Study on hemostatic activities of the rhizome of Paris bashanensis

Abstract

Context. Paris bashanensis Wang et Tang (Liliaceae) is widely used in traditional Chinese medicine for the treatment of injuries, fractures and hemorrhage in Hubei and Sichuan Province.

Objective: The n-BuOH extract of Paris bashanensis was investigated for hemostatic activity and chemical constituents in order to provide a basis for the application in folk use.

Materials and methods: The n-BuOH extract of P. bashanensis was divided into three eluents (30, 50 and 70% EtOH) by macroporous adsorptive resin D101. The bleeding time of breaking tail hemostasis and clotting time of capillary and slide method in mice were used extensively to screen the hemostasis properties after repetitive administration of these three fractions (100 mg/kg, 50 mg/kg) for 5 days (total of 5 times, once per day). The chemical compounds were analyzed by HPLC-UV.

Results: The inhibition rates in the bleeding time of 70, 50 and 30% n-BuOH ext. were 45, 32 and 21%, respectively. Using the slide method the decreasing rate of the clotting time of 70, 50 and 30% n-BuOH ext. were 71, 65 and 32% and in the experiment of capillary method, the inhibition rates were 43, 31 and 24%, respectively. A total of 70% n-BuOH ext. showed a high content of the pennogenin-type saponins by HPLC-UV.

Discussion and conclusions: The 70% n-BuOH ext. of P. bashanensis was found to contain high levels of pennogenin saponins, which may lead to a higher hemostatic activity. Combined with the hemostatic test, P. bashanensis could be used as a resource of hemostatic drug.

• Blood clotting time
• HPLC-UV
• pennogenin saponins
• steriodal saponins
• tail bleeding time

Introduction

The genus Paris (Liliaceae), including 24 plant species, is widely distributed in tropical and temperate regions of Europe and Asia, of which 19 species are distributed in southwest China (Li, 1998). Rhizoma Paridis, the rhizome of P. polyphylla Smith var. yunnanensis, found in southwest China, has been widely used in traditional Chinese medicine for the treatment of various diseases, particularly for the treatment of abnormal uterine bleeding, which is the main raw material for Chinese drug known as Gongxuening Capsule (Guo et al., 2008; Zhao & Shi, 2005). Other well-known prepared Chinese medicines-Yunnan Baiyao Powder is famous for hemostatic activity. Now the increasing application of Rhizoma Paridis has resulted in a shortage of wildlife resources. In our laboratory, a previous study compared the major activities of other species of genus Paris to find replacement plants, and the results showed that P. bashanensis could be used as a resource of hemostatic drug rather than other species (Liu et al., 2012).

However, the main active constituents and their hemostatic activity of P. bashanensis are not fully studied (Zhang et al., 2011). The rhizome of P. bashanensis is slender for the transitional characteristics of its habitat from south to north (Li, 1998). Different morphological characteristics may lead to different secondary metabolites.

In the present study, the n-BuOH extract of P. bashanensis was further suspended in water and subjected to column chromatography (CC) over D101 macroporous resin, eluted with H₂O, 30, 50, 70% EtOH. Each eluent (30, 50, 70% n-BuOH ext.) was investigated and compared for hemostatic and chemical constituents in order to provide a basis for the application in folk use and find a substitute resource of Rhizoma Paridis.

Materials and methods

Plant material

The rhizoma of Paris bashanensis was collected from Shennongjia, Hubei province, China, in October 2009, and authenticated by Prof. Wenyuan Gao from the School of Pharmaceutical Science and Technology, Tianjin University. A voucher specimen (20091013) has been deposited in the School of Pharmaceutical Science and Technology, Tianjin University, China.

