Accepted Manuscript Title: Synergistic effects of Rhizoma Paridis and Rhizoma Curcuma longa on different animal tumor models Author: Zhen Liu Wenyuan Gao Shuli Man Yao Zhang Hongfa Li Shanshan Wu Jingze Zhang Changxiao Liu PII: DOI: Reference:
S1382-6689(14)00111-2 http://dx.doi.org/doi:10.1016/j.etap.2014.04.026 ENVTOX 2003
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
22-10-2013 20-4-2014 24-4-2014
Please cite this article as: Liu, Z., Gao, W., Man, S., Zhang, Y., Li, H., Wu, S., Zhang, J., Liu, C.,Synergistic effects of Rhizoma Paridis and Rhizoma Curcuma longa on different animal tumor models, Environmental Toxicology and Pharmacology (2014), http://dx.doi.org/10.1016/j.etap.2014.04.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synergistic effects of Rhizoma Paridis and Rhizoma Curcuma longa on different animal tumor models
cr
Jingze Zhangc, Changxiao Liud
ip t
Zhen Liua, Wenyuan Gaoa*, Shuli Manb, Yao Zhanga, Hongfa Lia, Shanshan Wua,
a
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of
College of Biotechnology, Tianjin University of Science & Technology, Tianjin
an
b
us
Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China
300193, China
Department of Pharmacy, Medical College of Chinese People’s Armed Police Forces,
M
c
Tianjin 300162, China
Ac ce p
China
d
The State Key Laboratories of Pharmacodynamics and Pharmacokinetics, Tianjin,
te
d
* Corresponding author. Wenyuan Gao. School of Pharmaceutical Science and Technology, Tianjin University, No.92 Weijin Road, Nankai District, Tianjin, 300072, China. E-mail address: [email protected]. Telephone: 86-22-87401895; fax number: 86-22-8740 1895. 1
Page 1 of 29
Abstract Rhizoma Paridis saponins (RPS) with a good antitumor effect in clinical using
ip t
showed low bioavailability and toxicity. Combination of Rhizoma Curcuma longa with RPS, which called LouHuang preparation (LH), not only overcame the RPS
cr
limitations but also improved its anticancer effect. The median lethal dose (LD50) of
us
LH in mice was 3410.9 mg/kg by oral acute toxicity test. LH relieved the inhibition of RPS on the gastric emptying (70.13 ± 4.80% vs. 49.12 ± 8.06%). As for the antitumor
an
effect, the tumor weight/volume inhibition rate, tumor volume growth rate, and
M
water/food efficiency ratio were calculated. LH had the highest inhibition ratio of 57.07 ± 2.97% for H22 model, 43.22 ± 0.72% for S180 model, and 46.8 ± 0.97% for
d
EAC model, which were higher than RPS. Compared to ZiLongJin (ZLJ), a marked
te
antitumor drug in China, LH also had the higher inhibition rate for tumor weight and
Ac ce p
tumor volume growth, which weaker than CTX. The water/food efficiency ratio reflected the difference of the quality life of the mice bearing tumor cells or not. CTX attenuated body weight gain and increased food efficiency ratio compared to control group. LH did not affect the body weight or water/food intake. The active part of LH was RPS and turmeric polysaccharides with the inhibition of 58% and 47% on H22 and S180 tumor models. The research provided theoretical and practical basis for LH application. Keywords: Rhizoma Paridis saponins; Rhizoma Curcuma longa; turmeric polysaccharides; antitumor activity; transplanted tumor; acute toxicity
2
Page 2 of 29
1.
