Identification of two mitochondrial-targeting cyclometalated iridium(III) complexes as potent anti-glioma stem cells agents.

Y.R. Wu et al. / Food Science and Human Wellness 12 (2023) 536-545 536 Contents lists available at ScienceDirect Food Science and Human Wellness journal homepage: http://www.keaipublishing.com/en/journals/food-science-and-human-wellness A heteropolysaccharide from Rhodiola rosea L.: preparation, puriﬁcation and anti-tumor activities in H22-bearing mice Yaru Wu, Qing Wang, Huiping Liu*, Lulu Niu, Mengyu Li, Qi Jia State Key Laboratory of Food Nutrition and Safety, Key Laboratory of Food Nutrition and Safety, Ministry of Education of China, College of Food Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, China ARTICLE INFO ABSTRACT Article history: Received 17 December 2020 Received in revised form 1 March 2021 Accepted 5 June 2021 Available Online 15 August 2022 Numerous polysaccharides isolated from plants have been used to augment traditional drugs in the treatment of cancer. I n order to explore the influence to hepatocellular carcinoma, a novel cold water-soluble polysaccharide was separated from Rhodiola rosea L. root (RLP) and then its structure and anti-cancer activities were tested. The chemical compositions and high performance gel permeation chromatography (HPGPC) results indicated that RLP was an acid heteropolysaccharide with the molecular weight of about 1.15×106 Da. Furthermore, ion chromatography (IC), Fourier transform infrared (FT-IR) and nuclear magnetic resoance (NMR) further indicated that RLP was main composed of →2,4)-α-Rha(1→, →5)-α-L-Araf-(1→, α-D-Glu, →6)-β-D-Galp-(1→, β-D-Man and →4)-α-GalpA-(1→. In vivo antitumor activities of RLP were carried out by using H22 tumor-bearing mice model. The results shown that RLP (100 and 300 mg/kg) could inhibit tumor growth of H22 cells from 23.59% to 45.52% and protect thymuses and spleen without damage. In addition, according to cell cycle, AV-FITC/PI and JC-1, RLP could induce dose-dependent apoptosis of H22 cells via S phase arrested which was through a mitochondrial related pathway. Our data indicated that RLP has a broader application prospect in anti-tumor preparations. © 2023 Beijing Academy of Food Sciences. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: Polysaccharide Antitumor activity Rhodiola rosea L. Structure analysis 1. Introduction Rhodiola rosea L. which belongs to the plant family crassulaceae is a perennial herb and mainly grows in the harsh alpine environment of Tibet. The root of Rhodiola rosea L. known as “golden root” is a traditional medicinal and edible plant which is used to soak in water. And then, multiple active ingredients are isolated from that such as polysaccharide, salidroside, tyrosol, flavones, and so on [1,2]. Among them, polysaccharide is one of main active ingredients, which has the effects of antioxidant, immune regulation, anti- * Corresponding author at: Tianjin University of Science & Technology, Tianjin 300457, China. E-mail address: liuhuiping111@163.com (H.P. Liu) Peer review under responsibility of KeAi Communications Co., Ltd. inflammatory and anti-diabetic [3]. Furthermore, many researchers have shown that natural polysaccharides have an inhibitory effect on the proliferation of various tumor cells. Therefore, recent work have focus on the extraction of the functional component from medicinal and edible plants. In recent years, the anti-tumor researches of salidroside have achieved relatively mature results [4,5]. However, there are few researchers about the mechanism of large molecular weight Rhodiola rosea L. polysaccharide (RRP) which is isolated by traditional technology. Thus, it is particularly necessary to research the polysaccharide further because it can effectively promote the development of auxiliary therapy in anti-tumor drugs. The study of RRP structure was mainly focused on relatively small molecular weight components. Cheng et al. [6] isolated and purified the RRP by using traditional crafts (80 °C) and DEAE-52. And then, the molecular weight of RRP was calculated as 11.82 kDa by HPLC. More importantly, it was concluded that the RRP was Publishing services by Elsevier http://doi.org/10.1016/j.fshw.2022.07.056 2213-4530/© 2023 Beijing Academy of Food Sciences. