Study on the preparation process and evaluation of immune enhancement effect of dried extract powder from trevesia palmata leaves

Semi-chronic toxicity: Monitored for 90 consecutive days,

the lots of mice administered T. palmata extract powder at the dose

of 240 mg/kg/day and the dose of 720 mg/kg/day showed that: all

mice were healthy, weight gain well and regularly; no changes in the

hematological indices and the biochemical criteria for liver and

kidney function evaluation; no damages on liver, spleen and kidney

histopathology.

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chine(China), UV- Vis spectrophotometer, high-performance liquid chromatography system, hematological and biochemical analyzers... and other common reagents, chemicals, equipment. 5 2.2. Research method 2.2.1. Method of formulating the preparation process of dried extract powder from T. palmata leaves 2.2.1.1. Identification of tracer Investigate chemical composition groups by specific chemical reactions. T. palmata leaves (5kg) were ultrasound extracted with methanol solution; fractional extraction with solvents of increasing polarization. Isolate compounds by chromatographic methods. Detect compounds by ultraviolet light or using reagents, checking the purity of isolated compounds by combined methods such as: TLC, HPLC, and NMR. Determine the chemical structure based on physical and chemical parameters, compare with published data. 2.2.1.2. Methods of setting up the quality standard of pharmaceutical raw material for T. palmata leaves * Establish and validate SPN quantitative methods using UV-Vis - Principle: Total saponin in the leaves of T. palmata was quantified using UV-Vis spectroscopy after the Rosenthaler’s coloring reaction. SPN content was calculated according to AO. - Preparation: standard solution series: AO in MeOH concentration from 100 - 300 µg/mL; test solution: 5.0 g of T. palmata leaves, after chlorophyll removal, ultrasonically extract with MeOH in 2 hours at 50°C twice, collect the extract, add the solvent to 200 mL, dilute 2.5 times; filter through 0.45µm membrane before carrying out the reaction; blank determination: MeOH. - Carry out the Rosenthaler’s coloring reaction: Draw exactly 0.2 mL test solution (either standard or blank) into a test tube, then 6 add 0.2 mL of 5% vanillin solution in glacial acetic acid and 1.2 mL perchloric acid. Close the tubes and incubate at 70°C ± 1°C for a suitable period. Immerse the test tube in ice water, then transfer to a 5 mL volumetric flask, rinse the tube with ethyl acetate and add sufficient ethyl acetate to the mark. - Establish method: + Investigating maximum absorption: Scanning the spectra from 400 to 800 nm, select λmax. + Investigating the color reaction time: Perform the color reaction with a series of AO standard solutions with concentration: 92.50, 138.75, 185.0, 231.25, 277.50 µg/mL with incubation times of 10, 20, 30, 40 and 60 minutes respectively; repeat six times, selecting the most appropriate reaction time. - Validate the method: Determine dynamic linear range, precision and accuracy according to ICH guidelines. * Validate AO quantification method with HPLC - Chromatographic conditions: Chromatographic column: C18 column (4.6 × 250mm, 5µm); flow rate: 0.8 mL/min; injection volume: 10 µl; UV Detector: 215 nm wavelength; mobile phase: H2O/MeOH (5/95, v/v). - Preparation: standard solution series: AO in MeOH concentration from 10-100 µg/mL; sample: T. palmata, ultrasonically extract 5.0g leaves with MeOH x 2 times in 2 hours, get 250 mL, filter through 0.45µm membrane before injecting into the chromatographic system; blank determination: MeOH. - Validate the method: calculate module compatibility, method specificity, linear concentration range, precision and accuracy, limit 7 of detection (LOD) and limit of quantification (LOQ) according to ICH guidelines. * Method of setting up the institutional standards of pharmaceutical raw material for Trevesia palmata leaves Describe the medicinal material (plant macroscopic, microscopic, powder morphology), loss of weight due to drying, total ash, limits on heavy metals (Cd, Hg, As, Pb) according to the method of Vietnam Pharmacopoeia V. Qualitify oleanolic acid by TLC; quantify SPN by UV-Vis method, quantify AO by HPLC that have been verified. 