全 文 ：第 26卷第 4期
2006年 12月
林　产　化　学　与　工　业
Chem istry and Indus try of Fo res t Products
Vo.l 26 No. 4
Dec. 2006
Studies on Chem ical Constituents and Antmi icrob ial
Activities of Persmi mon Leaves
　　R eceived date:2005 - 05 - 08
　　 Foundation item:国家 “十五 ”攻关资助项目(2001BA502B0403)
　　 Biography:季志平(1963 -),男 ,陕西礼泉人 ,副研究员,主要从事植物资源利用研究;E-ma il:jizh ip ing207@yahoo. com. cn
　*C orrespondence Auther:苏印泉 ,教授 ,硕士生导师 ,从事林产资源的教学与研究;E-m ail:suy inquan@ pub lic. xa. sn. cn。
　 JIZhi-ping　　　SU Y in-quan　
JI Zhi-p ing, SU Y in-quan* , LÜPing-hui, Zhang Cun-li
(C ollege o f Fo re stry Sciences, Northw estA＆ F University, Yang ling 712100, China)
Abstrac t:Chem ical constituents of persimm on leaves and an tim icrobia l activ ities w ere
stud ied. Resu lts show ed that pe rsimmon leaves contained alka lo ids, sapon ins, am ino
ac ids, po lypep tide s, org anic acids, pheno lic com pounds and tann ins, saccharides,
ste ro ids, flavones, ca rdiac g ly co side s, anthraquinones, naphtha, etc. Resu lts o f
experim ents on antibacteria l and an tifungal activ ities show ed that ex tracts o f a lcohol and ace tic e ste rhad rem a rkab le bac te rio sta sis
e ffec ts against 6 bac te ria;aqueous ex tract wa s mo re effec tive aga inst fung i, and slight aga inst bacteria;pe tro leum e ther ex trac t
had no effe ct aga inst bac te ria and fung i;buty la lcohol ex trac t had diffe rent effects aga inst bacteria, but had no effe cts against
fung .i Results of bac te rio stasis activ itie s of acetic ester ex trac t unde r diffe rent concentrations showed tha t the h ighe r concen tra tion,
the stronger bacteriosta sis e ffect w as, and tha t the sequence o f bac te riostasis activ ities w as diffe rent unde r different concen tra tions.
K ey words:persimm on leave s;ex trac t;antim ic rob ial ac tivity
CLC num ber:TQ91　　　　　　　D ocum ent code:A　　　　　　　Art ic le ID:0253 - 2417(2006)04 -0087 - 05
柿叶 化 学成 分 及其 抑菌 活 性研 究
季志平 , 苏印泉 , 吕平会 , 张存莉
(西北农林科技大学林学院 , 陕西 杨陵 712100)
摘　要:对柿叶的化学成分进行了定性分析和抗菌活性的研究。通过系统预试 , 初步确定柿叶中含有生物碱 、皂苷 、氨基
酸 、多肽 、有机酸 、酚类和鞣质 、糖类 、甾体 、总黄酮 、强心苷 、蒽醌 、挥发油等物质。抑制活性试验结果显示:乙醇提取物和
乙酸乙酯萃取物对供试的 6种细菌有较强的抑制作用;水相对供试的 5种真菌有较强的抑制作用 , 而且对细菌有较小抑
制作用;石油醚相对真菌和细菌几乎无抑制作用;正丁醇萃取物对细菌有不同程度的抑制作用 , 而对真菌无抑制作用。
用不同质量浓度的乙酸乙酯提取物进行抑菌活性试验 ,结果显示:质量浓度越高 ,其抑菌能力越强 , 而且 , 在不同质量浓
度下 , 对测试的 6个细菌的抑制活性的大小顺序不一致。
关键词:柿叶;提取物;抑菌活性
Pe rsimmon(D iospyros kaki Linn. f. ) is an important fruit tree o f long h isto ry, w ide distribution and rich
resources in Ch ina. Its fruit has g rea t va lue, and its leaf, flow er, pee l, root and pedicel have been used in
medicine
[ 1]
. Researches show tha t pe rsimmon leaves are effective in dim in ishing inf lammation, clearing-aw ay
heat, intene rating blood vesse l, reducing b lood pressure, reducing b lood lipid, antiox idation, etc. [ 2-4] . B io-
active constituen ts in pe rsimmon leaves have been isola ted and iden tified as flavonoids, trite rpenes, pheno lic
compounds, alka lo ids, o rganic acid, e tc. [ 5-8] . In this study, antim icrobia l ac tiv ities of extracts from persim-
mon leaves w ere sy stematically studied through testing antim icrobial ac tiv ities of different extrac ts, and itw as
found that extrac ts o f different po larities had distinct discrepancy, an tim icrobia l activ ity w as corre lative to
88　 林　产　化　学　与　工　业 第 26卷
concentration o f ex trac.t The re sults provided sc ientific accordance for explo ita tion and u tilization of persimmon
leaves resources.
