Aqueous extracts of tree peony petals: renin and angiotensin I-converting enzyme inhibitory activities in different colours and flowering stages

Tree peony (Paeonia suffruticosa Andr.) is an ornamental and medicinal plant from China. Previous studies have detected novel blood pressure-regulating substances in this species, which potentiate its value of utilization. To explore these substances, the aqueous extracts of 7 different colours of tree peony petals were assessed for inhibitory activity on renin and angiotensin-converting enzyme (ACE). The results showed that the activity of dark-coloured samples was significantly stronger than that of light-coloured ones. Furthermore, the inhibitory activity of the red tree peony petals ‘Hong TaiYang’ on renin and ACE indicated a downward trend from bud compaction to the full opening stage. The antioxidant activities of the aqueous extracts, on one side, and the correlations between phenolics and flavonoids functionalities and total contents, on the other, were also evaluated. In this regard, the extracts of different samples had ABTS free radical scavenging capacities of 17.28–210.41 mg TE per g DW, DPPH radical scavenging capacities of 35.45–150.78 mg TE per g DW, iron ion reduction capacities of 16.66–150.77 mg TE per g DW, and total phenolic content of 23.94–150.78 mg GAE per g DW. Correlation analysis revealed that the renin and ACE inhibitory activities, the DPPH and ABTS free radical scavenging capacities, and the iron reduction ability of different sample extracts were positively correlated with total phenolic contents (p < 0.01). Finally, the aqueous phenolic compounds in the sample extracts tended to show strong renin and ACE inhibitory activities and therefore exhibit a potential auxiliary blood pressure control prospect.


