Fruits from Rosa roxburghii: A Valuable Bioresource of Potent Radical Scavengers and Novel Ursane-Type Triterpenoids

Four new ursane-type triterpenoids named rosaroxine A–D and 21 known compounds were identified from Rosa roxburghii fruits. The structures of all compounds were established by 1D and 2D NMR spectroscopy and mass spectrometry. The phenolics catechin (EC50 13.4 μM), quercetin (13.1 μM), gallic acid (10.0 μM), and protocatechuic acid (15.2 μM) were identified as powerful in vitro antioxidants with EC50 values lower than ascorbic acid (31.3 μM). The triterpenoids rosaroxine C (EC50 37.4; 40.3 μM) and 2-oxo-pomolic acid (16.6; 28.2) and the phenolics catechin (53.3; 29.0), quercetin (18.8; 33.1), and gallic acid (26.3; 40.0) exerted partly higher activities in the cyclo-oxygenase (COX 1/2) assay than the positive control acetaminophen (EC50 45.0; >100 μM). The triterpenoids rosaroxine C and 2-oxo-pomolic acid also performed well in the anti-aging assay using HaCaT cells. Quantification of the bioactive compounds by LC-MS revealed concentrations of 3.08 mg kg–1 rosaroxine C, 17.40 mg kg–1 2-oxo-pomolic acid, 76.29 mg kg–1 catechin, and 5.58 mg kg–1 protocatechuic acid in the dried fruits. Overall, this work provides detailed phytochemical information, and the results from the accomplished bioassay point toward health promoting properties of these fruits.


INTRODUCTION
Rosa roxburghii Tratt.(Rosaceae), or Cili, is widely distributed in the southwest of China, particularly in the provinces Guizhou and Yunnan.The fruits of this plant species play an important role in the daily diet of local people, and more than one hundred food and health products from Cili are manufactured.Despite its economic importance in the above-mentioned provinces, detailed phytochemical data about the fruits of this plant are scant.Hydrolyzable tannins, 1 polyphenols, 2 and triterpenoids 3 were described in the past decade.Recently, ascorbic acid derivatives together with flavonoid derivatives were reported. 4Performed bioassays of either extracts or single compounds of R. roxburghii exhibited radioprotective properties 5 and also radical scavenging activities. 6The polysaccharide fraction from R. roxburghii showed neurotrophic activity 7 , and Ellagitannins from R. roxburghii are able to suppress poly(I:C)-induced IL-8 production in human keratinocytes. 8Extracts from fruits of R. roxburghii also exerted lipid lowering effects in hyperlipidemic rats and antidiabetic effects, influencing the LDL and HDL as well as the cholesterol levels.
Since most of these reported bioactivities were assessed by using either crude extracts or not well separated fractions of these fruits, we performed a detailed phytochemical analysis of the crude methanolic fruit extract and identified 25 compounds.In addition to the identification of these compounds, we assessed potential antioxidative and antiinflammatory properties of selected compounds and quantified them in the fruits in order to provide as much information as possible for the use of these fruits.1) of 1 showed the occurrence of an olefinic proton (δ H 5.29), two hydroxymethine protons (δ H 3.92, 3.31), one hydroxymethylene proton (δ H 3.76, 3.70), and six methyl groups (δ H 1.30, 1.29, 0.97, 0.86, 0.79, 0.98) (Table 1), similar to the characteristics of euscaphic acid, 9 besides a hydroxymethylene group and the absence of the doublet methyl signal in compound 1.The 13 C and DEPT NMR data (   24.5, 22.4, 17.5, 16.8).These data suggest that compound 1 belongs to the group of ursane-type triterpenoids.Compared to the 13 C NMR spectrum of euscaphic acid, 9 the presumption that an oxygenated methylene signal in compound 1 replaced the doublet methyl (C-30) in euscaphic acid was supported.The cross-peaks visible in the HMBC spectrum from the methylene protons H-30 (δ H 3.70 and  3).On the basis of these data, the structure of 1 was established as shown in Figure 1.The key HMBC and 1 H− 1 H COSY correlations of 1 and 3−4 and ROESY correlations of 1 and 3 are given in Figure 2.
Compound 2 was obtained as a colorless solid.Its molecular formula was determined as C 30 H 46 O 7 from HRESIMS m/z = 517.3176[M−H] − as well as the NMR spectra, showing an additional degree of unsaturation in comparison to 1.The 1 H NMR spectra (Table 1) revealed the similarity to euscaphic acid, 9 due to the absence of the doublet methyl signal.Its 13 C NMR as well as HMBC spectra indicated two carbonyl carbons resonating at δ C 181.7 and the absence of the methyl group C-30.The HMBC cross-peaks from H-20 (δ H 1.59) and H-21 (δ H 2.24) to carbonyl supported a new carbonyl of C-30.The assignations of the other signals were performed by using the correlations visible in the HMBC and HSQC spectra.Thus, 2 was subsequently named rosaroxine B.
Compound 3 was obtained as a white, amorphous powder.Its molecular formula was determined as C 30 H 44 O 7 from HRESIMS and NMR data.The 1 H and 13 C NMR spectra of 3 were similar to those of 2-oxo-pomolic acid (5). 10 However, the signals of the doublet CH 3 -30 in compound 5 were absent in compound 3, and instead, a carbonyl (δ C 181.1, s) was observable in the 1 H and 13 C NMR spectra of 3. The HMBC correlation from H-20 (δ H = 2.28) to the carbonyl function supported this.The ROESY correlations of H-3 with H-5 (δ H 1.56) and H-23 (δ H 1.17) suggest that H-3 was α-oriented.Additionally, the chemical shifts of H-3 (δ H 3.99) and C-4 (δ C 46.5) were consistent with previously reported data (δ H 3.99 and δ C 45.3 at α-orientation, 3.50 and δ C 42.5 at βorientation). 11On the basis of these data, the chemical structure of 3 was established as shown in Figure 1.

