Selenium-decorated biocompatible honeycomb films with redox-switchable surface for controlling cell adhesion/detachment.

HYPOTHESIS: Selenium (Se)-containing compound is sensitive to redox stimulation, showing hydrophobic-hydrophilic reversible transition. Introduction of such compound into honeycomb film could confer on it redox-switchable surface wettability, which is expected to control cell adhesion/detachment behavior. EXPERIMENTS: Didodecyl selenide was designed and mixed with polystyrene to prepare honeycomb films using "breath figure" method. The film microstructures were characterized by scanning electron microscope and atomic force microscopy, and the arrangement of Se atoms in honeycomb film was determined by X-ray photoelectron spectroscopy and energy dispersive spectrometry. The variation of film wettability upon the alternating stimulation of H2O2 and Vc was examined. Then the cell adhesion, proliferation, and controlled detachment on honeycomb films were conducted. FINDINGS: The introduction of didodecyl selenide helps to form ordered honeycomb film, and Se atoms were found to located on the bottom, pore walls, and top surface of the film. The presence of didodecyl selenide not only greatly improves film biocompatibility by enhancing cell thioredoxin reductase activity, but also imparts the film with H2O2-/vitamin C-regulated tunable wettability that controls cell adhesion and detachment. H2O2 treatment produces a hydrophilic surface for cell adhesion and proliferation, whereas the addition of vitamin C generates hydrophobic surfaces and allows cells to detach while remaining alive with high activity.

The Effects of Static Magnetic Field on Orthodontic Tooth Movement in Mice.

The application of static magnetic field (SMF) has been considered an effective and noninvasive method to accelerate orthodontic tooth movement. The objective of this study was to explore the effects of SMF on orthodontic tooth movement in mice. A total of 105 Balb/c mice (body mass: 25-30 g) were divided into experimental group (SMF + force, 48), control group (force only, 48), and blank group (neither SMF nor force, 9). After the placement of orthodontic appliances, the experimental group was exposed to the SMF environment generated by Neodymium-iron-boron (NdFeB) magnets with an intensity of 20-204 mT. At 1, 3, 7, 14, 21, and 28 days after appliance insertion, eight animals in both experimental and control groups were sacrificed and the left maxillae were dissected to measure the distance of tooth movement, respectively. Meanwhile, the width of periodontal ligament (PDL), length of hyalinized zone, and the number of osteoclasts were evaluated by hematoxylin-eosin and tartrate-resistant acid phosphatase staining. We finally found that the experimental group demonstrated an enhanced rate and greater cumulative amount of tooth movement than the control group (0.2887 ± 0.0041 mm vs. 0.2114 ± 0.0089 mm, P < 0.05). On Days 7, 14, and 28, the experimental group also displayed a significantly greater width of PDL. Earlier formation and removal of the hyalinized zone, and significantly more osteoclasts were observed in the experimental group as well. The results suggested that SMF may be a promising nonsurgical intervention to accelerate orthodontic tooth movement. © 2021 Bioelectromagnetics Society.

Discovery of Eucalyptin C, derived from the fruits of Eucalyptus globulus Labill., as a novel selective PI3Kγ inhibitor for immunosuppressive treatment / 中国天然药物

The fruits of Eucalyptus globulus Labill. are known to have a plenty of medicinal properties, such as anti-tumor, anti-inflammatory, and immunosuppressive activity. Our previous study found that the phloroglucinol-sesquiterpene adducts in the fruits of E. globulus were immunosuppressive active constituents, especially Eucalyptin C (EuC). Phosphoinositide 3-kinases-γ (PI3Kγ) plays a pivotal role in T cell mediated excessive immune responses. In this study, EuC was first discovered to be a novel selective PI3Kγ inhibitor with an IC

Integrative Rehabilitation for Hypertrophic Olivary Degeneration after Operation of Brain Stem Cavernous Malformation: A Case Report / 中国康复理论与实践