Introduction
Rhizoma Paridis saponins (RPS), the purified mixture of steroidal saponins, have
ip t
been extensive studied on the extraction process, chemical compositions, serum pharmacochemistry and the pharmacological evaluation (Liu et al., 2012; Man et al.,
cr
2011; Man et al., 2009; Man et al., 2013). As for the hypotoxicity and gastric stimulus
us
side effect (Liu et al., 2012), its application was limited. To combat the shortcoming, we selected herb combination according to the Traditional Chinese medicine theory.
an
By systematic screening, we found water extract of Rhizoma Curcuma longa
M
improved the antitumor effect of RPS. We named the combination as LouHuang preparation (LH). In previously reported, the phenomenon was explained from the
d
absorption aspect and proved LH as the potent anticancer agent (Man et al., 2013).
te
The new study revealed that a certain dose of curcuminoids significantly increased
Ac ce p
absorption of RPS in the everted rat duodenum sac system, which supported the antineoplastic effect of LH (Man et al., 2013). Preclinical studies using relevant models are prerequisite to clinical testing of new anti-cancer drugs. For the xenografted tumor models, the relative tumor volume, the tumor growth index, the tumor growth delay index, and tumor growth speed are the criteria for analysis efficiency (Medioni et al., 2012). Only the tumor weight inhibition is used for syngeneic hosts tumor models. To increase analysis efficiency, we added two indexes including tumor growth speed and water/food efficiency ratio in this research. With the tumor inhibition, the antitumor effects of LH were investigated. From the
3
Page 3 of 29
perspective of the toxicity, we also evaluated the acute toxicity and gastric irritating of LH, which provide theoretical and practical basis for LH prospective use.
Materials and methods
ip t
2.
cr
2.1. Plant materials and drugs
us
The dried rhizoma of P. polyphylla Smith var. yunnanensis were collected from Lijiang, Yunnan province, China and the rhizoma of Curcuma longa L. were obtained from Anguo, Hebei province,
an
China. Both of them were identified by Prof. Gao (Tianjin University, China). The voucher specimens (GWCL201309 and GWJH201309) were deposited at the School of Pharmaceutical
M
Science and Technology at Tianjin University, Tianjin, China. Other drugs used in this study were
d
ZiLongJin (ZLJ, Tianjin new pharmaceutical group Limited by Share Ltd longshunrong
te
pharmaceutical factory), 10-Hydroxycamptothecin (HCPT, Wuhan Li Shizhen Pharmaceutical Co.,
Ac ce p
Ltd. China), and cyclophosphamide (CTX, Jiangsu Hengrui Medicine Co., Ltd. China).
2.2. Animals
Adult female Kunming mice (6-8 weeks) with body weight ranging from 18-20 g were purchased from Tianjin Experimental Animal Center (Tianjin, China, License No. SCXK (Jin) 2009-0002). The animals were housed in polycarbonate cages (ten animals in each cage) with white wood chips for bedding, and given free access to food and drinking water, under controlled temperature, humidity and photoperiod. This animal study was approved by the Institutional Animal Care and Use Committee of China, and institutional guidelines for animal welfare and experimental conduct were followed.
4
Page 4 of 29
2.3. Preparation of LH and turmeric polysaccharides Rhizoma Paridis (100 g) and Rhizoma Curcuma longa (100 g) were the formula of LH. Rhizoma
ip t
Paridis was processed as the same method previously reported and obtained the powder (the main compounds were Rhizoma Paridis saponins, RPS) (Liu et al., 2012). Rhizoma Curcuma longa was
cr
decocted three times in boiling water for 2 h (1:8, 1:6 and 1:6). The filtrate of the decoction was
us
concentrated under vacuum to yield the extract powder. LH was the mixture of the Rhizoma Paridis and Rhizoma Curcuma longa powders and was stored at desiccator for future analysis.
an
Turmeric polysaccharides were prepared as the following procedure:
(1) Rhizoma Curcumae Longae (150 g) were crushed into small pieces and were extracted twice
M
by water (1 g : 10 mL) at the temperature of 90°Ϲ (2 hours every time).
d
(2) The extraction was concentrated to a volume 1/10 for the original volume. Then 95% alcohol
te
was added to the extract and made the final concentration of alcohol was 75%. After static night,
Ac ce p
the solution were centrifuged for 20 min at 7500 r/min. Precipitate were obtained and washed with absolute alcohol, acetone and ether successively, and then refrigerate drying. Finally, turmeric polysaccharides were obtained (9 g). The combination of RPS and turmeric polysaccharides was named RTP.
