Are Cistanchis Tubulosa Extracts Safe in Healthy Food?

Cistanche tubulosa is a dried fleshy stem with scaly leaves of Cistanche tubulosa (Schrenk.) R. Wight, a perennial parasitic herb[1]. Cistanche tubulosa (Schrenk.) R. Wight is a perennial parasitic herb[1]. It has the effects of tonifying the kidney and yang, benefiting the essence and blood, moistening the intestines, and laxative[2], and it is also known as “desert ginseng”. Modern pharmacological studies have also shown that it has various pharmacological effects such as improving renal function, anti-fatigue, lowering blood pressure, anti-aging, and regulating immune function[3-4].

Cistanchis tubulosa has a long history of use in Xinjiang due to its remarkable pharmacological activity. In 2005, Japan's Ministry of Health and Welfare (MHLW) approved its food application. The development of related health food products has high application value. Is Cistanchis tubulosa extract a safe raw material for healthy food, and what is its safety and consumption dose?

In this study, the safety of Cistanchis tubulosa extract was evaluated by acute oral toxicity test, A-mes test, mouse bone marrow polymorphic erythrocyte micronucleus test, mouse spermatozoa deformity test, and teratogeny test, to provide an adequate scientific basis for its development and application.

1.Materials and Methods

1.1 MajorInstruments, Equipment and Reagents

MD200-3 upper dish electronic balance, electronic weighing, CXA Olympus double-pupil microscope, XTL-I type continuous zoom stereomicroscope, 500 paraffin rotary slicer, TS-12H biological tissue automatic dehydrator, BM-VI biological tissue freezing and embedding machine, LEIT220 microscope, Myriad BC-3000 hematocrit analyzer, Myriad BC-2800Vet hematocrit analyzer, Roche-400 automatic biochemistry. Blood cell analyzer, Myers BC-2800Vet blood cell analyzer, Roche-400 automatic biochemistry.

1.2 Methods

1.2.1 Acute Oral Toxicity Test

20 mice of Kunming breed, body mass 18~22 g, half male and half female. Weigh 25 g of Cistanchis tubulosa extract, add sterile distilled water to 100 mL, and then give the extract to the mice twice at intervals of 3h at the rate of 20 mL/kg-bw. After gastric gavage, the mice were observed continuously for 14d, and the toxicity and deaths were recorded.

1.2.2 Micronucleus Test of Mouse Bone Marrow Polycythemia Vera (PCE)

50 mice of Kunming breed, body mass 25~30 g, were randomly divided into 5 groups, half male and half female. The method of oral gavage was used twice at 24h intervals, and the groups of low, medium and high dose of Cistanchis tubulosa extract (1.5, 3.0 and 6.0 g/kg-bw), negative control (sterilized distilled water) and positive control (40mL/kg-bw cyclophosphamide, 10mL/kg-bw intraperitoneal injection) were set up.

 The animals were killed by cervical dislocation 6h after the last administration of the test substance, and the routine sampling and preparation of 10,000 slices were carried out. 1,000 bone marrow polymorphic erythrocytes were counted in each mouse, the number of polymorphic erythrocytes containing micronuclei was observed, and 200 polymorphic erythrocytes were counted, and the ratio of the number of polymorphic erythrocytes to the mature erythrocytes (PCE/RBC) was observed, and the rate of micronuclei was calculated as the rate of micronuclei (‰). Micronucleus rate (‰) = (number of polycytophilic erythrocytes with micronuclei/total number of polycytophilic erythrocytes) × 100%.

1.2.3 Mouse Sperm Deformity Test

Twenty-five male clean-grade Kunming breed mice, body mass 26-33 g, were randomly divided into five groups. The amount of test substance, solution preparation and gavage were the same as that of the micronucleus test, and the positive control (45 mL/kg-bw cyclophosphamide) was injected intraperitoneally at 11.25 mL/kg-bw once a day for 5 consecutive d. 30 days after the last administration of the test substance, the positive control was injected into the abdominal cavity at 11.25 mL/kg-bw. The animals were put to death 30 days after the last administration of the test material, and 5000 spermatozoa with complete structure were counted in the filtrate of the testes, stained with eosin, and the incidence of aberrant spermatozoa (%) was calculated. The incidence of aberrant spermatozoa (%) = (number of aberrant spermatozoa/total number of spermatozoa) × 100%.

1.2.4  Ames Test

The Ames test was performed with identified Salmonella typhimurium histidine-deficient strains TA97, TA98, TA100, TA102, and polychlorinated biphenyl (PCB)-induced rat liver microsomal enzymes (S9). There were five dose groups (0.008, 0.040, 0.200, 1.000, 5.000 mg/dish), spontaneous revertant group, solvent control (distilled water) group and positive control (mutagens) group. Positive agents: - S9: TA97 and TA98 were 2,4,7-trinitrofentanone (0.2 μg/dish), TA100 was sodium azide (1.5 μg/dish), and TA102 was mitomycin C (4 μg/dish). +S9: TA97, TA98 and TA100 were 2-aminophene (10 μg/dish), and TA102 was 1,8-dihydroxyanthraquinone (50 μg/dish). Observe the changes in the number of revertant colonies in each group.

1.2.5 Teratogenic Test

Clean grade SD 100 female rats, body mass 208~380 g, 60 male rats, body mass 302~450 g, mating ratio of 2:1 between male and female.

