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Abstract To evaluate the extraction process of Huidouba polysaccharides （HDBP） and its anti-diabetic activity， this study focused on the two contents as follows： extraction process of HDBP， which was optimized by orthogonal experiments using the extraction ratio of HDBP for the index； and bioactivity evaluation of HDBP on streptozotocin-induced diabetic rat model based on the histological， biochemical and enzyme linked immunosorbent assay （ELISA） method for histological observation， physiological and biochemical index such as fasting blood glucose （FBG）， nitric oxide （NO）， malondialdehyde （MDA）， super oxide dismutase （SOD） and insulin level detection in pancreas tissue respectively. The results showed that HDBP extraction ratio was the highest under following conditions： the extraction temperature at 90 ℃， the extraction time of 1.5 h， and the solid-liquid ratio at 1：10. Compared with the normal control group and the model group， histological observation， physiological and biochemical index and insulin level in pancreas tissue significantly increased and FBG significantly decreased according to the dosage administrated. HDBP affected Streptozotocin-induced diabetic rats.
Key words HDBP； Diabetic rat； Blood glucose； Oxidative stress
Diabetes is a common chronic disease characterized by high blood glucose levels， and its many complications have become a serious threat to human health[1-3]. Type-2 diabetes mellitus （T2DM） is mainly caused by relatively or absolutely insulin hyposecretion and its etiology and pathogenesis have not yet been fully elucidated. The drugs for diabetes mellitus treatment are mainly classified as insulin， insulin secretion enhancers， insulin sensitive agents， insulin secretion accelerant， nitric oxide synthase inhibitors， glyconeogensis inhibitors， the drugs effecting on the absorb of the carbohydrates and traditional Chinese medicine[4]. In recent years， with the emergence of insulin resistance， screening of new natural active ingredients from the traditional Chinese medicine for the treatment of diabetes has become an important way to develop more effective new drugs to treat diabetes. Huidouba is the cobweb secreted by a spider parasitized on the teaplant （Camellia sinensis） of Emei Mountain. In this study， optimum extraction process including the extraction temperature， extraction time and the solid-liquid ratio of HDBP was investigated based on its practical common use for prevention of diabetes and diabetic complications in folk. Meanwhile， the effect of HDBP on body weight， blood glucose， insulin， pancreatic malondialdehyde （MDA）， nitric oxide （NO） and superoxide dismutase （SOD） changes， and pancreatic histopathological changes of streptozotocin-induced diabetic rats were also studied to confirm the action of HDBP on diabetic rats and explore its mechanism to treat diabetes. Materials and Methods
Sample collection and drugs
Huidouba used in this study was purchased from Emei Mountain scenic area， Sichuan； Streptozotocin was purchased from Sigma-Aldrich company （batch number： S0130； and Metformin hydrochloride was purchased from Bristol-Myers Squibb Pharmaceuticals Limited （batch number： H20023370）.
Extraction process and content detection of HDBP
HDBP extraction ratio was chosen for the index to confirm the best extraction method based on the L25 （56） orthogonal experiment and the three factors namely the extraction temperature， the extraction time and the solid-liquid ratio were investigated， and five levels were selected for each factor. In accordance with the orthogonal experimental conditions designed before， 10 g of Huidouba was soaked in flask and extracted after 12 h in water bath， and then centrifuged for 15 min at 5 000 r/min， and the supernatant was concentrated to the 2/5 original volume under the negative pressure， and 3 times the volume of 95% ethanol was added with constant stirring slowly. After standing for 12 h before centrifugation at 8 000 r/min at 4 ℃ for 3 min， the precipitate was collected and washed by ethanol， acetone， and petroleum ether successively. Then， HDBP was dried in microwave oven and polysaccharide content， and the extraction rate were calculated by sulphate-anthrone method. Each experiment was repeated three times.