1　M aterials and me thods
1. 1　Expermi entmater ia ls
1. 1. 1　Plant samp le　Persimmon leaves we re co llected from no rthern slope o f Q ing ling in August, 2004,
dried in shade, crushed and passed the sample through 0. 420mm mesh sieve.
1. 1. 2　Bac te ria and fungi for experiment　S ix kinds o f bacteria w ere te sted in th is experiment, including:
S taphylococcus aureus, Bacilllus subtilis, B. cereus, E scherich ia coli, Proteus vulgaris and B. megathrium.
F ive kinds o f fung i are as fo llow s:Aspergillum f lauus, A. n iger, Trichoderma koningii, Pen icillium sp. and T.
lignorum. A ll these bacteria and fung iwe re supplied by theM icrobia l Labora tory ofNorthwestA＆F University.
1. 1. 3　Substra te　Substratum used fo r bacteria experimen t is beef album en agar:beef 3 g, peptone 20 g,
sodium ch lo ride 5 g, agar 18-20 g, wate r 1 000mL, pH va lue 7. 2-7. 4. Substra tum used for fung i experimen t
is po tato agar:pota to 200 g, sucro se 20 g, agar 20 g, and distilled w ater 1 000mL.
1. 1. 4　Reagents and instrumen ts　Reagents:A lcohol used in expe riment is industrial alcoho l, o thers are
petroleum ethe r, ace tic este r, bu ty lalcoho l, anhydrous e thanol, and methanol, ae ther, which w e re manufac-
tured in X ian Chem ical Reagents Factory. Instrum ents:filtration cy linder, SENCOR50 rotatory evapora to r,
SHB-Ⅲ rota to ry w a ter vacuum pump, CS1M-500D ultrasonic cleanout m achine, ZF-2 ultravio let analyze r,
evaporating dish (d=90mm), cultiva ting dish (d =90mm), FA2004 ba lance, 101-2 evaporator, LS-B50L
ve rtical p ressure vapor sterilizing disinfecto r, LRH-250A incubator, and a YJ-875 sterile-wo rk pla tfo rm.
1. 2　Methods
1. 2. 1　Prepa ra tion of samp le s fo r pretestings o f constituen ts[ 9] 　Preparation of experimental samp les:1)
W eigh out leaf powder 5 g, add distilled w ater 50mL, heat at 50-60℃ for 1 h, then filter to ob tain aqueous
ex trac.t 2)Weigh out leaf pow er 5 g, extrac tw ith 95% alcoho l to ob tain alcoho l ex tract, d ilu te w ith distilled
w a ter to 70%, pu t it in separating funne l, add equal volume o f pe tro leum e ther to ex tract out chlorophy l.l To
alcoho l part, add 95% alcoho l 50mL, after re flux fo r 1 h in ho t w ater bath, filter, d iv ide filtrate into tw o
portions of A and B. A is used for testings of organic ac ids, pheno lic compounds and tann ins;B is
concentrated into paste, then divided into C and D. To C , add hydrochloric acid (2 %), then filter to ge t
acid fraction fo r testing a lkaloid, disso lve the residue wh ich rema ins on filter paper w ith a lcohol fo r testing
flavones and an thraqu inone;disso lve D w ith ace tic este r, then put it in to separating funne l, add 5%N aOH to
alkaline, evapora te to dryness and dissolve itw ith 1-2 mL ethano l fo r testing coumarins, terpene, lactones,
cardiac g lycosides. 3)Weigh ou t leaf pow de r 1 g, add 10mL pe tro leum ether and extrac t fo r 2-3 h , filte r,
vo latilize the extrac t for testing steroids, triterpenes and naphtha.
1. 2. 2　Prepara tion o f samp les　C rushed and dried persimmon leaves are repea tedly leached w ith 70%
alcoho l fo r 48 h a t room temperature for 5 times, so lutions a re concentra ted to ge t e thano l ex tract(S), which
is partly used fo r testing bacteriostasis, the rest is suspended in w ater and ex tracted by pe troleum ethe r, acetic
ester, buty lalcoho l successive ly to ge t pe tro leum e ther extrac t(S-1), ace tic e ster extrac t(S-2), buty lalcoho l
ex tract(S-3) and aqueous ex tract (S-4). Aqueous solution of e thanol extrac t is partitioned w ith pe tro leum
ethe r to obtain ex tract S-1-1 from pe tro leum e ther phase, and ex tract S-1-2 from aqueous phase fo r tests o f
bacteriostasis after concentration.
