Antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of ethanol extract and pure flavonoids from Adinandra nitida leaves

Abstract

Context: Adinandra nitida Merr. ex. H.L. Li (Theaceae) is an indigenous plant in south China. Its leaves have been reported to have many curative effects such as reducing blood pressure, as well as antibacterial, antioxidant, and analgesic properties, which could be used in foods and medicines.

Objective: The antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of the main flavonoids and ethanol extract (EE) of A. nitida leaves were investigated for the first time.

Materials and methods: The main flavonoids of A. nitida leaves (camellianin A, camellianin B) were prepared and their contents in EE were determined by HPLC. The antioxidant activities of the samples were measured by DPPH radical scavenging assay and Rancimat test. The ACE inhibitory activities of the samples were carried out by using an assay kit with hippuryl-glycyl-glycine as substrate.

Results: The contents of camellianin A, camellianin B and apigenin in EE were determined as 41.98, 2.67, and 1.73%, respectively. The antioxidant activities of the flavonoids were far lower than that of EE in DPPH radical scavenging and Rancimat assays. However, the ACE-inhibitory activities of camellianin A, camellianin B and apigenin were higher than that of EE.

Discussion and conclusion: The flavonoid content of EE was more than 45%. The high activities of EE in DPPH scavenging and Rancimat assay could be mainly attributed to compounds other than flavonoids. However, the ACE-inhibitory activity of EE could be mainly attributed to the presence of the flavonoids.

Keywords::

• Adinandra nitida
• angiotensin converting enzyme (ACE)
• antioxidant activity
• camellianin A
• flavonoid

Introduction

Flavonoids, which exist ubiquitously in fruits, tea, vegetables, and medicinal plants, have been studied extensively and received great attention, since they are effective free radical scavengers and are assumed to be less toxic than synthetic antioxidants such as BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene), which are suspected of being carcinogenic and hepatotoxic (Burda & Oleszek, 2001; Zou et al., 2004). Today, more than 4000 kinds of flavonoids have been identified or synthesized. However, only rutin can be widely used in the fields of food and medicine (Yuan et al., 2008). Few plants contain sufficient amounts of flavonoids to achieve large-scale production, although flavonoids are widely distributed in plants.

Adinandra nitida Merr. ex H.L. Li (Theaceae), a wild plant in south China, is a flavonoid-rich plant source. Its leaves have been consumed as a health tea (Shiyacha) and herbal medicine for hundreds of years (Wang et al., 2003; Zhang et al., 2005). It is reported to have many curative effects such as reducing blood pressure, as well as antibacterial, antioxidant, and analgesic properties (Wang et al., 2003). According to our previous studies (Liu et al., 2008; Yuan et al., 2008, 2009), the total flavonoid content in A. nitida leaves was more than 20% with camellianin A as the main flavonoid. Today, A. nitida has been cultivated in Guangxi province, China, so there is the possibility of large-scale application of flavonoids from A. nitida leaves in functional food and medicine. However, so far, A. nitida leaves have not been completely studied. For example, it was always believed that the strong bioactivity of its ethanol extract was due to the high flavonoid content, but this result lacked the support of the corresponding experiment. Since pure camellianin A and camellianin B were not available, there were no reports on the determination of the main flavonoids of EE using HPLC as well as reports on the bioactivity comparison. In view of the above background, the ethanol extract and pure flavonoids were prepared from A. nitida leaves in this study and their antioxidant activities were evaluated by the commonly accepted assays such as DPPH radical scavenging and Rancimat antioxidant assays. Since A. nitida leaves are famous for their antihypertensive activity, the ACE-inhibitory activities of the samples were also investigated.

Materials and methods

Materials and chemicals

Leaves of A. nitida (2007 production, moisture content 9.3%) for this study were purchased in Pingle County, Guangxi Province, China and identified by Yousheng Zhang in Guangdong Academy of Agricultural Sciences. The voucher specimen (FC-2007-10) was deposited in the Laboratory of Food Chemistry of South China University of Technology. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) was the product of Sigma Chemicals (St. Louis, MO). Apigenin was obtained from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing). The assay kit for ACE activity was provided by Health Science (Beijing). Other chemicals were of analytical grade.

