Structural characterization and antioxidant activity of processed polysaccharides PCP-F1 from Polygonatum cyrtonema Hua.

Introduction Polygonatum cyrtonema Hua. (PC) is a traditional Chinese herb with a history of use in both food and medicine. For clinical use, processed PC pieces are most commonly used, while present research has focused on crude PC polysaccharides (PCPs). Methods In this study, a new polysaccharide, PCP-F1, with a molecular weight of 37.46 kDa, was separated from four-time processed PCPs by column chromatography and evaluated by antioxidant activity. It was composed of glucose, mannose, galactose, rhamnose, and galacturonic acid with a molar ratio of 3.5: 2.5: 1.3: 1.8: 0.8. Results and Discussion The methylation analysis and two-dimensional NMR measurement revealed that the configuration of PCP-F1 contained nine residues in the primary structural unit by the chain of →3)-α-D-Glcp, →2)-α-D-Glcp (6→, →1)-ꞵ-D-Glcp (2→, →2)-α-D-GalAp (3,4→, →1) -ꞵ-D-Manp (3→, →2)-α-D-Glcp (3→, branched for →3)-α-D-Glcp, →2)-ꞵ-D-Galp (4→, →1)-ꞵ-D-Glcp (2→, →2,4)-α-D-Manp (6→, →3)-α-L-Rhap (4→. Radical scavenging assays indicated that PCP-F1 could scavenge radicals with a high scavenging rate, suggesting PCP-F1 possesses good antioxidant activity. The study confirms the importance of processed PC and offers the potential for exploiting it as a functional food.


Introduction
The polygonata rhizoma "Huangjing" was first described in the Mingyi-Bielu.Among the three types of Huangjing, the rhizome of P. cyrtonema Hua. is the most commonly used for medicine and food (1,2).The chemical components of P. cyrtonema Hua.mainly include polysaccharides, steroidal saponins, alkaloids, and so on.Notably, the significant important polysaccharides component has various bioactivity, such as enhancing human immunity (3), antitumor (4), and improving superoxide dismutase activity (5), and are usually designed as the quality marker (Q-Marker) of P. cyrtonema Hua.It.Although P. cyrtonema Hua. is a type of traditional Chinese medicine (TCM), it can only be used as medicine after steaming to ensure its safety and efficacy (6).Additionally, the steaming process eliminates some side effects such as a numb tongue, and stimulation throat; more importantly, it enhances the pharmacological function.Currently, various processing methods for P. cyrtonema Hua.(PC) include single-steaming, wine-steamed, nine-steaming, and ninebasking (7,8).These methods impact the polysaccharide compositions."Nine-steaming and nine-basking" is a traditional approach derived from Lei-Gong-Paozhilun, later refined in Qianjin-Yi-Fang.Chemical changes occur after nine cycles, enhancing the tonifying effect and reducing throat irritation (9).Initial PC rhizomes lack GalA but gain it during steaming, with shorter steaming (2-4 h) yielding more immunologically active PCPs (10).HPLC-QTOF-MS/ MS revealed an oligosaccharide in steamed PC with fructose units linked by ꞵ-(2 → 1) or ꞵ-(2 → 6) patterns (11).Steaming elevates Gal while reducing Glc and Man, and GC-MS shows increased β-1, 4-Manp, and β-1, 4-Galp, resulting in heightened antioxidant activity (12).However, the structure-activity relationship of PCPs from the nine-steaming and nine-basking methods remains unexplored.
Reactive oxygen species (ROS) are present in living organisms and help maintain cell homeostasis (13).These compounds arise from the normal metabolism and oxidation of xenobiotics.Organisms use an antioxidant system to balance oxidative stress and protection.ROS, particularly radicals, pose a cell-damaging risk (14).To counter radical damage, organisms generate endogenous or exogenous antioxidant species.Some synthetic antioxidants like octyl gallate and propyl gallate are approved but face safety concerns (15).Notably, natural antioxidants are preferred, with hydroxyl groups of polysaccharides displaying radical scavenging and antioxidative functions (16).Moreover, properties of polysaccharides, like water solubility, size, monosaccharide composition, and structure, influence their activity (17).Given the intricate structural nature of polysaccharides in traditional Chinese medicine (TCM), marked by diverse monosaccharide types and compositions, linking modes, and molecular sizes, the task of conducting structural analyses on these polysaccharides becomes both captivating and daunting.
Following up on the above reports and continuing our interest in studying TCM (18-21), particularly Polygonatum Rhizoma (22)(23)(24)(25), we reported a novel polysaccharide, PCP-F1, prepared by using the nine-steaming and nine-basking approach.By methylation analysis combined with Congo red assay, a detailed structure was determined.Furthermore, two radical scavenging models were employed to assess the antioxidant effect of PCP-F1.Lastly, the correlation between antioxidant activity and corresponding structural characteristics of PCP-F1 was revealed.

