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AbstractIn order to investigate the effect of dwarfing inter stock on the structure of pear trees, investigations were made to the tree and group structure of the 4yearold spindleshaped ‘Huangguan’pear grafted with vigorous stock (Pyrus betulaefolia stock) and that grafted with the dwarfing inter stock ‘Zhong ai 1 hao’ (P. betulaefolia rootstock). The results showed that the 4yearold‘Huangguan’pear trees grafted with the dwarfing inter stock ‘Zhong ai 1 hao’ were 2.87 m high on average, with 70.70% of short braches, converted into 21 016.0 branches per 667 m2, 840.64 m2 of leaf area per 667 m2 with the leaf area coefficient of 1.75. On the other hand, for the ‘Huangguan’ trees grafted with vigorous stocks, the average tree height was 3.17 m, and the short branch proportion was 63.20%, converted into 15 806.4 branches per 667 m2, 719.28 m2 of leaf area per 667 m2 with the leaf area coefficient of 1.55. Therefore, the use of dwarfing inter stock ‘Zhong ai 1 hao’ had significant dwarfing effect on‘Huangguan’pear trees, which also showed significant yield increasing effect.
Key wordsPear; ‘Zhong ai 1 hao’ ; Spindleshaped; Tree structure
Received: June 20, 2018Accepted: November 7,2018
Supported by the China Agriculture Research System (Pear) (CARS2836); the National Natural Science Foundation of China (31601708); the Agricultural Stock Breeding Project of Shandong Province (2016LZGC034); the Youth Foundation of Shandong Academy of Agricultural Sciences (2015YQN40); the National Science and Technology Plan for Rural Areas of China (2014BAD16B034); the Project for Agricultural Science and Technology Innovation of Shandong Academy of Agricultural Sciences (CXGC2018F03); and the Key Science and Technology Innovation Project of Shandong Province (2018CXGC0208).
Shuwei WEI (1981-), male, P. R. China, associate researcher, devoted to research about pear cultivation and breeding, Email: weisw2007@163.com.
* Corresponding author. Shaomin WANG (1962-), male, P. R. China, researcher, devoted to research about fruit cultivation and breeding, Email: wsm@sdip.cn.
China is the largest pear producing country in the world, with both its planting area and fruit yield ranking the［1］. The tree structure and group structure of fruit trees are the main factors affecting orchard yield and quality. Dwarfing and compact planting is a kind of fruit culture technique to dwarf the trees and shorten the planting spacing by grafting dwarfing rootstocks (or dwarfing inter stock) to the selected dwarfing varieties (varieties with short branches) on the basis of manual dwarfing measures, thereby obtaining early high yield. It has become the general trend for the production and development of fruit trees in China. Currently, the use of dwarf stocks (or dwarf inter stocks) has become the main way to achieve the dwarfing and compact cultivation of pear trees. ‘Zhong ai 1 hao’ is a compact dwarfing stock selected from the natural seedlings of Jinxiang pear by Jiang et al.［2］ in 1980. Used as the dwarfing inter stock, it can make the cultivated pear varieties have dwarfing plants, early fruitsetting and early high yield. In this paper, investigations were made to the densely planted spindleshaped ‘Huangguan’ pear orchard which used ‘Zhong ai 1 hao’ as the inter stocks, with the aim to clarify the individual and group tree parameters of ‘Huangguan’ pear with the dwarf ‘Zhong ai 1 hao’ as the inter stocks for the dense planting of spindleshaped ‘Huangguan’ pear trees, thereby providing references for the dwarf and compact planting of pear trees. Materials and Methods
Survey orchard overview
In 2017, investigations were conducted at the Jinniushan Experimental Demonstration Base of (Shandong Institute of Fruit Research. The soil nutrient status of pear orchard was shown in Table 1, and the orchard was under routine management. The 4yearold ‘Huangguan’ pear trees were selected from the pear orchard in the base for the investigation and study, and all were of spindleshaped tree bodies. The grafting stocks were all Pyrus betulaefolia. The planting spacing for dwarf ‘Huangguan’ pear trees was 0.75 m×3 m. ‘Zhong ai 1 hao’ was used as the inter stocks with the stock length of 20-25 cm. the planting spacing for vigorous stocks of ‘Huanguan’ pear trees was 1 m×3 m. The test pear orchard had moderate management level. In 2017, the percent rate of high quality fruit was over 90% for both the dwarfing stocks and vigorous stocks of ‘Huangguan’ pear. The trees with similar loads and tree shapes were selected for the investigation, and there were 5 plants in a plot with 3 repetitions.
Survey indicators and methods
Tree structure parameters included both the basic structure parameters of tree height, trunk height, trunk girth, crown diameter form south to north, crown diameter from east to west, central trunk numbers, main branch numbers, main branch opening angle, but also the number of short, medium and long branches and their proportions, as well as the growth conditions of new shoots and leaves like the number of spring shoots and their length, the number of leaves on spring shoots, leaf number per plant, single leaf area, leaf area per plant. Group structure parameters included the parameters related with crown overlapping rate (between plants and lines), leaf area coefficients.