2.4. Acute oral toxicity study in mice Animals were housed in polycarbonate cages (ten animals in each cage) with white wood chips for bedding, and given free access to food and drinking water, under controlled temperature (23 ± 2°Ϲ), humidity (50 ± 10%) and photoperiod (12-h light/dark cycles). Animals were quarantined for 3 days before the experiment and were fasted overnight prior to oral administration. Mice were 5
Page 5 of 29
randomly divided into eight groups. Each group contained 10 mice. The control group received the vehicle (saline solution), and test groups received doses of 1000, 1495, 2236, 2500, 3535,
ip t
4204, 5000 mg/kg body weight of LH by gavage. The general behaviors of mice were observed continuously for 6 h after dosing and thereafter twice daily for mortality and once a day for
cr
clinical signs of toxicity (changes in gait, posture, skin, fur, eyes, or mucous membranes;
us
occurrence of diarrhea, unusual respiratory pattern, somnolence, clonic or tonic movement, etc.) for 14 days (Liu et al., 2012). After the observation period of 14 days, all animals were sacrificed
an
for further gross necropsy and LD50 values and the 95% confidence limits were calculated by
2.5. In vivo transplanted tumor model
M
SPSS software.
d
Three tumors of Hepatocarcinoma 22 (H22) liver ascites tumor, Sarcoma 180 (S180) ascites tumor,
te
and Ehrlich ascites tumor (EAC) in mice were obtain from Tianjin Institute of Traditional Chinese
Ac ce p
Medicine (Tianjin, China). The tumor cells from peritoneal cavity of tumor inoculated mice were suspended in saline solution. Under sterile condition, 0.2 mL of the tumor cells suspension (1×106 cells/mL) was inoculated into the right groin of mice. The mice randomly divided into different groups with 10 mice for each group 24 h later. RPS, LH, RTP and ZLJ were suspended in distilled water, while CTX and HCPT were dissolved in physiological saline. The doses for the different drugs in H22, S180 and EAC tumor models were shown in Table 1. Test samples (RPS, LH RTP and ZLJ) and distilled solution (negative control group) were administrated intragastrically every day. Chemotherapy drugs (positive control group, CTX and HCPT) were given intraperitoneally. During the experiments, the water and food intake were
6
Page 6 of 29
weighted every day, and the length (a) and width (b) of the tumor and body weight of each mouse were measured every 2 days from the third day. The tumor volume was calculated using the
ip t
formula, V=ab2/2. Twelve to fourteen days after the tumor inoculation, all the mice were sacrificed and the whole bodies, the segregated tumor, liver, spleen of the mice were weighted immediately.
cr
The formula of the food (water) efficiency ratio was [Increased weight/Food (water) intake] ×
us
100%. Compared with the control groups, the anti-tumor activity of the tested samples was expressed as an inhibition rate calculated as [(A-B)/A] × 100%, where A and B were the average
an
tumor weight/volume of the model and treated mice, respectively. The tumor growth rate was calculated as [Vn-V3/V3] × 100%, where V3 and Vn was the tumor volume of the third day and n
M
day. The liver and spleen indexes were calculated by the following formula: Liver index (g/kg) = Wliver/Wweight × 1000; Spleen index (g/kg) = Wspleen/Wweight × 1000, where Wliver, Wspleen
te
d
and Wweight were the average liver, spleen, and body weight of the mice (Li et al., 2008).
Ac ce p
2.6. Measurement of gastric emptying (GE) First the phenol red meal was prepared. Phenol red (50 mg; Sigma, St. Louis, MO) was diluted in 100 ml aqueous methylcellulose (1.5%) solution and used as a test meal. Methylcellulose was dispersed in hot water (80°C) under continuous stirring. The solution was then allowed to cool to 35°C, and the phenol red was added. Intensity and duration (5 h) of agitation were kept constant to obtain viscosities solutions. Sarcoma 180 (S180) solid tumor model was established by the above shown method (2.5 part). Fifty mice were randomly divided into five groups: Control group (saline solution), RPS groups (100, 200 mg/kg) and LH (100 mg/kg) were administered by oral route, and cyclophosphamide
7
Page 7 of 29
(CTX, 20 mg/kg) was given intraperitoneally. All the drugs were administrated intragastrically for 14 days. The animals were deprived of food for 24 h prior to the GE measurement, but allowed
ip t
water ad libitum until 2 h before the experiment. Forty minutes after the last drug administration, 0.3 mL phenol red was given orally to each
cr
mouse and the animals were euthanized by cervical dislocation immediately 20 min after gavage.
