53 (P < 0.05) and 10.38 (P < 0.01). These results suggest that the tuber could reduce blood lipids and protect the liver function in experimental hyperlipidemia rats ( Table 4).

Anti-Silicosis Activity
Silicosis is an important occupational disease caused by the inhalation of silica dust, and it is characterized by lung interstitial fibrosis. In 2007 and 2008, Wang et al. (2007Wang et al. ( , 2008 studied the effects of silica exposure on collagen synthesis in rat lungs and mechanisms of anti-oxidative stress by using a 60% ethanol extract of the tuber of G. conopsea. Silicotic animal models were established by surgically directing the tracheal instillation of silica into rat lungs. After administering the ethanol extract orally (8 g/kg, per day), the rats were sacrificed and the samples were collected to assay the relative index at 7, 14, 21, 28, and 60 days. The results showed that the extract could reduce the lung/body weight ratio of rats (8.6, 6.99, P < 0.01; 7.25, P < 0.05; 7.97, P < 0.05; and 9.75 mg/g, P < 0.05) relative to the model group (11.04, 9.28, 9.11, 11.98, and 13.91 mg/g), and the extract could also improve pathological changes in the lung. This treatment could also ameliorate silica-induced pulmonary fibrosis by decreasing the type I and type III collagen-positive area percentage in the lungs of rats exposed to silica at different time points. These studies also indicated that the ethanol extract could decrease the MDA content (4.78, P < 0.01; 5.39, P < 0.05; 5.48, P < 0.05; 5.29, P < 0.05; and 4.35 mmol/L) and increase the SOD (302.67, 243.95, P < 0.01; 293.38, P < 0.05; 277.74, P < 0.05; and 243.36 KU/L) and GPx (2199GPx ( .58, 2359.34, P < 0.01; 2538.66, P < 0.01; 2422.41, P < 0.05; and 2298.04 U/L) activities relative to that of the model group (6.43, 6.88, 6.98, 6.12, and 5.82 mmol/L, 314.84, 183.62, 219.41, 218.44, and 226.50 KU/L, and 2489.50, 1015.34, 1227.83, 1814.90, and 1867.38 U/L). In 2007 found that the ethanol extract could reduce the TNF-α integral optical density (86.86, 105.1, 122.09, 108, and 94.88, all P < 0.01) of rat lungs compared with that of the model group (116.98, 140.1, 220.19, 140.9, and 110.9) at 7, 14, 21, 28, and 60 days, respectively. Before and after treating with a 60% ethanol extract, Chen (2008) studied the differential gene expression profiles of rat lung tissue in the early stage exposure to silica. After treating with the extract (8 g/kg, per day, i.g.), there were 308 and 231 up-regulated and down-regulated genes among a total of 539 available genes compared with that of the model group. Up-regulated pathways might be associated with cell adhesion molecules, notch signaling pathways, and leukocyte transendothelial migration and cell communication. Down-regulated pathways might be related to genes in the linkage of complementary and coagulation cascades, hematopoietic cell lineages, urea cycle and Alzheimer's disease. The chip results were verified by using real-time PCR for SOD and HMOX genes with the same trends. Thus, the investigators indicated that the alcohol extract may inhibit silicosis progress during early exposure periods and attenuate airs acculitis and fibrosis of the lung. The results also showed that the ethanol extract (8 g/kg, per day, i.g.) has the reverse effect on silicosis, and this process involved the cathepsin D precursor SEC14-like protein 3 and peroxiredoxin-1 (Chen, 2009; Table 4).

