Identification of bioactive compounds and antioxidant activity in leaves and fruits of Actinidia arguta accessions from northeastern China

Actinidia arguta (Sieb. et Zucc.) Planch. ex Miq. is abundant of vitamin C and bioactive compounds with high antioxidant activities. In this study, eight wild A. arguta accessions from different areas in Northeast China were collected. Some bioactive compounds were examined on the different tissues of different germplasms including four kinds of leaves, petioles and fruits. The method of UPLC-MS was used to detect the flavonoid compounds. The results showed that some bioactive compounds including vitamin C, soluble sugar, free amino acid, total phenolics and flavonoids content showed significant differences between six tissues of A. arguta accessions and showed significant variability with maturity. In eight accessions, the highest vitamin C content was found in young apical leaves of ‘CBS-6’ (7.47 mg/g fresh weight), and the highest soluble sugar content was in fruits of ‘CJ-1’ (196.52 mg/g fresh weight) and the highest total phenolic content and total flavonoids content were in young apical leaves of ‘CBS-11’ (3.48 mg/g fresh weight) and of ‘CBS-3’ (2.00 mg/g fresh weight), respectively. Ten flavonoid compounds including kaempferol, isorhamnetin and quercetin were detected in leaves, petioles and fruits. The total content of flavonoids were highest in young apical leaves (10219.84 μg·g-1) and the lowest in fruits (78.75 μg·g-1). Based on the comparison of the contents of several bioactive compounds, the two accessions ‘CJ-1’ and ‘CBS-8’ had relatively outstanding performance, and in the comprehensive evaluation of the antioxidant activity among different tissues, the young leaves had the strongest antioxidant activity. These results highlighted the antioxidant potentialities of A. arguta leaves as a major source of phenolics and vitamin C as well as flavonoids. It provided a theoretical basis for the utilization of leaves of A. arguta.

Kiwifruit has been recorded in China for more than 2,000 years. It has rich nutritional value, strong protective activity and good medicinal value [10][11][12]. In recent years, many of the previous studies have focused on the fruits of A. arguta [13][14][15] and kiwifruit [16], their fruits have a high antioxidant capacity [17,18] and are also an important source of bioactive substances [19], such as high levels of vitamins, phenolics and flavonoids, etc. Some researchers have been studying flavonoid metabolites and other polyphenol content and evaluated the nutritional values of A. arguta fruits [20,21]. Yu et al. investigated the flavonoid compounds in A. chinensis and A. arguta fruits and identified a total of 125 flavonoids and 39 metabolites in two kiwi berries, which indicated that flavonoid components were abundant in kiwifruit and kiwi berry [22]. Although the bioactivity and antioxidant activity of the A. arguta fruit has been well characterized [4,23], there have been few studies investigating the bioactivity of A. arguta leaves.
A. arguta leaves are accumulated during the pruning each year, which are an affordable and abundant raw material as a kind of byproducts of farming. As early as 1977, A. arguta was recorded that it could be used as a medicinal food homologous plant. Its roots and leaves had the effects of clearing heat and promoting diuresis and strengthening stomach [24]. Cyboran et al. evidenced the effect of A. arguta leaf extract which could be used as an effective natural antioxidant protecting the body against external oxidizing agents and also be food additives or dietary supplements against food aging from oxidation-induced. So they are expected applicated in the cosmetic, medicine, and food industries because of rich bioactive compounds. [25]. Webby identified a new flavonol triglycoside and isolated the kaempferol analogues in leaves of A. arguta [26]. Almeida et al. evaluated the leaves of A. arguta regarding antioxidant and antimicrobial activity, as well as radical scavenging activity for the first time, identified and quantified phenolic compounds [19]. In this study, eight wild A. arguta accessions from different areas were collected. They were growing strong, six of them were female plants, the fruits were all green, and the quality was good, and there were two male accessions, with a large amount of blooms and concentrated flowering period. Using LC-MS/MS and a series of physiological and biochemical substances to determine the leaves at different developmental stages of eight accessions, it was expected that new germplasm could be screened out, which would lay the foundation for the utilization of leaves of A arguta.