Diabetic rats model copy and animal groups
A total of 70 male SD rats （200-250 g） were purchased from experimental animal center of west China hospital， Sichuan university， all experimental protocols were approved by the review committee for the use animal subjects of experimental animal center of west China hospital， Sichuan university. After 7 d of adaptive feeding， 60 rats were randomly selected for diabetic rats model copy with 65 mg/kg of streptozotocin solution by intraperitoneal injection， and the remaining 10 rats were treated with the same amount of citrate buffer. Seventy-two hours later， all the rats fasted， were unable to restrain the water for 8 h， blood samples were drawn from orbit for blood glucose determination， and rats with blood glucose over 16.8 mmol/L were designated diabetic rats and 60 rats were randomly divided into six groups with intragastric administration daily in accordance with Table 1. The model group and the normal control group were given the same amount of distilled water， and the positive control group fed with an aqueous solution of Metformin hydrochloride. Detection of FBG and body weight
After administration for 10， 20， 30 and 42 d， all the rats， fasted， were unable to restrain the water for 8 h， and blood samples were drawn from orbit for fasting blood glucose determination using glucose oxidase method. Meanwhile， the body weight of each group was measured respectively.
FINS， ISI and IS detection
After administration for 42 d， all the rats， fasted， were unable to restrain the water for 8 h， and blood samples were drawn from orbit for serum insulin levels detection using insulin ELISA kit according to the instructions. Meanwhile， FBG and FINS were measured， and ISI and IS were calculated according to the formula in reference[5].
Pancreatic MDA， NO and SOD activity
After administration for 42 d， all the rats， fasted， were unable to restrain the water for 8 h， pancreas tissue supernatant was prepared for MDA， NO and SOD detection according to the instructions， and the pancreas tissue supernatant concentration for SOD measurement was 1% and for MDA and NO was 10%.
Histological observation of pancreatic tissues
Pathological changes of pancreatic tissue were observed using paraffin technique with H.E staining.
Data statistics
Values are mean±SD （standard deviation）of three duplicates and SPSS19.0 statistical software is used for data analysis.
Results and analysis
Optimum extraction process of HDBP
The results showed that the extracting temperature was 90 ℃， the extraction time was 1.5 h and the solid-liquid ratio was 1∶10 in the optimum extraction process， which the extraction ratio of HDBP was chosen for the index.
FBG and body weight measurement
Body weights of each group were shown in Table 2. On the 10th d after intragastric administration， body weight of the HDBP treatment groups， the positive control group and the model group were significantly lower than that of the normal control group （P<0.01）. On the 30th day， body weights of HDBP-1 group and the positive control group were significantly higher than the model group （P<0.05）. On the 42th day， body weights of HDBP-1 group， HDBP-2 group and the positive control group were significantly higher than the model group （P<0.05 or P<0.01）， and body weight of HDBP-3 group and HDBP-4 were significantly higher than the model group， but the difference was not significant （P>0.05）. On the 10th day， after intragastric administration， body weight of each group was significantly lower than the body weight before grouping （P<0.05） beside the positive control group. On the 20th， 30th and 42th day after intragastric administration， body weight of each diabetic rat group restored gradually， and on the 42th day， body weight of HDBP-1 group was significantly higher than the 10th day （P<0.05）. During administration， body weight was not different obviously in each HDBP treatment groups （P>0.05） compared with the positive control group. Effect of HDBP on blood glucose in diabetic rats
Fasting glucose was detected on the 10th， 20th， 30th， and 42th day after HDBP treatment respectively， and the glucose levels of each group were shown in Table 3. On the 30th day after intragastric administration， fasting glucose of HDBP-1 group and the positive control group were significantly lower than the model group （P<0.05）. On the 42th day， FBG of HDBP-1 group， HDBP-2 group and the positive control group were significantly lower than the model group （P<0.05 or P<0.01）， and fasting glucose of HDBP-3 group and HDBP-4 were significantly lower than the model group， but the difference was not significant （P>0.05）. During administration， FBG of each HDBP treatment group， the positive control group and the model group increased significantly （P<0.01）. However， FBG in each HDBP treatment group was not different obviously （P>0.05） compared with the positive control group.
FINS， ISI and IS detection
Effect of HDBP on FINS， ISI and IS was shown in Table 4. On the 42th day after intragastric administration， FINS of the model group， each HDBP treatment group and the positive control group decreased significantly compared with the normal control group （P<0.01）. FINS of HDBP-1 group， HDBP-2 group， HDBP-3 group and the positive control group increased significantly （P<0.05 or P<0.01） and HDBP-4 group increased significantly but not different obviously compared with the model group（P>0.05）； FINS of each HDBP treatment groups was not different obviously compared with the positive control group （P>0.05）.