Tree height was measured by the sign post. The trunk girth was the perimeter of the trunk at 20 cm above the ground, and the trunk height was the vertical height from the ground to the 1st main branch of the first layer, both of which were measured with a tape. The crown diameters were the lengths of crowns from the south to the north and from the east to the west, also measured with a tape. The growth of new shoots was measured by randomly selecting 30 new shoots around the crown of each plant, the lengths of which were measured with a tape, and then the average length was calculated. Single leaf area=Leaf length×Leaf width÷2; Leaf area per plant=Single leaf area×Number of leaves per plant; Projected area of crown per plant=(π×North-south crown diameter×East-west crown diameter)÷4; Leaf area coefficient=Leaf area per plant÷Projected area of crown per plant; Orchard coverage (%)=(Projected area of crown per plant×Total number of plants)÷Total planting area×100; Crown overlapping rate (%)=(Single plant crown diameter-Average plant spacing)÷Average plant spacing×100. Table 1Soil nutrient status of the test orchard
Soil layercmpH valueOrganic matterg/kgTotal Ng/kgAvailable Kmg/kgAvailable Pmg/kgAvailable Bmg/kgAvailable Cumg/kgAvailable Femg/kgAvailable Znmg/kg
0-257.2816.131.10219.0049.000.666.0111.102.34
25-507.308.670.90156.0014.000.432.307.121.01
Data analysis
Data analysis was completed with Office Excel.
Results and Analysis
Effect of ‘Zhong ai 1 hao’ on the height and crown diameter of ‘Huangguan’ pear trees
The investigation showed that with ‘Zhong ai 1 hao’ as the inter stocks, the 4yearold spindleshaped ‘Huangguan’ pear tree had a tree height of 2.87 m, trunk height of 0.50 m, trunk girth of 17.0 cm, southnorth crown diameter of 1.15 m, easteast crown diameter of 1.58 m. The 4yearold ‘Huangguan’ pear tree grafted with vigorous stocks had a tree height of 3.17 m, trunk height of 0.54 m, trunk girth of 15.4 cm, southnorth crown diameter of 1.50 m, eastwest crown diameter of 1.78 m. Therefore, it could reduce tree height by using the dwarf inter stock ‘Zhong ai 1 hao’, which could also decrease the crown diameter (Table 2).
Table 2Effect of the inter stock of ‘Zhong ai 1 hao’ on the basic parameters of ‘Huangguan’ pear trees
CultivationpatternNumber ofplants per667 m2TreeheightmTrunkheightmTrunkgirthcmCrown diameterfrom southto north∥mCrown diameterfrom eastto west∥mMain branchesper plantnumberMain branchopeningangle∥°Compatibility (stockscionor rootstock withintermediate stock
Dwarfing stock2962.870.5017.01.151.588.085.0Good
Vigorous stock2223.170.5415.41.501.787.483.6Good
Effect of ‘Zhong ai 1 hao’ on the branch composition of ‘Huangguan’ pear trees
According to the investigation, there were 50.2 short branches in the 4yearold ‘Huangguan’ pear tree with ‘Zhong ai 1 hao’ as the inter stock, accounting to 70.70% of the total branch amount; 11.6 medium branches, accounting for 16.34%; 9.2 long branches, accounting for 12.96%. As for the vigorous stock grafted ‘Huangguan’ pear tree, there were 45 short branches, accounting fro 63.20% of the total branches; 9.6 medium branches, accounting fro 13.48%; 16.6 long branches, accounting for 23.31%. The ‘Huangguan’ pear tree grafted with dwarfing stocks and vigorous stocks had similar branch numbers per plant, but the branch numbers per 667 m2 of dwarf ‘Huangguan’ pear trees were much larger than those of the vigorous stocks grafted‘Huangguan’pear trees because there were more branches per 667 m2 on the dwarf‘Huangguan’pear trees (Table 3). Table 3Effect of the inter stock of ‘Zhong ai 1 hao’ on the branch composition of‘Huangguan’ pear trees
CultivationpatternNumber ofshortbranchesShort branchproportion%Number ofmediumbranchesMedium branchproportion%Number oflongbranchesLong branchproportion%Number offruitingbranchesFruiting branchproportion%Branchnumbersper plantBranchnumbersper 667 m2
Dwarfing stock50.270.7011.616.349.212.9632.041.4571.021 016.0
Vigorous stock45.063.209.613.4816.623.3142.459.8971.215 806.4
Effect of ‘Zhong ai 1 hao’ on the shoots and leaf growth of‘Huangguan’pear trees
According to the investigation, for the 4yearold‘Huangguan’ pear tree grafted with the inter stocks of ‘Zhong ai 1 hao’, the spring shoot was 17.40 cm long, the spring shoots number per plant was 43.8, leaf number per shoot was 9.80, leaf number per plant was 1 047, leaf area per plant was 2.84 m2. The converted spring shoots number per 667 m2 was 12 964.8, and leaf area per 667 m2 was 840.64 m2. In terms of the ‘Huangguan’ pear tree grafted with vigorous stocks, the spring shoot was 22.88 cm long, spring shoots number per plant was 52.6, leaf number per shoot was 7.62, leaf number per plant was 1 020, leaf area per plant was 3.24 m2. The converted spring shoots number per 667 m2 was 11 677.2, and leaf area per 667 m2 was 719.28 m2. Therefore, dwarf‘Huangguan’ pear tree had significantly higher converted spring shoot number per 667 m2, leaf number and leaf area than the ‘Huangguan’ pear tree grafted with vigorous stocks (Table 4).
(Continued on page 161)
Agricultural Biotechnology2019, 8（1）: 158-161
Inspection and Testing
Dengfeng ZOU et al. Determination of the Content of Kaempferol from Fermented Ginkgo (Ginkgo biloba L.) Leaves
Determination of the Content of Kaempferol from Fermented Ginkgo (Ginkgo biloba L.) Leaves
Dengfeng ZOU, Hua ZHU, Bowen JIANG, Xiaohua WANG*
Guilin Medical University, Guilin 541004, China
Abstract［Objectives］ This study was conducted to determine kaempferol content in ginkgo (Ginkgo biloba L.) leaves subjected to microbial fermentation.