2.2.1.3. Method of formulating the preparation process of dried extract powder from T. palmata leaves * Process of preparing extract - Investigate extraction method selection: ultrasonic extraction, percolation, reflux extraction, traditional decoction. - Investigate extraction process parameters: particle size of medicinal material, type of extraction solvent, DM/DL ratio and number of extraction times, extraction time. - After concentrating extract powder, remove the impurities; conduct testing and adjust the liquid extract powder based on the indices of solid ratio in the extract powder and SPN content. * Process of preparing dried extract powder from T. palmata leaves by spray drying method - Investigate factors/parameters: Spray drying excipients (Mal, Aer, lactose monohydrate, Starch 1500), excipient to solid ratio (10 - 30%), spray drying temperature and feed flow rate (120 - 150ºC, 20 - 40 mL/min), solid to feedstock ratio (10 - 20%). 8 - Basis for selection: sensory perception of powder, apparent density and compression index, weight loss due to drying, hygroscopic property, collection efficiency and spray drying efficiency. 2.2.2. Methods of researching to set up the institutional standard and evaluate the stability of dried extract powder from T. palmata leaves 2.2.2.1. Set up the institutional standard of dried extract powder from T. palmata leaves Form: Assessment by the sensory perception; weight loss due to drying, total ash, limits on heavy metals (Cd, Hg, As, Pb), limits on bacterial contamination according to methods in Vietnam Pharmacopoeia V. oleanolic acid qualification by TLC; SPN content quantification by UV-Vis method, AO content quantification by HPLC that have been validated. 2.2.2.2. Evaluate the stability of dried extract powder from T. palmata leaves - Storage conditions: Long-term test (temperature 30 ± 2ºC, humidity 75 ± 5%); accelerated aging (temperature 40 ± 2ºC, humidity 75 ± 5%). Sampling time: 0, 3, 6, 9, 12 months with long- term testing and 0, 3, 6 months with accelerated aging condition. Evaluation criteria: According to the institutional standard. 2.2.3. Method to study the immune enhancement effects and the toxicity of dried extract powder from T. palmata leaves on experimental animals 2.2.3.1. Evaluate the immune enhancement effect of dried extract powder from T. palmata leaves 9 * Evaluation model: Using an immunosuppression model by administering cyclophosphamide (Cyc) on mice: - Experimental mice were divided into 5 lots, 8 per lot: Lot 1 - Biological evidence: mice drank distilled water; Lot 2 - Model: Mice were injected with Cyc + drank distilled water; Lot 3 - Positive Control: Mice were injected with Cyc + oral levamisole at a dose of 100 mg/kg; Lot 4: The mice were injected with Cyc + drunk T. palmata extract powder at 1st dose (400 mg/kg); Lot 5: The mice were injected with Cyc + drunk T. palmata extract powder at 2nd dose (800 mg/kg). After injecting Cyc at the dose of 200 mg/kg in lots 2, 3, 4, 5, on the second day, the mice were drinking distilled water and the medicines for 7 days continuously. On day 8, kill the mice, draw blood and take lymphoid organs for testing. * Immune function tests: General indices: relative spleen weight, relative thymus weight; white blood cell count (WBC), lymphocyte and neutrophile cout, histology of thymus and spleen. Evaluation parameters of humoral and cell-mediated immunity: measurement of results of delayed hypersensitivity reactions to OA antigen by mouse feet thickness, IgG, cytokin IL-2 and TNF-α quantification. 2.2.3.2. Assessing the toxicity of dried extract powder from T. palmata leaves * Acute toxicity: conduct on the white mice (10 lots, 10 per lot), give T. palmata extract powder orally (dissolve in water) with increasing dose levels, determine the mortality rate in 72-hour period, continue to follow up to the end of the 7 days. LD50 was calculated using Excel software, then verified by Litchfield-Wilcoxon method. 