ISI of each HDBP treatment group， the positive control group and the model group decreased significantly compared with the normal control group （P<0.01）. ISI of each HDBP treatment group and the positive control group increased significantly but not different obviously compared with the model group （P>0.05）； and ISI of each HDBP treatment group was not different obviously compared with the positive control group （P>0.05）.
IS of each HDBP treatment group， the positive control group and the model group decreased significantly compared with the normal control group （P<0.01）. IS of each HDBP treatment group and the positive control group increased significantly but not different obviously compared with the model group （P>0.05）； and IS of each HDBP treatment groups was not different obviously compared with the positive control group （P>0.05）.
MDA， NO content and SOD activity Acknowledgements
All authors participated in designing experiments， analysis and interpretation of data equally. Zhihong Huang supervised the work and the manuscript. Chaoxi Chen critical revise the manuscript and approved the final version of manuscript. All authors read and approved the final manuscript.
（a） Normal control group； （b） and （c） Model group；（d） HDBP-1 group and （e） HDBP-2 group.
The number of islets reduced significantly， the islet volume became smaller， and many smaller islets and few medium-sized islets had abnormal or irregular fusiform shape in the microscopic field.
Cell density of islet reduced and cell nuclear was vague or disappeared. Heterochromatin was massive， distributed along the nuclear membrane and the nuclear structure was damaged with the characteristics of nuclear shrink and stained deep with hematoxylin.
The number of normal islets were large， no abnormal cells were present and the cell density was close to the normal group.
Pathological changes in the islet cells were reduced， and the islet cells tended to recover to the normal group
References
[1] EZURUIKE UF， PRIETO JM. The use of plants in the traditional management of diabetes in Nigeria： pharmacological and toxicological considerations[J]. J Ethnopharmacol， 2014， 155（2）： 857-924.
[2] STUMVOLL M， GOLDSTEIN BJ， VAN HAEFTEN TW. Type 2 diabetes： principles of pathogenesis and therapy[J]. Lancet， 2005， 365（9467）： 1333-1346.
[3] VIRALLY M， BLICKL JF， GIRARD J， et al. Type 2 diabetes mellitus： epidemiology， pathophysiology， unmet needs and therapeutical perspectives[J].Diabetes Metab， 2007， 33 （4）：231-244.
[4] ATTELE AS， ZHOU YP， XIE JT， et al. Antidiabetic effects of Panax ginseng berry extract and the identification of an effective component[J].Diabetes，2002， 51（6）： 1851-1858.
[5] HAFFNER SM， KENNEDY E， GONZALEZ C， et al. A prospective analysis of the HOMA model. the mexico city diabetes study[J]. Diabetes Care，1996， 19（10）： 1138-1141.
[6] SAGDIC O. Sensitivity of four pathogenic bacteria to Turkish thyme and wild marjoram hydrosols[J]. Lebensm Wiss Technol， 2003， 36（5）： 467-473.
[7] SIMMONS RA. Developmental origins of diabetes： The role of oxidative stress[J]. Best Pract Res Clin Endocrinol Metab， 2012， 26（5）： 701-708.
[8] MAUVAIS-JARVIS F， KAHN CR. Understanding the pathogenesis and treatment of insulin resistance and type 2 diabetes mellitus： what can we learn from transgenic and knockout mice[J]. Diabetes Metab， 2000， 26（6）： 433-448. [9] THALER JP， CUMMINGS DE. Minireview： Hormonal and metabolic mechanisms of diabetes remission after gastrointestinal surgery[J]. Endocrinology， 2009， 150（6）： 2518-2525.
[10] TERAUCHI Y， IWAMOTO K， TAMEMOTO H， et al. Development of non- insulin-dependent diabetes mellitus in the double knockout mice with disruption of insulin receptor substrate-1 and beta cell glucokinase genes. Genetic reconstitution of diabetes as a polygenic disease[J]. J Clin Invest， 1997， 99（5）： 861-866.
[11] JUNG CH， KIM BY， KIM KJ， et al. Contribution of subcutaneous abdominal fat on ultrasonography to carotid atherosclerosis in patients with type 2 diabetes mellitus[J]. Cardiovasc Diabetol， 2014， 13： 67.
