Investigation of the optimal medium and application strategy for foot-and-mouth disease vaccine antigen production.

AIMS: For the effective production of 146S particles, which determines foot-and-mouth disease (FMD) vaccine efficacy, we aimed to identify the optimal medium that is easy-to-use, productive and economically affordable for the large-scale production of FMD vaccine. METHODS AND RESULTS: Nine combinations of cell growth media and replacement media were tested for virus propagation. Apart from the replacement strategy, we tested a simple addition strategy involving the addition of 30% v/v of fresh medium to the total spent medium using the Cellvento BHK-200 (Vento). Unlike other tested media that produced poor yields of 146S particles when the spent media were not eliminated, Vento exhibited high productivity with the 30% addition strategy. CONCLUSIONS: Considering its lower price and media consumption compared to those of other media that require media replacement, the 30% addition strategy of Vento is highly effective. Furthermore, owing to its simple application strategy, it makes the scale-up process easy and helps in saving the time and labour involved in spent media removal. SIGNIFICANCE AND IMPACT OF THE STUDY: Through the first comparative assessment of commercial media for the 146S particle recovery, this study suggests the best practical medium for the industrial-scale production of FMD vaccines.

Isolation and characterization of human mesenchymal stem cells from gingival connective tissue.

BACKGROUND AND OBJECTIVE: The main purpose of this study was to isolate and characterize gingival connective tissue-derived mesenchymal stem cells (GMSCs). The secondary purpose was to present a modified isolation method for the GMSCs. MATERIAL AND METHODS: Collected healthy gingival tissue samples were de-epithelialized and minced into small fragments. The tissues were digested by dispase and collagenase IV for 30 min. The first digested cell suspension was discarded, and then additional digestion was performed to the remaining cells in the same solution for 90 min. The isolated cells from gingiva was incubated in 37°C humidified condition and observed by inverted microscope. Cytoskeletal morphology was evaluated by phalloidin immunofluorescence. Potency of the cells was tested by colony-forming unit fibroblast assay. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometric, immunofluorescence analysis. RESULTS: GMSCs showed spindle-shaped, fibroblast-like morphology, colony-forming abilities, adherence to plastic and multilineage differentiation (osteogenic, adipogenic, chondrogenic) potency. GMSCs expressed CD44, CD73, CD90 and CD105, but did not express CD14, CD45, CD34 and CD19 in flow cytometry. Expression of stem cell markers (SSEA-4, STRO-1, CD146, CD166 and CD271) and a mesenchymal marker (vimentin) were observed by immunofluorescence. CONCLUSIONS: In conclusion, we isolated and characterized stem cells from human gingival connective tissue with modified protocol. GMSCs showed multipotency with high proliferation and characteristics of mesenchymal stem cells. GMSCs are promising sources for tissue engineering and may be obtained during routine procedures under local anesthesia. Further research is needed to evaluate the potential of GSMCs' proliferation and cryopreservation.

Treatment of dentin hypersensitivity with a low-level laser-emitting toothbrush: double-blind randomised clinical trial of efficacy and safety.

Dentin hypersensitivity (DH) is defined as pain derived from exposed dentin in response to chemical, thermal, tactile, or osmotic stimuli that cannot be explained as having arisen from any other dental defect or disease. The aim of this trial was to test the efficacy and the safety of a low-level laser-emitting toothbrush on management of DH. A prospective, double blind, randomised clinical trial was designed; 96 individuals with hypersensitive teeth without caries or fracture were selected as subjects. The subjects were randomly allocated to either the test group with the 635 nm per 6 mW laser-emitting toothbrush, or the control group with the 635 nm per 12.9 µW light-emitting diode (LED) toothbrush. An air blast was applied with a dental air syringe held 3 mm away from the selected tooth and a visual analogue scale (VAS: 0-10) was used to quantify subjective pain. Assessments were completed at a screening visit and after 2-week and 4-week of using a test/control toothbrush. Results demonstrated that the use of both control and test toothbrushes resulted in decreased discomfort after 4 weeks. In the test group, pain intensity scores decreased from 5.8 ± 1.2 to 2.3 ± 1.6, and in the control group, the scores decreased from 6.4 ± 1.3 to 5.5 ± 2.0 (P < 0.05). This decrease was significantly greater in the test group. There were no significant adverse events or side effects. It was concluded that the use of the low-level laser emitting toothbrush is a safe and effective treatment option for the management of DH.