Vitamin C (Vc) content
Overall, the Vc content in the eight A. arguta accessions leaves at different stages of maturity performed significant differences. The Vc content in young tissues (eg. YAL, P and QL) was slightly higher than that of old tissues (eg. HL and ML), and declined with leaf maturity. A continuous decrease was observed in Vc content of eight accessions leaves as leaf maturity (from YAL to ML), such as 'CJ-1' where it ranged from 7.25 to 3.44 mg/g fresh weight from young apical leaves to mature leaves, as well as 'HY-1', 'BYS-13', 'CBS-8', etc. Interestingly, the Vc content in the petioles of young leaves was similar to that in young leaves, and three accessions were even higher than those in young leaves, such as 'BYS-13', 'BYS-5' and 'CBS-11'. On the other hand, Vc content in fruits was lower than those of immature leaves and petioles and higher than mature leaves. By comparing the Vc content of leaves, petioles and fruits of the eight accessions in Figure 1, it was found that the Vc contents in YAL leaves and fruits of 'CBS-6' were the highest among the eight accessions, being 7.47 mg/g and 4.47 mg/g fresh weight, respectively, followed by 'CBS-3' and 'CJ-1', while 'BYS-13' was the lowest.

Soluble sugar content (SSC)
The results of the SSC in six tissues of the eight accessions confirmed that the highest content was in the fruits, followed by the leaves, and the petioles had the lowest content (Fig. 2). While, the SSC in the leaves of each accession mostly increased with the gradual maturity of the leaves and the highest content of leaves was in mature leaves (ML). In 'CJ-1', the SSC in mature leaves (ML,156.85 mg/g) was lower than that in fruits, higher than that in other tissues, and 61.88% higher than that in petioles. The highest SSC (196.52 mg/g fresh weight) was obtained from the mature fruits of 'CJ-1'. The other five female accessions were similar to 'CJ-1', the highest SSC was in fruits and the lowest content in petioles. In male accessions, the highest SSC was in mature leaves. On the other hand, by comparing the SSC of the eight accessions in Figure 2, the content of 'CJ-1', 'HY-1', and 'CBS-6' in different tissues was relatively higher than the other five accessions, and 'CBS-11' was the lowest.

Free amino acid (FAA) content
The FAA content in young tissues (eg. YAL, P and QL) was significantly higher than that of old tissues (eg. HL and ML), and declined with leaf maturity, the lowest content was in fruits. In 'CJ-1', the content of FAA in petiole was the highest, which was 1.55 mg/g fresh weight, extremely higher than that in other tissues. The lowest content was observed in mature leaves and fruit (ML and F of 'CJ-1', 0.14 mg/g fresh weight), which was also the lowest among the eight accessions. Furthermore, it was found that the highest content was in QL of 'BYS-13' with 1.69 mg/g fresh weight, which was also the highest among the eight accessions. By comparing the FAA content of the eight accessions in Figure 3, it was found that the contents in P of 'CJ-1', and in YAL and QL of 'BYS-13' were higher than the other six accessions, while there was no significant difference among other six accessions.  Figure 3. Comparison of free amino acid content of different tissues in eight A. arguta accessions.