Objective To summarize the integrative rehabilitation treatment of traditional Chinese medicine (TCM) and Western medicine in hypertrophic olivary degeneration (HOD) secondary to the operation of brain stem cavernous malformation. Methods The clinical data of medication, rehabilitation and follow-up of a patient with HOD secondary to operation on brain stem cavernous malformation was retrospectively analyzed. Results Three months after operation, limb static and motor tremor, dysarthria, palate spasm, eye movement disorder and walking difficulty appeared. The patient was diagnosed as HOD according to clinical features and brain magnetic resonance imaging (MRI). He was treated with pertinence rehabilitation training combined with TCM including acupuncture and herbs. After integrative rehabilitation, he could stand and walk independently, the tremor was alleviated, the balance function improved, the activities of daily living improved, and the dosage of oral western medicine also decreased. Conclusion After intracranial surgery, secondary neurodegeneration and movement disorder may appear, and it could be improved by integrative rehabilitation treatment of TCM and Western medicine

Integrative Rehabilitation for Hypertrophic Olivary Degeneration after Operation of Brain Stem Cavernous Malformation: A Case Report / 中国康复理论与实践

Objective To summarize the integrative rehabilitation treatment of traditional Chinese medicine (TCM) and Western medicine in hypertrophic olivary degeneration (HOD) secondary to the operation of brain stem cavernous malformation. Methods The clinical data of medication, rehabilitation and follow-up of a patient with HOD secondary to operation on brain stem cavernous malformation was retrospectively analyzed. Results Three months after operation, limb static and motor tremor, dysarthria, palate spasm, eye movement disorder and walking difficulty appeared. The patient was diagnosed as HOD according to clinical features and brain magnetic resonance imaging (MRI). He was treated with pertinence rehabilitation training combined with TCM including acupuncture and herbs. After integrative rehabilitation, he could stand and walk independently, the tremor was alleviated, the balance function improved, the activities of daily living improved, and the dosage of oral western medicine also decreased. Conclusion After intracranial surgery, secondary neurodegeneration and movement disorder may appear, and it could be improved by integrative rehabilitation treatment of TCM and Western medicine

Discovery of LY3325656: A GPR142 agonist suitable for clinical testing in human.

The discovery and optimization of a novel series of GPR142 agonists are described. These led to the identification of compound 21 (LY3325656), which demonstrated anti-diabetic benefits in pre-clinical studies and ADME/PK properties suitable for human dosing. Compound 21 is the first GPR142 agonist molecule advancing to phase 1 clinic trials for the treatment of Type 2 diabetes.

[Correlative study of bufogenin constituents in venom of Bufo bufo gargarizans].

As known,simultaneous determination of various chemical indicators is one of the future trends in quality control of traditional Chinese medicines because of the extremely complex chemical compositions. This project is to screen the quality markers that can accurately control the quality of the Bufonis Venenum by exploring the intrinsic correlation of components. In this study,venom of Bufo bufo gargarizans from 17 different sources were used as research samples,and the contents of 7 bufogenin were determined by HPLC-DAD. Then,the data obtained were analyzed by Spearman correlation analysis and principal component analysis( PCA). In addition,a stepwise regression analysis was used to establish a predictive model for the contents of the seven bufogenin components( independent variable) and the total contents of the bufogenin( dependent variable). The results indicated that there is a significant positive correlation between the contents of telocinobufagin and cinobufotalin,and there is a significant positive correlation between the contents of bufalin,cinobufagin and resibufogenin. In contrast,the contents of telocinobufagin and cinobufotalin are negatively correlated with the contents of bufalin,cinobufagin and resibufogenin. However,the correlation between gamabufotalin and bufotalin and other components are not obvious. Furthermore,further study found that there is a correlation between the sum of the contents of bufalin,cinobufagin and telocinobufagin and the total contents of the bufogenin. In fact,the application of bufalin,cinobufagin and telocinobufagin as the quality control indicators of the Bufonis Venenum can better reflect the quality characteristics of the Bufonis Venenum compared with the previous quality control indicators. The conclusions will provide a reference for the revision of the quality standards of the Bufonis Venenum.

