The literature survey was performed to obtain information about distribution of the plants of BFC, which would be useful for choosing the sampling sites. According to Flora of China, Flora of Sichuan Province, and Flora of Yunnan Province and previous studies (Luo and Chen, 1996; Liu et al., 2008), 32 sites in Sichuan, Chongqing, and Yunnan provinces, which are the traditional producing regions of BFC, were chosen to perform a survey on plant resource of BFC during 2013–2014 (Table 1). In every site, there was one experienced and local herbalist who helped us to carry out resource investigation.

The field investigation was carried out in May, June, or July in 2013 and 2014. Field investigation was designed to assess the distribution, density and abundance of the plants of BFC (Delgado-Lemus et al., 2014). In order to estimate their distribution, the coordinate positions were firstly recorded using a Magellan Triton GPS400 in each site (Delgado-Lemus et al., 2014). The quadrat investigation was performed to assess the density and abundance of the plants of BFC as described previously (Zhang et al., 2015). Series of squares (quadrats) with a set size (4 × 4 m) were placed in each site. The mean density of the plants of BFC per quadrat was the number of the plants of BFC per quadrat. The resource density of the quadrat was calculated as: Resource density = total weight of bulbs/quadrat area (g/m²).

At each site, 5–15 plants were collected as specimens during field investigation. The specimens were prepared in the medicinal plants Herbarium of West China College of Pharmacy. The specimens were examined and identified by experts, and the voucher specimens with labels were deposited at the medicinal plants Herbarium. At each site, photographic records were taken to capture the field sites, growth habitat, and morphology of plants (Chekole et al., 2015). We also collected the bulbs from these sites. All samples were subjected to the described determination of content of the total alkaloid.

In this study, we visited six artificial cultivated bases and two individual cultivated plantations. The specific information about area, production, and market value were gathered by inquiring, personal interviewing, or direct surveying (Zhang et al., 2015).

The total alkaloid content was determined according to the previously described acid dye colorimetric method (Wang D. D. et al., 2014; Li et al., 2015). For alkaloid standard, a stock standard solution (0.1 mg/ml) of imperialine (in chloroform) was prepared and the stock solution was diluted to different working concentrations (0.0, 1.0, 2.0, 4.0, 8.0, 10.0 μg/ml).

A certain amount (about 2.0 g) of powdered sample was weighed, immersed in 3 ml of ammonia for 1 h, and then extracted under reflux with 40 ml of chloroform-methanol (3:1) solution for 2 h. The solution was filtered, and diluted to a final volume of 50 ml with chloroform-methanol solution. A certain volume (3–5 ml) of the solution was pipetted and evaporated, and the residue was dissolved in chloroform (10 ml) to get the sample solution. According to the absorption spectra of the standard solutions, 412 nm was chosen as the detection wavelength.

To measure total alkaloid content, 10 ml of each sample or working concentrations of standard solution was transferred into a separate funnel. Five milliliters of distilled water and 2 ml of bromocresol green buffer solution were added into it and shaken for 2 min. After 30 min, the chloroform layer was separated and collected. The absorbance of chloroform fraction was measured at 412 nm using Alpha-1900PC UV–Vis spectrophotometer (Shanghai Lab-Spectrum Instruments Co., Ltd., China). The experimental results were expressed as mean ± standard error of mean (SEM).

The distribution of wild plants of BFC is shown as Figure 1 and Table 1. To investigate the resource of BFC, we chose 32 traditional producing regions of BFC. In five sites investigated no BFC plants could be found. These sites are Guobalong and Gage Mountain in Renda Village in Luhuo county, Shuiwa Village and Ranwu Village in Xiangcheng county in Sichuan Province, Kari Mountain in Shangri-la in Yunnan province. The sites where plants of F. cirrhosa were found are Kangding, Daofu, Litang, Yajiang counties in Sichuan province and Lijiang county in Yunnan province (Table 1). The sites where plants of F. unibracteata were found are Yajiang, Songpan, Hongyuan, Aba, Rangtang, Litang, Mao counties in Sichuan province and Jiuzhi county in Qinghai province (Table 1). The sites where plants of F. wabuensis were found are Songpan and Mao counties in Sichuan province. The sites where plants of F. taipaiensis were found are Wuxi and Chengkou counties in Chongqing province (Table 1). We only found plants of F. delavayi in Yajiang county in Sichuan province. From the Table 1, the main species of BFC occurring in Sichuan province are F. cirrhosa, F. unibracteata, and F. wabuensis. The main species in Yunnan province is F. cirrhosa. The main species in Chongqing province is F. taipaiensis.

