Human Reproduction vol.6 no.l pp.158-165, 1991
Effect of stevioside on growth and reproduction*
Vithaya Yodyingyuad and Supranee Bunyawong Chulalongkorn University Primate Research Centre, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
The effect on growth and reproduction in hamsters of stevioside, which is extracted from stevia leaves (Stevia rebaudiana Bertoni) and is currently used as a non-caloric sweetener, was investigated. Four groups of 20 one-monthold hamsters (10 males and 10 females) were daily force-fed with stevioside (0.0, 0.5, 1.0 and 2.5 g/kg body wt/day, respectively). No abnormalities were found in growth and fertility in both sexes. All males mated females efficiently and successfully. Females showed normal 4-day oestrus cycles and became pregnant after mating. Each female was mated and allowed to bear three litters during the period of experiment. The duration of pregnancy, number of fetuses, as well as number of young delivered each time from females in the experimental groups were not significantly different from those in the control group. The young F] and F2 hamsters continuously receiving stevioside via drinking water until one month old and daily force-fed afterwards at the same doses as their parents showed normal growth and fertility. Histological examination of reproductive tissues from all three generations revealed no evidence of abnormality which could be linked to the effects of consuming stevioside. We conclude that stevioside at a dose as high as 2.5 g/kg body wt/day affects neither growth nor reproduction in hamsters. Key words: contraception/growth/hamster/stevioside
Introduction Stevia rebaudiana Bertoni is a shrub growing in the north-eastern border of Paraguay adjacent to Brazil (Soejarto et al., 1983). It has been used as a sweetener by the natives of that region for centuries (Lee, 1979; Inglett, 1981). The leaves of this plant contain stevioside, a sweet constituent which is 300 times as sweet as sucrose (Isima and Kakayama, 1976) and several other related sweet compounds such as rebaudioside A, rebaudioside B, steviol and steviolbioside (Kohda etal., 1976). Cultivation of Stevia rebaudiana has now spread to the south-east Asian countries including China, Korea, Japan and Thailand. The product is mostly for the Japanese market where stevioside is extracted and This work was funded in part by the Office of Research Affairs, Chulalongkorn University, in 1987. Additional support was provided by the Graduate School of Chulalongkorn University. The work had been presented (Yodyingyuad and Bunyawong, 1988) in preliminary form at the 14th Conference on Science and Technology of Thailand, 1988.
158
sold commercially. It has been used as a non-caloric sweetener in a variety of foods in Japan and Brazil (Sasaki, 1983; Yamada et al., 1985). Apart from its sweetness, the plant has also been reported to possess physiological and therapeutic properties. Some Paraguayan physicians prescribe it as a hypoglycaemic drug and Paraguayan Matto Grosso Indian tribes use it as an oral contraceptive (cited in Planas and Kuc, 1968). There were a few reports on toxicities of Stevia rebaudiana natural products in the literature. In an early report (Vignais et al., 1966) steviol was found to be a potent inhibitor of ATP synthetase in the isolated rat liver mitochondria. Further study (Wingard et al., 1980) suggested the likelihood that stevioside would be converted to steviol in vivo and the converted substance would subsequently be absorbed through the gastrointestinal wall. Recently, metabolically activated steviol was found to exhibit mutagenic activity (Pezzuto et al., 1985) when tested against Salmonella typhimurium. As a result of a combination of these reports, stevioside may be expected to exhibit significant toxicity in humans. However, this expectation differs from many other studies. Oral administration of large quantities of stevia extracts including stevioside revealed no acute or subacute toxic effect on mice (Akashi and Yokoyama, 1975) and rats (Lee et al., 1979; Yamada et al., 1985). It was once reported (Inglett, 1979) that stevioside was non-toxic and passed unchanged through human elimination channels. Moreover, no abnormalities attributable to consumed stevioside were observed on mating, fertility, or the development of fetuses (Mori et al., 1981). Neither did it promote urinary bladder carcinogenesis in rats (Hagiwara et al., 1984). Due to the controversy of these reports, this work was carried out to investigate the effect of stevioside on growth and reproduction in hamsters.
Materials and methods Materials White crystalline powder containing 90% total stevioside (Choli Co. Japan) was dissolved in distilled water to make a syrup at a concentration of 0.5 g/ml for force-feeding purposes. Based on a preliminary observation that female hamsters from day 15 of pregnancy onwards (late pregnancy) and during lactation consumed —20 ml of water per day per animal, subsequent dilutions of 0.5, 1.0 and 2.5 g/20 ml of water were also prepared for these animals in various appropriate groups. Animals and treatments Three successive generations of golden hamsters (Mesocricetus auratus) were used in this study. They were bred in the laboratory © Oxford University Press
Effect of stevioside on growth and reproduction
Table I. Changes of average body weight of three successive generations of male hamsters receiving various doses of stevioside during the period of rapid growth (30-120 days of age) Generation i Dose Body wt2 (g) at certain age (day) (g/kg body wt/day) 30 40 35