Extraction and isolation

The dried rhizoma of Paris bashanensis was powdered to a homogeneous size by a mill, sieved through a No. 40 mesh, and further dried at 40 °C in the oven for 2 h. The powder samples accurately weighed (100 g) were added to a round-bottomed flask containing 600 mL of 70% ethanol and the mixture was heated under reflux 3 times, 2 h for each time. After removal of the solvent under reduced pressure, the extract was suspended in water, with a yield of 11.23% (w/w), and then partitioned with petroleum ether, EtOAc and n-BuOH, subsequently leaving a residual water-soluble fraction. The n-BuOH soluble fraction (5.03 g) was suspended in water and subjected to column chromatography (CC) over D101 macroporous resin, eluted with 30 (1.74 g), 50 (2.32 g) and 70% (5.29 g) EtOH, to obtain three fractions.

Animals and drug administration

Animals and Kunming mice, weighing about 18–20 g, of SPF degree, were purchased from the Tianjin Experimental Animal Center (License No. SCXK (Jin) 2009-0002) and used in this trial. The animals were given free access to food and drinking water, under controlled temperature, humidity and photoperiod. Mice were allowed to be acclimated for 1 week. This animal study was approved by the Institutional Animal Care and Use Committee of China, and institutional guidelines for animal welfare and experimental conduct were followed. Yunnan Baiyao Powder was produced by Baiyao Group Co., Ltd. (Yunnan, China). All the drugs were dissolved in water for oral administration in mice.

Tail bleeding times

Tail transection bleeding time was determined according to the method described by Liu et al. (2012). Mice were given the extracts 5 days (once per day) and 40 min after the final oral administration the experiments were carried out. The mouse tail was transected at 5 mm from the tip and then the tail lesion was blotted with a filter paper every 30 s. The interval, from the time of the tail incision until the time that blood is no longer apparently transferred to the filter paper, was recorded as the bleeding time. Bleeding time beyond 600 s was considered as cut off time for the purpose of statistical analysis.

Blood clotting assays

Forty minutes after the final oral administration of the extracts (total of 5 times, once per day), the blood clotting assays were determined by two methods. The capillary method was described by Wang et al. (2012). The mouse blood samples (0.5 mL) were collected from the eye socket using a glass capillary, and the time interval required for blood coagulation inside the capillary was measured. In addition, the mouse blood samples (collected before) were spread onto the glass slides (slide method). This method used two drips of blood and one drip was used for repeat test. A stopwatch started immediately when the blood was dropped on a clean glass slide. At intervals of 30 s, the blood drip was gently teased to check for blood clot formation until the fibrin filament could be observed. The blood clotting time was recorded (Li et al., 2010). The experiments of all the tests were controlled at 27 °C with a humidity of 60%.

Statistical analysis

Data were expressed as means ± standard error (SEM) or percentage and analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Tests were performed using the SPSS 17.0 system. p Values less than or equal to 0.05 were considered to be statistically significant.

Reagents

HPLC-grade methanol and acetonitrile were purchased from Tedia (Fairfield, OH). Water was purified using a Milli-Q water purification system (Millipore, Molsheim, France). The other reagents were commercially available and of analytical purity. All solvents and samples were filtered through a 0.22 µm filter (Xinjinghua Co., Shanghai, China) before injecting into HPLC.

Analysis of Paris saponins by HPLC-UV

Each eluent (30, 50 and 70%) was made up to a concentration of 20 mg/mL in 80% methanol and filtered through a 0.45 μm membrane before being used for HPLC analysis. HPLC on Paris saponins were carried out on an Agilent 1100 liquid chromatograph system (Agilent Technologies, Santa Clara, CA), equipped with a quaternary pump, an online degasser, and a column temperature controller, coupled with G1322A, G1314AVWD as the detector. The analytical column temperature was kept at 35 °C, and detection wavelength was 203 nm. The samples were separated with a Kromasil RP-C18 column (4.6 mm × 250 mm, 5 µm, AKZO NOBEL, Sweden) using water (A) and acetonitrile (B) under gradient conditions (0–5 min, linear gradient 33–36% B; 5–12 min, linear gradient 36–45% B; 12–18 min, linear gradient 45–50% B; 18–42 min, linear gradient 50–43% B; 42–45 min, linear gradient 43–55% B; 45–70 min, linear gradient 55–100% B) as the mobile phase at a flow rate of 1 mL/min within 70 min. The injection volume was 20 µL. Peaks were assigned by comparing their retention time with that of each reference compound eluted in this mobile phase and by spiking samples with reference compounds (Man et al., 2010). Figure 2 shows HPLC chromatograms of 30 (A), 50 (B) 70% (C) n-BuOH ext. of P. bashanensis.