Table 2 shows the mean density of the plants of BFC per quadrat and resource density. The average number of the plants of BFC per quadrat is <7 except for the site in Ningyou Village in Jiuzhi county, Qinghai province. In addition, the average resource density is <22 mg/m² except for the sites in Lasang Mountain in Milong Village in Yajiang county, Sichuan province and Ningyou Village in Jiuzhi county, Qinghai province. The data reconfirm that the species in the Fritillaria genus are still at risk of depletion. The reason why the average resource density in Lasang Mountain in Yajiang county is higher than other sites may be that the species in this site is F. delavayi which price is much less than the other species. The reason for that in Ningyou Village in Jiuzhi county may be that this site is one of the main producing regions of Chinese Caterpillar Fungus, with a much higher price than FBC.

Plants of F. cirrhosa grow under forests, in alpine thickets, or on meadows, and flood lands. They are usually found on shady and moist slopes of valley (Figure 2A). The altitude range of occurrence is from 2,500 to 4,600 m. All altitude data were collected by using Magellan Triton GPS400. The plants of F. unibracteata are usually discovered in moist places of thickets or meadows (Figure 2B). Their altitude range is from 3,200 to 4,700 m. Plants of F. wabuensis grow under forests, or in alpine thickets (Figure 2C) and the altitude range of their occurrence is from 2,500 to 3,500 m. The plants of F. delavayi are usually discovered in sandy and gravelly places or on flood lands (Figure 2D). Their altitude range is often above 4,000 m. Plants of F. taipaiensis grow under forests, in hill thickets, or on grassy slopes (Figure 2E). Their altitude range is from 1,500 to 3,200 m. The habitat types of plants of F. cirrhosa and F. unibracteata are almost the same. The altitudes of plants of F. taipaiensis and F. wabuensis are lower than that of F. cirrhosa and F. unibracteata, and altitude of plants of F. taipaiensis is the lowest. F. taipaiensis and F. wabuensis are thus most suitable for artificial cultivation. The plants of F. delavayi grow in relatively harsh conditions with higher altitude, lower temperature and lower humidity.

F. cirrhosa (Cheng and Helen, 2000): As shown in Figure 3A, height range of stem is 15–65 cm. Seven to eleven leaves are opposite or sometimes 3- or 4-whorled and alternate. Shape of leaf blade is linear to linear-lanceolate (4–12 cm × 3–15 mm), which apex is often curved or cirrose. The inflorescence is (1-3)-flowered with 3 bracts, whose apex is curved or cirrose. Flowers are bell-shaped, usually nodding. The pedicel is much shorter than tepals. Tepals are yellow or yellowish green, slightly or heavily spotted or tessellated with purple, usually oblong-elliptic (3–5 × 1.2–1.8 cm). Nectaries are elliptic to ovate (3–5 × 2–3 mm), projecting abaxially. Length of stamens is 2–3 cm, filaments sometimes are slightly papillose. The style is 3-lobed with 3–5 mm lobes. Capsule is narrowly winged, and wings are 1–1.5 mm wide.

F. unibracteata (Cheng and Helen, 2000): As shown in Figure 3B, height range of stem is 15–50 cm. There are five to seven leaves, and the basal 2 leaves are usually opposite, others are opposite or alternate. The shape of leaf blade is linear to linear-lanceolate (3.6–5.5 cm × 3–5 mm), apex is not curved or cirrose. Inflorescence is (1-3)-flowered with 1 bract whose apex is acuminate. Flowers are bell-shaped, usually nodding. Pedicel is rather long. Tepals are blackish purple, tessellated with yellowish brown or sometimes with a colored, V-shaped stripe near apex. Nectaries are inconspicuous or strongly projecting abaxially. Length of stamens is 1.2–1.5 cm, filaments sometimes are papillose. The style is scarcely or shortly lobed with 0.5–2 mm lobes. The capsule is narrowly winged, and wings are about 1.1 mm wide.

