Anti-inflammatory and Cytotoxic Triterpenes from the Rot Roots of Panax notoginseng

Abstract Four new protopanaxatriol-type triterpenes (1–2) and glucosides (3–4), were isolated from the rot roots of Panax notoginseng (Burk.) Chen, along with four known ones (5–8). Their structures were elucidated on the basis of extensive spectroscopic analysis (HRESIMS, NMR, UV, IR, and OR) and acidic hydrolysis. The possible transformation pathway of these compounds were also speculated from ginsenoside Rg1. Compound 1, with a unique α,β-unsaturated ketene in its side chain, showed significant inhibitory effects against NO production on Murine macrophage cells (IC50 = 4.12 ± 0.20 μM) and comparable cytotoxicities against five human cancer cell lines (myeloid leukemia HL-60, lung cancer A-549 cells, hepatocellular carcinoma SMMC7721, breast cancer MCF-7, and colon cancer SW480) to positive control, cisplatin (DDP). Graphical Abstract Electronic supplementary material The online version of this article (10.1007/s13659-019-0211-4) contains supplementary material, which is available to authorized users.


Introduction
Panax notoginseng (Burk.) Chen (Araliaceae), a well-known member called Sanqi or Tianqi in Ginseng family for the treatment of cardiovascular diseases, has been domesticated and cultivated for more than 400 years in the southwest of China [1]. It is now used as one of the major raw materials for many traditional Chinese medicinal preparations, such as Pien Tze Huang, Yunnan Baiyao, Sanqi oral liquid, etc. [2]. Due to the increasing demands from medicinal industry and herbal market, the plantation of Sanqi has been enlarged sharply in recent decades. However, it is susceptible to various diseases during the plantation, because of the sensitive property to environmental factors. Among which, root rot caused mainly by microbial infections is the main destructive disease [3][4][5].
So far, more than 200 chemical compounds including triterpenoid saponins, flavonoids, amino acids, and so on, have been reported from the roots, stems, leaves, flower heads, fruits, and fruit pedicels, and ginsenosides are found to be the dominant active principles [6]. Previous HPLC-HRMS study revealed that the oxidation levels of constituents in roots of P. notoginseng are significantly increased after being infected by root rot diseases [7]. In order to clarify the chemical composition and explore the possible transformation mechanism in the rot roots of P. notoginseng, the minor chemical constituents of the rot root were studied in detail firstly. This led to the identification of four new (1)(2)(3)(4) and four known (5)(6)(7)(8) triterpenes and saponins (Fig. 1), and their possible transformation pathway were speculated in this paper. All the isolates were evaluated for their anti-inflammatory activity (inhibition NO production) on Murine macrophage cells and cytotoxicities against five human cancer cell lines.
Compound 1 is a triterpene aglycone with a terminal α,βunsaturated ketene on the side chain, whose diglycoside and triglycoside in flower buds of P. notoginseng and tetraglycoside in Xueshuantong Injection were reported previously by LC-MS(Q-TOF) analysis [24,25]. In the present study, compound 1 was isolated and identified with other three new compounds (2-4) and four known ones from the rot roots of P. notoginseng for the first time. Compounds 2 and 5 were a pair of diastereoisomers and separated simultaneously by semi-preparative HPLC.
Since the glycosides in Panax species were reported to have strong anti-inflammatory and anti-tumor activities [24][25][26][27][28][29][30], all the isolates were evaluated for their inhibitory effects against NO production on Murine macrophage cell line and cytotoxicities against five human cancer cells (myeloid leukemia HL-60, lung cancer A-549 cells, hepatocellular carcinoma SMMC7721, breast cancer MCF-7, and colon cancer SW480). Only compound 1 displayed significant inhibition of NO production with IC 50 value of 4.12 ± 0.2 μM (L-NMMA: IC 50 = 34.32 ± 1.20 μM) and obvious cytoxicities against all the five human cancer cell lines, comparable to the positive control, DDP (Table 3). At primary concentration of 50 μM for NO inhibition and 40 μM for cytotoxicity, the other compounds 2-8 displayed no obvious anti-inflammatory and cytotoxic activities.