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Y.R. Wu et al. / Food Science and Human Wellness 12 (2023) 536-545 consist of rhamnose, arabinose, xylose, mannose, glucose, galactose and galacturonic acid (1:2.71:0.16:0.21:0.11:0.58:0.14). Meanwhile, Xu et al. [7] improved the method of purification and got the RRP1 and RRP2 by using DEAE-52 and Sephadex G-100. Furthermore, the molecular weight of RRP1 and RRP2 was 5.5 kDa and 425.7 kDa. The structural information of RRP1 and RRP2 was then further confirmed by periodate oxidation, smith degradation, methylation analysis and NMR, and the results shown that RRP1 and RRP2 had a pyranose ring and uronic acid. More specifically, in RRP1, arabinose and glucose were connected by 1→3, and then mannose, rhamnose and galactose were mainly connected by 1→2, 6, 1→6, 1→2 and 1→4. In RRP2, The association between rhamnose, glucose and galactose was 1→3, and between mannose and arabinose was 1→2, 1→6 or 1→4. Regarding to the anti-tumor activity of RRP, the tissue and immune factors of mice have been studied extensively. However, further exploration of its mechanisms has been neglected. Cai et al. [8] puriﬁcated the polysaccharide (RRP-ws) and then established an S-180 sarcoma mice model to explore the immune regulation and antitumor activity of RRP-ws on sarcoma mice. The results shown that RRP-ws had a direct toxic effect on S-180 cells in vitro. In vivo, RRP-ws could inhibit growing of tumor cells while increasing index of spleen/thymus. In addition, RRP-ws also increased the production of interleukin-2 (IL-2), tumor necrosis factor α (TNF-α) and interferon-γ (IFN-γ) in serum and the ratio of CD+4/CD+8 in peripheral blood T lymphocytes. Many studies [9-11] have demonstrated that many cancers were caused by infection, chronic irritation, and inﬂammation, which might play a key role in the treatment of cancer. Pu et al. [12] suggested that Rhodiola rosea L. might affect tumor cells through two pathways, which were direct inhibition and inﬂammatory regulation. Besides, non-coding RNA fusions became the target of Rhodiola rosea L. for the treatment of cancer [13]. In addition, RRP has many other activities. The results (Cheng et al.) [6] shown that the intake of RRP could improve the influence of growth performance and nonspecific immunity on red swamp crayfish Procambarus clarkia. Meanwhile, it could enhance resistance to infection by A. hydrophila and exhibit high hypolipidaemic activities [14,15]. Yang et al. [16] studied the protective effect of freeze-thawed RRP on boar sperm. Xu et al. [7] studied its antioxidant and hepatoprotective activities, and then the unique advantages of RRP were appeared according to these researches. Hepatocellular caicinoma is the most common primary liver cancer, which is one of the most common fatal malignancies [17]. Currently, treatment is still limited to the poor prognosis and severe toxic side effect [18]. According to the isolated methods of RRP, high-temperature was convenient. While the extraction of long-term high-temperature would accelerate the degradation of polysaccharides and lead to changes in higher-level structures, which will affect the biological activities closely related to their structures [19,20]. The low temperature extraction process was relatively complicated and timeconsumed, while it could be mildly and maintain biological activities. Furthermore, few reports have been published on the low-temperature extraction of water-soluble RRP and their anti-tumor activity, which were closely related to their structural characteristics. In order to ﬁnd natural products against liver cancer, it was necessary to understand its plant polysaccharide composition and its anti-tumor potential. 537 In this work, a cold water-soluble and acidic polysaccharide, RLP, was isolated and purified from the root of Rhodiola rosea L. Moreover, its anti-tumor activities and possible mechanism of the RLP were against tumor in H22 tumor-bearing mice were also studied In vivo. This study provides comprehensive utilization of Rhodiola rosea L. and new ideas for the future research for the source of new natural anti-tumor drugs. 2. Materials and methods 2.1 Materials and reagent The roots of Rhodiola rosea L. were purchased from locial medical market (Xizang, China), which grown in the high mountain. T-series dextran (T-10, T-40, T-70, T-110, T-500, and T-2000), Propidium iodine (PI), and RIPA lysis buffer were purchased from Solarbio Co. (Beijing, China). Sephadex-G 200 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Test kits for bicinchoninic acid (BCA) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Cell cycle and apoptosis detection kit, Annexin V-FITC/PI Apoptosis detection kit were purchased from Beyotime Biotechnology (Shanghai, China). 