Determination and Comparison of Mineral Elements in the Leaves and Barks of Eucommia ulmoides Oliv.from Different Places of Zhangjiajie

ZHANG Lin-jie，PENG Sheng，ZHANG Chang-wei，WANG Zhi-hong，ZHOU Yun-lei，PENG Mi-jun*，ZHANG Shui-han

(1.Key Laboratory of Hunan Forest Products and Chemical Industry Engineering，Jishou University，Zhangjiajie 427000，China;2.Key Laboratory of Research and Development of New Drug of Chinese Traditional Medicine，Hunan Academy of Chinese Medicine，Changsha 410013，China)

As a unique Eucommiaceae single-genus plant in China，Eucommia ulmoides Oliv.is a kind of rare Chinese herbal medicine［1］.There are kinds of active ingredients in its leaves and barks such as lignans，iridoids，flavonoid，phenylpropanoids etc.［2-4］.They also contain the essential amino acids and mineral elements for human body［5-6］.E.ulmoides Oliv.is believed to be the highest quality natural antihypertensive drugs in the world［7-8］.With the development of science and technology，the living quality requirement of people becomes higher and higher.At the same time，the importance of mineral elements is known and valued by more and more people.Under the guidance of theory of traditional Chinese medicine(TCM)，domestic and overseas scholars found that the efficacy of traditional Chinese medicine had a close relationship with the kinds and contents of mineral elements by making a large number of clinical practice and research［9-13］.So analysis of the kinds and contents of different mineral elements could help to elucidate the pharmacological activity and toxic mechanism and evaluate the quality of traditional Chinese medicine.

1 Experimental

1.1 Reagents

Iron powder(99.99% ，Tianjin No.2 Chemical Reagent Factory);ZnSO4·7H2O，CuSO4·5H2O，MgSO4·7H2O，potassium chloride and calcium carbonate(GR，China National Medicines Corporation Ltd.Shanghai);hydrochloric acid and nitric acid(GR，Changsha Yuelu District Excellent High Purity Reagent Factory);Manganese(GR，Tianjin No.2 Chemical Reagent Factory).

1.2 Instruments

AA-680 FAAS(SHIMADZU，Japan);FW100 Stainless steel and high speed universal mill(Taisite，Tianjin);AEG-220 One Over Ten-thousand Analytical Balance(Shimadzu，Japan);SX-4-10 muffle furnace(Zhongda，Changsha);Electric stove;Porcelain crucible.

1.3 Digesting and detection method of the samples

Sample(0.5 g)was added to the porcelain crucible，carbonized until no smoke emitted，and then put into the muffle furnace for 5 h at 550±25℃.Affter that，5 mL 20%HCl was added and heated on the electric stove to dissolve the ash fully.The solution was transferred to 50 mL volumetric flask and brought to volume by ultra-pure water.Finally，the content of mineral elements in sample was determined by FAAS.

2 Results and Discussion

2.1 Samples

The leaves and barks of E.ulmoides Oliv.were collected from different places of Zhangjiajie.The details were presented in Table 1.After de-enzymed by microwave，dried in 50℃ and crushed，the samples were saved.

Table 1 The details of the places for collecting the samples

2.2 Working states of FAAS

The best working states of the FAAS while measuring different elements were presented in Table 2.

2.3 Standard curve and adding standard recovery for each element

The standard curve and recovery by adding standard for each element were presented in Table 3.The linearity and accuracy of the FAAS method were evaluated by the correlation coefficient and recovery.The values of r2for all the standard curves were more than 0.9990，and the correlations were good.The recoveries ranged from 98.99%to 103.0%，and they were qualified.

Table 2 The best working state of the FAAS while measuring different elements

Table 3 Standard curve and adding standard recovery for each element

2.4 The result for determination

The contents of mineral elements in the leaves and barks of E.ulmoides Oliv.from different places of Zhangjiajie were shown in Table 4.

