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@#Abstract: Objective - To investigate the effect of betulin on the cognition and emotion of rats with chronic aluminum induced Methods dementia and its influence on the expression of inflammatory factors in amygdala. The specific pathogen free female - - SD rats were randomly divided into control group, model group, intervention control group, low dose group and high dose group, with 10 rats in each group. The rats in the later four groups were given with 100 mg/kg body weight and 10.0 g/L aluminum chloride solution by gavage, while the rats in the control group were gavaged with the same volume of distilled water, once a day for 60 days. The rat model of dementia caused by chronic aluminum exposure was established. The rats in the intervention - - control group were given donepezil hydrochloride (0.5 g/L) by gavage, the rats in the low dose and high dose groups were given betulin (20.0 and 60.0 g/L) by gavage, and the rats in the control and model groups were given the same volume of distilled water - by gavage, once a day for four weeks. Anxiety like behavior was evaluated by Y maze new arm test and elevated cross maze test. - The depression like behavior was evaluated by forced swimming test. The amygdala of rats was isolated, and the expression of - - phosphorylated Tau (p Tau) protein andcell derived factor 1 (SDF 1), C X C chemokine receptor (CXCR) 3, CXCR4 and interferon inducible protein 10 (IP 10) was Results detected by Western blotting. In the behavioral experiments, the residence time in the new arm and the percentage of P P time in the open arm of the model group decreased (all <0.05), while the cumulative immobility time increased ( <0.05) compared with the control group. The residence time in the new arm and the percentage of time in the open arm increased in the - - P intervention control group, low dose group and high dose group (all <0.05), while the cumulative immobility time decreased (all P<0.05) compared with the model group. The residence time in the new arm and the percentage of time in the open arm increased - - P P in the low dose group and the high dose group (all <0.05), while the accumulated immobility time decreased (all <0.05) compared with the intervention control group. The residence time in the new arm and the percentage of time in the open arm P P - - increased (all <0.05), while the accumulated immobility time decreased ( <0.05) in the high dose group compared with the low - - - - - dose group. The relative expression of p Tau, βAPP, IL 1, IL 6, IL 8, CXCR3 and IP 10 increased, while the relative expression - P - of SDF 1 and CXCR4 decreased in the model group compared with the control group (all <0.05). The relative expression of p - - - - - Tau, βAPP, IL 1, IL 6, IL 8, IP 10 and CXCR3 decreased, while the relative expression of SDF 1 and CXCR4 increased in the - - P intervention control group, low dose group and high dose group compared with the model group (all <0.05). The relative - - - - - P - expression of p Tau, βAPP, IL 1, IL 6, IL 8, IP 10 and CXCR3 decreased (all <0.05), while the relative expression of SDF 1 - - and CXCR4 increased in the intervention control group, low dose group and high dose group compared with the intervention P Conclusion control group (all <0.05). Betulin can protect cognitive function and improve mood in chronic aluminum - exposure induced dementia model rats. The mechanism may be related to inhibiting the production of inflammatory factors and - chemokines in the amygdala of rats, thereby inhibiting the synthesis of p Tau protein and βAPP protein. βamyloid precursor protein (βAPP) was detected by immunohistochemistry. The
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Objective: To study the effect of betulin derivatives combination with 5-fluorouracil or hydrazine sulfate on the ROS generation, the SOD and LDH activity using rat blood, as well as the effect of combination drugs on Ehrlich carcinoma in experiments on mice. Methods: We used a chemiluminescence technique to study the ROS generation, and spectrophotometry to determine the MDA level and the SOD and LDH activity. The model of transplanted Ehrlich ascites carcinoma was investigated on mice using a cytological analysis of ascitic fluid cells according to Pappenheim`s method. Results: In vitro experiments on rat blood at the doses of 2, 5 and 10 μg per ml revealed the dose-dependent effect of combination drugs on the antioxidant properties. In plasma, the ROS generation and the MDA level increased by 10-300% in comparison with control at the doses of 5 and 10 μg per ml only. Still, the SOD and LDH activity in general increased by 10-130% in comparison with control under the action of the studied combination drugs. The study on mice showed the effectiveness of a combination of triterpenoids and cytostatics in Ehrlich ascites carcinoma therapy. The state and behavior of the animals improved, the volume of ascites fluid decreased by 40-50% after treatment for 10 d. Conclusion: The combination of betulin derivatives with cytostatics can be used as antitumor drugs in Ehrlich ascites carcinoma therapy that is due to metabolic plasticity, increased ROS generation in enhanced antioxidant enzyme protection.
