Measurement and Analysis of Calcium Content in Wild Chinese Dwarf Cherry ( Prunus humilis ) Fruits by Inductively Coupled Plasma-optical Emission Spectrometer

Different forms of calcium in wild Chinese dwarf cherry (Prunus humilis) fruits (two genotypes, smallfruit-type and big-fruit-type) were sequential extracted and measured by Inductively Coupled Plasma-Optical Emission Spectrometer (ICP-OES). The calcium content variation after postharvest storage was also studied, to provide the theory basis for the active nutritional ingredient evaluation of Chinese dwarf cherry (Prunus humilis). The results showed that, wild Chinese dwarf cherry (Prunus humilis) fruits in mature stage were rich in calcium nutrition element and mainly composed by water soluble calcium and calcium phosphate which absorbable to human. Absorbable calcium (including water soluble calcium and calcium phosphate) content in small-fruit-type (63.79%) was significantly higher than that of big-fruit-type (54.97%). After postharvest storage, the water soluble calcium content was increased while the calcium pectate and calcium oxalate content dropped. Especially to the bigfruit-type, the absorbable calcium content increased to 75.49% compared with just picking 54.97% (small-fruit-type increased from 63.79% to 68.86%). The study suggested that Chinese dwarf cherry (Prunus humilis) was a natural and biological calcium supplement to people; meanwhile, small-fruit-type was more suitable, especially after postharvest storage.


INTRODUCTION
Chinese dwarf cherry (Prunus humilis) is a kind of Chinese endemic wild species, which extensively distributed in north of China (Wang et al., 2005;Zhao et al., 2010).Their seeds are widely used in medicine as laxatives, diuretic and detumescence.Their fruits are rich in sugar, amino acid, vitamin C and several kinds of trace elements, especial the calcium element (Cao et al., 1999;Ma et al., 2007).It has been reported that the calcium content in Chinese dwarf cherry (Prunus humilis) is higher than many other fruits (Matkovic and Heaney, 1992;Weaver, 2000;Cashman, 2002).
It is well known that calcium as the essential element to human body is closely related to growth and development.So, we took the Chinese dwarf cherry (Prunus humilis) as materials to study the different forms of calcium contents in mature fruits as well as the calcium contents variation after postharvest storage, to provide a scientific basis for their further development and utilization.
Ultrapure water (18 mΩ) was obtained from MILLI-Q Biocel ultrapure water instrument (Millipore Company/America).A series of standard solution 0, 25, 50, 100 mg/L were prepared by 2% HNO 3 and 1000 mg/L Ca standard solution (provided by China national standard research center).All the vessels used were firstly soaked with 10% nitric acid solution for 24 h, ultrapure water flushed 3-4 times and dried.
Materials: Chinese dwarf cherry (Prunus humilis) were obtained from the wild population of YanShan mountains in Hebei province, north of China, about ten years old.According to the morphological differences, they can be divided into two genotypes, small leaf and small fruit type (small-fruit-type for short) and big leaf and big fruit type (big-fruit-type for short).The former leaves light green, single fruit weight about 5 g, the latter leaves dark green and single fruit weight about 10 g.

Sample preparation:
In maturation period (13 th August), randomly collected fully ripened Chinese dwarf cherry (Prunus humilis) fruits without mechanical wound and diseases.Each genotype selected three plants, each plant collected 45 fruits, repeated 3 times and then stored at room temperature about 25°C.In 13 th (picking day), 21 st (stored for a week) and 29 th (stored for two weeks), tested calcium content, respectively.
Different forms calcium was sequentially extracted according to Konishi and Kasai (1963) and improved as: deionized water, 1 mol/L NaCl, 0.348 mol/L CH 3 COOH and 1.625 mol/L HCl as extraction solution to extract water soluble calcium, calcium pectate, calcium phosphate and calcium oxalate, respectively.After smashed, accurately weighed about 10 g homogenate, mixed with the solvent mentioned above in a centrifuge tube and kept in a thermostat water bath about 25°C for 1 h, then centrifuged under 6000 r/min for 10 min.The supernatant solution was transferred to a 100 mL volumetric flask.The precipitate was washed by the extracted solvent 3~4 times, then mixed the washing liquid with supernatant solution, added some SrCl 2 solution to exclude the interference factor, at last added extracted solvent to 100 mL.The residue which the main component was calcium silicate (referred as residual calcium), was acid digested by the HNO 3 :HClO 4 (V: V = 1: 5).The calcium content was measured by the ICP-OES.All the data were analyzed by the SPSS software.

CONCLUSION
Chinese wild dwarf cherry (Prunus humilis) fruits in mature stage were rich in calcium nutrition element and mainly composed by water soluble calcium and calcium phosphate.The calcium content especial the absorbable calcium content in small-fruit-type (63.79%) was significantly higher than that of big-fruit-type (54.97%).After storage, the absorbable water soluble calcium content was increased while the calcium pectate and calcium oxalate contents dropped.Especially to the big-fruit-type, the absorbable calcium content rose to 75.49% compared with just picking 5 4 .97 % (small fruit increased to 68.86% from 63.79%), moreover calcium and phosphorus ratio is suitable (Guo and Wang, 2010).Therefore, after storage, Chinese dwarf cherry (Prunus humilis) fruits are appropriate for children and elder people to supplement calcium.
Fig. 1: The composition percentage change of diverse form calcium of Prunus humilis large fruits during storage period

Fig. 2 :
Fig. 2: The composition percentage change of diverse form calcium of Prunus humilis small fruits during storage period

Table 1 :
The content of diverse forms calcium in Prunus humilis fruits (X±S.D.)