Introduction
Hypertension is the most common chronic disease and the main risk factor for cardiovascular and cerebrovascular diseases. 1 The renin-angiotensin system (RAS) plays a crucial role in modulating blood pressure in the human body. Renin (EC 3.4.23.15), a rate-limiting enzyme, can hydrolyze the Nterminus of angiotensinogen to yield angiotensin I. Then, angiotensin I is further hydrolyzed to angiotensin II. Because of the catalysis of the angiotensin-converting enzyme (ACE; EC 3.4.15.1), these processes occur under strong vasoconstriction conditions that eventually increase blood pressure. 2 Because of this, inhibiting the activities of renin and ACE is considered an effective way to prevent and treat hypertension. At present, synthetic inhibitors, such as aliskiren for renin and captopril and enalapril for ACE, have become popular drugs to treat the hypertensive. However, there are evident side effects associated with the usage of these inhibitors, like skin rashes, dry cough, as well as taste disturbance, which are thought to be inevitable. 3 In this context, renin and ACE inhibitors derived from foodstuff have attracted the attention of researchers because they are easily absorbed and bear fewer side effects. 4 Recently, many renin and ACE inhibitory peptides obtained from plant or foodstuff sources have been investigated, including bean hydrolysates, 5 amaranth proteins, 6 beef hydrolysates, 7 axseed protein, 8,9 and bovine brinogen. 10 Although these food-derived peptides have provided new ideas for the prevention and treatment of hypertension, the efficacy of these peptides in the human body needs to be further veried. For example, some of them have been shown to easily decompose into inactive metabolites in vivo, which results in low bioavailability. 11 Therefore, the research and development of safe and effective renin and ACE inhibitors is a present necessity.
Some clinical experiments have indicated that increasing daily intake of phenols has a benecial effect in controlling hypertension. 12,13 This could be due to the polyphenols reducing the oxidative damage that blood vessels receive. It is worth noting that some phenolic compounds derived from plants have been reported to inhibit renin or ACE. For instance, saponin from soybean, 14 polyphenolic extracts of the green leafy vegetables Vernonia amygdalina and Gongronema latifolium, 15 and tea polyphenols 16 inhibit renin; on the other hand, polyphenols from tomato, 17 avonoids from the buds of rosa damascene, 18 and polyphenols from Indian gooseberry 19 inhibit ACE. In summary, these reports have shown that phenolics may regulate RAS by inhibiting renin and/or ACE and have a bene-cial effect on the treatment of hypertension.
Tree peony (Paeonia suffruticosa Andr.), a woody deciduous shrub (Paeoniaceae family) native to China, is considered a traditional ornamental and medicinal plant. 20 Now, tree peonies are cultivated all over the world, and more than 20 ha have been grown in China. Tree peony petals are beautiful and highly enriched in soluble sugars, avonoids, anthocyanins, and gallic acid, 21 which have become food materials and natural antioxidant resources to make yogurt, tea, cake, red wine, and essential oil. [22][23][24] Recently, an increasing number of researchers have paid attention to the chemical components and biological activities of tree peony petals. Some papers have used advanced technologies to separate and identify phenols and avonoids from tree peony petals and proved their antioxidant, anti-inammatory, and antibacterial activities. 25,26 In a previous study, we found that aqueous extracts of red and white tree peony petals had inhibitory activity on renin and ACE, but their inhibitory activity was signicantly different. 27 There is a diversity of colours among tree peony owers with nine categories: red, white, yellow, green, pink, purple, black, blue, and dual colours. Different phytochemicals are attributed to each ower colour, and dark petals contain more anthocyanins and avonoids. 21 During ower growth, phenolic compounds such as the secondary metabolites, including anthocyanins and carotenoids, accumulate at different stage. 28 Therefore, the content of bioactive substances varies with the owering stage. In this study, we investigated inhibitory activity in aqueous extracts of tree peony petals. In particular, we considered 7 different ower colours and assessed renin and ACE activities. Additionally, renin and ACE inhibitory activity in aqueous extracts of red tree peony 'Hong TaiYang' at 4 different owering stages was further valued. On the other hand, since oxidative damage in the blood vessels is closely related to the occurrence and development of hypertension, 12 the antioxidant activity of the samples was also investigated. Other assessments included detection of total phenols and avonoids and calculating correlations between the contents of total phenolics and their functionalities. This work could provide not only new ideas for exploring novel blood pressure regulatory factors but also a richer theoretical basis for further improving the functionalities of tree peony products with high-value utilization.
The tree peony petals used in the experiments were collected from the Fuxi tree peony planting base of He Ze, Shandong, China. The petals of different colours including the red 'Hong TaiYang' were classied according to different owering stages (Fig. 1). The tree peonies were transported on ice within 12 h, taken to a vacuum freeze dryer, and stored at À4 C.

Preparation of sample extracts
All freeze-dried samples of petals were ground to a ne powder with a pulverizer, and each sample powder (0.5 g) was dispersed in 20 mL of distilled water. Aer mixing completely, the solution was centrifuged for 10 min at 5000 rpm. The supernatant was collected and ltered through 0.45 mM membrane ltration. The ltrate was recovered for the subsequent experiment.

Renin inhibition assay
The determination of renin inhibition was based on the method by Li et al. 16 The human recombinant renin inhibitor screening kit was employed as follows: (1) blank: 20 mL of substrate, 160 mL of buffer, 10 mL of distilled water; (2) sample: 20 mL of substrate, 160 mL of buffer, 10 mL of sample solution. Then 10 mL of renin enzyme solution were added to the control and sample wells to start the reaction. Meanwhile, 10 mL of the buffer were added to the blank wells. Then, it was let rest at 37 C for 15 min.
The synthetic uorescence resonance energy transfer peptide utilized in this assay is the usual substrate for renin. It is linked to a uorophore at one end and a nonuorescent chromophore at the other. Aer the peptide is cleaved by renin, the product is highly uorescent and can be easily analysed by recording the uorescence intensity (FI) on a uorescence plate reader (Powerscan HT; BioTek Instruments, Inc., Winooski, VT, U.S.), with an excitation wavelength of 360 nm and an emission wavelength of 528 nm. The analyses were performed in triplicate. The renin inhibitory activity was calculated as follows: where: FI (blank) was the absorbance of blank, FI (sample) was the absorbance in presence of sample.