2.2 Isolation and purification of the Rhodiola rosea L. polysaccharide The dried roots of Rhodiola rosea L. were smashed into powder and passed through a 80 mesh sieve. The prepared Rhodiola rosea L. powder (50 g) were immersed in 30 times (m/V) ultrapure water and placed at 4 °C for three times (12 h each time). The mixed liquid was centrifuged (4 000 × g, 15 min) and then collect the supernatant. Next, it was frozen and thawed repeatedly to make the volume 1/3 of the original volume, following precipitated by anhydrous ethanol (95%) to 50%. Finally, after incubating at 4 °C overnight, the crude Rhodiola rosea L. polysaccharide were obtained by centrifuging (8 000 × g, 10 min). The crude polysaccharide re-dissolved in distilled water and deproteinated by Sevag (n-butanol and chloroform 1:4, V/V). After protein were removed, the mixture was dialyzed (mw 100 kDa) with distilled water for 72 h and lyophilized. The gained polysaccharide (20 mg/L) were further purified by Sephadex G-200 (16 mm × 40 cm). The column was eluted with ultrapure water at 0.1 mL/min, with 1 mL each tube. After measuring the carbohydrate content by the phenol-sulfuric acid method, the main fraction was collected and confirmed by high performance gel permeation chromatography (HPGPC). The pure polysaccharide, named as RLP, was obtained after lyophilization. 2.3 2.3.1 Characterization of RLP Chemical composition analysis The total carbohydrate content of RLP was tested by phenolsulfuric acid method using galactose as standard. The protein content was measured by using the Bradford method. The total uronic acid was measured by using m-hydroxydiphenyl method. Y.R. Wu et al. / Food Science and Human Wellness 12 (2023) 536-545 538 2.3.2 Molecular weight analysis The molecular weight of RLP was measured by HPGPC, a TSK-gel G4000PWxl column (7.8 mm × 300 mm) and a refractive index detector (RID). The mobile phase was ultrapure water at 0.6 mL/min and then the RLP (20 µL, 1 mg/mL) was injected and eluted under the same conditions with the column temperature at 30 °C. In order to calculate the molecular weight of RLP, a series of T-series dextran’s (T-10, T-40, T-70, T-110, T-500 and T-2000) were selected to establish the standard curve. 2.3.3 UV-visible and FT-IR spectroscopy analysis The RLP (1 mg/mL) was dissolved with distilled water and the ultraviolet spectrum scanning was performed in the range of 190 nm to 500 nm. The RLP (1 mg) and dried KBr powder (150 mg) were mixed fully and tableted. The mixed sample was measured by using FT-IR spectrometer ((Bruker VECTOR-22, Karlsruhe, Germany) at the range of 4 000-400 cm-1. 2.3.4 Monosaccharide composition analysis The RLP (5 mg) and the 1 mL trifluoroacetic acid (TFA, 2.0 mol/L) were added into oil bath tube. Then the mixture was oiled by oil bath under 110 °C for 3 h. After that, the degraded sample was blown by N2 until dried. Next, in order to remove the TFA, the methanol was added three times. Finally, the hydrolysates were dissolved with ultrapure water and adjusted to the final concentration of 10 mg/L for ion chromatography (IC) (Dionex ICS2500, Thermo, USA) analysis. D-glucose, D-galactose, L-rhamnose, D-xylose, D-mannose, and D-arabinose, D-glucuronic acid and D-galacturonic acid were used as standard up to 10 mg/L. 2.3.5 NMR analysis The sample was dissolved with D2O for 2 h and freeze dried for three times. The RLP (50 mg) was dissolved into D2O (0.5 mL) fully and transferred into NMR tube. The 1H, 13C, COSY, HSQC spectra were measured at room temperature on a Bruker Advance III 400M spectrometer (400 MHz; Bruker Co., German) [21]. 2.4 RLP (0.2 mL) with 100 or 300 mg/kg weight every day. After two weeks, the pre-cultured H22 cells (2 × 106 cell/mice) were injected into the right armpit of the mice expect the blank group to establish the H22 model [20]. Furthermore, the mice of blank, model and RLP group were administered continuously as above while the 5-Fu group (20 mg/kg) were intraperitoneally injected. All groups of mice were oral administrated for another 15 days. At the end of the experiment, all mice were weighed and collected blood from eyeball. Finally, each group of mice were sacrificed by cervical dislocation and dissected out the tumor issues immediately. Animals and animal experiments Female mice (8 weeks old, weighting (20 ± 2) g) were purchased from the Department of Experimental Animal, Academy of Military Medical Science, Beijing, China. All the mice were under barrier conditions in the Center of Experimental Animals at Tianjin University of Science and Technology. All mice were feed food and water freedom under temperature of (24 ± 1) °C, (50 ± 10)% relative humidity with a 12/12 h light/dark cycle. After adapting for a week, all mice were divided into five groups (10 mice/group) named as blank group, model group, low and high dose group, and fluorouracil (5-fu) group. The mice of blank group and model group were administered 0.2 mL water once each day. And the mice of low and high dose group were administered by Tumor inhibitory rate (%) = w1 − w2 × 100 w1 (1) Where w 1 represents the average tumor weight of the model group, w2 represents the tumor weight of the RLP groups. Spleen/thymus index (mg/g) = 2.5 Spleen/thymus weight (g) 1 000 (mg) × (2) Mice weight (g) 1 (g) Blood routine examination Two hundred microliter blood of each mouse was quickly mixed with anticoagulant (K 2EDTA, 7.5 µL), and then the sample was detected by XFA-6130 automatic blood analyzer after mixing gently. 