No less than 12 pregnant rats in each group were given at doses of 0.25, 0.50, and 1.00 g/kg-bw of Cistanchis tubiflora extract on the 7th to 16th day of gestation, each group was gavaged at 10 mL/kg-bw, and the solvent control was distilled water. On the 20th day of gestation, the uterus was dissected out and weighed, the number of stillbirths and live fetuses was recorded and examined, the body mass and length of the fetuses were recorded, and the appearance of the fetuses was examined for any abnormalities. 50% of the fetuses were subjected to skeletal examination, and the other 50% were subjected to visceral examination.

1.3 Statistical Processing

The data were transformed and statistically analyzed using PEMS3.1For Win-dows. A one-way ANOVA was used for overall comparison if the variance of the data was found to agree, and a Dunnett's test was used to compare the dosage groups with the control group if the variance was found to agree. If the variance was not homogeneous, a nonparametric (rank sum test) was used instead. The chi-square test was used for count data. The mouse bone marrow polychromatic erythrocyte micronucleus test and mouse spermatozoa aberration test were performed according to Poisson's distribution with a test level of α = 0.05. The test level was α=0.05.

2.Results

2 . 1 Acute oral Toxicity Test  

After gavage at a dose of 10 g/kg-bw, no adverse signs were observed for 14 d. There were no obvious symptoms of poisoning and no deaths. Anatomical examination of major organs showed no significant abnormal changes. The maximum tolerated dose (MTD) in acute oral toxicity test in both male and female mice was >10 g/kg-bw. The extract of Cistanchis tubulosa is of practically non-toxic grade.

2.2 Mouse Bone Marrow Polymorphophilic Erythrocyte Micronucleus Test

After gavage, the bone marrow micronucleus rate of mice in both male and female dosage groups did not change significantly (the number of PCEs examined was 5000), and the difference between the rate of erythrophilic micronuclei in each dosage group and that of the negative control group was not statistically significant (P > 0.05), while the rate of micronuclei in the positive control group was higher than that of the negative control group, with a statistically significant difference (P < 0.01), i.e., the results of the micronuclei test of Cistanches tuberculosis were negative. The results of the micronucleus test of Cistanche tubiflora extract were negative, as shown in Table 1.

2 . 3 Sperm Deformity Test in Mice

The sperm aberration rate of male mice was not significantly altered by each dose group, and the difference was not statistically significant when comparing with that of the negative control group (P > 0.05), while the sperm aberration rate of the positive control group was higher than that of the negative control group, and the difference was statistically significant (P < 0.01). The difference was statistically significant (P < 0.01), indicating that the extract of Cistanchis tubulosa did not produce any aberrant effect on the spermatozoa of mice, as shown in Table 2.

2.4 Ames Test    

The number of mutant colonies in each dose group was not more than two times of the number of spontaneous mutant colonies, and there was no dose-reflection relationship, while the number of spontaneous mutant colonies in the positive control group was significantly greater than that in the spontaneous mutant colonies. Therefore, no mutagenic effect could be observed in Salmonella typhimurium histolytic acid-deficient strains TA97, TA98, TA100, and TA1024 with or without the addition of S9, as shown in Table 3.

Table 1 Results of the micronucleus test for polychromatic erythrocytes from mouse bone marrow (x- ±s , n=5)

Note: Compared with the negative control group , * P < 0.05.

Table 2 Results of Sperm Deformity Test in Mice

Note: Compared with the negative control group , * P < 0.05.

Table 3 Results of the Ames test (x- ±s)

2 . 5 Teratogenic Test

Comparing the indicators and stillbirth rates of pregnant rats and fetuses in each dose group with those of the control group, the differences were not statistically significant (P > 0.05); no malformations were found in the internal organs of the fetuses in each group, but the skeletal examination of the animals in each dose group and the control group revealed that the sternum of individual fetuses was not fully ossified. According to the evaluation standard of teratogenicity test in GB15193-2003, no teratogenic effect was found in each dose group.

3.Conclusion

To provide a more adequate basis for the development of Cistanchis tubulosa related products, the safety of Cistanchis tubulosa extract was evaluated in this study. The acute oral toxicity test showed that the extract of Cistanchis tubulosa was practically non-toxic with a maximum tolerated dose (MTD) of >10 g/kg-bw; the results of three genotoxicity tests (Bone Marrow Micronucleus Test (BMMT), Sperm Abnormalities Test (SAT), and Ames Test (AMT)) were negative, indicating that the sample was non-mutagenic. The results of three genotoxicity tests (bone marrow micronucleus test, sperm deformity test, and Ames test) were all negative, indicating that the sample had no mutagenic effect. Cistanchis tubulosa extract has good safety and can be used to develop healthy food.

References:

[1] National Pharmacopoeia Commission. National Pharmacopoeia of the People's Republic of China: Part I [S]. 2010 Edition. Beijing: Chemical Industry Press, 2010: 126.

[2] Hou Zhi-Hua, Chang Guo-Wen. Progress of pharmacological studies on Cistanchiakis[J]. Journal of Chinese National Medicine, 2003, 9(4) : 3-4.

[3] JIN Xiulian, ZHANG Qingrong. Progress of Chemical Composition of Cistanchiakia[J]. Chinese Journal of Traditional Chinese Medicine, 1994, 19(11): 695-697.

[4] Li Yong, Xiong Yuanjun, Jia Xiaoguang, et al. Effects of Cistanchis tubulosa extract on blood pressure, erythrocyte membrane fluidity and whole blood viscosity in rats fed with high salt[J]. Natural Products Research and Development, 2009, 21(5): 220-222.