The amounts of total phenolics, flavonoids, and proanthocyanidins in ethanolic crude extract (ECE) and its sub-fractions of Receptaculum Nelumbinis were determined, ranging from 32.1 to 607.6 mg/g of extract, from 42.8 to 862.7 mg/g of extract, and from 10.6 to 331.0 mg/g of extract, respectively. As shown in Table 1, the total phenolic content of CE and its fractions decreased in the following order: BF > ECE > EAF > AF > PEF, the total flavonoid content in the order of n-butanol fraction (BF) > ethyl acetate fraction (EAF) > ECE > aqueous fraction (AF) > petroleum ether fraction (PEF). A similar distribution was found in the total proanthocyanidin contents. From among four different polarity fractions, BF had the highest total phenolic, flavonoid, and proanthocyanidin contents and EAF came next. Furthermore, the total phenolic and total flavonoid contents were much higher than total proanthocyanidin of CE and its fractions.

The radical scavenging activities of ECE and its fractions of Receptaculum Nelumbinis were evaluated using the methods of DPPH and ABTS radical scavenging activity assays, which have been widely used to test radical scavenging activity. As shown in Figure 1 and Table 2, the ECE and most of the sub-fractions showed obvious DPPH radical scavenging activity in a concentration-dependent manner. The scavenging ratios were improved with increasing sample concentration. Among the ECE of Receptaculum Nelumbinis and its fractions, BF (IC₅₀ = 5.4 μg/mL) exhibited the most efficient radical scavenging activity, higher than those of the other fractions and ECE (Table 2). Except PEF, all fractions possessed significantly (p < 0.05) stronger radical scavenging ability than that of butylated hydroxyl toluene (BHT) (IC₅₀ = 12.9 μg/mL).

Similar scavenging activity patterns were seen in the ABTS assay. The ABTS radical scavenging activity of CE and its fractions increased with the increase of concentrations (Figure 2). It was observed that the BF (IC₅₀ = 48.4 μg/mL) showed the highest radical scavenging activity of all the fractions (Table 2). ABTS radical scavenging activity of ECE and its fractions decreased in the following order: BF > EAF > ECE > BHT > AF > PEF (p < 0.05).

Based on the above results, activity-guided fractionation of the n-butanol fractions was carried out for the isolation of active constituents. Further fractionation and separation by silica gel and Sephadex LH-20 gel column chromatography methods yielded five flavonol glycosides. The structures of these compounds were identified as hyperoside (1), isoquercitrin (2), quercetin-3-O-β-d-glucuronide (3), isorhamnetin-3-O-β-d-galactoside (4) and syringetin-3-O-β-d-glucoside (5), respectively, by comparison of their spectroscopic data (¹H, ¹³C NMR and MS) with those reported in the literature. Compounds 2–5 are isolated and identified for the first time from Receptaculum Nelumbinis. Their structures are shown in Scheme 1.

Flavonoids are phenolic substances isolated from a wide range of vascular plants, with over 8000 individual compounds known, and have gained particular interest. Putative therapeutic effects of many traditional medicines may be ascribed to the presence of flavonoids, because of their broad pharmacological activity. The most important reported biological property of flavonoids is their antioxidant activity by scavenging radicals [9]. In this study the isolated compounds from n-butanol fraction of Receptaculum Nelumbinis were identified as five flavonol glycosides. The antioxidant activities of the five isolated flavonol glycosides were examined by using in the vitro antioxidant models described above, including DPPH and ABTS radical scavenging activities. As shown in Table 2, the scavenging effects of compounds 1–5 and BHT on DPPH and ABTS decreased in the following order: Compound 2 > Compound 1 > BHT > Compound 3 > Compound 4 > Compound 5 and Compound 2 > Compound 1 > BHT > Compound 3 > Compound 5 > Compound 4, respectively. Among these tested compounds, isoquercitrin (Compound 2) demonstrated highest DPPH and ABTS radical scavenging activities with IC₅₀ values of 5.2 ± 0.2 and 112.8 ± 0.8 μg/mL, respectively. The DPPH radical scavenging activity of isoquercitrin was two times greater than that of the BHT, and ABTS radical scavenging activity of isoquercitrin was similar to that of the BHT. Compounds 1 and 3 exhibited effective DPPH and ABTS radical scavenging activity but inferior to isoquercitrin, while compounds 4 and 5 showed relatively weak activity.