45
50
55
60
75
90
105
120
73.2 ± 7.93 69.7 ±566 72.8 ± 6 37 71.8 ± 8.15
79.0 ± 8.52 76.0 ± 6.41 77.1 ± 7.05 77.5 ±971
86.6 ± 8.63 857 ± 7.45 843 ± 8.69 86 5 ± 10.56
92.4 ± 7 89 93.6 ± 8.27 93 2 ± 899 90.6 ± 11.65
107 5 ± 14.38 102.6 ± 10.76 109.2 ± 14.94 103.1 ± 10.67
1139 ± 16.29 107.6 ± 14.13
122.6 ± 16.02 116.8 ± 14.55 130.1 ± 20.01 116.4 ±11.64
128.5 ± 14.12 121.2 ± 15.08 137.2 ± 20.15 123.3 ± 10.12
74.3 ± 4.95 76 3 ± 11.12 82 2 ± 9.32 74 7 ± 13.14
81.1 ± 3.67 85.4 ± 10.53 87.4 ± 10.20 79.8 ± 13 31
87.9 ± 4.65 93 9 ± 11 89 905 ± 1 0 00 85 2 ± 13 68
96.8 ± 6.41 106 7 ± 14.03 100 2 ± 10.33 95 7 ± 10.61
104.4b ± 5.25 118.0 b c d ± 10.34 104 6C ± 10.33 103. l d ± 10.89
108.6b ± 4.62 |225b.cd
114.8b ± 5 14 126.4 b c d
± 8 53
±890
59.5 ± 11.49
70 0 ± 6.18 63.2 ± 1041 76 7 ± 6.82 67 8 ± 1251
109 9C ± 9.64 109 4 d ± 12.05
115.3C ± 10.48 115.6d ± 11.48
60.2 ± 3.58 67.5c-d ± 6 33 57.3d ± 5.66 56.8C ± 13.01
67 0 ± 4.62 74.2c-d ±721 63.l d ± 7.78 62.9C ±1399
72.9 ± 4.86 80 4 c d ± 8.86 68. l d ± 9.10 68.6C ± 14.93
77 2 ± 5 43 85 l c ' d ± 7.45 70 2d ± 8.93 75 2C ± 14.67
81 6 ± 8 47 89 4d ± 8.49 73.7d ± 8.41 80.6 ± 16.56
86.0 ± 6.63 94 1 ± 8 13 82 8 ± 6 23 87 6 ± 14 61
90.8 ± 9 10 99 1
97 6 ± 8.25 104.0 ± 9.40 97 9 ± 5.93 98 0 ± 11.79
104.4
43.0 ± 7 27 43.6 ± 5.48 43.5 ± 5 58 46.7 ± 7 30
52.3 ± 7.01 51 2 ±651 52.4 ± 6 17 51 8
61.9 ± 7.34 61.1 ±664
±680
±690
44.7 ± 6.85 39.4 ± 9.91 43 0 ± 9.00 39.3 ± 7.70
50.2 ± 5.43 48.4 ± 11.16
61 1 ± 6.10 59.4 ± 10.78 67 7 ± 6 73
00
46 l b ± 6 87
Third
05
46.2 C
(F2)
1.0
53 5 ± 3 69 58.4c-d ± 6.19 49 5 d ± 4 14 47.5C ± 9.85
00 First
05
(*o>
1 0 2.5
0.0 Second
0.5
(F,)
1.0 25
± 4 89 44 l c ± 5.26 36. l b c e ± 8.88
2.5
55.5 ± 7.81 51 5 ± 10 51
62.4 ± 6 24 62.1
119.2 ± 19.92 113.9 ± 9.97
± 8 57 91 4 ± 6 69 93 7 ± 12.92
±700 108 3
±709 104 2 ± 6 29 103 4 ± 11.47
•"Mean ± SD from 10 animals. Comparisons are among groups of animals at the same ages, within each generation. b e ~ Figures in the same column with the same superscript are significantly different at the 5% level. Table II. Changes of average body weight of three successive generations of female hamsters receiving various doses of stevioside prior to mating (30-60 days of age) Generation
Dose (g/kg body wt/day)
0.0
Body wr3 (g) at certain age (day)
30
35 b
42.3 C ± 5.93 41 &> ± 5.06 52 2 b ' c ± 8.69
57.9 ± 8.58 53.2 ± 7.15 50.2 d ± 6.94 60.4 c ± 7.57
67.3 ± 7.69 64.3 ± 9.25 62.0 ± 6.75
54.4 b - c ± 11.73 44.8 b ± 5.25 42.7 C ± 5.44 38.4 d ± 10.48 47.4 ± 5.02 43.7 ± 5.54 42.2 ± 5.20 43.6 ± 6.59
40.2
±5 71 First
0.5
(Fo)
10 25
0.0 Second
0.5
1.0 2.5
0.0 Third
0.5
(F 2 )
1.0 2.5
a
40 b
45
50 b
55 b
60 b
66.1 ± 9.00
77.0 ± 7.04 72 2 ± 8.32 68.3 b ± 6.45 73.5 ± 9.79
± 6 34 79.2 ± 8.12 74.3b-c ± 4 99 83.8° ± 6 05
92.5 ± 7.66 87.2 ± 7.83 84.3 b c ± 5.50 91.3C ± 6.96
97 7 ± 6.27 97.8 ± 8.97 919 ± 7.72 97 5 ± 7.98
62.0bc ± 13.82 53.4 ± 7.03 52.0 b ± 5.96 47. T ± 9.55
66.0 ± 14.85 65.8 ± 9.32 64.9 ± 5.36 57.6 ± 12.39
74.4 ± 11.81 75.5 ± 11.15 73 3 ± 7.96 64.9 ± 13 21
80.6 ± 10.67 81.6 ± 12.90 78.2 ± 9.68 75.3 ± 10.82
84.5 ± 10.97 88.0 ± 13.71 84.2 ± 9.68 81.5 ± 10.83
92.5 ± 12.96 93.0 ± 13.90 87.6 ± 10.83 85.1 ± 10.60
54.4
59.7 b ± 4.99 67.7bc ± 5.38 56.7C ± 6.00 59.7 d ± 8.38
68.8 b ± 6.12 76.4bcd ± 5.02 66.4C ± 6.08 65.011 ± 9.12
75.2 ± 5 07 79.5 bc ± 5 08 71.l b ± 7.03 71.4C ± 900
79.6 ± 4.79 83.0 ± 4.99 76.6 ± 8.15 75.7 ± 9.78
87 3 ± 5.10 89.0 ± 5 93 83.9 ± 6.67 81.5 ± 11.86
± 4 01 56.4 ± 7.49 50.4 ± 6.29 52.3 ± 7.86
82.8
Mean ± SD from 10 animals. Comparisons are among groups of animals at the same ages, within each generation. " Figures in the same column with the same superscript are significantly different at the 5% level.
b d
159
V.Yodyingyuad and S.Bunyawong
OF,
12090 • 60 •
30 0
nogo • 60300 120(d)
9030
35
40
45
50
55
60
90
105
120
60-
Age (days) 30-
Fig. 1. Comparison of body wt at the same ages among groups of male hamsters in three successive generations receiving stevioside (a) 0.0, (b) 0.5, (c) 1.0 and (d) 2.5 g/kg body wt/day.
at the Biology Department, Faculty of Science, Chulalongkorn University. These animals were maintained under standard animal house conditions. The room temperature was 25 ± 1 °C with 14 h light (06.00-20.00) and 10 h dark (20.00-06.00). Rodent standard chow (Chareonpokaphan, Thailand) and tap water were provided ad libitum. The experiment was started with 80 one-month-old hamsters, 40 of each sex, weighing between 30 and 50 g. They were divided into one control and three experimental groups. Each group consisted of 10 males and 10 females. Animals of the same sex were kept in groups of five in each stainless-steel cage (18 x 10 x 8 in.). Examination of the oestrus cycle was done every morning. The day on which post-oestrus discharge was found was designated as day 1 of the cycle. From the age of 2 months, each female in heat (3 days after post-oestrus discharge was found) was paired in the evening with a male in the same group and observed for mating. The presence of sperm in the vaginal smear the following morning confirmed that mating was successful, and that day was designated as day 0 of pregnancy. Pregnant females were caged singly. These animals were orally force-fed daily, with either distilled water (control group) or stevioside syrup (experimental groups). Late pregnant and 160
0 30
35
40
45
50
55
60
Age (days)
Fig. 2. Comparison of body wt at the same ages among groups of female hamsters in three sucessive generations receiving stevioside (a) 0.0, (b) 0.5, (c) 1.0 and (d) 2.5 g/kg body wt/day.