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Betulinic acid is a natural pentacyclic pentane triterpenoid, usually isolated from the bark of Betula platyphylla, or in the form of free glycosides and glycosyl derivatives in various plants. A variety of derivatives can be obtained by modifying its chemical structure. Betulinic acid and its derivatives have certain regulatory effects in anti-tumor, antiviral, anti-inflammatory, analgesic, inhibition of cerebral nerve and vascular injury, and treatment of other common diseases. The category, pharmacological activities and related mechanisms of betulinic acid and its derivatives are reviewed in this paper, in order to provide a theoretical reference for the future application.
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Objective: To study betulin-3,28-diphosphate (BDP) water solubility improved by forming salt complexes with hydrophilic amino alcohols: meglumine as acidosis corrector and xymedon as the water-soluble antioxidant. Methods: We used 13C-, 31P-NMR, UV-spectroscopy and potentiometric titration to study the BDP-amine salt complexes formation and their solubility using HPLC-analysis. Results: The participation of xymedon in the proton transfer reaction with BDP in aqueous solutions was confirmed by the bathochromic shift of the carbonyl band from 299.1 nm to 304.2 nm, and by a hyperchromic effect (molar extinction ε from 8508 to 10 441 l·mol-1·cm-1) in UV-spectra. BDP complexation with meglumine was estimated by UV-spectral molar ratio method at 256 nm. Molar ratio of BDP-amine complexes (1:4) was proved by 31P-NMR. The chemical shift of phosphorus at C-3 atom of BDP (δ =-0.58 ppm) changed to+3.39 ppm, and at C-28 atom (δ =+0.28 ppm)–to+4.60 ppm. BDP solubility increased 100-600 fold according to HPLC-analysis. Conclusion: BDP interaction with amine in an aqueous solution was shown to proceed via a proton transfer due to relatively weak forces such as London forces, hydrogen bonding, electrostatic and hydrophobic interactions. In general, the formation of BDP salt complexes with amines in solution determines BDP water solubility. Water-soluble BDP enables to develop hydrophilic dosage forms.
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Objective: To analyze the effect of carbon monoxide (CO) on the growth of Betula platyphylla suspension cells and triterpenoid production. Methods: CO donor hematin of 5, 10, 20, and 30 μmol/L were added to eight-day-old B. platyphylla suspension cells, and the change of triterpenoid content and gene expression of FPPS, LUS, CAS1, CAS2, and β-AS related to the triterpenoid synthesis were analyzed by chemical colorimetry, high performance liquid chromatography and real-time PCR, respectively. Results: CO treatment did not have a significant effect on dry weight of B. platyphylla suspension cells except for 2-8 d at 20 μmol/L and 30 μmol/L CO treatment. CO treatment also did not have a significant effect on pH value and malondialdehyde content except for 30 μmol/L CO treatment. CO treatment significantly increased the content of triterpenoid, betulin and oleanolic acid. the content of triterpenoid, betulin and oleanolic acid were the highest after 1 d at 30 μmol/L CO treatment, 4 d at 10 μmol/L CO treatment and 2 d at 10 μmol/L CO treatment, which was 1.3, 1.5, and 4.2 times of the control, respectively. Triterpenoid production in B. platyphylla suspension cells induced by CO was further confirmed by key enzyme genes of FPPS, LUS, CAS1, CAS2, and β-AS related to the triterpenoid synthesis by RT-PCR. Conclusion: CO treatment could effectively enhance the triterpenoid production in B. platyphylla suspension cell culture.