Preparation of EE

The powdered leaves (~25.33 g) of A. nitida (moisture content 9.3%) were extracted with 500 mL of 63% ethanol in a water bath at 70°C for 30 min and then filtered. The filtrate was concentrated under vacuum at 45°C and freeze-dried (Four-Ring Science Instrument Plant, Beijing); about 11.62 g of the extract (EE) were obtained.

Preparation of camellianin A and camellianin B

Camellianin A was prepared by recrystallizing 10 times from water, according to our previous study (Liu et al., 2008). Camellianin B was prepared by using the following method: Camellianin A (900 mg) was mixed with methanol (20 mL) and K₂CO₃ (500 mg). After stirring for 30 min at 40°C, hydrochloric acid was added to make pH 7.0. The mixture was kept for 4 h at 4°C. The resultant white crystal was separated by filtration, washed with water and dried. As a result, a white powder (640 mg) was obtained.

Identification of flavonoids by UV, IR, NMR and ESI-MS

UV analysis was performed on a TU-1810PC spectrophotometer (Purkinje, China) and the spectra of the flavonoids in methanol were recorded and processed by UV Win 5.0.5 software (Purkinje). IR analysis was performed on a TENSOR 27 infrared spectrophotometer (Bruker, Ettlingen, Germany) and the data were recorded and processed by OPUS 4.0 software. ESI-MS² analyses were taken on a LCQ Deca XP MAX electrospray ionization mass spectrometer (Finnigan, Silicon Valley, CA, USA) in the negative ion mode. ¹³C-and ¹H-NMR spectra were recorded in DMSO-d₆ using a DRX-400 NMR spectrometer (Bruker) at 400 MHz.

Determination of the contents of three flavonoids in EE by HPLC

The standard mixture was prepared by mixing three standard solutions. For the calibration curves, the stock solutions were diluted with methanol in the concentration range from 28.65 to 573 μg/mL for camellianin A, from 4.6 to 92 μg/mL for camellianin B, and from 3.6 to 72 μg/mL for apigenin. All the solutions were stored at 4°C. About 34.7 mg of the ethanol extract was dissolved in methanol for HPLC analysis. The samples were separated on a reversed phase column, SunFire^™ C18 column (4.6 × 250 mm; 5 μm particle size) made by Waters (Milford, MA). The mobile phase consisted of water and methanol (1:1) with a flow rate of 1 mL/min. The column temperature was set at 30°C. The HPLC analysis was performed on a Waters Alliance HPLC system, which consisted of a Waters 2695 separations module and a Waters 2487 dual wavelength detector. The injection volume was 10 μL and the wavelengths for detection were set at 265 and 330 nm. Before HPLC analysis, all the samples were passed through a 0.45 μm millipore filter. The quantitative analysis of camellianin A, camellianin B and apigenin in the samples was based on an external standard. The chromatographic data were recorded and processed by Empower 2 software.

DPPH radical scavenging assay

The DPPH assay has been widely used for the measurement of free radical scavenging capacity of various natural products. The DPPH radical is a stable organic free radical with adsorption band at 515–528 nm. It loses this adsorption when accepting an electron or a free radical species, which results in a visually noticeable discoloration from purple to yellow (Sánchez-Moreno, 2002). The DPPH radical scavenging assay was measured by using the method of Sun and Ho (2005). Briefly, 2 mL of DPPH solution (0.2 mmol/L, in ethanol) was incubated with different concentrations of the sample. The reaction mixture was shaken and incubated in the dark for 30 min, at room temperature. The absorbance was read at 517 nm against ethanol. The control containing ethanol instead of the sample, and the blank containing ethanol instead of DPPH solution were also made. The tests were run in triplicate and the inhibition of the DPPH radical by the sample was calculated according to the following formula:

and the percentage of DPPH radical scavenging activity was plotted against the sample concentration to obtain the IC₅₀, defined as the concentration of sample necessary to cause 50% inhibition.