us
Total stomach was removed after cardiac and pylorus ligated. The stomach was cut into pieces and homogenized with its contents in 25 mL of 0.1 N NaOH. The homogenate was allowed to settle
an
for 1 h at room temperature, and 8 mL of the supernatant was added to 1 mL of 33% of trichloroacetic acid to precipitate proteins. After centrifugation (3000 rpm for 30 min at 4°Ϲ), 2
M
mL of 2 N NaOH were added to the supernatant and the amount of phenol red was determined from the absorbency at 560 nm. This correlates with the concentration of phenol red in the
te
d
stomach, which in turn depends on the gastric emptying. The gastric emptying rate was derived as GE = (1-X/Y)×100. X is the absorbance of phenol red recovered from the stomach of animals
Ac ce p
killed 20 min after test meal. Y is the mean absorbance of phenol red recovered from the stomachs of animals killed at 0 min following test meal (Sallam et al., 2011).
2.7. Structural characterization of turmeric polysaccharides by FTIR and SEM analysis
Fourier transform infrared (FT-IR) spectra were recorded with a Tensor 27 spectrophotometer (Bruker, Germany). The turmeric polysaccharides were blended with KBr powder and pressed into tablets before measurement. The vibrational transition frequencies were reported in wave numbers (cm-1), and the FT-IR spectra were recorded within the interval of 400~4000 cm-1, with a
resolution of 4 cm-1. The morphological feature of turmeric polysaccharides was observed with a 8
Page 8 of 29
scanning electron microscope (ESEM Philips XL-30), and the images were taken at an accelerating voltage of 20 kV.
ip t
2.8. Statistical analysis
cr
Data were expressed as means ± standard error (S.E.M.) or percentage and analyzed for statistical
significance using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Tests were
us
performed using SPSS 17.0 system. P-value less than or equal to 0.05 was considered to be
Results and Discussion
M
3.
an
statistically significant.
3.1. Acute oral toxicity and gastric emptying of LH
d
In Pharmacopoeia of the People’s Republic of China, many poisonous herbs are
te
routinely recorded and described as slightly toxic, toxic and highly toxic. Rhizoma
Ac ce p
Paridis belongs to slightly toxic herbal with the safe dosage of 3-9 g, while Rhizoma Curcuma longa as a homology of medicine and food has no toxicity with the safe dosage of 3-10 g (Pharmacopoeia, 2010). In our previous work, RPS had little toxicity with the LD50 dose 2.1 g/kg and inhibited the gastric of mice (Liu et al., 2012). When combined with Curcuma longa (that is LH), no mortality and treatment-related
adverse clinical signs was observed at 1000 mg/kg. Also at the dose no gross pathological changes were noted in any mice on necropsy. The acute toxicity increased progressively with increasing doses (Table 2). All of the mice were shown hypo-activity, crouching and insensitivity effects after given LH. Some adverse 9
Page 9 of 29
effects like diarrhea appeared immediately after treatment with higher doses. Low temperature and cyanosis around the lips and mouths were observed in the higher dose groups or before the death. For the death mice, gross necroscopy revealed the
ip t
same phenomenon as the previously reported such as air in the stomach, fluid in the
cr
intestinal tract (Liu et al., 2012). For the living mice, the adverse symptoms were
us
relieved in 1-3 days. Finally, no treatment-related gross pathological changes were observed in any organs of the living animals during necropsy after 14 days. The
an
calculated acute toxicity (LD50) of oral administered LH in mice was 3410.9 mg/kg, with 95% confidence limits of 2822.5-4391.7 mg/kg.