It is interesting to investigate the structure-activity relationship for compounds 1–5. Comparing their structures (Scheme 1), the main differences are the substituents at C-3′ and C-3. From comparison of the structures and activities of compounds 1, 2 and 3, it is inferred that the presence of a saccharide group at C-3 seems to have some, but little influence on antioxidant activity. By comparing compounds 1, 2, 3, 4 and 5, it is inferred that the presence of a –OH group at the C-3′ is the key factor that affects the activities. This is in agreement with previous studies: structure–activity relationship studies for the antioxidant efficacy of flavonoids have provided clear evidence that antioxidant activity depends on the catechol group in the B ring and the 2,3-double bond conjugated with the 4-oxo group [9].

Antioxidants are usually employed to prevent chronic and degenerative diseases by scavenging free radical intermediates or depressing the generation of free radicals [10,11]. It has been demonstrated that long-term treatment with Receptaculum Nelumbinis can prevent age-related increases in oxidation products and age-related deficits in cognitive functions that may be due to its free radical-scavenging and antioxidant activity, resulting from the presence of phenolic compounds in the extracts [12]. In addition, the antioxidant properties of Receptaculum Nelumbinis were believed to contribute to restore acetylcholine contents and acetylcholinesterase activities in the hippocampus and cerebral cortex of aged impaired animals [13]. The results of the present work also suggested that the activity of Receptaculum Nelumbinis could be explained, at least in part, by the presence of antioxidant flavonol glycosides. Further investigations, such as mechanistic studies of the antioxidant activity of flavonoids at both the molecular and cellular levels are underway.

NMR spectra were obtained with a Bruker DRX-400 spectrometer at 400 MHz for ¹H NMR and 100 MHz for ¹³C NMR. Peak positions are expressed in δ values with reference to TMS as internal standard, and coupling constants J in Hz. ESI-MS were recorded on a Varian MAT-212 mass spectrometer; column chromatography was performed on silica gel (200–300 mesh, Yantai, China) and Sephadex LH-20 (Pharmacia). The solvents used for isolation and purification were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Except butylated hydroxyl toluene (BHT) and aluminium trichloride, which were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China), all other chemicals and reagents used in antioxidant activity assay like Folin-Ciocalteu reagent, 2,2′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), gallic acid and catechin were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and solvents were of analytical grade.

Receptaculum Nelumbinis were collected from Fujian province of China and the voucher specimens were taxonomically identified based on morphological characteristics by J. Z. Wu and deposited in the Herbarium of the Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine in Fuzhou 350108, China.

The dried receptacles (21 kg) of N. nucifera were extracted with 75% ethanol two times under reflux, and the extracts were concentrated under reduced pressure. The residue obtained was suspended in water and successively partitioned with petroleum ether, ethyl acetate and n-BuOH, respectively. Each fraction was concentrated under reduced pressure to yield a petroleum ether fraction (PEF), an ethyl acetate fraction (EAF), a n-butanol fraction (BF) and a remainder (aqueous) fraction (AF). The n-BuOH extract (559 g) was subjected to silica gel column chromatography eluted with CH₂Cl₂-MeOH (50:1→30:1→20:1→10:1→5:1→3:1→1:1) and MeOH, and the resulting fractions were subjected to additional separation steps using a silica gel column and Sephadex LH-20 chromatography eluted with 85% methanol to yield compounds 1 (212 mg), 2 (180 mg), 3 (321 mg), 4 (26 mg) and Compound 5 (21 mg), respectively.