Table 4 Content of mineral elements in leaves and barks of E.ulmoides Oliv. μg·g-1

The content of Mg was the highest for all the leaves of E.ulmoides Oliv.，and followed by K，Ca，Zn，Fe，Cu and Mn.The content of Cu and Mn was almost the same.The leaves of No.3 contained the highest amounts of Fe and Zn，No.4 for K，No.2 for Mg，and No.1 for Ca.The lowest level of Fe was from No.5，Zn and Mg from No.1，K and Ca from No.2.

For the content of all the seven mineral elements in barks of E.ulmoides Oliv.，Mg was the highest，and followed by Ca，K，Fe，Zn，Cu and Mn.Compared with the other places，Zn and Cu was the most abundant in barks from places of No.3;Mn，Mg and Ca from No.4;K from No.1 and Fe from No.5.Barks from place of No.1 contained a minimum amount of Fe and Mg;No.2 for Zn;No.3 for Ca;No.4 for Cu and K;and No.5 for Mn.

2.5 Data statistical analysis and test

2.5.1 Statistical analysis and normal inspection for each mineral element The statistical analysis and K-S normal inspection are done with the SPSS software［14-16］.The results were presented in Table 5.

Table 5 The result of K-S test for each mineral element in the leaves and barks of E.ulmoides Oliv.

It could be seen from Table 4 that all the significance(p)were greater than 0.05.So it was believed that these data submitted to normal distribution.Skewness indicated that the contents of Zn，Mg and K in leaves and Mg in barks were skewed negatively distribution，while the others were skewed positively distribution.The SD of the mineral element contents both in the leaves and barks was absolutely great.This illustrated that the contents of mineral elements in the leaves and barks of E.ulmoides Oliv.from different places of Zhangjiajie were significantly different.

2.5.2 LSD test of mineral elements The LSD test results were showed in Tables 6 and 7.

Table 6 Divergence analysis of each mineral element in the leaves of E.ulmoides Oliv.

The analysis of variance for the contents of mineral elements in the samples was finished with SPSS software.The homogeneity test for variance showed that all the significance(p)was less than 0.05 which indicated the contents of mineral elements in the leaves and barks of E.ulmoides Oliv.from different places of Zhangjiajie were significantly different.

Table 7 Divergence analysis of each mineral element in the barks of Eucommia ulmoides Oliv.

It can be seen from Table 6 that the contents of Fe，Mn and Mg from different place of Zhangjiajie were significantly different(p ＜0.05).The content of Zn in the leaves from No.5 was significantly different from the leaves from the other places except No.3.Both the content of K in the leaves from No.1 and Ca from No.4 were significantly different from the leaves from the other places expect No.2.And there were no significant differences for the content of Cu.

The conclusion can be obtained from Table 7 that the contents of Fe，Mn and K from different place of Zhangjiajie were significantly different(p ＜0.05).The content of Zn in the barks collected from No.3 was significantly different from the barks collected from the other places except No.4.The content of Mg in the barks collected from No.4 was significantly different from the leaves collected from the other places except No.5.For the content of Ca in the barks collected from No.2，it was significantly different from the barks collected from the other places except No.1.And there were no statistically significant differences for the content of Cu.

Plants not only need essential nutrients，but also absorb and concentrate the mineral elements which are demanded by human body.These elements play important roles in healing or preventing ills.Fe can participate in the composition of hemoglobin，myoglobin，cytochrome and cytochrome oxidase.Zn helps to activate more than 200 kinds of enzyme and participates in the nucleic acid and energy metabolism.Cu is a special kind of catalyst in the body.It can do a favor in strengthening the body’s immune and defense function.Mn is a kind of anticancer element.Mg affects the activity of enzymes.K is the main source of cation of intracellular fluid.Ca can increase the density and reduce permeability of capillary wall in the form of Ca2+.The rich in the essential mineral elements helps to improve the medical value and research value of E.ulmoides Oliv.