Presence of Porphyromonas gingivalis and plasma cell dominance in gingival tissues with periodontitis.

OBJECTIVE: Porphyromonas gingivalis can invade and survive within its host epithelial cells. The aim of this study was to test our hypothesis that persistent presence of intracellular periodontal pathogens in gingival tissue causes the chronic inflammation and that an inappropriate immune response is a risk factor for periodontitis. METHODS: Together with the presence of P. gingivalis, the distribution of B cells, plasma cells, and CD4(+), CD8(+), and FOXP3(+) regulatory T cells was evaluated in gingival tissues from healthy (n = 7) and periodontitis (n = 8) sites by in situ hybridization and immunohistochemistry, respectively. RESULTS: Porphyromonas gingivalis was detected in proximity to inflammatory infiltrates in three and seven biopsies from the healthy and periodontitis sites, respectively. Compared with healthy sites, periodontal lesions contained a significantly increased number of each immune cell studied with a relative dominance of plasma cells over T cells. CONCLUSIONS: Persistent bacterial invasion of gingival tissues in combination with a plasma cell-dominant immune response may be involved in the pathogenesis of periodontitis.

Analysis of the attachment and differentiation of three-dimensional rotary wall vessel cultured human preosteoblasts on dental implant surfaces.

PURPOSE: The purpose of this study was to determine whether osseous tissues engineered in three-dimensional (3D) environments preserved their mineralizing capacity and retained biologic characteristics when cultured on dental implant surfaces. MATERIALS AND METHODS: Human preosteoblast cells were cultured in both 3D rotary wall vessels and on 2D tissue culture plastic plates for 3 days. Aggregates from the 3D chambers and cells from the 2D plates were collected and transferred to commercially pure titanium disks with either 600-grit polished or sandblasted surfaces. These were cultured for an additional 7 days. The aggregates and cells from the disks were collected and prepared for scanning electron microscopy for microscopic evaluation and atomic adsorption assays for mineral content analysis. Additionally, staining with Alizarin red S was performed to compare the mineralization amount and pattern in each group. Polymerase chain reaction analysis was performed to evaluate expression of osteogenic genes, including Runx2, FAK, bone morphogenetic protein 2, and osteocalcin. RESULTS: Cells from 3D rotary wall vessel cultures attached to implant surfaces and presented cell attachment and growth patterns similar to those of standard 2D cultured cells, showing evidence of radial and random growth, yet they formed multiple focal niches on implant surfaces out of which cells proliferated. The 3D cultured cells and osseous tissues retained higher amounts of mineral formed during the initial culture and showed a higher tendency toward mineralization on implant surfaces compared to standard cultured cells. The 3D cultured cells and osseous tissues on implant surfaces at 1 week showed higher key gene protein expression. RNA expression at 1 week was equivalent to that of standard cultured cells. CONCLUSION: Culture of human osteogenic cells and tissues in 3D rotary wall vessels may expedite the osseointegration process on dental implant surfaces, thus reducing the overall treatment time.

Gene delivery into ischemic myocardium by double-targeted lipoplexes with anti-myosin antibody and TAT peptide.

The treatment of myocardial ischemia using gene therapy is a rather novel but promising approach. Gene delivery to target cells may be enhanced by using double-targeted delivery systems simultaneously capable of extracellular accumulation and intracellular penetration. With this in mind, we have used low cationic liposomes-plasmid DNA complexes (lipoplexes) modified with cell-penetrating transactivating transcriptional activator (TAT) peptide (TATp) and/or with monoclonal anti-myosin monoclonal antibody 2G4 (mAb 2G4) specific toward cardiac myosin, for targeted gene delivery to ischemic myocardium. In vitro transfection of both normoxic and hypoxic cardiomyocytes was enhanced by the presence of TATp as determined by fluorescence microscopy and ELISA. The in vitro transfection was further enhanced by the additional modification with mAb 2G4 antibody in the case of hypoxic, but not normoxic cardiomyocytes. However, we did not observe a synergism between TATp and mAb 2G4 ligands under our experimental condition. In in vivo experiments, we have clearly demonstrated an increased accumulation of mAb 2G4-modified TATp lipoplexes in the ischemic rat myocardium and significantly enhanced transfection of cardiomyocytes in the ischemic zone. Thus, the genetic transformation of normoxic and hypoxic cardiomyocytes can be enhanced by using lipoplexes modified with TATp and/or mAb 2G4. Such complexes also demonstrate an increased accumulation in the ischemic myocardium and effective transfection of hypoxic cardiomyocytes in vivo.