10 * Semi-chronic toxicity: Conducted on white rats (3 lots, 10 per lot: physiological control group: drink distilled water; Lot 1: drink T. palmata extract powder at a dose of 240 mg/kg/day; Lot 2: drink T. palmata extract powder at a dose of 720 mg/kg/day). Evaluation criteria: general condition of rats, hematological and biochemical indices, macroscopic features of liver, spleen and kidney on day 90. CHAPTER 3: RESULTS 3.1. Results of establishing the preparation process of dried extract powder from T. palmata leaves 3.1.1. Result of determining the tracer Preliminary investigation of organic matter groups showed that saponins are the main components of T. palmata leaves. The results of extraction and isolation identified that six triterpenoid saponin compounds belonging to oleanolic and ursolic acid derivatives. Four known saponin compounds are oleanolic acid, ilekudinos ide C, kalopanaxsaponin H, and davisianoside B; two new compounds named acetyltrevesiasaponin A and acetyltrevesiasaponin B . 3.1.2. Result of setting up the institutional standard of pharmaceutical raw material for T. palmata leaves 3.1.2.1. Set up and validate quantitative methods * Saponin quantitative methods using UV-Vis - Establish method + Maximum absorption investigation: Scanning wavelengths from 400 to 800 nm, the maximum absorption was at 550 nm. 11 + Survey the color reaction time: Results showed that out of the samples with high correlation coefficients, the sample with the reaction time of 40 minutes had the highest optical absorbance. Therefore, our research team selected 40 minutes to be the time for carrying out the color reaction for further investigation. - Verify SPN quantitative method with UV-Vis + Determination of linear range: Results showed that within the concentration range of the AO investigation, the absorbance and the amount of AO had a linear correlation, with the correlation coefficient r2 = 0.9986 and the linear regression equation as Y = 0.0742 X - 0.0364. + Precision and accuracy: Results showed that the RSD values in a day and different days were less than 2.21%, meeting the requirement of the precision of the analytical method. The percentage of institutional standard found during the day and between different days varied from 92.67% to 97.11%, meeting the requirement for the accuracy of the analytical method. * AO quantification method with HPLC + Validate System suitability: Results showed that: RSD of retention time and peak area of oleanolic acid were less than 2%. The column efficiency was high at N> 20,000 and the peak asymmetric factor was about 1.15. Therefore, this method of analysis was compatible with the chromatographic system. + Method specificity: chromatograms of the sample had peak matching with retention time of AO standard; there was no peak in the chromatogram of the blank at the retention time of AO. Comparing the spectra of AO peak on the standard chromatogram 12 and the sample peak at the same retention time showed that these two spectra overlapped by 99% correlation, proving that the method was highly specific. + Linear concentration range: Results showed that within the concentration range of the investigation (from 11.13 to 111.25 µg/mL) there was a linear relationship between the peak area and the analyte concentration. The regression equation was y = 11333 x - 9726.9 with r2 = 0.9972. + Precision and accuracy: Results showed that the method had high precision with RSD values during a day and between different days were less than 3.3%, meeting the precision requirement for the analytical method. The recovery rates found during the day and between different days varied from 96.30 to 99.05%, meeting the accuracy requirements for the analytical method. + Limit of detection (LOD) and limit of quantitation (LOQ): results obtained LOD = 0.0122 µg/mL and LOQ = 0.04 µg/mL. 3.1.2.2. Results of setting up institutional standard for pharmaceutical raw material of T. palmata leaves Institutional standard of pharmaceutical