Genuineness is one of the main indexes to measure the quality of Chinese medicinal materials，which means that the effect of the Chinese medicinal material will be different once it deviates from its growing surroundings.The leaves and barks of E.ulmoides Oliv.from different places all contain Fe，Zn，Cu，Mn，Mg，K and Ca，which may be related to their own genetic characteristics.But the level of these mineral elements is different.Indeed the differences of the contents of Fe，Mn，Mg in leaves and Fe，Mn，K in barks are significant.This may be caused by its surroundings，altitude，light，rainfall etc.Their relationship needs further investigation and analysis.

3 Conclusions

3.1 Both the leaves and barks of E.ulmoides Oliv.were rich in mineral elements.And this will provide more positive proof for the medical and research value of E.ulmoides Oliv.

3.2 The general trend of mineral elements in the leaves of E.ulmoides Oliv.was:Mg＞K ＞Ca＞Zn＞Fe＞Mn≥Cu，while in the barks Mg＞Ca＞K ＞Fe＞Zn＞Mn≥Cu.The content of Cu and Mn was almost the same and very low both in the leaves and barks.

3.3 It was proved that the contents of Fe，Mn，Mg in leaves and Fe，Mn，K in barks were significantly different by LSD test.This may be the reason for the difference in pharmacological activity and quality of the leaves and barks of E.ulmoides Oliv.from different places.

［1］(魏)吳譜.神農本草經［M］.北京:人民衛生出版社，1963.

［2］DAI Xing-ping，HUANG Qiong，ZHOU Bo-ting，et al.Preparative isolation and purification of seven main antioxidants from Eucommia ulmoides Oliv.(Du-zhong)leaves using HSCCC guided by DPPH-HPLC experiment［J］.Food Chem，2013，139(1-4):563-570.

［3］ZHANG Qiang，SU Yin-quan，ZHANG Jing-fang.Seasonal Difference in antioxidant capacity and active compounds contents of Eucommia ulmoides oliver leaf［J］.Molecules，2013，18(2):1857-1868

［4］潘亞磊，翟遠坤，武祥龍，等.杜仲活性成分的提取及分離純化方法研究進展［J］.化學與生物工程，2012，29(2):1-4.

［5］周芳，劉韶，鄧夢茹，等.GC-MS/SIM法測定不同產地杜仲葉中的氨基酸［J］.中南藥學，2012，10(4):257-260.

［6］彭金年，彭湘君，李銀保，等.不同產地杜仲葉中金屬元素的含量及其變化趨勢分析［J］.廣東微量元素科學，2011，18(4):46-49.

［7］李健民，徐艷明，朱魁元，等.杜仲抗氧化生物活性研究進展［J］.中醫藥學報，2010，38(2):137-139.

［8］辛曉明，馮蕾，王浩，等.杜仲的化學成分及藥理活性研究進展［J］.醫學綜述，2007，13(19):1507-1509.

［9］曹治權.微量元素與中醫藥［M］.北京:中國中醫藥出版社，1993.

［10］秦俊法，陳磐華.中國的中藥微量元素研究.微量元素(Ⅰ)一切中藥的基本成分［J］.廣東微量元素科學，2010，17(11):1-18.

［11］秦俊法，林宣賢.中國的中藥微量元素研究.微量元素(Ⅱ)中藥有效成分的核心組分［J］.廣東微量元素科學，2010，17(12):1-12.

［12］秦俊法.中國的中藥微量元素研究.微量元素(Ⅲ)中藥性效量化的物質基礎［J］.廣東微量元素科學，2011，18(1):1-10.

［13］秦俊法.中國的中藥微量元素研究.微量元素(Ⅳ)匯通傳統中藥理論與現代科學理論的橋梁［J］.廣東微量元素科學，2011，18(2):1-12.

［14］鄧維斌，唐興艷，胡大權，等.SPSS19(中文版)統計分析實用教程［M］.北京:電子工業出版社，2012.

［15］陳勝可.SPSS統計分析從入門到精通［M］.北京:清華大學出版社，2010.

［16］王璐，王沁，等.SPSS19統計分析基礎、應用與實戰精粹［M］.北京:化學工業出版社，2012.