Measurement of antioxidant capacity by Rancimat test

In the Rancimat method, the sample is exposed to a stream of air at temperatures from 50–220°C. The volatile oxidation products (chiefly formic acid) are transferred to the measuring vessel by the air stream and absorbed there in the measuring solution (distilled water). When the conductivity of this measuring solution is recorded continuously an oxidation curve is obtained whose point of inflection is known as the induction time; this provides a good characteristic value for the oxidation stability. The antioxidant activities of the samples were performed on a 743 Rancimat analyzer (Metrohm, Herisau, Switzerland) according to the method of Proestos et al. (2006). The samples of lard oil (3 g) containing 0.02% of the flavonoids and the extracts were subjected to oxidation at 110°C (air flow 20 L/h). The induction periods (IP) were recorded automatically. The protection factors (PF) were calculated according to the following formula: (PF =IP_sample/IP_control).

ACE inhibition assay

ACE inhibition assay was carried out using an assay kit for ACE activity according to the method of Neels et al. (1983). The sample (0.1 mL) and human serum (0.1 mL) were mixed for 5 min. Then 0.1 mL of hippuryl-glycyl-glycine (30 mM) was added into the mixture. The obtained solution was incubated for 30 min at 37°C and mixed with diluted sulfuric acid (330 mM) and sodium tungstate (100 g/L). The above solution was centrifuged for 5 min at 12,000 g. The supernatant (0.1 mL) was mixed with 0.1 mL of borate buffer (100 mM, pH 9.6) followed 10 μL of trinitrobenzene sulfonate (TNBS, 60 mM) solution and stood for 30 min. The absorbance against a serum blank was measured by a BioRad-550 Microplate Reader provided by Bio-Rad Laboratories (Berkeley, CA). ACE inhibitory activity was calculated according to the following formula:

Statistical analysis

The data obtained in this study were expressed as the mean of three replicate determinations and standard deviation (SD). Statistical comparisons were carried out using Student’s t-test. P values of <0.05 were considered to be significant.

Results

Identification of camellianin A and camellianin B

By using UV, IR, ESI-MS and NMR, the flavonoids obtained in this study were identified as camellianin A and camellianin B.

Camellianin A (Figure 1). UV, λ_max(nm) (MeOH) 263, 330; IR bands (KBr disc): 3384 (-OH), 1733 (ester bond), 1630 (-C=O), 1579, 1516, 1495, 1454 (-Ar) cm⁻¹; ESI-MS² negative ion m/z: 681.92 ([M+NO₃]⁻), 654.87 ([M+Cl]⁻), 619.39 ([M-H]⁻), 578.08 ([M-Acetyl-H]⁻), 474.02 ([M-Rham-H]⁻), 269.12 ([M-Acetyl-Rham-Glu-H]⁻); ¹³C-NMR: 176.31 (C-4), 170.70 (C-7″), 162.82 (C-2), 161.37 (C-4′), 160.9 (C-7), 159.36 (C-9), 157.68 (C-5), 128.46 (C-2′ and C-6′), 121.98 (C-1′), 116.51 (C-3′ and C-5′), 107.95 (C-10), 106.43 (C-3), 100.28 (C-1′′), 99.75 (C-6), 97.79 (C-1′′′), 96.94 (C-8), 77.47 (C-4′′), 77.11 (C-3′′), 74.03 (C-5′′), 72.83 (C-2′′), 71.11 (C-3′′′), 71.02 (C-4′′′), 70.55 (C-2′′′), 69.25 (C-5′′′), 63.5 (C-6′′), 20.90 (C-8′′), 18.52 (C-6′′′); ¹H-NMR: 10.73 (1H, s, 7-OH), 10.2 (1H, s, 4′-OH), 7.86 (2H, d, J = 8.6 Hz, 2′- and 6′-H), 6.92 (2H, d, J = 8.6 Hz, 3′- and 5′-H), 6.62 (1H, s, 8-H), 6.53 (1H, s, 3-H), 6.5 (1H, s, 6-H), 5.51 (1H, d, J = 6.0 Hz, 1′′-H), 5.2 (1H, s, 1′′′-H), 3.16-4.64 (10H, m, Hs in sugar), 1.87 (3H, s, 8′′-H), 1.09 (3H, d, J = 6 Hz, 6′′′-H);

Figure 1.  Chemical structures of camellianin A and B.

