全 文 ：云南大学学报 (自然科学版) 1998 , 20 (3):166 ～ 169 CN 53-1045/N　ISSN 0258-7971
Journal of Yunnan University
Studies on The Antifertility Constituent of Hibiscus
rosa-sinensis L.(HR-1)①
Tu Yuanquan1)　Zhao Cuilan2)　Jiang Yan2)　Cui Yueli2)
(1)Kunming Station of Blood Center , 650011 , Kunming , PRC;
2)Department of Biology of Yunnan University , 650091 , Kunming , PRC)
Abstract　the petroleum ether Extract of Hibiscus rosa-sinensis flowers(HR-1)w as administrated at five differ-
ent dose levels(2 , 4 , 10 , 100 , 1 000 mg/(kg·d)from day 7 ～ 9 of gestation.The H R-1 caused reg ression and/o r re-
sorption of the fetuses.The early-pregnancy terminating effects was associated with a significant fall in peripheral plas-
ma levels of progesterone(P)and estradiol(E2), as measured by radioimmunoassay(RIA)on day 12.Histology exami-
na tion of the ovary exhibited signs of luteolysis.Luteoly sis and consequent fall of P and E2 had been suggested as the
plausible cause of termination of pregnancy.
Key words　Hibiscus rosa-sinensis flowers , mouse , early-pregnancy terminating , progesterone(P), estradio l(E2),
ovary
Considering some reports that ext racts of Hibiscus rosa-sinensis L.flowers have many-sided antifertility
effects(1 ～ 2), we undertook the project to investigate the inf luence of the ether ext ract , the most effective part
w ith reference to antifert ility effect(3).In earlier communication , our pro ject-group repo rted the antifertility
effect of HR-1 in vitro(4 ～ 5).Now we planed to study the ef fect as well as mode of action of HR-1 , during
early stage of gestation in animal models systematicly.Corpora luteal , est radiol and progesterone w ould be
studied on mechanism.
1　Materials and methods
1.1　Animals　Kunming mice weighing 25±3g were maintained under natural light and temperature , and
allow ed free access to balanced food and w ater.
1.2 　Drugs　Hibiscus rosa-sinensis f lowers w ere collected from south of Yunnan , preserved af ter ai r-dry.
Dried f low ers w ere ext racted wi th ethanol , concentrated and added water , then ex tracted by pet roleum ether.
Product ion HR-1 were inspected by thin layer chromatog raphy(TLC)and the major contents are chlorophy ll ,
analogy of β-sitosterol and fat acid , etc.
1.3　Experiment of early-pregnancy terminating　Female mice were caged w ith males , of proven fert ility in
the ratio of 2:1.The day on which vaginal plug w as obtained w as considered as day 1 of pregnancy.Pregnant
mice w ere allocated to 6 groups at random.HR-1 w as dissolved in 10% tween-20 and diluted to the desired
concentration.They received 10% tw een-20 and dist illed water(control)o r the drag at the dose levels of 2 , 4 ,
10 ,100 , 1 000 mg/kg .d from day 7 ～ 9.They w ere laparotomized and examined the condition of embryos
(based on size and g ross appearance)on day 12.The number of embryos w as recorded.
① The project is supported by the Applied Basic Research Foundation of Yunnan Province.Received 1998-03-02
1.4　Experiment of radioimmunoassay of estradiol and progesterone　Before laparotomized , the mice′s blood
w as collected in heparinized tubes by eye bottom puncture.Plasma was gotten after cent rifugation and stored
at -20℃.Plasma level of P and E2 w ere measured by RIA follow ing the methods described on the directions.
1.5　Experiment of microtomy of ovaries　At the time mice w ere laparotomized , the ovaries w ere dissected
out , trimmed of ex traneous tissues , fixed in Bouin′s fluid , processed for staining with hematoxy lin-eosin.His-
tology of the corpora lutea w as examined under microscope.
1.6　Analysis of data　Data was analyzed by student′s t test and a probability level of P <0.05 w as consid-
ered as statistically significant.
2　Results
2.1　Result 1　Results are showed in Table 1.
Tab 1.Effect of HR-1 on early pregnancy of mouse
concentr-
ation
mg/(kg·d)
pregnant
mice
(No.)
total
embryos
(No.)
total dead
embryos
(No.)
ra te of
dead
embryo s
/ %
total njured
embryos
(No.)
rate of
injured
embryos
(No.)
rate of
antifertility
/ %
average
embryos
(No.)
average nomal
embryos
(No.)
P value
0 12 132 6 45 — — 45 11.00±0.97 10.50±0.89 —
2 11 117 10 85 27 231 316 10.64±0.88 8.45±1.34 >0.05
4 11 128 29 226 36 281 50.76 11.54±0.56 5.64±1.69 >0.05
10 14 153 54 353 34 222 57.5 10.93±0.56 4.64±1.34 >0.05
100 10 109 73 670 20 183 853 10.90±0.85 1.60±0.89 <0.01
1000 10 101 72 713 26 257 970 10.10±0.66 1.20±0.73 <0.01
The morphological ex amination of the embryos showed that the HR-1 treated mice had embryos at differ-
ent stages of resorption , in comparison to normal healthy implantation sites in control group.In 100 and 1000
mg/(kg·d)g roup , a number of mice′s pregnancy terminated.While in some o thers although no degenerative
changes were apparent , size of the embryos appeared comparatively smaller and black spots appeared in many
of the inter-embryonic junctions.There w ere no statistical differences between treated g roups.