Comparison of three cell fixation methods for high content analysis assays utilizing quantum dots.

Semiconductor nanoparticles or quantum dots are being increasingly utilized as fluorescent probes in cell biology both in live and fixed cell assays. Quantum dots possess an immense potential for use in multiplexing assays that can be run on high content screening analysers. Depending on the nature of the biological target under investigation, experiments are frequently required on cells retaining an intact cell membrane or also on those that have been fixed and permeabilized to expose intracellular antigens. Fixation of cell lines before or after the addition of quantum dots may affect their localization, emission properties and stability. Using a high content analysis platform we perform a quantitative comparative analysis of three common fixation techniques in two different cell lines exposed to carboxylic acid stabilized CdTe quantum dots. Our study demonstrates that in prefixed and permeabilized cells, quantum dots are readily internalized regardless of cell type, and their intracellular location is primarily determined by the properties of the quantum dots themselves. However, if the fixation procedures are preformed on live cells previously incubated with quantum dots, other important factors have to be considered. The choice of the fixative significantly influences the fluorescent characteristics of the quantum dots. Fixatives, regardless of their chemical nature, negatively affected quantum dots fluorescence intensity. Comparative analysis of gluteraldehyde, methanol and paraformaldehyde demonstrated that 2% paraformaldehyde was the fixative of choice. The presence of protein in the media did not significantly alter the quantum dot fluorescence. This study indicates that multiplexing assays utilizing quantum dots, despite being a cutting edge tool for high content cell imaging, still require careful consideration of the basic steps in biological sample processing.

Three-dimensional culture environments enhance osteoblast differentiation.

PURPOSE: In previous work from our laboratory, we demonstrated that the three-dimensional (3D) cell cultures developed in simulated microgravity environments enhanced osseous-like aggregate formation and accelerated preosteoblast cell differentiation. Thus, as described here, we hypothesize that aggregate formation and mineralization would occur with fewer than 10 x 10(6) cells as previously described. MATERIALS AND METHODS: Human preosteoblastic cells were cultured at different concentrations in a rotary wall vessel to simulate microgravity for 7 days. Aggregate size was assessed, and mineralization and collagen expression detected using Von Kossa and Masson Trichrome staining. Scanning electron microscopy was used for structural and elemental analysis. Immunohistochemistry was used to detect expression of the osteogenic markers BSPII and osteopontin (OP). RESULTS: Size and calcium expression were dependent upon cultured cell number (p < 0.01). Calcium and collagen expression were detected throughout the aggregate, but organization was independent of cell number. Aggregates had similar microscopic structural patterns demonstrating organized development. Presence of BSPII and OP showed that the aggregates share common differentiation proteins with in vivo bone formation. CONCLUSIONS: These results may lead to novel bone engineering techniques associated with dental treatment.

Cholesterol enhances platelet-derived growth factor-BB-induced [Ca2+]i and DNA synthesis in rat aortic smooth muscle cells.

In the present study, we describe possible mechanisms by which hypercholesterolemia may contribute to the development of cardiovascular diseases. Treatment of rat aortic smooth muscle cells for 20 hours with cholesterol-rich liposomes (500 micrograms/mL cholesterol, 100 micrograms/mL low-density lipoprotein) resulted in a 76 +/- 12% increase in total cholesterol content. The effects of cholesterol enrichment were examined by determination of changes in cell membrane fluidity. Fluidity of the cholesterol-enriched cell membranes was decreased at all temperatures between 15 degrees C and 40 degrees C. Changes in membrane fluidity in whole cell membranes represented changes in fluidity of microsomal membranes isolated by Percoll gradient ultracentrifugation. The basal [Ca2+]i and the maximal platelet-derived growth factor (PDGF)-BB-induced [Ca2+]i was elevated by 30% and 90% in cholesterol-enriched cells, respectively. In contrast, the resting pH, and the PDGF-BB-induced stimulation of the Na+/H+ exchange were not affected in cholesterol-enriched cells. The effect of PDGF-BB on [3H]thymidine incorporation in cholesterol-enriched cells was elevated by 40% in comparison with untreated cells. Our findings show that cellular cholesterol may be involved in the development of vascular diseases via modulation of the PDGF-induced increase in [Ca2+]i and DNA synthesis in vascular smooth muscle cells.