Assay for ACE inhibition
The determination of ACE inhibition was performed according to Li et al. 29 The aqueous extract of the samples was appropriately diluted and a 96 well microtiter plate was used as a reaction container. Then, 15 mL of the sample solution (in the control reaction solution, this was replaced with distilled water) was mixed with 30 mL of 4.66 mmol L À1 of HHL (previously dissolved in 0.6 mol L À1 NaCl-0.4 mol L À1 phosphate buffer, pH 8.5). Subsequently, 30 mL of 12.5 mU mL À1 ACE enzyme solution were added (for the blank of the sample reaction solution and the control reaction solution, distilled water was used instead), and the reaction was taken to a microplate mixer at 37 C for 1 h. To stop the enzyme reaction, 120 mL of 1.2 mol L À1 NaOH solution were added, followed by 30 mL of 2% OPA solution (dissolved in methanol). The latter was mixed and le resting at room temperature for 20 min. The derivatization reaction was terminated with 30 mL of 6 mol L À1 HCl solution. Finally, uorescence absorption intensity was measured with an excitation wavelength of 340 nm, emission wavelength of 455 nm, and a slit width of 5 nm. The ACE inhibition rate of the samples was calculated as follows: where I 1 indicated the uorescence absorption intensity of the sample reaction solution in the presence of the ACE inhibitor; and I 2 specied that the control reaction solution had no ACE inhibition.
2.5 Determination of total antioxidant capacity 2.5.1 Ferric reducing antioxidant power (FRAP) assay. The FRAP assay was carried out according to the procedure described by Benzie et al. 30 Briey, TPTZ solution was prepared as follows: 25 mL of 0.3 mol L À1 acetate buffer and 2.5 mL of 10 mmol L À1 TPTZ working solution were mixed, and then 2.5 mL of a 20 mmol L À1 FeCl 3 solution was added. Successively, 1.8 mL of the TPTZ solution was taken and added to 10 mL of sample extract and mixed with 1 mL of distilled water. The reaction occurred at 37 C for 10 min. Finally, the absorbance was determined at 593 nm. The standard curve was constructed using Trolox solution (0.03125-1 mg mL À1 ) and the results were expressed as Trolox per g dry of owers (mg TE per g DW).
2.5.2 DPPH free radical scavenging assay. The antioxidant activities of the samples were analysed by investigating their potential to scavenge the DPPH free radical. For this purpose, the method by Cai et al. 31 was employed with some modications, as will be described hereon. Samples at a concentration of 5 mg mL À1 were evenly mixed with 200 mL of 0.08 mg mL À1 DPPH ethanol solution and then incubated for 30 min in the darkness. The absorbance was measured at 517 nm. In the blank control, the same volume of distilled water was used instead of the sample. The nal result was expressed as antioxidant capacity, this was Trolox per g dry of ower (mg TE per g DW). The scavenging rate of DPPH was calculated according to the following formula: where I was DPPH scavenging rate; A c was the blank absorbance; A i was the sample absorbance; A j was the absorbance without DPPH. 2.5.3 Determination of ABTS + scavenging capacity. Antioxidant activity in the samples was analysed by investigating their potential to scavenge the ABTS + radicals. We used the method described by Ozgen et al. 32 Briey, ABTS reagent was prepared by mixing 5 mL of 7 mmol L À1 ABTS stock solution with 88 mL of 140 mmol L À1 potassium persulfate and then letting rest in the dark for 12 h. Then, the mixture was diluted with ethanol before measuring absorbance at 732 nm. Finally, 25 mL of the sample extract were added to 2 mL of ABTS solution, and let rest for 6 min in the dark to subsequently determine absorbance. Trolox solution (nal concentration 0.03125-1 mg mL À1 ) was used as a reference standard. The results were expressed as Trolox per g dry of ower (mg TE per g DW). The scavenging rate of ABTS was calculated based on the following formula: where I was ABTS scavenging rate; A 0 was the blank absorbance; A 1 was the absorbance of the test sample.