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Aims: In our search for new antiplasmodial agents, in vitro antiplasmodial activities of the crude extracts and isolated pure compounds were determined. In addition to the in vitro assays, in vivo acute toxicity of the crude extracts was investigated to assess the safety of the plants. Furthermore, structure elucidation of the pure compounds was also carried out to determine the identity of the isolated compounds.Study Design: Extraction of the root crude extracts of Euclea latideus was done using four solvents: hexane, dichloromethane, ethyl acetate and methanol. Isolation and purification were carried out on only the dichloromethane and ethyl acetate crude extracts.Methodology: Four solvent; hexane, dichloromethane, ethyl acetate and methanol were used to carry out the extraction process of the crude samples. Isolation and purification of crude extracts were achieved using chromatographic techniques which included column and thin layer chromatography (TLC). The characterization of the isolated compounds was determined using NMR spectroscopic techniques. In vitro antiplasmodial activity was performed on two strains of Plasmodium falciparum (chloroquine [CQ]-sensitive 3D7 and CQ-resistant Dd2 strains) using a non-radioactive fluorescence-based SYBR Green 1 assay technique. Lorke's method of acute toxicity was used to determine the in vivo acute toxicity of the crude extracts in mice.Results: Results of acute toxicity studies showed that all crude extracts of E. latideus had LD50 > 5000 mg/kg and therefore regarded as a non-toxic plant. The four crude extracts of E. latideus had good activityWith range of (IC50) 3D7: (9.75-38.21) µg/mL and Dd2: (2.78-38.93) µg/mL. The resistance indices for E. latideus crude extracts ranged between 0.10- 1.43, suggesting that some of the extracts had equal promise against the CQ resistant strain of P. falciparum. Isolation resulted in the identification of three known compounds which include; three triterpenoids Lupeol (EL1), betulin (EL2), 3?-(5-hydroxyferuloyl)lup-20(30)-ene (EL3 ). Among the pure compounds EL2 had the highest activity against on both strains (IC50) 3D7: 1.64 ± 0.02 µg/mL and Dd2: 7.69 ± 1.21 µg/mL while Lupeol (EL1) displayed moderate activity with (IC50) 3D7: 23.91 ± 0.05 µg/mL, Dd2: 25.14 ± 0.01 µg/mL. The antiplasmodial activity of the crude extracts and pure compounds were significantly different (P < 0.05) from that of the reference standards (chloroquine diphosphate and mefloquine hydrochloride). Both the crude extracts IC50 (2.78-38.93) µg/mL and pure compounds IC50 (1.64-25.14) µg/mL showed a significant decrease in activity compared to the reference standards (0.0056-0.0440) µg/mL. Significant difference (P < 0.05) also existed between the antiplasmodial activities of the crude extracts, which showed the same trend with that of the pure compounds.Conclusion: The results show that the root crude extracts and pure compounds of the plant have good antiplasmodial activity and low toxicity which can be exploited for malaria therapy. Therefore, this justifies their ethnomedicinal use of the plant by the local communities of Butebo Sub-County, in Pallisa District in Eastern Uganda in the treatment of malaria.
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We investigated whether betulin affects the gene expression, secretion and proteolytic activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective effect of betulin. Rabbit articular chondrocytes were cultured and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of betulin on IL-1β-induced secretion and proteolytic activity of MMP-3 was investigated using western blot analysis and casein zymography, respectively. Effect of betulin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) betulin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) betulin inhibited the secretion and proteolytic activity of MMP-3; (3) betulin suppressed the production of MMP-3 protein in vivo. These results suggest that betulin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes.
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Animals , Rats , Blotting, Western , Caseins , Chondrocytes , Collagen Type II , Gene Expression , Knee Joint , Knee , Osteoarthritis , ThrombospondinsABSTRACT
Objective: Marine fungi play an important role in human and animal health, leading compounds to new drug discoveries and prospects for their bioactivity potential. Materials and Methods: Paecilomyces WE3-F was isolated from marine sediment (Red Sea, Shalateen, Egypt). Fungal isolate was screened for their antagonistic activity against four Gram-positive (Bacillus cereus, Lesteria monocytogenes, Micrococcus luteus and Staphylococcu aureus) and four Gram-negative (Aeromonas hydrophila, Flavobacteruim sp, Pseudomonas aeruginosa and Vibrio cholera,) pathogenic bacteria. Paecilomyces WE3-F was identified using 18S rRNA technology. Seven factors were chosen to be screened for bioactivity using the Placket Burman experimental design: sucrose, yeast extract, Na NO3, temperature, initial pH, inoculum size, and incubation period. Results: Among conditional factors, acidic pH and 1.5 ml inoculum size favored the bioactive metabolites. Furthermore, a number of solvents have been experimented for the extraction of the bioactive metabolite(s). Dichloromethane (DCM) crude extract from the fermentation broth of a marine Paecilomyces WE3-F showed the highest activity with averages of 26 and 24 mm against G-ve and G+ve, respectively. Under optimal culture conditions, the maximum extractable compound concentration in a 10-L culture medium reached 83.4 mg/L. Based on data obtained by thin layer chromatogram (TLC), gas chromatography - mass spectrum (GC-MS) and Fourier Transform Infrared (FTIR) the major compound, betulin was structurally identified. Conclusions: The isolated marine Paecilomyces WE3-F, therefore, showed the ability to produce a betulin yield after optimal operating conditions for antibacterial potential.