Camellianin B (Figure 1). UV, λ_max(nm) (MeOH) 262, 330; IR bands (KBr disc): 3382 (-OH), 1634 (-C=O), 1598.07, 1580, 1509, 1444 (-Ar) cm⁻¹; ESI-MS² negative ion m/z: 639.93 ([M+NO₃]⁻), 613.02 ([M+Cl]⁻), 577.37 ([M-H]⁻), 431.09 ([M-Rham-H]⁻), 269.18 ([M-Rham-Glu-H]⁻); ¹³C-NMR: 175.95 (C-4), 164.07 (C-2), 161.15 (C-4′), 160.35 (C-7), 159.35 (C-9), 158.01 (C-5), 128.23 (C-2′ and C-6′), 121.79 (C-1′), 116.38 (C-3′ and C-5′), 107.23 (C-10), 106.21 (C-3), 100.0 (C-1′′), 99.78 (C-6), 98.1 (C-1′′′), 96.79 (C-8), 77.49 (C-4′′), 77.23 (C-3′′), 76.91 (C-5′′), 72.75 (C-2′′), 71.0 (C-3′′′), 70.84 (C-4′′′), 70.35 (C-2′′′), 68.95 (C-5′′′), 61.15 (C-6′′), 18.41(C-6′′′); ¹H-NMR: 7.82 (2H, d, J = 8.6 Hz, 2′- and 6′-H), 6.88 (2H, d, J = 8.6 Hz, 3′- and 5′-H), 6.55 (1H, s, 8-H), 6.49 (1H, s, 3-H), 6.44 (1H, s, 6-H), 5.26 (1H, d, J = 6.6 Hz, 1′′-H), 5.2 (1H, s, 1′′′-H), 3.0-5.0 (10H, m, Hs in sugar), 1.04 (3H, d, J = 6.0 Hz, 6′′′-H).

The contents of camellianin A, camellianin B and apigenin in EE

With camellianin A, camellianin B and apigenin as the standards, the contents of the main flavonoids in EE were measured by HPLC (Figure 2). The contents of camellianin A, camellianin B and apigenin in EE were determined as 41.98 ± 0.23, 2.67 ± 0.05 and 1.73 ± 0.01%, respectively.

Figure 2.  HPLC profiles of standards (A) and EE (B) at 265 nm: (1) Camellianin B;(2) Camellianin A; (3) Apigenin.

DPPH radical scavenging activity

Figure 3 illustrated the DPPH scavenging activities of EE and the flavonoids found in EE. All the samples showed a dose-dependent manner in scavenging DPPH radicals. The IC₅₀ values of EE, camellianin A, camellianin B and apigenin were 14.74 μg/mL, 1.62 mg/mL, 1.8 mg/mL, and 0.95 mg/mL, respectively.

Figure 3.  DPPH radical scavenging activity of camellianin A, camellianin B, apigenin and EE.

Rancimat antioxidant test

As shown in Figure 4, the PF values of EE, camellianin A, camellianin B and apigenin were 1.49, 1.07, 1.05 and 1.26, respectively. The antioxidant performance of the samples coincided with that in DPPH scavenging assay.

Figure 4.  Antioxidant activity of camellianin A, camellianin B, apigenin and EE in Rancimat test.

ACE inhibitory activity

In this study, a sensitive colorimetric procedure was used for the assay of ACE in serum. Serum is incubated with hippuryl-glycyl-glycine, the liberated glycyl-glycine is derivatized with a borate-buffered trinitrobenzene sulfonate solution to form trinitrophenyl-glycyl-glycine, the maximum absorbance of which was at 420 nm. As shown in Figure 5, all the samples exhibited the ACE inhibitory activity. And the inhibitory activities of three flavonoids were slightly higher than that of EE. At 500 μg/mL, the ACE inhibitory activities of EE, camellianin A, camellianin B and apigenin were 29.7%, 30.16%, 40.68%, and 30.27%, respectively. The antioxidant activities of three flavonoids were slightly higher than that of EE.