Correlative study of bufogenin constituents in venom of Bufo bufo gargarizans / 中国中药杂志

As known,simultaneous determination of various chemical indicators is one of the future trends in quality control of traditional Chinese medicines because of the extremely complex chemical compositions. This project is to screen the quality markers that can accurately control the quality of the Bufonis Venenum by exploring the intrinsic correlation of components. In this study,venom of Bufo bufo gargarizans from 17 different sources were used as research samples,and the contents of 7 bufogenin were determined by HPLC-DAD. Then,the data obtained were analyzed by Spearman correlation analysis and principal component analysis( PCA). In addition,a stepwise regression analysis was used to establish a predictive model for the contents of the seven bufogenin components( independent variable) and the total contents of the bufogenin( dependent variable). The results indicated that there is a significant positive correlation between the contents of telocinobufagin and cinobufotalin,and there is a significant positive correlation between the contents of bufalin,cinobufagin and resibufogenin. In contrast,the contents of telocinobufagin and cinobufotalin are negatively correlated with the contents of bufalin,cinobufagin and resibufogenin. However,the correlation between gamabufotalin and bufotalin and other components are not obvious. Furthermore,further study found that there is a correlation between the sum of the contents of bufalin,cinobufagin and telocinobufagin and the total contents of the bufogenin. In fact,the application of bufalin,cinobufagin and telocinobufagin as the quality control indicators of the Bufonis Venenum can better reflect the quality characteristics of the Bufonis Venenum compared with the previous quality control indicators. The conclusions will provide a reference for the revision of the quality standards of the Bufonis Venenum.

Effect of static magnetic field on pain level and expression of P2X3 receptors in the trigeminal ganglion in mice following experimental tooth movement.

Recent research has demonstrated that static magnetic fields (SMF) can generate an analgesic effect in different conditions. The present study explored effects of SMF on pain levels and expressions of P2X3 receptors in trigeminal ganglion (TG) in mice after experimental tooth movement (tooth movement induced by springs between teeth). Experiments were performed in male mice (body mass: 25-30 g) and divided into SMF + force group, force group, and no force group. Exposure time was over 22 h per day. Mouse Grimace Scale was used for evaluating orofacial pain levels during experimental tooth movement at 4 h and 1, 3, 7, and 14 days. Meanwhile, expression levels of P2X3 receptors in the TG were evaluated by immunohistochemistry and western blotting at same time points. We finally found that during experimental tooth movement, pain levels of mice peaked at 3 days, and then decreased. While pain levels of mice were reduced in the SMF environment at 4 h, 1 and 3 days, there was a significant difference at 1 and 3 days. Meanwhile, under the action of SMF, expression levels of P2X3 receptors in TG were significantly lower at 4 h, 3 and 7 days. These results suggest that SMF can reduce pain levels in mice, and down-regulate P2X3 receptors in TG. Bioelectromagnetics. 38:22-30, 2017. © 2016 Wiley Periodicals, Inc.

Current advances in orthodontic pain.

Orthodontic pain is an inflammatory pain that is initiated by orthodontic force-induced vascular occlusion followed by a cascade of inflammatory responses, including vascular changes, the recruitment of inflammatory and immune cells, and the release of neurogenic and pro-inflammatory mediators. Ultimately, endogenous analgesic mechanisms check the inflammatory response and the sensation of pain subsides. The orthodontic pain signal, once received by periodontal sensory endings, reaches the sensory cortex for pain perception through three-order neurons: the trigeminal neuron at the trigeminal ganglia, the trigeminal nucleus caudalis at the medulla oblongata and the ventroposterior nucleus at the thalamus. Many brain areas participate in the emotion, cognition and memory of orthodontic pain, including the insular cortex, amygdala, hippocampus, locus coeruleus and hypothalamus. A built-in analgesic neural pathway-periaqueductal grey and dorsal raphe-has an important role in alleviating orthodontic pain. Currently, several treatment modalities have been applied for the relief of orthodontic pain, including pharmacological, mechanical and behavioural approaches and low-level laser therapy. The effectiveness of nonsteroidal anti-inflammatory drugs for pain relief has been validated, but its effects on tooth movement are controversial. However, more studies are needed to verify the effectiveness of other modalities. Furthermore, gene therapy is a novel, viable and promising modality for alleviating orthodontic pain in the future.

Identification of compounds in an anti-fibrosis Chinese medicine (Fufang Biejia Ruangan Pill) and its absorbed components in rat biofluids and liver by UPLC-MS.