PREPARATIONS AND QUALITATIVE AND QUANTITATIVE ANALYSIS
G. conopsea has been widely used as a folk medicine in the treatment or prevention of diseases for thousands of years in China, but it has not been listed in the Chinese pharmacopeia because there is a lack of scientific quality standards to control the quality of the tuber since 1977 (Committee for the Pharmacopoeia of P.R. China, 1977China, , 2000Chinese Materia Editorial Committee, State Chinese Medicine Administration Bureau, 2002). Recently, experts have attempted to formulate the proper quality standard by analyzing the active compounds and determining the contents of plants from different habitats with different chromatographic equipment. Now, as a strategy to control the quality of folk medicines, chemical fingerprint analyses have been accepted by the WHO (1991), State Food and Drug Administration of China (2000), and other authorities, which has been recognized as a rapid and reliable method of identifying and qualifying herbal medicines. Cai et al. (2006) first developed a highperformance liquid chromatography-diode array detectiontandem mass spectrometry (HPLC-DAD-MS n ) method for the chemical fingerprint analysis and rapid identification of major compounds in G. conopsea tubers. An HPLC separation was performed by using a linear gradient at room temperature (20 • C) and a flow rate of 0.7 ml/min with an Inertsil C 18 ODS-3 column. The gradient elution started with a methanol: water mixture (20:80, v/v), and the methanol content was increased to 100% within 60 min; the detection wavelength for fingerprint analysis was 270 nm. Adenosine, 4-hydroxybenzyl alcohol, 4hydroxybenzyl aldehyde, dactylorhin B, loroglossin, dactylorhin A, and militarine were well-separated and identified from the tuber of 10 samples, which were collected from Sichuan, Qinghai, and Hebei Provinces and Tibet Autonomous Region of China and Nepal. Xue et al. (2013) also studied the fingerprint of G. conopsea by using HPLC. Their samples were separated on a Kromasil C 18 (4.6 × 250 mm, 5 µm) column and gradienteluted with a mixture of methanol and water (0.04% phosphoric acid) at a flow rate 0.7 ml/min; the detection wavelength was set at 222 nm, and the column temperature was 30 • C. The results showed that 13 common peaks were selected, and the method validation met the technical requirements of a fingerprint. The similarity of 12 samples from the different regions of the Xizang Autonomous Region and Qinghai Province was 0.904-0.989. The above two methods are simple, practical and reliable. The combined use of the two fingerprints confirmed the identification and quality assessment of G. conopsea. In 2009, after investigating the optimal extraction method, Li et al. (2009) firstly developed a HPLC method for the simultaneous quantification of five glucosyloxybenzyl 2-isobutylmalates in the tubers of G. conopsea, which was collected at five regions in China. The optimal extraction conditions was the direct reflux with 70% ethanol for 1 h, and the compounds are separated on an Agilent Hydrosphere C18 (150 × 4.6 mm i.d., 5 µm) column using a mobile phase of acetonitrile-water including 0.3% acetic acid (adjusted with 36% acetic acid) with gradient elution at a flow rate of 1.0 mL/min. Detection is set at a UV wavelength of 221.5 nm. The recovery of the method is 97.7-101.0%, and linearity (r > 0.9998) is obtained for all the analytes. The results showed that the contents of dactylorhin B, dactylorhin E, loroglossin, dactylorhin A, and militarine were Region), 3.103 mg/g (Chengduo, Qinghai Province), 2.734 mg/g (Bomi County, Xizang Autonomous Region), and 2.268 mg/g (Beishan Forest Farm, Huzhu County, Qinghai Province; Xue et al., 2011).
Meanwhile, the content of four marker constituents of the tubers with the climatic factors were determined and analyzed by HPLC in 2014. The results showed that the samples collected from Milin County in the Xizang Autonomous Region had the highest gastrodin content at 2.725 mg/g, and the Shennongjia region of Hubei Province had lowest content at 0.374 mg/g. The samples collected from Kangding County of Sichuan Province, Aba region of Sichuan Province and Naqu County of the Xizang Autonomous Region had the highest contents of adenosine, 4-hydroxybenzyl alcohol and 4-hydroxybenzaldehyde at 1.485, 1.505, and 1.048 mg/g, respectively. These findings suggest that the average temperature and annual average variations in monthly temperature change along with seasonal variations, and the highest temperature, variations in the scope of the annual temperature, average temperature of the most humid season, average temperature of the hottest season, annual rainfall, rainfall during the driest season, rainfall during the coldest season, and latitude and altitude of the habitat can have an effect on the active ingredient content and quality of the tuber of G. conopsea .
The study results also indicated that the samples collected from Huzhu County of Qinghai Province had the highest contents of gastrodin (4.9242 mg/g), dactylorhin A (8.2274 mg/g), and militarine (7.4645 mg/g; Yao and Lin, 2014).

TOXICITY
In 2007, Bai and Zheng (2007) evaluated the toxicity of the tuber of G. conopsea. In acute toxicity test, after administering the tuber (1.00, 2.15, 4.64, and 10 g/kg, i.g.) to mice and rats, the behavior, and growth of the animals did not change after 2 weeks compared to the control group (P > 0.05), and the LD 50 was more than 10 g/kg. The genotoxicity of the medicine was then studied, and the results showed that the rates of bone marrow and sperm abnormalities were not changed compared with that of the control group, but the sperm abnormality rates of the group administered a common drug (cyclophosphamide, 30 mg/kg) changed markedly (P < 0.01). And after administering the tuber (1.67, 3.33, and 6.67 g/kg, i.g) to rats for 30 days, the parameters of blood routine, biochemical indexes, organ coefficient, and organ pathology of rats haven't marked changed compared the control group (P > 0.05). So they thought that the tuber didn't present the apparent toxicity.

CONCLUSIONS AND FUTURE PERSPECTIVES
As an important traditional medicine in China, the tuber of G. conopsea has been historically used in a number of clinical applications. However, lack of scientific quality standard to control the quality of the tuber has been hampered to the development of this plant. Up to now although investigators have begun studying and developing the quality of the tuber and its preparations by analyzing the marker compounds and determining the contents of plants from different habitats, a scientific method to control the quality of these products has not yet been developed and approved by government. Thus, a characteristic chemical and biological index should be established to monitor and evaluate quality of samples and maintain their clinical and pharmaceutical stability, and the method of accurately controlling the quality of the tuber in traditional medicine and preparations should be studied further.
With the excavation and abundant use of G. conopsea, as well as the over-grazing and disorder tourism resulted in the habitat destruction, the resources of this plant were rapid decreased. At the same time, because of the low reproductive capacity of this plant, once the resources and habitat have been destroyed, the species does not recover easily. In 2000, G. conopsea has been listed in the grade section of endangered species (Gesang and Gesang, 2010). Recently, investigators have begun studying and developing methods of cultivating this plant, such as tissue culture and artificial breeding, but this technology is still not applied in industry (Bao et al., 2008). Therefore, the sustainable use of G. conopsea is necessary to investigate. Of course, along with the decrease of resources, the studies on the aerial part should be paid more attention, especially for finding new active compounds and bioactivities.
In a word, phytochemical and pharmacological studies of G. conopsea have received great interest, and an increasing number of extracts and active compounds have been isolated that have demonstrated tonifying activity, anti-viral activity and immunoregulatory activity, among others. However, validating the correlations of the ethnomedicinal uses and pharmacological effects should be carried out further, and the toxicity of this plant also should be studied systematically. Meanwhile, the poor quality control, decreased resources, the increasing gap between more experience-based traditional uses and less evidence-based clinical trials for the tuber of G. conopsea has created challenges. Therefore, this plant should be studied and developed further, especially in the resource conservation.

AUTHOR CONTRIBUTIONS
XS and JZ conceived the review; XS, XG, XM wrote the manuscript; HP collected the literatures; and YL edited the manuscript. All the authors read and approved the final version of the manuscript.