lactating females received stevioside via drinking water. The amounts of stevioside given to those in the three experimental groups were 0.5, 1.0 and 2.5 g/kg body wt/day respectively. After giving birth, females were allowed to feed their young for 1 month before being separated for a 2-week rest. Thereafter they were ready for their next mating. Each female was mated to produce a total of three litters. The same numbers of young F, and F2 hamsters (40 animals of each sex from each generation), presumably continuously receiving stevioside via drinking water given to their mother until the time of weaning, were divided and treated with the same procedure. Observation Individual body wts were recorded at 5-day intervals during the period of rapid growth (30-120 days of age). In females, due to variation of the body wt caused by pregnancy, only their body wts before mating (30—60 days of age) were recorded. Regularity of the oestrus cycle of each female was examined every morning. For those females allowed to mate, the presence of sperm in the
Effect of stevioside on growth and reproduction
Table III. Average duration and litter size of the three pregnancies in three successive generations of female hamsters receiving various doses of stevioside Generation
Dose (g/kg body wt/day)
1st pregnancy Duration8
0.0
16.1 ± 0.11
Frist
0.5
16.0 ± 0.00
(Fo)
1.0
16.0 ± 0.00
2.5
16.1
0.0
16.0 ± 0.00
Second
05
16.3 ± 0.16
(F|)
1.0
16.1 ± 0.11
25
16.1 ± 0.11
0.0
16.0 ± 0.00
Third
0.5
16.0 ± 0.00
(F2)
1.0
16.1 ± 0.11
2.5
16.2 ± 0.14
±0.11
2nd pregnancy Duration11
Litter size" (range) 8.2 ± 1 81 (5-10) 7.8 ± 1.62 (5-10) 8.1 ± 1 85 (6-12) 8.5 ± 1.58 (7-12)
16.2 ± 0.14
8.0 ± 1.70 (5-10) 8.6 ± 1.35 (6-10) 8.5 ± 1.90 (4-10) 7.6 ± 2.84 (4-12)
16.2 ± 0.14
16.0 ± 0.00 16.2 ± 0.14 16.1 ± 0.11
160 ± 000 16 1 ±0 11 160 ± 000
16.1 ± 0 11
8.1 ± 1 37 (5-10) 7.7 ± 1.70 (6-11) 7.9 ± 2.28 (4-12) 7.2 ± 1.93 (4-10)
16.3 ± 0.16 16.1 ± 0.11 16.1 ±0.11
Litter sizea (range)
3rd pregnancy Duration"
10.0 ± 2.36 7-13) 7.8 ± 1.87 (4-11) 7.4 ± 1.96 (5-11) 9.2 ± 1.75 (7-12)
16.0 ± 0.00
7.9 ± 2.47 (5-13) 9.4 ± 2.01 (6-12) 8.5 ± 2.22 (5-12) 7.8 ± 1.23 (6-10)
16.1 ± 0.00
7.3 ± 2.31 (4-11) 8.7 ± 2 21 (5-12) 7.9 ± 2 02 (5-11) 7.5 ± 1.84 (5-10)
16.0 ± 0.00 16.2 ± 0.14 16.0 ± 0.00
16.1 ± 0.11 (6-11) 16.0 ± 0.00 16.1 ± 0.11
16.0 ± 0.00 16.1 ±0 11 16.0 ± 0 00 16.1 ± 0 11
Litter sizea (range) 8.9 ± 2.18 (6-12) 7 1 ± 1.45 (5-9) 7.4 ± 1.26 (5-9) 7.7 ± 1.49 (6-10) 7.2 ± 1.32 (5-9) 8.8 ± 1.55 8.9 ± 1.52 (7-12) 8.2 ± 1 81 (6-11) 7 4 ± 1.35 (5-10) 8.3 ± 1.70 (6-11) 7.2 ± 1.93 (4-11) 7.6 ± 2.07 (5-12)
"Mean ± SD from 10 animals. Neither the average duration of pregnancy nor litter size among groups in each generation were significantly different from each other.
vaginal smears was taken as the indication of successful mating. Duration of pregnancy and the number of young from each pregnant animal were recorded. To find out the actual number of fetuses in each pregnancy, separate groups of pregnant Fj and F2 females, of the same numbers and treatments, were laparotomized on day 8 of pregnancy and the number of fetuses in each animal was recorded. Soon after the weaning of the third litter, breeding hamsters of both sexes were killed and their reproductive organs (ovaries, oviducts and uterus from females; testes, epididymes, vas deferens and prostate glands from males) were removed for histological observation. These tissues were fixed in Bouin, sectioned and stained with haematoxylin and eosin. Statistical analysis The values shown in all the tables and figures are expressed as mean ± SD (calculated from the total number of animals in each group) at the age indicated. Analysis of variance was employed to determine any statistical differences between the control and the groups consuming stevioside. Results Growth The increases in body wt during the period of rapid growth are shown in Tables I and II (males and females, respectively). In the first generation (Fo), the average growth of males
Table IV. Average numbei• of fetuses in uteri of day 8 pregnant F, and F2 female hamsters receiving 'various doses of stevioside, at the time of laparotomy Dose (g/kg body wt/day) 0.0 0.5 1.0 2.5
Number of fetuses'1 (range) F,
F2
15.4 ± 0.45 (14-18) 14 9 ± 0.53 (13-16) 15.1 ± 0.46 (13-17) 15.4 ± 0.39 (14-18)
15.0 ± 0.47 (12-17) 15.0 ± 0.42 (13-17) 14.7 ± 0.47 (13-17) 14.8 ± 0.77 (10-18)
a
Mean ± SD from 10 animals. No significant differences among groups were observed.
receiving various doses of stevioside did not differ significantly between each male (Table I). Occasional significant differences of growth were observed in females receiving different doses of stevioside, but the differences were clearly not related to the amount of stevioside consumed. Furthermore, their body wts reached the same level at 60 days of age (Table II). In the second generation (F,), no significant difference of the body wt was observed among groups of males receiving various doses of stevioside until 90 days of age. Thereafter, growth of males in the group receiving stevioside 0.5 g/kg body wt/day was significantly higher than those in the other groups 161
V.Yodyingyuad and S.Bunyawong
animals (Figure lc). Males receiving stevioside 2.5 g/kg body wt/day in all three generations had about the same body wts up to 60 days of age. At 75-120 days of age, growth of the F2 was significantly lower than the F o (P < 0.01), but not the F b except at 120 days of age (Figure Id). The same comparison among female hamsters was carried out. In the control groups, the body weight at 30 days of age of the F) was significantly higher than that of the F o (P < 0.01). However, the F o became significantly heavier than the F2 and/or F, (P < 0.05) at 5 5 - 6 0 days of age (Figure 2a). Females of all three generations which received the same dose of stevioside, either 0.5 or 1.0 g/kg body wt/day, were not significantly different in their body wts (Figure 2b and 2c). Growth of the F| and F2 females in groups receiving stevioside 2.5 g/kg body wt/day were about the same. Their body wts were both occasionally significantly lower than that of the F o (P < 0.05, Figure 2d).
Fig. 3. Implantations at the time of laparotomy on day 8 of pregnancy. (A) F2 control. (B) F2 receiving stevioside 2.5 g/kg body wt/day.
(P < 0.05). Nevertheless, male hamsters receiving stevioside 1.0 and 2.5 g/kg body wt/day grew the same amount as the controls (Table I). Growth of females up to 60 days of age did not differ significantly between the various treatment groups (Table II). In the third generation (F2), males in the control group and those in the groups receiving stevioside 0.5 and 1.0 g/kg body wt/day started off with somewhat higher body wts than those in the group receiving stevioside 2.5 g/kg body wt/day. Up to 55—60 days of age, the body wt of males receiving stevioside 0.5 g/kg body wt/day increased rapidly and was significantly higher than animals receiving 1.0 and 2.5 g/kg body wt/day (P < 0.05), although it was not significantly different from the control. However, at 75—120 days of age, no significant differences of body wts were observed in all groups of male animals (Table I). In females, apart from the significantly higher body wt of those receiving stevioside 0.5 g/kg body wt/day than others at 40—50 days of age, the body wts before and after that time were all roughly the same (Table II). Growth of male hamsters at the same age consuming the same dose of stevioside were compared in the three generations. In the control groups, the body wt of F o hamsters was significantly higher than the F, and F2s at 55-120 days of age, while that of F|S was higher than F2s at 75-120 days of age (Figure la). Males receiving stevioside 0.5 g/kg body wt/day in all three generations grew at about the same rate until 75 days of age. Thereafter, the F,, as well as the F o at 105- 120 days of age, grew significantly better than the F2 (Figure lb). The increases of body wts of F o and F| males receiving 1.0 g/kg body wt/day were about the same until 75 days of age, and both were significantly higher than that of F2s at 45—75 days of age. At 90—120 days of age, body wts of the F| and F 2 hamsters were similar, and both were significantly lower than that of F o 162
Fertility The mating performance of animals in all three generations receiving stevioside up to 2.5 g/kg body wt/day was not different from the controls. Males efficiently mounted the females in heat when they were put together and plenty of spermatozoa could be found in the vaginal smear performed on the following morning in every case. Females showed normal 4-day oestrus cycles and became pregnant after mating. Most litters were delivered on day 16 (except a few cases on day 17) of pregnancy. The average duration of pregnancy as well as the number of young delivered in each group are summarized in Table ITJ. Their litter sizes ranged from four to 13. The average litter size from each group of 10 animals—among various dose groups—varied from seven to 10. Comparisons of the average litter sizes obtained (i) among groups of females obtained of the same generation receiving different doses of stevioside, (ii) among the three pregnancies of each group and (iii) among groups of females of different generations receiving the same dose of stevioside were not significantly different from each other. There were no differences in the average numbers of fetuses among various groups of animals receiving different doses of stevioside at the time of laparotomy on day 8 of pregnancy. The number of implantations ranged from 10 to 18 in the experimental groups, while there were 12-18 in the control group (Table IV; Figure 3). Histology Histological observations of reproductive tissues from all experimental groups, both male and female, did not differ from those of the controls. Spermatogenesis in the seminiferous tubules of males receiving doses of stevioside as high as 2.5 g/kg body wt/day was normal (Figure 4a). Epididymes were full of spermatozoa (Figure 4b). Epithelia lining the mucous membrane of the vas deferens (Figure 4c) and prostate glands (Figure 4d) were both actively producing secretions. In the females, ovaries of all experimental animals contained various stages of follicles and corpora lutea (Figure 5a) as seen in the controls. Their uterine endometrial tissues looked normal (Figure 5b). In the oviducts, ciliated columnar epithelium lining the mucous membrane was observed in all groups (Figure 5c).