[12] BELLAN M， GUZZALONI G， RINALDI M， et al. Altered glucose metabolism rather than naive type 2 diabetes mellitus （T2DM） is related to vitamin D status in severe obesity[J]. Cardiovasc Diabetol， 2014， 13： 57.
[13] ADAIKALAKOTESWARI A， JAYASHRI R， SUKUMAR N， et al. Vitamin B12 deficiency is associated with adverse lipid profile in Europeans and Indians with type 2 diabetes[J]. Cardiovasc Diabetol， 2014， 13： 129.
Key words HDBP； Diabetic rat； Blood glucose； Oxidative stress
Diabetes is a common chronic disease characterized by high blood glucose levels， and its many complications have become a serious threat to human health[1-3]. Type-2 diabetes mellitus （T2DM） is mainly caused by relatively or absolutely insulin hyposecretion and its etiology and pathogenesis have not yet been fully elucidated. The drugs for diabetes mellitus treatment are mainly classified as insulin， insulin secretion enhancers， insulin sensitive agents， insulin secretion accelerant， nitric oxide synthase inhibitors， glyconeogensis inhibitors， the drugs effecting on the absorb of the carbohydrates and traditional Chinese medicine[4]. In recent years， with the emergence of insulin resistance， screening of new natural active ingredients from the traditional Chinese medicine for the treatment of diabetes has become an important way to develop more effective new drugs to treat diabetes. Huidouba is the cobweb secreted by a spider parasitized on the teaplant （Camellia sinensis） of Emei Mountain. In this study， optimum extraction process including the extraction temperature， extraction time and the solid-liquid ratio of HDBP was investigated based on its practical common use for prevention of diabetes and diabetic complications in folk. Meanwhile， the effect of HDBP on body weight， blood glucose， insulin， pancreatic malondialdehyde （MDA）， nitric oxide （NO） and superoxide dismutase （SOD） changes， and pancreatic histopathological changes of streptozotocin-induced diabetic rats were also studied to confirm the action of HDBP on diabetic rats and explore its mechanism to treat diabetes. Materials and Methods
Sample collection and drugs
Huidouba used in this study was purchased from Emei Mountain scenic area， Sichuan； Streptozotocin was purchased from Sigma-Aldrich company （batch number： S0130； and Metformin hydrochloride was purchased from Bristol-Myers Squibb Pharmaceuticals Limited （batch number： H20023370）.
Extraction process and content detection of HDBP
HDBP extraction ratio was chosen for the index to confirm the best extraction method based on the L25 （56） orthogonal experiment and the three factors namely the extraction temperature， the extraction time and the solid-liquid ratio were investigated， and five levels were selected for each factor. In accordance with the orthogonal experimental conditions designed before， 10 g of Huidouba was soaked in flask and extracted after 12 h in water bath， and then centrifuged for 15 min at 5 000 r/min， and the supernatant was concentrated to the 2/5 original volume under the negative pressure， and 3 times the volume of 95% ethanol was added with constant stirring slowly. After standing for 12 h before centrifugation at 8 000 r/min at 4 ℃ for 3 min， the precipitate was collected and washed by ethanol， acetone， and petroleum ether successively. Then， HDBP was dried in microwave oven and polysaccharide content， and the extraction rate were calculated by sulphate-anthrone method. Each experiment was repeated three times.
Diabetic rats model copy and animal groups
A total of 70 male SD rats （200-250 g） were purchased from experimental animal center of west China hospital， Sichuan university， all experimental protocols were approved by the review committee for the use animal subjects of experimental animal center of west China hospital， Sichuan university. After 7 d of adaptive feeding， 60 rats were randomly selected for diabetic rats model copy with 65 mg/kg of streptozotocin solution by intraperitoneal injection， and the remaining 10 rats were treated with the same amount of citrate buffer. Seventy-two hours later， all the rats fasted， were unable to restrain the water for 8 h， blood samples were drawn from orbit for blood glucose determination， and rats with blood glucose over 16.8 mmol/L were designated diabetic rats and 60 rats were randomly divided into six groups with intragastric administration daily in accordance with Table 1. The model group and the normal control group were given the same amount of distilled water， and the positive control group fed with an aqueous solution of Metformin hydrochloride. Detection of FBG and body weight
After administration for 10， 20， 30 and 42 d， all the rats， fasted， were unable to restrain the water for 8 h， and blood samples were drawn from orbit for fasting blood glucose determination using glucose oxidase method. Meanwhile， the body weight of each group was measured respectively.