Results

Effect on tail bleeding time and blood clotting time in mice

The results showed that the bleeding time in tail of the mice of the three fractions of P. bashanensis extract were all reduced as compared to the control group, but effective extents were not equal (Figure 1A). 70% n-BuOH ext. showed the most potent effect and the inhibition rate of the bleeding time was about 45%, followed by 50% n-BuOH ext. and 30% n-BuOH ext. (about 32 and 21%, respectively), and the inhibition rate of the three extracts were higher than 15% compared to control group. Similar effects were observed in blood coagulation tests. The time interval of blood coagulation could be greatly reduced by the administration of the extracts. Using the slide method (Figure 1B), the decreasing rate of the clotting time of 70% n-BuOH ext. and 50% n-BuOH ext. were about 71% and 65%, 30% n-BuOH ext. was 32%. In the experiment of capillary method (Figure 1C), the inhibition rate of the clotting time of 70% n-BuOH ext. was 43%, and 50% (31%) and 30% n-BuOH ext. (24%) were lower than 40% compared to control group. These results indicated that 70% n-BuOH ext. of P. bashanensis possessed promising effects in reducing the bleeding time and blood clotting time in normal condition. Yunnan Baiyao Powder, a commonly hemostatic, used as the positive control group could decrease the bleeding time by about 15%, and the clotting time by about 45% (capillary method) and 57% (slide method).

Figure 1. Regulatory effects of 30, 50 and 70% n-BuOH ext. on bleeding and coagulation time. Mice were treated orally with the water solvent (control group), Yunnan baiyao powder (150 mg/kg, positive group), and 30, 50 and 70% n-BuOH ext. (100 mg/kg, 50 mg/kg) for 5 days. (A) The measurement of blood clotting was represented by the bleeding time from the cut tail. (B) The blood coagulation time was determined by using the slide methods. (C) The blood coagulation time was determined by using the capillary methods. Data were expressed as time of bleeding or blood coagulation (s), mean ± SEM, n = 10, *p < 0.05, ** p < 0.01 compared with the control group.

Analysis of Paris saponins by HPLC-UV

The method was subsequently applied to a simultaneous determination of eight bioactive markers in 30, 50, 70% n-BuOH ext. of P. bashanensis. Figure 2 shows the HPLC chromatograms of 30 (A), 50 (B) and 70% (C) n-BuOH ext. Compared with the HPLC chromatograms of steroidal saponins standards (Liang et al., 2012), the HPLC chromatograms of C showed the high content of the 8 saponins, especially the pennogenin-type saponin-Paris H, Paris VI and Paris VII. B contained a lower content of the pennogenin-type saponins (Rt = 17–20 min) and more furostanol saponins (Rt = 6–12 min) than C (Man et al., 2010). B was deduced to possess a furostanol structure based on TLC examination using the Ehrlich reagent and confirmed by the peak time (6–12 min). A with lower content of steroidal saponins showed lower hemostatic activity. The analysis of Paris saponins results showed the main activity constituents were total steroidal saponins (C) in rhizome of P. bashanensis and the main type of steroidal saponins was pennogenin-type saponins. Paris VII (tetraglycosides), PGRR (triglycosides) and Paris H (triglycosides) bearing (1→2)- or (1→4)-, α-l-Rha→β-d-Glc linkage always showed a higher activity. Together, these data indicate that the pennogenin-type saponins are mainly responsible for hemostatic activity.