Effect of stevioside on growth and reproduction
mj^K^^K..
se
*se
Fig. 4. Cross-sections of various reproductive tissues from male hamsters receiving stevioside 2.5 g/kg body wt/day. Spermatogenesis in the seminiferous tubule was normal (a), spermatozoa were tightly packed in the epididymes (b), epithelia lining the mucous membrane of vas deferens (c) and prostate gland (d) were actively producing secretions, ce, columnar epithelium; cce, ciliated columnar epithelium; sp, spermatozoa; se, secretion.
Discussion Since cyclamates were banned for use as a non-nutritive sweetener in the United States and saccharine was suspected of being a carcinogen in 1980, attempts have been made to find safe, highsweetness quality, non-nutritive sweeteners to replace these substances. Apart from aspartame, stevioside and some of its analogues were found to be potential candidates for application to food (DuBois and Stephenson, 1985). To investigate the effects of stevioside on growth and reproduction, we started our experiments by feeding this substance to the first experimental group of hamsters at 0.5 g/kg body wt/day since it had been shown in an earlier chronic toxicity
study (Yamada et ai, 1985) that stevioside at 550 mg/kg body wt/day taken for 2 years caused no effects in rats. Two other higher dose groups (1.0 and 2.5 g/kg body wt/day, respectively) were designed to find out whether its toxicity could be observed. Results were quite astonishing. Stevioside at a dose as high as 2.5 g/kg body wt/day did not do any harm to these animals. No serious effects of the substance were observed and not a single hamster died of its poisoning. Although the average body wt of females receiving stevioside 1.0 g/kg body wt/day in the first generation was sometimes lower than the others, it was only occasional. There was no decrease of body wt in females receiving stevioside 2.5 g/kg body wt/day; besides, there were no 163
V.Yodyingyuad and S.Bunyawong
en
cl
Fig. 5. Cross-sections of various reproductive tissues from female hamsters receiving stevioside 2.5 g/kg body wt/day, after weaning of the third litter. Various stages of follicular development and functioning copora lutea in the ovary were observed (a), uterine endometrium looked normal (b) and ciliated columnar epithelium lining the mucus membrane of the oviduct was in evidence (c). ce, columnar epithelium; cce, ciliated columnar epithelium; cl, corpus luteum; en, endometrium; f, follicle; st, stroma.
differences of the average body wt among most groups of male hamsters receiving different doses of stevioside. Hence change in body wt (which is an indication of growth) was not related to the amount of stevioside consumed. The occasional higher body wt of F| and F2 animals in groups receiving stevioside 0.5 g/kg body wt/day than other groups in their own generations may lead to the hypothesis that this is a growth stimulating dose for hamsters. However, such a phenomenon was not observed in animals in the first generation, and the average body wts beyond 60 days of age in most groups of animals were at the same level. Thus, it is unlikely that stevioside has any effect on growth. The decreased body wts of animals in the F) and F2 generations did 164
not seem to be the effect of stevioside consumed either, as the body wt of those in the control groups in both generations were also decreased. It was reported (Falconer, 1976) that inbreeding among animals caused poor health. This might be one of the reasons. Nevertheless, results in this study support the earlier finding (Yamada et al., 1985) that stevioside had no effect on growth. The fertility of hamsters was completely unaffected by daily consumption of stevioside at doses as high as 2.5 g/kg body wt/day. Gametogenesis and functions of reproductive tracts were normal in both sexes as judged from their histologies. Both the duration of pregnancy and the number of implanted fetuses were
Effect of stevioside on growth and reproduction
unaffected by the stevioside consumed. The lower number of live young born at term in each litter than the number of implantations observed at the time or laparotomy on day 8 of pregnancy could not be due to the effect of stevioside, as this also occurred in the control animals. We conclude that stevioside at a dose as high as 2.5 g/kg body wt/day affects neither growth nor reproduction in hamsters. If this is true in other mammalian species including human, this substance will be of great benefit to industry and medicine, and can be used more widely as a non-caloric sweetener in a variety of foods and drinks as already seen in Japan and Brazil.
Wingard,R.E.,Jr, Brown,J.P., Enderlin,F.E., Dale,J.A., Hale,R.L. and Seitz.E.T. (1980) Intestinal degradation and absorption of the glycosidic sweeteners stevioside and rebaudioside A. Experientia, 36, 519-520. Yamada,A., Ohgaki,S., Noda.T. and Shimizu,M. (1985) Chronic toxicity study of dietary stevia extracts in F344 rats. J. Food Hyg. Soc. Japan, 26, 169-183. Received on August 14, 1990; accepted on October 26, 1990
Acknowledgements We are grateful to Professor M.R.Puttipongse Varavudhi for his support of this work. Thanks to Professor Somsak Dumronglert and Mr Ekachai Dendeevanich for providing us with the stevioside used in this study.
