Converting ginsenosides from stems and leaves of Panax notoginseng by microwave processing and improving their anticoagulant and anticancer activities

A microwave processing technology was applied to degrade saponins from the stems and leaves of Panax notoginseng. Six transformation products (1–6), named 20(S)-ginsenoside Rg3 (1), 20(R)-ginsenoside Rg3 (2), notoginsenoside SFt3 (3), ginsenoside Rk1 (4), ginsenoside Rg5 (5), and 20(S)-ginsenoside Rh2 (6) were isolated and identified from a microwave processed extract of the stems and leaves of P. notoginseng (MEL). This transformation method was also applied for producing the minor ginsenosides in flowers, seeds and pedicels of P. notoginseng. The extract and compounds 1–6 in MEL were evaluated in vitro for anticancer and anticoagulant activities. The results showed that the MEL extract and transformation products had outstanding inhibitory activities against human cervical cancer Hela and lung cancer A549 cells. The strongest inhibitory effect was observed for 20(S)-Rh2 (6) with an IC50 value of 8.23 μM in Hela cells. Moreover, the results showed that the MEL significantly prolonged prothrombin time in a concentration-dependent manner. The anticoagulant effect of the MEL improved with the increased contents of Rk1, Rg5, and SFt3.


Introduction
Panax notoginseng (Burk.) F. H. Chen has been widely used as a herbal medicine in China. It was recorded in the Compendium of Materia Medica that the stems and leaves of P. notoginseng (PNL) were traditionally used to treat bone fractures, eliminate swelling, and stop bleeding. The PNL is also used as a functional food in folk medicine, as PNL is rich in nutrients and characterized by high protein, high dietary ber, and low fat. 1 Fresh leaves are suitable for eating as a vegetable and dried as a tea. 2 More than 40 chemical constituents have been isolated from the PNL. The main components are protopanaxadiol type saponins, such as notoginsenosides Fa, Fc, and Fe, and the ginsenosides Rb 1 , Rc, Rb 2 , and Rb 3 (Fig. 1). 3 The saponins have gained prominence owing to their potential pharmaceutical values of analgesic, hypolipidemic, anticancer, anti-arrhythmic, anticoagulatory, anti-inammatory, and anti-aging activities. 4,5 The interesting structures are less-polar saponins losing some glycosyl moieties such as 20(S/R)-ginsenosides Rg 3 and Rh 2 because of more effective anticancer activities. 6,7 The Shenyi capsule containing ginsenoside Rg 3 was clinically applied for treating non-small cell lung cancer in China. 8 The common methods used for producing less-polar saponins are acid-base degradation, 9 enzymatic degradation, 10 microbial transformation, 11 and steam processing. 12 At present, microwave processing method as a novel "green" solvent extraction technology is developed to increase the content of minor ginsenosides from white ginseng. 13,14 This green extraction process is oen used for extracting natural products owing to a faster extraction rate, less consumption of organic solvent, and lower costs of sample preparation, 15,16 but more rarely in the case of transformation. The heat processing by microwave method for producing the minor ginsenosides gained high yields within about 60 minutes. 13 The microwave time was much shorter than used in the traditional heating method (over 20 hours). 12 We applied the microwave processing method to accelerate the transformation of minor ginsenosides from the roots of P. notoginseng and determined the optimal conversion conditions using response surface methodology. 17 The roots of P. notoginseng are traditionally used as medicine, but a huge amount of PNL is oen considered waste. In this study, the microwave method was applied to effectively obtain a microwave MeOH, three times for 45 min. Aer ltration and concentration, the extract of stems and leaves of P. notoginseng (EL, 11.25 g) was obtained with a yield of 22.5%. In the same way, the yields of the extracts of owers (EF), seeds (ES), and pedicels (EP) were 36.36%, 6.48% and 25.48%, respectively.
The extract samples from different parts of P. notoginseng (EL, EF, ES and EP) was treated using microwave processing method as reported previously. 17 160 mg of the extract was added to the water (10 mL) in a microwave extraction system (model no: MDS-6G) manufactured by Sineo microwave chemical technology Co. Ltd. (Shanghai, China). Considering the inuence of microwave temperature, microwave power and time, the temperature ranged from 60 C to 180 C, microwave power ranged from 300 W to 1000 W, and time ranged from 5 min to 50 min. Aer microwave treatment, the sample solution was centrifuged and the supernatant was added to the water for 10 mL. The obtained samples were ltered through a 0.45 mm lter membrane for HPLC analysis. The yields of transformation products were obtained from HPLC data.
M g is the content of transformation products and M s is total amount of the sample.