［Methods］ Bacillus licheniformis was selected for solidstate fermentation of ginkgo leaves, and the content of kaempferol in ginkgo leaves was determined by RPHPLC method. At first, methanol was used to extract flavonoid glycosides, which were then hydrolyzed by hydrochloric acid solution. HPLC was performed with Platisil ODS column C18 (150 mm×4.6 mm, 5 μm) using mobile phase Vmethanol∶Vwater (0.4% phosphoric acid solution) = 55∶45 at a flow rate of 1 ml/min, and the eluate was detected with a shimadzu HPLC ultraviolet detector at 360 nm. ［Results］ With kaempferol as the reference substance, the correlation coefficient was 0.999 2 in the range of 0.001 06-0.016 96 g/L. The content in the fermented product was less than that in the nonfermented product by 28%.
［Conclusions］ The method is simple, accurate, and is suitable for determination of kaempferol. This study will provide an experimental basis for the development and utilization of ginkgo.
Key wordsGinkgo biloba L.; Ginkgo leaves; Microbial fermentation; Content determination; HPLC; Kaempferol
Received: August 29, 2018Accepted: November 22, 2018
Supported by Guilin Science and Technology Bureau Project (20100305); Guangxi "2011 Collaborative Innovation Center"Zhuang Yao Medicine Collaborative Innovation Center Project (G2013［20］).
Dengfeng ZOU (1975-), male, P. R. China, professor, devoted to research about natural medicine.
*Corresponding author. Email: 437593157@qq.com.
Ginkgo (Ginkgo biloba L.) is also known as Gongsunshu, "living fossil" and "panda in the plant world"［1］. The main active components of ginkgo leaves (dry leaves) are flavonoids and ginkgo terpene lactones from which more than 160 kinds of effective active components of ginkgo flavonols including quercetin, kaempferol and isorhamnetin have been found［2］. As to studies on ginkgo lactones, EGb761 is the patented ginkgo leaf extract medicine developed the earliest by Germany Schwabe company, and its production standard is still regarded as the international ginkgo leaf product standard［3］. In this study, Bacillus licheniformis was selected through fermentation method to be the test strain for the microbial solidstate fermentation of ginkgo leaf powder, and combined with the characteristic that the molecular structure of ginkgo flavonoids has strongly absorption at 360 nm, the current commonly used HPLC method with accurate determination results was selected for the determination of kaempferol content. In lactones, the structure of total flavonoids is as Fig. 1.
Instruments and Materials
Ginkgo leaves were collected from the Medicinal Plant Garden of Dongcheng Campus of Guilin Medical University, and identified as G. biloba leaves by associate professor Huang from Guilin Medical University.
Instruments: Super clean bench; autoclave; constant temperature incubator; constant temperature shaker; Shimadzu chemical station; ODS column (150 mm×4.6 mm, 5 μm). Reagents: Czapek–Dox medium; agar medium; methanol (analytical pure); chromatographically pure methanol, phosphoric acid, etc.
Strain: B. licheniformis, purchased from BeNa Culture Collection.
Fig. 1Structure of total flavonoids
Experimental Methods
Preparation of seed liquid
A ring of activated B. licheniformis was picked from the slant medium and then inoculated in a 250 ml flask (containing 50 ml of sterile seed liquid). The inoculated strain was cultured in a constant temperature shaker for 24 h, at 36 ℃, with shaking at 180 r/min.
Solidstate fermentation of ginkgo leaves［4］
The seed solution was transferred with a pipette to a flask containing the fermentation medium, followed by solidstate fermentation in a constant temperature incubator. The inoculum size, liquidsolid ratio, initial pH, fermentation temperature and fermentation time were 15%, 1.2∶1, 7.0, 28 ℃, and 48 h, respectively.
Preparation of test solution
After the fermentation was completed, the fermentation product was sterilized in an autoclave, and 2 g of each of the fermented and unfermented samples was accurately weighed with an electronic balance, respectively, and placed in a Soxhlet extractor, respectively. Into each of Soxhlet extractor, five times of petroleum ether (30-60 ℃) was introduced, and reflux extraction was then performed for 1.5 h. The solvent was discarded, and the drug residue was evaporated and added with an appropriate amount of chloroform, and refluxextracted for 3 h to further remove the soluble polar impurities in the polar range. The solvent was discarded, and the drug residue was evaporated. Reflux extraction was performed again with methanol for 4 h, and the extract was evaporated. Finally, 25 ml of methanol25% hydrochloric acid (4∶1) solution was prepared and added, followed by reflux extraction in a water bath at 85 ℃ for 40 min, and after cooling, the extract was transferred to a 50 ml volumetric flask, added with methanol to constant volume and shaken uniformly. The solution was filtered with 0.45 μm filter membrane before injection.
Chromatographic conditions
Platisil ODS C column (150 mm×4.6 mm, 5 μm) and the mobile phase Vmethanol∶Vwater (0.4% phosphoric acid solution)=55∶45 were selected. The HPLC separation was performed with the column at a column temperature of 40 ℃ using the mobile phase at a flow rate of 1 ml/min. The sample size was 20 μl, and detection was performed with a Shimadzu HPLC ultraviolet detector at 360 nm. Preparation of kaempferol standard solution
For the standard solution of quercetin, 10.2 mg of quercetin reference substance was weighed into a 100 ml volumetric flask, and diluted with methanol to constant volume, obtaining a 0.052 0 g/L standard stock solution.
Drawing of standard curve［5］
Certain amounts of kaempferol standard stock solution (1.0, 2.0, 4.0, 8.0 and 16.0 ml) were pipetted into 20 ml volumetric flasks, respectively, and diluted with anhydrous methanol to constant volume. Then, 20 μl of each solution was injected and determined for peak area. The test results showed that the peak area of kaempferol A was in good linear relation with concentration C in the range of 0.001 06-0.016 96 g/L (as shown in Fig. 2). The regression equation was A=6.676×104C-37.75, R2=0.999 2.