FINS， ISI and IS detection
After administration for 42 d， all the rats， fasted， were unable to restrain the water for 8 h， and blood samples were drawn from orbit for serum insulin levels detection using insulin ELISA kit according to the instructions. Meanwhile， FBG and FINS were measured， and ISI and IS were calculated according to the formula in reference[5].
Pancreatic MDA， NO and SOD activity
After administration for 42 d， all the rats， fasted， were unable to restrain the water for 8 h， pancreas tissue supernatant was prepared for MDA， NO and SOD detection according to the instructions， and the pancreas tissue supernatant concentration for SOD measurement was 1% and for MDA and NO was 10%.
Histological observation of pancreatic tissues
Pathological changes of pancreatic tissue were observed using paraffin technique with H.E staining.
Data statistics
Values are mean±SD （standard deviation）of three duplicates and SPSS19.0 statistical software is used for data analysis.
Results and analysis
Optimum extraction process of HDBP
The results showed that the extracting temperature was 90 ℃， the extraction time was 1.5 h and the solid-liquid ratio was 1∶10 in the optimum extraction process， which the extraction ratio of HDBP was chosen for the index.
FBG and body weight measurement
Body weights of each group were shown in Table 2. On the 10th d after intragastric administration， body weight of the HDBP treatment groups， the positive control group and the model group were significantly lower than that of the normal control group （P<0.01）. On the 30th day， body weights of HDBP-1 group and the positive control group were significantly higher than the model group （P<0.05）. On the 42th day， body weights of HDBP-1 group， HDBP-2 group and the positive control group were significantly higher than the model group （P<0.05 or P<0.01）， and body weight of HDBP-3 group and HDBP-4 were significantly higher than the model group， but the difference was not significant （P>0.05）. On the 10th day， after intragastric administration， body weight of each group was significantly lower than the body weight before grouping （P<0.05） beside the positive control group. On the 20th， 30th and 42th day after intragastric administration， body weight of each diabetic rat group restored gradually， and on the 42th day， body weight of HDBP-1 group was significantly higher than the 10th day （P<0.05）. During administration， body weight was not different obviously in each HDBP treatment groups （P>0.05） compared with the positive control group. Effect of HDBP on blood glucose in diabetic rats
Fasting glucose was detected on the 10th， 20th， 30th， and 42th day after HDBP treatment respectively， and the glucose levels of each group were shown in Table 3. On the 30th day after intragastric administration， fasting glucose of HDBP-1 group and the positive control group were significantly lower than the model group （P<0.05）. On the 42th day， FBG of HDBP-1 group， HDBP-2 group and the positive control group were significantly lower than the model group （P<0.05 or P<0.01）， and fasting glucose of HDBP-3 group and HDBP-4 were significantly lower than the model group， but the difference was not significant （P>0.05）. During administration， FBG of each HDBP treatment group， the positive control group and the model group increased significantly （P<0.01）. However， FBG in each HDBP treatment group was not different obviously （P>0.05） compared with the positive control group.
FINS， ISI and IS detection
Effect of HDBP on FINS， ISI and IS was shown in Table 4. On the 42th day after intragastric administration， FINS of the model group， each HDBP treatment group and the positive control group decreased significantly compared with the normal control group （P<0.01）. FINS of HDBP-1 group， HDBP-2 group， HDBP-3 group and the positive control group increased significantly （P<0.05 or P<0.01） and HDBP-4 group increased significantly but not different obviously compared with the model group（P>0.05）； FINS of each HDBP treatment groups was not different obviously compared with the positive control group （P>0.05）.
ISI of each HDBP treatment group， the positive control group and the model group decreased significantly compared with the normal control group （P<0.01）. ISI of each HDBP treatment group and the positive control group increased significantly but not different obviously compared with the model group （P>0.05）； and ISI of each HDBP treatment group was not different obviously compared with the positive control group （P>0.05）.
IS of each HDBP treatment group， the positive control group and the model group decreased significantly compared with the normal control group （P<0.01）. IS of each HDBP treatment group and the positive control group increased significantly but not different obviously compared with the model group （P>0.05）； and IS of each HDBP treatment groups was not different obviously compared with the positive control group （P>0.05）.