M
As for the gastric, gastric emptying of RPS and LH were measured once again (Liu et al., 2012) but using phenol red. Gastric emptying were significantly inhibited by RPS,
te
d
LH, and CTX (P
S1382-6689(14)00111-2 http://dx.doi.org/doi:10.1016/j.etap.2014.04.026 ENVTOX 2003
To appear in:
Environmental Toxicology and Pharmacology
Received date: Revised date: Accepted date:
22-10-2013 20-4-2014 24-4-2014
Please cite this article as: Liu, Z., Gao, W., Man, S., Zhang, Y., Li, H., Wu, S., Zhang, J., Liu, C.,Synergistic effects of Rhizoma Paridis and Rhizoma Curcuma longa on different animal tumor models, Environmental Toxicology and Pharmacology (2014), http://dx.doi.org/10.1016/j.etap.2014.04.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Synergistic effects of Rhizoma Paridis and Rhizoma Curcuma longa on different animal tumor models
cr
Jingze Zhangc, Changxiao Liud
ip t
Zhen Liua, Wenyuan Gaoa*, Shuli Manb, Yao Zhanga, Hongfa Lia, Shanshan Wua,
a
Tianjin Key Laboratory for Modern Drug Delivery & High-Efficiency, School of
College of Biotechnology, Tianjin University of Science & Technology, Tianjin
an
b
us
Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China
300193, China
Department of Pharmacy, Medical College of Chinese People’s Armed Police Forces,
M
c
Tianjin 300162, China
Ac ce p
China
d
The State Key Laboratories of Pharmacodynamics and Pharmacokinetics, Tianjin,
te
d
* Corresponding author. Wenyuan Gao. School of Pharmaceutical Science and Technology, Tianjin University, No.92 Weijin Road, Nankai District, Tianjin, 300072, China. E-mail address: [email protected]. Telephone: 86-22-87401895; fax number: 86-22-8740 1895. 1
Page 1 of 29
Abstract Rhizoma Paridis saponins (RPS) with a good antitumor effect in clinical using
ip t
showed low bioavailability and toxicity. Combination of Rhizoma Curcuma longa with RPS, which called LouHuang preparation (LH), not only overcame the RPS
cr
limitations but also improved its anticancer effect. The median lethal dose (LD50) of
us
LH in mice was 3410.9 mg/kg by oral acute toxicity test. LH relieved the inhibition of RPS on the gastric emptying (70.13 ± 4.80% vs. 49.12 ± 8.06%). As for the antitumor
an
effect, the tumor weight/volume inhibition rate, tumor volume growth rate, and
M
water/food efficiency ratio were calculated. LH had the highest inhibition ratio of 57.07 ± 2.97% for H22 model, 43.22 ± 0.72% for S180 model, and 46.8 ± 0.97% for
d
EAC model, which were higher than RPS. Compared to ZiLongJin (ZLJ), a marked
te
antitumor drug in China, LH also had the higher inhibition rate for tumor weight and
Ac ce p
tumor volume growth, which weaker than CTX. The water/food efficiency ratio reflected the difference of the quality life of the mice bearing tumor cells or not. CTX attenuated body weight gain and increased food efficiency ratio compared to control group. LH did not affect the body weight or water/food intake. The active part of LH was RPS and turmeric polysaccharides with the inhibition of 58% and 47% on H22 and S180 tumor models. The research provided theoretical and practical basis for LH application. Keywords: Rhizoma Paridis saponins; Rhizoma Curcuma longa; turmeric polysaccharides; antitumor activity; transplanted tumor; acute toxicity
2
Page 2 of 29
1.
Introduction
Rhizoma Paridis saponins (RPS), the purified mixture of steroidal saponins, have
ip t
been extensive studied on the extraction process, chemical compositions, serum pharmacochemistry and the pharmacological evaluation (Liu et al., 2012; Man et al.,
cr
2011; Man et al., 2009; Man et al., 2013). As for the hypotoxicity and gastric stimulus
us
side effect (Liu et al., 2012), its application was limited. To combat the shortcoming, we selected herb combination according to the Traditional Chinese medicine theory.