References Akashi.H. and Yokoyama.S. (1975) Security of dried-leaves extracts of Stevia. Food Industry, 18, 34—43. DuBois,G.E. and Stephenson,R.A. (1985) Diterpenois sweeteners. Synthesis and sensory evaluation of stevioside analogues with improved organoleptic properties. J. Med. Chem., 28, 93—98. Falconer,D.C. (1976) Genetic aspects of breeding method. In Universities Federation for Animal Welfare (UFAW) (5th edn), Handbook on the Care and Management of Laboratory Animals. Churchill Livingstone, Edinburgh, pp. 7—26. Hagiwara,A., Fukushima,S. and Kitaori.M. (1984) Effects of the three sweeteners on rats urinary bladder carcinogenesis initiated by N-butylN-(4-hydroxybutyl)nitrosamine. Gann, 75, 763-768. Inglett,G.E. (1979) Potential intense sweeteners of natural origin. Health Sugar Substitutes (see Food Science and Technology Abstracts, 1981), 184-190. Inglett,G.E. (1981) Sweeteners—a review. Food Technol.,3S, 37-38. Isima,N. and Kakayama,O. (1976) Sensory evaluation of stevioside as a sweetener. Natl. Food Res. Inst., 31, 80—85. Kohda.H.R., Yamasaki,K., Murakami,K. and Tanaka.O. (1976) New sweet diterpene glucosides from Stevia rebaudiana. Phytochemistry, 15, 981-983. Lee.C.K. (1979) Carbohydrate sweeteners: structural requirements for taste. World Rev. Nutr. Diet., 33, 142-197. Lee,S.-J., Lee,K.R., Park,J.R., Kim,K.S. and Tchai,B.S. (1979) Safety of stevioside as a new sweetening source. Korean J. Food Sci. Technol., 11, 224-231. Mori,N., Sakanoue,M., Takeuchi,M., Shimpo,K. and Tanabe.T. (1981) Effect of stevioside on fertility in rats. J. Food Hyg. Soc. Jpn, 22, 409-414. Pezzuto.J.M., Compadre,C.M., Swanson.S.M., Nanayakkara,N.P.D. and Kinghom.A.D. (1985) Metabolically activated steviol the aglycone of stevioside, is mutagenic. Proc. Natl. Acad. Sci. USA, 82, 2478-2482. Planas,G.M. and Kuc,J. (1968) Contraceptive properties of Stevia rebaudiana. Science, 162, 1007. Sasaki,K. (1983) Use of stevioside sweeteners in drinks. New Food Industry, 25, 38-43. Soejarto.D.D., Compadre.C.M., Medon.P.J., Kamath,S.K. and Kinghorn,A.D. (1983) Potential sweetening agents of plant origin n. Field search for sweet-tasting stevia species. Econ. Bot., 37, 71 - 7 9 . Vignais.P.V., Duee.E.D. Vignais.P.M. and Huet,J. (1966) Effects of atractyligenin and its structural analogues on oxidative phosphorylation and on the translocation of adenine nucleotides in mitochondria. Biochim. Biophys. Acta, 118, 465-483.
165
Effect of stevioside on growth and reproduction*
Vithaya Yodyingyuad and Supranee Bunyawong Chulalongkorn University Primate Research Centre, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
The effect on growth and reproduction in hamsters of stevioside, which is extracted from stevia leaves (Stevia rebaudiana Bertoni) and is currently used as a non-caloric sweetener, was investigated. Four groups of 20 one-monthold hamsters (10 males and 10 females) were daily force-fed with stevioside (0.0, 0.5, 1.0 and 2.5 g/kg body wt/day, respectively). No abnormalities were found in growth and fertility in both sexes. All males mated females efficiently and successfully. Females showed normal 4-day oestrus cycles and became pregnant after mating. Each female was mated and allowed to bear three litters during the period of experiment. The duration of pregnancy, number of fetuses, as well as number of young delivered each time from females in the experimental groups were not significantly different from those in the control group. The young F] and F2 hamsters continuously receiving stevioside via drinking water until one month old and daily force-fed afterwards at the same doses as their parents showed normal growth and fertility. Histological examination of reproductive tissues from all three generations revealed no evidence of abnormality which could be linked to the effects of consuming stevioside. We conclude that stevioside at a dose as high as 2.5 g/kg body wt/day affects neither growth nor reproduction in hamsters. Key words: contraception/growth/hamster/stevioside
Introduction Stevia rebaudiana Bertoni is a shrub growing in the north-eastern border of Paraguay adjacent to Brazil (Soejarto et al., 1983). It has been used as a sweetener by the natives of that region for centuries (Lee, 1979; Inglett, 1981). The leaves of this plant contain stevioside, a sweet constituent which is 300 times as sweet as sucrose (Isima and Kakayama, 1976) and several other related sweet compounds such as rebaudioside A, rebaudioside B, steviol and steviolbioside (Kohda etal., 1976). Cultivation of Stevia rebaudiana has now spread to the south-east Asian countries including China, Korea, Japan and Thailand. The product is mostly for the Japanese market where stevioside is extracted and This work was funded in part by the Office of Research Affairs, Chulalongkorn University, in 1987. Additional support was provided by the Graduate School of Chulalongkorn University. The work had been presented (Yodyingyuad and Bunyawong, 1988) in preliminary form at the 14th Conference on Science and Technology of Thailand, 1988.
158
sold commercially. It has been used as a non-caloric sweetener in a variety of foods in Japan and Brazil (Sasaki, 1983; Yamada et al., 1985). Apart from its sweetness, the plant has also been reported to possess physiological and therapeutic properties. Some Paraguayan physicians prescribe it as a hypoglycaemic drug and Paraguayan Matto Grosso Indian tribes use it as an oral contraceptive (cited in Planas and Kuc, 1968). There were a few reports on toxicities of Stevia rebaudiana natural products in the literature. In an early report (Vignais et al., 1966) steviol was found to be a potent inhibitor of ATP synthetase in the isolated rat liver mitochondria. Further study (Wingard et al., 1980) suggested the likelihood that stevioside would be converted to steviol in vivo and the converted substance would subsequently be absorbed through the gastrointestinal wall. Recently, metabolically activated steviol was found to exhibit mutagenic activity (Pezzuto et al., 1985) when tested against Salmonella typhimurium. As a result of a combination of these reports, stevioside may be expected to exhibit significant toxicity in humans. However, this expectation differs from many other studies. Oral administration of large quantities of stevia extracts including stevioside revealed no acute or subacute toxic effect on mice (Akashi and Yokoyama, 1975) and rats (Lee et al., 1979; Yamada et al., 1985). It was once reported (Inglett, 1979) that stevioside was non-toxic and passed unchanged through human elimination channels. Moreover, no abnormalities attributable to consumed stevioside were observed on mating, fertility, or the development of fetuses (Mori et al., 1981). Neither did it promote urinary bladder carcinogenesis in rats (Hagiwara et al., 1984). Due to the controversy of these reports, this work was carried out to investigate the effect of stevioside on growth and reproduction in hamsters.
