Characterization and optimization of lyophilization and storage conditions of Leech saliva extract from the tropical leech Hirudinaria manillensis.

The medicinal Malaysian leeches have been used in traditional medicine to treat many different ailments. In this study, leech saliva extract (LSE) was collected from the medicinal Malaysian leech Hirudinaria manillensis. Gel electrophoresis of LSE was carried out to estimate the peptide and protein molecular weights of its content. Results showed that LSE contains more than 60 peptides and proteins with molecular masses ranging from 1.9-250kDa. Thrombin time assay in vitro was employed to assess the collected LSE antithrombin activity. First, to study its stability, LSE was lyophilized under the following different conditions: pre-freezing temperature, type of container and lyophilization cycle. Pre-freezed LSE sample at -20°C and lyophilized for 24 hours retained about 100-95% of its original biological activities. Second, the LSE antithrombin activity was monitored for a period of six months. Storage temperature, type of the container and photosensitivity effects on antithrombin activity of the lyophilized (solid state) and non-lyophilized (liquid state) were investigated. Results showed that storage temperature drastically affected the biological activity of LSE with -20 °C as the optimum temperature. Samples stored at ambient temperature and +4 °C were light photosensitive and adversely affected when stored in polypropylene tubes. Lyophilized samples were more stable than non-lyophilized ones over the period of study. To sum up, in order to have a biologically active stock of LSE, it has to be lyophilized for no more than 24 hours following freezing at -20°C and has to be stored at -20°C in glass tubes protected from light.

[The standard operating procedure of the standardization breeding for Poecilobdella manillensis].

OBJECTIVE: To prepare Standard Operating Procedure (SOP) for the artificial breeding technology of Poecilobdella manillensi. METHODS: The environment of producing area and breeding technology, the control of diseases, reserving seeds for breeding, harvesting and processing,the standard of the product quality and supervision for the standardization breeding of Poecilobdella Manillensis was studied. RESULTS: The average yield of vital specimen of Poecilobdella Manillensis was 420.88 kg/mu, the natural hirudin in 1kg of the living specimen of Poecilobdella Manillensis contained 430,000 AT-U, and the quality index and hygienic standard of all products complied with the regulations and standards of the State. CONCLUSION: SOP is applicable for the breeding of Poecilobdella manillensi for Guangxi district.

[Study on formulation of naoxuekang dispersible tablets].

OBJECTIVE: To study the formulation of Naoxuekang dispersible tablets. METHODS: The formulation was determined by pre-processing leech extractum prior to a series of experiments used to screen excipients like bulking agents and disintegrants and so on, and by adding disintegrants within and without. RESULTS: Microcrystalline cellulose was determined as the bulking agent, and carboxymethyl cellulose and low-substituted hydroxypropyl cellulose were determined as the disintegrants of Naoxuekang dispersible tablet formula. The average disintegration time and nitrogen content of one tablet were 52 seconds and 5.47 milligrams, respectively. Also disperse homogeneity, weight variation and microbial limit all met the requirements in Ch. P. CONCLUSION: The prepared Naoxuekang dispersible tablet is reasonable in formula, feasible in technology, which meets the quality standards.

[Study of anticoagulant activity of ethanol extracts from leech in vitro].

OBJECTIVE: To study the anticoagulant activity of different portions of leech ethanol extracts (LEEs). METHODS: Anticoagulant activity was determined by measuring prothrombin time(PT), thrombin time (TT), activated partial throboplstin time (APTT) and fibrinogen coagulation time (FCT). RESULTS: PT, TT, APTT and FCT were remarkably prolonged by ethyl acetate portion of LEEs. Portions of petroleum ethrer, n-butanol and water extracted from LEEs was much weaker on anticoagulant activity. CONCLUSION: The anticoagulant effect of ethyl acetate extract portion of LEEs is the strongest among the four portions, and this results may be from its inhibitory effect on thrombin-catalyzed fibrinogen hydrolysis.

[Inhibition effects of compound leech eye drops on apoptosis of lens epithelial cells and expressions of Bcl-2 and Bax genes in rats].