Liver fibrosis represents a major public health problem worldwide. To date, antifibrotic treatment of fibrosis still remains an unconquered area for western medicine. Fufang Biejia Ruangan Pill (FFBJ) is the first anti-fibrosis drug approved by the China State Food and Drug Administration, and has been demonstrated to have a good antifibrotic efficacy in China. However, the chemical constituents of FFBJ and the absorption and distribution of it in vivo remain unclear, which restricts its research on bioactive components identification and mechanisms of action. In this study, ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) combined with ultra-performance liquid chromatography/triple quadrupole mass spectrometry (UPLC/QqQ-MS) was applied to identify compounds in FFBJ and its absorbed components in rat serum, liver and urine samples after intragastric administration of FFBJ. As a result, a total of 32 Chinese material medica components including organic acids, terpenoids, flavonoids, phenylpropanoids and alkaloids, were identified or tentatively characterized, while the distribution of 10 prototype compounds in rat serum, liver and urine were discovered. The identified constituents in FFBJ and the distribution of prototype compounds in rat serum, liver and urine are help for understanding the material bases of its therapeutic effects.

[Modeling of thermal degradation of linarin during concentration process].

Ganmaoling granule, with annual sale of over one billion yuan, is the first brand of domestic cold medicine sales. As the only traditonal Chinese medicine（TCM） quality control indicator of Ganmaoling granule, linarin is thermally unstable. Its content will be changed significantly during the production process, which would then affect the quality of the finished product. In this paper, the law of degradation of linarin was investigated. The experimental results showed that degradation reaction of linarin belongs to the first reaction characteristics. The effective methods to reduce the loss of linarin would be realized fortunately by strictly controlling the heating temperature or shortening the heating time.

[Study on balance of process of alcohol precipitation of ganmaoling granules].

Ganmaoling granule is the first brand of domestic cold medicine sales, but its preparation method and process control parameters are relatively rough. Therefore it is urgent to upgrade the technologies of large varieties of traditional Chinese medicine (TCM). This paper focused on the balance between the remove of impurity and the retention of linarin during the process of alcohol precipitation of Ganmaoling granules. The effects of four factors on the process were investigated via single factor experiments. The results showed that the precipitating period, the initial ethanol concentration and the final ethanol concentration had a great effect on retention of linarin while the initial density of the extract has not. Similarly, the initial ethanol concentration, the final ethanol concentration and the initial extract density have a great effect on the yield of dry extract while the time of alcohol precipitation has not. The parameters of alcohol precipitation of Ganmaoling granules were optimized as 16 h of precipitating period, 95% ethanol as the initial reagent, 70% of the final ethanol concentration, and 1.10 of the initial extract density.

[On-line monitoring of extraction of coldrine by near-infrared spectrum].

Extraction of the four Chinese herbals is the beginning step of the production process of coldrine granules and influences on drug quality significantly. In this paper, the on-line near infrared spectrum was collected during the extraction process of coldrine and then pre-processed by the first derivative. Partial least square regression (PLSR) model was developed for the quantity indicators of linarin, chlorogenic acid and solid content, according to results of both HPLC and weight-loss as reference methods. The correlation coefficient, root mean square error of cross-validation (RMSECV), root mean square error of calibration (RMSEC) and root mean square error of prediction (RMSEP) were used to optimize model parameters and confirm their performance. Correlation coefficients of three quality control indicator models reached more than 0.95.Values of RMSEC of linarin, chloroenic acid and solid content were 0.010 4, 0.009 34 and 0.055 5, respectively. And the values of RMSEP were 0.009 47, 0.142 and 0.008 42, respectively. The models, built on-line analyze data, revealed that the correlation coefficients of predicted values and measured values were greater than 0.97 and values of RSEP of linarin, chloroenic acid and solid content were 8.14%, 8.17% and 9.86%, respectively. The results showed that the NIR method could achieve the on-line detection and real-time monitoring of multi-indexes during the extraction process of coldrine. The technology could be used for drug quality control in the process of practical production, reducing the batch differences and ensuring pharmaceutical quality stability. In addition, it could provide real-time production data for subsequent product quality backtracking.

Study on balance of process of alcohol precipitation of ganmaoling granules / 中国中药杂志

Ganmaoling granule is the first brand of domestic cold medicine sales, but its preparation method and process control parameters are relatively rough. Therefore it is urgent to upgrade the technologies of large varieties of traditional Chinese medicine (TCM). This paper focused on the balance between the remove of impurity and the retention of linarin during the process of alcohol precipitation of Ganmaoling granules. The effects of four factors on the process were investigated via single factor experiments. The results showed that the precipitating period, the initial ethanol concentration and the final ethanol concentration had a great effect on retention of linarin while the initial density of the extract has not. Similarly, the initial ethanol concentration, the final ethanol concentration and the initial extract density have a great effect on the yield of dry extract while the time of alcohol precipitation has not. The parameters of alcohol precipitation of Ganmaoling granules were optimized as 16 h of precipitating period, 95% ethanol as the initial reagent, 70% of the final ethanol concentration, and 1.10 of the initial extract density.