Fig. 2Quercetin standard curve
Calculation of ginkgo kaempferol content
The fermented and unfermented samples were subjected to qualitative and quantitative analysis by HPLC combined with kaempferol standard. According to the kaempferol standard curve and the regression equation, the kaempferol contents of the fermented and unfermented samples were compared.
Methodological investigation
Repetitive experiment
The fermentation and unfermented samples were injected repeatedly for 5 times, and the results are shown in the above two tables. The RSD was 1.25% and 0.67%, respectively. The repeatability was good.
Table 1Determination results of repetitive experiment
Fermented sample Peak area of quercetin∥mv*sRSD∥%
19331.25
2936
3929
4927
5932
Unfermented samplePeak area ofquercetin∥mv*sRSD∥%
1128 00.67
2128 7
3128 4
4127 8
5127 5
Precision test
The reference solution was prepared according to the method under "Preparation of test solution", and injected for 5 times according to the chromatographic conditions under "Chromatographic conditions", and the chromatogram was recorded. According to the relative peak area of kaempferol and the peak height and symmetrical factor, the RSD of the peak area was 0.23%, and the results are shown in Table 2. The test results showed that the precision of this method is good.
Stability test
The prepared sample solution was determined at 0, 4, 8, and 24 h, respectively, and the relative peak area (RSD<2.4%) was investigated. It was further known that the differences in content were almost inconspicuous. Therefore, the stability of the sample solution was good within 24 h. Results and Analysis
Determination of quercetin content in ginkgo leaves
According to the kaempferol standard curve and the regression equation, the concentration of kaempferol in the test solution of the unfermented sample was calculated to be 0.018 6 g/L, and the concentration of kaempferol in the test solution of the fermentation sample was calculated to be 0.013 4 g/L. It could be seen that the concentration of kaempferol in the test solution was reduced by 28% compared with the unfermented product.
System suitability
The quercetin reference solution, the unfermented test solution, the fermented test solution and the blank solution were determined according to the chromatographic conditions under "Chromatographic conditions". The chromatograms are shown in the figures as Fig. 3-Fig. 6.
Table 2Results of precision test (n=5)
SamplePeak area of quercetin∥mv*sPeak height∥mvSymmetrical factorAverage peak areaRSD∥%
1719 01680.8317 6800.23
2720 41700.82
3720 11700.83
4719 71690.84
5718 81670.83
Fig. 3Quercetin reference solution
Fig. 4Test solution of the unfermented sample
Fig. 5Test solution of the fermented sample
Agricultural Biotechnology2019
In the determination of the reference solution, the peak shape was complete, and there was no interference peak. For the unfermented test solution, the peaks of the main components were completely separated, the resolution was 2.24, and the theoretical plate number was 315 6. As to fermented test solution, the resolution was 2.33, and the theoretical plate number was 2 963. The blank solvent had no interference on the determination of the content in the test solutions.
Selection of detection wavelength
Because of the C6C3C6 structure of ginkgo flavonoids, the UV spectra of the ginkgo leaves were scanned to have a major absorption peak in the range of 300-400 nm, with methanol as blank, but the maximum absorption wavelengths of the three main flavonoid aglycones were slightly different. Combined with the chromatographic conditions in the references［6-8］, this study chose 360 nm as the detection wavelength.
Fig. 6Blank solution
Exploration of mobile phase
It was found through experiments that the main component could not be effectively separated when the ratio of the mobile phase (methanol∶water) is relatively large (such as 85∶15), and with the gradual reduction of the ratio, the detection time is prolonged, and the problem is improved. Under the improved condition, measured aglycones could be separated from other flavonoids, showing sharp peak and reduced tailing. From the results obtained, it was known that the separation effect was better under the condition of VmethanolVwater (0.4% phosphoric acid aqueous solution)=55∶45 system, so this ratio was selected as the experimental mobile phase. Determination of hydrolysis solution concentration
In view of the stability of the sample, it is necessary to pay attention to the selection of the concentration of the hydrolysis solution, so as to avoid large deviation of the results caused by excessive loss. According to the chemical nature of the parent nucleus of total flavonoid glycosides, toohigh or toolow acidity of hydrochloric acid would lead to a lower content. In combination with the Chinese Pharmacopoeia (2015 edition) and references, methanol25% hydrochloric acid solution (4∶1) was selected. The mixed solution had a good hydrolysis effect.
Hydrolysis time
According to literature reports and pharmacopoeia standards, flavonoid glycosides can be completely hydrolyzed into aglycones under the conditions of this study, and there were no significant differences in the content of main aglycones obtained after 60 to 120 min of hydrolysis. However, it is common to adopt a hydrolysis time of 30 min. After several times of extraction for improving the conditions, taking accidental error into account, it was finally decided to hydrolyze sample for 40 min.
Hydrolysis temperature
It has been reported in literatures that the best suitable hydrolysis temperature is 85 ℃, as shown in Table 4.
Table 4Selection of hydrolysis temperature
Hydrolysis temperature∥℃80859095100
Total flavonoid content∥%1.291.411.341.35
Through Table 4, it could be seen that when selecting the water temperature of 85 ℃ for hydrolysis of flavonoid glycoside, the yield was the highest.
Conclusions and Discussion
The preliminary investigation in this study revealed that B. licheniformis was selected as the fermentation strain, and the kaempferol content was lower in the microbial fermentation product of ginkgo leaves than in the unfermented ginkgo leaves. The effects of fermentation of ginkgo leaves on other effective active components still need further experimental exploration.