F. wabuensis (Cheng and Helen, 2000): As shown in Figure 3C, height range of stem is 50–115 cm. There are 12–28 leaves, and the basal 2 leaves are usually opposite, others are whorled or alternate. Shape of leaf blade is linear-lanceolate (6.5–16.5 cm × 6–25 mm), the apex is not curved or cirrose. The inflorescence is (1-7)-flowered with 1-4 bracts whose apex is acuminate. Flowers are bell-shaped, usually nodding. Tepals are yellowish green, spotted with or without blackish purple. Nectaries are elliptic to ovate (5–8 × 2–3 mm), projecting abaxially. The style is 3-lobed with 3–5 mm lobes. The capsule is narrowly winged, and wings are 1–2 mm wide.

F. delavayi (Cheng and Helen, 2000): As shown in Figure 3D, height range of stem is 10–35 cm. Five to seven leaves are closely arranged in middle or distal part of the stem, alternate or subopposite. The shape of the leaf blade is ovate or ovate-elliptic (2–7 × 1–3 cm), apex often is obtuse or rounded. The inflorescence is 1-flowered without bract. Flowers are bell-shaped, usually nodding. The pedicel is long. Tepals are yellowish, spotted or tessellated with reddish brown, narrowly elliptic or oblong-elliptic (3.2–4.5 × 1.2–1.8 cm). Nectaries are inconspicuous. Length of stamens is 1.6–2.2 cm, filaments are glabrous. Anthers are basifixed. The style is 3-lobed with 0.5–5 mm lobes. The capsule is narrowly winged.

F. taipaiensis (Cheng and Helen, 2000): As shown in Figure 3E, height range of stem is 20–100 cm. Five to twenty leaves are usually opposite or sometimes middle and distal ones also whorled and alternate. The shape of the leaf blade is linear to linear-lanceolate (5–13 cm × 3–12 mm), apex often is not curved. The inflorescence is (1-3)-flowered with 3 bracts whose apex is often curved. Flowers are bell-shaped, usually nodding. The pedicel is 2–4 cm. Tepals are yellowish green, densely spotted purple, narrowly oblong or obovate-oblong (2.5–5 × 0.6–1.8 cm). Nectaries are slightly projecting abaxially. Length of stamens is about 3/5 as long as repals, filaments are slightly papillose distally. The style is 3-lobed with 2–4 mm lobes. The capsule is winged, and wings are 0.5–2.0 mm wide.

Because production of BFC obtained from the wild field has fallen short of consumption and the price is growing higher in recent decades, the artificial cultivation of BFC has been gradually developed by the traditional Chinese medicinal cultivation companies (Wang et al., 2014a). In this work, we investigated six artificial cultivated bases and two individual cultivated plantations (Figure 4). As shown in Table 3, the cultivated bases or plantations are located in Kangding, Mao, Songpan, and Hongyuan counties in Sichuan province, and Wuxi county in Chongqing province. The species in cultivation include F. cirrhosa, F. unibracteata, F. wabuensis, and F. taipaiensis, which grow in relatively easier habitats compared to other species. We also found that lots of farmers in Wuxi county cultivate F. taipaiensis in gardens or surrounding the home yard. The income of selling bulbs of F. taipaiensis is one of the important economic sources for the region. Enwei Plateau Medicinal Plant Fostering Base and New Lotus Chuanbeimu Cultivation Base are the two largest bases among the investigated bases covering an area of about 400 hm² and their productivity is up to 180 t. However, the production of BFC from artificial cultivation is far short of what is needed (Liu et al., 2008). So, at present wild populations is still the main source of BFC in the market, which may further aggravate BFC wild resource depletion.

The calibration curve was drawn with six standard solutions at concentrations ranging from 0 to 10.43 μg/ml. The calibration curve showed a good linearity with the correlation coefficient being r = 0.9984. The regression equation was Y = 17.5668X + 0.1477, where Y and X correspond to the concentration and absorbance, respectively. The data about the content of total alkaloid in different samples are listed in Figure 5 and Table 1. The results show that samples from Mingzhong Village in Chengkou county, Huangcaoping Chuanbeimu Cultivation Base in Wuxi county, Chongqing province and from Enwei Plateau Medicinal Plant Fostering Base in Kangding county, New Lotus Chuanbeimu Cultivation Base in Songpan and Mao counties, Sichuan province have the higher content of total alkaloids. These samples are from artificial cultivation bases or individual plantation, which suggests that the content of total alkaloid of bulbs from artificial cultivation is higher than that from wild fields. The reason is unknown so far.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.