Anticancer activity in vitro
2.6.1 Cell lines and culture. Human cervical cancer (HeLa) and human lung cancer (A549) cell lines were obtained from the Cell Bank of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. HeLa cells and A549 cells were cultivated in RPMI Media 1640 (RPMI1640) containing 10% fetal bovine serum, 100 U mL À1 penicillin, 0.1 mg mL À1 streptomycin and 2 mmol L À1 L-glutamine. All cultures were maintained with the aeration of 5% CO 2 at 37 C.
2.6.2 Cell cytotoxicity assay. The cytotoxicity of EL, MEL and compounds 1-6 on two cancer cell lines was determined by MTT assay. 22 The cells were seeded in ve-well plates (1 Â 10 4 cells per well) and incubated at 37 C for 24 h. The cells were then treated with EL, MEL, cisplatin and compounds 1-6 at different concentrations (0.1-100 mmol L À1 ). Cisplatin was used as the positive control. Aer 48 h, 20 mL MTT (5 mg mL À1 ) was added to each well and the cells were incubated for 4 h. The supernatant was removed and then 150 mL of DMSO was added to each well. Aer the plates were shaken at room temperature for 10 min, the absorbance was measured by PHERA star FSX Microplate Reader (BMG Labtech, Offenburg, Germany) at a wavelength of 570 nm. The half maximal inhibitory concentration (IC 50 ) against cancer cells was obtained by curve tting of a sigmoidal dosage-response curve using nonlinear regression model.

Anticoagulant activity in vitro
2.7.1 Animals. Kunming mice (18-22 g) were purchased by Changsha Tianqin Biotechnology Co., China (Qualied no. SCXK 2014-0011). The mice were maintained at a temperature of 25 AE 2 C, a humidity of 62 AE 2%, and a 12 h dark/light cycle, with unrestricted access to food and water. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Kunming University of Science and Technology, and were approved by the Animal Ethics Committee of Kunming University of Science and Technology.
2.7.2 Plasma preparation. The plasma was obtained from mice whole blood mixed with 0.109 mol L À1 sodium citrate (9 : 1 ratio of blood to citrate, v/v) by centrifugation at 3000 Â g for 15 min.
2.7.3 Blood plasma clotting assays. Prothrombin time (PT) was determined with a coagulation analyzer (XN06-IV, Aierfu, Wuhan, China). 100 mL of a solution containing plasma (50 mL) and EL, MEL and compounds 1-6 (50 mL) were incubated at 37 C for 3 min. The samples of EL and MEL were diluted with puried water to give the following concentrations: 25, 12.5, 5 mg mL À1 . The concentration of compounds 1-6 is 1 mg mL À1 . Puried water was used as negative control. The PT assay reagent (100 mL) was added to the mixed samples, and the clotting time was recorded by the analyzer. All clotting tests were performed with three individual replicates.

Statistical analysis
Statistical analyses were performed with SPSS 19.0 soware. All data were expressed as mean AE standard deviation (SD). The values of p < 0.05 were considered to be statistically signicant, and p < 0.01 and p < 0.001 being very signicant.

Results and discussion
3.1 Purication and identication of the transformation products from the stems and leaves of P. notoginseng using the microwave processing method The MEL was identied by ultra-high performance liquid chromatography-quantitative time of ight mass spectroscopy (QTOF-MS) in positive mode. The total ion chromatogram of the MEL is shown in Fig. 4. Table 2 Table 1 The results of linear regression of transformation product (1-6) (n ¼ 6)