Hyperoside (1): Yellow powder; C₂₁H₂₀O₁₂; ESI-MS: m/z 463 [M − H]^{−; 1}H NMR (MeOD, 400 Hz): δ 5.15 (1H, d, J = 7.2 Hz, H-1″), 6.20 (1H, d, J = 2.0 Hz, H-6), 6.39 (1H, d, J = 2.0 Hz, H-8), 6.86 (1H, d, J = 8.4 Hz, H-5′), 7.58 (1H, dd, J = 2.0, 8.4 Hz, H-6′), 7.83 (1H, d, J = 2.0, H-2′). ¹³C NMR (MeOD, 100 MHz): δ 179.8 (C-4), 163.3 (C-5), 159.1 (C-7), 159.1 (C-9), 158.8 (C-2), 150.3 (C-4′), 146.1 (C-3′), 136.1 (C-3), 123.2 (C-1′), 123.2 (C-6′), 118.1 (C-5′), 116.4 (C-2′), 105.9 (C-10), 105.7 (C-1″), 100.3 (C-6), 95.1 (C-8), 77.5 (C-5″), 75.4 (C-3″), 73.5 (C-2″), 70.3 (C-4″), 62.5 (C-6″). These data are identical with those for Hyperin reported elsewhere [14].

Isoquercitrin (2): Yellow powder; C₂₁H₂₀O₁₂; ESI-MS: m/z 463 [M − H]^{−; 1}H NMR (MeOD, 400 Hz) δ 3.20–3.78 (5H, m, H-2″, 3″, 4″, 5″ and 6″), 5.24 (1H, d, J = 7.6 Hz, H-1″), 6. 23 (1H, d, J = 2.1 Hz, H-6), 6.42 (1H, d, J = 2.1 Hz, H-8), 6.88 (1H, d, J = 8.5 Hz, H-5′), 7.58 (1H, dd, J = 2.2, 8.5 Hz, H-6′), 7.72 (1H, d, J = 2.2 Hz, H-2′). ¹³C NMR (MeOD, 100 Hz) δ: 179.8 (C-4), 167.0 (C-7), 163.4 (C-5), 159.1 (C-2), 158.7 (C-9), 149.8 (C-4′), 145.9 (C-3′), 135.7 (C-3), 123.2 (C-1′), 123.1 (C-6′), 117. 6 (C-5′), 116.0 (C-2′), 105.8 (C-10), 104.4 (C-1″), 99.9 (C-6), 94.8 (C-8), 78.8 (C-5″), 78.2 (C-3″), 75.8 (C-2″), 71.3 (C-4″), 62.4 (C-6″). These data are identical with those for isoquercitrin reported elsewhere [15].

Quercetin-3-O-β-d-glucuronide (3): Yellow powder; C₂₁H₂₇O₁₃; ESI-MS: m/z 477 [M − H]^{−; 1}H NMR (MeOD, 400 Hz) δ: 8.08 ( 1H, br s, H-2′), 7.43 ( 1H, d, J =8.1 Hz, H-6′), 6.83 (1H, d, J = 8.1 Hz, H-5′), 6.37 (1H, br s, H-8), 6.19 (1H, br s, H-6), 5.33 (1H, d, J = 6.5 Hz, H-1″). ¹³C NMR (MeOD, 100 MHz) δ: 177.6 (C-4), 172.7 (C-6″), 164.7 (C-7), 161.0 (C-5), 157.1 (C-2), 156.5 (C-9), 148.5 (C-4′), 144. 9 (C-3′), 133. 8 (C-3), 121.1 (C-1′), 120.6(C-6′), 117.3 (C-2′), 115.4 (C-5′), 103.7 (C-10), 102.3 (C-1″), 99.0 (C-6), 93.7 (C-8), 76.5(C-3″), 74.7 (C-5″), 74.0 (C-2″), 71.8 (C-4″). These data are identical with those for quercetin-3-O-β-d-glucuronide reported elsewhere [16].

