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The fruits of C. pinnatifida used in this study were Chuncheon 1, Chuncheon 8, Chuncheon 15, Jungsun and Poechun which were selected from Korea and Maban, Changgu, Keumsung and Daekeumsung which were Chinese cultivar. C. pinnatifida fruits grown in the National Institute of Forest Science (Suwon, Korea) were collected in October after mature.

The sugars content levels (sucrose, glucose, and fructose) in the samples were analyzed by a reported LC method with some modification (Godin et al., 2011). A total of 2g of freeze-dried hawthorn powders was extracted with 100mL of water in a cooled ultrasonic bath (40kHz) for 60 min. Then the extract was centrifuged (15,000 rpm, 4°C, 10 min) and the supernatant was separated with membrane filter (0.45 um) and transferred to vials. Samples were analyzed on a Dionex Ultimate 300 HPLC system. A Sugar-pak (waters, 300×6.5mm) column with deionized water as the mobile phase was used for separation of the sugars. The flow rate were optimized as 0.5mL/min and a Shodex RI-101 detector was used for identification. Quantification was performed on the basis of linear calibration plots of the logarithm of peak areas versus the logarithm of concentrations. The concentrations were expressed in grams per 100g of distilled water (DW). The summary of HPLC conditions used for free sugar analysis in this study were shown in Table 1.

The freeze-dried hawthorn powders of 3g were transferred to Eppendorf tube with 5 ml of distilled water. Then the extract was centrifuged (15,000 rpm, 4°C, 10 min) and the supernatant was separated with membrane filter (0.45 um) and transferred to vials. Organic acids in hawthorn fruit were analyzed by a Dionex Ultimate 300 HPLC system. The chromatographic separation used for organic acid detection employed 0.01 N H₂SO₄ as the mobile phase, Aminex 87H (300×10mm) column (Bio-Rad, USA) with a flow rate of 0.5ml/min, samples organic acids were detected with a RI detector (ERC, RefractoMax, Japan).

Sweetness index in this study was calculated based on the amount and sweetness properties of individual carbohydrate in fruit (Keutgen and Pawelzik, 2007). The contribution of each carbohydrate was calculated, based on the fact that fructose is 2.30 and sucrose 1.35 times sweeter than glucose. Accordingly, sweetness index=1.0 (glucose) + 2.30 (fructose) +1.35 (sucrose).

All experiments were performed at least three times and the results were reported as mean±SD. Data were statistically evaluated by one-way ANOVA analysis and Duncan’s multiple range test. Results of tables and figure were presented as mean±standard deviation.

Generally, the sugar composition is one of the most important parameters for food energy level. In Table 2, we listed the HPLC quantitative analytical data of the free sugars in the hawthorn fruit. The fruits of five different hawthorn clones selected from Korea and four Chinese cultivars were used in this study. Glucose, sorbitol and sucrose were the major sugar components of the hawthorn fruit.

Glucose contents are between 48.90 and 179.34g/100g, and sucrose contents are between 188.12 and 0.00g/100g, respectively. The highest sucrose content of hawthorn fruit was 188.12g/100g in Daekeumsung cultivar.

Because the relationship between fructose and glucose content was about 1:1, which is typical for berry fruit, (Keutgen and Pawelzik, 2007) and the same results were observed in this study. It is well known that fruit sweetness depends not only on the content of each sugar, but also on the ratios of the main individual sugars (Zheng et al., 2016). It is also found that the sweetness index, which plays important role of taste in hawthorn fruit (Fig. 1).

Fig. 1. 
Sweetness index of the fruit of hawthorn selected clones and Chinese cultivars. (CH1: Chuncheon 1, CH8: Chuncheon 8, CH15: Chuncheon 15, JS: Jungsun, PC: Poechun, MB: Maban, CG: Changgu, KS: Keumsung, DKS: Daekeumsung).

The highest sweetness of hawthorn fruit was 579.52 in Poecheon clone while the lowest sweetness of hawthorn fruit was 285.59 in Chuncheon 8 clone.

Hawthorn fruits have long been used in traditional Korean, Chinese and European medicine, and are widely consumed as food, in the form of juice, drink, jam and canned fruit. Due to the significant amounts of various organic acids, including caffeic acid, malic acid and tartaric acid, contained in the hawthorn fruits (Gao et al., 1995), the components in hawthorn preparations, such as hawthorn drink, might exist in acidic condition.

Organic acids represent further compounds contributing to taste and flavour of hawthorn fruit. Three major organic acids in hawthorn fruit, citric acid, malic acid, and shikimic acid, were detected by HPLC. The organic acids of the fruit of hawthorn clones and Chinese cultivars are shown in Table 3. The results showed that citric acid showed was the main organic acid in hawthorn fruit and followed by malic acid and shikimic acid. While, Albertini et al. (2006) reported that citric acid dominated in lemons, limes and orange, the malic acid was the main organic acid in hawthorn fruit. Citric acid content in Poechun clone fruit was the highest as 157.50g/100g. Citric acid can be used for proper mineral supplementation of food and as a flavoring ingredient. It is also reported that citric acid ensures proper functioning of the kidneys and prevents the formation of kidney stones. Recently, it is found that organic acids have been used in food preservation and as a new generation of growth promotors instead of antibiotics (Kim and Rhee, 2015). Shikimic acid was the third-most common organic acid, and occupied 0.67~0.22g/100g according to clones and cultivars. A similar trend was reported for strawberry and jujube fruit (Keutgen and Pawelzik, 2007; Park and Kim, 2016). Malic acid contents in Daekeumsung cultivar was the highest as 34.12g/100g.

In this study, we analyzed the free sugars and organic acid using HPLC. To our knowledge, this is the first study investigating the content of free sugars and organic acids in the fruit of five different hawthorn clones selected from Korea and four Chinese hawthorn cultivars. From the results of this study we found that the glucose sorbitol and sucrose were the major sugar components of the hawthorn fruit. The sweetness index was also calculated from the content of free sugars. The highest sweetness of hawthorn fruit was 579.52 in Pocheon clone while the lowest sweetness of hawthorn fruit was 285.59 in Chuncheon 8 clone.

Three major organic acids in hawthorn fruit were citric acid, malic acid, and shikimic acid. The highest citric acid and malic acid content in hawthorn fruit were 157.50g/100g (Poechun clone) and 34.12g/100g (Daekeumsung cultivar), respectively. The results of this study would be helpful for using food and functional food products, due to the beneficial effects of free sugar and organic acid for human health such as antioxidants and anticarcinogenic properties.