an
By systematic screening, we found water extract of Rhizoma Curcuma longa
M
improved the antitumor effect of RPS. We named the combination as LouHuang preparation (LH). In previously reported, the phenomenon was explained from the
d
absorption aspect and proved LH as the potent anticancer agent (Man et al., 2013).
te
The new study revealed that a certain dose of curcuminoids significantly increased
Ac ce p
absorption of RPS in the everted rat duodenum sac system, which supported the antineoplastic effect of LH (Man et al., 2013). Preclinical studies using relevant models are prerequisite to clinical testing of new anti-cancer drugs. For the xenografted tumor models, the relative tumor volume, the tumor growth index, the tumor growth delay index, and tumor growth speed are the criteria for analysis efficiency (Medioni et al., 2012). Only the tumor weight inhibition is used for syngeneic hosts tumor models. To increase analysis efficiency, we added two indexes including tumor growth speed and water/food efficiency ratio in this research. With the tumor inhibition, the antitumor effects of LH were investigated. From the
3
Page 3 of 29
perspective of the toxicity, we also evaluated the acute toxicity and gastric irritating of LH, which provide theoretical and practical basis for LH prospective use.
Materials and methods
ip t
2.
cr
2.1. Plant materials and drugs
us
The dried rhizoma of P. polyphylla Smith var. yunnanensis were collected from Lijiang, Yunnan province, China and the rhizoma of Curcuma longa L. were obtained from Anguo, Hebei province,
an
China. Both of them were identified by Prof. Gao (Tianjin University, China). The voucher specimens (GWCL201309 and GWJH201309) were deposited at the School of Pharmaceutical
M
Science and Technology at Tianjin University, Tianjin, China. Other drugs used in this study were
d
ZiLongJin (ZLJ, Tianjin new pharmaceutical group Limited by Share Ltd longshunrong
te
pharmaceutical factory), 10-Hydroxycamptothecin (HCPT, Wuhan Li Shizhen Pharmaceutical Co.,
Ac ce p
Ltd. China), and cyclophosphamide (CTX, Jiangsu Hengrui Medicine Co., Ltd. China).
2.2. Animals
Adult female Kunming mice (6-8 weeks) with body weight ranging from 18-20 g were purchased from Tianjin Experimental Animal Center (Tianjin, China, License No. SCXK (Jin) 2009-0002). The animals were housed in polycarbonate cages (ten animals in each cage) with white wood chips for bedding, and given free access to food and drinking water, under controlled temperature, humidity and photoperiod. This animal study was approved by the Institutional Animal Care and Use Committee of China, and institutional guidelines for animal welfare and experimental conduct were followed.
4
Page 4 of 29
2.3. Preparation of LH and turmeric polysaccharides Rhizoma Paridis (100 g) and Rhizoma Curcuma longa (100 g) were the formula of LH. Rhizoma
ip t
Paridis was processed as the same method previously reported and obtained the powder (the main compounds were Rhizoma Paridis saponins, RPS) (Liu et al., 2012). Rhizoma Curcuma longa was
cr
decocted three times in boiling water for 2 h (1:8, 1:6 and 1:6). The filtrate of the decoction was
us
concentrated under vacuum to yield the extract powder. LH was the mixture of the Rhizoma Paridis and Rhizoma Curcuma longa powders and was stored at desiccator for future analysis.
an
Turmeric polysaccharides were prepared as the following procedure:
(1) Rhizoma Curcumae Longae (150 g) were crushed into small pieces and were extracted twice
M
by water (1 g : 10 mL) at the temperature of 90°Ϲ (2 hours every time).
d
(2) The extraction was concentrated to a volume 1/10 for the original volume. Then 95% alcohol
te
was added to the extract and made the final concentration of alcohol was 75%. After static night,
Ac ce p
the solution were centrifuged for 20 min at 7500 r/min. Precipitate were obtained and washed with absolute alcohol, acetone and ether successively, and then refrigerate drying. Finally, turmeric polysaccharides were obtained (9 g). The combination of RPS and turmeric polysaccharides was named RTP.
