Chemical assessment of wild Paris rhizome from Southwest China

Chonglou is a well known traditional Chinese medicine for anti-cancer and traumatic injuries. The major sources of Chonglou are from genus Paris. The present investigation aimed to determine contents of active steroidal saponins in Paris plants collected from different locations of Southwest China. The results showed that polyphyllin I and polyphyllin VII could be detected in almost all samples. However, polyphyllin II and polyphyllin VI were not detected in the research. The results indicated that besides Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis, used as medicine traditionally, other Paris species in Southwest China also have medicinal value. Ranges of bioactive compounds in P. polyphylla var. yunnanensis from different parts of the plants were wide. Coefficient of variation of polyphyllin I, polyphyllin II, polyphyllin VII, and total steroidal saponins were 53.4132, 43.9341, 88.2673, and 60.9082%, respectively. Varieties of compounds type and contents in Paris plants would be related to habitats.


INTRODUCTION
Traditional Chinese medicine (TCM) is getting more understood and popular worldwide in different medical purpose.Nowadays, TCM is playing an important role in healthcare system in several Asian countries (Elena et al., 2010).Medicinal plants are an essential part of TCM.These plants have been used to treat various diseases for more than 8000 years (Drǎsar and Moravcova, 2004).Recently, a large number of bioactive compounds were found from medicinal plants and these compounds are the key compounds of TCM development and utilization (Calvin and Chen, 2011).Steroidal saponins were isolated from various plants.These chemical compounds have bioactivities for anticancer, antibacterial, antihypertensive, antivirus, relieve inflammation, sedative, and anthelmintic activity.Hence, steroidal saponins are termed as important natural products (Deng et al., 1999;Sun et al., 2002).
Paris, belonging to the family Trilliaceae, is a genus of about 24 species distributed from Europe to Eastern Asia.There are 22 Paris species in China.The diversity center of this genus is located in Southwest China (Li, 1986;Li, 1998).The rhizomes of several species of Paris, such as Paris polyphylla var.yunnanensis and P. polyphylla var.chinensis are famous traditional Chinese herb named Chonglou.Paris plants as medicine in folk of China have a history of thousands of years (Li, 1998), and are widely used to treat parotitis, fractures, snakebite, hemostasis, and abscess (Committee for the Pharmacopoeia of P. R. China).It is also the main raw material of some well-know Chinese patent drug, such as "Yunnan Baiyao", "GongXue Ning", etc (Li, 1998;Zhang et al., 2011).The quality of Chonglou is been measured by bioactive compound contents.Polyphyllin I, polyphyllin II, polyphyllin VI, and polyphyllin VII are a group of steroidal saponins, and important components of Paris genus (Committee for the Pharmacopoeia of P.R. China, 2010; Zhang et al., 2011).Previous study revealed that polyphyllin has powerful pharmacological activities on homeostatic, spermicidal, and analgesic (Li, 1998).In recent years, researchers found that polyphyllin I was a potential anti-cancer drug as the functions of cytotoxic and apoptosis (Lee et al., 2005;Ong et al., 2008;Siu et al., 2008).P. polyphylla var.yunnanensis and P. polyphylla var.chinensis are the main materials of medicine in the official records (Committee for the Pharmacopoeia of P.R. China, 2010).However, many other Paris plants could be found in markets in China.To study contents and distribution of important bioactive compounds in Paris genus might help us to understand differences of Paris medicinal materials from different source and to improve conservation and utilization of Paris genus resources.The aim of this study was to describe the variation of the contents of main active compounds (polyphyllin I, polyphyllin II, polyphyllin VI, and polyphyllin VII) in some medicinal Paris plants from Southwest China.

Plant
The plant materials included 12 species collected from 43 locations in wild habitats of Yunnan, Guizhou, and Guangxi in Southwest China (Table 1).

Preparation of samples
The rhizomes of Paris plants were dried under the temperature of 50°C and ground to fine powder using a pestle mortar.