2.6 Cell cycle analysis Tumor issues (0.2 g) were polished by using saline (0.9%) to obtain the cell suspension, next washed with saline for three times. Then the cell suspension was added into the cold 70% ethanol to fixed over 18 h. At the end of fixing, the cell suspension was centrifuged (1 000 × g, 10 min) and washed by using saline for three times to remove ethanol. The mixture (500 µL) of RNase and PI were added into the cell precipitation in dark under 4 °C. After passing cell sieve, the cell cycle was analyzed by using flow cytometer (Becton Dicknson, USA). 2.7 Annexin V-FITC/PI staining assay Tumor issues (0.2 g) were polished under low temperature by using PBS to obtain the cell suspension. After centrifuging (1 000 × g, 10 min), the 1 × binding buffer (100 µL) were added into the cell precipitation. Next the AV-FITC (5 µL) was added into the cell suspension. Then mixing them, in dark, PI (5-10 µL) was added and then incubated for 15 min. The mixture was made up to 500 µL and detected by ﬂow cytomenter in 1 h. 2.8 Mitochondrial membrane potential detection The mitochondrial membrane potential (ΔΨm) was detected by using the fluorescent probe JC-1. The method of obtaining the cell suspension was the same as above. After that, the cell suspension was treated by the JC-1 working solution according to the assay kit (JC-1) instruction (Solarbio Beijing, China). The ΔΨm was detected by ﬂow cytomenter. Y.R. Wu et al. / Food Science and Human Wellness 12 (2023) 536-545 Statistical analysis Experimental data were presented as mean ± standard deviation and an inter-group test was used. The statistical analysis was made by SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Signiﬁcant differences and standard deviation were established through analysis of variance (ANOVA). *P < 0.05 was considered as signiﬁcant, and ** P < 0.01 was considered as extremely signiﬁcant. 3. Results and discussion 3.1 Purification and physicochemical properties of RLP In this study, the crude cold water-soluble polysaccharide was obtained from Rhodiola rosea L. root (cRLP) after alcohol precipitating (4 °C) and deproteinizing. The yield of cRLP was 2.58% (m/m). The cRLP was then purified by Sephadex G-200, resulted in a final yield of (21.32 ± 0.46)% based on dried cRLP powder. The results of chemical compositions shown that RLP was consist of total sugar (92.35 ± 0.21)% and uronic acid (27.68 ± 0.45)% which indicated that the RLP was acid polysaccharide. The result of acid polysaccharide was consistent with the research of Xu et al. [7]. As shown in the Fig. 1A, there were no significant peak in 260 nm and 280 nm in the UV-visible spectrum which indicated that RLP had no nucleic acid and protein [31]. 0.6 A Absorbance 0.5 0.4 0.3 0.2 0.0 175 200 225 250 275 300 325 350 375 400 Wavelength (nm) 9.211 6 000 B 5 000 4 000 3 000 2 000 1 000 0 4 6 8 10 12 14 16 18 Time (min) 70 C 60 50 40 30 20 3 420.45 10 0 4 000 3 500 3 000 2 500 2 000 1 500 1 000 500 Wavenumbers (cm1) Fig. 1 Puriﬁcation, composition of RLP was carried out by various instruments (A) UV-visible spectrum; (B) HPGPC chromatogram; (C) FT-IR spectrum. 3.2 Primary structure of RLP 3.2.1 3.2.2 Monosaccharide composition analysis As shown in Fig. 2, the sugar composition and molar ratio of RLP were identiﬁed and shown by IC analysis that RLP was mainly consist of Rhamnose (Rha), Arabinose (Ara), Galactose (Gal), Glucose (Glu), Xylose (Xyl), Mannose (Man), Galacturonic acid in a molar ratio of 1:3.33:2.87:5.62:0.49:0.32:4.50, which suggested that the RLP was an acidic polysaccharide. The results of conﬁrming uronic acid were consistent with the method by m-hydroxydiphenyl. At the same time, according to monosaccharide composition, the results also conﬁrmed the difference of RLP compared with others [7], which could further conﬁrm the novelty of RLP. 1 740.63 1 621.58 1 422.28 1 378.15 1 260.04 1 149.23 1 077.34 917.67 1 024.68 890.28 829.31 Transmittance (%) 2 2 960.31 nRIU 0.1 of polysaccharide. The HPGPC spectrum of RLP (Fig. 1B) shown that RLP was homogeneous polysaccharide. The average molecular weight of RLP was calculated to 1.15 × 106 Da by a calibration with standard regression lg mw = -0.352T + 9.334 (R2 = 0.996 6). The functional groups of RLP could be deﬁned by FT-IR analysis. As shown in the Fig. 1C, there was a strong major absorption peak at −1 3 420.45 cm , which revealed the O-H stretching vibrations. And the −1 C-H variable angle vibration were relatively weak at 2 960.31 cm −1 −1 and 1 422.28 cm . The absorption band at 1 621.58 cm and 1 740.63 cm−1 were resulted from the presence of C=O and COOH. Furthermore, the absorption band at 1 260.04 cm−1 and 1 378.15 cm-1 