material was proposed to have the following criteria: Description of pharmaceutical raw material: plant macroscopic, microscopic, powder morphology; weight loss due to drying (≤12%); total ash (10%); limits on heavy metals based on AAS method (As ≤ 1.0 mg/kg; Cd ≤ 1.0 mg/kg; Hg ≤ 0.1 mg/kg; Pb ≤ 3.0 mg/kg); oleanolic acid qualitation: according to TLC method; Quantification: SPN content ≥ 15 mg/g calculated based on AO by UV - Vis, AO content ≥ 750 µg/g by HPLC. 13 3.1.3. Results of formulating the process of preparation dried extract powder from T. palmata leaves 3.1.3.1. The process of preparing extract From the results of the investigation, the parameters of the extraction process of T. palmata leaf extract by ultrasonic extraction method were selected as follows: Table 3.18. Parameters of the extraction process of T. palmata leaves by ultrasonic extraction method No. Method/parameter Equipment/parameter 1 Ultrasonic extraction SM30-CEP device 2 Dried powder of T. palmata leaves Particle Size 0.5 - 1mm 3 Extraction solvent Ethanol 50% 4 Extraction temperatura 70ºC 5 Number of extraction Two times 6 DM/DL ratio 15mL/1gr/time x two times 7 Extraction time 90 minutes/time x two times Proceed to extract the scale of 2 kg/batch for 3 different batches. Concentrate the extract, remove the impurities (filter through thick cloth), evaporate the solvent (70ºC, reduced pressure, concentrate to 3:1 extract, settle for 24h, decant). The residue was added with EtOH 5 times, settle, decant, filter adjustably to 3:1 extract) The results obtained were as follows: 14 Table 3.20. Results of extract, concentrate and impurity removal of 3:1 extract of T. palmata leaves Index T.palmata leaves Extract 3:1 extract before removing impurity 3:1 extract after removing impurity Weight, volume 5.48 kg 170.0 L 1.83 kg 1.83 kg SPN(1) 22390 µg/g 683,98 µg/mL 156.00 ± 4.61 mg/g 205.67 ± 4.45 mg/g SPN weight 123,28 g 116,28 g 102,33 g 94.77 g The rate of dried residue in extract 35.93% 25.24% Extraction, extract concentration efficiency based on SPN 83.44% - Extraction, concentration and impurity removal efficiency based on SPN 77.27% SPN enrichment rate 131.84% SPN(1): SPN in 1 gram of extract solid. Results showed that: SPN content in 3:1 extract before removing the impurities was 156,00 ± 4.61 mg/g. After removing the impurities, concentrating and adjusting to 3:1 extract, the total saponin content was 205.67 ± 4.45 mg/g. The rate of SPN enrichment was 131.84%. 3.1.3.2. Prepare dried extract powder from T. palmata leaves by spray drying method From the survey results, the formula and parameters of the preparation process of dried extract powder from T. palmata leaves by spray drying method were selected as the followings: 15 Table 3.29. Formulation and parameters for the preparation process of dried extract powder from T. palmata leaves by spray drying method No. Parameter name Parameter 1 Liquid extract for spray drying Liquid extract (3:1) of T. palmata leaves with excipient 2 Spray drying excipient Aerosil 3 Excipient to solid ratio 30% 4 Inlet temperature 130 ± 2ºC 5 Feed flow rate 30 mL/min 6 CR/DP ratio 14% 7 Atomization pressure 2 Bar The established process to prepare dried extract powder of T. palmata leaves is described in the following diagram: 16 Figure 3.12. Scheme of the preparation stages of 3:1 liquid extract from T. palmata leaves ĐĐR extracts 1+2 1st ultrasonic extraction ĐĐR extract 1 ĐĐR extract 2 ĐĐR grounds 2 ĐĐR grounds 1 2nd ultrasonic extraction DM/DL = 15ml/1g 700C, 90 mins Concentrate, solvent recollect DM/DL = 15ml/1g 700C, 90 mins EtOH collection 3:1 ĐĐR liquid extract Fluid content1 Residue 1 EtOH 96º Fluid content 2 Residue 2 Settle, decant Settle, decant 3:1 ĐĐR liquid extract Decant, filter Concentrate, solvent recollect EtOH 50º Testing: - SPN: 165 - 245 mg/g - Solid proportion: 20 - 30% ĐĐR powder Meet the TCCS Decant, filter 17 Figure 3.13. Summary cheme of preparation stage of dried extract powder from T. palmata leaves by spay drying method 3.2. Results of setting up the quality standard and assessing the stability 