OBJECTIVE: To investigate the inhibition effects of compound leech eye drops (Co-SZ) on apoptosis of lens epithelial cells (LECs) induced by hydrogen peroxide (H2O2) and the expressions of apoptosis-related genes Bcl-2 and Bax in rats. METHODS: All fresh transparent LECs in SD rats were bathed in culture medium with H2O2 in vitro, meanwhile Co-SZ were added in the culture medium. All LECs were incubated for 24 hours. The apoptosis rate of LECs was determined by terminal deoxynucleotidyl transferase mediated biotin-dUTP nick end labeling method (TUNEL). The changes of LEC ultrastructure and the formation of apoptotic body were observed by transmission electron microscopy. The expressions of apoptosis-related genes Bcl-2 and Bax were detected by streptavdin-peroxidase-biotin method. RESULTS: The apoptosis rate of LECs in the Co-SZ-treated group was significantly lower than that in the H2O2-treated group. The changes of apoptotic LEC ultrastructure in the Co-SZ-treated group were less than those in the H2O2-treated group. The expression of Bcl-2 protein was up-regulated and the expression of Bax protein was down-regulated in the Co-SZ-treated group as compared with the H2O2-treated group. CONCLUSION: The LEC apoptosis induced by H2O2 can be inhibited by Co-SZ. The molecular mechanisms of Co-SZ in inhibiting LEC apoptosis may be related to regulating the expressions of apoptosis-related genes Bcl-2 and Bax.

[Residues analysis of pesticides and heavy metals in Whitmania pigra and its breeding base].

OBJECTIVE: To give a reference to the safe and standardized production of Whitmania pigra, through the comparative research on residues of heavy metals and pesticide in W. pigra, breeding base soil and breeding water . METHOD: Residues of heavy metals and pesticide in W. Pigra, soil and water were determined by atomic absorption spectrophotometers, atomic fluorescence spectrophotometers and GC-ECD. RESULT AND CONCLUSION: With the exclusion of Pb residue, the residues of HCH, DDT and other heavy metals in W. pigra, soil and water in breding base met the requirements of the national standards. We suggest that the residues limits of animal drugs like W. pigra should be listed in a separate category when the national standards are revised in the fature.

[Effect of gufuhuo decoction on local apoptosis of steroid-induced necrosis of femoral head].

OBJECTIVE: To observe the bone specimen of SANFH (steroid-induced avascular necrosis of femoral head) and the effect of Gufuhuo decoction on local apoptosis of SANFH. METHOD: Model rabbits of necrosis of femoral head, made by intragluteal injection with prednisone acetate, were killed after 6 weeks to study the apoptosis by means of TUNEL. RESULT: A great number of faded osteocytes and osteoblasts existed in specimen of SANFH. There was significant difference in apoptosis indexes between model and control group, Gufuhuo decoction and Xianlinggubao group (P < 0.05). CONCLUSION: In the course of SANFH, the necrosis results from the interaction of apoptosis and necrosis of steroid-induced osteocytes and osteoblasts. Gufuhuo decoction has some effects on improving apoptosis and necrosis of SANFH.

[Study in the mechanism of Shuxuetong injection on antithrombosis and thrombolysis].

OBJECTIVE: To study the effect of Shuxuetong injection on cerebral tissue and brain microvascular endothelial cell(BMEC) in secreting tissue-type plasminogen activator (tPA). METHOD: The experiment in vivo was designed for three groups: sham-operation group, focal cerebral ischemia model group, Shuxuetong injection-treated group. The gene expression and the activity of tPA on brains were detected by RT-PCR and chromogenic substrate analysis respectively after treatment for five days; The experiment in vitro: The cultured BMEC were divided into experimental group and control group. The Shuxuetong injection were added into experimental group and the BMECs in the experimatal groups were treated for 24 h. The gene expression and the activity of tPA on cultured BMEC were detected by RT-PCR and chromogenic substrate analysis respectively. RESULT: The gene expression and the content of tPA on both cerebral tissue and BMEC in the experimatal groups were significantly higher than that in the control groups. CONCLUSION: Shuxuetong injection could accelerate secretion of tPA, and therefore enhance fibrinolysis.

[The effect of hirudo on proteinuria, lipid metabolism and coagulation system in the patients with chronic glomerulonephritis].

Renal biopsy was performed in 31 cases of primary glomerulonephritis and the effect of Hirudo in these patients observed. The results revealed that proteinuria decreased significantly, serum albumin increased significantly and cholesterol, triglyceride reduced significantly 4 weeks after treatment with Hirudo (P < 0.01 or P < 0.05); Fibrinogen and platelet aggregation reduced significantly (P < 0.01). However, platelet count, partial thromboplastin time, bleeding and clotting time did not change (P > 0.05); urine NAGase decreased significantly (P < 0.01). It is concluded that Hirudo may decrease proteinuria and alleviate renal parenchymal damage.

[Comparison among 5 different extracting methods of Whitmania pigra whitman].