Compounds
Calibration curve  which were identied by matching molecular formulas with reported data. 3,4 Six transformation products were puried from the MEL, and their structures were determined by comparing their spectroscopic data with those reported in the literature (Fig. 2). The transformation products were identied as 20(S)-ginsenoside Rg 3 (1), 18 20(R)-ginsenoside Rg 3 (2), 18 notoginsenoside SFt 3 (3), 19 ginsenoside Rk 1 (4), 20 ginsenoside Rg 5 (5), 20 and 20(S)-ginsenoside Rh 2 (6). 21 3.2 Impact of microwave processing conditions on the yields of the transformation products Microwave processing as a "green and innovative" technique has been widely used for preservation, 25 transformation, 13,14 and extraction 26 in research and industrial elds. The microwave processing technique has advantages of less time and energy as well as fewer solvents. 16 In our study, this microwave processing method was applied to degrade the major saponins in the PNL and to produce the rare saponins with fewer sugar moieties. Microwave heating occurs within the molecule, and the glycosidic bonds in the polar molecules break easily. 15 The major saponins in PNL were hydrolyzed at the glucosyl residue of C-20 to produce ginsenosides 20(S)-Rg 3 , 20(R)-Rg 3 , 20(S)-Rh 2 , Rk 1 , Rg 5 , and notoginsenoside SFt 3 in the MEL with an increase in microwave power and temperature. The three factors of microwave power, temperature, and time were assessed for yields of the ve transformation products by HPLC analysis aer microwave processing (Fig. 5).
Microwave power was an important factor in the transformation process. When microwave temperature and time were set to 135 C and 15 min, microwave power was varied from 300 to 1000 W to evaluate its impact on the yields of the transformation products (Fig. 5A). The highest yield of each transformation product was reached with microwave power of 500 W. The yields of the ve transformation products in the MEL were in the order of Rg 5 > SFt 3 > 20(R)-Rg 3 > 20(S)-Rg 3 > 20(S)-Rh 2 . When the microwave temperature range was from 60-180 C for 20 min, the yields of the transformation products increased gradually (Fig. 5B). When the temperature was 150 C, the yields of the transformation products were the highest in the following order: Rg 5 > SFt 3 > 20(R)-Rg 3 > 20(S)-Rg 3 > 20(S)-Rh 2 . According to the yields of the transformation products, a suitable time for this microwave processing method was 15-30 min (Fig. 5C). The yields of the transformation products were in the following order: Rg 5 > SFt 3 > 20(R)-Rg 3 > 20(S)-Rg 3 > 20(S)-Rh 2 . It was reported that the contents of the primary degradation products 20(S)-Rh 1 , 20(R)-Rh 1 , Rk 3 , Rh 4 , 20(S)-Rg 3 , 20(R)-Rg 3 , Rk 1 , and Rg 5 for dried notoginseng steamed at 120 C increase rapidly aer 12 h. 12 A similar yield was achieved in 20 min when the microwave processing method was used. Transformation products are obtained by microwave processing with less energy and less waste compared with the steaming method. According to the yields of the transformation products aer microwave processing, the optimum conditions of microwave power, temperature, and time were 500 W, 150 C, and 20 min, respectively. 3.3 Applying the microwave processing method to different parts of P. notoginseng The results of saponin transformation in different parts of P. notoginseng (stems and leaves, owers, seeds, and pedicels) using microwave processing are shown in Fig. 3 and 6. High performance liquid chromatography chromatograms of stems and leaves, owers, seeds, and pedicels of P. notoginseng revealed similar typical ginsenosides as the PNL, consisting of notoginsenosides Fa and Fc, and ginsenosides Rc, Rb 1 , Rb 2 , and Rb 3 (Fig. 3A-D). Aer microwave processing, six newly transformed products of 20(S)-Rg 3 , 20(R)-Rg 3 , SFt 3 , Rk 1 , Rg 5 , and 20(S)-Rh 2 were generated from the MEL, owers (MEF), seeds (MES), and pedicels (MEP) of P. notoginseng (Fig. 3E-H). As shown in Fig. 6, the Rg 5 content in the microwave-treated extracts from different parts of P. notoginseng was the highest and the Rh 2 content was the lowest. Under the same microwave processing conditions (500 W, 500 C, and 20 min) the yields of Rg 5 in the microwave transformed extracts from different parts of P. notoginseng were in the order of: MEL > MEP > MES > MEF. The 20(S)-Rh 2 contents in the MEL and MES were 2.58% and 0.36% respectively, while 20(S)-Rh 2 was not detected in the MEP or MEF. Three special saponins of notoginsenoside Fe, ginsenoside Rd 2 , and gypenoside IX were detected in the PNL (Fig. 3A), which easily lost the glycosyl group at C-20 to generate 20(S)-Rh 2 . This result indicates that 20(S)-Rh 2 content in the MEL was much higher than that in the MEP, MES, or MEF.

Transformation mechanism for producing the minor ginsenosides using the microwave processing method
Microwave heating occurs within molecules, which is described as "internal heating". 15 If the microwave energy matches with the molecular bond energy, the reactive activity of glycoside bond in the molecule increased and easy to break. 15,27 In our paper, major saponins lost the glycosyl residues and produced the minor ginsenosides with increased microwave power and temperature. The proposed transformation pathway of saponins in PNL by the microwave processing were shown in Fig. 7. The major saponins, e.g., ginsenosides Rb 1 , Rd, Rc, Rb 2 , and Rb 3 easily lost the glycosyl residue at C-20 to produce 20(S)/ (R)-Rg 3 , and then formed Rk 1 and Rg 5 through dehydration at C-20. Notoginsenosides Fa and Fc were hydrolyzed and dehydrated at C-20 to obtain notoginsenoside SFt 3 . When the microwave temperature was increased, a small amount of 20(S)/ (R)-Rg 3 was converted to 20(S)/(R)-Rh 2 through loss of a glucosyl group at C-3. In addition, the three compounds of notoginsenoside Fe, ginsenoside Rd 2 and gypenoside IX rstly removed the arabinose or xylose residues at C-3 to produce ginsenoside F 2 , and continued to lose one glucose residue at C-20 to obtain 20(S)-Rh 2 . This indicated that the content of 20(S)-Rh 2 was much higher than that of 20(R)-Rh 2 in the microwave processing. When the glycosyl residues at C-20 were lost, 20(S/R) epimers such as 20(S/R)-Rg 3 and 20(S/R)-Rh 2 were obtained by the selective attack of the OH group. 28 The