1. 2. 3　Preparation of suspended-bacterium-liqu id　Activate bacteria o r fung i in inc lined fitting, pu t
8-10mL distilled w ater in test tube, kill bacterium for 30m in under the condition o f 0. 11MPa, 121℃ to
obtain asepticw ate r. Pu t burned inoculator into aseptic wa ter, then sc rape cu lture substratum to fetch bacte ria
第 4期 季志平 , 等:柿叶化学成分及其抑菌活性研究 89　
or fung i, fetch bacteria 1-2 rings, fe tch fungi 3-4 rings, pu t fe tched bac teria o r fungi into aseptic w a te r,
shake su fficiently to obta in suspended-bacterium-liqu id.
1. 2. 4　Bac te riostasis experiment[ 10] 　Coo l the k illed-bacterium substra te to 45-50 ℃, and put it in to cu lti-
va ting dish under aseptic condition, each 10-15 mL, after coo led to room tempe ratu re, suck the suspended-
bacterium-liqu id w ith asep tic sucker, and add 0. 2mL into each dish, daub w ith asep tic spatula, then d ip
aseptic filte r paper (d =6mm) in ex tract so lu tion for 30 s, smoo th it on the su rface of substrate afte r drip-
ping, pu t 3-5 pieces in each dish, each sample is repeated 3 times, pu t the dish in the rmostat and cu ltivate
for 24 h at 28℃ (fo r bacteria) or 72 h at 26-28℃( for fung i), measure bacterio stasis diame terw ith decus-
sating method, then ca lculate the percentage o f bacterio stasis based on formu la.
2　Result and ana lysis
2. 1　Testings of active chem ical constituents
Result on testing s o f active chem ica l constituents in pe rsimmon leaves show ed that pe rsimmon leaves
contained alka lo ids, saponins, am ino ac ids, po lypeptides, organic ac ids, pheno lic compounds and tannins,
saccharides, stero ids, flavones, cardiac g ly cosides, anthraquinones and naphtha (Tab le 1).
Tab le 1　Testing resu lt of an tibacterial constituen ts1)
com pounds　　　　 reagen ts　　　　 resu lts
alkaloid
HgI2 2KI
B iI3 KI;S iO 2 12WO 3 +
sapon in s foam test;L iebeerm ann-Burch ard reaction +
am ino acid /polypep tide /protein B iuret reaction;Ninhyd rin reaction +
organ ic acids
pH value testing paper
brom oph enol b lue
+
ph enolic com pounds and tann ins
1% FeC l3;ge latin;gelatin +FeC l3
Lieberm an reagen t
+
-
polysaccharides glycosides Feh ling reagents;HC l hyd rolysis;Mo lish reagent +
steroid and triterpene phosphoru sm olybdic acid;Sa lkow ski reaction +
f lavones HC l-Zn reaction;NH3 reaction;5% A lC l3 aqueous solution +
coum arin /terpene / lactone
coup ling reaction;ring-opening and closing reaction
fluorescence tes t;co lor reaction
+
card iac glycosides Kedd le reagent;Ba ljet reaction +
an th raquinone
alka lescence tes t
Feigl react ion
H2 SO 4 react ion;1% boric acid
+
+
naph tha
appearance in spection
25% phospho-mo lybd ic acid +
1) -:negative reaction;+:posi tive reaction;b lank:no sign ifican t reaction
2. 2　Antmi icrob ial ac tivities of extracts from persmi mon leaves
O f the seven ex tracts, S and S-2 had remarkab le bacte riostasis against S. aureus, B. subtilis, B. cereus,
E. coli, P. vu lgaris, B. megathrium , but had no bacterio stasis aga instA. niger andA. flauus. S had remark-
able bacteriostasis againstP. sp. , T. koningii, T. lignorum , S-2 had slight bacteriostasis against T. konin-
gii, T. lignorum , bu t had no bacte riostasis against P. sp. S-1 had nearly no bacteriostasis against bacte ria
and fung .i S-3 had slight bacteriostasis aga inst bac te ria, but had no bacteriostasis against fung.i S-4 had
remarkable bacte riostasis aga inst bacte ria and fung.i S-1-1 had no bac teriostasis againstP. sp. , T. kon ing ii,
S. aureus and E. coli, bu t had slight bac teriostasis aga instB. subtilis, B. megathrium , T. lignorum , and
had remarkab le bacteriostasis againstB. cereus, A. niger, T. koning ii. S-1-2 had slight bacte riostasis against
90　 林　产　化　学　与　工　业 第 26卷
bacteria, bu t had no bacte riosasis against fung i(Tab le 2).