Immobilization of poly(ethylene glycol) or its sulfonate onto polymer surfaces by ozone oxidation.

A novel surface modification method has been developed to improve biocompatibility of polymeric biomaterials. This approach involves ozonation and then followed by graft polymerization with acrylates containing PEG, sulfonated PEG or by coupling of PEG derivatives. All the reactions were confirmed by ATR FT-IR and ESCA. The degree of ozonation measured by the iodide method was dependent on the ozone permeability of the polymers used. Surface hydrophilicity was investigated by measuring the contact angles. Ozonation itself yielded a slight increase in hydrophilicity and a decrease in platelet adhesion, but PEG immobilization showed a significant effect on surface hydrophilicity and platelet adhesion to confirm well-known PEG's passivity which minimize the adhesion of blood components on polymer surfaces. Both graft polymerization and coupling were effective for PU. In contrast, only grafting gave enough yields for PMMA and silicone. Platelet adhesion results demonstrated that all PEG modified surfaces adsorbed lower platelet adhesion than untreated or ozonated ones. Polymers coupled with sulfonated PEG exhibited the lowest platelet adhesion when compared with control and PEG coupled ones by virtue of the synergistic effect of non-adhesive PEG and negatively charged SO3 groups. This PEG or sulfonated PEG immobilization technology using ozonation is relatively simple for introducing uniform surface modification and therefore very useful for practical application of blood contacting medical devices.

Oleanolic acid and ursolic acid stabilize liposomal membranes.

The effects of oleanolic acid (OA) and ursolic acid (UA) on the fluidity and stability of dipalmitoyl phosphatidylcholine (DPPC) liposomal membrane were monitored by measuring the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene labeled in the liposomal membrane and the leakage of calcein from the probe-encapsulated liposomes. The experiments with the liposomes made of DPPC and OA or UA showed that OA and UA exhibited a moderate fluidity-modulating effect for the liquid-crystalline liposomal membrane, and a strong condensing effect for both crystalline and liquid-crystalline liposomal membranes. Their effects were comparable to those of cholesterol. These results suggest that their fluidity-modulating and condensing effects might have some implications in their biological functions.

[Bone pain, polydipsia, polyuria]. / Knochenschmerzen, Polydipsie, Polyurie.

This 20 year old man suffered increasingly from multifocal bone- and back pain over the last 6 months. Painful weakness of the left leg with dysesthesia of the 4th and 5th toe, a weight loss of 15 kg and polydipsia and pollakiuria had developed. The clinical workup disclosed hard tumors in the right mandible and tibia, a waddling gait with bilateral sign of Trendelenburg, reduced muscular force in the left leg with missing achilles tendon reflex and a loss of sensibility in the distal S1 segment, epigastric tenderness on pressure and hypertension with a value of 160/100 mmHg. X-rays revealed multiple cystic bone lesions at all sites. Hypercalcemia and massively elevated parathyroid hormone were measured. Since the parathyroids were enlarged on sonography, primary hyperthyroidism with fibrosing osteitis v. Recklinghausen was diagnosed.

Outbreaks and diagnosis of foot-and-mouth disease serotype O in the Republic of Korea, April-June 2010.

Thirteen outbreaks of foot-and-mouth disease (FMD) were reported in pigs and cattle in Korea between 8 April and 4 June 2010. The FMD virus (FMDV) isolates were of serotype O, indicating that they were related to the virus strains of the Southeast Asia topotype that are circulating in East Asian countries. Animals carrying the viruses were identified by reverse transcriptase-polymerase chain reaction (RT-PCR) during a 29-day period between 8 April and 6 May, 2010. Prior to this outbreak, these FMDVs had not been detected in Korea and may therefore have been introduced from neighbouring countries into Ganghwa Island and subsequently spread inland to other areas, including Gimpo, Chungju and Cheongyang. Tests conducted to lift restrictions on animal movements lead to detection of two additional FMD-positive farms. Through appropriate responses, including swift diagnoses and culling policies, Korea was able to quickly regain its recognition as being free of FMD, without vaccination, by the World Organization for Animal Health (OIE) on 27 September 2010.