Materials and methods Materials White crystalline powder containing 90% total stevioside (Choli Co. Japan) was dissolved in distilled water to make a syrup at a concentration of 0.5 g/ml for force-feeding purposes. Based on a preliminary observation that female hamsters from day 15 of pregnancy onwards (late pregnancy) and during lactation consumed —20 ml of water per day per animal, subsequent dilutions of 0.5, 1.0 and 2.5 g/20 ml of water were also prepared for these animals in various appropriate groups. Animals and treatments Three successive generations of golden hamsters (Mesocricetus auratus) were used in this study. They were bred in the laboratory © Oxford University Press
Effect of stevioside on growth and reproduction
Table I. Changes of average body weight of three successive generations of male hamsters receiving various doses of stevioside during the period of rapid growth (30-120 days of age) Generation i Dose Body wt2 (g) at certain age (day) (g/kg body wt/day) 30 40 35
45
50
55
60
75
90
105
120
73.2 ± 7.93 69.7 ±566 72.8 ± 6 37 71.8 ± 8.15
79.0 ± 8.52 76.0 ± 6.41 77.1 ± 7.05 77.5 ±971
86.6 ± 8.63 857 ± 7.45 843 ± 8.69 86 5 ± 10.56
92.4 ± 7 89 93.6 ± 8.27 93 2 ± 899 90.6 ± 11.65
107 5 ± 14.38 102.6 ± 10.76 109.2 ± 14.94 103.1 ± 10.67
1139 ± 16.29 107.6 ± 14.13
122.6 ± 16.02 116.8 ± 14.55 130.1 ± 20.01 116.4 ±11.64
128.5 ± 14.12 121.2 ± 15.08 137.2 ± 20.15 123.3 ± 10.12
74.3 ± 4.95 76 3 ± 11.12 82 2 ± 9.32 74 7 ± 13.14
81.1 ± 3.67 85.4 ± 10.53 87.4 ± 10.20 79.8 ± 13 31
87.9 ± 4.65 93 9 ± 11 89 905 ± 1 0 00 85 2 ± 13 68
96.8 ± 6.41 106 7 ± 14.03 100 2 ± 10.33 95 7 ± 10.61
104.4b ± 5.25 118.0 b c d ± 10.34 104 6C ± 10.33 103. l d ± 10.89
108.6b ± 4.62 |225b.cd
114.8b ± 5 14 126.4 b c d
± 8 53
±890
59.5 ± 11.49
70 0 ± 6.18 63.2 ± 1041 76 7 ± 6.82 67 8 ± 1251
109 9C ± 9.64 109 4 d ± 12.05
115.3C ± 10.48 115.6d ± 11.48
60.2 ± 3.58 67.5c-d ± 6 33 57.3d ± 5.66 56.8C ± 13.01
67 0 ± 4.62 74.2c-d ±721 63.l d ± 7.78 62.9C ±1399
72.9 ± 4.86 80 4 c d ± 8.86 68. l d ± 9.10 68.6C ± 14.93
77 2 ± 5 43 85 l c ' d ± 7.45 70 2d ± 8.93 75 2C ± 14.67
81 6 ± 8 47 89 4d ± 8.49 73.7d ± 8.41 80.6 ± 16.56
86.0 ± 6.63 94 1 ± 8 13 82 8 ± 6 23 87 6 ± 14 61
90.8 ± 9 10 99 1
97 6 ± 8.25 104.0 ± 9.40 97 9 ± 5.93 98 0 ± 11.79
104.4
43.0 ± 7 27 43.6 ± 5.48 43.5 ± 5 58 46.7 ± 7 30
52.3 ± 7.01 51 2 ±651 52.4 ± 6 17 51 8
61.9 ± 7.34 61.1 ±664
±680
±690
44.7 ± 6.85 39.4 ± 9.91 43 0 ± 9.00 39.3 ± 7.70
50.2 ± 5.43 48.4 ± 11.16
61 1 ± 6.10 59.4 ± 10.78 67 7 ± 6 73
00
46 l b ± 6 87
Third
05
46.2 C
(F2)
1.0
53 5 ± 3 69 58.4c-d ± 6.19 49 5 d ± 4 14 47.5C ± 9.85
00 First
05
(*o>
1 0 2.5
0.0 Second
0.5
(F,)
1.0 25
± 4 89 44 l c ± 5.26 36. l b c e ± 8.88
2.5
55.5 ± 7.81 51 5 ± 10 51
62.4 ± 6 24 62.1
119.2 ± 19.92 113.9 ± 9.97
± 8 57 91 4 ± 6 69 93 7 ± 12.92
±700 108 3
±709 104 2 ± 6 29 103 4 ± 11.47
•"Mean ± SD from 10 animals. Comparisons are among groups of animals at the same ages, within each generation. b e ~ Figures in the same column with the same superscript are significantly different at the 5% level. Table II. Changes of average body weight of three successive generations of female hamsters receiving various doses of stevioside prior to mating (30-60 days of age) Generation
Dose (g/kg body wt/day)
0.0
Body wr3 (g) at certain age (day)
30
35 b
42.3 C ± 5.93 41 &> ± 5.06 52 2 b ' c ± 8.69
57.9 ± 8.58 53.2 ± 7.15 50.2 d ± 6.94 60.4 c ± 7.57
67.3 ± 7.69 64.3 ± 9.25 62.0 ± 6.75
54.4 b - c ± 11.73 44.8 b ± 5.25 42.7 C ± 5.44 38.4 d ± 10.48 47.4 ± 5.02 43.7 ± 5.54 42.2 ± 5.20 43.6 ± 6.59
40.2
±5 71 First
0.5
(Fo)
10 25
0.0 Second
0.5
1.0 2.5
0.0 Third
0.5
(F 2 )
1.0 2.5
a
40 b
45
50 b
55 b
60 b
66.1 ± 9.00
77.0 ± 7.04 72 2 ± 8.32 68.3 b ± 6.45 73.5 ± 9.79
± 6 34 79.2 ± 8.12 74.3b-c ± 4 99 83.8° ± 6 05
92.5 ± 7.66 87.2 ± 7.83 84.3 b c ± 5.50 91.3C ± 6.96
97 7 ± 6.27 97.8 ± 8.97 919 ± 7.72 97 5 ± 7.98
62.0bc ± 13.82 53.4 ± 7.03 52.0 b ± 5.96 47. T ± 9.55
66.0 ± 14.85 65.8 ± 9.32 64.9 ± 5.36 57.6 ± 12.39
74.4 ± 11.81 75.5 ± 11.15 73 3 ± 7.96 64.9 ± 13 21
80.6 ± 10.67 81.6 ± 12.90 78.2 ± 9.68 75.3 ± 10.82
84.5 ± 10.97 88.0 ± 13.71 84.2 ± 9.68 81.5 ± 10.83
92.5 ± 12.96 93.0 ± 13.90 87.6 ± 10.83 85.1 ± 10.60
54.4
59.7 b ± 4.99 67.7bc ± 5.38 56.7C ± 6.00 59.7 d ± 8.38
68.8 b ± 6.12 76.4bcd ± 5.02 66.4C ± 6.08 65.011 ± 9.12
75.2 ± 5 07 79.5 bc ± 5 08 71.l b ± 7.03 71.4C ± 900
79.6 ± 4.79 83.0 ± 4.99 76.6 ± 8.15 75.7 ± 9.78
87 3 ± 5.10 89.0 ± 5 93 83.9 ± 6.67 81.5 ± 11.86
± 4 01 56.4 ± 7.49 50.4 ± 6.29 52.3 ± 7.86
82.8
Mean ± SD from 10 animals. Comparisons are among groups of animals at the same ages, within each generation. " Figures in the same column with the same superscript are significantly different at the 5% level.
b d
159
V.Yodyingyuad and S.Bunyawong
OF,
12090 • 60 •
30 0
nogo • 60300 120(d)
9030
35
40
45
50
55
60
90
105
120
60-
Age (days) 30-
Fig. 1. Comparison of body wt at the same ages among groups of male hamsters in three successive generations receiving stevioside (a) 0.0, (b) 0.5, (c) 1.0 and (d) 2.5 g/kg body wt/day.
at the Biology Department, Faculty of Science, Chulalongkorn University. These animals were maintained under standard animal house conditions. The room temperature was 25 ± 1 °C with 14 h light (06.00-20.00) and 10 h dark (20.00-06.00). Rodent standard chow (Chareonpokaphan, Thailand) and tap water were provided ad libitum. The experiment was started with 80 one-month-old hamsters, 40 of each sex, weighing between 30 and 50 g. They were divided into one control and three experimental groups. Each group consisted of 10 males and 10 females. Animals of the same sex were kept in groups of five in each stainless-steel cage (18 x 10 x 8 in.). Examination of the oestrus cycle was done every morning. The day on which post-oestrus discharge was found was designated as day 1 of the cycle. From the age of 2 months, each female in heat (3 days after post-oestrus discharge was found) was paired in the evening with a male in the same group and observed for mating. The presence of sperm in the vaginal smear the following morning confirmed that mating was successful, and that day was designated as day 0 of pregnancy. Pregnant females were caged singly. These animals were orally force-fed daily, with either distilled water (control group) or stevioside syrup (experimental groups). Late pregnant and 160
0 30
35
40
45
50
55
60
Age (days)
Fig. 2. Comparison of body wt at the same ages among groups of female hamsters in three sucessive generations receiving stevioside (a) 0.0, (b) 0.5, (c) 1.0 and (d) 2.5 g/kg body wt/day.
