Study On The Active Constituents Of Bacopa Monnieri

"Jia Ma Chi Xian", the whole plant of Bacopa monnieri (L.)Wettst., is a perennial creeper in wet, damp and marshy areas. It is slightly sweet and light in taste, and cool in property according to the theory of Traditional Chinese Medicine. Studies show that the ethanol extract of whole plant of Bacopa monnieri (BM) can contract the womb of animals in vivo, relax skeletal muscle and smooth muscle. It also has cardiotonic and diuretic actions.BM is also an important constituent of Ayurvedic materia medical in Indian with reported memory-enhancing, anti-inflammatory, analgesic, antipyreticing., sedative and anti-epileptic properties. While BM has been reported to have many actions, its memory-enhancing effect has attracted most attention. BM in Ayurveda is classified as medhyarasayana, a drug used to improve memory and intellect (rnedhya). BM finds extensive used in Ayurveda as a brain tonic, restorative in debilitated conditions and for the promotion of longevity. Extensive investigation indicate that the ethanol extract of BM facilitated learning acquisition, improved retention of learning (memory) and delayed the extinction of learning tasks in several experimental paradigms in rats. The ethanol extract of BM can distinctly improve the vision memory, learning ability and memory confirmation in mice, and has actions of sedation, stabilization, anti-anxiety and anti-fatigue. Furthermore, it has good endurance and no sides effects. Pharmacological studies of the ethanol extract of the plant showed that which can significantly decrease the step-down errors of mice and latency time of electrical shock zone. The extract function by adding noradrenalin (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) to the hippocamp, striatum corpora and cerebral cortex of mice, so the concentration of Ach is indirectly altered, and other neural transmission systems are thus affected to improve the cognitive ability. The extract can increase the activity of protein kinase of the hippocamp, and increase the activities of antioxidant enzymes, such as SOD, catalase (CAT) and glutathione peroxidase (GPX), which can clear oxygen radicals, so that the cognitiveability of mice can be improved. Attempts have been made to isolate and define the different constituents of the ethanol extract of the plant, to explore chemical components in BM and clarify the material foundation and mechanism of its memory-enhancing effect.In view of the increasing interest on the neuropharmacological action of this herb, we are undertaking preliminary pharmacological studies on BM collected in Fujian Province.The results showed that the methanol extract of BM has significant anti-depressant activity in the most commonly paradigms of models depression namely, the forced swimming test and the tail suspension test in mice. The extract significantly reduced immobility period suggesting anti-depressant activity and the activity was compared with the vehicle drug fluoxetine in these tests. Furthermore, the step-down test in mice showed that the methanol extract of BM can significantly decrease the step-down errors of mice and latency time of electrical shock zone, which indicate that the extract can facilitated learning acquisition, improved retention of learning (memory) and delayed the extinction of learning tasks obviously.These results showed that the methanol extract of BM have a siginificantly variety of neuropharmacological actions, and thus warrant systematic study on their chemical components and intensive study on the mechanism. Through the pharmacological tests of multi-index, multi-animals models and safety evaluation, monomeric components were screened out which were obvious in drug-effect and safe. Artificial synthesis and structure optimization are to be performed to facilitate future studies. These efforts will lay a scientific references and material foundations for developing anti-depressant and anti-AD drugs with our own independent property right.On account of that, we are undertaking a thorough chemical re-investigation of the methanol extract of the plant, using non-polar macroreticular resin, sephadex LH-20, ODS, HPLC and silica gel chromatography. Forty-six compounds were isolated and the structures of them were elucidated by chemical and spectural technology (infrared spectroscopy IR, mass spectroscopy MS, nuclear magnetic resonance NMR, ultraviolet spectroscopy UV).The bioassay-guided method are used to screen effective extract fractions andactive monomers of BM on the level of intact animal and molecular pharmacology, using the animal models with step down test, forced swimming test, tail suspension test and MTT test. The results showed that the neuropharmacological activities of the herb are associated with the polar extract containing mainly saponins as a complex mixture.We found that the monomers of Bacopasaponin C and Bacopaside VII have significent effect on