2.2　Result 2　The results showed in Table 2
Tab 2.The ef fects of HR-1 on plasma levels of estradiol and progesterone in early pregnant mice
concentration
mg/(kg·d)
mice
(No.)
E2
(pg/ ml) P value
mice
(No.)
P
(ng/ ml) P value
0 6 210.83±16.00 — 7 15.49±0.91 —
2 6 169.17±15.13 >0.05 5 13.16±0.68 >0.05
4 5 110.00±16.78 <0.05 6 9.23±0.65 <0.05
10 6 101.25±10.70 <0.01 8 8.90±0.92 <0.01
100 5 80.50±6.76 <0.01 5 4.50±0.85 <0.001
1 000 6 67.92±7.59 <0.01 7 3.70±0.44 <0.001
167第 3 期　　　　　　　　　　　　　　涂源泉等:扶桑花抗生育活性成分 HR-1 的研究　　　　　　　　　　　　　　
Compared with the control , the levels in treated groups all reduced.AsHR-1 doses rose , plasma levels of E2 and
P fell.Except the 2 mg/(kg·d)group , there w ere significant drop of E2 content.For P levels , the t reated
g roups of 100 and 1 000 mg/(kg·d)fell very significant ly (P<0.001).
2.3　Result 3　The results showed in Fig.1
Fig 1.The ef fects of HR-1 on corpora lutea
a Con trol　　b Treated w ith 10 mg/(kg·d)
The histological picture(Fig.1)of the control ovary show ed presence of large corpora lutea ,w ith prominent
nuclei in the well-developed lutein cells.But the corpora lutea of the ex tract-t reated groups revealed typical
signs of regression.It w as characterized by leucocy tic and connective tissue infilt ration , foamy appearance of
the cytoplasm , vague cellular outline and disappearance of column-like arrangement of lutea cells.
3　Discussion
Earlier studies have wi tnessed the contraception ef fect of the ex tract of Hibiscus rosa-sinensis flow ers in
vi tro.Present investigation w as undertaken to assess the effect in vivo.The ex tract HR-1 resulted in the termi-
nat ion of pregnancy in 97% of the 1 000mg/(kg·d)treated mice.Clearly the f low er of Hibiscus rosa-sinensis
is a prospective antifertility medicine.The dose maybe high , but in our later study , a w hi te cry stallize HR-5
had been purified from HR-1.The activity of HR-5 w as higher than HR-1.Through further progress , it can be
say analogy of β-Si tosterol is the majo r active material of HR-1.
Regular development of all the events leading to pregnancy , is chief ly under the direct command of est ro-
gen-progesterone interplay at the cellular level.Extracts of Hibiscus rosa-sinensis f low ers have been reported to
have ant iest rogenic effect (6 ～ 7).Estrogen has been credi ted w ith it s abili ty to increase the progesterone re-
ceptor concentrat ion in mammalian uterus.Progesterone is designated as “ the hormone of pregnancy.”In ab-
sences of P most gestation could never be maintained.In our experiments , the failure of pregnancy maybe due
to the signif icantly decreases of E2 and P content of the HR-1-t reated mice.P and E2 are secreted f rom ovary.
Normal function of corpus lutea is the essent ial facto r to maintain the pregnancy.Luteoly tic degeneration of
the ex tract-t reated ovary w as evident by histological picture of the ovary.This luteolysis evidence may be con-
sidered as the causal facto r behind antifertili ty effect of HR-1.
What mechanisms were be involved behind ex t ract-induced luteolysis remains to be determined , LH ,pro-
lactin and FSH have been claimed to constitute the luteot ropic complex in pregnant mouse.In vitro , HR-1 can
reduce the secretion of HCG of cultured human villa s membrane.There w as some homology betw een LH and
HCG.On male rat , the benzene ext ract had contraception ef fects through pi tuitary.So it may be assumed that
168 　　　　　云南大学学报(自然科学版)　　　　　　　　　　　　　　　　第 20 卷
by w ay of action on pitui tary ,HR-1 induced it ef fect by interfered w ith the funct ion of lutea , low ering the pe-
ripheral level of P and E2 and thus terminated the early-pregnancy.
Acknowledgment Thanks to M r.Li Kaiyuan and Ms.Zhou M ingdong fo r their technical assistance.
References
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扶桑花抗生育活性成分 HR-1的研究
涂源泉1)　　赵翠兰2)　　江　燕2)　　崔悦礼2)
(1)昆明市中心血站 , 昆明 , 650011;2)云南大学生物系 ,昆明 , 650091;第一作者 24岁 , 女 ,硕士)
摘要　本文研究了扶桑花(Hibiscus rosa-sinensis)石油醚溶物 HR-1的抗生育活性.对妊娠早期小鼠
灌胃 HR-1(剂量为 2 ,4 , 10 ,100 ,1 000 mg/(kg·d)可抗早孕;放免测定血清 E2 和 P 表明 ,给药组 E2 和 P
均比对照组水平降低 ,可能是参与了抗早孕作用;卵巢组织学观察说明黄体在给药后受损.
关键词　扶桑花 ,石油醚提取物 ,小鼠 ,抗早孕 ,雌二醇(E2),孕酮(P),黄体
分类号　Q492.5
169第 3 期　　　　　　　　　　　　　　涂源泉等:扶桑花抗生育活性成分 HR-1 的研究