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Objective To explore the mechanism and regulative function of betulin on anti-aging genes related of normol human dermal fibroblasts (ESF-1), and to reveal the repairing mechanism of betulin on wrinkles. Methods The cultured cell in vitro were divided into the control group, the estradiol group and the betulin high, medium and low dose groups. Competence of ESF-1 cells was detected by MTT. Expression of mRNA of collagen type I and Ⅲ (ColⅠand Col Ⅲ), tissue inhibitors of matrix metalloproteinase 1 and 2 (TIMP-1 and TIMP-2), matrix metalloproteinase 1 (MMP-1) was detected by RT-PCR. Results Compared with the control group, the proliferation of ESF-1 cells (0.455 ± 0.037, 0.445 ± 0.040 vs. 0.385 ± 0.601) in the estradiol group and the medial-dose betulin group were increased (P<0.05). Compared with the control group, the expression of mRNA of ColⅠ (0.960 ± 0.012, 0.929 ± 0.015 vs. 0.842 ± 0.014), Col Ⅲ (0.892 ± 0.009, 0.824 ± 0.022 vs. 0.768 ± 0.025), TIMP-1 (0.938 ± 0.026, 0.878 ± 0.035 vs. 0.796 ± 0.022), TIMP-2 (0.557 ± 0.025, 0.506 ± 0.036 vs. 0.436 ± 0.063) in the estradiol group and the medial-dose betulin group were increased (all P<0.01). Conclusion Betulin based on increasing the competence of ESF-1 cells, promoting collagen synthesis and the expression level of related-proteinase is to suspend the development of wrinkles.
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Objective: To develop an HPLC-MS/MS method for the determination of jujuboside A, jujuboside B, spinosin, betulinic acid, betulin, rutin, apigenin, hesperidin, and naringin in Ziziphi Spinosae Semen. Methods: Analysis was performed on a Sapphire C18 column (150 mm × 4.6 mm, 5 μm) eluted with acetonitrile and 0.1% methanoic acid solution containing 1 mmol/L ammonium acetate in a gradient program. The flow rate was 1 mL/min, the injection volume was 10 μL, and the column temperature was 30℃. The multiple-reaction monitoring scanning (MRM) was employed for the quantification with switching electrospray ion source polarity in negative mode. The ion spray voltage was set at -4 500 V and the turbo spray temperature was maintained at 650℃. Results: The regression equations showing linear relationships between peak areas and contents of each compound were obtained. The average recoveries of the compounds ranged from 98.04% to 101.5% and the precision in terms of RSD was in the range of 0.03%-0.74%. Conclusion: The method is simple, rapid, and sensitive. It could be used as a quantitative determination method for the nine components in Ziziphi Spinosae Semen.
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Objective: To study the chemical constituents in the whole herb of Lycopodium japonicum. Methods: The chemical constituents were separated and purified by silica gel, Sephadex LH-20 column chromatography, and preparative HPLC. Their structures were identified by various spectroscopic analyses. Results: Thirteen compounds were obtained from the whole herb of L. japonicum by the chromatographic methods on silica gel, ODS, and Sephadex LH-20 column, and preparative HPLC. According to physicochemical properties and spectral data, these compounds were identified as betulin (1), di-(2-ethylhexyl) phthalate (2), α-onocerin (3), 16-oxo-3α-hydroxyserrat-14-en-21β-ol (4), 3-epilycoclavanol (5), (24S)-24-methyl cholesterol (6), lycopodiin A (7), tomentosanan B (8), α-obscurine (9), lycoclavanol (10), serratenediol (11), 1, 2-diarylpropane-3-ol (12), and 13, 13-ethylenedioxy-15, 16-dinwlabd-7-en-6β-ol (13). Conclusion: Compounds 1, 2, 6, 8, and 11-13 are isolated from this plant for the first time. Compounds 2, 6, 8, and 12 are isolated from the plants of this genus for the first time.
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Objective: To investigate the chemical constituents in the dried leaves of Sabia fasciculata. Methods: Column chromatography such as silica gel, Sephadex LH-20, and preparative HPLC were used to isolate and purify the compounds. Spectroscopic methods like MS, 1H-NMR and 13C-NMR, and physical constants were used to elucidate their structures. Results: Fifteen compounds were isolated from 95% ethanol extracts of S. fasciculata, including seven pentacyclic triterpenoids, such as methyl-3-oxo-olean-12-ene-28-oate (1), betulin (2), 3-oxo-olean-Δ11,13(18)-diene (3), oleanolic acid (4), imberic acid (5), pseudo- ginsenoside RP1 (6), and chikusetsusaponin IVa (7); three flavonoids, such as quercetin (8), rutin (9), and mutabiloside (10); three alkaloids, such as fuseine (11), N-p-feruloyltyramine (12), and N-trans-coumaroyl tyramine (13); two steroids, such as β-sitosterol (14) and β-daucosterol (15). Conclusion: All the compounds are isolated from the plant for the first time. Compounds 1, 5-7, and 10 are isolated from the plants of Sabia Colelbr. for the first time.