Physicochemical properties of processed PCPs total sugar, acid sugar, and FT-IR spectroscopy
The content of total sugar and acid sugar is often used as the quality control index of traditional Chinese medicine.As shown in Figure 1A, the 0-Z PCPs had the highest total sugar content of 90.87 ± 1.77%.As the number of steaming increases, the total sugar content decreases gradually.In the 2-Z PCPs, the total sugar content is 68.89 ± 2.14% and tends to stabilize at 66.24 ± 1.77% in the 4-Z PCPs.On the other hand, 0-Z PCPs had a content of acid sugar of 6.35 ± 0.43% which is the lowest in the processed PCPs, as shown in Figure 1B.In the 2-Z PCPs and the 4-Z PCPs, the content of acid sugar is 6.57 ± 0.37 and 11.20 ± 0.48%, respectively.Subsequently, increasing the number of steaming times did not cause a significant increase in the content.
The FT-IR method is commonly used for analyzing polysaccharide structures (26).By analyzing the FT-IR spectrum of processed PCPs, we were able to characterize their structural characteristics.According to Figure 1C, the spectra of processed PCPs had similar absorption characteristics, indicating that the steaming process did not significantly affect the substituent groups.It was confirmed that a wide peak of polysaccharides was located at 3,403 cm −1 , which was attributed to the -OH group in the polysaccharides.In addition, the peak width gradually widened at 2-Z with the increase of steaming times but became sharp again from the beginning of 4-Z.The peak 2,929 cm −1 was assigned to the C-H asymmetric tensile vibration (27), and the weak peak located at 1,719 cm −1 was attributable to the vibration of symmetry C=O (28).It is noteworthy that the C-O-C glycosidic bond, as well as the C-C and C-OH stretch vibration bonds, are primarily observed between 1,200 and 1,000 cm −1 .These two signals at 1,057 and 1,129 cm −1 were identified as stretching vibrations of the pyranose ring (29).In addition, the peaks at 933 and 891 cm −1 were ascribed to β-type glycosidic and α-glycosidic bonds, respectively (30).

pH measurement and antioxidant activity
When the pH decreased, electron transfer occurs very quickly in an ionized solution due to electron availability (31, 32).As presented in Figure 2A, the pH value of 0-Z PCPs solution was 6.46 ± 0.04, while the pH value of 3-Z PCPs solution was 4.58 ± 0.04, indicating a marked increase in the acidity of the solution.Nevertheless, the pH value was 4.27 ± 0.05 in the solution of 4-Z PCPs.Even if the number of steaming is further increased, the pH value of the corresponding solution of processed PCPs tends to stabilize around 4.0, which may be because the acid substance was gradually stable after 4-Z (33).
The occurrence of some diseases is closely related to the existence of inflammation.However, high concentrations of radicals in the body can lead to the occurrence and development of inflammation (34).Herein, we carefully investigated the antioxidant activity of 10 kinds of processed PCPs (0-Z, 1-Z, 2-Z, 3-Z, 4-Z, 5-Z, 6-Z, 7-Z, 8-Z, and 9-Z PCPs) in the range of 0.2-1.0 and 2.0-10.0mg/mL to determine the higher activity portion.Antioxidant assays were performed by two models: scavenging DPPH radicals and hydroxyl radicals.The 10 kinds of processed PCPs showed effective antioxidant activity and the 4-Z PCPs exhibited higher radical-scavenging abilities in both models.The DPPH radical scavenging rate of processed PCPs was presented in Figures 2B,C as follows: As a positive control, the Vc group had the highest radical scavenging rate.
The scavenging rate was 0.0% ± 0.0 in the 0-Z PCPs group with the concentration was 0.2 mg/mL; increasing the concentration to 0.6 mg/ mL drastically increased the scavenging rate of 0-Z PCPs to 45.2% ± 1.0, while when the concentration is 1.0 mg/mL, the scavenging result was somewhat affected.Meanwhile, 4-Z PCPs have a scavenging rate of 54.1% ± 1.4 at the concentration of 0.2 mg/mL, obviously higher than 0-Z PCPs and slightly lower than the 5-Z PCPs group.When the concentration of each processed PCPs increases to 0.6 mg/mL, the highest scavenging rate was found in the 4-Z PCPs group, and a similar result was also found when the concentration of analyte is 1.0 mg/mL.Subsequently, in the hydroxyl radical assays, we observed a similar phenomenon on the radical scavenging effect.Combining the results of the two models, 4-Z PCPs had a more robust antioxidant performance in all the processed PCPs groups.The EC50 of DPPH and hydroxyl radicals scavenging rate of 4-Z PCPs were 0.049 and 1.95 mg/mL, respectively.