Diagnosis and control measures of the 2010 outbreak of foot-and-mouth disease A type in the Republic of Korea.

In January 2010, foot-and-mouth disease (FMD) occurred for the first time in 8 years in Korea. The outbreaks were because of A serotype, different from the O type, which had occurred previously in 2000 and 2002. The FMD outbreaks were identified in seven farms, consisting of six cattle farms where viruses were detected and one deer farm where only FMDV antibody was detected. The seven farms were within 9.3 km of each other. All susceptible animals within 10 km radius of the outbreak farms were placed under movement restrictions for 3-11 weeks. No vaccination took place to facilitate the clinical observation of infected animals and virus detection. After clinical observations and serological tests within the control zones showed no evidence of FMD infection, the movement restrictions were lifted, followed by FMD-free declaration (23 March) at 80 days after the first outbreak on 2 January. This communication describes the outbreak of FMD A serotype, and control measures applied to eradicate the disease in Korea.

Osteoblast differentiation is enhanced in rotary cell culture simulated microgravity environments.

PURPOSE: As the aging population increases, more people will become reliant on regenerative dental medicine for implant therapy. The objective of this study was to test the hypothesis that 3D rotary cell culture (RCC) environments created by simulated microgravity would enhance osteogenic gene expression using integrin mediated pathways. MATERIALS AND METHODS: Human embryonic palatal mesenchymal (HEPM, ATCC 1486) pre-osteoblasts were cultured in either RCC to create 3D environments or in 2D monolayers for 72 hours. Gross phenotypic analysis was performed using Alizarin Red S staining for calcium and microscopy. Real-time PCR analysis was used to detect differences in osteoblast gene expression. Aggregates developed in 3D RCC environments were treated with or without antibody to the collagen-I integrin receptor alpha2beta1 to determine whether this molecular pathway might contribute to the development of a mineralized matrix. RESULTS: Microscopic analysis demonstrated that RCC environments promoted 3D aggregate formation by 72 hours without any scaffold. The mass appeared osseous-like with a white, shiny, translucent surface. The center was amorphous with areas of vacuolization, tubule-like structures, and fibrous-like extensions. Real-time PCR data showed that 3D environments enhanced osteogenic gene expression as compared with 2D monolayer culturing conditions. At 72 hours, changes in levels of osteogenic gene expression were noted. Cbfa1, a necessary transcription factor for osteoblast differentiation, was expressed 33% higher (p= 0.26); Collagen 1, 69% higher (p= 0.05); Osterix, 49% higher (p= 0.001); and BSPII, 54% higher (p= 0.001) than osteoblasts cultured for 72 hours in standard 2D monolayer conditions. When cultured in the presence of collagen alpha2beta1 integrin receptor antibody, 3D aggregates had decreased levels of mineralization as compared with non-treated aggregates. CONCLUSION: RCC enhances osteoblast differentiation using integrin mediated pathways.

Cholesterol displacement from membrane phospholipids by hexadecanol.

Adding cholesterol to monolayers of certain phospholipids drives the separation of liquid-ordered from liquid-disordered domains. The ordered phases appear to contain stoichiometric complexes of cholesterol and phospholipid. Furthermore, it has been suggested that the cholesterol in these complexes has a low chemical activity compared to that of the free sterol; i.e., that in excess of the phospholipid binding capacity. We have now tested the hypothesis that the membrane intercalator 1-hexadecanol (HD) similarly associates with phospholipids and thereby displaces the complexed cholesterol. HD introduced into monolayers of pure dimyristoylphosphatidylcholine generated highly condensed (stable and solid) domains. In contrast, the phase behavior of mixed monolayers of the phospholipid, sterol, and alcohol suggested that HD could substitute for cholesterol mole for mole in promoting liquid-ordered domains. We also found that the transfer of cholesterol from mixed monolayers to aqueous cyclodextrin was greatly stimulated by the presence of HD, but only at levels sufficient to competitively displace the sterol from the phospholipid. This enhanced efflux was interpreted to reflect an increase in uncomplexed cholesterol. We conclude that HD forms complexes with dimyristoylphosphatidylcholine that are surprisingly similar to those of cholesterol. HD competitively displaces cholesterol from the phospholipid and thereby increases its chemical activity.