lactating females received stevioside via drinking water. The amounts of stevioside given to those in the three experimental groups were 0.5, 1.0 and 2.5 g/kg body wt/day respectively. After giving birth, females were allowed to feed their young for 1 month before being separated for a 2-week rest. Thereafter they were ready for their next mating. Each female was mated to produce a total of three litters. The same numbers of young F, and F2 hamsters (40 animals of each sex from each generation), presumably continuously receiving stevioside via drinking water given to their mother until the time of weaning, were divided and treated with the same procedure. Observation Individual body wts were recorded at 5-day intervals during the period of rapid growth (30-120 days of age). In females, due to variation of the body wt caused by pregnancy, only their body wts before mating (30—60 days of age) were recorded. Regularity of the oestrus cycle of each female was examined every morning. For those females allowed to mate, the presence of sperm in the
Effect of stevioside on growth and reproduction
Table III. Average duration and litter size of the three pregnancies in three successive generations of female hamsters receiving various doses of stevioside Generation
Dose (g/kg body wt/day)
1st pregnancy Duration8
0.0
16.1 ± 0.11
Frist
0.5
16.0 ± 0.00
(Fo)
1.0
16.0 ± 0.00
2.5
16.1
0.0
16.0 ± 0.00
Second
05
16.3 ± 0.16
(F|)
1.0
16.1 ± 0.11
25
16.1 ± 0.11
0.0
16.0 ± 0.00
Third
0.5
16.0 ± 0.00
(F2)
1.0
16.1 ± 0.11
2.5
16.2 ± 0.14
±0.11
2nd pregnancy Duration11
Litter size" (range) 8.2 ± 1 81 (5-10) 7.8 ± 1.62 (5-10) 8.1 ± 1 85 (6-12) 8.5 ± 1.58 (7-12)
16.2 ± 0.14
8.0 ± 1.70 (5-10) 8.6 ± 1.35 (6-10) 8.5 ± 1.90 (4-10) 7.6 ± 2.84 (4-12)
16.2 ± 0.14
16.0 ± 0.00 16.2 ± 0.14 16.1 ± 0.11
160 ± 000 16 1 ±0 11 160 ± 000
16.1 ± 0 11
8.1 ± 1 37 (5-10) 7.7 ± 1.70 (6-11) 7.9 ± 2.28 (4-12) 7.2 ± 1.93 (4-10)
16.3 ± 0.16 16.1 ± 0.11 16.1 ±0.11
Litter sizea (range)
3rd pregnancy Duration"
10.0 ± 2.36 7-13) 7.8 ± 1.87 (4-11) 7.4 ± 1.96 (5-11) 9.2 ± 1.75 (7-12)
16.0 ± 0.00
7.9 ± 2.47 (5-13) 9.4 ± 2.01 (6-12) 8.5 ± 2.22 (5-12) 7.8 ± 1.23 (6-10)
16.1 ± 0.00
7.3 ± 2.31 (4-11) 8.7 ± 2 21 (5-12) 7.9 ± 2 02 (5-11) 7.5 ± 1.84 (5-10)
16.0 ± 0.00 16.2 ± 0.14 16.0 ± 0.00
16.1 ± 0.11 (6-11) 16.0 ± 0.00 16.1 ± 0.11
16.0 ± 0.00 16.1 ±0 11 16.0 ± 0 00 16.1 ± 0 11
Litter sizea (range) 8.9 ± 2.18 (6-12) 7 1 ± 1.45 (5-9) 7.4 ± 1.26 (5-9) 7.7 ± 1.49 (6-10) 7.2 ± 1.32 (5-9) 8.8 ± 1.55 8.9 ± 1.52 (7-12) 8.2 ± 1 81 (6-11) 7 4 ± 1.35 (5-10) 8.3 ± 1.70 (6-11) 7.2 ± 1.93 (4-11) 7.6 ± 2.07 (5-12)
"Mean ± SD from 10 animals. Neither the average duration of pregnancy nor litter size among groups in each generation were significantly different from each other.
vaginal smears was taken as the indication of successful mating. Duration of pregnancy and the number of young from each pregnant animal were recorded. To find out the actual number of fetuses in each pregnancy, separate groups of pregnant Fj and F2 females, of the same numbers and treatments, were laparotomized on day 8 of pregnancy and the number of fetuses in each animal was recorded. Soon after the weaning of the third litter, breeding hamsters of both sexes were killed and their reproductive organs (ovaries, oviducts and uterus from females; testes, epididymes, vas deferens and prostate glands from males) were removed for histological observation. These tissues were fixed in Bouin, sectioned and stained with haematoxylin and eosin. Statistical analysis The values shown in all the tables and figures are expressed as mean ± SD (calculated from the total number of animals in each group) at the age indicated. Analysis of variance was employed to determine any statistical differences between the control and the groups consuming stevioside. Results Growth The increases in body wt during the period of rapid growth are shown in Tables I and II (males and females, respectively). In the first generation (Fo), the average growth of males
Table IV. Average numbei• of fetuses in uteri of day 8 pregnant F, and F2 female hamsters receiving 'various doses of stevioside, at the time of laparotomy Dose (g/kg body wt/day) 0.0 0.5 1.0 2.5
Number of fetuses'1 (range) F,
F2
15.4 ± 0.45 (14-18) 14 9 ± 0.53 (13-16) 15.1 ± 0.46 (13-17) 15.4 ± 0.39 (14-18)
15.0 ± 0.47 (12-17) 15.0 ± 0.42 (13-17) 14.7 ± 0.47 (13-17) 14.8 ± 0.77 (10-18)
a
Mean ± SD from 10 animals. No significant differences among groups were observed.
receiving various doses of stevioside did not differ significantly between each male (Table I). Occasional significant differences of growth were observed in females receiving different doses of stevioside, but the differences were clearly not related to the amount of stevioside consumed. Furthermore, their body wts reached the same level at 60 days of age (Table II). In the second generation (F,), no significant difference of the body wt was observed among groups of males receiving various doses of stevioside until 90 days of age. Thereafter, growth of males in the group receiving stevioside 0.5 g/kg body wt/day was significantly higher than those in the other groups 161
V.Yodyingyuad and S.Bunyawong
animals (Figure lc). Males receiving stevioside 2.5 g/kg body wt/day in all three generations had about the same body wts up to 60 days of age. At 75-120 days of age, growth of the F2 was significantly lower than the F o (P < 0.01), but not the F b except at 120 days of age (Figure Id). The same comparison among female hamsters was carried out. In the control groups, the body weight at 30 days of age of the F) was significantly higher than that of the F o (P < 0.01). However, the F o became significantly heavier than the F2 and/or F, (P < 0.05) at 5 5 - 6 0 days of age (Figure 2a). Females of all three generations which received the same dose of stevioside, either 0.5 or 1.0 g/kg body wt/day, were not significantly different in their body wts (Figure 2b and 2c). Growth of the F| and F2 females in groups receiving stevioside 2.5 g/kg body wt/day were about the same. Their body wts were both occasionally significantly lower than that of the F o (P < 0.05, Figure 2d).
Fig. 3. Implantations at the time of laparotomy on day 8 of pregnancy. (A) F2 control. (B) F2 receiving stevioside 2.5 g/kg body wt/day.