3.2.1. Results of setting up the quality standard Conduct on 3 lots of dried extract powder (each lot included 2 batches of extract, each batch used 2 kg of pharmaceutical raw materials); The proposed result of the institutional standard of dried T. palmata extract powder include the following criteria: properties Feedstock Dried extract of ĐĐR Package 3:1 ĐĐR liquid extract Spray drying Inlet temp. 130 ± 20C Atomization pressure: 2 Bar - Add Aer, water: Aer to solid ratio: 30%, CR/DP: 14% - Stirring homogeneously Feed flow (30ml/mins) Testing according to TCCS 18 (softly dried powder, pale yellowish brown, homogeneous, bitter taste, typical smell); weight loss due to drying (≤5%); total ash (15%); limits on heavy metals by AAS method (As ≤ 1.0 mg/kg; Cd ≤ 1.0 mg/kg; Hg ≤ 0.1 mg/kg; Pb ≤ 3.0 mg/kg); bacterial contamination (agar plate method according to Vietnamese Pharmacopoeia V); oleanolic acid qualification: according to TLC method; SPN content quantification ≥ 100 mg/g and ≤ 200 mg/g calculated according to AO by UV - Vis method; AO content quantification ≥ 7.5 mg/g and ≤ 12,5 mg/g by HPLC. 3.2.2. Results of the evaluation of the stability of dried extract powder from T. palmata leaves Results showed that all evaluation criteria after 6 months in the accelerated aging condition and 12 months in long-term condition were all seen little changes and still reached the institutional standard of pharmaceutical. 3.3. Results of evaluating the immune enhancement effects and the toxicity of dried extract powder from T. palmata leaves on experimental animals 3.3.1. Evaluate the immune enhancement effect - On the general immunological indices: Dried extract powder from T. palmata leaves at both doses of 400 and 800 mg/kg had no effect of increasing the volume of lymphoid organs, but caused increased lymphocytes. T. palmata extract powder at 1st dose increased the number of peripheral white blood cells and increases the number of neutrophils. Whereas 2nd dose reduced the number of white blood cells and neutrophils. 19 - On humoral immunity indicators: Two doses of T. palmata extract powder both had the effect of increasing the concentration of IgG in peripheral blood. - On the cell-mediated immunity indices: T. palmata extract powder at both doses had the effect of increasing the secretion of IL- 2, 1st dose reduced TNF-α secretion, whereas 2nd dose caused increased secretion of TNF-α. 3.3.2. Assess toxicity 3.3.2.1. Acute toxicity: Results showed that the mortality rate of mice and the level of dose of dried T. palmata extract powder was strongly correlated with correlation coefficient r² = 0.9707 and the regression equation was y = 4.738e0.016x. Then, LD50 = 10.54 (8.79 - 12.65) g of T. palmata extract powder/kg, which was 26.35 times higher than the estimated effective dose. 3.3.2.2. Semi-chronic toxicity: Monitored for 90 consecutive days, the lots of mice administered T. palmata extract powder at the dose of 240 mg/kg/day and the dose of 720 mg/kg/day showed that: all mice were healthy, weight gain well and regularly; no changes in the hematological indices and the biochemical criteria for liver and kidney function evaluation; no damages on liver, spleen and kidney histopathology. CHAPTER 4: DISCUSSION 4.1. About formulating preparation process 4.1.1. Identify tracer groups Identified the main group of compounds in the leaves of T. palmata as saponins. The thesis had isolated six triterpenoid saponin compounds; including four known saponins and two new 20 compounds; contributing to the chemistry database for this species. SPNs were a group of substances with many immunological effects that have been studied. Among the known compounds, kalopanaxsaponin H has been studied for a number of biological effects such as antitumor and cancer cell-selective toxicity. Oleanolic acid has been proven a lot of pharmacological effects related to immunity such as anti-cancer, anti-diabetes, anti-inflammatory, liver protection, antioxidant, antibacterial, ... Therefore our thesis used total SPN and AO as tracers. 