Antioxidant Activities of Extract and Fractions from Receptaculum Nelumbinis and Related Flavonol Glycosides

The antioxidant activities of ethanolic crude extract (ECE) and its four different solvent sub-fractions (namely, petroleum ether fraction (PEF), ethyl acetate fraction (EAF), n-butanol fraction (BF) and the aqueous fraction (AF) from the receptacles of Nelumbo nucifera Gaertn. (Receptaculum Nelumbinis) were investigated using two in vitro antioxidant assays. BF showed the highest total phenolic content (607.6 mg/g gallic acid equivalents), total flavonoid content (862.7 mg/g rutin equivalents) and total proanthocyanidin content (331.0 mg/g catechin equivalents), accompanied with the highest antioxidant activity compared to other fractions through 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radical scavenging assays. Five flavonol glycosides, namely 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) were isolated from the Receptaculum Nelumbinis. Compounds 2–5 were isolated for the first time from the Receptaculum Nelumbinis. The five isolated flavone glycosides, particularly compounds 1–3, demonstrated significant DPPH and ABTS radical scavenging activity, with IC50 values of 8.9 ± 0.2, 5.2 ± 0.2, 7.5 ± 0.1 for DPPH and 114.2 ± 1.7, 112.8 ± 0.8, 172.5 ± 0.7 μg/mL for ABTS, respectively. These results suggest that Receptaculum Nelumbinis has strong antioxidant potential and may be potentially used as a safe and inexpensive bioactive source of natural antioxidants.


Total Flavonoids and Total Phenolic Content
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.

Radical Scavenging Activities
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 (IC 50 = 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) (IC 50 = 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 (IC 50 = 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).

Structural Determination of the Isolated Compounds from n-Butanol Fraction (BF)
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)

Antioxidant Activities of the Isolated Compounds from n-Butanol Fraction (BF)
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 IC 50 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.

General
NMR spectra were obtained with a Bruker DRX-400 spectrometer at 400 MHz for 1 H NMR and 100 MHz for 13

Plant Material
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.

Determination of Total Phenols, Total Flavonoids and Total Proanthocyanidin
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.

DPPH Radical-Scavenging Activity
The DPPH radical scavenging activities of the crude extract of Receptaculum Nelumbinis and its fractions were determined according to the previously described method [4]. The DPPH radical scavenging activity was calculated by the following formula: Scavenging ability (%) = (1 − A sample /A blank ) × 100%, where A blank is the absorbance of the blank reaction (using 2 mL of methanol instead of the tested sample) and A sample is the absorbance of the tested sample. For each sample, its concentration with 50% scavenging ability on the DPPH radicals (IC 50 ) was determined by interpolation of linear regression analysis. BHT was used as positive control.

ABTS Radical-Scavenging Activity
The ability of the crude extract and fractions of Receptaculum Nelumbinis to scavenge ABTS radicals were determined according to ABTS decolorization assay as previously described [4]. The ability of individual Receptaculum Nelumbinis extracts to scavenge ABTS radicals (%) and the IC 50 were calculated as described in the DPPH radical scavenging assay. BHT was used as positive control.

Statistical Analysis
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.

Conclusions
This study was designed to evaluate the total phenolic, flavonoid and proanthocyanidin contents and antioxidant activity of ECE and its sub-fractions of Receptaculum Nelumbinis. The BF sub-fraction showed the highest total phenolic and flavonoids contents, exhibited the highest free-radical scavenging activities. Five flavonol glycosides were isolated and their antioxidant activities were evaluated using DPPH and ABTS models. Hyperoside (1), isoquercitrin (2) and quercetin-3-O-β-D-glucuronide (3) showed effective activity and the structure-activity relation investigation indicated that the catechol group in the B ring affected the antioxidant activity. Being the by-products of lotus food production, the utilization of Receptaculum Nelumbinis is still low: it is usually discarded as waste. The obvious antioxidant activity in Receptaculum Nelumbinis confirmed its important role in the bioactivity of whole lotus and suggested that the Receptaculum Nelumbinis removal may induce a more significant nutrient loss. Until now, the Receptaculum Nelumbinis has not received much attention with respect to recycling it for the antioxidants instead of disposing it due to the lack of commercial application of these wastes. Our findings not only provide an added value to this regional bio-resource, but could also facilitate its development as a safe and inexpensive bioactive source of natural antioxidants, especially for the fast growing functional food industry nowadays.