(P < 0.05). Nevertheless, male hamsters receiving stevioside 1.0 and 2.5 g/kg body wt/day grew the same amount as the controls (Table I). Growth of females up to 60 days of age did not differ significantly between the various treatment groups (Table II). In the third generation (F2), males in the control group and those in the groups receiving stevioside 0.5 and 1.0 g/kg body wt/day started off with somewhat higher body wts than those in the group receiving stevioside 2.5 g/kg body wt/day. Up to 55—60 days of age, the body wt of males receiving stevioside 0.5 g/kg body wt/day increased rapidly and was significantly higher than animals receiving 1.0 and 2.5 g/kg body wt/day (P < 0.05), although it was not significantly different from the control. However, at 75—120 days of age, no significant differences of body wts were observed in all groups of male animals (Table I). In females, apart from the significantly higher body wt of those receiving stevioside 0.5 g/kg body wt/day than others at 40—50 days of age, the body wts before and after that time were all roughly the same (Table II). Growth of male hamsters at the same age consuming the same dose of stevioside were compared in the three generations. In the control groups, the body wt of F o hamsters was significantly higher than the F, and F2s at 55-120 days of age, while that of F|S was higher than F2s at 75-120 days of age (Figure la). Males receiving stevioside 0.5 g/kg body wt/day in all three generations grew at about the same rate until 75 days of age. Thereafter, the F,, as well as the F o at 105- 120 days of age, grew significantly better than the F2 (Figure lb). The increases of body wts of F o and F| males receiving 1.0 g/kg body wt/day were about the same until 75 days of age, and both were significantly higher than that of F2s at 45—75 days of age. At 90—120 days of age, body wts of the F| and F 2 hamsters were similar, and both were significantly lower than that of F o 162
Fertility The mating performance of animals in all three generations receiving stevioside up to 2.5 g/kg body wt/day was not different from the controls. Males efficiently mounted the females in heat when they were put together and plenty of spermatozoa could be found in the vaginal smear performed on the following morning in every case. Females showed normal 4-day oestrus cycles and became pregnant after mating. Most litters were delivered on day 16 (except a few cases on day 17) of pregnancy. The average duration of pregnancy as well as the number of young delivered in each group are summarized in Table ITJ. Their litter sizes ranged from four to 13. The average litter size from each group of 10 animals—among various dose groups—varied from seven to 10. Comparisons of the average litter sizes obtained (i) among groups of females obtained of the same generation receiving different doses of stevioside, (ii) among the three pregnancies of each group and (iii) among groups of females of different generations receiving the same dose of stevioside were not significantly different from each other. There were no differences in the average numbers of fetuses among various groups of animals receiving different doses of stevioside at the time of laparotomy on day 8 of pregnancy. The number of implantations ranged from 10 to 18 in the experimental groups, while there were 12-18 in the control group (Table IV; Figure 3). Histology Histological observations of reproductive tissues from all experimental groups, both male and female, did not differ from those of the controls. Spermatogenesis in the seminiferous tubules of males receiving doses of stevioside as high as 2.5 g/kg body wt/day was normal (Figure 4a). Epididymes were full of spermatozoa (Figure 4b). Epithelia lining the mucous membrane of the vas deferens (Figure 4c) and prostate glands (Figure 4d) were both actively producing secretions. In the females, ovaries of all experimental animals contained various stages of follicles and corpora lutea (Figure 5a) as seen in the controls. Their uterine endometrial tissues looked normal (Figure 5b). In the oviducts, ciliated columnar epithelium lining the mucous membrane was observed in all groups (Figure 5c).
Effect of stevioside on growth and reproduction
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se
*se
Fig. 4. Cross-sections of various reproductive tissues from male hamsters receiving stevioside 2.5 g/kg body wt/day. Spermatogenesis in the seminiferous tubule was normal (a), spermatozoa were tightly packed in the epididymes (b), epithelia lining the mucous membrane of vas deferens (c) and prostate gland (d) were actively producing secretions, ce, columnar epithelium; cce, ciliated columnar epithelium; sp, spermatozoa; se, secretion.
Discussion Since cyclamates were banned for use as a non-nutritive sweetener in the United States and saccharine was suspected of being a carcinogen in 1980, attempts have been made to find safe, highsweetness quality, non-nutritive sweeteners to replace these substances. Apart from aspartame, stevioside and some of its analogues were found to be potential candidates for application to food (DuBois and Stephenson, 1985). To investigate the effects of stevioside on growth and reproduction, we started our experiments by feeding this substance to the first experimental group of hamsters at 0.5 g/kg body wt/day since it had been shown in an earlier chronic toxicity
study (Yamada et ai, 1985) that stevioside at 550 mg/kg body wt/day taken for 2 years caused no effects in rats. Two other higher dose groups (1.0 and 2.5 g/kg body wt/day, respectively) were designed to find out whether its toxicity could be observed. Results were quite astonishing. Stevioside at a dose as high as 2.5 g/kg body wt/day did not do any harm to these animals. No serious effects of the substance were observed and not a single hamster died of its poisoning. Although the average body wt of females receiving stevioside 1.0 g/kg body wt/day in the first generation was sometimes lower than the others, it was only occasional. There was no decrease of body wt in females receiving stevioside 2.5 g/kg body wt/day; besides, there were no 163
V.Yodyingyuad and S.Bunyawong
en
cl
Fig. 5. Cross-sections of various reproductive tissues from female hamsters receiving stevioside 2.5 g/kg body wt/day, after weaning of the third litter. Various stages of follicular development and functioning copora lutea in the ovary were observed (a), uterine endometrium looked normal (b) and ciliated columnar epithelium lining the mucus membrane of the oviduct was in evidence (c). ce, columnar epithelium; cce, ciliated columnar epithelium; cl, corpus luteum; en, endometrium; f, follicle; st, stroma.
differences of the average body wt among most groups of male hamsters receiving different doses of stevioside. Hence change in body wt (which is an indication of growth) was not related to the amount of stevioside consumed. The occasional higher body wt of F| and F2 animals in groups receiving stevioside 0.5 g/kg body wt/day than other groups in their own generations may lead to the hypothesis that this is a growth stimulating dose for hamsters. However, such a phenomenon was not observed in animals in the first generation, and the average body wts beyond 60 days of age in most groups of animals were at the same level. Thus, it is unlikely that stevioside has any effect on growth. The decreased body wts of animals in the F) and F2 generations did 164
not seem to be the effect of stevioside consumed either, as the body wt of those in the control groups in both generations were also decreased. It was reported (Falconer, 1976) that inbreeding among animals caused poor health. This might be one of the reasons. Nevertheless, results in this study support the earlier finding (Yamada et al., 1985) that stevioside had no effect on growth. The fertility of hamsters was completely unaffected by daily consumption of stevioside at doses as high as 2.5 g/kg body wt/day. Gametogenesis and functions of reproductive tracts were normal in both sexes as judged from their histologies. Both the duration of pregnancy and the number of implanted fetuses were
Effect of stevioside on growth and reproduction
unaffected by the stevioside consumed. The lower number of live young born at term in each litter than the number of implantations observed at the time or laparotomy on day 8 of pregnancy could not be due to the effect of stevioside, as this also occurred in the control animals. We conclude that stevioside at a dose as high as 2.5 g/kg body wt/day affects neither growth nor reproduction in hamsters. If this is true in other mammalian species including human, this substance will be of great benefit to industry and medicine, and can be used more widely as a non-caloric sweetener in a variety of foods and drinks as already seen in Japan and Brazil.
Wingard,R.E.,Jr, Brown,J.P., Enderlin,F.E., Dale,J.A., Hale,R.L. and Seitz.E.T. (1980) Intestinal degradation and absorption of the glycosidic sweeteners stevioside and rebaudioside A. Experientia, 36, 519-520. Yamada,A., Ohgaki,S., Noda.T. and Shimizu,M. (1985) Chronic toxicity study of dietary stevia extracts in F344 rats. J. Food Hyg. Soc. Japan, 26, 169-183. Received on August 14, 1990; accepted on October 26, 1990
Acknowledgements We are grateful to Professor M.R.Puttipongse Varavudhi for his support of this work. Thanks to Professor Somsak Dumronglert and Mr Ekachai Dendeevanich for providing us with the stevioside used in this study.
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