References
［1］ ZHAO H, ZHANG HM, DONG YM, et al. Optimization on extraction conditions of flavonoids from Ginkgo biloba leaf using response surface methodology［J］. Journal of Beijing Technology and Business University: Natural science Edition, 2010, 28(3): 27-31, 43. (in Chinese)
［2］ WEI XL, QU YW, LIANG JY. Research progress of ginkgo［J］. Strait Pharmaceutical Journal, 2013, 25(3): 1-8. (in Chinese)
［3］ XU YF, ZHANG LJ, SONG XB. Research progress on Ginkgo biloba extract［J］. Drug Evaluation Research, 2010, 33(6): 452-256. (in Chinese)
［4］ GAO SF. Study on preparation of new biological feed by fermenting ginkgo leaves［D］. Nanjing: Nanjing Forestry University, 2014. (in Chinese)
［5］ ZOU DF, FAN RF, HUANG X. The optimization of ultrasoni extraction technology of total flavonoids in leaves of Mallotus of Mallo apelta by response surface analysis methodology［J］. Medicinal Plant, 2018, 9(3): 68-72.
［6］ GAO YY, TANG DQ, YIN XX, et al. Simultaneous determination of five flavonoids in Ginkgo biloba extract by high performance liquid chromatograpy［J］. Acta Academiae Medicinae Xuzhou, 2008, 28(5): 307-310. (in Chinese)
［7］ QIAN ZY, FU J, HU M. HPLC detection of ginkgo flavonoids and its discussion［J］. Journal of China Pharmaceutical University, 1996, 27(4): 253. (in Chinese)
［8］ FENG P, SUN M, ZHANG LP. Study on extraction techniques of five flavonoids in ginkgo leaves［J］. Applied Chemical Industry, 2013, 42(6): 1090. (in Chinese)
Key wordsPear; ‘Zhong ai 1 hao’ ; Spindleshaped; Tree structure
Received: June 20, 2018Accepted: November 7,2018
Supported by the China Agriculture Research System (Pear) (CARS2836); the National Natural Science Foundation of China (31601708); the Agricultural Stock Breeding Project of Shandong Province (2016LZGC034); the Youth Foundation of Shandong Academy of Agricultural Sciences (2015YQN40); the National Science and Technology Plan for Rural Areas of China (2014BAD16B034); the Project for Agricultural Science and Technology Innovation of Shandong Academy of Agricultural Sciences (CXGC2018F03); and the Key Science and Technology Innovation Project of Shandong Province (2018CXGC0208).
Shuwei WEI (1981-), male, P. R. China, associate researcher, devoted to research about pear cultivation and breeding, Email: weisw2007@163.com.
* Corresponding author. Shaomin WANG (1962-), male, P. R. China, researcher, devoted to research about fruit cultivation and breeding, Email: wsm@sdip.cn.
China is the largest pear producing country in the world, with both its planting area and fruit yield ranking the［1］. The tree structure and group structure of fruit trees are the main factors affecting orchard yield and quality. Dwarfing and compact planting is a kind of fruit culture technique to dwarf the trees and shorten the planting spacing by grafting dwarfing rootstocks (or dwarfing inter stock) to the selected dwarfing varieties (varieties with short branches) on the basis of manual dwarfing measures, thereby obtaining early high yield. It has become the general trend for the production and development of fruit trees in China. Currently, the use of dwarf stocks (or dwarf inter stocks) has become the main way to achieve the dwarfing and compact cultivation of pear trees. ‘Zhong ai 1 hao’ is a compact dwarfing stock selected from the natural seedlings of Jinxiang pear by Jiang et al.［2］ in 1980. Used as the dwarfing inter stock, it can make the cultivated pear varieties have dwarfing plants, early fruitsetting and early high yield. In this paper, investigations were made to the densely planted spindleshaped ‘Huangguan’ pear orchard which used ‘Zhong ai 1 hao’ as the inter stocks, with the aim to clarify the individual and group tree parameters of ‘Huangguan’ pear with the dwarf ‘Zhong ai 1 hao’ as the inter stocks for the dense planting of spindleshaped ‘Huangguan’ pear trees, thereby providing references for the dwarf and compact planting of pear trees. Materials and Methods
Survey orchard overview
In 2017, investigations were conducted at the Jinniushan Experimental Demonstration Base of (Shandong Institute of Fruit Research. The soil nutrient status of pear orchard was shown in Table 1, and the orchard was under routine management. The 4yearold ‘Huangguan’ pear trees were selected from the pear orchard in the base for the investigation and study, and all were of spindleshaped tree bodies. The grafting stocks were all Pyrus betulaefolia. The planting spacing for dwarf ‘Huangguan’ pear trees was 0.75 m×3 m. ‘Zhong ai 1 hao’ was used as the inter stocks with the stock length of 20-25 cm. the planting spacing for vigorous stocks of ‘Huanguan’ pear trees was 1 m×3 m. The test pear orchard had moderate management level. In 2017, the percent rate of high quality fruit was over 90% for both the dwarfing stocks and vigorous stocks of ‘Huangguan’ pear. The trees with similar loads and tree shapes were selected for the investigation, and there were 5 plants in a plot with 3 repetitions.
Survey indicators and methods
Tree structure parameters included both the basic structure parameters of tree height, trunk height, trunk girth, crown diameter form south to north, crown diameter from east to west, central trunk numbers, main branch numbers, main branch opening angle, but also the number of short, medium and long branches and their proportions, as well as the growth conditions of new shoots and leaves like the number of spring shoots and their length, the number of leaves on spring shoots, leaf number per plant, single leaf area, leaf area per plant. Group structure parameters included the parameters related with crown overlapping rate (between plants and lines), leaf area coefficients.