Figure 5.  ACE inhibitory activity of camellianin A, camellianin B, apigenin and EE (Error bars show the standard deviation of three determinations).

Discussion

A. nitida has great commercial interest for the food and phytopharmaceutical market, but few reports have been published about it. Zhang et al. (2005) reported the identification of flavonoids in leaves of A. nitida by HPLC-MS. But in their report, the names and chemical structures of camellianin A and camellianin B were confused. For example, the flavone with m/z 619 was named as camellianin B in their report and its structure was also expressed as apigenin-rham-glc-OCCH₃ (Glc: glucose; Rham: rhamnose), which did not coincide with the results of our ESI-MS² analysis and the original report about the identification of camellianin A and camellianin B (Cheng et al., 1987).

For lack of camellianin A and camellianin B, the established quality control method in the study of Zhang et al. (2005) was based on the quantitative analysis of three minor flavonoids (apigenin, epicatechin and rholifolin). In our previous study (Yuan et al., 2009), the method of preparing camellianin A and camellianin B by using high-speed counter-current chromatography (HSCCC) was established. But for the low content of camellianin B in A. nitida leaves, it was difficult to obtain enough camellianin B by HSCCC. In this study, we invented a method to obtain large-scale production of camellianin B by removing the acetyl group of camellianin A in the reaction system of methanol and K₂CO₃. With camellianin A, camellianin B and apigenin as the standards, the contents of the main flavonoids in EE were measured by HPLC. The HPLC result showed that A. nitida leaves were rich in flavonoids and the total flavonoid content of its ethanol extract was more than 45%.

In previous reports (Liu et al., 2008; Yu & Chen 1997), the high antioxidant activity of EE was attributed to the high flavonoid content, which lacked the support of the experiment. In the DPPH radical scavenging and Rancimat assays it was found that, although the flavonoid content in EE was more than 45%, the antioxidant activities of camellianin A, camellianin B and apigenin were far lower than that of EE, which suggested the high antioxidant activity of EE should mainly be attributed to the other compounds instead of the flavonoids.

The synthesized ACE inhibitors, such as captopril, enalapril, alacepril, and lisinopril, are currently used extensively in the treatment of essential hypertension and heart failure in humans. However, these synthetic drugs were believed to have certain side effects, such as cough, taste disturbances and skin rashes (Je et al., 2005). Therefore, the search for ACE inhibitors from foods has become a major area of research. In this study, the ACE-inhibitory activities of three flavonoids were slightly higher than that of EE, which suggested that the ACE-inhibitory activity of EE could be mainly attributed to the presence of the flavonoids. It is known that the active site of ACE consisted of three parts: a carboxylate binding functionality such as the guanidinium group of arginine, a pocket that accommodates a hydrophobic side chain of C-terminal amino acid residues and a zinc ion (Loizzo et al., 2007). It was suggested that the flavonoids showed in vitro activity via the generation of chelate complexes within the active centre of ACE (Wagner et al., 1991). The free hydroxyl groups of the phenolic compounds were also suggested to be the important structural moieties to chelate the zinc ions, thus inactivating the ACE activity (Chen & Lin, 1992). So camellianin A, camellianin B and apigenin could show the ACE-inhibitory activity, which could explain the antihypertensive activity of A. nitida leaves.

To sum up, the flavonoid content of A. nitida leaves was so high that the flavonoid content of its ethanol extract was more than 45%. The high antioxidant activities of the ethanol extract in DPPH scavenging and Rancimat assays could be mainly attributed to compounds other than the flavonoids. However, the ACE inhibition activity of EE could be mainly attributed to the presence of the flavonoids. The results obtained in this study could become the initial base of the application of A. nitida leaves in the field of food and medicine.

Declaration of interest

Financial support provided by the National Natural Science Foundation of China (No.20806029), Foundation of Henan Educational Committee (No. 2009A550005) and the Science and Technology Plan of Henan institute of science and technology (No.7040) was greatly appreciated.