2.4. Acute oral toxicity study in mice Animals were housed in polycarbonate cages (ten animals in each cage) with white wood chips for bedding, and given free access to food and drinking water, under controlled temperature (23 ± 2°Ϲ), humidity (50 ± 10%) and photoperiod (12-h light/dark cycles). Animals were quarantined for 3 days before the experiment and were fasted overnight prior to oral administration. Mice were 5
Page 5 of 29
randomly divided into eight groups. Each group contained 10 mice. The control group received the vehicle (saline solution), and test groups received doses of 1000, 1495, 2236, 2500, 3535,
ip t
4204, 5000 mg/kg body weight of LH by gavage. The general behaviors of mice were observed continuously for 6 h after dosing and thereafter twice daily for mortality and once a day for
cr
clinical signs of toxicity (changes in gait, posture, skin, fur, eyes, or mucous membranes;
us
occurrence of diarrhea, unusual respiratory pattern, somnolence, clonic or tonic movement, etc.) for 14 days (Liu et al., 2012). After the observation period of 14 days, all animals were sacrificed
an
for further gross necropsy and LD50 values and the 95% confidence limits were calculated by
2.5. In vivo transplanted tumor model
M
SPSS software.
d
Three tumors of Hepatocarcinoma 22 (H22) liver ascites tumor, Sarcoma 180 (S180) ascites tumor,
te
and Ehrlich ascites tumor (EAC) in mice were obtain from Tianjin Institute of Traditional Chinese
Ac ce p
Medicine (Tianjin, China). The tumor cells from peritoneal cavity of tumor inoculated mice were suspended in saline solution. Under sterile condition, 0.2 mL of the tumor cells suspension (1×106 cells/mL) was inoculated into the right groin of mice. The mice randomly divided into different groups with 10 mice for each group 24 h later. RPS, LH, RTP and ZLJ were suspended in distilled water, while CTX and HCPT were dissolved in physiological saline. The doses for the different drugs in H22, S180 and EAC tumor models were shown in Table 1. Test samples (RPS, LH RTP and ZLJ) and distilled solution (negative control group) were administrated intragastrically every day. Chemotherapy drugs (positive control group, CTX and HCPT) were given intraperitoneally. During the experiments, the water and food intake were
6
Page 6 of 29
weighted every day, and the length (a) and width (b) of the tumor and body weight of each mouse were measured every 2 days from the third day. The tumor volume was calculated using the
ip t
formula, V=ab2/2. Twelve to fourteen days after the tumor inoculation, all the mice were sacrificed and the whole bodies, the segregated tumor, liver, spleen of the mice were weighted immediately.
cr
The formula of the food (water) efficiency ratio was [Increased weight/Food (water) intake] ×
us
100%. Compared with the control groups, the anti-tumor activity of the tested samples was expressed as an inhibition rate calculated as [(A-B)/A] × 100%, where A and B were the average
an
tumor weight/volume of the model and treated mice, respectively. The tumor growth rate was calculated as [Vn-V3/V3] × 100%, where V3 and Vn was the tumor volume of the third day and n
M
day. The liver and spleen indexes were calculated by the following formula: Liver index (g/kg) = Wliver/Wweight × 1000; Spleen index (g/kg) = Wspleen/Wweight × 1000, where Wliver, Wspleen
te
d
and Wweight were the average liver, spleen, and body weight of the mice (Li et al., 2008).
Ac ce p
2.6. Measurement of gastric emptying (GE) First the phenol red meal was prepared. Phenol red (50 mg; Sigma, St. Louis, MO) was diluted in 100 ml aqueous methylcellulose (1.5%) solution and used as a test meal. Methylcellulose was dispersed in hot water (80°C) under continuous stirring. The solution was then allowed to cool to 35°C, and the phenol red was added. Intensity and duration (5 h) of agitation were kept constant to obtain viscosities solutions. Sarcoma 180 (S180) solid tumor model was established by the above shown method (2.5 part). Fifty mice were randomly divided into five groups: Control group (saline solution), RPS groups (100, 200 mg/kg) and LH (100 mg/kg) were administered by oral route, and cyclophosphamide
7
Page 7 of 29
(CTX, 20 mg/kg) was given intraperitoneally. All the drugs were administrated intragastrically for 14 days. The animals were deprived of food for 24 h prior to the GE measurement, but allowed
ip t
water ad libitum until 2 h before the experiment. Forty minutes after the last drug administration, 0.3 mL phenol red was given orally to each
cr
mouse and the animals were euthanized by cervical dislocation immediately 20 min after gavage.