Bioassays. 2.3.1. Radical Scavenging Activities.
Bearing in mind that the fruits of R. roxburghii are a part of the regular diet by local people, we systematically assessed potential health promoting properties of identified compounds.An initial screening of all identified compounds including the ethyl acetate and water layer indicated potent radical scavenging activities of compounds 3, 5, 15, 16, 21, and 22 as well as the ethyl acetate and water phases.These compounds were further selected for testing the antiinflammatory, hepatoprotective, anti-aging, and antioxidant activities.For the screening and evaluation of antioxidant activity of obtained extracts and pure compounds, the widely used DPPH assay was performed, with ascorbic acid as a positive control.The ethyl acetate and water fractions and the purified compounds 15, 16, 21, and 22 showed powerful free radical scavenging effects in comparison to ascorbic acid (Table 3).The potent radical scavenging activities of the water and ethyl acetate extracts are in line with the data from R. roxburghii 6 and other Rose species, e.g., R. davurica Pall, 31 due to the high concentration of the water-soluble ascorbic acid as well as other antioxidants in the aqueous extracts. 4,32.3.2.COX-1/2 Assay.In the COX-1/COX-2 assays, both triterpenoids 3 and 5 exerted significant activities in comparison to the positive control acetaminophen (Table 3).A possible keto/enol tautomeric effect at C-2 may explain this effect even though no aromatic ring system is present in both compounds.

Anti-aging and Hepatoprotective
Assays.The antiaging effects were assessed by using HaCat cells.The results disclosed rosaroxine C (3), 2-oxo-pomolic acid (5), and protocatechuic acid (22) as the most active compounds (Figure 3, B2).According to a previous study 33 protocatechuic acid ( 22) is regarded as a natural anti-aging agent; therefore, this result is not a surprise.In contrast, the positive effects of the triterpenoids rosaroxine C (3) and 2-oxo-pomolic acid (5)  in this assay were somehow unexpected.A possible keto/enol tautomeric effect at C-2 involving adjacent carbons may explain the observed effects.The selected samples 3, 5, 15, 16, 21, 22, the ethyl acetate, and water phase were also used to test the cell viability of HepG2 cells at the tested concentration of 100 μM.The obtained results exhibited no protective effects in this assay (Figure 3, B1).