Tree height was measured by the sign post. The trunk girth was the perimeter of the trunk at 20 cm above the ground, and the trunk height was the vertical height from the ground to the 1st main branch of the first layer, both of which were measured with a tape. The crown diameters were the lengths of crowns from the south to the north and from the east to the west, also measured with a tape. The growth of new shoots was measured by randomly selecting 30 new shoots around the crown of each plant, the lengths of which were measured with a tape, and then the average length was calculated. Single leaf area=Leaf length×Leaf width÷2; Leaf area per plant=Single leaf area×Number of leaves per plant; Projected area of crown per plant=(π×North-south crown diameter×East-west crown diameter)÷4; Leaf area coefficient=Leaf area per plant÷Projected area of crown per plant; Orchard coverage (%)=(Projected area of crown per plant×Total number of plants)÷Total planting area×100; Crown overlapping rate (%)=(Single plant crown diameter-Average plant spacing)÷Average plant spacing×100. Table 1Soil nutrient status of the test orchard
Soil layercmpH valueOrganic matterg/kgTotal Ng/kgAvailable Kmg/kgAvailable Pmg/kgAvailable Bmg/kgAvailable Cumg/kgAvailable Femg/kgAvailable Znmg/kg
0-257.2816.131.10219.0049.000.666.0111.102.34
25-507.308.670.90156.0014.000.432.307.121.01
Data analysis
Data analysis was completed with Office Excel.
Results and Analysis
Effect of ‘Zhong ai 1 hao’ on the height and crown diameter of ‘Huangguan’ pear trees
The investigation showed that with ‘Zhong ai 1 hao’ as the inter stocks, the 4yearold spindleshaped ‘Huangguan’ pear tree had a tree height of 2.87 m, trunk height of 0.50 m, trunk girth of 17.0 cm, southnorth crown diameter of 1.15 m, easteast crown diameter of 1.58 m. The 4yearold ‘Huangguan’ pear tree grafted with vigorous stocks had a tree height of 3.17 m, trunk height of 0.54 m, trunk girth of 15.4 cm, southnorth crown diameter of 1.50 m, eastwest crown diameter of 1.78 m. Therefore, it could reduce tree height by using the dwarf inter stock ‘Zhong ai 1 hao’, which could also decrease the crown diameter (Table 2).
Table 2Effect of the inter stock of ‘Zhong ai 1 hao’ on the basic parameters of ‘Huangguan’ pear trees
CultivationpatternNumber ofplants per667 m2TreeheightmTrunkheightmTrunkgirthcmCrown diameterfrom southto north∥mCrown diameterfrom eastto west∥mMain branchesper plantnumberMain branchopeningangle∥°Compatibility (stockscionor rootstock withintermediate stock
Dwarfing stock2962.870.5017.01.151.588.085.0Good
Vigorous stock2223.170.5415.41.501.787.483.6Good
Effect of ‘Zhong ai 1 hao’ on the branch composition of ‘Huangguan’ pear trees
According to the investigation, there were 50.2 short branches in the 4yearold ‘Huangguan’ pear tree with ‘Zhong ai 1 hao’ as the inter stock, accounting to 70.70% of the total branch amount; 11.6 medium branches, accounting for 16.34%; 9.2 long branches, accounting for 12.96%. As for the vigorous stock grafted ‘Huangguan’ pear tree, there were 45 short branches, accounting fro 63.20% of the total branches; 9.6 medium branches, accounting fro 13.48%; 16.6 long branches, accounting for 23.31%. The ‘Huangguan’ pear tree grafted with dwarfing stocks and vigorous stocks had similar branch numbers per plant, but the branch numbers per 667 m2 of dwarf ‘Huangguan’ pear trees were much larger than those of the vigorous stocks grafted‘Huangguan’pear trees because there were more branches per 667 m2 on the dwarf‘Huangguan’pear trees (Table 3). Table 3Effect of the inter stock of ‘Zhong ai 1 hao’ on the branch composition of‘Huangguan’ pear trees
CultivationpatternNumber ofshortbranchesShort branchproportion%Number ofmediumbranchesMedium branchproportion%Number oflongbranchesLong branchproportion%Number offruitingbranchesFruiting branchproportion%Branchnumbersper plantBranchnumbersper 667 m2
Dwarfing stock50.270.7011.616.349.212.9632.041.4571.021 016.0
Vigorous stock45.063.209.613.4816.623.3142.459.8971.215 806.4
Effect of ‘Zhong ai 1 hao’ on the shoots and leaf growth of‘Huangguan’pear trees
According to the investigation, for the 4yearold‘Huangguan’ pear tree grafted with the inter stocks of ‘Zhong ai 1 hao’, the spring shoot was 17.40 cm long, the spring shoots number per plant was 43.8, leaf number per shoot was 9.80, leaf number per plant was 1 047, leaf area per plant was 2.84 m2. The converted spring shoots number per 667 m2 was 12 964.8, and leaf area per 667 m2 was 840.64 m2. In terms of the ‘Huangguan’ pear tree grafted with vigorous stocks, the spring shoot was 22.88 cm long, spring shoots number per plant was 52.6, leaf number per shoot was 7.62, leaf number per plant was 1 020, leaf area per plant was 3.24 m2. The converted spring shoots number per 667 m2 was 11 677.2, and leaf area per 667 m2 was 719.28 m2. Therefore, dwarf‘Huangguan’ pear tree had significantly higher converted spring shoot number per 667 m2, leaf number and leaf area than the ‘Huangguan’ pear tree grafted with vigorous stocks (Table 4).
(Continued on page 161)
Agricultural Biotechnology2019, 8（1）: 158-161
Inspection and Testing
Dengfeng ZOU et al. Determination of the Content of Kaempferol from Fermented Ginkgo (Ginkgo biloba L.) Leaves
Determination of the Content of Kaempferol from Fermented Ginkgo (Ginkgo biloba L.) Leaves
Dengfeng ZOU, Hua ZHU, Bowen JIANG, Xiaohua WANG*
Guilin Medical University, Guilin 541004, China
Abstract［Objectives］ This study was conducted to determine kaempferol content in ginkgo (Ginkgo biloba L.) leaves subjected to microbial fermentation.