MDA， NO content and SOD activity Acknowledgements
All authors participated in designing experiments， analysis and interpretation of data equally. Zhihong Huang supervised the work and the manuscript. Chaoxi Chen critical revise the manuscript and approved the final version of manuscript. All authors read and approved the final manuscript.
（a） Normal control group； （b） and （c） Model group；（d） HDBP-1 group and （e） HDBP-2 group.
The number of islets reduced significantly， the islet volume became smaller， and many smaller islets and few medium-sized islets had abnormal or irregular fusiform shape in the microscopic field.
Cell density of islet reduced and cell nuclear was vague or disappeared. Heterochromatin was massive， distributed along the nuclear membrane and the nuclear structure was damaged with the characteristics of nuclear shrink and stained deep with hematoxylin.
The number of normal islets were large， no abnormal cells were present and the cell density was close to the normal group.
Pathological changes in the islet cells were reduced， and the islet cells tended to recover to the normal group
References
[1] EZURUIKE UF， PRIETO JM. The use of plants in the traditional management of diabetes in Nigeria： pharmacological and toxicological considerations[J]. J Ethnopharmacol， 2014， 155（2）： 857-924.
[2] STUMVOLL M， GOLDSTEIN BJ， VAN HAEFTEN TW. Type 2 diabetes： principles of pathogenesis and therapy[J]. Lancet， 2005， 365（9467）： 1333-1346.
[3] VIRALLY M， BLICKL JF， GIRARD J， et al. Type 2 diabetes mellitus： epidemiology， pathophysiology， unmet needs and therapeutical perspectives[J].Diabetes Metab， 2007， 33 （4）：231-244.
[4] ATTELE AS， ZHOU YP， XIE JT， et al. Antidiabetic effects of Panax ginseng berry extract and the identification of an effective component[J].Diabetes，2002， 51（6）： 1851-1858.
[5] HAFFNER SM， KENNEDY E， GONZALEZ C， et al. A prospective analysis of the HOMA model. the mexico city diabetes study[J]. Diabetes Care，1996， 19（10）： 1138-1141.
[6] SAGDIC O. Sensitivity of four pathogenic bacteria to Turkish thyme and wild marjoram hydrosols[J]. Lebensm Wiss Technol， 2003， 36（5）： 467-473.
[7] SIMMONS RA. Developmental origins of diabetes： The role of oxidative stress[J]. Best Pract Res Clin Endocrinol Metab， 2012， 26（5）： 701-708.
[8] MAUVAIS-JARVIS F， KAHN CR. Understanding the pathogenesis and treatment of insulin resistance and type 2 diabetes mellitus： what can we learn from transgenic and knockout mice[J]. Diabetes Metab， 2000， 26（6）： 433-448. [9] THALER JP， CUMMINGS DE. Minireview： Hormonal and metabolic mechanisms of diabetes remission after gastrointestinal surgery[J]. Endocrinology， 2009， 150（6）： 2518-2525.
[10] TERAUCHI Y， IWAMOTO K， TAMEMOTO H， et al. Development of non- insulin-dependent diabetes mellitus in the double knockout mice with disruption of insulin receptor substrate-1 and beta cell glucokinase genes. Genetic reconstitution of diabetes as a polygenic disease[J]. J Clin Invest， 1997， 99（5）： 861-866.
[11] JUNG CH， KIM BY， KIM KJ， et al. Contribution of subcutaneous abdominal fat on ultrasonography to carotid atherosclerosis in patients with type 2 diabetes mellitus[J]. Cardiovasc Diabetol， 2014， 13： 67.
[12] BELLAN M， GUZZALONI G， RINALDI M， et al. Altered glucose metabolism rather than naive type 2 diabetes mellitus （T2DM） is related to vitamin D status in severe obesity[J]. Cardiovasc Diabetol， 2014， 13： 57.
[13] ADAIKALAKOTESWARI A， JAYASHRI R， SUKUMAR N， et al. Vitamin B12 deficiency is associated with adverse lipid profile in Europeans and Indians with type 2 diabetes[J]. Cardiovasc Diabetol， 2014， 13： 129.