General Experimental Procedures
Optical rotations were measured with a P-1020 polarimeter (JASCO, Tokyo, Japan). IR spectra were measured on Bio-Rad FTS-135 series spectrometer or NICOLLET iS10 with OMNIC 9.8.372 software. UV spectra were recorded on a Shimadzu UV2401A ultra-violet-visible spectrophotometer. HRESIMS spectra were run on an API QSTAR

Plant Material
The

Extraction and Isolation
The fresh rot roots of P. notoginseng (70 kg) were cut from the healthy part, air-dried under room temperature, and then extracted with CH 3 OH three times. After removal of the organic solvent, the methanol extract (4.0 kg) was subjected to macro-porous resin D101 CC (250 × 30 cm) eluting with H 2 O to remove most of the saccharides and then washing with MeOH to give a crude saponins fraction (2.7 kg), which was further fractionated on a silica gel CC (250 × 30 cm), eluting with CHCl 3 -CH 3 OH (7:3), to afford three fractions (Fr. A-C). TLC and HPLC analysis showed Fr. A (220.8 g) containing mainly notoginsenoside R 1 and ginsenosides Rg 1 , Re, Rd and Rb 1 .

Acid Hydrolysis of Compounds 3 and 4
Compounds 3 and 4 (each 2.0 mg) were hydrolyzed in 2 M HCl (3 mL) at 65 °C for 4 h, respectively. The reaction mixture was extracted with CH 2 Cl 2 , three times (3 × 3 mL). The aqueous layer was neutralized with 2 M NaOH and dried to produce a monosaccharide mixture. Next, l-cysteine methyl ester hydrochloride (1.0 mg) was added to a no aqueous pyridine solution of the sugar mixture (1.0 mL) and kept at 60 °C for 1 h. After this, trimethylsilylimidazole (1.0 mL) was added to the reaction mixture and kept at 60 °C for 30 min, followed the extraction with n-hexane (1 × 2 mL). The n-hexane layer was subjected to GC analysis, run on a Agilent Technologies HP5890 gas chromatograph, equipped with a HP-5 quartz capillary column (30 mm × 0.32 mm × 0.25 mm) and a H 2 flame ionization detector with the following conditions: column temperature, 180-280 °C; programmed increase, 3 °C/min; carrier gas, N 2 (1.5 mL/min); injector and detector temperature, 250 °C; injection volume, 2.0 μL; and split ratio 1/50. The configuration of the sugar moiety was determined by comparing the retention time with the derivatives of the authentic samples.
The retention times of d-/l-glucose were 20.418/20.825 min, and the configuration of the respective sugar moiety from compounds 3 and 4 was determined as d-glucose.

Cytotoxic Assay
Five human cancer cell lines, myeloid leukemia HL-60, lung cancer A-549 cells, hepatocellular carcinoma SMMC7721, breast cancer MCF-7, and colon cancer SW480, were used in the cytotoxic assay. All the cells were cultured in RPMI-1640 or DMEM medium (Hyclone, USA), supplemented with 10% fetal bovine serum (Hyclone, USA). The cytotoxicity assay was performed according to the MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxy-phenyl)-2-(4-sulfopheny)-2H-tetrazolium] method in 96-well microplates [33]. Briefly, adherent cells (100 μL) was seeded into each well of 96-well cell culture plates and allowed to adhere for 24 h before drug addition, while suspended cells were seeded just before drug addition, each tumor cell line was exposed to the test compound dissolved in DMSO in triplicates for 48 h at 37 °C, with DDP and Taxol (Sigma, USA) as positive controls. After the incubation, 20 μL MTS and 100 μL medium were added to each well after removal of 100 μL medium, and the incubation continued for 4 h at 37 °C. The optical density was measured at 492 nm using a Multiskan FC plate reader (Thermo Scientific, USA). Concentration of a compound inhibiting 50% of cell growth (IC 50 ) was calculated by the Reed and Muench method.

Conclusion
In summary, eight PPT-type triterpenes (1-2, 5, 8) and saponins (3-4, 6-7) were isolated from the rot roots of P. notoginseng for the first time. Among them, 1-4 are new compounds. Their structures were determined on the basis of the extensive spectroscopic analyses and acidic hydrolysis. Compound 1 with a novel terminal ketene at C-24 to C-26 showed significant cytotoxic and anti-inflammatory activities.
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