Total phenolic content (TPC)
A continuous decrease was observed in TPC of the eight accessions in leaves as leaf maturity (from YAL to ML). While the highest content was in fruits of six female accessions, and the values in petioles of each accession were the lowest. In 'CJ-1', the TPC in fruits was higher than that in leaves and petioles with significant differences, that in petioles was the lowest (0.37 mg/g fresh weight), and the highest content in fruits was 2.96 mg/g fresh weight, the difference was eight times. By comparing the TPC of the eight accessions in Figure 4, it was found that the contents of different tissues in 'BYS-5' were the highest in fruits (3.25 mg/g fresh weight), while 'BYS-13' were lower than the other accessions, meanwhile the highest value (3.48 mg/g) among the eight accessions was observed in the YAL of 'CBS-11'.  Overall, the TFC in the eight A. arguta accessions leaves performed continuous decrease with the leaf maturity. The TFC of the young leaves were higher than that of the old leaves, while those in petioles were higher than fruits, but lower than leaves among the eight accessions, the lowest content was in fruits. The TFC of YAL in the eight accessions were highest, followed by QL, HL and ML. When compared to ML among the eight accessions, the TFC of YAL was higher from 1.83-fold to 3.41-fold. In 'CJ-1', the TFC in different tissues were in the order of leaf>petiole>fruit, and the content in YAL (1.83 mg/g) was 6.3 folds higher than that in fruit (F, 0.29 mg/g). By comparing the TFC of the eight accessions ( Figure 5), it was found that there was no significant difference among the eight accessions; 'CJ-1' 'CBS-8' and 'CBS-3' were slightly higher than the other six accessions.

Antioxidant activity determinations
2.2.1. ABTS free radical scavenging assay It was observed that ABTS radical scavenging activity in the eight A. arguta accessions leaves performed continuous decrease with the leaf maturity. The ABTS radical scavenging activity of the young leaves were higher than that of the old leaves, and higher than that of petioles and fruits, the lowest values were in fruits. Interestingly, ABTS radical scavenging activity in the petioles of young leaves was almost the lowest not similar to that in young leaves. The ABTS radical scavenging activity of YAL in the eight accessions were highest, followed by QL, HL and ML, such as 'CJ-1' where it ranged from 41.51 to 14.90 mmol/g fresh weight from young apical leaves to mature leaves, as well as 'HY-1', 'BYS-13', 'BYS-5' and 'CBS-6', were also showed similar trend. On the other hand, in 'CJ-1', except for ML, the ABTS radical scavenging activity of leaves was greater than that of petioles and fruits, and the ABTS radical scavenging activity of YAL (41.51 mmol/g) was 2.5 times the fruit (16.65 mmol/g). By comparing ABTS radical scavenging activity of the eight accessions ( Figure 6), it was found that 'CJ-1' was slightly higher than the other accessions and the lowest was 'BYS-13'. The highest value in fruits was in 'CBS-3', that was 18.51 mmol/g.

DPPH free radical scavenging assay
It was observed that DPPH radical scavenging activity in the eight A. arguta accessions leaves showed continuous decrease with the leaf maturity as similar to that of ABTS radical scavenging activity. The DPPH radical scavenging activity of the young leaves and petioles was higher than that of the old leaves. While the values in fruits were only higher than mature leaves. The DPPH radical scavenging activity of YAL in eight accessions was highest except 'BYS-13', 'CBS-3' and 'CBS-8', followed by QL, HL and ML, such as 'CJ-1' where it ranged from 91.22 to 71.29 µmol TE/L from young apical leaves to mature leaves. In 'CJ-1', the DPPH radical scavenging activity of YAL (91.22 µmol TE/L) was 1.3 times than the fruit, and the value of fruits (71.29 µmol TE/L) was only higher than ML (65.60 µmol TE/L). By comparing DPPH radical scavenging activity of leaves and petioles with different maturity of the eight accessions (Figure 7), it was found that 'CJ-1' was slightly higher than the other accessions, while there was no significant difference among these accessions.

Correlation between bioactive compounds and antioxidant activity
To identify the relationship between the main bioactive substances in the leaves of A. arguta and their relationship with the ability to scavenge free radicals, a correlation analysis was done and the results were presented in Table 1. The Vc content and the soluble sugar content had significantly The results showed that Vc content, FAA content as well as TPC and TFC were significantly positively correlated with DPPH free radical scavenging rate (r2=0.989, 0.952, 0.986, 0.987, respectively; p<0.05), while significant negative correlation was found between soluble sugar content and DPPH free radical scavenging rate (r2 = -0.967; p<0.05). A strong positive correlation was found between TPC and ABTS free radical scavenging rate (r2=0.956; p<0.05). Regarding the ABTS and DPPH radical scavenging activities, the correlation determined was positive (r2=0.970; p<0.05).