Contents of total phenolics
Total phenolic content (TPC) was determined by the Folin-Ciocalteu method according to McDonald et al. 33 with minor modications. Briey, 100 mL of the sample solution was added to 100 mL of 10% Folin-Ciocalteu solution and incubated at 37 C. Aer 10 min, 100 mL of 10% Na 2 CO 3 was added and the mixture was le to rest at 37 C for 60 min in the dark. The absorbance was measured at 750 nm. The standard curve was set using various concentrations of gallic acid in distilled water. Total phenolic content (TPC) was expressed as mg gallic acid equivalent (GAE) per g dried weight (DW).

Content of avonoids
The total content of avonoids (TF) was analysed using the method described by Pourmorad et al. 34 Briey, 100 mL of extract were mixed with 625 mL of distilled water and 375 mL of 5% NaNO 2 solution. Aer 6 min, 75 mL of a 10% AlCl 3 $6H 2 O solution were added. The mixture was le to rest for another 5 min before the addition of 250 mL of 1 M NaOH. The absorbance was measured at 510 nm using a spectrophotometer. Results were expressed as mg of catechin equivalents per g of dry weight (mg CE per g DW).

Statistical analysis
Data were expressed as means AE standard errors aer triplicate evaluation. The statistical soware employed was the IBM SPSS Statistics (version 19.0). Duncan's multiple range test was used to evaluate differences among samples. Differences at p < 0.05 were considered statistically signicant.

Renin and ACE inhibitory activities in tree peony petals of different colours and owering stages
The renin inhibitory activity experiment was carried out at a sample concentration of 0.27 mg mL À1 , while the ACE inhibitory activity test was at a sample concentration of 0.59 mg mL À1 . Results according to each ower colour were shown in Fig. 2 20% and 25.70%). The above indicated that, in tree peony petals, differences in ower colour and corresponding chemical composition signicantly inuenced the inhibitory ability of renin and ACE. The different owering stages of red tree peony 'Hong Taiyang' and their relation to renin and ACE were shown in Fig. 2. The inhibitory activities of the two enzymes showed a decreasing trend from ower bud to fully open. In particular, there were signicant differences in the rst three stages: the inhibition rates of renin and ACE were 62.53% and 73.34% (respectively) in the compact stage of ower bud, 58.45% and 64.30% in the loose stage of ower bud, 49.76% and 55.34% in the half opening stage, and 46.74% and 56.53% when fully open.
In previous studies, we found that the aqueous extract of red tree peony petals had signicant inhibitory activity over renin and ACE, with an IC 50 of 0.08 mg mL À1 for renin, and 0.23 mg mL À1 for ACE. 27 Other reported plant extracts that inhibit these enzymes were prepared from Vernonia amygdalina (IC 50 of 0.513 and 0.413 mg mL À1 for renin and ACE, respectively), 15 green tea (IC 50 of 0.48 mg mL À1 for renin 16 ), and green soybean (protease hydrolysate; IC 50 0.14-1.14 mg mL À1 (ref. 35) for ACE). Contrary to these other sources, tree peony petals could be regarded as an important source of natural renin and ACE inhibitory substances. In this study, aer comparing the inhibitory activity among owers of different colours, the results indicated that red, black, purple, green, and yellow tree peony petals had the highest inhibitory activity. Moreover, petals in the bud stage had superior inhibitory activities than those in full bloom.
3.2 Total antioxidant capacity in tree peony petals of different colours and owering stages DPPH, ABTS, and FRAP analyses were used to evaluate the antioxidant capacity of samples. The antioxidant activities of tree peony petals of different colours and red tree peony 'Hong Taiyang' at different owering stages were listed in Fig. 3. The iron ion reduction ability of tree peony petals of different colours ranged from 16.66 mg TE per g DW to 150.77 mg TE per g DW (9-fold difference). The strongest iron ion reduction capacity was 150.77 mg TE per g DW for Black 'Wujin Yaohui'   The seven tree peony petals of different colours showed a certain degree of antioxidant capacity, but the difference was signicant. The antioxidant capacity order was black > purple > green > yellow > red > pink > white. For red tree peony 'Hong Taiyang', this capacity decreased from bud compaction to full bloom. Roses with an important antioxidant capacity have also shown their best antioxidant activity at the bud stage. 36 The antioxidant properties of tree peony petals are attributed to phenolics and avonoids. 21 In the few articles that have also investigated the antioxidant capacity of these owers at different colours, their results indicated that dark petals had signicantly higher values than that of light petals, 21,37 which agrees with our ndings. Given the high antioxidant activity found in tree peony petal extracts, they can be regarded as valuable natural antioxidant sources, and be even applied to food, fragrance, and cosmetic products. It should be noted that the antioxidant capacity of the samples was positively correlated with the inhibition activity over renin and ACE. As displayed in Table 1, the correlation coefficients for inhibition of renin with DPPH, ABTS, and FRAP were 0.98, 0.95, and 0.94, respectively. The correspondent three values for ACE were 0.96, 0.99, and 0.96.