Isorhamnetin-3-O-β-d-galactoside (4): Yellow powder; C₂₁H₁₇O₁₃; ESI-MS: m/z 477 [M − H]^{−; 1}H NMR (DMSO-d₆, 400 MHz): δ 8.04(1H, d, J = 2.0 Hz, H-2′), 7.51(1H, dd, J = 2.0, 9.0 Hz, H-6′), 6.90 (1H, d, J = 9.0 Hz, H-5′), 6.46 (1H, d, J = 2.0 Hz, H-8), 6.22 (1H, d, J = 2.0 Hz, H-6), 5.50 (1H, d, J = 7.5 Hz, H-1″). ¹³C NMR (DMSO-d₆, 100 MHz): δ 177.6 (C-4), 164.4 (C-7), 161.4 (C-5), 156.5 (C-2), 156.4 (C-9), 149.6 (C-4′), 147.2 (C-3′), 133.2 (C-3), 122.0 (C-1′), 121.2 (C-6′), 115.3 (C-5′), 113.6 (C-2′), 104.1 (C-10), 101.8 (C-1″), 98.9 (C-6), 93.9 (C-8), 76.0 (C-5″), 73.3 (C-3″), 71.4 (C-2″), 68.1 (C-4″), 57.0 (3′-OCH₃), 60.5 (C-6″). These data are identical with those for isorhamnetin-3-O-β-d-galactoside reported elsewhere [17].

Syringetin-3-O-β-d-glucoside (5): Yellow powder; C₂₃H₂₄O₁₃; ESI-MS: m/z 507 [M − H]^{−; 1}H NMR (MeOD, 400 Hz): δ 7.51 (2H, s, H-2′, 6′), 6.14 (1H, d, J = 2.0 Hz, H-8), 6.00 (1H, d, J = 2.0 Hz, H-7), 5.23 (1H, d, J = 7.0 Hz, H-1″), 3.91 (6H, s, 3′,5′-OCH₃). ¹³C NMR (MeOD, 100 MHz): δ 178.4 (C-4),165.1 (C-7), 162.7 (C-5), 159.5 (C-9), 157.4 (C-2), 149.3 (C-3′,5′), 141.0 (C-4′), 135.4 (C-3), 121.4 (C-1′), 108.3 (C-2′,6′), 104.7 (C-6), 103.6 (C-1″), 103.1(C-10), 97.5 (C-8), 78.8 (C-5″), 78.5 (C-3″), 76.3 (C-2″), 71.8 C-4″), 62.8 (C-6″), 57.4 (3′,5′-OCH₃). These data are identical with those for syringetin-3-O-β-d-glucoside reported elsewhere [18].

The total phenolic content of the crude extract and its fractions were determined using Folin-Ciocaleteu assay according to the previously described method [19]. Gallic acid (GA) was used as standard and the results were calculated as gallic acid equivalents (mg/g) of lotus receptacle extract through the calibration curve of gallic acid. The total flavonoid content was determined using a colorimetric assay according to the previously described method with some modifications [20]. The results were calculated and expressed as rutin equivalents (mg) per gram of lotus receptacle extract using the calibration curve of rutin. The total proanthocyanidin content was determined according to the modified previously described procedure [21], using (+)-catechin as a standard and the results were expressed as (+)-catechin equivalents in microgram per gram of lotus receptacles extract.

All the experiments were conducted in triplicates, and the data were presented as mean ± SD. SPSS (version 16.0; SPSS Inc.: Chicago, IL, USA, 2007) and Origin (version 8.0; Microcal Software Inc.: Northampton, MA, USA, 2007) were used to process the results, which were analyzed by one-way ANOVA and Tukey-HSD at p < 0.05 to detect significant differences among groups.