Analysis of Flavonoids Compounds Using LC-MS/MS
By LC-UV-MS analysis, the composition of flavonoids in A arguta could be identified by the UV-vis spectrum, elution order, retention time and MS fragmentation pattern by reference with published data [27]. A total of ten peaks were detected in the flavonoid extract of A. arguta, which were identified by the retention time in LC-MS/MS system, elution order, λmax in the visible region, main MS 2 fragments and molecular ion ( Figure S1). Some components had similar absorption UV spectra. The maximum absorption peaks were at 240-280 nm and 330-380 nm. Therefore, it was inferred that these components belonged to the flavonol glycoside compound ( Figure S1). As shown in Figure S1 (Table 2). It was preliminarily determined to be aglycone components belonging to isorhamnetin, quercetin and kaempferol. From the above information, it could be inferred that the compound was replaced by one glycoside and two glycosides. On the basis of the UV-vis spectrum, retention time, main MS 2 fragments and molecular ion, F1, F2 and F3 were tentatively established as Kaempferol The content of ten flavonoid compounds in different tissues of 'CJ-1' was quantitatively analyzed (Table 4). Quercetin -3-O-rhamnoglycoside was among the highest content of total flavonoids in the five tissues, accounting for 32.62% in YAL, 29.71% in QL, 31.58% in ML, 58.95% in P, and 56.55% in F, respectively. Kaempferol, isorhamnetin, and quercetin compounds were found in leaves (YAL, QL, HL and ML), petioles (P) and fruits (F). The highest level of flavonoid accumulation was detected in leaves, and flavonoids in P and F were 151.63 µg·g -1 and 78.75 µg·g -1 , respectively. It varied significantly in different types of leaves, which was 10219.84 µg·g -1 , 4977.83 µg·g -1 , 3986.94 µg·g -1 and 350.55 µg·g -1 in YAL, QL, HL and ML, respectively. The highest total flavonoid content was found in YAL and the flavonoid in YAL, which was 29.2 folds of that in ML, 67.4 folds of that in P and 129.8 folds of that in F. On the other hand, it was interesting to find that the concentration of some flavonoids from different tissues, such as isorhamnetin-3-O-glucoside, kaempferol-3-O-Rutinoside and quercetin-3-O-glucoside, showed significant differences in different maturity. Quercetin compounds were the most widely distributed in A. arguta (Table 3). However, the levels of isorhamnetin and kaempferol compounds were higher than those of quercetins in HL. The content of isorhamnetin compounds in YAL was the highest followed by quercetin and kaempferol compounds. Among the flavonoids in QL, kaempferol compounds were present at the highest level, followed by isorhamnetin and quercetin compounds (relatively low). The content of isorhamnetin compounds was the highest in HL, followed by kaempferol and quercetin compounds. The content of isorhamnetin compounds was highest in ML, followed by quercetin and kaempferol compounds. However, the levels of quercetin compounds were the highest in P and F, followed by isorhamnetin and kaempferol compounds.

Plant Materials
Eight A. arguta accessions were collected from Northeast of China, which were A. arguta cv.
No       water, followed by Folin-Ciocalteu reagent (250 µL) and then 7% sodium carbonate (2.5 mL) was temperature for 1.5h. The absorbance at 760 nm was recorded using spectrophotometer. The gallic acid was used as standard.

Total flavonoids content (TFC)
added every 6 min, respectively. The distilled water was used to make total volume to 10 mL, which 113 kept for 10 min. The absorbance at 510 nm was recorded using spectrophotometer. The rutin was used 114 as standard. The result was represented as mg /g fresh weight (mg/g . FW).