The effectiveness of low-level laser therapy in accelerating orthodontic tooth movement: a meta-analysis.

Low-level laser therapy is claimed to accelerate bone remodeling. The aim of this meta-analysis was to critically appraise current evidence and to determine the effectiveness of low-level laser therapy in accelerating orthodontic tooth movement. PubMed, Web of Knowledge, Embase, CENTRAL, ProQuest Dissertations &Theses, and SIGLE were electronically searched from Jan 1990 to Jun 2013. Article screening, data extraction, assessment of risk of bias and evaluation of evidence quality through GRADE were conducted independently and in duplicate by two reviewer authors. Outcome of interest in this meta-analysis was accumulative moved distance (AMD). Meta-analyses were performed in Comprehensive Meta-Analysis Version 2.2.064 (Biostat, Englewood, NJ, USA). Finally, five studies were included in this meta-analysis. The meta-analysis revealed that the pooled difference in mean (DM) was 0.33 [95 % CI: (0.03-0.64)], 0.76 [95 % CI: (-0.14, 1.65)] and 0.43 [95 % CI: (-0.05, 0.91)] for AMD within 1 month, AMD within 2 months and AMD within 3 months, respectively. However, significant heterogeneities and instability of the pooled results were detected. Moreover, publication bias was found for AMD within 3 months. The subgroup analysis on the wavelength of 780 nm revealed that the pooled DM of AMD were 0.54 (95 % CI = 0.18-0.91), 1.11 (95 % CI = 0.91-1.31) and 1.25 (95 % CI = 0.68-1.82) for 1, 2, and 3 months, respectively. For the output power of 20 mW, the subgroup analysis showed that the pooled DM of AMD was 0.45 (95 % CI = 0.26-0.64), 1.11 (95 % CI = 0.91-1.31), and 1.25 (95 % CI = 0.68-1.82) for 1, 2, and 3 months, respectively. Weak evidence suggests that low-level laser irradiations at the wavelength of 780 nm, at the fluence of 5 J/cm(2) and/or the output power of 20 mW could accelerate orthodontic tooth movement within 2 months and 3 months. However, we cannot determine its effectiveness within 1 month due to potential measurement errors.

Biofeedback treatment for sleep bruxism: a systematic review.

PURPOSE: The aim of this systematic review was to evaluate the efficacy of any biofeedback treatment on sleep bruxism. METHODS: We searched the Cochrane Central Register of Controlled Trials, MEDLINE, Embase, ISI Web of Science, System for Information on Grey Literature in Europe, Chinese Biomedical Literature Database, and PsycINFO up to October 2012 for randomized controlled trials and controlled clinical trials involving biofeedback treatment for sleep bruxism. Reference lists of relevant studies were hand searched. Quality assessment and data extraction were performed by two reviewers independently. RESULTS: Seven eligible studies involving 240 participants were finally included. Three of them had moderate risk of bias, and four had high risk of bias. In an electromyographic-measured sleep bruxism episode, meta-analysis showed no significant difference between contingent electrical stimulation and blank control (95% confidence interval = -12.33, 3.38, P = 0.26). Moreover, five studies reported electromyographic activity index. Due to the diversity of biofeedback modalities (auditory, electrical, and visual stimulus) and controls (splint, occlusal adjustment, etc.), these data were unable to be pooled, so only qualitative description was provided. CONCLUSIONS: In the current stage, there is no powerful evidence to support the use of biofeedback technology on sleep bruxism treatment. Contingent electrical stimulation which is defined as a kind of biofeedback modality shows no effect on reducing sleep bruxism episode compared with the no-treatment group. Although many studies support the efficacy of biofeedback treatment, more large sample-sized randomized controlled trials which adopt uniform outcome index are necessitated to verify its application.

[Screening based on response surface methodology of multi-fractions traditional Chinese medicine with anti-influenza virus neuraminidase activity: take shuanghuanglian injection as an example].