Antiproliferative effect of the transformation products on the cancer cell lines
The antiproliferative activities of the PNL extract before and aer microwave processing, and transformation products 1-6 were evaluated in two human cancer cell lines (HeLa and A549) by the MTT assay. The results are expressed as the IC 50 of the half maximal inhibitory concentration against cancer cells. As shown in Table 3, the extract of stems and leaves of P. notoginseng (EL) had no inhibitory effect on human cervical cancer (Hela) or human lung cancer (A549), and no IC 50 values were obtained. Aer the EL microwave treatment, microwave processing of the MEL resulted in better inhibitory activity against Hela and A549 cells, with IC 50 values of 42.97 and 50.47 mg mL À1 , respectively. Microwave processing of ginseng enhances its anticancer activity due to the increased content of the ginsenosides Rg 3 , Rg 5 , and Rk 1 .  To conrm the antiproliferative effect of the transformation products, the morphologies in Hela and A549 cells treated with ginsenoside 20(S)-Rh 2 was observed under a microscope (Fig. 8).
In the treatment of Hela and A549 cells, 20(S)-Rh 2 at the concentration of 1 mM had no signicant effect on cell morphology. While increasing concentration to 10 or 25 mM, 20(S)-Rh 2 induced cell body shrinkage and death in Hela and A549 cells.

The anticoagulant effect of the transformation products in vitro
According to previous reports, different parts of P. notoginseng have anticoagulant activity, and the leaves possess stronger anticoagulant activity among them. 32 The components of the PNL changed dramatically aer the microwave processing treatment (Fig. 3), which may have resulted in the change in anticoagulant activity. Therefore, the effects of the EL, MEL and transformation products 1-6 on anticoagulation were evaluated by the PT assay (Fig. 9). The PT value reects the activity of the  coagulation system in vitro. 33 As shown in Fig. 9A, the EL and MEL had signicantly prolonged clotting times in a concentration-dependent manner (p < 0.001). The anticoagulant activity of the MEL was better than that of the EL aer comparing the extracts of the PNL before and aer the microwave treatment. Further studies showed that the degradation products of ginsenosides Rk 1 and Rg 5 , as well as notoginsenoside SFt 3 prolonged coagulation time, while ginsenosides 20 (S/R)-Rg 3 shortened coagulation time (Fig. 9B). The structure-activity results revealed that the hydroxy-substituted compounds at C-20 (20 (S/R)-Rg 3 ) had the hemostatic activities, while Rk 1 , SFt 3 and Rg 5 with double bond at D20 (21) or D20(22) played the anticoagulant activities. The anticoagulant activity of the MEL increased due to increases in the concentrations of Rk 1 , Rg 5 , and SFt 3 . This result was consistent with previous ndings that ginsenosides Rk 1 and Rg 5 inhibit arachidonic acid-induced platelet aggregation in a dose dependent manner. 34

Conclusion
In summary, microwave processing was applied to produce rare saponins in the PNL. Six transformation products of ginsenosides 20(S)-Rg 3 , 20(R)-Rg 3 , Rk 1 , Rg 5 , 20(S)-Rh 2 , and notoginsenoside SFt 3 were isolated from the MEL. Microwave power, temperature, and time affected the yields of the six transformation products. This transformation method was widely applied for producing the minor ginsenosides in stems and leaves, owers, seeds and pedicels of P. notoginseng. The MEL had an obvious inhibitory effect on human cervical cancer and human lung cancer cell lines. The MEL had a stronger anticoagulant effect due to increased contents of ginsenosides Rk 1 , Rg 5 , and notoginsenoside SFt 3 . Therefore, the PNL may be a good source of anticancer and anticoagulant therapeutics aer microwave processing.  . Compared with the negative control group: *p < 0.05, **p < 0.01, ***p < 0.001. EL group compared with MEL group: Dp < 0.05, DDp < 0.01, DDDp < 0.001.