Whitmania pigra whitman is one of the traditional Chinese drugs commonly used in clinical practice for Blood circulation promoting and Stasis relieving, its active principle was unknown yet. In this report, five extracting methods have been studied based on its extract's anticoagulant and antithrombotic activity in vitro. The results have revealed that the extracts using different extracting methods showed different anticoagulant and anti-thrombotic activity. Among them, the extracts produced by water-boiling and alcohol-prepitating have shown the strongest anticoagulant activity and good antithrombotic effect.

[Preventive and therapeutic effects of hirudo on incipient acute tubular necrosis in rats].

Male Sprague-Dawley rats, weighing 180-250 g and depleted with water for 16 h, were injected with glycerol (im) to induce acute tubular necrosis, and then divided into groups given blood-activating and stasis-removing drug, Hirudo solution (GH) tap water (GW), verapamil (GV) and none (GSDW) in incipient stage separately. It was observed that levels of BUN increased at 24th and 48th h after administration of glycerol and levels of Bcr increased at 3rd, 24th and 48th h after injecting glycerol in GH were significantly lower than those increased in GW and GSDW (P less than 0.05-P less than 0.001), but roughly similar to those in GV (P greater than 0.05-P greater than 0.5). Renal histopathological damage under light microscope and electron-microscope in GH at 3rd and 24th h after administration of glycerol were also less severe than those in GW and GSDW. The results suggested that Hirudo could exert a preventive and therapeutic effects on incipient acute tubular necrosis induced by glycerol in rats.

[Experimental study of hirudo and Salvia miltiorrhiza on smooth muscle cell hyperplasia after balloon angioplasty].

OBJECTIVE: To find the preventive method of restenosis after transluminal angioplasty (TA). METHODS: Sixty rabbit iliac arteries of 30 rabbits were divided randomly into three groups, group A, B and C, 20 in each. After experimental atherosclerotic stenosis and TA performing, ordinary forage plus 2 g of hirudo powder was fed to the group A every day, to the group B, 2 g Salvia miltiorrhiza powder was added and to the group C, ordinary forage only. Angiography and pathomorphological examination were carried out 30 days later. RESULTS: (1) Hirudo significantly reduced the thickness of iliac intima as compared with the other two groups (P < 0.05), the thickness in the group A, B and C was 40.1 +/- 9.8 microns, 48.2 +/- 8.2 microns and 69.3 +/- 9.2 microns respectively. (2) The incidence of restenosis after TA in both group A (31.4%) and group B (48.4%) were significantly lower than that in the group C (62.8%, P < 0.05). And significant difference was also found in comparison of group A and group B (P < 0.05). (3) Ultrastructure change examined by transmission electron microscopy showed that less rough endoplasmic reticulum and more myofilaments in well differentiated smooth muscle cell (SMC) of the arterial intima in the group A and B, while in the group C, more rough endoplasmic reticulum, more synthetic cells were seen in SMC. CONCLUSIONS: Hirudo and red sage root could effectively inhibit the hyperplasia of SMC in arterial intima and reduce the intima thickness and the incidence of restenosis after TA significantly, thus might play a role in the prevention of restenosis after TA. The effect of hirudo is more potent than that of red sage root.

[Clinical study of shuizhi-dahuang mixture in treating schizophrenics with blood stasis syndrome].

A clinical study of 67 female schizophrenics was conducted. Thirty two patients of them treated with the Shuizhi (leech)-Dahuang (rhubarb) mixture mainly with low dosage of antipsychotic drugs (combined therapy group), while other 35 cases were treated with antipsychotic drugs only (control group). The result showed that their overall therapeutic effects were similar and the combined therapy group could reduce the dosages of antipsychotic drugs and its side effects, and tended to normalize the hemorheologic indices.

[Clinical study on combined treatment of shuizhi tuyuan powder and nifedipine in treating hypertension patients complicated with left ventricular hypertrophy].

Patients with essential hypertension complicated with left ventricular hypertrophy (LVH) confirmed by ultrasonic cardiography were divided randomly into observation group and control group. Both groups were treated with nifedipine. Shuizhi Tuyuan Powder (SZTYP, consisted of Hirudo nipponia and Eupolyphaga sinensis) was given in addition to the observation group. The therapeutic course for the two groups were 6 months. Results showed that after one course of treatment, the myocardial weight index lowered from 136.8 +/- 7.5 g/m2 to 130.5 +/- 6.4 g/m2 in observation group, while in control group, it lowered from 136.7 +/- 7.4 g/m2 to 134.3 +/- 6.2 g/m2, the difference between the two groups was significant (P < 0.01). The symptoms were relieved in part of the patients with early stage of cerebrovascular disease treated with SZTYP. The results suggested that SZTYP combined with nifedipine has active curative effect on essential hypertension patients complicated by LVH and part patients in early stage of cerebrovascular disease.