［Methods］ Bacillus licheniformis was selected for solidstate fermentation of ginkgo leaves, and the content of kaempferol in ginkgo leaves was determined by RPHPLC method. At first, methanol was used to extract flavonoid glycosides, which were then hydrolyzed by hydrochloric acid solution. HPLC was performed with Platisil ODS column C18 (150 mm×4.6 mm, 5 μm) using mobile phase Vmethanol∶Vwater (0.4% phosphoric acid solution) = 55∶45 at a flow rate of 1 ml/min, and the eluate was detected with a shimadzu HPLC ultraviolet detector at 360 nm. ［Results］ With kaempferol as the reference substance, the correlation coefficient was 0.999 2 in the range of 0.001 06-0.016 96 g/L. The content in the fermented product was less than that in the nonfermented product by 28%.
［Conclusions］ The method is simple, accurate, and is suitable for determination of kaempferol. This study will provide an experimental basis for the development and utilization of ginkgo.
Key wordsGinkgo biloba L.; Ginkgo leaves; Microbial fermentation; Content determination; HPLC; Kaempferol
Received: August 29, 2018Accepted: November 22, 2018
Supported by Guilin Science and Technology Bureau Project (20100305); Guangxi "2011 Collaborative Innovation Center"Zhuang Yao Medicine Collaborative Innovation Center Project (G2013［20］).
Dengfeng ZOU (1975-), male, P. R. China, professor, devoted to research about natural medicine.
*Corresponding author. Email: 437593157@qq.com.
Ginkgo (Ginkgo biloba L.) is also known as Gongsunshu, "living fossil" and "panda in the plant world"［1］. The main active components of ginkgo leaves (dry leaves) are flavonoids and ginkgo terpene lactones from which more than 160 kinds of effective active components of ginkgo flavonols including quercetin, kaempferol and isorhamnetin have been found［2］. As to studies on ginkgo lactones, EGb761 is the patented ginkgo leaf extract medicine developed the earliest by Germany Schwabe company, and its production standard is still regarded as the international ginkgo leaf product standard［3］. In this study, Bacillus licheniformis was selected through fermentation method to be the test strain for the microbial solidstate fermentation of ginkgo leaf powder, and combined with the characteristic that the molecular structure of ginkgo flavonoids has strongly absorption at 360 nm, the current commonly used HPLC method with accurate determination results was selected for the determination of kaempferol content. In lactones, the structure of total flavonoids is as Fig. 1.
Instruments and Materials
Ginkgo leaves were collected from the Medicinal Plant Garden of Dongcheng Campus of Guilin Medical University, and identified as G. biloba leaves by associate professor Huang from Guilin Medical University.
Instruments: Super clean bench; autoclave; constant temperature incubator; constant temperature shaker; Shimadzu chemical station; ODS column (150 mm×4.6 mm, 5 μm). Reagents: Czapek–Dox medium; agar medium; methanol (analytical pure); chromatographically pure methanol, phosphoric acid, etc.
Strain: B. licheniformis, purchased from BeNa Culture Collection.
Fig. 1Structure of total flavonoids
Experimental Methods
Preparation of seed liquid
A ring of activated B. licheniformis was picked from the slant medium and then inoculated in a 250 ml flask (containing 50 ml of sterile seed liquid). The inoculated strain was cultured in a constant temperature shaker for 24 h, at 36 ℃, with shaking at 180 r/min.
Solidstate fermentation of ginkgo leaves［4］
The seed solution was transferred with a pipette to a flask containing the fermentation medium, followed by solidstate fermentation in a constant temperature incubator. The inoculum size, liquidsolid ratio, initial pH, fermentation temperature and fermentation time were 15%, 1.2∶1, 7.0, 28 ℃, and 48 h, respectively.
Preparation of test solution
After the fermentation was completed, the fermentation product was sterilized in an autoclave, and 2 g of each of the fermented and unfermented samples was accurately weighed with an electronic balance, respectively, and placed in a Soxhlet extractor, respectively. Into each of Soxhlet extractor, five times of petroleum ether (30-60 ℃) was introduced, and reflux extraction was then performed for 1.5 h. The solvent was discarded, and the drug residue was evaporated and added with an appropriate amount of chloroform, and refluxextracted for 3 h to further remove the soluble polar impurities in the polar range. The solvent was discarded, and the drug residue was evaporated. Reflux extraction was performed again with methanol for 4 h, and the extract was evaporated. Finally, 25 ml of methanol25% hydrochloric acid (4∶1) solution was prepared and added, followed by reflux extraction in a water bath at 85 ℃ for 40 min, and after cooling, the extract was transferred to a 50 ml volumetric flask, added with methanol to constant volume and shaken uniformly. The solution was filtered with 0.45 μm filter membrane before injection.
Chromatographic conditions
Platisil ODS C column (150 mm×4.6 mm, 5 μm) and the mobile phase Vmethanol∶Vwater (0.4% phosphoric acid solution)=55∶45 were selected. The HPLC separation was performed with the column at a column temperature of 40 ℃ using the mobile phase at a flow rate of 1 ml/min. The sample size was 20 μl, and detection was performed with a Shimadzu HPLC ultraviolet detector at 360 nm. Preparation of kaempferol standard solution
For the standard solution of quercetin, 10.2 mg of quercetin reference substance was weighed into a 100 ml volumetric flask, and diluted with methanol to constant volume, obtaining a 0.052 0 g/L standard stock solution.
Drawing of standard curve［5］
Certain amounts of kaempferol standard stock solution (1.0, 2.0, 4.0, 8.0 and 16.0 ml) were pipetted into 20 ml volumetric flasks, respectively, and diluted with anhydrous methanol to constant volume. Then, 20 μl of each solution was injected and determined for peak area. The test results showed that the peak area of kaempferol A was in good linear relation with concentration C in the range of 0.001 06-0.016 96 g/L (as shown in Fig. 2). The regression equation was A=6.676×104C-37.75, R2=0.999 2.