Tab le 2　Bacteriestasis resu lts of extract of persimm on leaves1)
bacteria or fungi S S-1 S-2 S-3 S-4 S-1-1 S-1-2
S. aureus ++ 　
-
++ 　+ ++ 　 - 　+
B. subti lis +++ 　+ +++ ++ ++ 　+
　
+
B. cereus +++ 　
-
+++ 　+ ++ ++ 　+
E. coli. ++ 　
-
++ 　+ 　+ 　 - 　+
P. vulga ris +++ 　+ ++ 　+ ++ 　+
　
+
B. mega thrium +++ 　
-
++ 　+ 　+ 　+ 　+
A. flauu s 　 - 　
-
　 - 　 - +++ 　 - 　
-
A. n iger 　 - 　
-
　 - 　 - +++ ++ 　
-
T. kon ing ii. ++ 　
-
　+ 　 - ++ ++ 　
-
P. sp. ++ 　
-
　 - 　 - +++ 　 - 　
-
T. lignorum ++ 　+ 　+ 　 - ++ 　 -
　
-
1) -:no bacteriostasis ef fect, +:d < 8. 0mm , ++:d = 8. 0-10. 0mm , +++:d > 10mm;con cen tration of S sam ple is 100 g /L;others
are 25 g /L
2. 3　Bacteriostasis effects of extracts under d ifferent concentrations
Test bacterio stasis activities of ace tic ester ex tract under different concentra tions, resu lts showed tha t
acetic e ster ex tract had different bacteriostasis aga inst diffe rent bac te ria under the same concentration, and the
highe r concen tration, the stronger bacte riostasis effect w as, but the sequence of bacte riostasis activities w as
diffe rent under diffe rent concentrations. The sequence of S-2-1 (100 g /L) was:B. subtilis >B. megath-
rium =E. coli>B. cereus>S. aureus>P. vulgaris;the sequence o f S-2-2 (50 g /L) was:E. coli >B.
cereus>B. subtilis>S. aureus>B. mega thrium >P. vulgaris;the sequence o f S-2-3 (25 g /L) was:P.
vulgaris>S. aureus>B. mega thrium >B. subtilis>E. coli =B. cereus;the sequence of S-2-4 (12. 5 g /L)
was:B. subtilis=S aureus>B. mega thrium >E. coli =B. cereus =P. vulgaris(Table 3).
Tab le 3　Bac ter iostasis effects of extracts under d ifferen t concen tration s1) mm
bacteria
S-2-1
100 g /L
S-2-2
50 g /L
S-2-3
25 g /L
S-2-4
12. 5 g /L
con trast
S. aureus 13. 8 13. 1 12. 2 10. 4 0
B. subti lis 18. 7 13. 4 10. 6 10. 4 0
B. cereus 14. 4 13. 8 10. 2 8. 8 0
E. co li. 15. 5 14. 5 10. 2 8. 7 0
P. vulga ris 13. 0 11. 6 12. 7 8. 7 0
B. mega thrium 15. 5 12. 7 11. 1 9. 2 0
1) data in the tab le express d iam eter of b acteriostasis circle
3　Conclusion and discussion
　　Among the ex tracts o f persimmon leavesw ith different pola rities, ex tracts o f e thano l and acetic este r have
significant effect against 6 tested bacteria, but have sligh t effect against fung i;aqueous ex tract is more effec-
tive against 5 tested fungi, and slight against bacteria;pe tro leum e ther ex tract has slight effect against B.
m egathrium and T. lignorum , bu t has no e ffec ts against o thers;bu ty la lcohol ex tract has different effects
against bacteria, the strongest aga instB. subtilis, bu t has no e ffec t against fung.i Resu lts o f analysis show ed
that there a re abundant organic compounds of sapon ins, flavones, coumarins in the ex tract of the persimmon
leaves, wh ich exhib ited strong inhibitory activities. How ever, ex tracts of ethano l, ace tic ester, buty lalcoho l
have no t show n inhibitory activity as anticipa ted, the left aqueous ex tract has ce rtain bacterio static effects
against different bacteria, and strong effects against fung.i Results show ed tha t there must coexist secondary
me tabo lic produc ts w ith contrary effects in persimmon leaves. Furthermo re po lysaccharides in the leavesm ay
have v ital importan t activity, so thatmo re research w orks should be done in the fu tu re.
第 4期 季志平 , 等:柿叶化学成分及其抑菌活性研究 91　
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