[Effects of leech and ground beetle powder on hemorheology and blood lipid of ischemic stroke].

In order to improve blood supply for the brain, restore the functions of the cerebral cells and the limbs, and increas the curative rate, the leech (Hirudo nipponica and ground beetle Eupolyphage sinensis), powder (LGBP) to the patients according to the principle of promoting the blood circulation to remove the stasis was administered, and the clinical observation and experimental study was conducted. Its effects were compared with those of Western medicines. The results showed that after medication of LGBP, the blood flow of brain significantly increased, the hypoxia was improved, blood viscosity and blood lipid were lowered and thrombosis was inhibited in vitro or in vivo. No toxic side-effects caused by LGBP was found.

[The comparison of contents of uracil, xanthine and hypoxanthine in five species of leech].

Three constituents(uracil, xanthine, hypoxanthine) were isolated from five species of leech and determined by reversed phase HPLC. The column employed was Shim-pack CLC-ODS C18(150 mm x 6 mm). The mobile phase was 0.05 mol/l ammonium phophate dibasic solution(pH = 8.4). The flow rate was 0.8 ml/min and detection was effected at 254 nm. This method is accurate, rapid and reproducible. Analytical data for five species and Whitmania pigra samples from different places were given.

[Investigation and experimental study on the standards and quality of leech pieces].

It has been confirmed by investigation that Whitmania pigra is the main medicinal breed of leech, and filiform pieces are the best for extraction. The quality of leech processed by 3 different methods has been studied qualitatively and quantitatively in terms of amounts of extraction, TLC, electrophoresis of protein and anti-thrombin action.

[Experimental studies on anticoagulant activity of different extract of leech in vitro].

OBJECTIVE: To study the anticoagulant activity of different extracts of Leech. METHODS: Prothrombin time (PT), thrombin time (TT) and activated partial thromboplastin time (APTT) were determined by coagulometer for blood samples collected from abdominal vein of Wistar rat. RESULTS: PT, TT, APTT were prolonged remarkably by ethyl acetate part of Leech. PT was prolonged remarkably by hexane part and aqueous solution part of Leech. APTT was prolonged remarkably by n-Butylalcohol part of Leech. CONCLUSION: The different extracts of Leech have obvious anticoagulant effects. There are new anticoagulant components different from hirudin in Leech.

[Study of protective effect of leech, radix Salviae miltiorrhizae and its composite recipe on vascular endothelial cells in rats with blood stasis syndrome].

OBJECTIVE: To study the protective effect for Chinese medicine of promoting blood circulation and removing blood stasis (PBCRBS) on vascular endothelial cells (VEC) in rats, aiming at further research on the mechanism of blood stasis syndrome and PBCRBS. METHOD: Establishing a model of blood stasis with endothelial damage by means of giving rats an injection of adrenalin and making it swim in ice-cold water, then measuring the number of circulating endothelial cells (CEC) in whole blood and rheology. Moreover observing the change of the above indexes after the rats have taken Leech, Radix salviae miltrorrhizae (RSM) and its composite recipe for 5 days or 10 days. RESULT: It could increase the number of CEC accompanied by whole blood viscosity, fibrinogen and hematocrit in rats with blood stasis. But for groups taking Chinese medicine, the number of CEC was decreased distinctively and blood rheology was improved. CONCLUSION: The rat with blood stasis syndrome suffered from obvious injury of VEC. The Leech, RSM and its composite recipe could protect VEC.

[Preliminary studies on process and ultramicro-pulverization on pharmacodynamics of Leech].

OBJECTIVE: To compare pharmacodynamics of decoction and powder of Leech; To identify effect of pharmacodynamics of Leech with new methods of process and ultrmicro-pulverization. METHOD: The size of ultramicor-powder of Leech and new method processed Leech is determined by BT-1500 size distributive instrument. It is compared that the pharmacodynamics of Leech decoction and powder as well as ultramicro-powder with new method processed of 1/2 decoction dose by anticoagulation and antithrombus action of mice. The safety of different samples is identified. RESULT: The pharmacodynamics of samples is as ultramicro-powder of Leech with new method processed > ultrmicro-powder of Leech > powder of Leech > decoction of Leech. CONCLUSION: The ultramicro-powder of Leech with new method processed has better pharmacodynamic effect and the smell of Leech is improved.