indicated the bending of C-H and C-O bond of carbohyydrates. −1 It was reported that the several bands (1 200-1 000 cm ) were characteristic for C-O and C-C stretching vibration [23]. In addition, −1 the three band at 1 149.23, 1 077.34 and 1 024.68 cm were due to the pyranose which represented galactose, mannose and glucose [24]. −1 −1 The absorption peaks at 917.67 cm and 890.28 cm proved that there were both α-conﬁguration and β-conﬁguration in RLP [25]. The −1 −1 characteristic absorption peaks at 890.28 cm and 829.31 cm further indicated that RLP contained mannose and galactopyranose. Peaks −1 −1 at 1 625.11 cm and 1 743.09 cm were from the carboxyl and ester carbonyl, respectively. The degree of esterification (DE) value was estimated at 19% according to the equation A1 743.09/(A1 743.09 + A1 625.11) (where A referred to the peak area), which indicated that RLP was a low methoxylated pectin. Singthong et al. [26] established calibration curve and obtained the DE value according to titrimetric method. Compared with this method and FT-IR, they confirmed that FT-IR was a reliable method for determining the DE value of pectins and there was no signiﬁcant difference. HPGPC and FT-IR results HPGPC was an effective method to measure the molecular weight nC 2.9 539 160 140 120 100 80 60 40 20 0 20 40 STD RLP 1 0 5 2 3 10 45 6 9 7 15 20 Time (min) 8 25 30 Fig. 2 Ion chromatogram of standard monosaccharide (above) and RLP monosaccharide (below): (1) Fucose (Fuc); (2) Rhamnose (Rha); (3) Arabinose (Ara); (4) Galactose (Gal); (5) Glucose (Glu); (6) Xylose (Xyl); (7) Mannose (Man); (8) Galacturonic acid (GalA); (9) Glucuronic acid (GluA). Y.R. Wu et al. / Food Science and Human Wellness 12 (2023) 536-545 NMR of RLP 77.16/4.08, 72.32/3.58, 63.92/3.96, and the linkages was 1→6, which was consistent with 1D NMR and previous researchers. And in rhamnose, the chemical shifts H-2/H-1, H-2/H-3, H-4/H-3, H-4/H-5 were at δ 4.09/5.21, 4.09/3.92, 3.80/3.92, 3.80/3.78, 1.31/3.78, which indicated the linkages of →2,4)-α-Rha (1→ according to the previous studies [36]. The other chemical shifts of monosaccharide were difﬁcult to obtain because of its big molecular weight and the more structure analysis needed to be explored further. Many studies have indicated that the linkages of (1→6)-β-DGalp and α-D-Glu might be the basis reason for immune activity [37]. In addition, compared with the study of literature [8], the presence of →5)-α-L-Araf-(1→ of RLP might be the main structure basis to improve biological activities, which was the unique linkages of RLP under low temperature conditions. Table 1 Assignments of 1H and 13C NMR spectra for RLP. Residues Linkages →5)-α-L-Araf-(1→ A B →6)-β-D-Galp-(1→ →2,4)-α-Rha(1→ C →4)-α-GalpA-(1→ D 3.3 1 3.3.1 2 3 4 5 6 107.75 84.03 80.20 80.92 63.26 - H 5.19 - C 107.40 71.24 74.97 77.16 72.32 63.92 4.11 3.69 3.96 4.58 C 107.18 76.70 74.97 80.30 79.89 17.69 3.58 3.96 H 5.21 3.80 3.78 1.31 C 99.87 69.20 79.58 68.56 - 170.70 H 5.08 - 3.89 3.92 4.08 3.82 H 4.09 3.91 4.24 4.38 4.06 Effect of RLP on transplanted cell growth As shown in Table 2, at the beginning of experiment, the weight of mice was maintained at about 25 g, and there was no signiﬁcant difference between the dose group. The ﬁnal body masss of groups apart from 5-Fu were increased. Especially, the reason why final weight of model group increased significantly was the Abundance 400 350 300 250 200 150 100 50 0 C Antitumor effect on H22-bearing mice B 1.36 2.28 2.20 2.74 3.54 4.42 4.40 4.32 4.24 4.07 3.96 3.90 3.85 3.84 A 5.50 5.36 5.29 5.26 5.22 5.19 The NMR spectra of 1H and 13C of RLP were presented in Figs. 3A and B. As shown in Fig. 3A, in the 1H spectrum of RLP, the proton signals almost all appeared in the narrow range of δ 3.56-5.38, which was a typical polysaccharide signal. Generally, then proton signal δ > 4.9 was α-configuration while β-configuration in range of δ 4.3-4.9. And according to FT-IR, ion chromatography and previous research, we could infer that the proton H-1 signals at δ 5.36, 5.26 and 5.19 proved the presence of α-D-Glu, α-D-Gal and α-L-Araf (residue A) residues The presence of α-D-Glu, α-D-Gal and α-L-Araf (residue A) residues could be inferred from the proton H-1 signal at δ 5.36, 5.26 and 5.19 [27-29]. In addition, the signal at δ 4.60, 4.40-4.42, and 4.34 was assigned to β-D-Gal (residue B), β-D-Man and β-D-Xyl [30,31]. And the proton signal peak at δ 1.37 in the high ﬁeld indicated the presence of Rha (residue C). It could be seen from the Fig. 3B that there was an obvious absorption peak at low field δ 170.70, which indicated that RLP contained uronic acid. Accordingly, in the anomeric carbon region, the faint signals at δ 100 and 100.46 indicated the presence of β-D-Man and β-DXyl [29] which was consistent with the result of monosaccharide analysis. And