Total sugar, acid sugar, homogeneity, and FT-IR spectroscopy
The total sugar contents of PCP-F0, PCP-F0.05,PCP-F0.1, and PCP-F1 were 85.65 ± 1.30, 76.28 ± 1.77, 83.53 + 1.53, and 82.06 ± 0.35%, respectively.And the acid sugar contents of PCP-F0, PCP-F0.05,PCP-F0.1, and PCP-F1 were 4.73 ± 0.54, 5.95 ± 0.10, 6.14 ± 0.26, and 7.20 ± 0.39%, respectively (Figures 4A,B).There was no obvious absorption peak of polysaccharide PCP-F1 at 260 and 280 nm in Supplementary Figure S3, indicating that the amount of protein and nucleic acid impurity has been reduced to the lowest level, which is less than the minimum detection limit (35).The The physicochemical property analysis.The total sugar content (A), the uronic acid content (B), and FT-IR spectroscopy (C) of Polygonatum cyrtonema Hua. are processed by different times of repeated steaming (0-Z indicates no steam process; 1-Z-9-Z is the number of steaming times.Tested for three times at 25°C, p < 0.05.To understand the types of functional groups contained in molecule PCP-F1 and how they are connected, infrared spectroscopy is used.As shown in Figure 4D, the stretching vibration of O-H residues appears at 3,403, 1,626, and 1,452 cm −1 , which are the bending vibration peaks of-COOH (27).The absorption peaks at 1,131 cm −1 represent the asymmetric stretching vibration of C-O-C (36).The absorption peak at 1,026 cm −1 belongs to the C-O-H bond.In addition, the absorption peaks at 880 and 835 cm −1 belong to β-configuration pyranose (30).

Partial acid hydrolysis
Due to the complexity of polysaccharide structure, the partial acid hydrolysis of PCP-F1 was performed further to determine the distribution of monosaccharides.In polysaccharide skeletons, the hydrolysis of the branch chain is a priority than that of the backbone (27).In this study, PCP-F1 was divided into four subcomponents (H-1, H-2, H-3, and H-4), and the monosaccharide components of these subcomponents are shown in Table 2. Due to the Man, Gal, The characterization and physicochemical property analysis of samples (PCPs).The total sugar content (A), the uronic acid content (B), HPGPC (C), and FTIR spectra of PCP-F1 (D), (Tested three times at 25°C, p < 0.05).and Glc being the main monosaccharide composition of H-1, they are presumed to locate in the side chain.When the concentration of TFA increases to 0.1 M, the contents of Glc and Gal in H-2 are higher than others.Meanwhile, GalA was determined in H-2.In the structurally stable H-4, the Glc, Man, and Gal were found, indicating that they should be located in the core structure of PCP-F1.