This study aimed to establish a novel method to screen out the combined components of multi-fractions traditional Chinese medicine (TCM), so that the internal relationship between multi-ingredients could be objectively assessed and the proportioning ratio could be optimized. Taking antiviral effect on neuraminidase activity of influenza virus as the evaluating indicator and using Box-Behnken response surface methodology, the main effective ingredients of Shuanghuanglian injection (SHL) were screened. Meanwhile, the relationship between active ingredients was discussed. Taking SHL as a comparison, the optimum proportioning ratio was predicted. The results indicated that chlorogenic acid, cryptochlorogenic acid, caffeic acid and baicalin have comparatively strong antiviral activity against influenza virus. Moreover, antagonistic action existed between chlorogenic acid and cryptochlorogenic acid, whereas synergistic action between caffeic acid and other components. The optimum proportioning ratio resulted from fitted model is: chlorogenic acid, cryptochlorogenic acid, caffeic acid and baicalin (107 microg x mL(-1) : 279 microg x mL(-1) : 7.99 microg x mL(-1) : 92 microg x mL(-1)). The antiviral activity of the recombined components is stronger than that of SHL, which was consistent with the experiment results (P < 0.05). Box-Behnken response surface methodology has the advantages of general-screening, high-performance and accurate-prediction etc, which is appropriate for screening the combined components of multi-fractions TCM and the optimization of the proportioning ratio. The proposed method can serve as a technological support for the development of modern multi-fractions TCM.

Screening based on response surface methodology of multi-fractions traditional Chinese medicine with anti-influenza virus neuraminidase activity: take shuanghuanglian injection as an example / 药学学报

This study aimed to establish a novel method to screen out the combined components of multi-fractions traditional Chinese medicine (TCM), so that the internal relationship between multi-ingredients could be objectively assessed and the proportioning ratio could be optimized. Taking antiviral effect on neuraminidase activity of influenza virus as the evaluating indicator and using Box-Behnken response surface methodology, the main effective ingredients of Shuanghuanglian injection (SHL) were screened. Meanwhile, the relationship between active ingredients was discussed. Taking SHL as a comparison, the optimum proportioning ratio was predicted. The results indicated that chlorogenic acid, cryptochlorogenic acid, caffeic acid and baicalin have comparatively strong antiviral activity against influenza virus. Moreover, antagonistic action existed between chlorogenic acid and cryptochlorogenic acid, whereas synergistic action between caffeic acid and other components. The optimum proportioning ratio resulted from fitted model is: chlorogenic acid, cryptochlorogenic acid, caffeic acid and baicalin (107 microg x mL(-1) : 279 microg x mL(-1) : 7.99 microg x mL(-1) : 92 microg x mL(-1)). The antiviral activity of the recombined components is stronger than that of SHL, which was consistent with the experiment results (P < 0.05). Box-Behnken response surface methodology has the advantages of general-screening, high-performance and accurate-prediction etc, which is appropriate for screening the combined components of multi-fractions TCM and the optimization of the proportioning ratio. The proposed method can serve as a technological support for the development of modern multi-fractions TCM.

[Optimization of alcohol precipitation process for extract of Carthamus tinctorius by multiple guidelines grading method].

OBJECTIVE: To investigate the optimal alcohol precipitation parameters for extract of Carthamus tinctorius. METHODS: The effects of different factors on the transfer rate of hydroxy safflower yellow A (HSYA) was studied via single factor experiments, including the final alcohol concentration of the liquor, the speed of stirring, the initial density of the extract, the temperature and the pH of the liquor. Based on the results of single factor experiments, the final alcohol concentration of the liquor, the speed of stirring, the initial density of the extract and the pH of the liquor were studied by an orthogonal test and a multiple guidelines grading method, and the transfer rate of HSYA, the yield and the purity of extract in the supernatant were used as comprehensive evaluation index. RESULT: The optimal alcohol precipitation process of Carthamus tinctorius extract was as follows: the final alcohol concentration of the liquor 50%, the speed of stirring 500 r/min, the initial density of the extract 1.15 g/ml and the pH of the liquor 5.0. CONCLUSION: The proposed alcohol precipitation process is convenient and steady with high transfer rate of HSYA, high yield and purity of extract in the supernatant.