Extraction
The rhizomes powder (0.5000 g) was extracted with 25 ml of 98.0% alcohol under reflux in water bath for 45 min.1.5 ml of the extract was transferred into 2 ml centrifuge tube, then centrifuged at 16,000 rpm for 10 min after cooled to room temperature.

Chemicals and reagents
The standards (polyphyllin I, polyphyllin II, polyphyllin VI, and polyphyllin VII) were purchased from the National Institute for Control of Pharmaceutical and Biological Products (Beijing, China).Acetonitrile (high performance liquid chromatography (HPLC) grade) was purchased from TEDIA (Ohio, USA).Purified water (HPLC grade) was produced by Milli-Q system (Massachusetts, USA).Other reagents were all of analytical grade.

Preparation of standard solutions
Pure standards of polyphyllin I, polyphyllin II, polyphyllin VI, and polyphyllin VII were introduced as external standards in the analysis.Standard solutions with different concentrations were dispensed in methanol.All standard solutions for HPLC were filtered using 0.25 μm syringe filter before injection.

Qualitative analysis
Standard of polyphyllin I, polyphyllin II, polyphyllin VI, and polyphyllin VII were tested on HPLC.Six repeated measurements were then given to each sample.The range of standard curve of polyphyllin I and polyphyllin II was 0.01 to 1. 0 mg/ml.The range of standard curve of polyphyllin VI and polyphyllin VII was 0.002 to 0.1 mg/ml.Chromatograms and calibrations are shown in Figure 1 and Table 2.
Table 3 shows the HPLC analysis results.Polyphyllin I and VII have been detected in almost all samples however,  Total steroidal contents of these species were all more than 6.00 mg/g (Committee for the Pharmacopoeia of P. R. China, 2010).Among four compounds, polyphyllin I had the highest mean content (11.01 mg/g) followed by polyphyllin VI (2.97 mg/g), polyphyllin II, (1.35 mg/g) and polyphyllin VI which had the lowest mean content (0.68 mg/g).It implied that polyphyllin I was the most important part of steroidal saponins in Paris plant.In recent years, strong anti-cancer effect of polyphyllin I was proved (Ma et al., 2009, Man et al., 2010;Kong et al., 2010).Besides the traditional efficacy, Paris plants would become a new medicine of anti-cancer drug.P. polyphylla var.yunnanensis, P. polyphylla var.chinensis and other Pairs genus resources which had high content of polyphyllin I should be paid attention and conserved.Most of the bioactive compounds of TCM were secondary metabolites.Quality of TCM depends on the accumulation of these compounds (Su et al., 2005).By comparison (Table 4), our result showed that the ranges of contents of bioactive compounds in P. polyphylla var.yunnanensis from different area were wide.Polyphyllin VII had the highest coefficient of variation (CV) which was 89.12%, followed by polyphyllin I (53.39) and polyphyllin II (43.87).Steroidal saponins varied widely in P. polyphylla var.yunnanensis from different locations.
Most bioactive compounds were secondary metabolites (Huang and Guo, 2007).They play an important role in adaptation of plant to change of habitat and overcoming stress constrains (Edreva et al., 2008).The result gave a clue to take further research on the relationship between steroidal saponins in Paris and their living environment.

Conclusion
The content of steroidal saponins in Paris rhizome was polyphyllini> polyphyllin II> polyphyllin VI> polyphyllin VII.Polyphyllin I was the main composition of steroidal saponins.The result implied Paris plant may be a new medicine of anti-cancer drug.There were wide variation of content of bioactive compound in P. polyphylla var.yunnanensis from different location (Table 4).This might have a correlation with the habitat and it need further research.

Table 3 .
Contents of detected compounds in Paris plants (mg/g).

Table 4 .
Contents of detected compounds in P. polyphylla var.yunnanensis samples (mg/g) from different locations in Southwest China.Paris plants, other species, such as Paris caobangensis, Paris mairei, P. polyphylla var.alba, P. polyphylla, and Paris fargesii, would be judged to have medicinal value too.