Figure 2. HPLC chromatograms of 30 (A), 50 (B), 70% (C) n-BuOH ext. of P. bashanensis. The peaks corresponding to Paris VII (1), PGRR {(25R)-5-en-spirost-3,17-diol-3-O-α-l-rhamnopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyanoside} (2), Paris H (3), Paris VI (4), Paris II (5), Gracillin (6) and Paris I (7) were identified.

Discussion

Steroidal saponins are widely distributed in the genus Paris and have many pharmacologic actions and biological activities, such as antitumor, hemostatic, antibacterial, anti-inflammatory and immunoregulatory (Wu et al., 2004). Previous data suggest that P. bashanensis could be used as a resource for hemostatic drugs rather than other species (Liu et al., 2012). P. bashanensis is not fully researched. So, this study is the first to report P. bashanensis from the view point of hemostatic bioactivities and chemical components.

The bleeding time of breaking tail hemostasis and clotting time of capillary and slide method in mice are used extensively to screen the hemostasis and thrombosis properties of medicinal herbs. These convenient methods could reflect the whole process of blood coagulation since blood was eluted from the micrangium (Li et al., 2008). In our study, P. bashanensis showed hemostatic activity by reducing the values of bleeding time and clotting time, which was consistant with the report by Li et al. (2008). We found that the 70% n-BuOH ext. of P. bashanensis was shown more effective than the other groups. In order to explain the relationship between the hemostatic activity and chemical constituents, these extracts were further studied by the analysis of Paris saponins using HPLC-UV.

The n-BuOH fraction, with abundant steroidal saponins, was further divided into three eluents (30, 50 and 70% EtOH) by macroporous adsorptive resin D101. The chemical study showed that each eluent may contain different types of steroidal saponins. However, it was also dependent on the genus of Paris (Man et al., 2010). More pennogenin-type saponins were identified from the 70% n-BuOH ext. of P. bashanensis, especially the Paris VII, PGRR and Paris H, and that may lead to the higher hemostatic activity. The hemostatic of steroidal saponins was dependent on their structures, especially on the nature, number and sequence of the sugars in the saponins, while extending sugar units of pennogenin glycosides from diglycoside to triglycosides, and then to tetraglycosides, also significantly enhanced their activities. Their effects upon hemostasis and platelet function were evaluated and identified as the active ingredients of total steroidal saponins by Fu et al. (2008).

The furostanol saponins, which bear an additional monosaccharide at the other side of the aglycone (compared to the spirostanol saponins mentioned above), were not hemolytic at all and had little effect upon platelet aggregation. This 50% n-BuOH ext. showed lower hemostatic activity because of the less active spirostanol saponins and more furostanol saponins than 70% n-BuOH ext. 30% n-BuOH ext. with lower content of spirostanol saponins and furostanol saponins was the last.

P. bashanensis, belonging to the subgenus Paris, was found to contain high levels of pennogenin saponins, compared with the P. polyphylla Smith var. chinensis and P. polyphylla Smith var. yunnanensis which belong to the subgenus Daiswa (Li, 1998). Due to the different growth environment of P. bashanensis, its morphology and chemical constituents have changed a lot. The results of evolution showed that biosynthesis approach of pennogenin glycosides were increased in P. bashanensis and other species in the subgenus Paris.

Conclusions

In our study, hemostatic activity of P. bashanensis was further studied. More pennogenin-type saponins of total saponins from the 70% n-BuOH ext. of P. bashanensis, especially Paris VII and Paris H, may lead to the higher hemostatic activity. Combined with the hemostatic test, P. bashanensis could be used as a resource of hemostatic drug.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Acknowledgement

Financial support from the Doctoral Scientific Fund Project of the Ministry of Education of China (Grant No. 20090032110059) is gratefully acknowledged.