then, the anomeric carbon C-1 signals of δ 107.18/5.36 and δ 107.75/5.26 suggested the presence of α-D-Glu and α-L-Araf [31]. The signal at δ 107.40/4.60 indicated the presence of →6)-β-D-Galp-(1→, which suggested that there had a substituent on C-4 and the chemical shift would move to low field. The signal absorption peak in the high ﬁeld region of δ 16.86 was the Rhamnose, which further confirmed the result of 1H [33,34]. As shown in Fig. 3C, the chemical shifts of cross absorption peaks H-2/H-1, H-2/ H-3, H-4/H-3, H-4/H-5 in arabinose were at δ 4.11/5.19, 4.11/3.96, 4.24/3.96, 4.24/3.82. Subsequently, the C-1, C-2, C-3, C-4, C-5 signals from Fig. 3D of arabinose were identiﬁed at δ 107.75, 84.03, 85.3, 79.3, 83.42, which indicated the existences of 1→5 glycosidic linkages in arabinose [35]. Combined with COSY and HSQC, the chemical shifts of C-1/H-1, C-2/H-2, C-3/H-3, C-4/H-4, C-5/H-5, C-6/H-6 in galactose were at δ 107.40/4.58, 71.24/3.69, 74.97/3.91, 20.58 16.86 3.2.3 101.75 107.40 107.18 84.36 84.17 82.56 81.55 81.10 79.36 77.01 76.84 67.15 61.38 53.16 540 110 100 90 80 70 60 50 δ f1 190 170 150 130 110 90 70 50 30 10 δ f1 5.8 5.2 4.6 4.0 3.4 2.8 2.2 1.6 1.0 δ f1 D C B4/B5 B6/B5 B4/B3 C4/C5 A4/A5A2/A3 B2/B3 A4/A3 C2/C3 C4/C3 B6 D4 B4 C2 B5/C3 A5 D2 B5 D3 δ f2 C1 A1 A3 C4 B1 C6 5.5 5.3 5.1 4.9 4.7 4.5 4.3 4.1 3.9 3.7 3.5 3.3 3.1 δ f2 5.5 5.3 5.1 4.9 4.7 4.5 4.3 4.1 3.9 3.7 3.5 3.3 3.1 δ f2 Fig. 3 The 1D and 2D NMR spectrum of RLP. (A)1H spectrum; (B) 13C spectrum; (C) COSY spectrum; (D) HSQC spectrum. 55 65 75 85 95 105 115 δ f1 A4 A2 D1 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 B2 C5 55 65 75 85 95 105 115 δ f1 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 δ f2 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 δ f1 A2/A1 C2/C1 C6/C5 δ f1 B2/B1 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 Y.R. Wu et al. / Food Science and Human Wellness 12 (2023) 536-545 5-Fu on the body’s immune system. In contrast, this study has shown that RLP treatment had no toxic side effect and could inhibitory the growth of tumor cells. A 2.5 Tumor weight (g) inﬁnite proliferation of H22 cells. In contrast, the average weight in RLP groups were obviously descended compared to model group (P < 0.05), which indicated that RLP treatment exhibited the tumor inhibitory effect and few negative impacts. Additionally, compared to the blank group, the mice in RLP groups had normal body mass, healthy appetite and shiny hair. Meanwhile, significant anorexia, lethargy, and thinning hair were observed about mice in the 5-Fu group. And the body mass in 5-Fu group were significant lower compared to model group (P < 0.01), indicating that 5-Fu had toxic and side effect while inhibiting tumor cells. 541 2.0 * 1.5 ** 1.0 ** 0.5 0.0 Model group 5-Fu 100 mg/kg 300 mg/kg Table 2 Effect of RLP on body mass, number, and tumor rate of mice. Dose (mg/kg) Blank group Model group RLP RLP 5-Fu 0 0 100 300 10 Body mass (g) Start End 25.16 ± 0.66 33.68 ± 1.34 25.12 ± 0.68 37.52 ± 1.85 25.76 ± 1.14 32.76 ± 1.50* 25.01 ± 1.59 33.09 ± 1.25* 25.38 ± 1.38 24.63 ± 1.96** 2.5 TIR (%) Tumor volume (cm3) Groups RLP group 23.59 45.52 75.12 Notes: * P < 0.05 compared to model group was considered as signiﬁcant; ** P < 0.01 compared to model group was considered as extremely signiﬁcant. 2.0 1.5 * 1.0 0.0 * ** 0.5 Model group 5-Fu 100 mg/kg 300 mg/kg RLP group 12 Organ index (mg/g) Under normal physiological conditions, the weight and function of immune organs were related to the number of immune cells [38]. Therefore, being important immune organs of the body, the thymus and spleen could be used to obtain organ index and assess the strength of the body’s immune function to a certain extent [39]. Many studies have found that the atrophy of thymus and splenomegaly were often accompanied by developing of tumors cells [40]. To further obtain the inhibitory activity of RLP, the average weight, volume of tumor, the index of spleen, and thymus were noted. It was shown in Fig. 4A and B that the average weight and volume of tumor in model group were signiﬁcant higher compared to RLP dose groups. And the antitumor effect of high dose group (300 mg/kg) was superior to low dose group (100 mg/kg), with the tumor inhibition ratio of 45.52% and 23.59%, which suggested that RLP oral administration could effectively suppress the growth of H22 cell in a dose-dependent manner. Similarly, the literature [8] found a water-soluble polysaccharide from Rhodiola rosea, which could signiﬁcantly inhibitory the growth of S-180 cells with inhibition ratio of 37.61%. And then, the index of spleen and thymus of H22 mice in different groups were show in Fig. 4C. The results shown that the thymus index of the mice in the model group was signiﬁcantly reduced compared to blank group (P < 0.05), while