memory-enhancing at the dosage of 100mg/kg and 50mg/kg, versus vehicle group P < 0.01 and P < 0.001, respectively. When the mice is adminitrated with the dose 100 mg/kg, the compound Bacopaside I showed the strongest antidepressant activity (P < 0.01) among the tested compounds. At the dosage of 100g/ml, the cytotoxic ratio of Bacopaside I against human tumor cell lines of K-562. A-549 PC-3VL HCT-8 and SHG-44 are 100% in vitro. Furthermore, the inhibitory of Bacopaside I to K-562 is 61.69% at the dosage of 0.01 ug/ml.An LC-MS/MS-based method has been developed for characterization and quantification of four major activity saponins (Bacopaside I , II > VD and Bacopasaponin C) in two chromatographic runs in the whole plant of BM. Optimization of the ionination process was performed with electrospray and atmospheric pressure chemical ionization techniques in both positive and negative ion modes. Postive ion ESI was adopted for generation of the precursor deprotonated molecules to achieve the best ionization sensitivity for the analytes. In addition, the most abundant fragment ion was chosen for each analyte to give the best CID sensitivity. Because some of the saponin derivatives are isomeric, complete resolution for the whole analytes was achieved both chromatographically and mass spectroscopically. Good linearity over the rang 20 to 5 ug/ml for the analytes was observed. The accuracy and precision for intraday and interday at nominal low, intermediate, and high concentration were determinated. The lower limits of determination and quantitation for the test compounds were 0.1 to 1.0 u.g/ml and 0.2 to 2.0 ug/ml on-column. After the validation, the applicability of the method for determination of these chemicals present in the plant of BM has been demonstrated. The sensitivity and specificity of the technique will be the basis of a method for theaccurate quantification of the saponins in the herb.The structures of compounds isolated and purified from BM were elucidated as: BM-1: p-hydroxy benzenemethanol BM-2: /?-hydroxyl benzoic acid BM-3: A5'24(28)-ergostadien-3-y9-ol BM-4: ursolicacidBM-5: lupeol, lupo-20(29)-ene-3-yff-ol BM-6: 28-hydroxyllupeol BM-7: betulinic acid BM-8: stigmasterolBM-9: stigmasterol-3-0-/?-￡>-glucopyranoside BM-10: 3-0-stigmasterol-(6-0-palmitoyl)-/^Z)-glucopyranoside BM-11: /?-sitosterol BM-12: /?-daucosterin BM-13: Ampelozigenin,16,22:16,30-diepoxydammar-24(24')-methylene-3,15,20-triol BM-14: BacopasideB,3,4-dihydroxyphenylethyl alcohol (2-O-feruloyl)-^-D-glucopyranoside BM-15: BacopasideC,phenylethyl alcohol [5-0-/^hydroxybenzoyl-/?-D-apiofuranosyl(l—>2)]-/?-jD-glucopyranoside BM-16: 3,4-dimethoxycinnamic acid BM-17: feruloyl sucrose BM-18: rosavin BM-19: stearicacid BM-20: H-octacosanoic acid BM-21: /i-pentatetracontanoic acid BM-22: quercetin BM-23: apigeninBM-24: apigenin-7-O-glucopyranosiduronic acidBM-25: luteolinBM-26: luteolin-7-O-glucosideBM-27: luteolin-7-O-glucopyranosiduronic acidBM-28: Bacopaside I ,3-0-{6-Osulphonyl-/?-D-glucopyranosyl(l—+3)[a-Z,-arabinofuranosyl(1 —?2)]-ar-Z-arabinopyranosyl} pseudojujubogenin BM-29: Bacopaside II,3-O-^-D-glucopyranosyl(l -*3)-[or-L-arabinofuranosyl(l —>2)]-y5-I>-glucop-yranosyl pseudojuj ubogenin BM-30: Bacopaside III,3-0-[a-I-arabinofuranosyl(l —?2)-/?-D-glucopyranosyl] jujubogenin BM-31: Bacopaside IV,3-0[/#-￡>-glucopyranosyl( 1 —*3)- a-Z-arabinofuranosyl] jujubogenin BM-32: Bacopaside V,3-O-[/?-Z)-glucopyranosyl(l —?3)-a-Z-arabinofuranosyl] pseudojujubogenin BM-3 3: Bacopasaponin A,3-0- a-Z-arabinopyranosyl-20-C?- a-L-arabinopyranosyl j uj ubogenin BM-34: Bacopasaponin B,3-O-[cr-￡-arabinofuranosyl(l—?2)-y5-D-arabinopyranosyl]pseudojujubogenin BM-35: Bacopasaponin C,3-O-{^-D-glucopyranosyl(l—>3)[flr-I-arabinofuranosyl(l—*2)]or-I-arabino-pyranosyl} pseudojujubogenin BM-3 6: Bacopasaponin D,3-O-[(2r-Z-arabinofuranosyl(l —?2)-/?-Z)-glucopyranosyl] pseudojujubogenin BM-37: BacosideA3,3-O-/?-￡>-glucopyranosyl(l—>3)-[a-I-arabinofuranosyl(l—?2)yranosyl jujubogeninBM-38: Bacopaside Ai,3-O-[a-Z,-arabinofuranosyl( 1 -^3)-y3-D-arabinopyranosyl] jujubogenin BM-39: Bacopaside G,3-<9-[a-Z-arabinofuranosyl( 1 —*2)-/?-￡>-arabinopyranosyl] jujubogenin BM-40: Zizyotin,3-0-ar-L-arabinopyranosyl jujubogenin BM-41: 1 -octan-3-O-^-D-glucopyranoside BM-42: Bacopaside VI,3-O-[6-(9-sulphonyl-/0-D-glucopyranosyl( 1 —>3)]-a-Z,-arabinopyranosylpseudojujubogenin BM-43: Bacopaside VD,3 -O- {/9-￡>-glucopyranosyl( 1 —*3) [ or-Z,-arabinofuranosyl( 1 —*2)] or-Z,-arabino-pyranosyl }jujubogenin BM-44: Bacopaside VD1,3-0- {/?-￡>-glucopyranosyl( 1 —O) [ a-￡-arabinofuranosyl( 1 —+2)]/?-Z)-glucop-yranosyl}-20-a-X-arabinopyranosyl jujubogenin BM-45: Bacopaside K,3-0-{6-0-sulphonyl-/?-.D-glucopyranosyl(l—?3)[a-Z,-arabinoftiranosyl(l-*2)]a-I-arabinopyranosyl} jujubogenin BM-46: loliolide (digiprolactone)Among them, BM-41, BM-42, BM-43, BM-44 and BM-45 are new compounds, andBM-l~6> 9> 10, 12, 13, 16-23, 25, 26, 40, 46 were isolated from this herb for the first time.