Fig. 2Quercetin standard curve
Calculation of ginkgo kaempferol content
The fermented and unfermented samples were subjected to qualitative and quantitative analysis by HPLC combined with kaempferol standard. According to the kaempferol standard curve and the regression equation, the kaempferol contents of the fermented and unfermented samples were compared.
Methodological investigation
Repetitive experiment
The fermentation and unfermented samples were injected repeatedly for 5 times, and the results are shown in the above two tables. The RSD was 1.25% and 0.67%, respectively. The repeatability was good.
Table 1Determination results of repetitive experiment
Fermented sample Peak area of quercetin∥mv*sRSD∥%
19331.25
2936
3929
4927
5932
Unfermented samplePeak area ofquercetin∥mv*sRSD∥%
1128 00.67
2128 7
3128 4
4127 8
5127 5
Precision test
The reference solution was prepared according to the method under "Preparation of test solution", and injected for 5 times according to the chromatographic conditions under "Chromatographic conditions", and the chromatogram was recorded. According to the relative peak area of kaempferol and the peak height and symmetrical factor, the RSD of the peak area was 0.23%, and the results are shown in Table 2. The test results showed that the precision of this method is good.
Stability test
The prepared sample solution was determined at 0, 4, 8, and 24 h, respectively, and the relative peak area (RSD<2.4%) was investigated. It was further known that the differences in content were almost inconspicuous. Therefore, the stability of the sample solution was good within 24 h. Results and Analysis
Determination of quercetin content in ginkgo leaves
According to the kaempferol standard curve and the regression equation, the concentration of kaempferol in the test solution of the unfermented sample was calculated to be 0.018 6 g/L, and the concentration of kaempferol in the test solution of the fermentation sample was calculated to be 0.013 4 g/L. It could be seen that the concentration of kaempferol in the test solution was reduced by 28% compared with the unfermented product.
System suitability
The quercetin reference solution, the unfermented test solution, the fermented test solution and the blank solution were determined according to the chromatographic conditions under "Chromatographic conditions". The chromatograms are shown in the figures as Fig. 3-Fig. 6.
Table 2Results of precision test (n=5)
SamplePeak area of quercetin∥mv*sPeak height∥mvSymmetrical factorAverage peak areaRSD∥%
1719 01680.8317 6800.23
2720 41700.82
3720 11700.83
4719 71690.84
5718 81670.83
Fig. 3Quercetin reference solution
Fig. 4Test solution of the unfermented sample
Fig. 5Test solution of the fermented sample
Agricultural Biotechnology2019
In the determination of the reference solution, the peak shape was complete, and there was no interference peak. For the unfermented test solution, the peaks of the main components were completely separated, the resolution was 2.24, and the theoretical plate number was 315 6. As to fermented test solution, the resolution was 2.33, and the theoretical plate number was 2 963. The blank solvent had no interference on the determination of the content in the test solutions.
Selection of detection wavelength
Because of the C6C3C6 structure of ginkgo flavonoids, the UV spectra of the ginkgo leaves were scanned to have a major absorption peak in the range of 300-400 nm, with methanol as blank, but the maximum absorption wavelengths of the three main flavonoid aglycones were slightly different. Combined with the chromatographic conditions in the references［6-8］, this study chose 360 nm as the detection wavelength.
Fig. 6Blank solution
Exploration of mobile phase
It was found through experiments that the main component could not be effectively separated when the ratio of the mobile phase (methanol∶water) is relatively large (such as 85∶15), and with the gradual reduction of the ratio, the detection time is prolonged, and the problem is improved. Under the improved condition, measured aglycones could be separated from other flavonoids, showing sharp peak and reduced tailing. From the results obtained, it was known that the separation effect was better under the condition of VmethanolVwater (0.4% phosphoric acid aqueous solution)=55∶45 system, so this ratio was selected as the experimental mobile phase. Determination of hydrolysis solution concentration
In view of the stability of the sample, it is necessary to pay attention to the selection of the concentration of the hydrolysis solution, so as to avoid large deviation of the results caused by excessive loss. According to the chemical nature of the parent nucleus of total flavonoid glycosides, toohigh or toolow acidity of hydrochloric acid would lead to a lower content. In combination with the Chinese Pharmacopoeia (2015 edition) and references, methanol25% hydrochloric acid solution (4∶1) was selected. The mixed solution had a good hydrolysis effect.
Hydrolysis time
According to literature reports and pharmacopoeia standards, flavonoid glycosides can be completely hydrolyzed into aglycones under the conditions of this study, and there were no significant differences in the content of main aglycones obtained after 60 to 120 min of hydrolysis. However, it is common to adopt a hydrolysis time of 30 min. After several times of extraction for improving the conditions, taking accidental error into account, it was finally decided to hydrolyze sample for 40 min.
Hydrolysis temperature
It has been reported in literatures that the best suitable hydrolysis temperature is 85 ℃, as shown in Table 4.
Table 4Selection of hydrolysis temperature
Hydrolysis temperature∥℃80859095100
Total flavonoid content∥%1.291.411.341.35
Through Table 4, it could be seen that when selecting the water temperature of 85 ℃ for hydrolysis of flavonoid glycoside, the yield was the highest.
Conclusions and Discussion
The preliminary investigation in this study revealed that B. licheniformis was selected as the fermentation strain, and the kaempferol content was lower in the microbial fermentation product of ginkgo leaves than in the unfermented ginkgo leaves. The effects of fermentation of ginkgo leaves on other effective active components still need further experimental exploration.
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