the spleen index was significantly increased (P < 0.05), which indicated that the attack of tumor cells severely could damage the thymus and spleen of the tumor-bearing mice. Compared with the model group, the thymus and spleen indexes of H22 mice treated with RLP were significantly improved (P < 0.05) with a dose correlation, which indicated that RLP oral administration could effectively protect the immune organs of mice from attacking of H22 cells [41]. Being a conventional chemotherapeutic drug, 5-Fu was widely used in clinical H22 treatment and had obvious present inhibitory activities with sorafenib. Our results also shown that the immune organ index of H22 tumor-bearing mice treated with 5-Fu was significantly lower than that of blank group with inhibition ratio of 75.12% (P < 0.05), which further illustrated the toxic side effects of B 10 C Blank Model RLP 100 mg/kg RLP 300 mg/kg 5-Fu 20 mg/kg # * 8 6 * 4 2 0 # Spleen index Organ * * * Thymus index Fig. 4 The antitumor effect of RLP on H22 bearing mice. (A) The effect of RLP on weight of tumor at different dose groups; (B) The effect of RLP on volume of tumor at different dose groups; (C) Effects of RLP on spleen index and thymus index. * P < 0.05 indicated signiﬁcant from model group; ** P < 0.01 indicated extremely signiﬁcant from model group. # P < 0.05 indicated signiﬁcant from blank group. 3.3.2 Blood routine examination The occurrence and development of tumors were not only caused lesions in tissues and organs, but also caused changes in blood components. The blood routine test results of each group of mice were shown in Table 3. Compared with the blank group, the Leucocytes and Platelets in the model group increased signiﬁcantly (P < 0.05), while Erythrocytes and Hemoglobin were all reduced (P < 0.05), which indicated that the normal growth and proliferation of H22 cells could cause inﬂammation, anemia and immunosuppression in tumorbearing mice. Each examination in 5-Fu group were all lower than that in model group (P < 0.05), which fully veriﬁed the serious side effects of 5-Fu on the body. While compared with model group, each examination has been greatly improved after treating RLP, especially closed to the normal index (RLP, 300 mg/kg). The results suggested that RLP could maintain the examination at normal levels and alleviate the additional damage caused by tumor cells so that it could prevent immune dysfunction in tumor-bearing mice and inhibit the malignant proliferation of H22 cells effectively. Y.R. Wu et al. / Food Science and Human Wellness 12 (2023) 536-545 542 Table 3 Blood routine examination of H22 mice. Units Blank Model 5-Fu Leucocytes 109/L 4.56 ± 0.56 13.26 ± 1.08# Erythrocytes 1012/L 8.96 ± 0.25 5.96 ± 0.96# Hemoglobin g/L Platelets 9 # 176.96 ± 9.65 10 /L 120.56 ± 8.65 300 3.26 ± 0.26* 7.95 ± 0.61* 5.07 ± 1.16* 4.88 ± 0.37* 8.32 ± 0.71* 101.39 ± 4.82 # 340.26 ± 14.64 RLP (mg/kg) 100 590.43 ± 25.26 9.25 ± 0.64* * 170.49 ± 5.35* * 361.75 ± 14.98* 154.26 ± 6.23 * 321.51 ± 15.64 436.25 ± 19.60 P < 0.05 indicated signiﬁcant compared to blank group; P < 0.05 indicated signiﬁcant compared to model group. * 3.3.3 Cell cycle assay 3.3.4 The cell cycle arrest could affect tumor growth and induce apoptosis of tumor cell. Many studies have shown that many antitumor drugs can block the cell cycle at a speciﬁc site and thus induce apoptosis [13]. Cell cycle was composed of G0/G1, S and G2/M, which defined on the DNA content of cell. From the Fig. 5, a dose-dependent decrease was also observed in G1/G0 phase from 51.55% (model group) to 45.63% (RLP, 100 mg/kg), 41.45% (RLP, 300 mg/kg) and 36.90% (5-Fu group) as well as in G2/M phase (from 17.23% to 6.06%). The reduction of synthesized RNA and ribosomes in G1/G0 indicated that RLP could induce the disruption of preparing substance and energy [42]. More importantly, the RLP treatment could result in accumulation of cells proportion in S phase with a significantly increase from 39.2% (RLP, 100 mg/kg) to 52.65% (RLP, 300 mg/kg), which suggested that the cell cycle arrest could cause by disruption of DNA replication after RLP treatment [18,43]. These results indicated that RLP could induce tumor cell apoptosis by blocking solid tumor cells in S phase and the synthesis of energy and substances, thereby achieving the purpose of inhibiting tumor growth. Cell apoptosis assay by Annexin V-FITC/PI In the process of inducing apoptosis, the phosphatidylserine (PS) of tumor cells will transfer from the inner surface to outside of the plasma membrane. Annexin V, as a Ca2+-dependent phospholipid binding protein, was found to have high afﬁnity with PS and could be combined with propidium iodide (PI) to distinguish normal cells, apoptotic cells, and necrotic cells [44]. In order to conﬁrm the above cell morphological results, Annexin V-FITC/PI