NMR analysis
Nuclear Magnetic Resonance (NMR) spectroscopy is a widely used and important technique for ascertaining distinct structural attributes.It is employed for verifying monosaccharide classifications, distinguishing between α-and β-anomeric arrangements, and unraveling details about glycosidic connections (38-40).Herein, the 13 C and 1 H NMR spectra of residues A to I were analyzed through 2D NMR.Regarding the information gleaned from the 13 C and 1 H spectra of PCP-F1, the specific signals corresponding to different residues are outlined as follows.
Within the 1H NMR spectrum depicted in Supplementary Figure S5A, the presence of a signal at δ H 4.62 ppm suggests the presence of a β-configured residue within the PCP-F1 structure.This signal is attributed to H-1 of residue D (38).Moreover, the regions between δ H 4.05 and δ H 3.43 ppm were responsible for H-2 to H-6 of residues (A, B, C, D, E, F, G, H, and I) presented in PCP-F1 (39).In 13 C NMR spectra depicted in Supplementary Figure S5B, nine peaks at δ C 106.47, 92.68, 104.33, 104.28, 102.29, 96.37, 106.06, 103.78, and 104.35 ppm were assigned as the anomeric signals situated at C-1 of residues A-I, respectively.In the more elevated field segment, the resonance at δ C 166.73 ppm was attributed to the carbonyl group of the uronic acid moiety (22).
The HMBC spectrum facilitated the analysis of correlations among nine residues ( A, B, C, D, E, F, G, H, and I).As depicted in Figure 6D, a reciprocal relationship was observed between the H-2 signal of residue A (δ H 4.00 ppm) and the C-6 of residue H (δ C 77.91 ppm), as well as between the H-6 signal of residue H (δ H 3.43 ppm) and the C-2 of residue A (δ C 71.76 ppm), signifying a presence of a 2,6-linkage between residue A and H. Similarly, a  Lastly, a 2,3-linkage between residue E and I was established, as evidenced by the correlation between the C-2 signal of residue E (δ C 77.39 ppm) and the H-3 of residue I (δ H 3.82 ppm), along with the correlation between the H-2 signal of residue E (δ H 3.98 ppm) and the C-3 of residue I (δ C 88.05 ppm).A comprehensive assignment of PCP-F1 was outlined in Table 3.

Congo red experiment
The triple helix structure of PCP-F1 was characterized by Congo red experiment.Under weakly alkaline conditions, the triple helical polysaccharide can form a complex with Congo red, and compared with Congo red solution, the maximum absorption wavelength will be red-shifted (41).With the increase of concentration of alkali, the complex triple helix structure will be destroyed, leading to the decrease of the maximum absorption wavelength.As shown in Figure 7A, the maximum absorption wavelength of PCP-F1 was 496 nm.Compared with the Congo red solution, the maximum absorption wavelength of PCP-F1 polysaccharide showed a weak red-shift trend, indicating that no triple helix conformation was formed.

Physicochemical properties of processed PCPs
The "nine-steaming and nine-basking" approach is one of the traditional methods of processing precious and nourishing Chinese medicine.Subjecting the herb to multiple rounds of steaming and drying, typically around nine cycles, imparts a gently "warm" quality to it.This transformation renders the herb apt for alleviating symptoms associated with "cold" conditions.In most cases, raw rhizomes are not employed in their untreated state.Instead, the practice of Nine-Steam Nine-Bask is favored, as it effectively augments the herb's tonic attributes via a series of sequential steaming and drying processes.Through the analysis of processed PCPs, including total sugar, acid sugar and FT-IR spectroscopy, and pH measurement, and combined with the result of antioxidant activity, 4-Z PCPs displayed a better antioxidant performance in all the processed PCPs group and thus were selected for the next experiment.

Structure characteristics of PCP-F1
Under the action of stomach acid, large molecular-weight polysaccharides are easily degraded into small molecular-weight polysaccharides, oligosaccharides, and even monosaccharides.For monosaccharide composition, galactose has a positive effect on hydroxyl radical scavenging activity, while galacturonic acid and rhamnose have a negative effect.Mannose, glucuronic acid, and glucose have no significant effect on antioxidant activity (43).In addition, β-structured polysaccharides in the form of pyranose have higher antioxidant activity (44).The PCP-F1 contains a large amount of Glc, Man, Gal, and a small amount of Rha, GalA, and small contents of Glc, Man, and Gal were β configuration.
Previous studies reported the change and dynamic analysis of monosaccharide composition and content of processed PCPs (10)(11)(12)(45)(46)(47), but there was no further structural analysis.First, we used the procedure of steaming and basking nine times, combined with the water extraction and alcohol precipitation method to obtain the crude processed PCPs.By radical scavenging assay, the 4-Z PCPs showed the best activity.The DEAE-52 cellulose and Sephadex G-200 column were used to yield PCP-F1.Finally, UV, HPGPC, IR, GC-MS, and NMR were used to analyze the structure of PCP-F1.The UV showed that PCP-F1 had no nucleic acid or protein.Monosaccharide composition analysis The structural analysis of PCP-F1.The Congo red (A) and putative structure (B).(Tested three times at 25°C, p < 0.05).showed that PCP-F1 is mainly composed of Glc, Man, Gal, Rha, and GlcA, consistent with the results of methylation analysis and partial acid hydrolysis.Combined with the above results, the structure of PCP-F1 was suggested:

The structure and antioxidant activity relationship
The relationship between the structure of polysaccharides and their antioxidant activity has been studied and analyzed.DPPH radical scavenging is in terms of the hydrogen supply ability of antioxidants, and polysaccharides play an antioxidant role by providing H+ to free radicals to stop the oxidation reaction (48).The content of uronic acid can be an important indicator of the antioxidant activity of polysaccharides, that is, polysaccharides with high uronic acid content often have strong antioxidant activity.Moreover, polysaccharides with high content in GalA can exhibit significant antioxidant activity, possibly due to their-COOH functional groups (49).The increase of galactose content in polysaccharides may enhance their antioxidant activity.The number of active hydroxyl groups in polysaccharide structure positively correlates with its ability to scavenge radicals (50).In addition, molecular weight is another significantly vital factor affecting antioxidant activity, and polysaccharides with molecular weights ranging from 4,000 to 100,000 Da exhibit high DPPH scavenging activity.Low-molecularweight polysaccharides may have more reducing hydroxyl groups at their ends, which can accept and scavenge free radicals (51).
Purification of the 4-Z PCPs was performed on a DEAE-52 Cellulose column (3.5 cm × 30 cm), with gradient NaCl solutions (0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 M).In vitro antioxidant activities were used to screen the active fraction, and the elution fractions, PCP-F1 (from 0.1 M NaCl) was then separated using deionized water on a Sephadex G-200 column (1.6 cm × 40 cm), and was gathered for further analysis.

Characterization and structural analysis 4.3.1. pH experiments and FT-IR spectrum of processed PCPs
Processed PCPs solution with a concentration of 1 mg/mL was prepared and tested at 25°C by Starter 3100 pH meter.Processed PCPs and PCP-F1 were, respectively, mixed with dried potassium bromide (1, 100, w/w) to ground and pressed in a vacuum at 25°C.A Nicolet 5700 IR spectrometer given corresponding spectra in the wavelength region of 4,000-400 cm −1 .

Total sugar and uronic acid determination of processed PCPs
The uronic acid content and total sugar content of processed PCPs were measured by the anthrone-sulfuric acid and m-hydroxyphenyl methods, respectively (27,30).PCP-F1 solution (1 mg/mL) was prepared and scanned by a SHIMADZU spectrophotometer at a wavelength range of 200-800 nm at 25°C.Monosaccharide composition analysis was conducted employing Gas Chromatography-Single Quadrupole Mass Spectrometry (GC-MS) methodology.Briefly, PCP-F1 (20 mg) was introduced into a solution of trifluoroacetic acid (TFA) with a concentration of 2 mol/L and a volume of 2 mL.Subsequently, hydrolysis was performed at 100°C for a duration of 6 h, followed by evaporation of the solvent under reduced pressure.The acidhydrolyzed analyte, dissolved in anhydrous DMSO (2 mL), was combined stepwise with NaOH powder (60 mg) in a flask.Following stirring at 35°C for 30 min, methylating reagent CH 3 I (1 mL) was carefully added drop by drop.The reaction proceeded in darkness for 12 h, after which it was halted by introducing ultrapure water (2 mL).The resulting methylated sample was subjected to extraction using dichloromethane and subsequently subjected to analysis via GC-MS.PCP-F1 homogeneity was determined based on a previous method (22).

Methylation analysis of PCP-F1
Glycosidic linkage analysis of PCP-F1 was conducted with a slight modification to a previously documented method (35,37,52).The initial step involved reducing the uronic acid to a neutral sugar prior to methylation analysis.To achieve this, 10 mg of dried PCP-F1 was fully dissolved in anhydrous DMSO (2 mL).Subsequently, NaOH (60 mg) was added, and the reaction mixture was stirred at 35°C for a span of 2 h.Following this, a slow addition of 1 mL of CH 3 I took place.The reaction proceeded under dark conditions for 12 h.Termination of the reaction occurred by adding ultrapure water (2 mL).The resultant methylated sample underwent extraction using dichloromethane and was washed thrice with ultrapure water.Complete methylation of PCP-F1 was confirmed by the disappearance of peaks in the infrared spectrum within the range of 3,200-3,700 cm −1 .The ensuing procedure involved hydrolyzing the mixture using 2 mL of 2 M TFA at 100°C for a period of 6 h.Post removal of excess TFA, NaBH 4 (30 mg) and a solution of 0.05 M NaOH (1 mL) were sequentially introduced.After a reaction time of 12 h, 100 μL of acetic acid was added, followed by solvent removal under vacuum.Subsequently, a mixture of pyridine and acetic anhydride (1 mL each) was added, sealed, and stirred at 90°C for an additional 2 h.The reacted solution was subjected to extraction using dichloromethane and subsequently analyzed using the GC-MS technique.