General Information.
Optical rotations were measured with a RUDOLPH APVI-6 automatic polarimeter.UV spectra were recorded on a Shimadzu 2401A spectrophotometer.CD data were recorded with an Applied Photophysics V100 (Agilent, USA).1D and 2D NMR spectra were acquired using Bruker AVANCE III-600 MHz spectrometers with SiMe 4 as an internal standard.MS data were obtained using a Shimadzu UPLC-IT-TOF (Shimadzu Corp., Kyoto, Japan).
3.4.Cyclo-oxygenase (COX-1/2) Inhibition Assay.The inhibition activity of tested compounds against COX-1 and COX-2 was determined by using the COX (ovine) colorimetric inhibitor screening assay kit (Cayman, USA, item number: 760110) according to the manufacturer's protocol.Each compound was assayed in triplicate, and GraphPad Prism 8 software was used to calculate EC 50 values.All statistical analyses were performed by Microsoft Excel.
3.5.DPPH Assay.The DPPH radical scavenging capacity assay was performed according to a previously described method with slight modifications. 34Briefly, the tested compounds or extracts were dissolved in a small volume of DMSO to generate a 10 mM or 100 mg mL −1 (for the extracts) stock solution and diluted to generate the final testing solution with methanol.The 0.2 mM fresh DPPH methanol solution was prepared daily.The 90 μL of methanolic solution of DPPH was added to 60 μL of tested compound solution with different concentrations (or 60 μL of methanol as blank) in 96-well plates and allowed to react for 30 min at ambient temperature in the dark.The absorbance (OD) was measured at 517 nm on an Infinite M200 Pro (Tecan, Austria) microplate reader.The level of the DPPH• scavenging activity was calculated as The EC 50 values were calculated by using GraphPad Prism 8 software 4.6.
3.6.Quantification of Compounds 3, 5, 15, 16, 21, and 22 by LC-MS.The selected triterpenoids 3 and 5 as well as the phenolics 15, 16, 21, and 22 were quantified by employing the external standard method.The previously purified compounds were dissolved in methanol to reach a concentration of up to 2000 μg mL −1 .From these stock solutions, dilution series from 2000 to 220 μg mL −1 were prepared (three standards per sample; for the concentration of each standard see Supporting Information S27) and measured by LC-MS.The crude extract was diluted to 2.5 mg mL −1 for compounds 3, 5, 15, 21, and 22 and 10 mg mL −1 for compound 16.From the obtained data, the concentration of these compounds in the plant material was calculated.

Anti-aging Bioassay (UV−B Irradiation Model and Viability Assay).
Human HaCaT cells were cultured routinely in DMEM (high glucose) supplemented with 10% fetal bovine serum and 1% penicillin−streptomycin solution in a humidified incubator at 37 °C and 5% CO 2 . 19All cells were cultured in culture dishes, and the medium was changed every day.The cells were cultured when the cell density reached 80%.Briefly, the HaCaT cells were cultured for 24 h after plating at a density of 4 × 10 4 cells/100 μL in 96-well plates.Subsequently, cells were irradiated according to the grouping using a 20 W UV−B lamp (SiTing, Shanghai, CN) at a distance of 10 cm for 5 s.The irradiation dose was calculated to be approximately 0.80 J cm −2 .Cells were divided into the following groups: control, UV−B alone, and UV−B + samples (25 μM).After UV−B irradiation, HaCaT cells continued to culture for 24 h with the medium containing compounds to be measured.The effects of those compounds on cell protection were determined by the MTS assay (BestBio, Shanghai, CN).After incubation, cells in each well were treated with 20 μL MTS solution for 1 h.The absorbance at 490 nm was measured directly with a microplate reader (FlexStation3, Molecular Devices, CA, US).Cell viability was expressed as the ratio percentage of MTS after deducting background value, assuming that the absorbance of control cells with deducting background absorbance was 100%.
3.7.1.In Vitro SOD Activity.The superoxide dismutase type assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, CN) was used to detect whether the compounds in R. roxburghii possess a direct promoting effect on the SOD enzymatic activity.Briefly, the cell samples were extracted with lysate buffer after the cells grew to 80% in one cell dish; 25 μM compounds were added to the experimental wells; and the same volume of distilled water was added to the control wells to maintain a constant final volume.Finally, SOD enzyme activity was detected according to the manufacturer's instructions.Adherent HaCaT cells were incubated in a sixwell plate at a density of 1 × 10 6 cells per well for 24 h.According to the UV−B irradiation model, the cells were treated with UV−B irradiation and different compounds.After 24 h, the total protein was extracted by cell lysis buffer.After the protein concentration of all samples is unified, the enzyme activity is detected according to the instruction of the assay kit.
3.8.Hepatoprotective Assay.HepG2 cells were seeded into 96-well tissue culture dishes at 5 × 10 3 cells/well and cultured overnight in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin antibiotics at 37 °C in a humidified 5% CO 2 incubator.The cells were then incubated with test compounds at 50 μM or extracts at 200 mg mL −1 for 4 h before acetaminophen (APAP) exposure.After this pretreatment, the experimental group was treated with 20 mM APAP together with the compounds; the APAP-only group was treated with 20 mM neomycin and an equivalent volume of DMSO; and the control group was treated with an equivalent volume of DMSO without APAP or test compounds.After another 24 h of culture, the cell viability was then measured using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, USA). 12.9.Isolated Compounds.