double staining was further used to detect the apoptosis-inducing effect of RLP [45]. As shown in Fig. 6, with the increasing of RLP dose, the proportion of living cells (Annexin V-/PI-) decreased from 96.6% to 71.7%, 31.2% and 26.3%, respectively. In addition, the total apoptosis rate of tumor cells (including early and late apoptosis) increased signiﬁcantly from 2.36% to 57.90% (P < 0.01). And after treating RLP, the proportion of early apoptosis cells (Annexin V+/PI-) increased from 2.25% (model group) to 15.4% (RLP, 300 mg/kg), as well as the proportion of late apoptosis cells (Annexin V+/PI+) from 0.12% (model group) to 42.4% (RLP, 300 mg/kg), which shown a dose-dependent manner [47]. These data further confirmed that RLP could induce apoptosis on H22 tumor cells and further caused changes in apoptosis morphology, which was consistent with the results of TIR and cell cycle. A 400 400 Dip G1 Dip S 100 0 150 100 200 400 600 800 1 000 FL2-A Model 0 200 0 0 200 400 600 800 1 000 FL2-A 5-Fu Blank Dip G1 Dip S 200 100 50 0 250 Dip G1 Dip S 300 Count 200 Dip G1 Dip S 200 Count Count 300 Cell number 250 Count # Examinations 150 100 50 0 0 200 400 600 800 1 000 FL2-A RLP 100 mg/kg 0 200 400 600 800 1 000 FL2-A RLP 300 mg/kg DNA content Cell percentage (%) 125 B S G2/M G0/G1 100 75 50 25 0 Model 5-Fu 100 mg/kg 300 mg/kg RLP group Fig. 5 (A) Effect of RLP on cell cycle and apoptotic rates of the tumor in mice; (B) Bar graph of cell population about G0/G1, S, G2/M. Y.R. Wu et al. / Food Science and Human Wellness 12 (2023) 536-545 543 A Content 104 Q2 0.117% 104 104 Q2 51.1% 104 Q2 4.53% 103 103 103 103 102 102 102 102 101 100 101 101 Q3 96.6% 100 Q4 2.25% 101 102 Model 103 100 104 Q3 26.3% 100 Q4 14.6% 101 102 5-Fu 103 100 104 Q2 42.4% 101 Q3 71.7% 100 Q4 20.9% 101 102 103 RLP 100 mg/kg 104 100 Q3 31.2% 100 Q4 15.4% 101 102 103 RLP 300 mg/kg 104 Apoptosis proportion (%) Annexin V-FITC Fig. 6 100 B 80 Model group 5-Fu group RLP (100 mg/kg) RLP (300 mg/kg) * 60 * 40 20 * * * * 0 Viable cells * * * Annexin V-FITC /PI Group + Annexin V-FITC+/PI+ (A) The distribution effect of RLP on viable cells, apoptotic cells, and death cells of tumor issues in different groups; (B) Bar chart of apoptosis percentage. * P < 0.05 compared to model group. A 100 80 150 40 50 20 0 100 101 102 FL1-H Model 103 104 0 100 M1 M2 M2 M1 60 100 120 150 Content M2 M1 M2 M1 200 Content 200 Content Content Cell contents 250 100 50 101 102 FL1-H 5-Fu 103 60 30 0 100 104 90 101 102 103 FL1-H RLP 100 mg/kg 104 0 100 101 102 103 FL1-H RLP 300 mg/kg 104 JC-1 Loss of ∆Ψm 100 B * 80 ** ** 60 40 20 0 Model 5-Fu 100 mg/kg 300 mg/kg RLP group Fig. 7 (A) Histograms of the Rh123-stained tumor issue by ﬂow cytometry; (B) Representative bar graph of the mitochondrial membrane potential changes in H22 cells. 3.3.5 Mitochondrial membrane potential detection As an important organelle of multicellular organisms, mitochondria play an important role in the signal cascade of apoptosis [17]. More and more evidence has shown that the loss of mitochondrial membrane potential might lead to mitochondrial dysfunction, which was essential in the process of apoptosis induced by polysaccharide [46]. As shown in Fig. 7, the mitochondrial membrane potential decreased in a dose-dependent manner after treating with different concentrations of RLP, from 84.6% (model group) to 68.87% (RLP, 100 mg/kg) and 42.26% (RLP, 300 mg/kg). In addition, the ΔΨm in 5-Fu was signiﬁcantly lower compared to the model group. According to the results, it could be concluded that RLP might induce apoptosis of H22 tumor-bearing mice cells through the mitochondrial apoptosis pathway [47]. Y.R. Wu et al. / Food Science and Human Wellness 12 (2023) 536-545 544 4. Conclusions In this work, a novel cold water-soluble polysaccharide was isolated and purified from Rhodiola rosea L. root (RLP). Furthermore, its structure and anti-cancer activities In vivo were further researched. In addition, the structure of RLP was tested by using HPGPC, FT-IR, IC, NMR, and SEM. More importantly, the growth of tumor cells and immune organs were observed in H22bearing mice after administration of RLP, and then the cell cycle and apoptosis were detected by AV-FITC/PI and JC-1. The key ﬁndings of this study could be summarized as follows: (I) RLP was an acid heteropolysaccharide with the molecular weight of about 1.15 × 106 Da. (II) RLP was composed of Rha, Ara, Gal, Glu, Xyl, Man, GalA in a molar ratio of 1:3.33:2.87:5.62:0.49:0.32:4.50. (III) RLP was a pyranose containing α- and β-configuration, which was consist of→2,4)-α-Rha(1→, →5)-α-L-Araf-(1→, α-D-Glu, →6)-β-D-Galp-(1→, β-D-Man and →4)-α-GalpA-(1→. (IV) RLP could reduce the damage of cancer for spleen and thymus and inhibit the growth of H22 cells (45.52%). 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