Partial acid hydrolysis of PCP-F1
According to a previous method (27), partial acid hydrolysis of PCP-F1 was performed.20 mg PCP-F1 was hydrolyzed with 2 mL TFA for 1 h (0.05 M, 100°C).TFA was removed, and four volumes of ethanol (95%) were added to the hydrolysate.The sample was stored at 4°C overnight for centrifugation (5,000 rpm, 5 min), the supernatant was collected and named H-1, and the precipitation was hydrolyzed again with 2 mL TFA (0.1 M, 100°C) for 1 h.Similarly, after removing the TFA with methanol, the supernatant was obtained and named H-2.The precipitation was separated and hydrolyzed with 2 mL TFA with a concentration of 0.5 mol/L at 100°C for 1 h to get H-3 and the final precipitated H-4.The H-1, H-2, H-3, and H-4 samples were completely hydrolyzed using 2 mL TFA (2 M, 100°C) for 6 h to further analysis.

NMR spectra of PCP-F1
Nuclear magnetic resonance (NMR) measurements were performed using 0.6 mL of D 2 O dissolved with 20 mg of dry polysaccharide PCP-F1 at 25°C.A 600 Hz NMR spectrometer was utilized to record the 1D 1 H NMR and 13 C NMR, as well as the 2D 1 H-1 H COSY, HSQC, HMBC, and NOESY spectra of the PCP-F1.

Antioxidant assay 4.4.1. DPPH free radical scavenging
The capacity of scavenging DPPH radicals of processed PCPs and purified 4-Z PCPs, including PCP-F0, PCP-F0.05,PCP-F0.1, and PCP-F1, were detected according to a previous report with slight modifications (36).Briefly, first, fresh DPPH (0.2 mM in methanol, 2.0 mL) and PCPs solutions (2.0 mL) at various concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL) were prepared.Second, the reaction mixture of prepared DPPH and PCPs solutions at various concentrations was thoroughly stirred and hatched at 25°C for 30 min under dark conditions.The UV-2550 UV spectrophotometer recorded the absorbance of the mixture at a wavelength of 517 nm.Finally, the activity of DPPH radical scavenging was calculated.

FIGURE 2
FIGURE 2The characterization and antioxidant activities analysis.The pH analysis of different processed PCPs (A), DPPH radical scavenging experiments of different processed PCPs (B), and Hydroxyl radical scavenging experiments of different processed PCPs (C), (Tested for three times at 25°C, p < 0.05).
positive interaction was noted between the C-3 signal of residue B (δ C 74.42 ppm) and the H-6 of residue F (δ C 3.61 ppm), and between the C-6 signal of residue F (δ C 71.53 ppm) and the H-3 of residue B (δ H 3.56 ppm), indicating a 3,6-linkage between residue B and F. Further examinations revealed the existence of 1,2-linkages between residue G and both F and H.The C-1 signal of residue G (δ C 106.06 ppm) displayed a positive correlation with the H-2 of residue F (δ H 3.96 ppm), and the C-2 signal of residue F (δ C 74.92 ppm) exhibited a correlation with the H-1 of residue G (δ H 4.65 ppm), illustrating the 1,2-linkages between residue G and F. Additionally, the C-1 signal of residue G (δ C 106.06 ppm) showed a positive correlation with the H-2 of residue H (δ H 3.62 ppm), and the C-2 signal of residue H (δ C 73.09 ppm) correlated with the H-1 of residue G (δ H 4.65 ppm), indicating 1,2-linkages between residue G and H.The linkage type between residue B and D was determined to be a 3,4-linkage based on the highly correlated H-3 signal of residue B (δ H 3.56 ppm) and the C-4 of residue D (δ C 73.17 ppm), as well as the correlation between the C-3 signal of residue B (δ C 74.42 ppm) and the H-4 of residue D (δ H 3.96 ppm).Similarly, the linkage type between residue G and D was identified as a 1,2-linkage, with the H-1 signal of residue G (δ H 4.65 ppm) correlating with the C-2 of residue D (δ C 77.41 ppm), and the C-1 signal of residue G (δ C 106.06 ppm) showing correlation with the H-2 of residue D (δ H 4.02 ppm).

TABLE 2
Monosaccharide composition of the fractions after partial acid hydrolysis.

TABLE 3
Signal assignment in the 1 H and13C NMR spectra of PCP-F1.