CONCLUSIONS
In this work, we could show that mature rosehips from R. roxburghii contain a diversity of ursane-type triterpenoids, which may contribute to the taste of these fruits 35 and phenolics belonging to several subclasses of compounds derived from the shikimic acid pathway.Some of the identified compounds exhibited excellent bioactivities in vitro, especially with regard to the radical scavenging activities.Taking the presence of ascorbic acid derivatives as reported recently 4 into account, these fruits are a rich source of potent antioxidants.The regular uptake of these fruits by the local population in China suggests a positive effect on the health of consumers due to the identified constituents.These results contribute to the phytochemical knowledge of R. roxburghii and indicate possible health-promoting properties of these fruits.However, further studies are needed to elaborate these effects.

Figure 1 .
Figure 1.Chemical structures of compounds isolated from Rosa roxburghii fruits.
C NMR spectra of compound 4 were almost identical with those of 3, except the methylene signal (δ C 54.3) present in compound 3, which was substituted by a signal of a hydroxymethine (δ C 85.5) in compound 4. Its molecular formula was determined as C 30 H 44 O 8 deduced from the HRESIMS data in combination with the NMR data, indicating one OH group more than in compound 3.The HMBC correlations from H-1 (δ H 4.10) to C-25 (δ C 12.4), C-2 (δ C 212.2), and C-10 (δ C 50.1) confirmed this OH group at position C-1.The configuration of this OH group should be βorientated, established by the ROESY correlation of H-1 with H-5 as that of 3. Compounds 3 and 4 were subsequently named as rosaroxine C and rosaroxine D. The NMR and MS spectra of compounds 1−4 are given in the Supporting Information (Figures S1−S4).

μm 3 . 2 . 3 . 3 .
grain size) column was used with a detection wavelength of 210−300 nm and a flow rate of 1.0 mL min −1 .The injection volume was 10 μL, and the column temperature was 30 °C.MeOH is used as the mobile phase A, and water is used as the mobile phase B. The gradient elution program was as follows: 0−5 min, 5% A; 5−25 min, 5%−95% A; 25−30 min, 95% A; 30−35 min, 100% A. For quantification, the following parameters were used: Agilent Zorbax SB C18 column (4.6 × 150 mm, 5 μm, grain size); column temperature: 30 °C; flow rate: 1.0 mL min −1 , injection volume: 10 μL.Gradient of elution: 0−25 min 5% methanol, 95% water, isocratic elution.Mass spectrum parameters: Dual ESI source; atomization pressure of 30 psi; drying gas flow rate: 8 L min −1 ; drying temperature: 350 °C; ion source pressure: 3500 V; capillary pressure: 175 V; taper hole voltage: 65 V; level 4 pole voltage: 750 V; acquisition mode: Auto MS/MS; MS full scan range m/z: 100−1700; MS/MS full scan range m/z: 50−1700; acquisition frequency: 1 spectra s −1 ; data storage mode: Centroid.Plant Material.Mature fruits of Rosa roxburghii were collected in October 2021 in Panzhou, Guizhou Province, People's Republic of China, and identified by Dr. Jie Cai.The voucher specimen (Cai20211002) was preserved in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences.Extraction and Isolation.Twenty kilogram of fresh fruits of Rosa roxburghii were crushed and extracted with 200 L of MeOH at room temperature and filtered, and the solvent was evaporated in vacuo.The concentrated extract (1 kg) was partitioned between water and ethyl acetate (EtOAc), and the organic solvent was removed under reduced pressure.The mother liquor (100 g) was further subjected to column chromatography (CC) over silica gel (1 kg) eluted with

2.1. Structure Elucidation.
Compound 1 was obtained as a colorless solid.Its molecular formula C 30 H 48 O 6 with ten