us
Total stomach was removed after cardiac and pylorus ligated. The stomach was cut into pieces and homogenized with its contents in 25 mL of 0.1 N NaOH. The homogenate was allowed to settle
an
for 1 h at room temperature, and 8 mL of the supernatant was added to 1 mL of 33% of trichloroacetic acid to precipitate proteins. After centrifugation (3000 rpm for 30 min at 4°Ϲ), 2
M
mL of 2 N NaOH were added to the supernatant and the amount of phenol red was determined from the absorbency at 560 nm. This correlates with the concentration of phenol red in the
te
d
stomach, which in turn depends on the gastric emptying. The gastric emptying rate was derived as GE = (1-X/Y)×100. X is the absorbance of phenol red recovered from the stomach of animals
Ac ce p
killed 20 min after test meal. Y is the mean absorbance of phenol red recovered from the stomachs of animals killed at 0 min following test meal (Sallam et al., 2011).
2.7. Structural characterization of turmeric polysaccharides by FTIR and SEM analysis
Fourier transform infrared (FT-IR) spectra were recorded with a Tensor 27 spectrophotometer (Bruker, Germany). The turmeric polysaccharides were blended with KBr powder and pressed into tablets before measurement. The vibrational transition frequencies were reported in wave numbers (cm-1), and the FT-IR spectra were recorded within the interval of 400~4000 cm-1, with a
resolution of 4 cm-1. The morphological feature of turmeric polysaccharides was observed with a 8
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scanning electron microscope (ESEM Philips XL-30), and the images were taken at an accelerating voltage of 20 kV.
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2.8. Statistical analysis
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Data were expressed as means ± standard error (S.E.M.) or percentage and analyzed for statistical
significance using one-way analysis of variance (ANOVA) followed by Dunnett’s test. Tests were
us
performed using SPSS 17.0 system. P-value less than or equal to 0.05 was considered to be
Results and Discussion
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3.
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statistically significant.
3.1. Acute oral toxicity and gastric emptying of LH
d
In Pharmacopoeia of the People’s Republic of China, many poisonous herbs are
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routinely recorded and described as slightly toxic, toxic and highly toxic. Rhizoma
Ac ce p
Paridis belongs to slightly toxic herbal with the safe dosage of 3-9 g, while Rhizoma Curcuma longa as a homology of medicine and food has no toxicity with the safe dosage of 3-10 g (Pharmacopoeia, 2010). In our previous work, RPS had little toxicity with the LD50 dose 2.1 g/kg and inhibited the gastric of mice (Liu et al., 2012). When combined with Curcuma longa (that is LH), no mortality and treatment-related
adverse clinical signs was observed at 1000 mg/kg. Also at the dose no gross pathological changes were noted in any mice on necropsy. The acute toxicity increased progressively with increasing doses (Table 2). All of the mice were shown hypo-activity, crouching and insensitivity effects after given LH. Some adverse 9
Page 9 of 29
effects like diarrhea appeared immediately after treatment with higher doses. Low temperature and cyanosis around the lips and mouths were observed in the higher dose groups or before the death. For the death mice, gross necroscopy revealed the
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same phenomenon as the previously reported such as air in the stomach, fluid in the
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intestinal tract (Liu et al., 2012). For the living mice, the adverse symptoms were
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relieved in 1-3 days. Finally, no treatment-related gross pathological changes were observed in any organs of the living animals during necropsy after 14 days. The
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calculated acute toxicity (LD50) of oral administered LH in mice was 3410.9 mg/kg, with 95% confidence limits of 2822.5-4391.7 mg/kg.
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As for the gastric, gastric emptying of RPS and LH were measured once again (Liu et al., 2012) but using phenol red. Gastric emptying were significantly inhibited by RPS,
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d
LH, and CTX (P