TPC and TF in tree peony petals of different colours and owering stages
The TPC and TF in tree peony petals of different colours and red tree peony 'Hong Taiyang' petals at different owering stages were shown in Fig. 4. The content of phenolic compounds (mg g À1 ) in aqueous extracts was expressed in gallic acid equivalents (GAE   The content of phenols in tree peony owers was relatively high compared to other plants. 38 The phenolic compounds in peony owers were antioxidant, anti-inammatory, antibacterial, and prolong the life of nematodes. 21,37,39 Various studies have determined that owers rich in phenolic compounds can inhibit some chronic diseases. 40,41 It is worth noting that renin and ACE inhibitory activities have been detected in polyphenols extracted from soybeans (saponins), 14 black and oolong tea, 16 tomato, 17 and avonoids from rosebuds. 18 These studies denote that polyphenols can be regarded as potential renin and ACE inhibitors. In this study, we found signicant differences among tree peony petals of different colours regarding renin/ ACE inhibition capacities and antioxidant activities. All these activities showed signicant correlations with polyphenol content ( Table 1). The correlation coefficients between total phenol content and renin or ACE inhibition were 0.95 and 0.96, respectively, while the correlation values between total phenol content and DPPH, ABTS free radical scavenging capacity, and iron ion reduction ability were 0.98, 0.98, and 0.99, respectively. These ndings support polyphenols as relevant renin and ACE inhibitors.

Conclusions
This study found that aqueous extracts of red, white, yellow, green, pink, purple, and black tree peony petals had a certain degree of inhibitory effect against renin and ACE. However, the inhibitory activity was signicantly different among colours; the order of strength was: black > purple > green > red > yellow > pink > white. In red 'Hong Taiyang' petals, the inhibitory activities against renin and ACE decreased from bud compaction to the full opening stage. In addition, a certain degree of antioxidant capacity was detected in the evaluated aqueous extracts, and the samples with higher renin and ACE inhibitory activities also exhibited greater ABTS, DPPH free radical scavenging ability, and iron ion reduction ability. Notably, all the functional activities mentioned above were positively correlated with total phenol content (p < 0.01). Our ndings evidenced that the phenolic presented in tree peony petals was the main active component responsible for the difference in renin and ACE inhibitory activities. In conclusion, this work provided a broader range of options for the development of renin and ACE inhibitors and a richer theoretical basis to further improve the functional value system of tree peony. For this purpose, it seems necessary to realize its high-value utilization and promote the development of an industry. In the future, more active inhibitors of renin and ACE could be further explored, isolated, puried and identied from aqueous extracts of tree peony petals.

Conflicts of interest
The authors declare that they have no conicts of interest.