4.1.2. Choose two quantitative methods Our thesis used two quantitative methods: SPN was quantified by UV-Vis spectroscopy method, and AO was quantified by HPLC method. This is necessary because it will contribute to increased rigor and accuracy in product quality assessment. The formulation and validation of both quantitative methods were carried out in accordance with ICH guidelines, showing that the quantitative process met the requirements of the analytical method. 4.1.3. The institutional standard of T. palmata leaves Currently, in Vietnam and in the world, there have not been many research publications on T. palmata, there is no treatise on T. palmata in the pharmacopeias. In this study, we proposed the institutional standard based on samples collected in 5 provinces to evaluate the quality of raw T. palmata leaves. 4.1.4. The process of preparing extract powder from T. palmata leaves Ultrasonic extraction has been being applied more and more at laboratory to industry. Comparison between extraction methods 21 shows that ultrasonic extraction method has significantly improved extraction efficiency but at lower temperature and shorter time. In this study, ultrasonic extraction was used to investigate with the survey parameters: particle size of pharmaceutical raw material, type of extraction solvent, DM/DL ratio, extraction temperature and time. Within the framework of the thesis, ultrasonic extraction equipment had fixed the frequency of ultrasonic waves as 60 kHz, thereby the influence of ultrasonic frequency (a factor with great influence on extraction efficiency) had not been investigated. 4.1.5. Regarding extraction, concentration equipment and results of 3: 1 extract preparation After selecting parameters of extraction procedure, extract 3 consecutive batches, each batch of 2kg on 30-liter SM30 extraction device, 60kHz ultrasonic frequency, 8 ultrasound heads, 400W ultrasound power. Ultrasonic devices are typically characterized by ultrasonic frequencies (usually in the range of 20 - 100kHz) and ultrasonic power, but the structure of the device also affects the extraction process. In this study, the ultrasonic device with the ultrasound heads is fixed to the ultrasonic frequency at 60kHz, so it is not possible to investigate the effects of ultrasonic frequencies. Therefore, when scaling up equipment with other ultrasonic frequencies, it is necessary to calculate and re-evaluate the process parameters to achieve the desired extraction purpose. 4.1.6. Prepare dried extract powder from T. palmata leaves by spray drying In this thesis, the spray-drying method was selected to prepare dried extract powder from T. palmata leaves because this is a drying technique with many advantages and currently being widely applied 22 even at the industrial scale domestically. In order to develop the preparation process of dried extract powder from T. palmata leaves by spray-drying method, it is necessary to examine the parameters of the device (inlet gas temperature, feed flow rate) and the parameters spraying feed (solid ratio, type, excipient ratio). The parameters of the product to be assessed were: weight loss due to drying, hygroscopic property, apparent density, flowability, spray drying efficiency, active ingredient content, and efficiency. - The excipients selected for the survey were Mal, Aer, a mixture of Mal and Aer, Starch 1500, lactose. Aer was chosen because it has the effect of protecting active ingredients, regulating the flowability, improving the moisture absorption ... - Results of the investigations showed that when the Aer ratio increased, both the spray drying efficiency and other physicochemical indicators of the product were improved, the collection efficiency of the active ingredient also increased slightly, proving that Aer showed certain protective ability for active ingredients, but the total saponin content tended to decrease, this is due to the concentration dilution phenomena. In this study, selecting an excipient ratio of 30% was appropriate. - Investigation results showed that spray drying formulas at d

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