Purification of glycosylphosphatidylinositol-anchored proteins by modified triton X-114 partitioning and preparative gel electrophoresis.

Triton X-114 phase partitioning has frequently been used to obtain preparations enriched in glycosylphosphatidylinositol (GPI)-anchored proteins and other hydrophobic proteins from crude cellular homogenates. We have developed a new modification of this phase-partitioning technique which allows two distinct GPI-anchored proteins of Tetrahymena mimbres to be separated from other hydrophobic as well as hydrophilic proteins and recovered in approximately 90% yield. The unique feature of the new method is a 24-h incubation of the first Triton X-114 extract at -20 degrees C. This improves the partitioning of GPI-anchored proteins into the detergent phase while promoting the aggregation of other hydrophobic proteins. Individual GPI-anchored proteins in the detergent phase are then purified to near homogeneity by one-step preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. By substituting Triton X-114/water mixtures for the conventional SDS buffer used to collect proteins eluting from the preparative gel, the purified GPI-anchored protein can be rapidly concentrated from relatively large volumes of eluate by phase partitioning at 32 degrees C. The method is also effective in separating mammalian GPI-anchored alkaline phosphatase from other proteins. It is likely to be of general utility in characterizing the GPI anchor structures associated with nonabundant and abundant GPI-anchored proteins coexisting within the same cell type.

Reliability of non-invasively acquired human genomic DNA as a substrate for real-time PCR-assisted analysis of genetic polymorphisms.

Molecular epidemiological studies require high numbers of participants. The combination of an non-invasive access to human DNA with a rapid genotyping analysis, e.g. by use of LightCycler assisted real-time polymerase chain reaction (PCR), can be helpful in conducting such trials. The aim of our study was to define, for the first time, the use of LightCycler technology in analysis of non-invasively derived DNA. DNA extracted from blood, mouthwash and buccal cytobrush samples from 100 volunteers was analyzed for the genotypes of cytochrome P450 CYP1B1, and glutathione S-transferases GSTT1, GSTM1 and GSTP1. The median amounts of DNA isolated from blood, mouthwash and buccal cytobrush samples were 95, 11 and 8 microg, respectively. While genotyping for CYP1B1 codon 432 polymorphism and GSTP1 codon 105 polymorphism resulted in a complete correspondence for all three modes of sampling, the identification of individuals with null-genotype for GSTT1 or GSTM1 failed in some cases due to atypical courses of the corresponding melting curves, leading to high false-positive rates in the group of non-invasively derived samples. Thus, the results presented here call for caution in using LightCycler assisted real-time PCR in non-invasively collected samples, at least when appropriate control strategies are not implemented.

Formation of protoberberine-type alkaloids by the tubers of somatic embryo-derived plants of Corydalis yanhusuo.

The effects of 0.5 - 5 mg/l abscisic acid [ABA], 0.5 - 10 mg/l (2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol [paclobutrazol] and 0.5 - 2 mg/l alpha-cyclopropyl-alpha-(4-methoxyphenyl)-5-pyrimidinemethanol [ancymidol], 0.5 - 5 mg/l gibberellic acid [GA(3)] and 15 - 100 mg/l polyethylene glycol [PEG] 4000 supplemented in half-strength Murashige and Skoog's (MS) medium on the production of the two major protoberberine-type alkaloids (D,L-tetrahydropalmatine and D-corydaline) by the tubers of somatic embryo-derived plants of Corydalis yanhusuo were examined. Somatic embryo derived plants were also maintained for 6 months on half-strength MS medium containing 0.1 mg/l GA(3) or 0.5 mg/l paclobutrazol. The alkaloid contents were determined by high performance liquid chromatography (HPLC). The analysis revealed that the contents of D,L-tetrahydropalmatine and D-corydaline in the tubers of somatic embryo-derived plants were higher than the marketed crude drug and varied with growth regulator/PEG-4000 treatment and the age of the plant.